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Part 1. Synthesis of arylalkylguanidines as dopamine agonists. Part 2, Section A. Modifications of trimetoquinol and the effects on /3-adrenergic and thromboxane2 receptor A systems. Section B. Approaches to the asymmetric synthesis of irreversibly binding iodinated derivatives of trimetoquinol
Christoff, Jeffrey James, Ph.D.
The Ohio State University, 1993
300 N. Zeeb Rd. Ann Arbor, M I 48106
Part 1: Synthesis of Arylalkylguanidines as Dopamine Agonists
Part 2, Section A: Modifications of Trimetoquinol and the Effects on
B-Adrenergic and Thromboxane A2 Receptor Systems
Section B: Approaches to the Asymmetric Synthesis of Irreversibly Binding
lodinated Derivatives of Trimetoquinol
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
By
Jeffrey J. Christoff, B.S Pharm., R.Ph.
* * * * *
The Ohio State University
1993
Dissertation Committee: Approved by
Duane D. Miller, Ph.D.
Robert W. Brueggemeier, Ph.D. Duane D. Miller, Ph.D., co-advisor
Larry W. Robertson, Ph.D.
Dennis R. Feller, Ph.D. Robert W. Brueggemeier, Ph.D., co-advisor College of Pharmacy Dedicated to Cindy
The Time is Now Ours to Share
ii ACKNOWLEDGEMENTS
I wish to thank:
Dr. Duane D. Miller for his support and guidance throughout my graduate career.
Dr. Robert W. Brueggemeier for his advice and support while serving as co-advisor.
The dissertation committee for their input in preparing this document.
Dr. Dennis Feller and Dr. Norman Uretsky for their collaborative efforts.
Gamal Shams, Karl Romstedt, Paul Fraundorfer, Tahira Farooqui, Longping Lei, and Fernando Rodriguez for their hard work evaluating my compounds.
Jill (Golder) O’Reilly for her friendship, advice and tolerance.
Carl Lovely, Pat Cyr, Jeff Herndon, Kazu Matsumoto, Ron Hill, Yasser Abdel-Ghany, and Meri Slavica for their suggestions and criticisms.
Nancy (Katlic) Gilbert for her expert advice and aid in preparing slides and posters.
Kim Risch, Joan Dandrea, Kathy Brooks, and Carol Settles for their assistance with numerous predicaments.
Jack Fowble, John Miller, and Bruce Posey for their assistance with instrumental and computer complications.
Luke Bradley for his invaluable work on the Trimetoquinol project.
Damon Sharp and Margie Centeno for steadily supplying me with clean glassware. Dr. B’s research group for adopting me as one of their own and tolerating synthetic organic chemistry during group meetings.
The entire Division of Medicinal Chemistry and Pharmacognosy for allowing me to mooch whatever, whenever I needed it.
Dr. Doskotch for his objective opinions and comments.
LAGNAFs, Home Platelets, Heavily Sedated and Topliss Dancers for providing a physical and emotional diversion from work.
The National Institutes of Health, American Foundation for Pharmaceutical Education, College of Pharmacy, and Graduate School for financial support.
iv VITA
Aug. 14, 1965 Born - Pittsburgh, Pennsylvania
May 8, 1988 B.S. Pharmacy, Duquesne University Pittsburgh, Pennsylvania
May 1988 - Aug. 1988 Research Intensive Summer Intern Merck, Sharp & Dohme Research Laboratories West Point, Pennsylvania
Sept. 1988 - Aug. 1989 University Fellow The Ohio State University Columbus, Ohio
Sept. 1988 - Oct. 1993 Academic Challenge Fellow The Ohio State University Columbus, Ohio
Sept. 1989 - Aug. 1990 Graduate Teaching Assistant The Ohio State University Columbus, Ohio
Sept. 1990 - Oct. 1993 Graduate Research Associate The Ohio State University Columbus, Ohio
Sept. 1991 - Oct. 1993 American Foundation for Pharmaceutical Education Fellow The Ohio State University Columbus, Ohio
v PUBLICATIONS AND PRESENTATIONS
1. Katdare, A.V.; Keller, K.O.; Christoff, J.J.; Bavitz, J.F. Evaluation of Dissolution Characteristics of an Encapsulated Water Soluble Tablet Granulation. Drug Dev. Ind. Pharm. 1990, 16(7), 1109-1119.
2. Christoff, J.J.; Harrold, M.W.; Miller, D.D.; Fraundorfer, P.; Romstedt, K.J.; Feller, D.R. Modification of the Trimethoxybenzyl Substituent of Trimetoquinol and its Effects on (3-Adrenergic and Thromboxane A2 Receptor Systems. Presented at the 25th Annual Graduate Student Symposium in Medicinal Chemistry, Ann Arbor, Ml, June 28-30, 1992. Session 2, First Presentation.
3. Christoff, J.J.; Miller, D.D.; Farooqui, T.; Uretsky, N. Arylalkylguanidines as Dopaminergic Agonists. Presented at the 26th Annual Graduate Student Symposium in Medicinal Chemistry, West Lafayette, IN, June 27- 29, 1993. Fifth Poster.
4. Christoff, J.J.; Miller, D.D.; Farooqui, T.; Uretsky, N. Synthesis and Biological Evaluation of Arylalkylguanidines as Dopaminergic Agonists. Presented at the 206th National Meeting of the American Chemical Society, Chicago, IL, August 22-27, 1993. Paper MEDI 170.
FIELDS OF STUDY
Major Field: Pharmacy
Studies in : Synthetic Medicinal Chemistry
vi TABLE OF CONTENTS
PAGE
DEDICATION ...... ii
ACKNOWLEDGEMENTS ...... iii
VITA ...... v
LIST OF TABLES...... xi
LIST OF FIGURES...... xiii
LIST OF SCHEMES...... xvi
Part 1 Synthesis of Arylalkylguanidines as Dopamine Agonists ...... 1
CHAPTER I INTRODUCTION ...... 2 1.1 Background ...... 2 1.2 Location and Distribution of Dopamine ...... 5 1.3 Dopamine Receptor Subtypes ...... 7 1.4 Therapeutic Uses of Dopamine Agonists ...... 11 1.5 Classifications of Dopamine A g o n ists ...... 14 1.5.1 Phenylethylamines ...... 15 1.5.2 Conformationally Restricted Analogues of Dopamine 17 1.5.2.1 Miscellaneous Semi-rigid Analogues ...... 18 1.5.2.2 Aminotetralins ...... 20 1.5.2.3 Aminoindanes ...... 22 1.5.2.4 Benzoquinolines ...... 24 1.5.2.5 Nitrogen Heterocycles ...... 25 1.5.3 Naturally Occurring Dopamine A gonists ...... 26 1.5.3.1 A p o rp h in e s ...... 27 1.5.3.2 Ergot Alkaloids ...... 28
vii 1.5.4 Dopamine Bioisosteres ...... 29 1.5.4.1 Permanently Charged Derivatives ...... 30 1.5.4.2 Permanently Uncharged Derivatives ...... 32 1.6 Drug-Receptor Interactions ...... 33 1.6.1 Hydrogen Bonding ...... 33 1.6.2 Ionic Bonding ...... 34 1.6.3 Reinforced Ionic Bonding ...... 34 1.7 Summary ...... 35
CHAPTER II STATEMENT OF THE PROBLEM AND OBJECTIVES ...... 37
CHAPTER III RESULTS AND DISCUSSION ...... 46 3.1 Chemistry ...... 46 3.2 Biological Evaluation ...... 72 3.3 Summary ...... 76
CHAPTER IV EXPERIMENTAL ...... 78
Part 2 Section A Modifications of Trimetoquinol and the Effects on 13-Adrenergic and Thromboxane A2 Receptor Systems ...... 128
CHAPTER V INTRODUCTION ...... 129 5.1 6-Adrenergic Agonism ...... 129 5.1.1 Sympathetic Nervous System ...... 130 5.1.1.1 Catecholamine Biosynthesis ...... 131 5.1.1.2 Catecholamine Metabolism and Reuptake ...... 132 5.1.2 6-Adrenergic Receptors ...... 133 5.1.3 Molecular Biology of (3-Adrenergic R eceptors ...... 138 5.1.4 SAR of 6-Adrenergic Agonists ...... 142 5.1.5 Therapeutic Uses of 8-Adrenergic Agonists ...... 146 5.2 Thromboxane A2 Antagonism ...... 148 5.2.1 Platelet P h y s io lo g y ...... 150 5.2.2 Platelet Activation of Hemostasis ...... 150 5.2.2.1 Thromboxane A2 Platelet Activation ...... 152 5.2.3 Platelet Involvement in Cardiovascular Disease ...... 155 5.2.4 Thromboxane A2 Antagonists ...... 157 5.2.4.1 Prostanoid Antagonists ...... 158
viii 5.2.4.2 Non-prostanoid Antagonists ...... 160 5.2.5 Molecular Biology of Thromboxane A2 R eceptor ...... 162 5.3 Trimetoquinol and Derivatives ...... 162 5.3.1 N-Alkylated Trimetoquinol D erivatives ...... 164 5.3.2 Modified Tetrahydroisoquinoline TMQ Derivatives ...... 165 5.3.3 1-Substituted 1,2,3,4-Tetrahydroisoquinoline Derivatives ... 169 5.4 Irreversible Binding of D rugs ...... 173 5.4.1 Affinity Labeling ...... 174 5.4.2 Photoaffinity Labeling ...... 175
CHAPTER VI STATEMENT OF THE PROBLEM AND OBJECTIVES ...... 178 6.1 N-Phenylethyl Derivative ...... 179 6.2 Amidine Derivative ...... 181 6.3 lodinated Derivatives ...... 182 6.3.1 Retrosynthetic Strategy of lodinated Affinity and Photoaffinity Labels ...... 184
CHAPTER VII RESULTS AND DISCUSSION ...... 191 7.1 Chemistry ...... 191 7.1.1 N-Phenylethyl-Trimetoquinol ...... 191 7.1.2 Amidine-Trimetoquinol ...... 195 7.1.3 Modified Trimetoquinol Derivatives ...... 201 7.1.4 N-Protected Trimetoquinol Derivatives ...... 207 7.2 Biological Evaluation ...... 215 7.3 Summary ...... 219
CHAPTER VIII EXPERIMENTAL ...... 221
Part 2 Section B Approaches to the Asymmetric Synthesis of Irreversibly Binding lodinated Derivatives of Trimetoquinol ...... 273
CHAPTER IX INTRODUCTION ...... 274 9.1 Synthesis of Chiral 1-(Benzyl)-1,2,3,4-Tetrahydroisoquinolines . . . 274 9.1.1 Separation of D iastereom ers ...... 274 9.1.1.1 Achiral Chromatography of Diastereomeric Derivatives 276 9.1.1.2 Fractional Recrystallization of Diastereomeric Salts . . . 276 9.1.2 Enzymatic Resolution of Racemic Amines ...... 277 9.1.3 Chiral HPLC of Racemic Mixtures ...... 278 9.1.4 Asymmetric Synthesis of 1-Substituted THIQs ...... 281 9.1.4.1 Alkylation of Chiral Formamidines ...... 281 9.1.4.2 Asymmetric Catalytic Hydrogenation ...... 282
CHAPTER X STATEMENT OF THE PROBLEM AND OBJECTIVES ...... 284 10.1 Chiral H P L C...... 284 10.2 Enzymatic Resolution ...... 285 10.3 Chiral Formamidine Synthesis ...... 286 10.4 Asymmetric Catalytic Hydrogenation ...... 287
CHAPTER XI RESULTS AND DISCUSSION ...... 289 11.1 Chiral HPLC...... 289 11.2 Enzymatic Resolution ...... 292 11.3 Asymmetric Synthesis ...... 294 11.3.1 Chiral Formamidine S y n th e s is ...... 294 11.3.2 Asymmetric Catalytic Hydrogenation ...... 306 11.4 Summary ...... 319
CHAPTER XII EXPERIMENTAL ...... 321
BIBLIOGRAPHY ...... 373
x LIST OF TABLES
TABLE PAGE
1. Inhibition of K+-Evoked Release of [3H]Acetylcholine from Striatal Slices by Dopamine, Dimethyldopamine, and Permanently-Charged Analogues ...... 39
2. Effect of Chlorpromazine and Analogues on the Binding of [3H]Spiperone to D2 Dopaminergic Receptors ...... 41
3. Potencies of Dopaminergic Agonists for the Inhibition of K+-Evoked Release of [3H]Acetylcholine from Striatal Slices 74
4. Apparent Equilibrium Binding Dissociation Constants of Dopaminergic Agonists for the Inhibition of [3H]Spiperone in NaCI Free Medium ...... 76
5. Tissues Containing B-Adrenergic Receptors ...... 135
6. Effect of Isoproterenol and Trimetoquinol in Guinea Pig Right Atria and Trachea ...... 179
7. Effect of N-Benzyltrimetoquinol Analogues on U46619-lnduced Platelet Aggregation and Serotonin Secretion ...... 180
8. Binding Affinities (pK,) of Selected Trimetoquinol Analogues on Human B-Adrenergic Receptors Expressed in E. Coli...... 183
9. Binding Affinities (KiP pM) of Selected Trimetoquinol Analogues in Human Platelets (HP), Rat Vascular Endothelial Cells (RVEC) and Rat Vascular Smooth Muscle Cells (RVSMC) ...... 183
10. Comparative Adrenergic Agonist Activities of Trimetoquinol and N-Phenylethyl-Trimetoquinol on Guinea Pig Right Atrial (3,) and Tracheal (B2) Tissues ...... 215
xi 11. Comparative Potencies of Trimetoquinol Derivatives for the Inhibition of U46619 (1 n-M) Induced Platelet Aggregation and Serotonin Secretion ...... 216
12. Comparative Adrenergic Agonist Activities of Trimetoquinol and Amidine Trimetoquinol on Guinea Pig Tracheal (32) Tissue . . 217
13. Comparative Adrenergic Agonist Activities of Trimetoquinol Analogues on Guinea Pig Right Atrial (I3J and Tracheal (I32) Tissues ...... 218
14. Reaction Conditions for Catalytic Hydrogenation ...... 308
xii LIST OF FIGURES
FIGURE PAGE
1. The Biosynthesis of Catecholamines ...... 3
2. Dopamine Metabolism ...... 4
3. Selective D, and D2 Agonists and Antagonists ...... 7
4. a-and 3-Rotamers of Dopamine ...... 17
5. Physiological Equilibrium of Dopamine’s Amino Group ...... 29
6. Physiological Forms of Dopamine ...... 30
7. Reinforced Ionic Bonding of Dopamine ...... 35
8. Potential Drug-Receptor Interactions of Amino, Permanently Charged, and Permanently Uncharged Functional Groups ...... 39
9. Potential Drug-Receptor Interactions of Hydazinium Functionalities ...... 40
10. Reinforced Ionic Bonding of Guanidine Functionalities with Aspartate Residues ...... 42
11. General Approach to the Synthesis of Primary Guanidines ...... 46
12. Potential Mechanisms for Decomposition During the Synthesis of 3,4-Dihydroxbenzylguanidine 85 ...... 53
13. Partial 1FI NMR Spectrum of the Crude Bicarbonate Salt Containing Both 3,4-Bis(benzyloxy)benzylguanidine and its Amine Precursor ...... 61 14. Possible Mechanism for Decomposition of 3,4-Dihydroxbenzylguanidine 86 During the Photolysis of its 2-Nitrobenzyloxy Protected Precursor 136 ...... 63
15. Schematic Representation of Agonist and Antagonist Evaluation at the D2 Receptor ...... 73
16. Resonance Forms of a Primary Guanidine ...... 75
17. Metabolism of Adrenergic Catecholamines ...... 133
18. Classical Selective and Non-Selective 3-Adrenergic Agonists and Antagonists ...... 136
19. Transmembrane Topology of the 6-Adrenoceptor ...... 139
20. Cross Section Arrangement Transmembrane Helices ...... 140
21. Molecular Modeling Interactions of Norepinephrine with the 62-Adrenergic Receptor ...... 141
22. Schematic Diagram Illustrating the Esson-Stedman Hypothesis for the Binding of Dopamine and the Stereoisomers of Norepinephrine ...... 143
23. Biosynthesis of Thromboxane A2 via the Arachidonic Acid Cascade ...... 149
24. Thromboxane A2 Activation of Platelet Aggregation and Secretion...... 153
25. Prostaglandin Dependent Pathway of Platelet Activation ...... 155
26. Affinity and Photoaffinity Labeling ...... 174
27. Affinity and Photoaffinity Labels ...... 176
28. Tautomeric Forms of Amidine-TMQ 250...... 181
29. Partial 1H NMR Spectrum of N-Phenylacetamide 274 in CDCI3. . . 194
30. Partial 1H NMR Spectrum of N-Phenylethylamine 275 in CDCI3. . . 194
xiv 31. Mechanism for Trimethylsilyl Iodide Cleavage of Methylurethanes ...... 208
32. Rationale for the Stability of Amides in the Presence of Trimethylsilyl Iodide ...... 209
33. Partial 1H NMR Spectrum of Nitro Urethane 307 in CDCI3 at 298, 313 and 325 K ...... 211
34. Partial 1H NMR Spectrum of 4-Hydroxy-3-nitro Urethane 312 in CDCI3 at 298 and 323 K ...... 214
35. Resolution of Diasteromeric Derivatives ...... 275
36. Chiral Derivatizing Reagents for Diasteromeric Separations 277
37. Chiral Stationary Phases ...... 280
38. Methodology for cx-Lithiation of Formamidines ...... 282
39. Chiral HPLC Chromatogram for 8-Fluoro-TMQ Derivative 318. . . 290
40. HPLC Chromatogram of Aminourethane 308 on an Analytical ChiralcelR OD Column ...... 291
41. Chiral HPLC of (±) Trimetoquinol Naphthamide 352 ...... 301
42. TAGIT HPLC Chromatogram for (±)-Dibenzyloxytrimetoquinol Derivative 271 ...... 302
43. Partial 1H NMR Spectra of Methoxy Acetamide 379 ...... 313
44. Partial 1H NMR Spectrum of (S)-4-Nitro Acetamide 384 ...... 317
xv LIST OF SCHEMES
SCHEME PAGE
I. Previous Synthesis of 2-(3,4-Dihydroxyphenyl)ethylguanidine 8 6 ...... 47
II. Approach to the Synthesis of 2-[3,4-Bis(benzyloxy)phenyl]ethylguanidine 100 ...... 48
III. Synthesis of 2-(3,4-Dihydroxyphenyl)ethylquanidine 86. ... 49
IV. Synthesis of 3-(3,4-Dihydroxyphenyl)propylguanidine 87. . 51
V. Approach to the Synthesis of 3.4-Dihydroxybenzylguanidine 85 from 3.4-Dimethoxvbenzylquanidine 109...... 52
VI. Approach to the Synthesis of 3-Hydroxybenzylguanidine 88 from 3-Methoxybenzylguanidine 112 ...... 54
VII. Synthesis of Vanillylguanidine 90 ...... 55
VIII. Approach to the Synthesis of 3-Hydroxybenzylguanidine 88 from 3-Hydroxybenzylamine 116 ...... 56
IX. Synthesis of Isovanillylguanidine 91 ...... 57
X. Approach to the Synthesis of THIQ-guanidine 89 from its Dimethoxy Derivative 122...... 58
XI. Synthesis of THIQ-guanidine 89 from 6,7-Dihydroxy-1,2,3,4-THIQ 123...... 59
XII. Approach to the Synthesis of 3,4-Dihyroxybenzylguanidine 85 from 3.4-Dihvdroxvbenzvlamine 124...... 59
xvi XIII. Approach to the Synthesis of 3.4-Bis(benzyloxy)benzylguanidine 129...... 60
XIV. Synthesis of 3.4-Bis(2-nitrobenzyloxy)benzylguanidine 136...... 62
XV. Approach to the Synthesis of 3.4-Bis(methoxymethoxy)benzylguanidine 139 ...... 64
XVI. Approach to the Synthesis of [(2,2-Dimethyl- 1.3-benzodioxole)-5-yl]methylguanidine 145...... 65
XVII. Synthesis of (Spiro[1,3-Benzodioxole-2,1’cyclohexane]- 5-yl)methylguanidine Bicarbonate 150 ...... 67
XVIII. Unexpected Synthesis of the Bisamine 151 ...... 68
XIX. Unexpected Synthesis of 3.4-Dihydroxybenzylguanidine 85 ...... 69
XX. Synthesis of 3,4-Dihydroxbenzylguanidines 155 and 156. . . 70
XXI. Synthesis of 3,4-Dihydroxybenzylguanidine 85 from 3.4-Bis(benzyloxy)benzylguanidine 157 ...... 71
XXII. Synthesis of 3,4-Dihydroxyphenylguanidine 84 ...... 72
XXIII. Retrosynthesis of 4-Substituted Diiodotrimetoquinol Irreversible Labels ...... 187
XXIV. Retrosynthesis of 3-Substituted lodotrimetoquinol Irreversible Labels ...... 188
XXV. Synthesis of N-Phenylethyl-Trimetoquinol 249 ...... 193
XXVI. Retrosynthetic Approach to Urea 276 and Thiourea 277. . . 195
XXVII. Synthesis of Urea 276 from 3.4-Dibenzyloxyphenylethylamine and Urethane 278 ...... 196
XXVIII. Synthesis of Urea 276 from Trimethoxyaniline and Urethane 281 ...... 197
xvii XXIX. Synthesis of Amidine 283 via Thiourea 277...... 198
XXX. Synthesis of Isocyanate 285 ...... 199
XXXI. Synthesis of Amidine-TMQ 250 from Urea 276 ...... 200
XXXII. General Synthesis of Catecholamines 262, 263, and 266 ...... 202
XXXIII. Attempted Synthesis of lodinated Analogues 255 and 258...... 204
XXXIV. Synthesis of lodoacids 298 and 299...... 204
XXXV. Attempted Synthesis of lodo-THIQs 255 and 258 from the Corresponding Amides ...... 205
XXXVI. Attempted Synthesis of Phenylacetic Acid 304 by lodination of Phenylacetic Acid 303...... 206
XXXVII. Synthesis of 3-lodo-4-methoxy-5-nitrophenylacetic Acid 304...... 207
XXXVIII. Synthesis of Methoxyiodoamides 305 and 306...... 207
XXXIX. Synthesis of Methyl Urethanes 307-309 ...... 210
XL. Synthesis of lodourethanes 310 and 311 ...... 212
XLI. Synthesis of 4-Hydroxy-3-iodo-5-nitro- Urethane 313...... 213
XLII. Reduction of an Achiral Alkene with a Chiral Catalyst 283
XLIII. Chiral Catalytic Hydrogenation of Dehydro Trimetoquinol Derivative 315 ...... 283
XLIV. Synthesis of 2,2,2-Trifluoroethyl Butyrate 314...... 293
XLV. Retrosynthetic Strategy for Synthesis of 1-Benzyl Substituted THIQs via Chiral Formamidines 294
XLVI. Synthesis of Tetrahydroisoquinoline 334 ...... 295
xviii XLVII. Synthesis of (S)-Chiral Formamidine 340 ...... 296
XLVIII. Synthesis of (R)-Chiral Formamidine 344 ...... 297
XLIX. Synthesis of 3,4,5-Trimethoxybenzyl Bromide 347 ...... 297
L. Chiral Formamidine Synthesis of (S)-Trimetoquinol 209. . . 298
LI. Chiral Formamidine Synthesis of (R)-Dibenzyloxy Trimetoquinol Derivative 351 ...... 299
Lll. Synthesis of Trimetoquinol Naphthamide 352 ...... 301
LIU. In situ Generation of TMQ-TAGIT Diastereomers ...... 302
LIV. Synthesis of 4-Acetamidobenzyl Bromide 356 ...... 303
LV. Synthesis of 4-Acetoxybenzyl Bromide 360 ...... 303
LVI. Synthesis of N-(4-Bromomethylphenyl)phthalimide 364. . . . 304
LVII. Synthesis of 4-Methoxymethoxybenzyl Bromide 367 ...... 305
LVIII. Synthesis of Dibenzyioxy Enamide 315...... 307
LIX. Reported Literature Syntheses of Ru(BINAP) Catalysts. . . . 307
LX. Synthesis of Methoxy Enamides 376 and 377 ...... 309
LXI. Thermal Isomerization of (E)-Enamide 377 to (Z)-Enamide 376 ...... 310
LXII. Synthesis of Trimetoquinol Derivatives by Chiral Catalytic Hydrogenation ...... 313
LXII I. Synthesis of (±) Dibenzyioxy Acetamide 380 ...... 314
LXIV. Synthesis of (±)-Methoxy Trimetoquinol Derivative 382. ... 314
LXV. Hydrazinolysis of Benzyloxy Trimetoquinol Derivative 349 ...... 315
xix LXVI. Synthesis of (S)-4-Nitro Trimetoquinol Derivative 384 by Chiral Catalytic Hydrogenation ...... 316
LXVII. Attempted Synthesis of 4-Acetoxy-3-nitro Enamide 330. ... 318
LXVI 11. Attempted Synthesis of 4-Nitro Enurethane 332 ...... 318
xx Part 1
Synthesis of Arylalkylguanidines as Dopamine Agonists
1 CHAPTER I
INTRODUCTION
1.1 Background
Dopamine (DA) 1 is an endogenous central nervous system (CNS) neurotransmitter implicated in many debilitating neurological disorders. Prior to the elucidation of dopamine’s neurotransmitter activity [1], the primary role of dopamine was thought to be that of a biosynthetic precursor for the production
OH
1 2 R = H
3 r = c h 3 of the catecholamines norepinephrine (NE) 2 and epinephrine (EPI) 3
(Figure 1). The biosynthesis of DA and subsequent catecholamines begins with the oxidation of the aromatic amino acid L-tyrosine to L-dihydroxyphenylalanine
(L-DOPA). This reaction is the rate-limiting step of catecholamine biosynthesis
2 3
Tyrosine Hydroxlase
COOH COOH
L-Tyrosine L-DOPA
HO NH2 L-Aromatic Amino Dopamine Acid Decarboxylase no beta-Hydroxylase
DA
OH OH
HO NH2 Phenylethanolamine ^*0 NHCH3 N-Methyltransferase HO HO
NE EPI
Figure 1. The Biosynthesis of Catecholamines. and is catalyzed by tyrosine hydroxylase (TH) [2]. Decarboxylation of L-DOPA to yield DA is accomplished by L-aromatic amino acid decarboxylase (also known as dopa decarboxylase). In dopaminergic nerve terminals, DA is stored in vesicles until released by depolarization. In adrenergic nerve terminals, NE is produced by oxidizing DA with dopamine 3-hydroxylase (DBH).
N-Methylation of NE by phenylethanolamine N-methyl transferase (PNMT) produces EPI [3]. These biogenic amines are biosynthesized in both catecholaminergic nerves and the adrenal medulla.
Identification of DA in the CNS [4,5,6,7] suggested that this biogenic amine has neurological activity. Dopamine is synthesized and released from dopaminergic neurons for interaction with both presynaptic (autoreceptors) and postsynaptic receptors. Neurotransmitter activity is terminated by a combination of reuptake into the presynaptic nerve terminus and metabolism within the synapse. Primarily, uptake is the more important mode of deactivation and occurs by two mechanisms. High affinity uptake is a sodium dependent, stereoselective process referred to as the "uptake, mechanism". There is also
V a low affinity, sodium independent process referred to as the "uptake2 mechanism" [8].
HO ho-M 2. Aldehydre Dehydrogenase zxrr DA DOPAC
Catechol-O-Methyl Catechol-O-Methyl Transferase Transferase
c h 3o . . CH qO . . AU NH2 1. Monoamine oxidase XT' 2. Aldehydre Dehydrogenase ■ jonr 3-MT HVA
Figure 2. Dopamine Metabolism.
Dopamine is metabolized principally by the enzymes monoamine oxidase
(MAO) and catechol O-methyl transferase (COMT) by one of two routes
(Figure 2). Oxidative deamination by MAO leads to
3,4-dihydroxyphenylacetaldehyde which is subsequently oxidized by aldehyde dehydrogenase (AD) to 3,4-dihydroxyphenylacetic acid (DOPAC). Further methylation of DOPAC by COMT produces homovanillic acid (HVA).
Alternatively, methylation of dopamine by COMT yields 3-methoxytyramine
which is subsequently oxidized by MAO and AD to give HVA [8].
1.2 Location and Distribution of Dopamine
Dopamine is found in much greater amounts than the adrenergic
catecholamines in CNS pathways [4,5,9]. Dopaminergic neurons are classified
in three groups according to the length of the fibers :
1. Ultrashort Systems. The neurons in this system consist of the
interplexiform amacrine-like neurons linking the inner and outer plexiform
layers of the retina and the periglomerular dopamine cells of the olfactory
bulb which link mitral cell dendrites in sperate adjacent glomeruli [10].
2. Intermediate-Length Systems. These neurons include the
tuberohypophysial dopamine cells which project from the arcuate and
periventricular nuclei of the hypothalamus to the intermediate lobe of the
pituitary and into the median eminence (tuberofundibular system), the
incertohypothalamic neurons which link the dorsal and posterior
hypothalamus with the dorsal anterior hypothalamus and lateral septal
nuclei, and the medullary periventricular group which includes neurons
in the perimeter of the dorsal motor nucleus of the vagus nerve, the
nucleus tractus solitarius, and neurons in the tegmental radiation of the
periaqueductal grey matter [10]. 3. Long-Length Systems. These systems link neurons from the ventral,
tegmental and substantia nigra to the three targets: the nigrostriatal
system (caudate nucleus and putamen), the mesocortical system (medial
prefrontal, cingulate, and entorhinal areas), and the mesolimbic system
(regions of the septum, olfactory tubercle, nucleus accumbens septi,
amygdaloid complex, and the piriform cortex) [10]. The nigrostriatal
system contains 80% of the dopaminergic neurons and is involved in the
regulation of motor activity via the extrapyramidal system (EPS).
Parkinson’s disease results from the degeneration of the nigrostriatal
neurons and subsequent loss of striatal dopamine [11].
Dopamine receptors are also found in the peripheral nervous system.
Neuronal tissues in the heart, lung, kidney, liver, Gl tract, male reproductive tract, and both endocrine and exocrine glands [12,13] respond to dopamine, and can be blocked by dopaminergic antagonists [13,14],
Dopamine produces its activity in many peripheral tissues by its action at both dopaminergic and adrenergic receptors. For example, low dose infusion of dopamine has a vasodilatcry effect on renal vasculature via dopaminergic receptors causing an increase in renal blood flow. Intermediate doses of dopamine increase cardiac output by an inotropic stimulation of cardiac
B^adrenergic receptors. Higher doses of dopamine produce vasoconstrictive effects on the peripheral vasculature through the activation of ex-adrenergic
receptors [3].
1.3 Dopamine Receptor Subtypes
Dopamine receptors were initially classified into two types, D! and D2
based on functional and pharmacological criteria [15]. Activation of adenylate cyclase with production of cyclic adenine monophosphate (cAMP) was associated with D, receptors while an inhibition of or no effect on cAMP production was associated with D2 receptors [15]. In the past, selective D, and
D2 agonists and antagonists (Figure 3) have verified the existence of two
Agonist Antagonist
HO Cl D i NH N-CHg HO HO
SKF 38393 SCH 23390
H
H » CH2CH2CH3
LY 141865 Sulpiride
Figure 3. Selective D, and D2 Agonists and Antagonists. 8 receptor subtypes [16]. However, these receptor selectivities are once again somewhat arbitrary designations because of the recent development of molecular biology techniques such as gene cloning. A total of five dopaminergic receptor subtypes have now been isolated [17]. These receptors are part of the GTP-binding protein-coupled receptor (GPCR) family which also contains a-adrenergic, B-adrenergic, seratonergic, and muscarinic receptors as well as many other receptors [18]. Although five receptor subtypes have been identified, these subtypes still display similarities to the original two subtype system. Thus, the five subtypes have been organized into Dt and D2 subfamilies [17].
The D, subfamily consists of the Dand D5 subtypes [17] . The Dt subfamily is linked to the activation of adenylate cyclase with subsequent production of cAMP [19], The human D, receptor was first cloned in 1990 by four groups simultaneously [17]. This receptor consists of 446 amino acids having a predicted topology similar to other GPCRs [20], This receptor was characterized based on criteria which included tissue distribution of the receptor’s mRNA, a pharmacological profile consistent with the Dreceptor when transfected into COS-7 cells, and subsequent activation of adenylate cyclase [20,21,22]. The D5 receptor consists of 477 amino acids with a strong homology to the D t receptor. This receptor displays a 10-fold higher affinity for dopamine and is primarily localized within the limbic regions of the brain [23]. The D2 receptor subfamily consists of the D2) D3, and D4 receptors [17].
The D2 subfamily inhibits the enzyme adenylate cyclase preventing subsequent production of cAMP [19]. The rat D2 receptor was the first dopamine receptor cloned utilizing a B-adrenergic receptor gene as the hybridization probe. This receptor was characterized by the relationship between its amino acid sequence and other GPCRs, the tissue distribution of the receptor, and the pharmacological profile when transfected into cells [24]. The human form of this receptor has been subsequently cloned [25,26,27,28].
D2 receptors exists in two forms that are derived from alternative splicing of the same gene [25,29,30]. These two forms, designated D2S (D2 short) and D2L
(D2 long) are pharmacologically identical, yet they differ by 29 amino acids within the third cytoplasmic loop, the portion of the receptor coupled to the
G-protein [25,29].
The second member of the D2 subfamily is the D3 receptor. This receptor has been cloned from both rat [31] and human [32] cDNA and genomic libraries by a combination of reverse transcription and polymerase chain reaction. This receptor is located in the ventral striatum and other limbic structures with little overlap with the D2 receptor. In the substantia nigra and the ventral tegmental area, studies showed that both D2 and D3 receptors are expressed. The D3 receptor has only 52% homology with the D2 receptor, but this increases to 75% if only the transmembrane domains are considered.
There are also a number of isoforms for the D3 receptor, but unlike the D2S and D2L receptor isoforms, the D3 isoforms are not functional [32]. Dopamine has a 20-fold higher affinity for D3 than D2, and several dopamine agonists and antagonists also have a higher affinity for D3. These higher affinities for the D3 receptor as well as the differences in distribution between the receptors suggests the possibility of selective drugs for the receptors [32].
1 The third member of the D2 subfamily is the D4 receptor. This receptor has been cloned from human cDNA using the rat D2 receptor cDNA as the probe [33]. This receptor is located in the frontal cortex, midbrain, amygdala, and medulla with lower levels in the striatum and olfactory tubercle. The D4 receptor has only 41% and 39% homology to the D2 and D3 receptors respectively, but this increases to 56% for both receptors if only the transmembrane regions are considered. The pharmacological profile of the D4 receptor is based on a hybrid-gene DNA since a full length receptor cDKlA was unobtainable. The affinities of many dopamine agonists and antagonists for binding to this mutant receptor were similar to or lower than the affinities for the
D2 receptor. Clozapine, however had a one order higher affinity for the D4 receptor [33].
Presynaptic dopamine receptors are known as autoreceptors.
Autoreceptors have the ability to regulate dopamine synthesis and depolarization-evoked release of the neurotransmitter. Stimulation of autoreceptors by dopaminergic agonists eventually causes the inhibition of the enzyme tyrosine hydroxylase and thus inhibits the rate of dopamine synthesis. 11
Activation of somatodendritic autoreceptors also produces a membrane
hyperpolarization, thus inhibiting neuronal firing. Both D2 and D3 receptors may function as autoreceptors [32].
1.4 Therapeutic Uses of Dopamine Agonists
Parkinson’s disease is a syndrome of diverse etiology characterized by the degeneration of pigmented cells in the substantia nigra. The biochemical and pathological characteristics of Parkinson’s include a diminished nigrostriatal content of dopamine and a loss in number of pigmented cells in the substantia nigra. The clinical manifestations of the progressive disease are the triad of resting tremor, bradykinesia, and rigidity. The disease begins in a mild form, usually only a slight hand tremor. At first, the disease progresses unilaterally, but eventually there is bilateral involvement with full-blown clinical symptoms
[34].
There are several animal models for studying Parkinson’s disease [34].
Initially, reserpine was used as a biogenic amine depleter producing some
Parkinson’s-like symptoms. This model is not very accurate because
Parkinson’s is a progressive, irreversible degenerative disease while reserpinization is a completely reversible process [34]. A better model for studying Parkinson’s is the use of 6-hydroxydopamine (6-OHDA) 4 to cause unilateral or bilateral destruction of nigrostriatal neurons. The para-dihydroxy groups of 6-OHDA undergo rapid oxidation to form several highly reactive species responsible for neurotoxicity. Rats unilaterally treated with 6-OHDA respond with a predictable contralateral circling behavior when treated with direct-acting dopamine agonists. Bilaterally treated rats have extensive behavioral deficits including bradykinesia. Parkinsonian-like rats treated with direct-acting dopamine agonists show significant improvement in motor function compared to untreated controls [34], A third model for Parkinson’s disease involves nigrostriatal degradation produced by peripheral administration of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 5 [34]. This neurotoxic drug was found as an impurity in the illicit preparation of the analgesic 1 -methyl-
4-phenyl-4-proprionoxypiperidine (MPPP) 6. Drug abusers who inadvertently administered MPPP tainted with the impurity MPTP suffered an irreversible, rapid Parkinson’s-like syndrome [34]. Mice repeatedly treated with MPTP eventually develop impaired motor function (Parkinsonian state). Other rodents, such as rats and guinea pigs are relatively resistant to MPTP [34]. It’s believed that the biological half-life of the metabolite 1-methyl-4-phenylpyridinium ion
(MPP+) 7 correlates well with the destruction of the nigrostriatal neurons. 13
Humans and other primates have extremely long half-lives of MPP+, while mice
(2 h) and rats (15 min) have relatively short half-lives. This explains the need for only a few doses of MPTP in primates, several larger doses in mice, and many mega-doses in rats to produce Parkinson’s like symptoms [34].
Current therapy for Parkinson’s disease utilizes L-DOPA 8, the biochemical precursor of dopamine. This drug is utilized in the treatment of
Parkinson’s as a substitute for dopamine. Dopamine is rapidly metabolized in the periphery and poorly penetrates the blood brain barrier (BBB), thus making it useless in treating a CMS disorder. L-DOPA is transported across the BBB by a neutral amino acid pump and converted to dopamine in neurons that contain L-aromatic amino acid decarboxylase. Administration of L-DOPA often requires the co-administration of the peripheral dopa decarboxylase inhibitor, carbidopa, 9 to prevent peripheral conversion of L-DOPA to dopamine.
Bromocriptine 10 and pergolide H are ergot alkaloids utilized as dopamine agonists in the treatment of Parkinson’s disease [35]. Unfortunately, dopamine agonists only alleviate the symptoms of the disease rather than curing the underlying cause of the disease. Current therapeutic drugs have several inherent problems. First, these drugs lose their effectiveness as the disease progresses. Secondly, it is difficult to titrate the optimal dose of dopamine agonists because subtherapeutic dosing cannot control the symptomatology while excessive dosing produces excessive stimulation and unwanted side effects. In addition, these drugs also have activity at other 14 CH3 HO NH2 H0 v|<^sr/ \ Z NHNH j
COOH COOH HO HO 8 9
OH
HN CHoSCH
CH
HN 10 HN Br
receptors, so more selective dopamine agonists are necessary to properly control the symptoms of Parkinson’s.
Dopamine agonists are also useful in treating other disorders associated with inappropriate or inadequate dopaminergic function. Bromocriptine is utilized for the treatment of endocrine disorders such as hyperprolactinemia, unwanted lactation, and infertility [36]. Dopamine is utilized for its dopaminergic effects on renal blood vessels to improve renal perfusion [3].
1.5 Classifications of Dopamine Agonists
In an effort to understand and treat disorders caused by insufficient dopaminergic function, many dopamine agonists have been synthesized. These 15 compounds constitute many types of structures which can be grouped together by chemical or structural similarities. Unfortunately, many different assay systems (in vitro and in vivo) in several animal models are utilized in evaluating the efficacy of these compounds. In addition, pharmacological systems are not available for evaluating the effects of dopamine agonists on the recently isolated dopamine receptors. Therefore, comparisons of functional and binding properties for both selective and nonselective agonists and antagonists should be made with extreme caution.
1.5.1 Phenvlethvlamines
Dopamine is a dihydroxylated phenylethylamine, and this class includes all acyclic modifications which retain the phenylethylamine backbone. Many simple modifications such as alkylation and homologation of the ethyl side chain have resulted in a loss or attenuation of activity [37]. Monoalkylation of the nitrogen typically reduced activity except for N-methyldopamine (epinine) 12, which retains its potency in displacing [3H]-spiperone from D2 sites [38],
N-Disubstituted analogues such as N,N-di-n-propyldopamine 1_3 and
N-phenylethyl-N-n-propyldopamine 14 displayed the "N-n-propyl phenomenon"
[37]. The addition of two substituents on nitrogen in which one is an n-propyl group retains DA activity even if one substituent is quite large. This phenomenon is not related to hydrophobicity since lower and higher homologs fail to produce a pattern of enhanced or depressed activity [8], Removal of the 16
^ nr 1r2
X
X R, r 2 12 OHH c h 3
13 OH CH2CH2CH3 c h 2c h 2c h 3
14 OH c h 2c h 2c h 3 CH2CH2Ph 15 H H H 16 H CH2CH2CH3 CH2CH2CH3
17 H c h 2c h 2c h 3 CH2CH2Ph 18 F H H 19 F CH2CH2CH3 CH2CH2CH3
20 F c h 2c h 2c h 3 CH2CH2Ph
21 Cl c h 2c h 2c h 3 CH2CH2Ph
22 Cl c h 2c h 2c h 3 CH2CH2(4-OH)Ph
para-hydroxyl group to produce m-tyramine.15, N.N-di-n-propvl-m-tvramine 16. and N-phenylethyl-N-n-propyl-m-tyramine 17 resulted in relatively potent D2 agonists [37,39,40]. Thus, the catechol is not be absolutely necessary for activity at D2 dopaminergic receptors. This idea is confirmed by more recent work which shows that replacement of the para-hydroxy with halogens such as fluorine [41] in compounds 18, 19, and 20 or chlorine [42] in compounds
21 and 22 retains or improves dopamine activity at the D2 receptor. The
N-phenylethyl substituent offers the possibility for selectivity in phenylethylamine agonist drugs [42]. 17
1.5.2 Conformationallv Restricted Analogues of Dopamine
Dopamine is a flexible molecule which may exist in a number of conformations in solution or at the receptor. Dopamine may exist in either the a- or B-rotamer. In the a-rotamer, the meta hydroxyl group is projected over the ethylamine chain while in the 3-rotamer, the hydroxyl group extends away from the ethylamine chain (Figure 4) [37]. The x-ray crystal structure has been determined for dopamine hydrochloride [43], but as with the rotameric conformations, this conformation is only suggestive of a possible conformation when the drug is in solution or at the receptor. In order to fully understand the
OH OHnu
I NH2
OH
alpha-rotamer beta-rotamer
Figure 4. a- and B-Rotamers of Dopamine. 18
interaction of the drug with the receptor, structurally defined molecules are
required. These molecules rigidity the ethyl side-chain by incorporation of the
two carbons into a ring leaving the amino group exocyclic or by incorporation
of the ethylamine side-chain into a heterocyclic ring.
1.5.2.1 Miscellaneous Semi-rigid Analogues
Rigidification of the ethyl side chain by ring formation can position the
amino group in an exocyclic manner or incorporate the amine in a heterocyclic
ring. Both cis- and trans-cyclopropyl analogues 23 and 24 have been
synthesized and evaluated as dopamine agonists [44]. These analogues are devoid of agonist activity. It is believed that the cyclopropyl ring restricts the
HO OH HO OH
NH2 23 24
HO OH HO OH
NH2 25 26 aromatic ring into a position which does not allow the dopamine backbone to
adopt either the a- or 13-rotameric conformation [44],
The cis- and trans-cyclobutyl analogues 25 and 26 of dopamine have
also been synthesized and evaluated for their biological activity [45,46],
The trans-cyclobutyl derivative 26 was much more potent than the cis-isomer
25 in preventing [3H]-dopamine from binding to the dopamine receptor.
Conformational analysis of the trans-isomer 26 showed that the dihedral angle
between the amino and catechol groups exist varied from 110-160 0 [46], This
approximates the 180 0 of the anti-conformation of the a- and B-rotamers of
dopamine. Conformational analysis of the cis-isomer 25 approximates that of
syn-conformation and provides a rationale for its poor binding to the receptor.
Incorporation of the ethylamino portion into an endocyclic six-membered
ring produces the 3-phenylpiperidines. One of the more potent analogues of this class is 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP) 27 [47]. The
pharmacological profile of this molecule is very complex. The lone chiral center
ch2ch2ch3 20
produces a pair of enantiomers with different activity profiles. The
(R)-(+)-isomer acts as a D2 postsynaptic agonist and a weak D! postsynaptic
agonist while the (S)-(-)-isomer acts as a weak DJD2 postsynaptic antagonist
[48]. Both enantiomers act as autoreceptor agonists. Recent piperidine ring
modifications such as conversion to morpholine derivative 28 [49] or
thiomorpholine derivative 29 [50] have confirmed previous reports that the
3-phenylpiperidine moiety is indespensible for high potency and selectivity at
dopaminergic receptors [37].
1.5.2.2 Aminotetralins
The aminotetralins are dopamine analogues where the ethylamine
side-chain is tethered at the ex-carbon to the aromatic ring by an ethylene
bridge. The basic examples this class are 2-amino-5,6-dihydroxytetralin
(5,6-ADTN) 30 and its isomer 2-amino-6,7-dihydroxytetralin ( 6,7 -ADTN) 31.
These compounds are conformationally restricted analogues of dopamine in which 30 represents a rigid cx-rotamer while 31_ represents a rigid (3-rotamer.
Both ADTN isomers are potent dopamine agonists, but consistent structure- activity relationships have not been successful [37], These compounds are also fragments of the naturally occurring apomorphine 32 and isoapomorphine 33.
Isoapomorphine is inactive as a dopaminergic, yet its fragmented derivative 31 is active. This means that there is some steric bulk in isoapomorphine 33 which does allow for receptor interactions. 21
Ri
r 2
CH
R Ri r 2
30 OHOH H 32 31 H OHOH 33
N.N-Dialkysubstituted ADTN derivatives have substantially different activities as compared to their parent compounds [37]. The N,N-di-n-propyl derivative 34 has dopaminergic and ex-adrenergic activity [51]. The
6,7-dihydroxy isomer 35 is much less potent as a dopaminergic agonist [52],
Monophenolic compounds 36, 37, 38 showed consistent structural-activity relationships (SAR). 5-Hydroxy-ATN 36 was the most potent derivative, followed by the 7-hydroxy 38 and the 6-hydroxy compounds 37 [53]. This is not surprising because the 5-hydroxy derivative 36 is a rigid cx-rotamer of m- tyramine and the 7-hydroxy derivative 38 is a rigid 6-rotamer. In addition, the
"N-n-propyl phenomenon" is also evident for this class of monophenolic compounds [54]. 22
R Ri
N i c h2ch2ch3
R Ri r2 34 OH OH H 35 H OH OH 36 OH H H 37 H OH H 38 H H OH
1.5.2.3 Aminoindanes
The aminoindanes are dopamine analogs in which the ethylamine side chain is tethered at the a-carbon to the aromatic ring with a methylene bridge.
The basic examples of this class are 2-amino-4,5-dihydroxyindane 39 and its isomer 2-amino-5,6-dihydroxyindane 40. These compounds are conformationally restricted analogues of dopamine in which 39 represents a rigid a-rotamer while 40 represents a rigid B-rotamer. These aminoindanes are much less potent than their corresponding tetralins as dopamine agonists.
There is a significant change in the spatial relationship between the amino group and the catechol group when comparing the indanes to the tetralins.
These compounds do not overlap as well with the naturally occurring apomorphine 32 and isoapomorphine 33. Tetralins 30 and 31 have very good 23 overlap with the respective aporphines [55] and are much more potent than the indanes as dopaminergic agonists. N,N-Dialkysubstituted indane derivatives
have substantially different activities. The 4,5-dihydroxy-N.N-di-n-propyl derivative 41 has dopaminergic activity, but the 5,6-dihydroxy isomer 42 has very weak activity [56]. Monophenolic compounds 43 and 44 showed that the
4-hydroxy derivative 43 was much more potent than the 5-hydroxy derivative 44
[57]. Resolution of the enantiomers of the 43 revealed that the (R)-isomer
is 100-fold more potent than the corresponding (S)-isomer [58]. This suggests that fragmented analogs of the aporphines, such as the indanes and tetralins, should have the same stereochemistry as (R)-apomorphine [58].
R r i r 2 R, 39 HOHOH H 40 HHOH OH 41 CH2CH2CH3 OH OH H
42 c h 2c h 2c h 3 HOH OH
43 c h 2c h 2c h 3 OHH H
44 c h 2c h 2c h 3 HOH H 24
1.5.2.4 Benzoquinolines
The benzoquinolines are an extension of the aminotetralins in which the
exocyclic amino group has been tethered to the saturated ring by a propyl
bridge. The octahydrobenzo[g]quinolines and octahydrobenzo[f]quinolines are
rigid dopamine analogues in which the saturated rings can be either cis or trans fused. Generally, dopaminergic activity resides in the trans fused ring system and alkylation of the nitrogen to produce tertiary amines greatly enhances activity [37]. In the benzo[g]quinoline series 45-48. the 6,7-dihydroxy compounds 45 and 47 corresponding to the a-rotamer of dopamine are much more active than the 6-rotameric 7,8-dihydroxy compounds 46 and 48 [52]. In the benzo[f]quinoline series 49-52, the 7,8-dihydroxy compounds 49 and 51
R
R r i r 2 r 3 45 H OH OH H 49 46 H H OH OH 50 47 CH2CH2CH3 OHOH H 51
48 c h 2c h 2c h 3 H OHOH 52 25
corresponding to the a-rotamer of dopamine are much more active than the
B-rotameric 8,9-dihydroxy compounds 50 and 52. The "N-n-propyl
phenomenon" is evident with enhanced activity for the tertiary derivatives of
both benzoquinoline series [37].
1.5.2.5 Nitrogen Heterocvcles
Cylcization of the ethylamine side chain into various heterocyclic ring
systems produces a wide range of compounds with greatly varied activity at dopaminergic receptors. Closure of the ethylamine chain into the seven
membered-ring tetrahydro-3-benzazepine 53 produced a potent dopamine agonist [59]. Incorporation of a 1-phenyl substituent as in SKF 38393 54
[60] or fenoldopam 55 [61] greatly enhanced agonist activity and also produced D, selectivity. Compound 54 has been converted to the D, selective antagonists SCH 23390 56 and SKF 83566 57 by substituting the 7-hydroxyl group with a halogen and methylation of the secondary amine. Optical resolution of the enantiomers of 54, 55, and 56 shows that the agonist or antagonist activity resides in the (R)-isomer [59].
Tetrahydroisoquinoline 58 is a very weak dopaminergic agonist [62].
Incorporation of the amino group into a tetrahydroisoquinoline 59 which is closely related to 3-phenylpiperidine 27 generates a functional agonist [63].
Compound 59 is conformationally mobile, so an attempt to rigidity this analog in to a conformationally restricted derivative yielded the 26 hexahydrobenzo[a]phenanthradine 60 [64]. This tetracycle is a high potency
D! selective agonist. For the benzo[a]phenanthradines, alkylation of the
nitrogen decreases the potency and full agonist activity in comparison to the parent secondary amine [65].
HO HO NH N-R HOXOH HO HO
53
R2
54 X = R1 = H 56 x = ci
55 X = Ci R, = OH 57 X = Br
HO HO HO
H HOIX!" HO HO N'
58
59 60
1.5.3 Naturally Occurring Dopamine Agonists
The aporphines and the ergot alkaloids are naturally occurring dopamine agonists. These polycyclic compounds are very complex, and numerous fragmentation studies have tried to define the portions of these molecules responsible for their dopaminergic activity [8]. 27
1.5.3.1 Aporphines
Apomorphine 32 is the classical example from the aporphine class. This compound contains the a-rotamer of dopamine within its structure.
Isoapomorphine 33 contains the 6-rotamer of dopamine within its structure, but isoapomorphine is inactive as a dopamine agonist. This simple example does not rule out activity for the 6-rotamer, because the fragmented isoapomorphine derivative 31 from the aminotetralins is an active dopaminergic agonist. Thus, isoapomorphine and the related octahydrobenzo[g]quinoline 61 have significant steric interactions that do not occur for aminotetralin 31 The "N-n-propyl phenomenon" is evident in the aporphine class as well, since
N-n-propylnorapomorphine 62 is much more potent than apomorphine
[66,67]. Monophenolic aporphines 63 and 64 are active agonists with the
11-hydroxy derivative 63 distinctly more potent than the 10-hydroxy derivative
64 [68].
n CH2CH2CH:
R R1 62 OH OH
63 OH H
64 H OH 28
1.5.3.2 Ergot Alkaloids
The ergot alkaloids are truly a unique class of dopamine agonists. These
compounds can be divided into two groups, the ergolines and the ergopeptines.
The ergolines pergolide jM and lisuride 65 are tetracyclic molecules currently
used in the treatment of Parkinson’s disease [69]. These compounds lack the tricyclic peptide portion of the ergopeptines of which the classical example
is bromocriptine 10. As a group, the ergot alkaloids have a number of
pharmacological actions including dopamine and serotonin receptor activity, vasoconstrictive and hallucinogenic properties, and oxytocic effects [8]. Their dopaminergic activity is related to their interactions at D,, D2 or both receptors.
The ergot alkaloids inhibit prolactin secretion, decrease dopamine turnover in the CNS, displace dopaminergic ligands from receptor sites, and show agonist activity in many animal models [8].
NH
CH
HN 65 29
1.5.4 Dopamine Bioisosteres
Bioisosteres are structural groups that have chemical or physical
similarities, and produce a similar biological response [70], Dopamine has
many functionalities that can be considered independently or jointly for
bioisosteric replacement. These functionalities are the hydroxy groups, the
catechol ring, the phenyl ring, the ethyl side chain or the amino terminus.
Many dopamine agonists are active as either a primary, secondary or
tertiary amine. Each of these amines can undergo proton exchange with
biological fluids at physiological pH producing an equilibrium of amine and
ammonium compounds (Figure 5). The mode of binding between the amine
terminus and the receptor is an area of great debate. It has been suggested that ligands bind to the receptor in a charged form [71], an uncharged form
[72], or an uncharged form in which the lone pair of electrons on nitrogen are
important for interaction with the receptor [73]. In order to address which form of dopamine interacts with the receptor, permanently charged and
permanently uncharged derivatives of dopamine have been synthesized
[71,74,75],
Figure 5. Physiological Equilibrium of Dopamine’s Amino Group. 1.5.4.1 Permanently Charged Derivatives
Dopamine undergoes a rapid equilibrium between its five charged forms and one uncharged form at physiological pH (Figure 6). Both the catechol ring and the amino terminus are ionizable in biological fluids. The amino terminus has a pKa of 9.74 and predominantly exists in its charged form at physiological pH [76]. In order to determine the ionization state of the amine terminus when dopamine interacts with the receptor, several permanently charged derivatives of dopamine 66-68 have been prepared [71,74,75,77].
N,N,N-trimethyldopamine 66, the dimethylsulfonium analog 67, and the dimethylselenonium analog 68 are all weakly active agonists in functional and binding studies [78,79,80] when compared to dopamine. This is strong evidence that the amino terminus of dopamine can interact with the receptor in
Figure 6. Physiological Forms of Dopamine. 31 its charged form. In addition, permanently charged derivatives of dopamine antagonists 69-75 have also been prepared and evaluated for their biological activity. These compounds are al! active compounds with weaker activity than the corresponding amine [81,82,83].
SeMe
67 68
o
N i N Cl S R 73 r = c h 3
74 r = c h 2c h 3 69 r = c h 2n +(Ch3)3
70 R = c™ 2 ^ 8
71 R = NHN+(CH3)3 U x i L I \ ^ ^ o c h3 72 r = c h 2n +(c h 3)2n h 2 75 32
1.5.4.2 Permanently Uncharged Derivatives
As previously stated previously, the amine terminus can exist in a charged or uncharged form at physiological pH [76]. Since it had been proposed that the uncharged form of the amino terminus of dopamine was important for receptor interactions [72,73], permanently uncharged bioisosteres of dopamine may interact with the receptor as well. Bioisosteric replacement of the amino group in dopamine with a methylsulfide group 76 or a methylselenide group 77 has been completed [75], In each case, the permanently uncharged derivative was inactive as a dopamine agonist in functional and binding studies [84], Likewise, an uncharged tetrahydrothiophene analog of sulpiride 78 was inactive as a dopamine antagonist [82]. The lack of activity of the permanently uncharged analogues
SeMe
76 77
78 33
of dopamine and sulpiride is very strong evidence to disprove the hypothesis that the amine terminus of dopamine interacts with the receptor in its unionized form.
1.6 Druq-Receptor Interactions
Drugs interact with receptors in a highly specific manner. In order to achieve a discrete, meaningful interaction at the extremely low concentrations of both drug and receptor in the human body, a number of attractive forces stabilize the binding of structurally specific drugs to their respective receptors
(or enzymes). These forces include covalent bonding, electrostatic interactions
(ionic bonding), ion-dipole and dipole-dipole interactions, hydrogen bonding, charge-transfer interactions, hydrophobic interactions, and van der Walls interactions [85]. The interactions that are important for the binding of the amine terminus of dopamine to the dopamine receptors will be discussed.
1.6.1 Hydrogen Bonding
Hydrogen bonds are a dipole-dipole interaction formed between a highly electronegative atom containing free electrons (H-bond acceptor) and a highly electronegative atom containing a covalently bonded hydrogen (H-bond donor).
The only significant atoms to partake in hydrogen bonding are nitrogen, oxygen and fluorine. The high electronegativity of oxygen or nitrogen as compared to hydrogen polarizes the covalent bond in hydroxyl and amino groups. Thus, 34
hydrogen has a partial positive charge which is strongly attracted to the non
bonding electrons of electronegative atoms. The hydrogen bond is unique
because hydrogen is the only atom able to maintain a positive charge at
physiological pH while remaining covalently bonded to another atom. The
strength of a hydrogen bond depends upon the distance between the
electronegative atoms and the colinearity of the bond. Typically the AG for
hydrogen bonding is 3 to 5 kcal/mole [85]. Hydrogen bonding is very important
in maintaining the tertiary structures of proteins and nucleic acids [86].
1.6.2 Ionic Bonding
Ionic bonding is the mutual attraction of opposite charges. This
interaction can be effective at distances much further than those required by other interactions, but the strength of the ionic bond does diminish by the square of the distance between the charges. A simple ionic interaction can
provide a AG of about 5 kcal/mol [85].
1.6.3 Reinforced Ionic Bonding
A reinforced ionic bond is a sum of two interactions in which one is an ionic bond. Typically, the second interaction is a hydrogen bond. As for dopamine (Figure 7), the interaction of the charged amino terminus (a hydrogen containing cation) with an ionized carboxylate residue (anion) of the receptor 35 can be rationalized as a reinforced ionic bond. A reinforced ionic bond can
provide a AG up to 10 kcal/mol [85].
HO H HO N + - H
H reinforced ionic bond O O
Receptor
Figure 7. Reinforced Ionic Bonding of Dopamine
1.7 Summary
From the previous discussion, a number of observations about dopamine, the dopamine receptors, and dopamine agonists can be made:
1. Dopamine is a CNS neurotransmitter implicated in the debilitating
neurological disorder, Parkinson’s disease.
2. Multiple receptor subtypes have recently been cloned compounding the
already confusing situation for the rational design of selective dopamine
agonists. Considerable research will be required to reevaluate the
selectivity of dopaminergic agonists and antagonists with these recently
identified receptor subtypes. Current therapeutic treatments for Parkinson’s disease offer limited value and suffer a loss of efficacy as the disease progresses.
A wide variety of dopamine agonists in various chemical classes have been synthesized. Although a large amount of work has been accomplished on structural activity relationships of dopamine agonists, a limited amount of information is available on the optimal interactions between the drugs and the receptor to produce agonist activity. It is known that the catechol functionality, the "meta" hydroxyl group, or a some hydrogen bonding isostere is important for agonist activity.
N-Dialkylation in which one alkyl group is an n-propyl substituent offers a significant increase in activity for agonist via the "N-n-propyl phenomenon". N-Alkylation with a phenylethyl substituent offers some
D2 selectivity. Finally, information about the preferred rotamer for binding to the receptor is inconclusive since many a-rotamers and a number of
13-rotamers are dopaminergic agonists.
Studies with permanently charged and permanently uncharged dopamine bioisosteres strongly suggest that dopamine interacts with its receptor in a charged form via a reinforced ionic bond. t
CHAPTER 18
STATEMENT OF THE PROBLEM AND OBJECTIVES
Parkinson’s disease is a progressive, debilitating disease for which
current therapeutic drug intervention has had minimal impact [35], This disease
is manifested by the degeneration of nigrostriatal dopaminergic neurons
resulting in impaired neurotransmitter function [34]. Current therapy consists of
replacing the lost function of the neurotransmitter with dopamine agonists.
However, this treatment is of limited value due to the nature of the disease and lack of therapeutic selectivity of these drugs [34,35,36]. In order to improve on the design of more selective and effective therapeutic agents, a comprehensive
knowledge of the drug-receptor interactions is required. A detailed understanding of these interactions can be extremely valuable for the design of selective therapeutic agents that demonstrate enhanced potency with fewer side effects than currently available drugs.
Dopamine 1 is a biogenic amine which exists in a charged (protonated amine) and uncharged (free base amine) equilibrium at physiological pH
(Figure 5). Recent molecular biology and molecular modeling studies have postulated that a conserved aspartate residue (Asp114 in the D2 receptor) is
37 responsible for the binding of the amino side chain of dopamine through an ionic interaction [87,88,89]. In the past, there were several theories which postulated the interaction of the amine terminus of dopamine with its receptor [73,74,75]. Permanently charged 66-68 and uncharged 76 and 77 bioisosteric replacements of this amine terminus provide substantial evidence that the charged form of dopamine is responsible for interactions with the receptor [80,84]. Although the permanently charged compounds are active, they are also less potent than both dopamine and dimethyldopamine 79
(Table 1) [80]. The potential difference in activities between the amines and the permanently charged analogues is that the catecholamines are capable of accepting a proton from biological fluids to become charged. In this ammonium form, dopamine agonists have been postulated to interact with the dopamine receptor via a reinforced ionic bond [90], Protonated primary, secondary and tertiary amines can bind to the receptor with both hydrogen and ionic bonding
N(CH3)2
N(CH3)2 Table 1.
Inhibition of K+-Evoked Release of [3H]Acetylcholine from Striatal Slices by Dopamine, Dimethyldopamine, and Permanently-Charged Analogues.
Compd. # Description EC^fn/M) 95% C.i. (pM)
1 DA 1 .0 1 0.59 ■• 1.69
79 DA-Me 2 0.06 0.03 •■ 0 . 1 2
6 6 DA-Me3+ 7.49 3.83 ■• 14.7 67 DA-SMe2+ 22.7 16.8 ■■ 30.7
6 8 DA-SeMe2+ 8.99 5.94 ■■ 13.6 while the permanently charged analogues can only undergo ionic interactions
(Figure 8) [90]. Thus, compounds which can undergo both ionic and hydrogen bonding interactions may have enhanced potency as dopamine agonists [90].
Similar studies with permanently charged 69.70 and 73-75 and uncharged 78 nitrogen bioisosteric dopamine antagonists in comparison to chlorpromazine 80 and sulpiride 81 support these findings [81,82]. These
H CH, CHo / (Drug) ------i|+ — H (Drug) — s+ (Drug) / I \ CH, reinforced H ionic Ionic bond bond - .O ° - w ° Receptor X Receptor Receptor Figure 8. Potential Drug-Receptor Interactions of Amino, Permanently Charged, and Permanently Uncharged Functional Groups. tertiary amine receptor antagonists can also interact with the receptor via a
reinforced ionic bond while the permanently charged derivatives can only
interact by means of an ionic bond [90]. The similarities of these studies
support the hypothesis that dopamine agonists and antagonists bind to a
common site on the receptor [90,91]
c h 3 c h 3 I H (Drug) N N+—CH3 (Drug) N+— I H CH3 reinforced reinforced ionic * ionic bond O O bond
Receptor Receptor
Figure 9. Potential Drug-Receptor Interactions of Hydazinium Functionalities.
In an effort to study other functionalities which are capable of both ionic
and hydrogen bonding, two hydrazinium analogues 71 and 72 of chlorpromazine
were prepared [83], The analogues contain a permanently charged nitrogen
atom with an adjacent nitrogen atom capable of hydrogen bonding (Figure 9).
These compounds are dopamine antagonists with an affinity for the receptor
between that of the tertiary amine and the permanently charged antagonists
(Table 2) [83]. Thus, the adjacent hydrogen bonding group restores some of the potency lost by a permanently charged derivative [83], The synthesis of
hydrazinium analogues of dopamine 82 and 83 is currently in progress [92], 41
CH, CH NH, \ +. CH 3 a + CH
82 83
The objective of this research was to design and synthesize dopamine
analogues which have the potential to interact with the receptor via a reinforced
ionic bond in which the functional group is capable of both ionic and hydrogen
bonding. In addition, this functional group must have the proton which
undergoes hydrogen bonding directly attached to the cationic group. The guanidine functionality is a highly basic group which is almost completely
ionized at physiological pH. This group, if appropriately oriented within the receptor, is capable of forming a reinforced ionic bond which contains two
hydrogen bonds with the aspartate residue of the receptor (Figure 10).
Table 2.
Effect of Chlorpromazine and Analogues on the Binding of [3H]Spiperone to D2 Dopaminergic Receptors.
Compd. # Description K, ( i x M )
80 CPZ 0.05 ± 0.01 69 CPZ-N+ 8.57 ± 0.52
71 CPZ-NN* 4.88 ±0.16
72 CPZ-NPN 0.82 ± 0.06 A similar example of this interaction has been recently postulated. Guanosine triphosphate (GTP), which inherently contains guanidine within its structure, is believed to bind to an aspartate residue (Asp138) in elongation factor Tu (EF-Tu) by a similar arrangement (Figure 10) [93].
o
reinforced reinforced ionic ionic bond bond R
Receptor GTP-EF Tu binding
Figure 10. Reinforced Ionic Bonding of Guanidine Functionalities with Aspartate Residues.
Because of the variability of the dopamine binding site to accept multiple agonists with conflicting steric requirements, a receptor model has been proposed in which the charged amino terminus can interact with either resonance extreme of a carboxyl group [94], A guanidine analog of dopamine with the appropriate length between the aromatic ring and the guanidine functionality may be able to bind to both oxygens of the carboxyl group resulting in enhanced affinity. Thus, the proposed target compounds are the catechol guanidines 84-87 which have a varying chain length from zero to three carbons. In this homologous series, the aromatic guanidine 84 has an external nitrogen and the ethyl derivative 86 has an internal nitrogen that is 43 analogous to the nitrogen atom in dopamine 1_. Benzylic guanidine 85 has the delocalized charge of the guanidine group centered at approximately the same distance from the aromatic ring as the charged amino group in dopamine.
Propyl derivative 87 is expected to have too large of a distance from the aromatic ring to the charged functionality to properly bind to the receptor.
During the course of the preparation of this series, numerous synthetic difficulties were encountered for the synthesis of benzylic guanidine 85.
NH .N H 2
84 n = 0 85 n = 1 86 n = 2 87 n = 3
NH
NH 88 89
NH NH
90 91 44
In order to circumvent these difficulties, multiple protecting group strategies
were explored. In addition, a number of benzylic derivatives 88-91 were also
investigated in an effort to understand the difficulties encountered during the
preparation of the benzylic guanidine derivative. These compounds were
prepared to provide insight on the chemical instability of the benzylic guanidine
or to provide an alternate benzylic containing derivative for biological evaluation.
Ethyl guanidine 86 has been evaluated as an MAO inhibitor [95] and
as an inhibitor of platelet aggregation [96], but its synthesis was not reported.
This compound has been previously synthesized in our lab with preliminary
evaluation as a dopaminergic agonist [97]. This compound was shown to be
a direct acting dopamine agonist which could be blocked by the antagonist
sulpiride [97],
A related compound, imidazoline 92 (DHBI), was ineffective as a dopamine agonist since it lacked activity in the rabbit renal artery assay and did
not stimulate adenylate cyclase [98],
Aryl guanidine 94 has been prepared as its hydrochloride salt and
evaluated as a vasoconstrictor [99], but it has not been evaluated as a dopamine agonist. Two similar classes of compounds have been prepared for
potential dopaminergic activity [100,101]. Quinazolines analogues 93 and 94 were found to be inactive as dopamine agonists in the dog renal artery assay with 93 displaying weak antagonist activity [100], Imidazoline 95 caused an increase in coronary blood flow in the dog which was antagonized by a dopamine antagonist [102]. However, this compound was later shown to be produce its effects through a-adrenergic mechanisms [103].
Even though several guanidines and related derivatives have appeared in the literature, there has not been a systematic review of arylalkylguanidines as dopamine agonists. Therefore, a homologous series of arylalkylguanidines and related derivatives have been synthesized for biological evaluation as potential dopaminergic agonists. These compounds will be assessed for their ability to bind to and activate D2 dopamine receptors. CHAPTER ill
RESULTS AND DISCUSSION
3.1 Chemistry
Several functionalizing reagents such as cyanamide [99],
2-ethyl-2-thiopseudourea hydrobromide [112], aminoiminomethanesulfonic acid
[111], and 1 H-pyrazole-1-carboxamidine hydrochloride [104] have been used
, NH NH2
R° (CH2)n RO (CH^n U ^Y| functionalize NH
RO ro
n = 0,1,2,3 R = protecting goup or H
Methods of functionalization
1.) 50% aq. H2NCN 2.) SEt 3.) S03H 4.) '— N H z N ^ N H HBr H2 N ^ N H H2N
Figure 11. General Approach to the Synthesis of Primary Guanidines.
46 47 for the conversion of primary amines to guanidines (Figure 11). Our approach to the synthesis of the homologous catecholguanidine series 84-87 required the investigation of several functionalizing reagents with protected and free catecholamines for the successful synthesis of the catecholguanidines
(Figure 11).
Ethylguanidine 86 has been reported in the literature (95,96), but its synthesis has not been described. This compound has been previously synthesized in our lab as shown in Scheme I [97], This method utilized an excess of functionalizing reagent and required the conversion of the intermediate sulfate salt to its bicarbonate salt 97 for isolation [97]. Several researchers from our group attempted to apply this technique to the synthesis
Scheme I.
Previous Synthesis of 2-(3,4-Dihydroxyphenyl)ethylguanidine 86 (modified from 97).
h2o 97 96 2. NaHC0 3
reflux
8 6 48 of the other target compounds without success [92]. Therefore, it was necessary to develop a methodology which may allow for the synthesis of the entire series of compounds.
The initial approach for an alternate synthetic route began with the conversion of the free base of 3,4-dibenzyloxyphenethylamine hydrochloride 98 to its respective guanidine with the sulfonic acid functionalizing reagent 99 in anhydrous acetonitrile (Scheme II) [111]. Neither the insoluble product 100 nor its free base yielded acceptable spectral or elemental data. Several attempts to convert the crude material or its free base to the catechol by catalytic hydrogenation or by refluxing in (1:1) hydrochloric acid/methanol were unsuccessful. Consequently, the previously described methodology [97] was reinvestigated.
Upon the recommendation of Beuchamp [105], the relative ratios of the amine and functionalizing reagent were changed so that the functionalizing
Scheme II.
Approach to the Synthesis of 2-[3,4-Bis(benzyloxy)phenyl]ethylguanidine 100.
NH2 HCI NaOH/CH2CI2
2. SOgH n h h2s o 3
h2n ^ n h 98 1 0 0 99 ch3cn reflux Scheme III. 4 9
Synthesis of 2-(3,4-Dihydroxyphenyl)ethylguanidine 86.
SEt
CH3O _ _ NH, Y NH HBr
h2o 101 103
HO HN NH2 48% HBr n NH HBr reflux HO J)
86
reagent became the limiting reagent. An aqueous solution of excess
3,4-dimethoxyphenylethylamine 101 was heated with pseudothiourea 102 to
produce the desired 3,4-dimethoxyphenylethylguanidine 103 upon workup
(Scheme III). Deprotection of the methoxy groups with 48% hydrobromic acid
gave the desired catecholguanidine 86 in high yield.
The general workup of the reaction mixture involved dilution with water,
extraction with several volumes of ether to remove ethyl mercaptan and excess free base amine, and evaporation of the aqueous layer. Trituration in acetone
attempted to produce a solid precipitate. The collected solid was recrystallized from either an alcohol, water, alcohol/diethyl ether, or acetonitrile/diethyl ether.
If a solid failed to form in acetone, the viscous oil was recrystallized as described above. If the oil failed to crystallize, it was dissolved in hot water 50 and poured into saturated sodium bicarbonate. The crystallization of protected and deprotected guanidines was extremely difficult, usually involving several attempts even in an appropriate solvent system. Conversion of the starting materials to a guanidine derivative was verified by 13C NMR spectroscopy and fast atom bombardment (FAB) mass spectrometry. The carbon of the guanidine group has a characteristic 13C resonance at 157 ppm while its related carbon of 2-ethyl-2-thiopseudourea 102 is at 173 ppm. FAB mass spectrometry typically produced only the guanidine peak (m + 1) as the base peak while electron impact mass spectrometry caused a rapid decomposition of the guanidine group such that a molecular mass was not detected.
The use of thiopseudourea 102 was also applied to the synthesis of propylguanidine 87 (Scheme IV). 3,4-Dihydroxyphenylpropionic acid 104 was converted to its acid chloride with thionyl chloride in dichloromethane. The crude acid chloride was added dropwise to concentrated ammonium hydroxide to produce amide 105. This amide was reduced with lithium aluminum hydride in tetrahydrofuran to yield amine 106 as its free base after distillation. However, many attempts to functionalize this amine by heating with thiopseudourea 102 in water or isopropanol failed to produce an isolatable guanidine hydrobromide salt. The crude guanidine was converted to its free base and was triturated with saturated sodium bicarbonate to yield guanidine bicarbonate 107. Removal of the methoxy groups with 48% hydrobromic acid produced the desired propylguanidine 87. 51
Scheme IV.
Synthesis of 3-(3,4-Dihydroxyphenyl)propylguanidine 87.
V w CH ° v ^ s ^ s^ A oh 1. SOCIa/CH2a 2 NH, LAH ► THF CH30 2 conc NH4OH cch h 3<3o 104 105 SEt
1- H z N '^ N H HBr NH H2C 0 3 c h 3o . . _ CH30 ^ JJ ^ 102 v^ Vjr^-^NH^NH 2
CH30 ^ 5 - / ------* CH30 3 IPOH 1 0 6 2. „ sat. . NaHC03mu™ 1 0 7
NH -HBr
48% HBr ^ HO'Y ^Y^^N H ^N H 2
reflux H O ' 87
This methodology was applied to the benzylguanidine derivative 85 with partial success (Scheme V). 3.4-Dimethoxvbenzvlamine 108 was heated with thiopseudourea 102 in water to produce benzylguanidine 109. However, deprotection of the methoxy groups with 48% hydrobromic acid resulted in immediate, severe decomposition. Deprotection with either boron tribromide
(BBrg) in dichloromethane or trimethylsilyl iodide (TMSI) in acetonitrile also resulted in decomposition. The use of demethylating reagents is limited to those which can be easily removed by evaporation or organic workup 52
Scheme V.
Approach to the Synthesis of 3,4-Dihydroxybenzylguanidine 85 from 3,4-Dimethoxybenzylguanidine 109.
SEt
H2N '^ i-NH HBr NH HBr CH30 _ _ _ CH30 . . II 'v ^ N '0 ‘nh2 102 NH2
c h 3o h2o c h 3o 108 109
NH
48% HBr or BBr3/CH2CI2 or x # \ -:r r N H X N H 2 tm s i/c h 3c n 85
(HBr, BBr3, TMSI) since the desired product will be insoluble in organic solvents.
In order to investigate the reasons for the severe decomposition so that an appropriate protecting group can be utilized, a pair bf guanidine derivatives were prepared.
There are two possible explanations for the decomposition during the demethylation reactions. The first explanation is that the strongly nucleophilic reagents attack at the benzylic position displacing the guanidine (Figure 12).
The second rationale is that the guanidine functionality is lost due to thermal or chemical instability in the presence of the para-hydroxyl group (Figure 12).
These problems can be investigated individually by preparing
3-hydroxybenzylguanidine 88 and vanillylguanidine 90. The preparation of
3-hydroxybenzylguanidine 88 removes the possibility of the para-hydroxyl group 53
»NH2+ c h3o CH3O NH 'v j c h3o H— 'Z S 3 Figure 12. Potential Mechanisms for Decomposition During the Synthesis of 3,4-Dihydroxbenzylguanidine 85. to cause decomposition via quinoid formation. Therefore, any decomposition would be related to the nucleophilic attack at the ex-carbon. Vanillylguanidine 90 retains the para-hydroxyl group. For this compound, decomposition could be related to chemical or thermal incompatibility of the para-hydroxyl and a-guanadino groups. 3-Methoxybenzylamine 110 was heated with thiopseudourea 102 in acetonitrile and subsequently poured into a solution of saturated sodium bicarbonate to produce benzylguanidine bicarbonate 111 (Scheme VI). This bicarbonate salt could be converted to the hydrobromide salt 112 by stirring with 48% hydrobromic acid at 0 °C. Several attempts to demethylate either salt with 48% hydrobromic acid, saturated hydrogen bromide (g) in glacial acetic acid or boron tribromide in dichloromethane resulted in severe decomposition. This is strong evidence that these reagents are also attacking the benzylic carbon with concurrent loss of the guanidine. For the preparation of vanillylguanidine 90, commercially available vanillylamine hydrochloride 113 was converted to its free base with one Scheme VI. 54 Approach to the Synthesis of 3-Hydroxybenzylguanidine 88 from 3-Methoxybenzylguanidine 112. 1,)HoN^NH- r HBr 102 NH h2c°3 ch3o ^ ^ ch3o II — 'S^^''^NH^NH2 HBr/MeOH ch3cn 'ST^V/Xf -► k^ 2.) sat NaHC 03 0°C 110 111 NH HBr NH HBr CH3° J| HO JJ >Y^V^s'NH^NH2 48 % HBr or W N^V^NH^NH 2 k-^ HBr (g)/HOAc or A k"^ 112 BBr3/CH2CI2 88 equivalent of sodium methoxide in methanol followed by the careful addition of diethyl ether. Filtration and evaporation of the filtrate provided the solid free base. This amine was immediately dissolved in acetonitrile and heated with thiopseudourea 102 to form vanillylguanidine 90 (Scheme VII). Since this final reaction involves heating the mixture to reflux for 2 h, the para-hydroxyl group and a-guanidine are compatible within the same structure under thermal conditions. Therefore, the decomposition described above is related to the nucleophilic attack at the benzylic carbon. Other protecting groups which can be removed under less nucleophilic conditions are required for the synthesis of benzylguanidine 85. Scheme VII. Synthesis of Vanillylguanidine 90. 1. NaOMe/MeOH; NH -HBr CHa° HCI Et2° r 2‘ H2N ^ N H -HBr 113 102 90 CH3CN The successful synthesis of vanillylguanidine 90 and the synthetic difficulties involved in preparing benzylic guanidine 85 prompted the synthesis of other guanidines containing a benzylic fragment that may offer insight into the potential activity of benzylic guanidine 85. Since it is know that the mefa-hydroxyl group is more important than the para-hydroxyl group for receptor activity [37,39,40], there was a continued effort to synthesize 3-hydroxybenzylguanidine 88. In addition, isovanillylguanidine 91_ is expected to be more active than vanillylguanidine 90 as a dopamine agonist. However, methylation of the para-hydroxyl group may result in a substantial loss of affinity, so a derivative which contains a catechol group would be preferable. This derivative is the tetrahydroisoquinoline compound 89 which tethers the guanidine to the aromatic ring by an ethyl bridge. Since the mefa-hydroxyl group is only hydroxyl group seen in several dopamine agonists [37,39,40], a second attempt to synthesize benzylguanidine 56 Scheme VIII. Approach to the Synthesis of 3-Hydroxybenzylguanidine 88 from 3-Hydroxybenzylamine 116. .OH o H2 HO HO H NH2OHHCI H Raney Nickel NaOH/95% EtOH EtOH 114 NH HBr NH ^NH2 ch3cn 116 88 88 was investigated. This effort began with the conversion of 3-hvdroxvbenzaldehvde 114 to the oxime 115 with hydroxylamine hydrochloride (Scheme VIII). The oxime was reduced by catalytic hydrogenation with Raney nickel in ethanol to produce the phenolic amine 116 which was isolated as the free base. The crude amine was heated with thiopseudourea 102 in acetonitrile, but a solid product was unable to be isolated. For this particular compound, the guanidine cannot be isolated as a bicarbonate salt because the phenolic group ionizes in saturated sodium bicarbonate. The preparation of isovanillylguanidine 91_ begins with the conversion of isovanillin 117 to its oxime with hydroxylamine hydrochloride followed by dehydration with acetic anhydride to form benzonitrile 118 (Scheme IX). This Scheme IX. 57 Synthesis of Isovaniilylguanidine 91_. 0it 1.H2NOHHCI i,n2Nwn nw NaOH/95% EtOH AC° BH 3 /THF CH3 O 2. Ac20 CH3q 117 118 SEt H z N ^ N H HBr NH HBr “ y V " " * 102 r H0 Y Y " h NA n h2 ch3o CH3CN c „ 3o 119 91 nitrile is reduced with borane-tetrahydrofuran complex to the phenolic amine 119 which was used without isolation. This amine was heated with thiopseudourea 102 in acetonitrile to form isovaniilylguanidine 91. The first approach to the synthesis of tetrahydroisoquinoline 89 started with the synthesis of amine 121 from 3,4-dimethoxyphenethylamine 120, formaldehyde and formic acid with isolation of the amine as its oxalate salt (Scheme X). This amine was converted to its free base and heated with thiopseudourea 102 in water to produce the protected guanidine 122. Unfortunately, the saturated ring did not improve the stability of the benzylic guanidine portion because deprotection of the methoxy groups with 48% Scheme X. 58 Approach to the Synthesis of THIQ-guanidine 89 from its Dimethoxy Derivative 122. MeO 1. CH20 / HCOOH 50° C MeO M e O NH2 2- oxa,ic acld -2H2 ° MeO A ^ v ^ N H H X 120 121 HX = oxalic acid i 1. NaOH/CHCI3 Me° HBr or ^ , HO, 48% HBr or \ f 2. sEt MeO A ^ V n y HBr(g)/HOAcHBr(g)/HOAc / \ n ^ n h 2 H^NHHBr 122 HNHBr gg HNHBr 102 h2o hydrobromic acid or saturated hydrogen bromide (g) in glacial acetic acid resulted in decomposition. An alternate route to guanidine 89 began with the demethylation of the f free base of amine 121 by 48% hydrobromic acid to yield catecholamine 123 (Scheme XI). This amine hydrobromide was reacted with 50% aqueous cyanamideand recrystallized several times from ethanol/diethyl ether to remove unreacted cyanamide and successfully produce catechol guanidine 89. This same technique was pursued for the synthesis of benzylic guanidine 85 from 3,4-dihydroxbenylamine hydrobromide 124 but failed to produce a solid product (Scheme XII). At this point numerous attempts to synthesize benzylic guanidine 85 were unsuccessful. Several protecting groups were investigated for the successful 59 Scheme XI. Synthesis of THIQ-guanidine 89 from 6,7-Dihydroxy-1,2,3,4-THIQ 123. 1. NaOH I CHCI3 121 123 HX = oxalic acid nh 2 cn/h2o HN HBr 89 Scheme XII. Approach to the Synthesis of 3,4-Dihyroxybenzylguanidine 85 from 3,4-Dihydroxybenzylamine 124. NH HBr nh 2 cn/h2o 124 85 synthesis of the desired compound. The groups included: benzyl ethers, o-nitrobenzyl ethers, methoxymethyl ethers, an acetonide group, and a cyclohexylidene ketal [106]. The benzyl ether protecting group was utilized as described above in the unsuccessful synthesis of ethylguanidine 86 . However, it was believed that this protecting strategy failed due to the inability to isolate an appropriate salt of the Scheme XIII. Approach to the Synthesis of 3,4-Bis(benzyloxy)benzylguanidine 129. o 'OH BnQ „ H Bn0 I I IJ NaOH/ 95% EtOH I 1 ^ Bno Bno 1 2 5 1 2 6 6 0 0 N ^ vjj^ CN I.BH3/THF BnO 2. HCI (g)/ MeOH 1 2 8 1 2 7 NH H2C 03 BnO ___ Jl 1. NaOH/CH2CI2 \ / II NH NH2 X - - X X ' 1 2. SEt " - ^N ^N H -H Br 102 CH3CN 3. sat NaHC03 protected guanidine. Since benzyl ethers can be mildly cleaved by catalytic hydrogenation [106], this protecting group was evaluated for the synthesis of benzylic guanidine 85. 3,4-Dibenzyloxybenaldehyde 125 was allowed to react with hydroxylamine hydrochloride to form oxime 126 (Scheme XIII). This oxime was dehydrated with acetic anhydride to form benzonitrile 127. Reduction of the nitrile with borane-tetrahydrofuran complex provided benzvlamine 128 which could be isolated as its hydrochloride salt. Several reactions of the benzylamine with thiopseudourea 102 in water or acetonitrile failed to produce and isolatable 61 hydrobromide salt. Therefore, the crude reaction mixtures were poured into a solution of saturated sodium bicarbonate to produce a bicarbonate solid which contained both the benzyl amine and the benzyl guanidine as determined by 1H NMR spectroscopy. The 1H NMR spectrum of the bicarbonate precipitate in CD3OD/DCI showed the benzylic protons of the both the guanidine (6 = 4.30) and the amine (6 = 4.02) in a 5:1 ratio (Figure 13). This mixture of compounds was unable to be separated. Another protecting group which can be mildly cleaved is the o-nitrobenzyl ether [106]. This group is a photolabile group which has been used for the successful protection of nucleotides [107] and the amino acid tyrosine [108]. The synthetic pathway begins with the conversion of A L 4.0 PPM Figure 13. Partial 1H NMR Spectrum of the Crude Bicarbonate Salt Containing Both 3,4-Bis(benzyloxy)benzylguanidineand its Amine Precursor. Scheme XIV. 62 Synthesis of 3,4-Bis(2-nitrobenzyloxy)benzylguanidine 136. o CH3O NH2OH -HCI CH3O H ------> IJ NaOH/ 95% EtOH CH3O CH3O 130 NO2 CH2Br CH3° CN B B r ^ CH2 CI2 CH3O XX 0°C - RT k2c o 3 /d m f 132 o-NOzBnO Qjyj 0 -NO2BnO 1. BH3 /THF NH2 1. NaOH/CH 2CI2 ► o-N02BnO 2. HCI/MeOH o-N02BnO Hj.N'^NHHBr 134 135 -JQ2 c h 3c n NH HBr NH HBr o-N 02BnO . II u r. 11 y . y Y" nh‘ nh* o-N 02BnO MeOH j f \ h o ^ 5 ^ 136 86 3.4-dimethoxvbenzaldehvde 130 to oxime 131 with hydroxylamine hydrochloride (Scheme XIV). The oxime was dehydrated to nitrile 132 with acetic anhydride and demethylated with boron tribromide in dichloromethane to yield 3.4-dihydroxybenzonitrile 133. This intermediate was alkylated with o-nitrobenzyl bromide and potassium carbonate in dimethylformamide to produce benzonitrile 134. The nitrile was reduced with borane-tetrahydrofuran complex to benzylamine 135 and isolated as its hydrochloride salt. The free 63 hv MeOH OHC O + CHO O::ox Figure 14. Possible Mechanism for Decomposition of 3,4-Dihydroxbenzylguanidine 86 During the Photolysis of its 2-Nitrobenzyloxy Protected Precursor 136. base of this benzylamine was heated with thiopseudourea 102 in acetonitrile to form the protected guanidine 136. Unfortunately, deprotection of this derivative with uv light in methanol under argon resulted in decomposition. This is apparently caused by the production of an oxidative intermediate [109,110] which subsequently oxidizes the catechol moiety (Figure 14). Therefore, this protecting group is unable to be used with catechols. The first of several acetal protecting groups which were investigated is the methoxymethyl (MOM) ether. 3.4-Dihvdroxvbenzonitrile 133 was converted to the MOM protected nitrile 137 with sodium hyrdide and chloromethyl methyl ether in tetrahydrofuran and dimethylformamide to yield a clear, colorless oil (Scheme XV). The nitrile was reduced with borane-tetrahydrofuran complex to provide benzylamine 138 which was used without purification. This amine was 64 Scheme XV. Approach to the Synthesis of 3,4-Bis(methoxymethoxy)benzylguanidine 139. HO>^^CN MOMO ^ .^CN y ' iJ 1. NaH THF/DMF |" || BH3/THF ------* MOMO ^ nu 2. CICH2OCH3 133i SEt 137 1. NH HoCOo MOMO _ _ H2N‘^'NH HBr MOMO ■« v y ^ nh2 102 x / n h ^ n h 2 MOMO CH CN ATM0M0 3 2. sat NaHC03 138 139 heated with thiopseudourea 102 in acetonitrile in an attempt to form the protected guanidine 139. but the hydrobromide salt could not be isolated. Again, the 1H NMR of the crude reaction rfiixture showed the benzylic protons of both the guanidine (6 4.17 in D20) and the amine (6 3.97 in D20) in a 6:1 ratio. The crude guanidine was poured into saturated sodium bicarbonate, but a solid precipitate did not form. With several heteroatoms within its structure, MOM-protected guanidine 139 can form several hydrogen bonding interactions which may be sufficient for solubilizing the bicarbonate salt. Therefore, an acetal which has fewer heteroatoms is required to form a guanidine bicarbonate salt in the absence of a successful isolation of a hydrobromide salt. The acetonide protecting group is a ketal which contains fewer heteroatoms than the methoxymethyl ethers. This protection strategy employed 65 an alternate approach in protection as compared to the o-nitrobenzyl ethers and methoxymethyl ethers because ketalization of 3,4-dihvdroxvbenzonitrile 133 was unsuccessful. Catechol 140 was ketalized to benzodioxole 141 with 2,2-dimethoxypropane and a catalytic amount of p-toluenesulfonic acid in benzene (Scheme XVI). This compound was brominated with N-bromosuccinimide in dimethylformamide to form arylbromide 142. A copper mediated cyanide-halogen exchange with copper(l) cyanide in dimethylformamide provided nitrile 143. Reduction of the nitrile with borane- Scheme XVI. Approach to the Synthesis of [(2,2-DimethyM ,3-benzodioxole)-5-yl]methylguanidine 145. CH3 C(OCH3 )2 CH3 NBS TsOH DMF 140 141 CuCN 1. BH3 /THF 2 . oxalic acid - 2 ^ 0 142 143 NH H2 C03 NH2 HX 1- NaOH/CHCI 3 144 H2 N ^ N H HBr 145 HX = oxalic acid 102 CH3CN 3. sat. NaHC0 3 66 tetrahydrofuran complex provided amine 144 which could be isolated as a stable oxalate salt. The free base of the amine was heated with thiopseudourea 102 in an attempt to form the protected guanidine 145. but this compound also could not be isolated as its hydrobromide salt. The crude reaction mixture was poured into saturated sodium bicarbonate but failed to form a precipitate. Since the ketal protecting groups failed to form a guanidine bicarbonate precipitate while the benzyl ether protecting group trapped the amine within the guanidine bicarbonate precipitate, there may be a relationship between too little hydrophobicity (ketals) and too much hydrophobicity (benzyl ethers) for the successful formation of a pure bicarbonate salt. A ketal group which may have the proper hydrophobicity is the cyclohexylidene ketal protecting group. The preparation of this protecting group is analogous to the acetonide strategy. Catechol 140 was ketalized with cyclohexanone and a catalytic amount of p-toluenesulfonic acid with azeotropic removal of water in toluene to form spirobenzodioxole 146 (Scheme XVII). Bromination with N-bromosuccinimide in dimethylformamide provided spirobromide 147. A copper mediated cyanide-halogen exchange with copper(l) cyanide in dimethylformamide provided soironitrile 148. The nitrile was reduced with borane-tetrahydrofuran complex to provide spiroamine 149 which could be isolated as a stable oxalate salt. An alternate preparation of the spiroamine involved the catalytic hydrogenation of spironitrile 148 with Raney nickel in ammonia saturated ethanol to provide spiroamine 149 as its free base. Scheme XVII. 67 Synthesis of (Spiro[1,3-Benzodioxole-2l1’cyclohexane]-5-yl)methylguanidine Bicarbonate 150. HO o° NBS DMF HO TsOH toluene oco 140 146 CuCN -CN 1. BH3/THF oca" DMF 'o 2 . oxalic acid ‘ 2 ^ 0 147 148 NH H2 C 0 3 1. NaOH/CHCI 3 NH2 is H N ^ N ^ SEt ocxr: HX L ocxr H zN ^N H 149 102 150 HX = oxalic acid c h 3c n 3. sat. NaHC0 3 The free base of the spiroamine was heated with thiopseudourea 102 and subsequently poured into saturated sodium bicarbonate to form spiroguanidine 150. Several attempts to remove the ketal protecting group were successful (catechol FeCI3 test was positive) but the salt either could not be isolated (acetate salt was extremely hygroscopic) or decomposed when concentrated (hydrochloride, hydobromide, nitrate or sulfate salts). This protecting group appeared to be promising, so several attempts were made to isolate another salt derivative. The first effort to isolate an alternate salt involved the use of spiroamine 149 as the limiting reagent. Upon prolonged heating (16 h vs 2 h) with 68 Scheme XVIII. Unexpected Synthesis of the Bisamine 151, 1. NaOH/CHCI 3 2. SEt l"^N 102 151 149 HX = oxalic acid CH 3 CN reflux 16 h thiopseudourea 102 in acetonitrile, a solid product deposited from the reaction mixture. This material was recrystallized twice from ethanol and identified as bisbenzylamine 151 by FAB mass spectrometry and 13C NMR spectroscopy (Scheme XVIII). Therefore prolonged heating could not provide the desired guanidine salt. A second attempt to isolate an alternate salt was to convert the bicarbonate salt 150 to its hydrobromide derivative 152. This effort involved the use of 1.1 equivalents of hydrobromic acid in methanol with guanidine bicarbonate 150 (Scheme XIX). After several days of crystallizing from ethanol/diethyl ether a solid was collected and identified as the desired benzylguanidine 85 in very low yield. Unfortunately, this procedure was unable to be duplicated. In addition, several other mineral acid salts (hydrochloric, hydroiodic, nitric and sulfuric) were unsuccessful for the isolation of an alternate salt. This prompted the study of organic acids as potential salt derivatives. Scheme XIX. Unexpected Synthesis of 3,4-Dihydroxybenzylguanidine 85. NH HBr n h h 2 c o 3 48% HBr (1.1 eg.) MeOH 0 °C 150 152 NH -HBr A NH2 H O '^ 5 ^ 85 Oxalic acid and maleic acid were simultaneously investigated in an effort to form guanidine salts. After several attempts, the following strategy was found to be reproducible. Guanidine bicarbonate 150 was converted to its free base with 5% sodium hydroxide and chloroform (Scheme XX). The guanidine free base was dissolved in a small volume of chloroform and added dropwise to a concentrated ethanolic solution of the desired acid (oxalic or maleic). The resulting clear solution was diluted with diethyl ether until cloudy. Each day for one week, diethyl ether was added until the solution became cloudy. This procedure successfully provided spiroguanidine oxalate 153 and spiroguanidine maleate 154 in moderate yield. These salts contain a second carboxyl group which is not involved in salt formation, and may be used as an acid catalyst for the hydrolysis of the ketal group. By simply heating these salts in water, Scheme XX. 70 Synthesis of 3,4-Dihydroxbenzylguanidines 155 and 156. NH HX n h h2c o 3 1. NaOH/CHCI3 NH2 ______► 2. CHCI3 ; HX/Et0H/Et20 150 HX = oxalic acid 153 (oxalic acid) or maleic acid 154 (maleic acid) NH -HX reflux 155 (oxalic acid) 156 (maleic acid) catechol guanidine oxalate 155 and catecholguanidine maleate 156 are produced. During the synthesis of benzylguanidines 155 and 156. synthetic efforts continued for the production of 3,4-benzyloxybenzylguanidine hydrobromide. After numerous attempts to isolate the desired hydrobromide salt failed, a modified procedure was required. Following guanidine formation, careful dilution of the crude reaction mixture with diethyl ether precipitated the desiredguanidine 157 (Scheme XXI). Once isolated, this protected guanidine was smoothly converted to benzylguanidine 85 by catalytic hydrogenation with 10% palladium on carbon in ethanol. The final derivative of the homologous series to be synthesized is arylguanidine 84- 3,4-Dimethoxyaniline 158 did not react sufficiently with Scheme XXI. 71 Synthesis of 3,4-Dihydroxybenzylguanidine 85 from 3,4-Bis(benzyloxy)benzylguanidine 157. 1- ?Et NH -HBr BnO k BnO ^ . II n ^ v ^ nh2 h2n ^ nh HBr nh2 BnO CH3CNCH-jCN Bno 128 2 Et2° 157 NH -HBr Ho HO. 10% Pd/C EtOH HO 85 thiopseudourea 102. so an alternate strategy was explored. The aniline was converted to its hydrobromide salt 159 with 1.1 equivalents of 48% hydrobromic acid in methanol and isolated after precipitation with diethyl ether (Scheme XXII). The hydrobromide salt was heated with 50% aqueous cyanamide in ethanol and upon cooling at 4 °C, deposited arylguanidine 160. The methoxy protecting groups were removed by heating with 48% hydrobromic acid to form guanidine 84- Scheme XXII. Synthesis of 3,4-Dihydroxyphenylguanidine 84. ch3o NH2 HBr 5 0 % aq. H2 NCN n h 2 48% HBr (1.1 eq.) CH3° EtOH ch3o Me0H/Et20 CH 3O 0°C 158 159 48% HBr reflux 160 84 3.2 Biological Evaluation The biological evaluation of these compounds is currently in progress. These drugs will be evaluated for their binding and functional properties with the D2 receptor [78,79,80,84], A schematic diagram illustrates the dopaminergic control of a cholinergic neuron in the striatum (Figure 15). Striatal dopaminergic neurons release dopamine which binds to the postsynaptic receptor located on the cholinergic neuron. Activation of this receptor inhibits the cholinergic neuron from releasing acetylcholine. If dopamine storage is depleted by reserpine and its biosynthesis is inhibited by cx-methyl-p-tyrosine, exogenously added agonists can be evaluated for their ability to inhibit [3H]acetylcholine release. In addition, if these agonists are functioning at the D2 receptor, their function will be inhibited by D2 antagonists such as sulpiride 81_. If they are antagonists, their function will be attenuated by the administration of agonists such as 73 Antagonist Reserplne + alpha-mpt DA V ^fA ch Agonist Figure 15. Schematic Representation of Agonist and Antagonist Evaluation at the D2 Receptor. apomorphine 32. This schematic also demonstrates the pharmacological model for evaluating binding affinity. Dopamine agonists and antagonists with affinity for the D2 receptor will inhibit [3H]spiperone binding to this receptor. Binding affinity is evaluated in the presence and the absence of sodium chloride. Sodium chloride converts sites of high affinity to those with low affinity and alters classical dopamine agonist binding. Agonists are evaluated for their similarities to dopamine in binding to this two site model. The functional ability of ethylguanidine 86 and propylguanidine 87 to inhibit the potassium-evoked release of [3H]acetylcholine in striatal slices is shown in Table 3. These agonists are compared to dopamine 1_, dimethyldopamine 79, and the permanently charged agonists 66-68. The guanidine derivatives have dopaminergic agonist activity similar to the Table 3. Potencies of Dopaminergic Agonists for the Inhibition of K+-Evoked Release of [3H]Acetylcholine from Striatal Slices. >mpd # Description EC,n(L!Mi 95% C.l. i u M ) 1 DA 1 .0 1 0.59-1.69 79 DM DA 0.06 0.03-0.12 6 6 DA-Me3+ 7.49 3.83-14.7 6 8 DA-SeMe2+ 8.99 5.94-13.6 67 DA-SMe2+ 22.7 16.8-30.7 8 6 DHPEG 7.47 5.09-11.29 87 DHPPG 1 0 .1 0 5.88-17.78 103 DMPEG NAa NA 107 DMPPG NA NA aNA = No Agonist Activity permanently charged derivatives. The weak agonist activity of the guanidine derivatives in comparison to dopamine and dimethyldopamine is not surprising. These compounds have a greater distance between the center of charge in the guanidine functionality and the catechol ring in comparison to the distance from the primary amine to the catechol in dopamine. The ethyl and propylguanidines may not properly fit into the ligand binding pocket of the receptor because of this increased distance. A second rationale for this loss of activity may be related to the distribution of the positive over the guanidine group. The protonated guanidine can exist in three resonance forms to distribute this charge over all three nitrogen atoms (Figure 16). 75 nh2 nh2+ nh2 ««------► II «----- R n h + ^ n h2 r — n h ^ n h 2 R— nh^^nh2+ Figure 16. Resonance Forms of a Primary Guanidine. Enhanced activity is expected from arylguanidine 84, benzylguanidine 85, and THIQ-guanidine 89 in comparison these derivatives because of the shorter distance between the charge and catechol ring. The relative activities of vanillylguanidine 90 and isovaniilylguanidine 91 in comparison to benzylguanidine 85 are expected to be lower due to methylation. The relative order of activity is expected to be benzylguanidine 85 > isovaniilylguanidine 91 > vanillylguanidine 90. As expected, the dimethoxylated derivatives 103 and 107 showed no agonist activity. Several derivatives were also evaluated for their ability to displace [3H]spiperone in sodium chloride free medium (Table 4). In general, these guanidines displayed a weaker affinity for the receptor as compared to dopamine. The rank order potency of these compounds is dopamine 1 > propylguanidine 87 > arylguanidine 84 > ethylguanidine 86. This progression does not show any expected relationship to the length of the alkyl chain. In addition, arylguanidine 84 displays a one site binding model while the other derivatives show a two site model comparable to dopamine. Evaluation of benzylguanidine 85, THIQ-guanidine 89, vanillylguanidine 90, and 76 Table 4. Apparent Equilibrium Binding Dissociation Constants of Dopaminergic Agonists for the Inhibition of [3H]Spiperone in NaCI Free Medium Compd # Description Kh(^i M) K|(nM) % H 1 DA 4.2 ± 0.7 (20) 84.2 ± 12.2 (20) 40 ± 2.6 DHPG 48.22 ± 7.54 (4) one site only 85 DHPMG NDa ND 8 6 DHPEG 77.4 ± 32.3 (5) 620.9 ± 289.7 (5) 47 ± 9.2 87 DHPPG 19.8 ± 1.2 (3) 243.1 ± 94.6 (3) 38 ± 2.0 103 DMPEG 630.2 ± (3) one site only 160 DMPG 884.1 ± 212.5 (3) one site only aND = Not Determined isovaniilylguanidine 91 as well as the binding studies in the presence of sodium chloride are currently in progress. 3.3 Summary 1. The synthesis of a homologous series of arylalkylguanidines 84-87 has been completed. The synthesis of each derivative was essentially completed on an individual basis requiring either 2-ethyl-2-thiopseudourea hydrobromide or cyanamide as the functionalizing agent. The synthesis of benzylguanidine 85 was successfully completed utilizing by using benzyl ethers or a cyclohexylidene ketal as the protecting group. Harsh nucleophilic deprotecting agents caused severe decomposition of benzylic guanidines by displacing the guanidine functionality. Several derivatives 89-91 containing the benzyiic skeleton with their structures were also synthesized. Preliminary evaluation of the ethyl 86 and propyl 87 derivatives shows that these compounds are weak agonists as compared to dopamine in functional and binding studies. This loss of activity may be related to the delocalization of the positive charge over the three nitrogen atoms. CHAPTER IV EXPERIMENTAL Melting points are uncorrected and were determined with a Thomas- Hoover melting point apparatus. NMR spectra were obtained at The Ohio State University College of Pharmacy, with either an IBM NR-250 FTNMR or and IBM AF-270 FTNMR spectrometers and are reported in parts per million relative to tetramethylsilane. Mass spectra were obtained at The Ohio State College of Pharmacy with a Kratos MS25RFA mass spectrometer or at The Ohio State University Chemical Instrument Center with either a VG 70-250S, Nicolet FTMS-2000, or Finnigan MAT-900 mass spectrometers. Fast atom bombardment mass spectroscopy (FAB MS) utilized 3-nitrobenzyl alcohol as solvent unless otherwise noted. Infrared spectra were obtained at The Ohio State University College of Pharmacy with an Analect RFX-40 FTIR spectrometer. Elemental Analysis were performed by Oneida Research Services, Inc., (Whitesboro, NY) or by Galbraith Laboratories, Inc., (Knoxville, TN) within ± 0.4% of the theoretical values. Anhydrous tetrahydrofuran was dried and stored over sodium with benzophenone as an indicator. All other reagents were used as received from commercial suppliers. 78 Aminoiminomethanesulfonic acid, (99) : In a manner similar to the procedure of Miller [111], 30% hydrogen peroxide (40 mL) was placed in a 500 mL 3-neck flask fitted with a thermometer and a 250 mL addition funnel. After cooling with an ice bath to 0 °C, acetic anhydride (25 mL) was slowly added dropwise. Upon completion, a second portion of acetic anhydride (10 mL) containing concentrated sulfuric acid (2 drops) was slowly added dropwise (avoids violent reaction from direct addition of acid). Finally, a third portion of acetic anhydride (40 mL) was slowly added dropwise, and the mixture was allowed to warm to room temperature by stirring overnight. The mixture was diluted with methanol (150 mL) and cooled with an ice bath to an internal temperature of 10-20 °C. Thiourea (5.0 g, 65.7 mmol) was dissolved in a 4:1 mixture of methanol:water (125 mL). This solution was added at such a rate to maintain an internal temperature of 10-20 °C. Upon complete addition, the ice bath was removed and the reaction allowed to warm to room temperature overnight. The few crystals that were formed at this point (sulfinic acid derivative [111]) were filtered and discarded. The filtrate was concentrated in vacuo to about 30 mL. The deposited crystals were collected by vacuum filtration, and washed with cold methanol to yield 6.05 g (74%) of a white solid, dp 129-130 (with bubbling, lit. [111] dp 131-131.5 with bubbling). The filtrate was placed in the freezer, deposited crystals were collected as before to yield an additional 500 mg (total yield 80%) of a white solid, dp 129-130 (with bubbling). IR (KBr) 1230, 1057 cm'1. SEt HsN^NH -HBr S-Ethvlthiopseudourea hvdrobromide, (102) . Prepared according to the procedure of Alam [112], thiourea (30.0 g, 0.394 mol) was suspended in a mixture of ethyl bromide (73.5 mL, 0.985 mol) and ethanol (50 mL) and heated to reflux for 6 h (until clear). The solution was cooled to room temperature and diluted with diethyl ether (500 mL). The deposited crystals were collected by vacuum filtration to yield 69.6 g (95%) of a white solid, mp 85-87 °C (lit. [112] mp 85-87 °C). 1H NMR (D20) : 6 3.18 (q, J=7.4Hz, 2H, C l-y, 1.38 (t, J= 7.4 Hz, 3H, CH3). 13C NMR (D20) : 6 172.7, 26.9, 14.6. 2-(3.4-Dimethoxvphenv0ethvlquanidine hvdrobromide. (103) S-Ethylthiopseudourea hydrobromide (7.7 g, 40 mmol) was placed in a 50 mL rb flask and melted in an oil bath at 140 °C. The temperature was lowered to 110-120 °C, and a solution of 3,4-dimethoxyphenylethylamine (9.0 g, 50 mmol) in water (6.5 mL) was added portionwise (4 x ca. 2 mL) every 15 min. Upon complete addition, the mixture was heated for a total of 2 h, cooled to room temperature, and evaporated in vacuo to a yellow oil. This oil was triturated in acetone (50 mL) to produce a white suspension. The solid was collected by vacuum filtration and recrystallized from ethanol/diethyl ether to yield 6.52 g (52%) in three crops. An analytical sample was prepared by a second recrystallization from ethanol/diethyl ether to yield a white solid, mp 171-3 °C (personal communication [105], mp 172-174 °C). 1H NMR (D20) : 6 6.82-6.67 (m, 3H, ArH), 3.67 (s, 3H, OCH3), 3.66 (s, 3H, OCH3), 3.25 (t, J= 6.6 Hz, 2H, CH2), 2.65 (t, J = 6.6 Hz, 2H, CH2). 13C NMR (D20) : 6 157.7, 149.2, 148.0, 132.6, 122.5, 113.9, 113.4, 56.9, 43.4, 34.7. FAB MS m/z 224.1 (m + 1 - HBr, base). Analysis for C11H18N30 2 : calc. C, 43.43; H, 5.96; N, 13.81; found C, 43.53; H, 6.06; N, 13.82. 2-(3.4-DihvdroxvphenvDethvlquanidine hvdrobromide. (86) : Guanidine 103 (3 g, 9.9 mmol) was dissolved in 48% hydrobromic acid (15 mL) under argon and heated to reflux for 6 h. The solution was cooled to room temperature and evaporated in vacuo to a solid. The solid was recrystallized from ethanol/diethyl ether to yield 2.60 g (96%) of a white solid, mp 141-144 °C. A portion of this material (1.0 g) was recrystallized a second time from ethanol/diethyl ether to yield 965 mg (97%) of a white solid, mp 142-144 °C (personal communication [105], mp 142-144 °C). ’H NMR (D20) ; 6 6.73-6.53 (m, 3H, ArH), 3.20 (t, J = 6.3 Hz, 2H, O y , 2.57 (t, J = 6.3 Hz, 2H, O y . 13C NMR (D20) : 6 156.7, 144.0, 142.6, 131.0, 121.1, 116.6, 116.3, 42.4, 33.4. FAB MS m/z 196.1 (m + 4 1 - HBr, base). Analysis for C9H14N30 2Br1 : calc. C, 39.15; H, 5.11; N, 15.22; found C, 39.05; H, 5.09; N, 15.15. o 3-(3.4-Dimethoxyphenvnproprionamide. (105) To a solution of 3-(3,4-dimethoxyphenyl)propionic acid (20.0 g, 95 mmol) in dichloromethane (100 mL) was added thionyl chloride (10.4 mL, 143 mmol), a catalytic amount of dimethylformamide (2 drops), and the entire mixture was heated to reflux for 5 h. The solution was evaporated in vacuo, and the resulting oil was rinsed with dichloromethane (50 mL) and evaporated in vacuo. The resulting oil was added dropwise to an ice cold solution of concentrated ammonium hydroxide (28%, 200 mL) with vigorous stirring. The precipitated solid was collected by vacuum filtration to yield 18.35 g (92%). An analytical sample was prepared by recrystallizing from toluene to give a tan solid, mp 119-21 °C (lit. [113] mp 121-22 °C). 1H NMR (CDCI/TMS) : 6 6.81-6.73 (m, 3H, ArH), 5.92 (bs, 1H, 83 NH), 5.60 (bs, 1H, NH), 3.86 (s, 3H, CH30), 3.85 (s, 3H, CH30), 2.91 (t, J= 7.6 Hz, 2H, ArCH2) 2.51 (t, J = 7.6 Hz, 2H, CH2CO). c h3o 'NH, CH30 3-(3,4-Dimethoxvphenvl)propylam8ne, (106) : To a suspension of lithium aluminum hydride (6.16 g, 162 mmol) in tetrahydrofuran (100 mL) at 0 °C was added a solution of propylamide 105 (17.0 g, 81 mmol) in tetrahydrofuran (300 mL) dropwise over 90 min. Upon complete addition, the ice bath was removed, and the suspension was heated to reflux for 2 h. The mixture was cooled to room temperature and stirred at this temperature for 48 h. The suspension was cooled to 0 °C, and quenched by the addition of 15% aqueous sodium hydroxide (24 mL) over 60 min. The suspension was filtered, the layers of the filtrate were separated, the organic layer was washed with brine, and dried over magnesium sulfate. Solvent was removed in vacuo to yield 12.78 g (81%) of a yellow oil. A portion of this oil was distilled in vacuo to yield a light yellow oil bp 100-20 °C / 0.5-2 mm Hg (lit. [113] bp 158-61 °C / 15 mm Hg). 1H NMR (CDCI/TMS, free base) : 6 6.81 -6.69 (m, 3H, ArH), 3.88 (s, 3H, CH30), 3.87 (s, 3H, CH30), 2.73 (t, J = 7.5 Hz, 2H, C H ^H .,), 2.59 (t, J = 7.5 Hz, 2H, ArCH2), 1.72 (m, 2H, ArCH2Chy, 1.30 (bs, 2H, NH2). 84 NH H2 C 0 3 3-(3.4-Dimethoxyphenvnpropvlquanidine bicarbonate salt (107) : S-Ethylthiopseudourea hydrobromide (3.94 g, 21.3 mmol) was placed in a 50 mL rb flask and melted in an oil bath at 120 °C. The temperature was lowered to 100 °C, and a solution of 3,4-dimethoxyphenylpropylamine 106 (4.38 g, 22.4 mmol) in isopropanol (10 mL) was added portionwise (4 x ca. 3 mL) every 15 min. Upon complete addition, the mixture was heated for a total of 2 h, cooled to room temperature, and evaporated in vacuo to a yellow oil. After many attempts to crystallize the oil, it was converted to its free base with 10% aqueous sodium hydroxide (100 mL) and dichloromethane (100 mL), and the layers were separated. The organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo. The resulting foam was triturated in a solution of saturated sodium bicarbonate (50 mL) and the precipitate collected by vacuum filtration to yield 2.13 g (37%) of a yellow solid, dp 123-6 °C (with bubbling). 1H NMR (CD3OD/DCI) : 6 6.86 (d, J= 8.2 Hz, 1H, ArH), 6.83 (d, J = 2.0 Hz, 1H, ArH), 6.75 (dd, J= 8.2 and 2.0 Hz, 1H, ArH), 3.81 (s, 3H, CH30), 3.79 (s, 3H, CH30), 3.17 (t, J= 7.0 Hz, 2H, CK.NH), 2.64 (t, J= 8.0 Hz, 2H, ArCH2), 1.88 (m, 2H, ArCH2CHJ. 13C NMR (CD3OD/DCI) : 6 158.8, 150.6, 149.0, 135.5, 121.7, 113.9, 113.7, 56.8, 56.7, 41.7, 33.1, 31.6. FAB MS m/z 238.2 (m + 1 - H2C03l base). Analysis for C13H21N30 5 : calc. C, 52.17; H, 7.07; N, 14.04; found C, 52.45; H, 7.04; N, 13.72. NH HBr 3-(3,4-Dihvdroxvphenvl)propylquanidine hvdrobromide, (87) : Guanidine 107 (1.0 g, 3.7 mmol) was dissolved in 48% hydrobromic acid (5 mL) under argon and heated to reflux for 4 h. The solution was cooled to room temperature and evaporated in vacuo to a solid. The solid was recrystallized twice from ethanol to yield 732 mg (68%) of a tan solid, mp 171-73 °C in two crops. 1H NMR (D20) : 6 6.73 (d, J= 8.0 Hz, 1H, ArH), 6.66 (d, J = 1.7 Hz, 1H, ArH), 6.57 (dd, J= 8.0 and 1.7 Hz, 1H, ArH), 3.01 (t, J= 6.8 Hz, 2H, ArCH2), 2.44 (t, J= 6.6 Hz, 2H, CKTJH), 1.72 (m, 2H, ArCH2Chy. 13C NMR (D20) : 6 157.5, 144.8, 142.8, 135.3, 121.5, 117.2, 117.1, 41.3, 32.1, 30.3. FAB MS m/z 210.1 (m + 1 - HBr, base). Analysis for C^H^NgCXBr,: calc. C, 41.39; H, 5.56; N, 14.48; found C, 41.53; H, 5.35; N, 14.40. 86 NH -HBr 3,4-Dimethoxvbenzvlquanidine hvdrobromide, (109) : S-Ethylthiopseudourea hydrobromide (7.7 g, 40 mmol) was melted in a 50 mL rb flask at 100-110 °C. A solution of 3,4-dimethoxybenzylamine (8.36 g, 50 mmol) in water (16 mL) was added portionwise (4 x ca. 5 mL) every 15 min. Upon complete addition, the solution was heated for a total of 2 h. The solution was cooled to room temperature, and the solvent was evaporated in vacuo. The resulting yellow oil was triturated in acetone (15 mL) to form a precipitate. The solid was collected by vacuum filtration, the mother liquor was concentrated in vacuo. The process was repeated with acetone (8 mL) and the resulting solid was combined with the first crop. The combined material was recrystallized from ethanol/diethyl ether to yield 4.31 g (36%) of a white solid, mp 155-156 °C. 1H NMR (D20) : 6 6.90-6.79 (m, 3H, ArH), 4.20 (s, 2H, CH2), 3.69 (s, 6H, CH30). 13C NMR (D20) : 6 157.8, 149.5, 148.9, 130.1, 120.9, 113.2, 112.1, 56.8, 45.3. FAB MS m/z 210.2 (m + 1 - HBr, base), 151.1 (m + 1 - HBr - C ^ N g , 69%). Analysis for C^H^NgOgBr, : calc. C, 41.39; H, 5.56; N, 14.48; found C, 41.75; H, 5.69; N, 14.53. 87 NH H2 CO3 3-Methoxvbenzvlquanidine bicarbonate salt (111) : 3-Methoxybenzylamine (3.5 g, 25.5 mmol) was added portionwise (4 x ca. 1 mL) every 15 min to a solution of S-ethylthiopseudourea hydrobromide (4.3 g, 23.2 mmol) in acetonitrile (10 mL) heated to reflux. Upon complete addition, the amine flask was rinsed with acetonitrile (5 mL) and the rinse was added to the reaction mixture. The mixture was heated to reflux for a total of 2 h and subsequently cooled to room temperature. The contents were diluted with water (20 mL) and extracted with diethyl ether (3 x 40 mL). The aqueous layer was evaporated in vacuo to an oil. After many attempts to recrystallize, the oil was dissolved in hot water (20 mL) and poured into a saturated solution of sodium bicarbonate (20 mL). The resulting precipitate was collected by vacuum filtration and washed with water to yield 3.73 g of a white solid, dp 139-141 °C (with bubbling). 1H NMR (D20/DCI) : 6 6.96-6.89 (m, 1H, ArH), 6.55-6.49 (m, 3H, ArH), 3.95 (s, 2H, CH2), 3.37 (s, 3H, CH30). 13C NMR (D20/DCI) : 6 160.1, 157.8, 138.5, 131.0, 120.5, 114.3, 113.6, 56.4, 45.3. FAB MS m/z 180.1 (m + 1 - H2C0 3, base), 121.1 (m + 1 - H2C0 3 - C ^ N ^ 21%). Analysis for C10H15N3°4 : calc- c ' 49-79; H, 6.27; N, 17.42; found C, 50.15; H, 6.17; N, 17.13. 88 NH -HBr 3-Methoxvbenzvlquanidine hvdrobromide, (112) : Bicarbonate salt 111 (1.0 g, 4.1 mmol) was added to an ice cold solution of 48% hydrobromic acid (2 mL) with vigorous stirring. Upon stirring for 5 min, the evolution of gas had ceased, and the solution was evaporated in vacuo at room temperature. The resulting solid was recrystallized from methanol/diethyl ether to yield 735 mg (68%) of a tan solid, mp 115-7 °C. 1H NMR (D20) : 6 7.21 (t, J = 7.9 Hz, 1H, ArH), 6.82-6.77 (m, 3H, ArH), 4.24 (s, 2H, CH2), 3.66 (s, 3H, CH30). 13C NMR (D20) : 6 160.3, 157.9, 138.9, 131.3, 120.7, 114.4, 113.7, 56.5, 45.4. FAB MS m/z 180.1 (m + 1 - HBr, base), 121.1 (m + 1 - HBr - C ^ N g , 23%). Analysis 1 for C gH ^N gO ^ : calc. C, 41.56; H, 5.43; N, 16.15; found C, 41.46; H, 5.37; N, 16.01. NH HBr 4-Hvdroxv-3-methoxvbenzvlquanidine hvdrobromide. (90) : A solution of 4-hydroxy-3-methoxybenzylamine hydrochloride (1.0 g, 5.3 mmol) in methanol (15 mL) was converted to its free base by adding a 25% solution of sodium 89 methoxide in methanol (1.2 mL, 5.3 mmol) at room temperature under argon. The solution was stirred for 1 h and diluted with diethyl ether (50 mL). The suspension was filtered, and the filtrate was evaporated in vacuo to a solid. This solid was immediately dissolved in acetonitrile (10 mL), and charged with S-ethylthiopseudourea hydrobromide (780 mg, 4.2 mmol). The mixture was heated to reflux for a total of 2 h. The hot solution was immediately diluted with diethyl ether until it became cloudy and allowed to cool to room temperature overnight. The precipitated crystals were filtered to yield 230 mg (16%) of yellow needles, mp 182-4 °C. The mother liquor was diluted with water (20 mL), extracted with diethyl ether (3 x 20 mL) and the aqueous layer was evaporated in vacuo. The resulting yellow oil was recrystallized from ethanol/diethyl ether to yield 626 mg (59% total yield) of a second crop of a yellow solid, mp 182-4 °C. 1H NMR (DzO) : 6 6.86-6.76 (m, 3H, ArH), 4.21 (s, 2H, CHjj), 3.74 (s, 3H, CH30). 13C NMR (DzO) : 6 157.9, 148.7, 145.7, 129.6, 121.3, 116.8, 112.9, 57.3, 45.5, FAB MS m/z 196.2 (m + 1 - HBr, base). Analysis for C9H14N30 2Br1 : calc. C, 39.15; H, 5.11; N, 15.22; found C, 39.54; H, 5.21; N, 15.14. 90 3-Hvdroxvbenzaldehvde oxime. (115) : In a manner similar to Buck [114], a solution of hydroxylamine hydrochloride (4.17 g, 60 mmol) in water (5 mL) was added to a solution of 3-hydroxybenzaldehyde (6.11 g, 50 mmol) in warm 95% ethanol. Aqueous 30% sodium hydroxide (10 mL, 75 mmol) was added, and the mixture was heated to reflux for 2 h and subsequently cooled to room temperature. Solvent was removed in vacuo, and the resulting oil was dissolved in ethyl acetate (250 mL). This solution was washed with saturated sodium bicarbonate (2 x 250 mL), brine (250 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The solid residue was recrystallized from toluene to yield 6.05 g (88%) of a brown solid, mp 86-87.5 °C (lit. [115] mp 90 °C). 1H NMR (CDCI/TMS/DMSO) : 6 11.02 (s, 1H, =NOH), 9.35 (s, 1H, ArOH), 7.99 (s, 1H, CH=N), 7.18-7.12 (m, 1H, ArH), 7.02-6.94 (m, 2H, ArH), 6.77 (d, J= 8.0 and 2.0 Hz, 1H, ArH). Q T NH2 3-Hvdroxvbenzvlamine. (116) : Raney Nickel catalyst (0.15 mL slurry) was added to an ethanolic solution (30 mL) of oxime 115 (2.0 g, 15 mmol) in a Parr bottle. The mixture was shaken for 36 h at room temperature and 50 psi of hydrogen on a Parr hydrogenator. Upon completion, the reaction mixture was filtered, and the filtrate was evaporated in vacuo. The resulting solid was recrystallized from methanol to yield 1.72 g (96%) of a light brown solid, mp 162-6 °C. A portion of this material was decolorized by heating with charcoal in a large volume of methanol. The hot mixture was filtered and evaporated in vacuo to give a white solid, mp 164-6 °C (lit. [116] mp 173-4 °C). 1H NMR (DzO/DCI) : 6 7.12-7.05 (m, 1H, ArH), 6.75-6.67 (m, 3H, ArH), 3.86 (s, 2H, CH2). To verify that the material obtained is the primary amine, a portion of the material (10 mg) was converted to N-(3-acetoxybenzyl)-acetamide by stirring overnight with acetic anhydride (1 mL) and pyridine (1 mL) at room temperature. The mixture was diluted with ethyl acetate (10 mL) and washed with 1.2 N HCI (3 x 10 mL), brine (10 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was characterized by 1H NMR spectroscopy. 1H NMR (CDCy : 6 7.37-7.30 (m, 1H, ArH), 7.13 (d, J= 7.8 Hz, 1H, ArH), 7.01-6.98 (m, 2H, ArH), 5.93 (bs, 1H, NH), 4.41 (d, J= 5.7 Hz, 2H, CH2), 2.29 (d, J= 0.7 Hz, 3H, NHCOCHy, 2.01 (s, 3H, CH3C0 2). 92 AcO CH30 3-Acetoxv-4-methoxvbenzonitrile. (118) : To a suspension of 3-hydroxy-4- methoxybenzaldehyde (5.0 g, 33 mmol) in 95% ethanol (25 mL) was added a solution of hydroxylamine hydrochloride (2.75 g, 40 mmol) in water (5 mL), a solution of 50% aqueous sodium hydroxide (3.9 mL, 49 mmol) and the mixture was heated to reflux for 3 h. The solution was cooled to room temperature, evaporated in vacuo, and dissolved in a mixture of ethyl acetate (200 mL) and 1.2 N hydrochloric acid (200 mL). The layers were separated and, the organic layer was washed with 1.2 N HCI (200 mL), brine (200 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo to yield 4.84 g (88%) of a solid that was used without purification. The oxime was dissolved in acetic anhydride (20 mL) and heated to reflux for 3 h. The solution was cooled to room temperature, and poured into an ice slurry (ca. 100 mL). The ice slurry was extracted with ethyl acetate (2 x 50 mL), the organics extracts were combined, washed with 1.2 N HCI (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting solid was recrystallized from water to yield 4.43 g (80% from oxime) of yellow needles, mp 110-111 °C (lit. [117] m.p. 116-117 °C). 1H NMR (CDCI/TMS) : 6 7.53 (dd, J = 8.5 and 93 1.9 Hz, 1H, ArH), 7.33 (d, J= 1.9 Hz, 1H, ArH), 7.01 (d, J= 8.5 Hz, 1H, ArH), 3.89 (s, 3H, CH30), 2.33 (s, 3H, CH3C02). 3-Hvdroxv-4-methoxvbenzvlamine hydrochloride. (119) : A 1.0 M solution of borane-tetrahydrofuran complex (20 mL, 20 mmol) under argon was charged with benzonitrile 118 (1.0 g, 5.2 mmol). The solution was heated to reflux overnight and cooled to room temperature. Methanol (20 mL) was carefully added over 30 min and the solution was heated to reflux for an additional 30 min. The solution was cooled to room temperature and evaporated in vacuo to yield 800 mg (quantitative) of an off white solid, mp 133-138 °C (lit. [118] 150-158 °C) that was used without further purification. An portion of this solid (200 mg) was dissolved in a mixture of methanol (5 mL) and diethyl ether (25 mL). Anhydrous hydrogen chloride (g) was bubbled into the solution and the resulting precipitate was collected to yield 175 mg (71%) of a white solid, mp 169-70 °C (lit. [119] mp 195-198 °C). 1H NMR (D20) : 6 6.91 (d, J= 8.9 Hz, 1H, ArH), 6.84-6.81 (m, 2H, ArH), 3.91 (s, 2H, CH2), 3.72 (s, 3H, CH30). FAB MS (GT-13) m/z 154.1 (m + 1, 51%), 137.1 (m + 1 - NH3, base). Analysis for CgH^^OjjCI, : calc. C, 50.67; H, 6.38; N, 7.39; found C, 50.29; H, 6.27; N, 7.39. 94 NH HBr 3-Hvdroxv-4-methoxvbenzvlquanidine hvdrobromide, (91) : The crude free ? base of 3-hydroxy-4-methoxybenzylamine hydrochloride 119 (500 mg, 3.26 mmol) was dissolved in acetonitrile (10 mL), charged with S-ethylthiopseudourea (544 mg, 2.94 mmol) and heated to reflux for 2 h. The hot solution was diluted with diethyl ether until the mixture became cloudy, and allowed to cool to room temperature overnight. The precipitated crystals were collected to yield 165 mg (20%) of a white solid, mp 146-7 °C. The mother liquor was diluted with water (10 mL), and extracted with diethyl ether (3 x 10 mL). The aqueous layer was evaporated to an oil which was recrystallized from ethanol/diethyl ether to yield and additional 225 mg (48% total yield) of a white solid, mp 142-4 °C. 1H NMR (D20) : 6 6.85 (d, J = 8.9 Hz, 1H, ArH), 6.69-6.72 (m, 2H, ArH), 4.13 (s, 2H, CH2), 3.68 (s, 3H, CH30). 13C NMR (D20) : 6 158.0, 148.2, 146.3, 130.4, 120.5, 115.5, 114.1, 57.3, 45.1. FAB MS m/z 196.1 (m + 1 - HBr, base). Analysis for CgH^NgCXjB^ : calc. C, 39.15; H, 5.11; N, 15.22; found C, 39.02; H, 5.05; N, 15.03. 95 MeO NH (COOH)2 MeO 6,7-Dimethoxv-1,2,3,4-tetrahydroisoquinoline oxalate salt (121) : To a solution of 3,4-dimethoxyphenylethylamine (10.0 g, 55 mmol) in formic acid (20 mL) was added a 37% (w/w) solution of formaldehyde (4.12 mL, 55 mmol). The mixture was heated at 50 °C for 24 h, cooled to room temperature, and evaporated in vacuo. The resulting oil was dissolved in ethanol (50 mL) and added dropwise to a solution of oxalic acid dihydrate (13.9 g, 110 mmol) in ethanol (50 mL) with vigorous stirring. The resulting solid was collected by vacuum filtration to yield 9.14 g (58%) of a white solid, dp 215-215.5 °C (with bubbling, lit. [120] mp 214-215 °C). 1H NMR (free base, CDCI/TMS) : 6 6.58 (s, 1H, ArH), 6.50 (s, 1H, ArH), 3.93 (s, 2H, ArCH2N), 3.90 (s, 3H, CH30), 3.84 (s, 3H, CH30), 3.11 (t, J = 5.8 Hz, 2H, ArCH2CH>N), 2.70 (t, J = 5.8 Hz, 2H, ArCHLjCHgN), 2.23 (bs, 1H, NH). HN -HBr 6.7-Dimethoxv-3,4-dihvdro-2(1 H)-isoquinol8necarboximideamide hvdrobromide. (122) : Tetrahydroisoquinoline oxalate 121 (5.0 g, 17.7 mmol) was converted to its free base by stirring with 5% aqueous sodium hydroxide (50 mL) and chloroform (50 mL) for. 60 min. The layers were separated, and the organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was added portionwise (4 x ca. 1 mL) every 15 min to a solution of S-ethylthiopseudourea hydrobromide (2.94 g, 15.9 mmol) in water (5 mL) that was heated to reflux. The amine flask was rinsed with hot water (4 mL) and added to the reaction flask. The reaction was heated for a total of 2 h. The mixture was allowed to cool to room temperature and deposited a white solid. The solid was collected by vacuum filtration and recrystallized from water/ethanol to yield 1.7 g (30%) of a white solid, mp 258-259 °C in 3 crops. 1HI NMR (D20) : 6 6.60 (s, 1H, ArH), 6.55 (s, 1H, ArH), 4.16 (s, 2H, ArCH2N), 3.61 (s, 6H, 2 x CH30), 3.22 (t, J = 5.8 Hz, 2H, A rC H ^ I^ N ), 2.59 (t, J = 5.8 Hz, 2H, ArCH,CH2N). 13C NMR (D20) : 6 157.0, 148.4, 148.0, 128.0, 124.4, 112.5, 110.7, 56.9, 56.8, 47.0, 44.2, 28.0. FAB MS m/z 236.1 (m + 1 - HBr, base). Analysis for C12H18N30 2Br1 : calc. C, 45.58; H, 5.74; N, 13.29; found C, 45.48; H, 5.73; N, 13.16. 6.7-Dihvdroxv-1.2.3.4-tetrahvdroisoquinoline hvdrobromide. (123) Tetrahydroisoquinoline oxalate 121 (2.0 g, 7.1 mmol) was converted to its free base by stirring with 5% aqueous sodium hydroxide (50 mL) and chloroform 97 (50 mL) for 60 min. The layers were separated, and the aqueous layer was extracted with chloroform (50 mL). The organic layers were combined and washed with water (50 mL) brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in 48% hydrobromic acid (8 mL) under argon and heated to reflux overnight. The mixture was cooled to room temperature and evaporated in vacuo. The resulting solid was triturated in hot ethanol (15 mL) and allowed to cool. The deposited solid was collected by vacuum filtration to yield 1.02 g (59%) of a white solid, mp 263-264 °C (lit. [121] m.p. 267-268 °C). 1H NMR (CD3OD) : 6 6.61 (s, 1H, ArH), 6.58 (s, 1H, ArH), 4.17 (s, 2H, ArCH2N), 3.42 (t, J= 6.4 Hz, 2H, ArCH2Oi,N), 2.94 (t, J = 6.4 Hz, 2H, A r C H ^ N ) . HN HBr 6,7-Dihvdroxv-3,4-dihvdro-2(1 H)-isoquinolinecarboximideamide hvdrobromide, (89) : To a solution of tetrahydroisoquinoline hvdrobromide 123 (500 mg, 2.03 mmol) in water (8 mL) was added a solution of 50% aqueous cyanamide (0.75 mL, 9.15 mmol) and the resulting mixture was heated to reflux overnight. The solution was cooled to room temperature, and evaporated in vacuo. The resulting oil was triturated in hot ethanol (25 mL) and filtered. The filtrate was recrystallized three times from ethanol/diethyl ether to yield 123 mg (21%) of an orange solid, m.p. 251-253 °C. 1H NMR (CD3OD) : 6 6.63 (s, 1H, ArH), 6.60 (s, 1H, ArH), 4.42 (2H, ArCH2N), 3.57 (t, J = 5.9 Hz, 2H, A rC H ^ J iN ), 2.81 (t, 2H, J = 5.9 Hz, ArChL>CH2N). 13C NMR (CD3OD) : 6 157.9, 146.0, 145.5, 126.7, 123.4, 115.8, 114.0, 47.6, 45.0, 28.5. FAB MS m/z 208.2 (m + 1 - HBr, base). Analysis for C ^ H ^ N ^ B r,: calc. C, 41.68; H, 4.90; N, 14.58; found C, 42.05; H, 5.07; N, 14.66. 3 . 4-Bis(benzvIoxv)benzaIdehvde oxime. (1261 3,4-bis(benzyloxy)benzaldehyde (10.0 g, 31.4 mmol) was suspended in 95% ethanol (100 mL) and charged with hydroxylamine hydrochloride (2.62 g, 37.7 mmol), sodium hydroxide (1.88 g, 47.1 mmol) and water (10 mL). The clear solution was heated to reflux for 3.5 h and cooled to room temperature. Solvent was evaporated in vacuo, and the resulting oil was partitioned between ethyl acetate (100 mL) and water (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL), and the organic layers were combined. The organic phase was washed with brine (100 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting solid was recrystallized from ethyl acetate/hexanes to yield 7.7 g (74%) of light brown needles, mp 86-87 °C (lit. [122] mp 86-87 °C). A second crop yielded 2.3 g (96% total yield) of 99 brown needles, mp 85-86 °C. 1H NMR (CDCI/TMS) : 6 8.05 (bs, 1H, OH), 8.02 (s, 1H, CH=N), 7.46-7.26 (m, 11H, ArH), 7.00 (dd, J= 8.3 and 1.9 Hz, 1H, ArH), 6.89 (d, J= 8.3 Hz, 1H, ArH), 5.17 (s, 2H, CH20), 5.15 (s, 2H, CH20). BnO BnO 3.4-Bis(benzvloxv)benzonitrile. (127) : Oxime 126 (7.0 g, 21.0 mmol) was dissolved in acetic anhydride (20 mL) and heated to reflux for 4 h. After cooling to room temperature, the solution was poured into an ice slurry (150 mL) and extracted with ethyl acetate (2 x 100 mL). The organics were combined, washed with water (2 x 100 mL), brine (100 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting orange oil was decolorized with charcoal and ethanol. The pale yellow ethanolic solution was evaporated in vacuo to a yellow oil which crystallized upon standing. After trituration in hexanes, the solid was collected by vacuum filtration to yield 6.18 g (93%) of a white solid, mp 53-54 °C (lit. [123] mp 62-63 °C). 1H NMR (CDCL/TMS) : 6 7.44-7.32 (m, 10H, ArH), 7.21 (dd, J= 8.4 and 1.9 Hz, 1H, ArH), 7.13 (d, J = 1.9 Hz, 1H, ArH), 6.93 (d, J = 8.4 Hz, 1H, ArH). El MS m/z 315.1261 (m+ calc. 315.1259, 7.1%), 224.07 m+ - C7H7, 33%), 91 (C7H7+, base). 100 3,4-Bis(benzvloxv)benzvlamine hydrochloride. (128) : Benzonitrile 127 (10.39 g, 33.0 mmol) was placed in a 250 mL rb flask under argon and charged with a 1.0 M solution of borane-tetrahydrofuran complex (100 mL, 100 mmol). The solution was heated to reflux overnight and subsequently cooled to room temperature. The mixture was carefully quenched with methanol (100 mL), and when the reaction ceased, the solution was heated to reflux for 60 min. After cooling to room temperature, solvent was evaporated in vacuo. The crude amine (9.50 g, 90%) was used without further purification. A portion of this material (1.5 g) was stirred with a saturated solution of hydrogen chloride (g) in methanol (10 mL) and subsequently diluted with diethyl ether (100 mL). The resulting precipitate was collected by vacuum filtration to yield 1.114 g (67%) of a white solid, mp 154-156 °C (lit. [122] mp 156-157 °C). 1H NMR (CD3OD) : 6 7.48 (m, 10H, ArH), 7.16 (d, J = 1.9 Hz, 1H, ArH), 7.07 (d, J = 8.2 Hz, 1H, ArH), 6.98 (dd, J= 8.2 and 1.9 Hz, 1H, ArH), 5.15 (s, 4H, ArCH20), 4.01 (s, 2H, ArCH2N). 101 3.4-Dimethoxvbenzaldehvde oxime, (131) : In a manner similar to the procedure of Buck [114], a solution of hydroxylamine hydrochloride (15.0 g, 0.215 mol) in water (20 mL) was added to a suspension of 3.4-dimethoxybenzaldehyde (30.0 g, 0.18 mol) in 95% ethanol (200 mL). Aqueous 50% sodium hydroxide (20 mL, 0.25 mol) was added, and the mixture was heated to reflux for 2 h. Upon cooling to room temperature, solvent was removed in vacuo, and the resulting oil was dissolved in ethyl acetate (125 mL). The organic layer was washed with saturated sodium bicarbonate (2 x 125 mL), brine (100 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil crystallized on standing to yield 26.35 g (81%) of a light yellow solid, mp 86-7 °C (lit. [124] 88-9 °C). 1H NMR (CDCI/TMS/DMSO) : 6 10.85 (s, 1H, NOH), 8.00 (s, 1H, CH=N), 7.20 (d, J= 1.8 Hz, 1H, ArH), 7.04 (dd, J = 8.2 and 1.8 Hz, 1H, ArH), 6.91 (d, J= 8.2 Hz, 1H, ArH), 3.82 (s, 6H, 2 x C H 30). 3.4-Dimethoxvbenzonitrile, (132) : Oxime 131 (5.0 g, 27.6 mmol) was dissolved in acetic anhydride (5 mL) and heated to reflux for 1.5 h. Upon cooling to room temperature, the solution was poured into an ice slurry (50 mL). The aqueous layer was extracted with dichloromethane (2 x 50 mL), and the organic extracts were combined. The solution was washed with brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was recrystallized from ethyl acetate/hexanes to yield 3.64 g (81%) of a white solid, mp 61-64 °C (lit. [125] mp 67-68 °C). 1H NMR (CDCI/TMS) : 6 7.29 (dd, J = 8.3 and 1.8 Hz, 1H, ArH), 7.08 (d, J= 1.8 Hz, 1H, ArH), 6.90 (d, J= 8.3 Hz, 1H, ArH), 3.94 (s, 3H, CH30), 3.91 (s, 3H, CH3Q). 3.4-Dihvdroxvbenzonitrile. (133) Una manner similar to that reported by Wick [126], benzonitrile 132 (1.0 g, 6.1 mmol) was dissolved in dichloromethane (5 mL) under argon and cooled to 0 °C with an ice bath. A solution of 1.0 M boron tribromide in dichloromethane (10 mL, 10 mmol) was added via a syringe. The ice bath was removed, the mixture was allowed to stir for 2 h at room 103 temperature before it was heated to reflux overnight. The solution was cooled to room temperature, and carefully quenched with methanol (10 mL). Solvent was removed in vacuo, and the resulting oil was dissolved in ethyl acetate (50 mL) and washed with 1.2 N HCI (50 mL). The aqueous layer was extracted with ethyl acetate (50 mL), and the organic extracts were combined. The organic portion was washed with brine (50 mL), dried over anhydrous sodium sulfate and evaporated in vacuo. The residue was triturated in hexanes to yield 740 mg (89%) of a light brown powder, mp 152-4 °C (lit. [127] 156 °C). ’H NMR (CDCI/TMS) : 6 7.20-7.16 (m, 2H, ArH), 6.93 (d, J = 8.6 Hz, 1H, ArH), 6.07 (bs, 1H, OH), 5.95 (bs, 1H, OH). IR (nujol) 3340 (OH), 2240 (ON) cm '1. 3.4-Bis(2-nitrobenzvloxv)benzonitrile, (134) : A foil wrapped flask containing a solution of benzonitrile 133 (500 mg, 3.7 mmol) in anhydrous dimethylformamide (10 mL) was charged with 2-nitrobenzylbromide (1.68 g, 7.8 mmol) and anhydrous potassium carbonate (1.06 g, 7.8 mmol). The mixture was stirred at room temperature overnight, diluted with water (25 mL), and extracted with ethyl acetate (3 x 20 mL). The organic extracts were combined, washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting solid was recrystallized from toluene to yield 910 mg (58%) of pale yellow needles, mp 191-191.5 °C. 1H NMR (CDCI/TMS) : 6 8.22-8.18 (m, 2H, NO^rH), 7.92-7.89 (m, 2H, N 0 2ArH), 7.73-7.67 (m, 2H, NOjArH), 7.57-7.51 (m, 2H, N 0 2ArH), 7.33 (dd, J = 8.4 and 1.9 Hz, 1H, ArH), 7.22 (d, J = 1.9 Hz, 1H, ArH), 7.03 (d, J = 8.4 Hz, 1H, ArH). El MS m/z 136.0 (C ^N A *. 94%). FAB MS m/z406.2 (m + 1, 1.3%), 232.1 (53%) 157.1 (base). Analysis for C21H15N30 6 : calc. C, 62.22; H, 3.73; N, 10.37; found C, 62.26; H, 3.85; N, 10.31. o -N 0 2BnO o -N 0 2BnO 3.4-Bis(2-nitrobenzvloxv)benzvlamine hydrochloride, (135) : Benzonitrile 134 (350 mg, 0.86 mmol) was placed in a 50 mL rb flask under argon and charged with a 1.0 M solution of borane-tetrahydrofuran complex (5 mL, 5 mmol). The solution was heated to reflux overnight and subsequently cooled to room temperature. The mixture was carefully quenched with methanol (5 mL), and when bubbling ceased, the mixture was heated to reflux for 30 min. After cooling to room temperature, solvent was evaporated in vacuo. The resulting oil was dissolved in dichloromethane (25 mL), washed with 5% aqueous sodium hydroxide (2 x 25 mL), brine (25 mL) and dried over anhydrous sodium sulfate. Solvent was evaporated in vacuo. A solution of the clear oil in methanol (2 mL) was diluted with a saturated solution of hydrogen chloride 105 (g)/methanol (2 mL) and allowed to stand overnight. Only a few crystals had deposited, so the mixture was diluted with diethyl ether (25 mL). The resulting precipitate was collected by vacuum filtration to yield 235 mg (61%) of a white solid, dp 215-217 °C (with darkening). ’H NMR (CD3OD) : 6 8.16-8.09 (m, 2H, NOgArH), 7.94-7.85 (m, 2H, NOgArH), 7.74-7.67 (m, 2H, NOgArH), 7.60-7.53 (m, 2H, NOgArH), 7.21 (d, J= 1.9 Hz, 1H, ArH), 7.12 (d, J = 8.3 Hz, 1H, ArH), 7.05 (dd, J = 8.3 and 1.9 Hz, 1H, ArH). FAB MS m/z 410.2 (m + 1 - HCI, 18%), 136.1 (C^e^Og, base). Analysis for C g ^^O g C ^ H20 calc. C, 54.38; H, 4.78; N, 9.06; found C, 54.42; H, 4.76; N, 8.90. NH HBr o-NOgBnO II ^ N H 2 o-N02BnQ 3.4-Bis(2-nitrobenzvloxv)benzvlquanidine hvdrobromide, (136) : Amine hydrochloride 135 (200 mg, 0.45 mmol) was converted to its free base with dichloromethane (20 mL) and 5% aqueous sodium hydroxide (20 mL). The organic layer was washed with brine (20 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The solid residue was suspended in acetonitrile (5 mL) and charged with S-ethylpseudothiourea hydrobromide (66 mg, 0.35 mmol). The mixture was heated to reflux for 2 h while being protected from light by foil. After cooling to room temperature, the deposited solid was collected by vacuum filtration and rinsed liberally with diethyl ether. 106 The solid was dissolved in hot ethanol (10 mL), filtered, and diluted with diethyl ether until the mixture became cloudy. The deposited solid was collected by vacuum filtration to yield 110 mg (58%) of a pale yellow solid, mp 190-192 °C. 1H NMR (CD3OD) : 6 8.14-8.09 (m, 2H, ArHN02), 7.91-7.86 (m, 2H, ArHN02), 7.77-7.66 (m, 2H, ArH N 02), 7.59-7.53 (m, 2H, ArHNCy, 7.09-7.04 (m, 2H, ArH), 6.94 (dd, J = 8.3 and 2.0 Hz, 1H, ArH), 5.50 (s, 2H, ArCH20), 5.49 (s, 2H, ArCH20), 4.32 (s, 2H, ArCH2N). 13C NMR (CD3OD) : 6 156.6, 147.7, 147.2, 147.1, 133.9, 133.8, 132.5, 130.5, 129.0, 124.6, 120.7, 114.7, 114.2, 67.3,43.7. FAB MS m/z 452.2 (m + 1 - HBr, base). Analysis for calc C, 49.64; H, 4.17; N, 13.16; found C, 49.60; H, 4.21; N, 12.76. CH3 OCH0 O _ X if^ CH30CH20 3.4-Bis(methoxvmethoxv)benzonitrile. (137) :ln a manner similar to the procedure of Winkle [128], sodium hydride (228 mg, 5.7 mmol) was washed with petroleum ether (bp 30-60 °C, 3 x 5 mL) and placed in a 3 neck flask fitted with a condenser, addition funnel and argon port. The washed solid was suspended in a mixture of tetrahydrofuran (10 mL) and dimethylformamide (2 mL). Benzonitrile 133 (350 mg, 2.59 mmol) was dissolved in tetrahydrofuran (10 mL) and added dropwise over 15 min. The resulting suspension was stirred for an additional 30 min before a solution of chloromethylmethyl ether (0.47 mL, 107 6.22 mmol) in tetrahydrofuran (5 mL) was added dropwise over a 10 min period. Upon complete addition, the suspension was stirred for an additional 2 h. The resulting suspension was diluted with water (50 mL) and diethyl ether (50 mL) with vigorous stirring, and the layers were separated. The organic layer was washed with 5% aqueous sodium hydroxide (2 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting pale yellow oil was subjected to silica gel chromatography with 20% ethyl acetate/hexanes as the eluent. Fractions were pooled and evaporated in vacuo to yield 520 mg (90%) of a clear, colorless oil. 1H NMR (CDCL/TMS) : 6 7.45 (d, J = 1.9 Hz, 1H, ArH), 7.30 (dd, J = 8.5 and 1.9 Hz, 1H, ArH), 7.22 (d, J = 8.5 Hz, 1H, ArH), 5.30 (s, 2H, CH2), 5.25 (s, 2H, CH2), 3.52 (s, 3H, CH30), 3.51 (s, 3H, CH30). El MS m/z 223.0840 (calc. 223.0844 m+, 5.9%), 45.0 (CH3OCH2+, base). IR (neat) 2227 (CN) cm'1. Analysis for C11H13N104 : calc. C, 59.19; H, 5.87; N, 6.28; found C, 59.63; H, 5.95; N, 6.32. NH2 (COOH)2 3.4-Bis(methoxvmethoxv)benzvlamine oxalate salt. (138) : Benzonitrile 137 (430 mg, 1.93 mmol) was placed in a 50 mL rb flask under argon and charged with a 1.0 M solution of borane-tetrahydrofuran complex (10 mL, 10 mmol). The solution was heated to reflux overnight and subsequently cooled to room temperature. The mixture,was carefully quenched with methanol (10 mL), and when bubbling ceased, the mixture was heated to reflux for 30 min. After cooling to room temperature, solvent was evaporated in vacuo. The resulting oil was dissolved in dichloromethane (25 mL), washed with 5% aqueous sodium hydroxide (2 x 25 mL), brine (25 mL) and dried over anhydrous sodium sulfate. Solvent was evaporated in vacuo. The clear oil was dissolved in diethyl ether (10 mL) and added dropwise to a solution of oxalic acid dihydrate (480 mg, 3.85 mmol) in diethyl ether (10 mL). After standing overnight, the precipitate was collected by vacuum filtration to yield 301 mg (49%) of a white solid, dp 115-118 °C (began to turn orange at 99 °C). 1H NMR (CD3OD) : 6 7.24 (d, J = 2.0 Hz, 1H, ArH), 7.16 (d, J = 8.3 Hz, 1H, ArH), 7.04 (dd, J= 8.3 and 2.0 Hz, 1H, ArH), 5.21 (s, 2H, OCH20), 5.19 (s, 2H, OCH20), 4.04 (s, 2H, CH2N), 3.49 (s, 3H, OCH3), 3.47 (s, 3H, OCH3). FAB MS m/z228.2 (m + 1, 8%), 211.1 (m + 1 - NH3, 67%), 167.1 (m + 1 - C2H7N10 1, 75%), 155.1 (m + 1 - C ^ N A , base). Analysis for C ^H ^^O g 1/2H20 : calc. C, 47.85; H, 6.18; N, 4.29; found C, 47.68; H, 5.93; N, 4.26. 2.2-Dimethvl-1.3-benzodioxole, (141) : Catechol (10.0 g, 91 mmol) was suspended in benzene (50 mL) and charged with 2,2-dimethoxypropane (20 mL, 109 163 mmol) and p-toluenesulfonic acid (5 mg). The mixture was heated to reflux for 48 h. After cooling to room temperature, solvent was evaporated in vacuo at room temperature. The resulting oil was triturated in petroleum ether (200 mL, bp 30-60°C) and passed through a silica gel filtering column. Solvent was evaporated in vacuo at room temperature to yield 12.35 g (90%) of a pale yellow liquid that was used without further purification. A portion of this material was purified by Kugelrohr distillation (bath temp. 75-80 °C / 30 mm Hg) to produce a clear, colorless oil (lit. [129] bp 75-78 °C / 23.5 mm Hg). 1H NMR (CDCI/TMS) : 6 6.79-6.70 (m, 4H, ArH), 1.66 (s, 6H, 2 x CH3). 5-Bromo-2.2-dimethvl-1.3-benzodioxole. (142) : To a solution of benzodioxole 141 (3.5 g, 23.3 mmol) in dimethylformamide (15 mL) was added N-bromosuccinimide (4.98 g, 28.0 mmol). The slightly exothermic reaction was stirred at room temperature for 72 h. Upon completion, the mixture was diluted with ethyl acetate (50 mL), washed with 5% sodium hydroxide (3 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting liquid was purified by Kugelrohr distillation (bath temp. 114-120 °C / 30 mm Hg) to yield 5.25 g (98%) of a clear, colorless liquid (lit. [130] bp 110 122 °C / 20 mm Hg). 1H NMR (CDCI/TMS) : 6 6.90-6.85 (m, 2H, ArH), 6.58 (d, J= 8.0 Hz, 1H, ArH), 1.66 (s, 3H, CH3), 1.65 (s, 3H, CH3). 5-Cvano-2,2-dimethvl-1,3-benzodioxole. (143) : To a solution of benzodioxole 142 (6.0 g, 26 mmol) in dimethylformamide (20 mL) was added cuprous cyanide (2.70 g, 30 mmol). The suspension was heated to reflux for 4 h. The hot, dark solution was immediately poured into a solution of sodium cyanide (5.0 g) in water (20 mL). The mixture was extracted with ethyl acetate (3 x 50 mL) and the organic extracts were combined. The organic layer was washed with a solution of sodium cyanide (2.5 g) in water (25 mL), water (2 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting solid was recrystallized from ethyl acetate/hexanes to yield 1.84 g (40%) of a white solid, mp 79-81 °C. 1H NMR (CDCI/TMS) : 6 7.16 (dd, J= 8.0 and 1.5 Hz, 1H, ArH), 6.94 (d, J= 1.5 Hz, 1H, ArH), 6.76 (d, J = 8.0 Hz, 1H, ArH), 1.70 (s, 6H, 2x CH3). El MS m/z 175.06322 (calc. 175.06333, m+, 36%), 160.04 (m+ - CH3, base), 135.03 (m+ - C3H4, 98%) Analysis for C^Hg^O., : calc. C, 68.56; H, 5.18; N, 8.00; found C, 68.49; H, 5.08; N, 8.20. 111 y x y ^ " " 2 (c o o h )2 5-Aminomethvl-2,2-dimethvl-1,3-benzodioxole oxalate salt, (144) Benzodioxole 143 (900 mg, 5.14 mmol) was placed in a 50 mL rb flask under argon, charged with a 1.0 M solution of borane-tetrahydrofuran complex (20 mL, 20 mmol), and heated to reflux overnight. After cooling to room temperature, the reaction was quenched by careful addition of methanol (10 mL). When bubbling ceased, the mixture was heated to reflux for 30 min. Upon cooling to room temperature, solvent was evaporated in vacuo. The resulting clear oil was dissolved in ethanol (10 mL) and added dropwise with stirring to a solution of oxalic acid dihydrate (1.295 g, 10.28 mmol) in ethanol (20 mL). The resulting precipitate was collected by vacuum filtration to yield 1.078 g (78%) of a white solid, dp 170-172 °C (with bubbling). 1H NMR (CD3OD) : 6 6.89-6.85 (m, 2H, ArH), 6.76 (d, J = 8.0 Hz, 1H, ArH), 3.99 (s, 2H, CH2), 1.64 (s, 6H, 2 x CH3). FAB MS /77 /z 180.1 (m + 1 - oxalic acid, 19%), 163.1 (m + 1 - oxalic acid, - NH3, base). Analysis for C ^H ^N ^-V ^O : calc. C, 52.65; H, 5.71; N, 5.12; found C, 52.77; H, 5.80; N, 4.94. 112 SpiroM ,3-benzodioxole-2,1 ’-cvclohexanel, (146) : Prepared according to the procedure of Cole [131], catechol (50g, 0.454 mol) was suspended in toluene (250 mL) under argon in a 500 mL rb flask. This suspension was charged with a catalytic amount of p-toluenesulfonic acid (5 mg) and cyclohexanone (47 mL, 0.454 mol). The mixture was heated to reflux for 24 h with azeotropic removal of water via a Dean-Stark trap. The solution was cooled to room temperature, evaporated in vacuo, and the resulting oil was triturated with of petroleum ether (500 mL, bp 35-60 °C). The mixture was filtered through a silica gel filtering column, evaporated in vacuo, and solidified on standing to yield 83.8 g (97%) of a white solid, mp 46-47.5. An analytical sample was prepared by Kugelrohr distillation (bath temp. 100-110 °C / 25 mm Hg) to yield a white solid, mp 47.5-49 °C (lit. [132] mp 47.5-49 °C). 1H NMR (CDCI/TMS): 6 6.76 (bs, 4H, ArH), 1.94-1.88 (m, 4H, CH2), 1.79-1.69 (m, 4H, CH2), 1.55-1.46 (m, 2H, CH2). 113 5-BromospiroH .3-benzodioxole-2,1 ’-cvclohexanel. (147) : In a manner similar to Mitchell [133], benzodioxole 146 (6.0 g, 31.5 mmol) was dissolved in DMF (75 mL) in a 100 mL rb flask and charged with N-bromosuccinimide (6.0 g, 33.7 mmol). After stirring at room temperature for 72 h, the solution was poured into ethyl acetate (100 mL) and 5% sodium hydroxide (100 mL). The layers were separated, and the organic layer was washed with 5% sodium hydroxide (100 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The oil solidified upon standing to yield 8.42 g (99%) of a white solid, mp 52-53°C. An analytical sample was prepared by Kugelrohr distillation (bath temp. 120-130 °C / 25 mm Hg) to yield a white solid, mp 55-56°C (lit. [134] mp 55-57°C). 1H NMR (CDCI/TMS) 6 6.90-6.86 (m, 2H, ArH), 6.59 (dd, J= 7.5 and 1.0 Hz, 1H, ArH), 1.91-1.86 (m, 4H, CH2), 1.76-1.67 (m, 4H, CH2), 1.53-1.46 (m, 2H, CH2). 114 5“Cvanospirori.3-benzodioxole-2,'r-cvcloxhexane) (148) : In a manner similar to Friedman [135], spirobromide 147 (2.5 g, 9.3 mmol) and copper(l) cyanide (0.96 g, 10.7 mmol) were dissolved in DMF (20 mL) and heated to reflux for 4 h. The hot solution was immediately poured into an aqueous solution of sodium cyanide (2.0 g/ 25 mL) and extracted with benzene (2 x 25 mL). The organic extracts were combined, washed with aqueous sodium cyanide (2.0 g/ 25 mL), water (2 x 25 mL) and dried over anhydrous sodium sulfate. The solution was evaporated in vacuo to a green oil. The desired product was isolated after column chromatography on silica gel with 5% ethyl acetate/hexanes as the eluent. Fractions were pooled and evaporated to yield 1.55 g (78%) of a white solid, mp 84-85°C. 1H NMR (CDCI/TMS) 6 7.15 (dd, J = 8.1 and 1.6 Hz, 1H, ArH), 6.94 (d, J - 1.6 Hz, 1H, ArH), 6.76 (d, J=8.1 Hz, 1H, ArH), 1.94-1.89 (m, 4H, CH2), 1.78-1.68 (m, 4H, CH2), 1.56-1.46 (m, 2H, CH2). FAB MS m/z 216.1 (m + 1, base,). Analysis for C13H13N10 2 : calc. C, 72.54; H, 6.09; N, 6.51; found C, 72.80; H, 6.21; N, 6.50. 115 ( y ) ( 0 j ^ j j ^ NH2 (C00H)2 5-Aminomethvlspiroli ,3-benzodioxole-2,1’-cvclohexanel oxalate salt (149) : Spironitrile 148 (5 g, 23.2 mmol) was placed in a 250 mL rb flask under argon, charged with a 1.0 M solution of borane-tetrahydrofuran complex (50 mL, 50 mmol), and heated to reflux overnight. The reaction mixture was cooled to room temperature, and methanol (50 mL) was cautiously added. After bubbling ceased, the mixture was heated to reflux for 1 h. The solution was cooled to room temperature and evaporated in vacuo. The colorless oil was dissolved in ethanol (25 mL) and added dropwise to a solution of oxalic acid dihydrate (5.86 g, 46.4 mmol) in ethanol (50 mL) with vigorous stirring. The precipitate was collected to yield 5.52 g (77%) of a white solid, mp 188-90 °C. 1H NMR (CD3OD) 6 6.87-6.84 (m, 2H, ArH), 6.76-6.73 (m, 1H, ArH), 3.98 (s, 2H, CH2), 1.90-1.86 (m, 4H, CH2), 1.76-1.67 (m, 4H, CH2), 1.56-1.51 (m, 2H, CH2). FAB MS m/z 219.13 (m + 1 - oxalic acid, 19%), 203.13 (m + 1 - oxalic acid - NH2, base). Analysis for C ^H ^^C y/T^O : calc. C, 57.41; H, 6.26; N, 4.46; found C, 57.40; H, 6.10; N, 4.32. 116 NH -H2C03 cx:xr,A""- (SpiroM .3-benzodioxole-2.1 ’-cvclohexanel-5-vh-methvlquanidine bicarbonate salt (1501 : Spironitrile 148 (2.5 g, 11.6 mmol) was dissolved in a saturated solution of ammonia (g) in ethanol (100 mL) in a Parr bottle. Raney Nickel catalyst (1 mL slurry) was added and the mixture was hydrogenated at 50 psi for 20 h. The mixture was filtered and the catalyst was washed with 10 mL ethanol. The filtrate was evaporated in vacuo to an oil (2.35 g, 92%) that was used without further purification. This oil (2.35 g, 10.7 mmol) was dissolved in acetonitrile (10 mL) and added portionwise (4 x ca. 3 mL) every 15 min to a solution of S-ethylpseudothiourea hydrobromide (1.78 g, 9.6 mmol) in acetonitrile (5 mL) heated to reflux. The mixture was heated for a total of 2 h. After cooling to room temperature, the reaction was diluted with water (25 mL), the aqueous layer was extracted with diethyl ether (3 x 50 mL), and evaporated in vacuo to an oil. The oil was redissolved in water (25 mL) and poured into a saturated solution of sodium bicarbonate (50 mL). The precipitate was collected by vacuum filtration to yield 2.46 g (79%) of an off white solid, dp 146-148 °C (with bubbling). 1H NMR (CD 3OD/DCI) : 6 6.78-6.68 (m, 3H, ArH), 4.28 (s, 2H, CH2), 1.89-1.84 (m, 4H, CH2), 1.75-1.67 (m, 4H, CH2), 1.55-1.48 (m, 2H, CH2), 13C NMR (CD3OD/DCI) 6158.6, 149.3, 148.7, 130.5, 121.5, 120.1, 109.2, 108.7, 117 46.0, 36.1, 25.5, 24.2. FAB MS m/z 262.2 (m + 1 - H2C 03, base) Analysis for C15H21N30 5 : calc. C, 55.72; H, 6.55; N, 13.00; found C, 55.75; H, 6.54; N, 12.68 . alternate procedure: Spiroamine 149 (3.5 g, 11.3 mmol) was converted to its free base with 5% sodium hydroxide (50 mL) and chloroform (50 mL) with vigorous stirring. The layers were separated, and the organic layer was washed with water (50 mL), brine (25 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo to an oil. The oil was dissolved in water (5 mL) and added portionwise (4 x ca. 1.5 mL) every 15 min to a solution of S-ethylthiopseudourea (1.67 g, 9.0 mmol) in water (5 mL) heated to reflux. Upon complete addition, the amine flask was rinsed with hot water (5 mL), the rinse was added to the reaction flask, and the mixture was heated for a total of 2 h. The hot reaction mixture was poured into a saturated solution of sodium bicarbonate (50 mL). The precipitate was collected by vacuum filtration, washed with water (25 mL) and then ether (25 mL) to yield 2.025 g (69%) of an off white powder, dp 146-8 °C (with bubbling). Spectroscopic data is consistent to the procedure described above. 118 bis((spiron,3-benzodioxole-2.1’-cvclohexanel-5-vl)methvl) amine hvdrobromide. (151) : A solution of the free base of spirobenzylamine 149 (1.0 g, 4.1 mmol) in acetonitrile (4 mL) was added to a solution of S-ethylthiopseudourea hydrobromide 102 (840 mg, 4.5 mmol) in acetonitrile (4 mL) heated to reflux. The resulting solution was heated to reflux overnight. A small amount of solid had formed. Upon cooling to room temperature, the reaction mixture deposited more material which was collected by vacuum filtration. The white solid was recrystallized twice from ethanol to yield 515 mg (45%) of a white solid, mp 218-219 °C. 1H NMR (CD 3OD) : 6 6.90-6.76 (m, 6H, ArH), 4.07 (s, 4H, 2 x CH 2N), 1.91 -1.87 (m, 8 H, CH2), 1.77-1.73 (m, 8 H, CH2), 1.54 1.52 (m, 4H, CH2). 13C NMR (CD3OD) : 6 149.9, 149.5, 125.3, 124.7, 120.7, 110.8, 109.5, 51.6, 36.1, 25.5, 24.2. FAB MS m/z 422.3 ( m + 1 - HBr, 22%), 203.1 (C13H150 2, base). Elemental analysis for the bisamine HBr shows excess nitrogen meaning that the bis amine has not been completely separated from a nitrogen containing compound such as guanidine. 119 NH HBr mX r A - 3,4-Dihvdoxvbenzvlquanidine hvdrobromide, (85) Spiroguanidine bicarbonate 150 (500 mg, 1.55 mmol) was suspended in methanol (4 mL) and cooled to 0 °C. Dilute hydrobromic acid (1.7 mL, 0.89 N in MeOH) was added dropwise, the ice bath was removed, and the solution stirred at room temperature for 4 h. The solvent was removed in vacuo at room temperature. The resulting oil was dissolved in minimal ethanol, and diethyl ether was added until the mixture became cloudy. The addition of ether was repeated every day for seven days. The deposited crystals were collected by vacuum filtration, washed with ether (5 mL), and dried in vacuo to yield 90 mg (22%) of a light yellow solid, mp 166-167 °C. 1H NMR (D 20) : 6 6.77 (dd, J= 8.2 and 1.8 Hz, 1H, ArH), 6.73 (d, J= 1.8 Hz, 1H, ArH), 6.55 (d, J= 8.2, 1H, ArH), 4.15 (s, 2H, CH2). 13C NMR 157.9, 145.3, 144.7, 129.6, 120.7, 117.5, 116.2, 45.2. FAB MS m/z 182.2 (m + 1 - HBr, base). Analysis for C 8 H12N30 2Br1V2H20 ; calc. C, 35.44; h, 4.83; N, 15.50; found C, 35.59; H, 4.49; N, 15.32. 120 NH -(COOH)2 U occrH N ^ N H 2 (SpiroH ,3-benzodioxole-2,1 ’-cvclohexanel-5-vl)methvlquanidine oxalate salt. (153) : Guanidine bicarbonate 150 (1.5 g, 4.64 mmol) was converted to its free base with 5% sodium hydroxide (20 mL) and chloroform (20 mL) in a separatory funnel. The aqueous layer was extracted with chloroform (20 mL), and the organic extracts were combined. The organic layer was washed with water (20 mL), brine (10 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. A portion of the resulting oil (600 mg, 2.3 mmol) was dissolved in chloroform (2.5 mL) and added dropwise to a solution of oxalic acid dihydrate (580 mg, 4.6 mmol) in ethanol (2.5 mL). Diethyl ether was added dropwise to this mixture until the solution became cloudy. The addition of diethyl ether was repeated every day for a total of seven days. The deposited crystals were collected by vacuum filtration, washed with diethyl ether (10 mL), and dried in vacuo to yield 324 mg (40%) of a white solid, mp 142-144 °C. 1H NMR (CD3OD) : 6 6.76-6.68 (m, 3H, ArH), 4.26 (s, 2H, CH2), 1.89-1.85 (m, 4H, CH2), 1.76-1.67 (m, 4H, CH2), 1.55-1.49 (m, 2H, CH2), 13C NMR (CD 3OD) : 6 166.5, 158.7, 149.4, 148.8, 130.6, 121.4, 120.1, 109.2, 108.7, 46.0, 36.1, 25.6, 24.2. FAB MS m/z 262.1 (m + 1 - oxalic acid, base), 203.1 (m + 1 - oxalic acid 121 - C,H5H3, 51%). Analysis for C 16H21N30 6 : calc. C, 54.70;, H, 6.02; N, 11.96; found C, 54.41; H, 5.95; N, 11.75. NH -HOOCCH=CHCOOH o:^r”A- (SpiroH,3-benzodioxole-2,1’-cvclohexanel-5-vl)methvlquanidine maleate salt. (154) : The oil (600 mg, 2.3 mmol) from the above conversion of guanidine bicarbonate 150 to its free base was dissolved in chloroform (2.5 mL) and added dropwise to a solution of maleic acid (534 mg, 4.6 mmol) in ethanol (2.5 mL). Diethyl ether was added dropwise to this mixture until the solution became cloudy. The addition of diethyl ether was repeated every day for a total of seven days. The deposited crystals were collected by vacuum filtration, washed with diethyl ether (10 mL), and dried in vacuo to yield 314 mg (36%) of a pale yellow solid, mp 138-140 °C. 1H NMR (CD 3OD) : 6 6.76-6.68 (m, 3H, ArH), 6.24 (s, 2H, -CH=), 4.26 (s, 2H, CH2), 1.90-1.85 (m, 4H, CH2), 1.76-1.67 (m, 4H, CH2), 1.56-1.51 (m, 2H, CH2), 13C NMR (CD3OD) : 6 170.8, 158.7, 149.4, 148.8, 136.7, 130.4, 121.4, 120.1, 109.2, 108.7, 46.1, 36.1, 25.5, 24.2. FAB MS m/z 262.2 (m + 1 - maleic acid, base). Analysis for C 16H21N30 6 : calc. C, 57.29;, H, 6.14; N, 11.13; found C, 57.27; H, 6.05; N, 11.07. 122 NH -(COOH)2 3.4-Dihvdoxvbenzvlquanidine oxalate salt, (155) : Spiroguanidine oxalate 153 (250 mg, 0.711 mmol) was suspended in degassed water (5 mL) and heated to reflux under argon for 24 h. The solution was cooled to room temperature and evacuated in vacuo to a solid. This solid was triturated in diethyl ether and collected by vacuum filtration to yield 183 mg (93%) of a white solid, mp 175-176 °C. 1H NMR (D20) : 6 6.75 (d, J= 8.1 Hz, 1H, ArH), 6.74 (d, J = 2.1 Hz, 1H, ArH), 6.63 (dd, J = 8.1 and 2.1 Hz, 1H, ArH), 4.21 (s, 2H, CH2). 13C NMR (D20) : 6 166.8, 158.7, 146.9, 146.4, 128.5, 120.1, 116.7, 115.9, 46.0. FAB MS m/z 182.1 (m + 1 - oxalic acid, base). Analysis for C10H13N3O61/4H20 : calc. C, 43.56; H, 4.94; N, 15.24; found C, 43.68; H, 4.75; N, 15.05. NH •HOOCCH=CHCOOH 3.4-Dihvdoxvbenzvlquanidine maleate salt (156) : Spiroguanidine maleate 154 (250 mg, 0.662 mmol) was suspended in degassed water (5 mL) and heated to reflux under argon for 48 h. The solution was cooled to room temperature and evacuated in vacuo to a solid. This solid was triturated in diethyl ether and collected by vacuum filtration to yield 179 mg (90%) of a pale yellow solid, mp 156-157 °C. 1H NMR (D20) : 8 6.77-6.74 (m, 2H, ArH), 6.64 (dd, J= 8.2 and 2.0 Hz, 1H, ArH), 6.24 (s, 2H, -CH=), 4.21 (s, 2H, CHJ. 13C NMR (D20) : 6 170.8, 158.6, 146.9, 146.5, 136.7, 128.8, 120.1, 116.7, 115.8, 46.0. FAB MS m/z 182.1 (m + 1 - maleic acid, base). Analysis for C12H15N3CV/4H20 : calc. C, 47.76; H, 5.18; N, 13.92; found C, 47.97; H, 5.16; N, 13.65. NH HBr 3.4-Bis(benzvloxv)benzvlauanidine hvdrobromide. (157) : A solution of benzylamine 128 (1.5 g, 4.7 mmol) in acetonitrile (10 mL) was charged with S-ethylthiopseudourea hydrobromide (695 mg, 3.8 mmol) and heated to reflux for 2 h. Upon cooling to room temperature, a small amount of solid had deposited. The reaction mixture was diluted with diethyl ether until it became cloudy. Upon standing, the mixture oiled. The oiled mixture was triturated overnight producing a suspension. The solid was collected by vacuum filtration and recrystallized from ethanol/diethyl ether (with trituration) to yield 1.14 g (69%) of a white solid, mp 114-115 °C. 1H NMR (CD3OD) : 8 7.46-7.28 (m, 10H, ArH), 7.03-7.01 (m, 2H, ArH), 6.86 (dd, J= 8.3 and 1.9 Hz, 1H, ArH), 5.14 (s, 2H, ArCH20), 5.12 (s, 2H, ArCH20), 4.28 (s, 2H, ArCH2N). 13C NMR 124 (CD3OD/DCI) : 6 158.7, 150.7, 150.3, 138.7, 130.9, 129.5, 129.4, 129.0, 128.9, 128.72, 128.69, 121.9, 117.0, 116.2, 72.8, 72.6, 45.8. FAB MS m/z 362.1 (m + 1, base). Analysis for C22H24N30 2Br1 : calculated C, 59.73; H, 5.47; N, 9.50; found C, 59.33; H, 5.35; N 9.12. NH HBr HO. ^ ^ ji. jn n h 2 3.4-dihvdroxvbenzvlquanidine hvdrobromide. (85) : A solution of benzvlquanidine 158 (300 mg, 0.68 mmol) in ethanol (10 mL) in a Parr bottle was charged with 10% palladium on carbon (30 mg). The suspension was shaken on a Parr hydrogenator at 60 psi at room temperature overnight. The catalyst was removed by gravity filtration and recrystallized from ethanol/diethyl ether (with trituration) to yield 155 mg (88%) of a white solid, dp 162-165 (with darkening) °C. 1H NMR and 13C NMR spectra are in agreement with the spectra of the 1/a hydrate on page 119. FAB MS m/z 182.1 (m + 1, base). Analysis for C aH ^N ^Br, : calculated C, 36.66; H, 4.62; N, 16.03; found C, 36.94; H, 4.62; N, 15.61. 125 3,4-Dimethoxyaniline hvdrobromide, (159) A solution of 3,4-dimethoxyaniline (1.0 g, 6.5 mmol) in methanol (5 mL) was cooled to 0 °C. Concentrated hydrobromic acid (0.75 mL, 6.68 mmol) was added with stirring. The deep purple solution was rapidly stirred for 5 min before it was quickly diluted with diethyl ether (50 mL). The precipitated product was collected after 1 h to yield 1.48 g (97%) of a deep purple solid, mp 217-219 °C. 1H NMR (D20): 6 6.97-6.81 (m, 3H, ArH), 3.73 (s, 3H, CH30), 3.72 (s, 3H, CH30). FAB MS /77/z(THIO-G/Gly) 154.1 (m + 1 - HBr, base). Analysis for CgH^^O ,,: calc. C, 41.05; H, 5.17; N, 5.98: found C, 41.26; H, 5.23; N, 5.96. 3,4-Dimethoxyphenvlquanidine hvdrobromide. (160) : In a manner similar to the procedure of Hughes [99], a solution of 50% aqueous cyanamide (0.5 mL, 6.4 mmol) was added to a solution of 159 (1.0 g, 4.27 mmol) in ethanol (4 mL , 68 mmol) under argon. After heating to reflux overnight (18 h), the mixture was cooled to room temperature and placed in the refrigerator for 24 h. The precipitated solid was collected by vacuum filtration and washed with diethyl 126 ether to yield 705 mg (60%) of a light purple solid, mp 233-234 °C. The mother liquor was evaporated in vacuo, and the oil was recrystallized from ethanol/diethyl ether to yield an additional 208 mg (77% total yield) of a light purple solid, mp 233-234 °C. 1H NMR (D20): 6 6.92 (d, J = 8.5 Hz, 1H, ArH), 6.83 (d, J= 2.0 Hz, 1H, ArH), 6.78 (dd, J= 8.5 and 2.0 Hz, 1H, ArH), 3.71 (s, 3H, CH30), 3.69 (s, 1H, CH30); 13C NMR (D20): 6 157.6, 149.7, 148.9, 127.9, 120.4, 113.2, 111.5, 56.7. FAB MS m/z 196.11 (m + 1 - HBr, base). Analysis for C9H14N30 2Br1: calc. C, 39.15; H, 5.11; N, 15.22; found C, 39.08; H, 5.11; N 15.13. 3.4-Dihvdroxyphenvlquanidine hvdrobromide. (84) : Guanidine 160 (500 mg, 1.81 mmol) was dissolved in concentrated hydrobromic acid (4 mL) under argon and heated to reflux for 4 h. The solution was cooled to room temperature and concentrated in vacuo. The solid residue was triturated in diethyl ether and filtered to yield 435 mg (97%) of crude product, mp 135-138 °C. A portion of this material (200 mg) was recrystallized from methanol/diethyl ether until a consistent melting point was obtained. Two successive recrystallizations yielded 146 mg (73%) of a light brown solid, mp 154-156 °C. 1H NMR (D20): 6 6.76 (d, J = 8.4 Hz, 1H, ArH), 6.66 (d, J= 2.4 127 Hz, 1H, ArH), 6.58 (dd, J = 8.4 and 2.4 Hz, 1H, ArH); 13C NMR (D20): 157.6, 145.6, 115.0, 127.3, 120.0, 117.5, 115.5. FAB MS m/z 168.08 (m + 1 - HBr, base). Analysis for C7H10N3O2Br1'1/4H2O: calc. C, 33.29; H, 4.19; N, 16.64: found C, 33.32; H, 3.97; N, 16.56. Part 2 Section A Modifications of Trimetoquinol and the Effects on G-Adrenergic and Thromboxane A2 Receptor Systems 128 CHAPTER V INTRODUCTION 5.1 B-Adrenerqic Aqonism Many drugs have specific effects on organs such as the heart, lungs and vasculature. One of the pathways involved in the regulation of the respiratory and cardiovascular systems is the sympathetic nervous system. The sympathetic nervous system, in cooperation with the parasympathetic nervous system, is involved in the homeostatic regulation of bodily functions. Stimulation of the sympathetic nervous system leads to a number of biochemical responses which are characterized by the "fight or flight" response. The "fight or flight" response is illustrated by an elevation of blood pressure and blood flow, an increased heart rate with an increased force of contraction, bronchodilation, and increased metabolism of carbohydrates and fatty acids [136,137]. Each of these responses results from the action of the sympathetic nervous system neurotransmitter, norepinephrine, on the appropriate tissue. Upon depolarization, the post ganglionic sympathetic neurons release norepinephrine for interaction with the postsynaptic receptors of various tissues. One class of postsynaptic receptors are the B-adrenergic receptors. These receptors have 129 130 discrete functions in selected tissues. Selective exploitation of f3-receptor subtypes in specific tissues can be beneficial for the treatment of many different diseases such as congestive heart failure, cardiac arrhythmias, asthma, and chronic obstructive pulmonary disease [138,139,140,141]. 5.1.1 Sympathetic Nervous System The autonomic nervous system regulates normal body function to maintain a constant internal environment [137]. This system is divided into two parts: the parasympathetic and the sympathetic nervous systems. These individual components of the autonomic nervous system are anatomically, physiologically, and biochemically different. In the sympathetic nervous system, the short preganglionic neurons originate in the thoracolumbar region of the spinal cord and synapse in the sympathetic ganglia with postganglionic neurons [137]. The long postganglionic neurons innervate three types of effector cells: smooth muscle, cardiac muscle, and exocrine glands. The neurotransmitters for the sympathetic nervous system are acetylcholine in the ganglia, and norepinephrine at the effector cell [137]. Neurons releasing norepinephrine (or epinephrine) have been designated as adrenergic nerves. Adrenergic nerves are responsible for the initiation and termination of neurotransmitter activity with the effector tissues. 131 5.1.1.1 Catecholamine Biosynthesis Sympathetic nerves synthesize and store norepinephrine. The biosynthesis of the adrenergic catecholamines norepinephrine and epinephrine starts with the amino acid L-tyrosine (Figure 1). This natural amino acid is actively transported from the blood stream to the CNS and other sympathetically innervated tissues. L-Tyrosine is oxidized to the catechol L-DOPA by tyrosine hydroxylase, the rate limiting enzyme in the biosynthesis of the catecholamines [2]. Tyrosine hydroxylase is a stereospecific enzyme requiring 0 2, Fe2+, and tetrahydrobiopterin as cofactors for its catalysis [142,143]. Subsequent decarboxylation of L-DOPA by L-aromatic amino acid decarboxylase (dopa decarboxylase) provides the neurotransmitter dopamine. L-Aromatic amino acid decarboxylase requires pyridoxal phosphate (Vitamin BG) as a cofactor [142]. To produce the adrenergic catecholamines, dopamine is oxidized by dopamine 6-hydroxylase to yield norepinephrine. Dopamine 0-hydroxylase is a Cu2+-containing enzyme which utilizes 0 2 and ascorbic acid as its cofactors [142]. N-Methylation of norepinephrine with phenylethanolamine N-methyltransferase yields epinephrine. Phenylethanolamine N-methyltransferase requires S-adenosyl methionine as its methyl donor [142], Once the neurotransmitter is synthesized, the neuron stores its in synaptic granules located in the nerve ending [142]. Upon nerve stimulation, the neurotransmitter is released into the synapse for subsequent interaction postsynaptic receptors. 132 5.1.1.2 Catecholamine Metabolism and Reuptake Neurotransmitter activity is terminated by two mechanisms; reuptake into the nerve terminal and neurotransmitter metabolism. Reuptake is the major process for termination of neurotransmitter activity. This process is a sodium dependent, stereoselective, active transport mechanism for L-norepinephrine termed "uptake 1" [142,144]. A second mechanism, "uptake 2", operates under high concentrations of norepinephrine. This process is less selective, removing compounds structurally related to norepinephrine as well. The second mechanism to terminate norepinephrine neurotransmitter activity is metabolism. The major enzymes involved in metabolism of norepinephrine and epinephrine are monoamine oxidase (MAO) and catechol-O- methyltransferase (COMT) (Figure 17). Monoamine oxidase, a flavin containing enzyme, converts the amine terminus into an aldehyde for subsequent reduction with aldehyde reductase or oxidation with aldehyde dehydrogenase. MAO is typically located in the neuron and thus limits its action to metabolizing free neurotransmitter within the nerve [142]. Catechol-O-methyl transferase is a synaptic and peripheral enzyme which requires S-adenosyl methionine and Mg2+ for the methylation of the meta-hydroxyl group of the catecholamines [142], Enzymatic methylation of the meta-hydroxy group reduces the activity of the norepinephrine 100-fold [145]. Sequential metabolism by these enzymes leads to multiple metabolic products (DOPGAL, DOPEG, DOMA, MOPEG, VMA, MPOGAL, metanephrine, and normetanephrine) [142]. 133 [MAO] OH OH OH NHHO HO HO [MAO] [MAO] HO HO HO NEDOPGAL EPI [ALD RED] [ALD DEHYD] OH OH HO, OH HO OH HO HO DOPEG DOMA [COMT] [COMT] [COMT] [COMT] OH OH OH OH HO HO MOPEG VMA [ALD RED] [ALD DEHYD] OH OH OH NH CH30 NHCH [MAO] [MAO] HO HO HO Normetanephrine Metanephrine MOPGAL t____ [MAO] Figure 17. Metabolism of Adrenergic Catecholamines. 5.1.2 (3-Adrenerqic Receptors An adrenergic drug is generically defined as a drug which interacts with postsynaptic receptors of effector tissues innervated by the sympathetic nervous system [144]. Norepinephrine may interact with a number of adrenergic receptors which have been classified into two types, a-receptors and (3-receptors [146]. 3-Receptors are G-protein coupled-receptors [18] which 134 OH 2 R = H 3 r = ch3 161 r = ch(ch3)2 have been traditionally subdivided into two subtypes, f3r and R2-receptors [147]. This classification was based on the rank order potency in cardiac, adipose, tracheal, and vascular tissues of several (3-adrenergic agonists including norepinephrine 2, epinephrine 3, and isoproterenol 161 [147]. Traditionally, (31-receptors, found in cardiac and adipose tissue, showed a rank order potency of isoproterenol > epinephrine = norepinephrine while the R2-receptors, found in tracheal and vascular smooth muscle, showed a rank order potency of isoproterenol > epinephrine > norepinephrine. These receptors are distributed in various tissues throughout the body (Table 5), but the standard example of a tissue containing Rr receptors is cardiac tissue while the standard example containing R2-receptors is the smooth muscle of the lung. Selective and nonselective R-adrenergic agonists and antagonists confirmed the original two-subtype classification (Figure 18). Recently, an increasing amount of evidence points to additional atypical R-adrenergic receptors. The adipose tissue R3-receptor [148,149,150] and other atypical R-receptors including intestinal Table 5. 135 Tissues Containing (3-Adrenergic Receptors (compiled from [9,137,151-153]). TISSUE PRIMARY RECEPTORS CARDIAC MUSCLE Bi BRONCHIAL SMOOTH MUSCLE 13, PULMONARY VESSELS SKELETAL BLOOD VESSELS CORONARY VESSELS I32 RENAL VESSELS 13, and (32 GLYCOGENOLYSIS (LIVER) I32 UTERUS I32 13-CELLS (PANCREAS) I32 DETRUSOR MUSCLE (BLADDER) I32 CILIARY MUSCLE OF THE EYE I32 RENIN SECRETION (KIDNEY) (3, and I32 ADIPOSE b3 SKELETAL MUSCLE ATYPICAL 13 Gl TRACT ATYPICAL 13 ENDOTHELIAL CELLS ATYPICAL 13 [151], skeletal muscle [152], endothelial cell [153], and heart tissue [154] have been investigated. Although Lands originally proposed that adipose tissue contained f3r receptors [147], some early studies conflicted with this proposal [155,156]. For example, a series of B-phenylethylamines 162-167 induced lipolysis in rat brown adipose tissue selectively as compared to their effects on f3r and 62-receptors [156], Their lipolytic activity in rat brown adipose is resistant to competitive 3r or f32-receptor antagonism [157]. 136 Agonist Antagonist non OH OH selective HO c h 3 HO Isoproterenol propranolol OH OH beta .ju c r^ r tazolol atenolol OH CH30 o h HO NH . CH3 NH yCHs > c H3 beta2 r ch3 c h 3 c h 3 c h 3 OH c h 3o terbutaline butoxamine Figure 18. Classical Selective and Non-Selective 6-Adrenergic Agonists and Antagonists. A number of other selective atypical 6-adrenergic agonists 168-171 have also been studied for their effects on various atypical 3-adrenergic tissues [158,159,160,161], 137 R1 i rfS c h3 0 * OH B, PL, 162 BRL 26830 H c o 2c h 3 163 BRL 33725 c f 3 C 0 2c h 3 164 BRL 35135 Cl o c h 2c o 2c h 165 BRL 28410 H c o 2c h 3 166 BRL 35113 c f 3 C 0 2CH3 167 BRL 37344 Cl o c h 2c o 2c h OH HO NH CH3 HO CGP-1217 7 SM-11044 1 6 8 1 6 9 OH n r 0- 1- " jOr" 1 7 0 101196,157 R = OCH2COOCH3 171 ICID7114 R = OCH2CONHCH2CH2OCH3 138 5.1.3 Molecular Biology of B-Adrenerqic Receptors The [3-adrenergic receptors are part of the G-protein coupled-receptor (GPCR) family [18]. Multiple subtypes of this family have been cloned [148,162,163,164] and bear a significant resemblance to the rhodopsin receptor [165,166]. The tertiary structures of 8-adrenergic 7receptors have been deduced from mechanistic and structural similarities to bacteriorhodopsin and hydropathicity analyses of their primary sequences [89,167,168]. These receptors are transmembrane spanning proteins composed of a hydrophilic extracellular amino terminus, seven hydrophobic a-helices which are embedded in the membrane and linked by alternating hydrophilic intracellular and extracellular loops, and a hydrophilic cytosolic carboxy terminus (Figure 19). The seven transmembrane a-helices contain stretches of 20-25 residues and are arranged in a bundle to produce the agonist ligand binding pocket (Figure 20) [168,169]. This ligand binding pocket is buried deep within the core of the receptor [170] Site-directed mutagenesis studies have yielded several critical residues for the binding of adrenergic agonists. All GPCRs which bind endogenous amines have a conserved aspartate residue [89,168]. For the 3-adrenergic receptors, this residue has been determined to be the Asp113 residue located on the third transmembrane helix [171]. This residue is involved in the binding the protonated amine portion of both agonists and antagonists. Substitutions of Asp113 with alanine (Ala), asparginine (Asn) or glutamic acid (Glu) residues, 139 0[5I1[1[n1©®©©®-nh2 Extracellular Region 190 ©©©<£>®( 100 i™®i i o®®®^ ® |s.s.©@ /V 300® ® © ®® _ Membrane Region 6 0 ® [5)130 400 330® 000 270 360 370 Intracellular Region 230 390 410 260 © 350 380 , COOH Figure 19. Transmembrane Topology of the 6-Adrenoceptor (Modified from Strader [168]). resulted in mutant receptors with greatly reduced affinity for agonists and antagonists [171]. In addition, the Glu113 mutant recognizes some antagonists as partial agonists [172]. This mutant receptor point mutation activity is not observed with the Asn113 mutant and shows that there is overlap between the agonist and antagonist binding domains for the cationic portion of ligands [172]. The GPCRs which bind catecholamine ligands (ex-adrenergic, 6-adrenergic, and dopaminergic receptors) have two conserved serine residues 140 VII Figure 20. Cross Section Arrangement Transmembrane Helices (Modified from Strader [171]. which are separated by a pair of amino acids [89,171]. These residues have been identified as Ser204 and Ser207 and are located on the fifth transmembrane helix [173]. Both residues are crucial for both binding and functional properties of the receptor. Replacement of either serine residue with alanine (Ala) resulted in decreased agonist affinity for the mutant receptors. This loss of activity is similar to that of monophenolic analogues when tested with the wild-type receptor [173]. Subsequent experiments of monophenolic derivatives with Ala204 and Ala207 mutants determined that Ser204 is associated by hydrogen bonding interactions with the meta-hydroxyl group of catecholamines, and Ser207 is associated with the para-hydroxyl group (Figure 20) [173], 141 m Phe 290 zzzz^TTrp Sen es K Ser, 204. * Asp 113 OH 0 S i Phe HO ^N.NH. H o \ 289 11 Pro 288 'Trp Phe 286 208 Helix 5 Helix 4 Helix 3 Helix 6 Figure 21. Molecular Modeling Interactions of Norepinephrine with the (32-Adrenergic Receptor (modified from Hibert [89]). Further mutagenesis studies revealed that replacement of Phe289 or Phe290 in the sixth helix with alanine (Ala) or methionine (Met), and replacement of Tyr326 in the seventh helix with leucine (Leu) suggests aromatic interactions with receptor agonists [174]. Other studies have shown that mutation of Asn318 with lysine (Lys) or Ser319 with alanine (Ala) influenced agonist binding affinity [174,175]. Molecular modeling of agonist-receptor interactions has proposed additional potential drug-receptor interactions including hydrogen 142 bonding to the (3-hydroxyl group and a number of hydrophobic interactions between aromatic residues of the receptor and the ligand (Figure 21) [89]. 5.1.4 SAR of (3-Adrenerqic Agonists Isoproterenol 161 is the classical 8-adrenergic agonist. This compound is therapeutically utilized primarily for its bronchodilatory (I32) properties [176]. This structure contains the requirements usually necessary for 6-adrenergic activity: (a) a phenylethylamine backbone; (b) hydrogen bonding functionalities in the 3- or the 3,4- positions of the phenyl ring; (c) an a-hydroxy group with the appropriate stereochemistry (R > S); and (d) a single alkyl substitution on the amine. Typically, large substituents on the amine enhance 6-adrenergic activity as compared to a-adrenergic activity [144]. Very few modifications can be made to the 6-hydroxyethyl fragment. The stereochemical influence of the 6-hydroxy group follows the Easson-Stedman hypothesis [177] in which the R-isomer > S-isomer = desoxy-isomer as is the case for the derivatives of norepinephrine (Figure 22). Alkylation of this side chain at the a-carbon may aid in 62-selectivity as with isoetharine 172 in comparison to the nonselective agonist, isoproterenol 161 [178]. For a-alkylation, the ethyl group is the most potent substitution while alkylation with methyl or propyl groups greatly reduces activity [179]. Modifications of the N-alkyl substituent can have a dramatic effect on receptor selectivity. The isopropyl group on isoproterenol 161 produces 6 vs 143 OH OH OH OH OH OH Ar Ar Ar H H H— H-bond H HO H ‘OOC •ooc •ooc Figure 22. Schematic Diagram Illustrating the Esson-Stedman Hypothesis for the Binding of Dopamine and the Stereoisomers of Norepinephrine. a selectivity, but isoproterenol is a non-selective B-agonist [178]. Conversion of this N-alkyl group to a t-butyl group as in N-t-butylnorepinephrine 173 produces B2-selectivity [178]. However, the N-alkylphenol group in dobutamine 174 and fenoterol 175 provides mixed results [179]. The catechol derivative dobutamine which lacks the B-hydroxy group is a Br selective agonist [178]. The resorcinol derivative fenoterol retains the B-hydroxy group and is a B2-selective agonist [179]. Another interesting compound with a unique N-alkyl group is trimetoquinol 176. This compound lacks the B-hydroxyl group and has the ethylamine tethered to the catechol ring by its N-alkyl substituent. Cyclization to an isoquinoline ring produces a new chiral center. 144 Tetrahydroisoquinolines are usually very weak agonists, yet trimetoquinol is a potent non-selective atypical 6-adrenergic agonist [179], Modification of the catechol nucleus can provide a number of advantages. Catechol drugs are quickly deactivated by catechol-O-methyltransferase (COMT) and are thus inactive orally. Conversion to a resorcinol ring as in fenoterol 175, metaproterenol 177, and terbutaline 178 provides longer-acting, oral bioavailable drugs [178,179], Other modifications include conversion of the meta-hydroxyl group to a sulfonamide group as in soterenol 179. or to a hydroxymethyl group as in albuterol 180. These compounds are long-acting, oral bioavailable drugs because they are not OH OH 172 173 OH 174 175 HO HO OCH3 176 o c h 3 o c h 3 145 substrates for COMT [179]. Bioavailability problems can be avoided for catechol-containing drugs by masking the functionality as an ester as in the prodrugs bitoliterol 181 and dipivifrin 182 [178,179]. A final modification of the catechol ring has been to convert it to a carbostyril ring system as in procaterol 183 or a pyridine ring as in pirobuterol 184 [179]. OH OH OH 177 r = ch(ch3)2 179 R = CH(CH3)2 r, = nhso2ch3 178 R = C(CH3)3 1 80 R = C(CH3)3 r, = CH2OH NHCH(CH3)2 NHCH. 181 182 NHCH(CH3)2 h o c h2 n NHC(CH3)3 183 184 146 5.1.5 Therapeutic Uses of 3-Adrenerqic Agonists 3-Adrenergic agonists have extensive therapeutic applications. Dobutamine 174 and sometimes isoproterenol 161 are used in congestive heart failure for inotropic and chronotropic responses of 31-receptors in the heart, and to a lesser extent, smooth muscle relaxant properties of 32-receptors in the blood vessels and the lungs to improve cardiovascular and pulmonary function [138]. Both of these drugs are also used in certain cardiac arrhythmias to restore the heart to a normal rhythm [139]. Selective f32-agonists such as terbutaline 178, albuterol 180, and prodrugs such as bitoleterol 181 are used in the treatment of asthma and chronic obstructive pulmonary disease for their bronchodilatory activity [140,141,176]. These drugs are frequently used for acute treatment only via the inhalation route [140,176], They have potentially severe side effects (reflex tachycardia, tremor, nervousness) resulting from their 3-adrenergic activity in other tissues. Patients also have a tendency to develop tolerance to these drugs with chronic administration [140,176]. Another therapeutic use for 32-selective agonists is in uterine relaxation for premature labor. Ritodrine 185 and terbutaline 178 are the preferred drugs for this condition [180]. Atypical 6-adrenergic agonists are currently under investigation as novel therapy for the treatment of protein wasting conditions. Repartitioning drugs such as clenbuterol 186 and cimaterol 187. both of which are 32-agonists have 1 8 6 R = C(CH3)3 Rt = r 2 = Cl 1 88 R, = R2 = H 187 r = ch(ch3)2 r^ c n r2 = h 189 r 1 = ci r 2 = nh2 been developed to promote protein accretion [161]. This condition, however, may actually be an atypical 6-adrenergic mediated event because other 62-selective agents are ineffective. Other unique atypical morpholine 6-agonists like BRL 46104 188 and BRL 47672 189 have also shown promise in protein wasting conditions [161]. Atypical 63-agonists have the potential to treat metabolic disorders such as obesity and type II diabetes, a convenient combination since many type II diabetics are also obese. The most promising candidates from this group are BRL 26830 162, BRL 35135 164, LY 99134 190, and RO 40-2148 191 [161]. These compounds stimulate thermogenic activity in adipose tissue, stimulate insulin production and release from the pancreas, and improve glucose tolerance of the tissues [161]. 148 Cl ci OH HO OH OCH2CH2OCH2CH3 191 5.2 Thromboxane A2 Antagonism Thromboxane A2 (TXA2) is a potent mediator of platelet aggregation and vasoconstriction [181]. It is an arachidonic acid metabolite biosynthesized via the arachidonic acid cascade (Figure 23). Various stimuli trigger the activation of phospholipase A2 which liberates arachidonic acid from cell phospholipids. Arachidonic acid can undergo two biosynthetic fates; conversion to prostaglandins, thromboxanes and prostacyclin or conversion to leukotrienes [181]. For the production of TXA2, arachidonic acid is oxidized by cyclooxygenase to the chemically unstable cyclic endoperoxide intermediate, PGG2. This short-lived intermediate is quickly converted to another unstable cyclic endoperoxide intermediate, PGH2. PGH2 is the common intermediate for the production of all prostaglandins via enzymatic and non-enzymatic routes. It is also the common intermediate for enzymatic conversion to the thromboxanes or to prostacyclin [181]. PGH2 is converted to thromboxane A2 by the enzyme thromboxane synthetase. TXA2 is a biologically unstable molecule (t,/2 = 30 seconds) which breaks down non-enzymatically to the inactive thromboxane B2 [182]. COOH arachidonic acid 5-Lipoxygenase cyclooxygenase 5-HETE O PGG, leukotrienes OOH prostaglandins PGH2 prostacyclin OH synthetase Thromboxane synthetase prostacyclin TXA, OH OH TXB2 HO OH Figure 23. Biosynthesis of Thromboxane A2 via the Arachidonic Acid Cascade. 150 5.2.1 Platelet Physiology Platelets are a disk-shaped blood product about 2-4 microns in diameter derived in the bone marrow from megakaryocytes. These cells are incapable of cell division, but they have many functional characteristics of whole cells [183]. The normal concentration of platelets in the blood is between 150,000 and 350,000 cells per microliter. The main function of platelets is to initiate the hemostatic process [183,184]. After vascular injury, the sequential steps of platelet adhesion, platelet activation, and platelet recruitment occur. Platelet deficiency is defined as less than 50,000 cells per microliter and is termed thrombocytopenia [183]. Platelets circulate in the blood with a half-life of 8 to 12 days before being removed by the tissue macrophage system, primarily by the spleen [183]. Under normal circumstances, platelets circulate with minimal interaction between themselves and other blood cells or the blood vessel walls. The cell membrane of the platelet contains glycoproteins which cause the platelets to repel normal endothelium yet adhere to injured blood vessels [183,185], 5.2.2 Platelet Activation of Hemostasis Hemostasis is the prevention of blood loss from damaged blood vessels [183]. When damaged, the capillary vessels contract to reduce blood flow. The injured vessel exposes collagen and other subendothelial structures causing platelet adhesion to the ruptured vessel. The adhering platelets undergo a 151 metamorphosis to form spherical bodies which begin to extend pseudopods. These activated platelets also stick to each other in what is know as platelet aggregation [184,185]. This platelet plug temporarily prevents blood loss, but it must be reinforced by fibrin deposition for long term effectiveness. Platelet plugs are sufficient to stop blood loss from minute injuries to smaller vessels such as capillaries, but larger vessels require fibrin clot formation to form a sturdy plug [183,184,185], The activated platelets undergo a number of biochemical processes. The metabolic activity of the platelets increases with eventual calcium mobilization. Calcium mobilization results in contractile protein phosphorylation of the platelet cytoskeleton. The cytoskeleton maintains platelet shape, and phosphorylation of the contractile proteins in conjunction with increased intracellular calcium leads to the morphological change in platelet structure [186]. A high intracellular concentration of cyclic AMP (cAMP), resulting from platelet inhibition, prevents platelet activation by decreasing the calcium mobilization. During platelet inhibition intracellular calcium is stored in calcium storage sites [184], Adhering platelets also begin a number of secretory processes. Lysosomes, dense granules (which contain preaggregating factors such as calcium, ADP, and serotonin), and a-granules (which contain adhesive proteins and repair factors) fuse with the platelet membrane for the release of these chemical modulators into the surrounding medium [184,185], In addition, 152 platelet adhesion activates thromboxane and prostaglandin synthesis [184]. Production of TXA2 is an important supplement to the physical activation of the platelets and can be considered as a positive feedback loop of platelet activation [184,185]. 5.2.2.1 Thromboxane A., Platelet Activation A detailed examination of thromboxane A2 production begins with platelet adhesion. Platelets have receptors for most aggregating agents on their membrane. Ligands for these receptors include thrombin, ADP, serotonin, epinephrine, TXA2, and platelet activating factor (PAF) [185] Collagen induction of platelet aggregation appears to be mediated by collagen binding to exposed glycoprotein lb on the platelet membrane. This collagen-glycoprotein bridge is anchored to the subendothelial constituents of the vessel by von Willebrand Factor (vWF) [185]. Exposure of the platelet membrane to the aggregating agents or collagen activates second messenger systems within the platelets. Generally, both systems operate simultaneously although the phosphoinositide pathway is stimulated by a number of the products such as thromboxane A2 from the arachidonic acid pathway [182,185], Interaction of an aggregating agent such as TXA2 with its receptor (Figure 24), activates phospholipase C [187,188]. Phospholipase C catalyzes the enzymatic hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 153 t x a 2 TMQ PHOSPHO- LIPASE C C a++ DAG release Protein Ca/Calmodulin KinaseI C Kinase I Substrates Substrates Platelet Aggregation and Secretion Figure 24. Thromboxane A2 Activation of Platelet Aggregation and Secretion. 154 triphosphate (IP3). Both of these products act as second messengers. In the platelet, DAG activates protein kinase C, a phosphorylating enzyme which influences many intracellular biochemical pathways including proteins involved in platelet secretion. Inositol triphosphate mobilizes calcium release from intracellular stores [187,188], The free cytosolic calcium interacts with calmodulin for subsequent kinase activity resulting in the phosphorylation of myosin light chains. The interaction of the myosin chains with actin filaments facilitates the metamorphosis of the cytoskeleton as well as the release of granule contents [187,188]. The second pathway involves the arachidonic acid cascade and the prostaglandin dependent pathway of platelet activation (Figure 25). Platelet phosphlipids are cleaved by phospholipase A2 to release arachidonic acid. This mediator is metabolized by cyclooxygenase to form the cyclic endoperoxides PGG2 and subsequently produces PGH2. PGH2 is a common intermediate the production of other prostaglandins, prostacyclin and the thromboxanes. In adhering platelets, PGH2 is converted by thromboxane A2 synthetase to TXA2 [187,188]. Aggregation induced by thromboxane A2 and other agonists occurs in two waves. The primary wave of aggregation results from the initiation of the phosphoinositide second messenger pathway and involves to formation of small aggregates. The primary wave is a reversible process characterized by the initial stages of aggregation [189]. The secondary wave includes the 155 inducers : Collagen, ADP, Epinephrine I Phospholipids | ------Phospholipase A 2 Arachidonic Acid | ------— — — Cyclooxygenase Cylcic Endoperoxides (PGG 2 ,PGH2 ) | ------Thromboxane A 2 Synthetase Thomboxane A 2 U46619 Secretion of Platelet Serotonin, ADP, ATP Aggregation (rom p|ate|et Dense Granules Figure 25. Prostaglandin Dependent Pathway of Platelet Activation. initiation of the prostaglandin pathway. The secondary wave is an irreversible process involves the formation of larger, denser aggregates with concurrent granule secretion [189]. 5.2.3 Platelet Involvement in Cardiovascular Disease Cardiovascular disease is the leading cause of death and a major cause for hospital admissions in the United States. Many types of cardiovascular disease are caused by hemostatic or thrombotic conditions. Since platelets are 156 OH HO 193 o c o > 194 195 involved in the initiation of hemostatic and thrombotic mechanisms, antiplatelet therapy is a popular approach to preventing various cardiovascular diseases. Unfortunately, very few therapeutic agents (aspirin 192, dipyridamole 193, ticlopidine 194. and sulfinpyrazone 195) are available for this type of therapy. The recent approach of thromboxane A2 antagonism as an antiplatelet therapy has received considerable attention [190,191]. Antiplatelet drugs, such as thromboxane A2 antagonists, prevent the platelets from either initiating or propagating the hemostatic process. This therapeutic approach has the potential for treating many diseases caused or exacerbated by platelet involvement in hemostasis. Atherosclerosis is a gradual narrowing of the blood vessels, and platelets are believed to be involved in the atherosclerotic plaque formation [192]. Blood vessels begin to narrow as 157 the plaque deposits on the vessel walls. As the plaque thickens, platelets can adhere to the growing plaque. Eventually, the vessel becomes occluded. Occlusion of the coronary vessels can result in an acute myocardial infarction. Occlusion of other blood vessels can precipitate arterial or venous thrombosis [193]. The inhibition of normal blood flow starves the dependent tissues of essential nutrients and allows the accumulation of cellular wastes. Preventing hemostasis and thrombosis has other benefits as well. Once an occlusion or thrombosis forms, there is a risk that a portion of the clot will embolize. Acute pulmonary emboli, peripheral arterial emboli, and cerebrovascular emboli (stroke, transient ischemic attacks) can be devastating [193]. The emboli can cause significant tissue death in an area distant to the original thrombosis. Emboli are also a concern for coronary by-pass and prosthetic valve replacement patients. These patients are highly susceptible to the formation of emboli. Since the heart directly supplies the brain with blood, these patients are extremely vulnerable to cerebrovascular emboli [193]. 5.2.4 Thromboxane A., Antagonists Receptor antagonism is an important rationale in drug design. For a biochemical mediator such as TXA2, an antagonist offers a number of advantages as compared to a TXA2 synthetase inhibitor [194]: 1. Antagonism of the receptor will not cause a biological shunting of the biosynthetic cascade to produce an excess of alternative products. In 158 the case of TXA2, a biological shunt can lead to excessive production of prostaglandins with undesired biological activities. In addition, TXA2 and its precursor PGH2 share the same receptor, so a receptor antagonist can prevent the activity of either ligand [194]. 2. Antagonists can be therapeutically useful after the initiation of TXA2 biosynthesis. A synthetase inhibitor can only be effective is given prior to TXA2 formation [194]. Thromboxane A2 receptors have been divided into two subtypes: the a-receptor subtype (aggregation) and the -t-receptor subtype (vascular smooth muscle tone) [195,196]. Thromboxane A2 has a short biological half-life and is ineffective for the evaluation of TXA2 antagonists. Therefore, synthetic TXA2 agonists such as U46619 196 are used to evaluate TXA2 antagonists. Competitive binding assays to determine receptor affinity are typically performed with [3H]SQ 29,548 197. 5.2.4.1 Prostanoid Antagonists Early research with thromboxane antagonists logically evolved around the design of prostanoid-like antagonists. Retention of the prostaglandin and thromboxane template led to the development of 13-azaprostanoic acid 198 [197] and pinane TXA2 199 [198] as TXA2 antagonists. Both derivatives were found to inhibit agonist induced platelet aggregation, and pinane TXA2 was also shown to be a TXA2 synthetase inhibitor [197,198], Modification of their U46619 SQ 29,548 1 9 6 1 9 7 COOH COOH OH 1 9 8 1 9 9 combined backbones was involved in the design of ONO-11120 200. a potent, selective antagonist to agonist induced platelet aggregation [199]. Recently, there has been a significant increase in the number of reports of both prostanoid and non-prostanoid thromboxane A2 antagonists [190,191], For example, the oxabicyclo[2,2.1]heptyl carboxylic acids 201 and 202 are potent selective receptor antagonists currently undergoing further evaluation as potential clinical candidates [200,201]. Another approach to antiplatelet therapy has been to design compounds with both TXA2 antagonist activity and TXA2 synthetase inhibition [202]. It has been shown with human volunteers that combination therapy with two separate agents had greater therapeutic benefit than either individually [203]. One such compound with activity as 160 a potent TXA2 synthetase inhibitor and a potent TXA2 receptor antagonist is the 8-[(arylsulfonyl)amino]octanoic acid 203 [202]. COOH COOH COOH COOH 5.2.4.2 Non-prostanoid Antagonists Relatively few effective non-prostanoid thromboxane A2 antagonists were originally reported as recent as only 10 years ago [204,205]. Trimetoquinol 176 has been shown to be a potent, stereoselective (R > S) inhibitor of platelet aggregation induced by arachidonic acid, collagen, U46619, and TXA2 in human platelets (a-subtype) and U46619 induced vasoconstriction in rat thoracic aorta [204,206]. Another original thromboxane A2 antagonist, sulotroban (BM 13,777) 204 has been reported to be a potent receptor antagonist [205,207]. Currently, many non-prostanoid thromboxane A2 antagonists are under investigation for the development of potent, long-acting, 161 orally effective drug candidates [190,191]. The azulene derivative KT2-962 205 has been reported to be 30 times more potent than sulotroban 204 [208]. Both the indole propionic acid (L-670,596) 206 and the imidazo[4,5-b]pyridine butanoic acid (UP 116-77) 207 are extremely potent orally active compounds [209,210]. Another novel non-prostanoid TXA2 antagonist that has been recently reported is dibenzoxepin 208 [211]. This is just another example of a rapidly growing list of a potent receptor TXA2 antagonists [190,191], 204 COOH 206 205 c h 3 '-O- 207 208 162 5.2.5 Molecular Biology of Thromboxane A., Receptor Until recently, very little was known about the thromboxane A2 receptor. The thromboxane A2/prostaglandin H2 receptor has been purified from human platelets by affinity chromatography [212], and the amino acid sequence of the human platelet TXA2 receptor has been resolved by gene cloning [213], This receptor is part of the G-protein coupled-receptor (GPCR) family. It contains 343 amino acids and forms a putative bundle containing seven transmembrane domains. Since this GPCR binds an anionic ligand, it has been postulated that Arg295, which is located on the seventh helix, may be important for binding the ligand [213]. This hypothesis has been supported by molecular modeling of the human receptor [214]. In addition, it has been proposed that the ligand binding pocket of this receptor contains a serine residue (Ser201) on the fifth helix important in binding the hydroxy group of TXA2 [214], Molecular modeling also shows that Ser201 and Arg295 are separated by a large hydrophobic pocket capable of binding a hydrophobic moiety [214]. 5.3 Trimetoquinol and Derivatives Trimetoquinol (TMQ) 176 is chiral tetrahydroisoquinoline utilized for its bronchodilatory activity in Japan [215,216]. This compound is both a potent bronchodilator (f32) and a potent inotropic/chronotropic agent (BJ [216,2171. Early investigations demonstrated that B-adrenergic activity is related to the stereoselective interaction of the (S)-(-)-isomer 209 with the tissue 163 [217]. The (S)-isomer has also been identified as a potent lipolytic agent [218]. This compound is a non-classical (3-adrenergic agonist for two reasons: 1. TMQ lacks the usual B-hydroxyl group seen in the classical agents such as isoproterenol 161. 2. The 3,4,5-trimethoxybenzyl group creates a chiral center on the tetrahydroisoquinoline nucleus. This is new chiral center is at a different position than that of the classical phenylethanolamines. This group can also be considered as the N-alkyl substituent and is a unique substitution in 13-adrenergic agonists. HO HO NH HO :xo OCH OCH ‘ OCH3 OCH3 o c h 3 209 (SM -)-TM Q 210 (R)-(+)-TMQ Trimetoquinol was also shown to be an antiplatelet aggregatory agent [219]. Surprisingly, the (R)-(+)-isomer 210 is a stereoselective thromboxane A2 antagonist [220], (R)-(+)-TMQ is a thromboxane A2 antagonist at both the a-subtype and -c-subtype TXA2 receptors [206]. Trimetoquinol is a unique 164 atypical thromboxane A2 antagonist, unrelated to other nonprostanoid antagonists [191]. The potent activity and unique structure of trimetoquinol in each receptor system has promoted the search for more potent and selective analogs of the parent derivative. Many modifications of trimetoquinol have been investigated and include, N-alkylation, modification of the tetrahydroisoquinoline ring, and modification of the 1-benzyl substituent. 5.3.1 N-Alkvlated Trimetoquinol Derivatives Two types of N-substituted TMQ derivatives were prepared for evaluation of their (3-adrenergic and thromboxane A2 antagonists properties. The first group of compounds synthesized were N-alkyl substituted analogues which contained small alkyl groups [221]. Methyl, (3-hydroxyethyl and 6-chloroethyl derivatives 211-213 of trimetoquinol displayed decreased potency in both receptor systems. N-Substitution produced compounds with equal or greater selectivity than TMQ in the 6-adrenergic system [222]. The second class of compounds synthesized were N-benzyl derivatives 214-220 [221,223]. In general, these tertiary amines were much less potent as 6-agonists as compared to TMQ and all analogs were moderately 62-selective regardless of their aromatic substituents [224]. These compounds were also less potent than TMQ as thromboxane A2 antagonists. However, the substitution pattern showed that electron-donating substituents increased potency in platelet aggregation in comparison to the unsubstituted or electron-withdrawing derivatives. This correlation was not evident for the inhibition of vascular contractile responses [223]. o c h 3 o c h 3 R R 211 c h 3 214 H 212 c h 2c h 2o h 215 4-CH3 213 c h 2c h 2c i 216 4-CI 217 4-OCH3 218 4-N 0 2 219 4-NH2 220 3,4-diCI 5.3.2 Modified Tetrahvdroisoquinoline TMQ Derivatives Many changes in the 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline nucleus have been investigated and include fragmentation of the isoquinoline nucleus, modification of the reduced ring, and ortho substitution of the catechol portion. 166 HO HO NH NHR HO H°)h o C Or HO OCH3 OCH3 OCH 221 222 223 r = h O C H 3 224 r = c h 3 225 r = ch2ch3 226 r = c h (c h 3)2 HO HO NH HO HO NH OCH OCH 2 2 7 OCH 2 2 8 OCH OCH3 Multiple fragmentations of the tetrahydroisoquinoline ring have been investigated. These fragmentations can occur by cleavage of the C4-C4a, C r C8a, or the N2-C3 bonds to produce compounds 221-226 [225,226,227]. In each case, fragmentation of the nucleus greatly reduced the 3-adrenergic activity as compared to the parent molecule [225,226,227]. Modifications of ther reduced ring of TMQ include migration of the trimethoxybenzyl substituent to the 4 position 227 (in effect, moving the nitrogen atom to the 3 position) and enlargement of the reduced ring to the benzazepine derivative 228 [228,229]. Migration of the benzyl substituent greatly reduced activity as compared to TMQ, but compound 227 is a selective 167 G2-agonist [228]. Enlargement of the ring to the benzazepine derivative 228 also greatly reduced G-adrenergic activity, but this derivative prevented phospholipase C induced platelet aggregation. However, this compound was inactive at preventing U46619-induced platelet aggregation. The catechol ring of trimetoquinol was modified in an effort to enhance G-receptor selectivity analogous to the observation of fluorine substitution with norepinephrine [230], In order to evaluate the electronic influence of similar substitutions in trimetoquinol, 5-substituted derivatives as well as the 8-substituted derivatives containing electron-withdrawing groups have been prepared [231,232]. Fluorinated derivatives 229 and 230 were shown to be equipotent to TMQ as a bronchorelaxant and also G2-selective as compared to TMQ [231]. These encouraging results of G2-selectivity prompted the synthesis of 5,8-difluoro-TMQ 231 [232], Unfortunately, 5,8-difluoro-TMQ lost potency as compared to TMQ and was also nonselective as a G-agonist. The rank order for G2-selectivity of this series is 8F-TMQ > 5F-TMQ > TMQ > 5,8-difluoro-TMQ [231,232], In contrast to the racemate, separation of the stereoisomers of 8F-TMQ showed that they were less potent than TMQ and also relatively non-selective G-agonists [232]. The thromboxane A2 antagonism of the fluorinated derivatives 229-231 is quite complex. The monofluorinated derivatives were less potent than TMQ for the inhibition of U46619-induced platelet aggregation, more or equipotent to TMQ in the inhibition of U46619-induced vasoconstriction, and x-subtype 168 HO NH HO OCH OCH OCH3 a a 229 F H 230 H F 231 F F 232 I H 233 H I 234 c f 3 H selective as compared to TMQ [233]. Difluoro-TMQ was much less potent than TMQ in each TXA2 system and only slightly x-subtype selective [232]. Thus, these compounds have a x-subtype selectivity rank order of 8F-TMQ > 5F-TMQ > 5,8-diF-TMQ > TMQ. The stereoisomers of 8F-TMQ were very interesting. Both isomers were less potent than TMQ for the inhibition of U46619 induced platelet aggregation. As expected, (R)-(-)-8F-TMQ was x-subtype selective and equipotent to TMQ in the inhibition of U46619 induced vasoconstriction [232]. Ironically, (S)-(+)-8F-TMQ was an agonist in rat aorta inducing vasoconstriction. This shows that optimal activity for TXA2 antagonism 169 resides with the (R)-isomer, but binding studies showed that fluorination greatly diminishes the receptor stereoselectivity [232,234]. lodinated 232-233, and trifluoromethyl 234 derivatives of TMQ were also evaluated as 13-agonists and TXA2 antagonists. The iodinated derivatives were very weak nonselective 3-agonists [232], Likewise, these compounds also lost potency as TXA2 antagonists, but they are x-subtype selective. In fact, 5I-TMQ was the most x-subtype selective antagonist developed [232]. Ironically, introduction of the 5-trifluoromethyl group produced a weak 3-antagonist and a very weak, x-subtype selective TXA2 antagonist [232], 5.3.3 1-Substituted 1.2,3.4-Tetrahvdroisoquinoline Derivatives Three classes of 1-substituted-1,2,3,4-tetrahydroisoquinolines were prepared for comparison to TMQ. These classes are 1,1-disubstituted- 1,2,3,4-tetrahydroisoquinolines, a-substituted-TMQ derivatives, and modified 3,4,5-trimethoxybenzyl-TMQ derivatives. Introduction of a second 1-alkyl substituent in TMQ produced interesting results. 1-Benzyl-TMQ 235 and 1-methyl-TMQ 236 were both inactive as 32-stimulants [235], However, 1-methyl-TMQ was weakly active as a 3r agonist, but 1-benzyl-TMQ was a selective 3r antagonist [235], Introduction of a second chiral center in the a-substituted-TMQ derivatives 237-241 attempted to enhance the stereoselectivity of TMQ for 3-receptors. a-Hydroxyl-TMQ diastereomers were weak non-selective 170 HO HO NH NH HO HO OCH CH OCH OCH OCH 2 3 5 2 3 6 6-agonists as compared to TMQ [236]. a-Methylation of trimetoquinol produced the erythro 239, threo 240, and dimethyl 241 derivatives. Both the erythro-methyl and the threo-methyl analogues were less active than TMQ on HO NH HO OCH OCH OCH3 Hi 237 H OH 238 OHH 239 H CH, 240 c h 3 H 241 c h 3 CH. 171 B1-receptors, and the dimethyl derivative was completely inactive [237,238]. In addition, erythro-methyl derivative 239 and dimethyl derivative 241 were less active than TMQ on f32-receptors. However, the threo-methyl derivative 240 was the most potent derivative on the (32-receptor. Each of the three methylated analogues can be considered (32-selective as compared to TMQ [237,238]. The final group of compounds is the modified 3,4,5-trimethoxybenzyl derivatives. Simple isomerization to 2,3,4- or 2,4,5-trimethoxybenzyl derivatives 242 and 243 reduced potency in (3-adrenergic receptor systems [239,240]. In addition, the 2,4,5-trimethoxybenzyl analog was an inhibitor of arachidonic acid-induced human platelet aggregation, although less potent than TMQ [240], HO HO NH NH HO HO OCH OCH OCH 2 4 2 2 4 3 172 A number of substitutions for the meta-methoxy groups of trimetoquinol 244-248 were investigated. These substitutions varied the electronic and lipophilic nature of the new group (amino, nitro, iodo, and hydrogen) in an attempt to influence receptor activity and selectivity in both f3-adrenergic and TXA2 receptor systems [241,242]. Only nitro-TMQ 245 had enhanced potency on Gr receptors as compared to TMQ, and all derivatives were only slightly f32-selective [242]. Both iodo-TMQ 246 and diiodo-TMQ 248 retained potency as TXA2 antagonists in comparison to TMQ for the inhibition of U46619 induced human platelet aggregation and ATP secretion [241,242]. Iodo-TMQ was also shown to protect mice in the mouse sudden death model [243], The iodinated derivatives of TMQ show promise as potential radioligands for the study thromboxane A2 receptor systems [241,242]. HO HO OCH3 244 OCH3 245 o c h 3 246 o c h 3 247 H o c h 3 248 173 5.4 Irreversible Binding of Drugs Drugs typically undergo many reversible interactions with proteins (receptors, enzymes, transport proteins, antibodies) including electrostatic interactions, ion-dipole and dipole-dipole interactions, hydrogen bonding, charge-transfer complexation, van der Walls forces, and hydrophobic interactions [85,244]. Multiple reversible associations can preserve the half-life of the drug-protein complex and provide sustained drug activity and subsequent therapeutic effects. An alternate means of sustaining drug-protein interactions is to covalently bond the drug to its target protein. Covalent bonds are very strong interactions which may be reversible or irreversible. Certain drugs which utilize covalent bonding in their association with a target protein are particularly useful. For example, aspirin acetylates the enzyme cyclooxygenase in platelets preventing the subsequent production of prostaglandins, thromboxanes and prostacyclin [245], Other examples of therapeutic irreversible covalent bonding include penicillin and cephalosporin antibiotics [245], DNA alkylators such as the nitrogen mustards and nitrosoureas [246], and the a-adrenergic B-haloalkylamine phenoxybenzamine [144]. The irreversible binding of drugs to proteins also has applications as a research tool. This methodology has been applied to the characterization, isolation and study of receptors, enzymes, regulatory proteins, and antibodies 174 [247,272]. The methods of irreversible drug binding can be classified as affinity and photoaffinity labeling [247]. 5.4.1 Affinity Labeling Affinity labelling of receptors requires a drug (D) which can interact with the receptor (R) in a reversible manner to form a drug-receptor complex (D--R). If an appropriately placed reactive species is incorporated into the drug, an irreversible alkylation can occur (Figure 26). Affinity Labeling R + D 7 ' R "D R D Photoaffinity Labeling hv R + D ^ * R-D ——► r ...d * ► R D* Figure 26. Affinity and Photoaffinity Labeling. Effective affinity labeling requires two conditions, selective binding and covalent bond formation [244], The affinity label is a chemically reactive species which retains as many of the structural features as possible of the original ligand. An appropriately placed electrophillic group (proximity effect) can form a covalent bond with the macromolecule. In addition, a marker (radioactive or fluorescent group) is required for detection of the covalently linked product [244]. 175 Covalent linkage of the reactive ligand may occur at a number of locations; the binding site, an adjacent site in which the affinity label blocks the binding site, or a nonspecific site [244]. In order to evaluate the success of affinity labelling, several experiments are required to verify site-specific labeling. Protection studies inhibit the rate of covalent modification due to a direct competition between the protector (agonist, antagonist, or reversible inhibitor) and the affinity label. Scavenger studies inhibit the quantity of site-specific covalent modification. Finally, nonspecific labeling studies determine the exclusivity of the affinity label for the binding site [244]. Most chemical labels are susceptible to hydrolysis. However, several functionalities are stable enough for successful affinity labeling. These include: a-halo or diazoamides, esters, and ketones, 3-chloroethylamines, isothiocyanates, aziridines, epoxides, sulfonyl fluorides, and Michael-acceptors (Figure 27) [244,248,249]. 5.4.2 Photoaffinitv Labeling Photoaffinity labelling of receptors requires a drug (D) which can interact with the receptor (R) in a reversible manner to form a drug-receptor complex (D--R). If this complex is photolyzed (hv), a reactive species is produced (D--R*) and irreversible alkylation can occur (Figure 26) [250,251]. Photoaffinity labels contain a specialized reactive species. The photoaffinity label is covalently inert until it is activated to a reactive species by 176 Affinity Labels o o ' X.a ' 'C, H 2Y .ACHN, alpha-haloacetamides H = NH, Y = Cl, Br.diazoacetamides X = NH beta-chloroethylamines alpha-haloesters X = O, Y = Cl, Br, I diazoesters X = O alpha-haloketones X = CH2, Y = Cl, Br, I diazolketones X = CH 2 O X it -N:C:S — S - F it yx O Isothiocyanates X = O, epoxides X = N, aziridines sulfonylfluorides OCH2 CH; Michael Acceptors Photoaffinity Labels O ^ A C^~N3 c h n 2 arylazides aryldiazirines diazolketones Figure 27. Affinity and Photoaffinity Labels. photolysis [250,251,252]. This offers several advantages as compared to affinity labelling. Nonspecific binding can be limited by allowing ligand-protein equilibration in the dark. This allows the inert ligand to diffuse to the binding site without covalently interacting with nonspecific sites. In addition, equilibration in the dark allows for the determination of reversible receptor 177 affinity for the photoaffinity ligand. This is not possible with chemical affinity labels [250]. Secondly, photoactivation generally produces a very reactive species capable of interactions unavailable to chemical affinity labels such as insertion to carbon-hydrogen bonds and carbon-carbon double bonds [251,252]. Conversely, there are also additional difficulties with photoaffinity labels. In addition to the protection, scavenger and nonspecific labeling studies required for the chemical affinity labels, photoaffinity labels require two additional experiments. The first experiment is the determination of protein viability following exposure to the light required for photolysis of the photoaffinity label. This experiment determines the potential for protein degradation upon exposure to light [250,251,252]. The second experiment requires supplementary nonspecific binding studies to determine non-specific labelling of a long-lived photogenerated species that may diffuse away from the binding site [250]. Several photoaffinity labels have been utilized and include the azido, diazirine, and the previously mentioned diazoketones and diazoacetamides (Figure 27) [248,251,252]. CHAPTER VI STATEMENT OF THE PROBLEM AND OBJECTIVES Trimetoquinol 176 is a potent, nonselective atypical 3-adrenergic agonist and nonprostanoid thromboxane A2 (TXA2) antagonist [216,219], This compound contains a 3,4,5-trimethoxybenzyl substituent which produces a chiral center that is unique among 3-agonists and nonprostanoid TXA2 antagonists. Biological studies reveal that the (S)-(-)-isomer is a stereoselective B-adrenergic agonist while the (R)-(+)-isomer is a stereoselective TXA2 antagonist [217,220], Several researchers have attempted to improve the biological profile of trimetoquinol by investigating several structural modifications [221,223, 225-229,231,232,235-237,240,241], These transformations attempted to improve potency and receptor subtype selectivity. Most of these modifications resulted in trimetoquinol analogues which either retained or lost potency as 3-agonists or thromboxane A2 antagonists [221,223,225-229,231,232, 235-237,240,241], Although these derivatives generally had reduced potency, enhanced receptor subtype selectivity was observed for a number of compounds [222,224,228,229,231,232,238,242,243], However, because of the reduced potency, very few modifications resulted in improved biological profiles, 178 Therefore, the first objective of this project was further investigate other modifications of trimetoquinol in an attempt to improve potency and establish receptor selectivity. 6.1 N-Phenvlethvl Derivative In general, N-substituted derivatives of trimetoquinol were less active as B-agonists and TXA2 antagonists than the parent compound (Tables 6 and 7) Table 6. Effect of Isoproterenol and Trimetoquinol in Guinea Pig Right Atria and Trachea (modified from 224,253). Compd # Description pD,a riaht atria (B,) pD, trachea (B,) 161 isoproterenol 7.69 ± 0.10 7.05 ±0.10 176 trimetoquinol (TMQ) 7.10 ± 0.08 7.05 ±0.10 209 (S)-(+)-TMQ 7.26 ± 0.20 7.10 ± 0.15 210 (R)-(-)-TMQ 5.47 ± 0.15 5.54 ± 0.10 214 N-benzyl-TMQ 4.34 ± 0.07 5.09 ± 0.12 215 N-4-methylbenzyl-TMQ 4.73 ± 0.13 4.87 ± 0.06 216 N-4-chlorobenzyl-TMQ 3.73 ± 0.11 4.89 ± 0.16 217 N-4-methoxybenzyl-TMQ 4.19 ± 0.10 5.32 ± 0.13 218 N-4-nitro-TMQ 4.71 ± 0.13 5.02 ± 0.17 219 N-4-amino-TMQ 5.54 ±0.12 6.18 ± 0.04 220 N-3,4-dichloro-TMQ 4.10 ± 0.30 5.32 ± 0.06 apD2 = - log ECgo, data represents the mean pD2 value ± S.E. Table 7. 180 Effect of N-Benzyltrimetoquinol Analogues on U46619-lnduced Platelet Aggregation and Serotonin Secretion [221,223], >mpd # Description IC s n IM ICsnjH M i agqreqation secretion 176 trimetoquinol (TMQ) 0.77 ± 0.09 0.75 ± 0.04 210 (R)-(-)-TMQ 0.71 ± 0.16 0.55 ± 0.13 209 (S)-(+)-TMQ 19.3 ± 2.8 24.5 ± 3.0 214 N-benzyi-TMQ 21.2 ± 0.4 96.8 ± 5.7 215 N-4-methyibenzyl-TMQ 55.9 ± 10.7 40.4 ± 9.0 216 N-4-chlorobenzyl-TMQ 119.3 ± 16.4 127.5 ± 19.5 217 N-4-methoxybenzyl-TMQ 50.2 ± 6.9 56.2 ± 7.0 218 N-4-nitro-TMQ 295.9 ± 25.7 268.9 ± 47.5 219 N-4-amino-TMQ 27.0 ± 6.2 27.1 ± 3.3 220 N-3,4-dichloro-TMQ 256.9 ± 13.6 264.5 ± 25.6 adata represents the mean IC^ value ± S.E. [221-224,253], However, a number of the benzyl compounds showed receptor subtype selectivity in 3-adrenergic (02 vs ft, selectivity) and TXA2 (a-subtype vs x-subtype) receptor systems [222,223,224], In an effort to regain receptor affinity while maintaining receptor subtype selectivity, the N-substituent was elongated to the phenylethyl homolog 249, The alkyl chain was lengthened to probe for a potential binding site in f3-adrenergic and thromboxane A2 receptors responsible for receptor subtype selectivity [254], 181 HO HO OCH 3 OCH 3 CH 3 O CH3 O 6.2 Amidine Derivative As previously mentioned, very few modifications of trimetoquinol resulted in enhanced potency. Trimetoquinol possess a chiral center which is unique to nonprostanoid antagonists and different from the benzylic hydroxy center found in classical 13-agonists. This chiral center shows a strong stereodependence for binding to R-adrenergic (S»R ) and TXA2 (R »S) receptor systems [217,220]. Since trimetoquinol lacks significant receptor subtype selectivity in each system, the planar amidine 250 was prepared to investigate the effect of a conformational restriction of the trimethoxybenzyl substituent. The combination HO HO OCH OCH 3 OCH3 CH 3 O CH3O Figure 28. Tautomeric Forms of Amidine-TMQ 250. 182 of a dihydroisoquinoline nucleus with a nitrogen substitution of the a-carbon in the benzyl substituent generates the amidine system. This amidine functionality lacks a chiral center and tautomerizes between the dihydroisoquinoline and a stilbene system (Figure 28). The planarity of the amidine will provide insight on the need for a chiral center and may have a substantial impact on receptor potency and selectivity. 6.3 lodinated Derivatives Trimetoquinol is one of the most potent derivatives of the 1-benzyl- 1,2,3,4-tetrahydroisoquinoline class with functional activity in both 6-adrenergic and TXA2 receptor systems [216,219]. The unique3,4,5-trimethoxybenzyl group is apparently important for receptor affinity and receptor activation. This substitution is not found in classical 6-adrenergic agonists or nonprostanoid TXA2 antagonists and may be significant in identifying important portions of the receptors for enhanced ligand affinity and/or function. Therefore, the second objective of this project was to design and synthesize trimetoquinol affinity and photoaffinity labels of the trimethoxybenzyl region to probe the receptors for this important binding site. Of the many trimetoquinol derivatives previously synthesized, very few have improved on the functional potency of trimetoquinol. However, two key derivatives have similar or enhanced affinity for human 6r and 62-adrenergic receptors expressed in E. coli. (Table 8) [253]. lodo- and diiodo-TMQ are 183 Table 8. Binding Affinities (pK3) of Selected Trimetoquinol Analogues on Human B-Adrenergic Receptors Expressed in E. Coli. (modified from 253). Compd # Description huB, huli, 176 rac-(±)-TMQ 7.98 ± 0.05 7.68 ± 0.06 209 (S)-(-)-TMQ 8.27 ± 0.03 7.67 ± 0.09 210 (R)-(+)-TMQ 5.45 ± 0.12 4.81 ± 0.09 246 iodo-TMQ 7.90 ± 0.07 8.57 ± 0.04 248 diiodo-TMQ 8.28 ± 0.10 9.23 ± 0.15 apKj = - log Kj, data represents the mean Kj value ± S.E., n = 4-8 Table 9. Binding Affinities (K3, jxM) of Selected Trimetoquinol Analogues in Human Platelets (HP), Rat Vascular Endothelial Cells (RVEC) and Rat Vascular Smooth Muscle Cells (RVSMC) (modified from 255). Compd # Description HP RVECRVSMC 176 rac-(±)-TMQ 0.38 ± 0.03 0.41 ± 0.10 0.46 ± 0.13 210 (R)-(+)-TMQ 0.14 ± 0.02 0.28 ± 0.05 0.26 ± 0.08 209 (S)-(-)-TMQ 130 ± 22 158 ± 16 233 ± 38 246 iodo-TMQ 0.080 ± 0.017 0.21 ± 0.06 0.25 ± 0.06 248 diiodo-TMQ 0.047 ± 0.007 0.089 ± 0.022 0.38 ± 0.07 3data represents the mean K, value ± S.E. excellent templates from which to form irreversibly binding derivatives because of their high affinity for the 0-adrenergic receptors. Fortuitously, these iodinated analogs also have high affinity for thromboxane A2 receptors in human platelets, rat vascular smooth muscle and rat vascular endothelium (Table 9) [255], 184 Therefore, the same affinity and photoaffinity labels designed for 8-receptors can be utilized to study TXA2 receptors as well. These irreversible trimetoquinol derivatives will be one of the few irreversible 8-agonists [256,257] and nonprostanoid antagonists [258] utilized to study the receptor systems. Typically, 8-antagonists [259,260,261,262,263,264] and prostanoid antagonists [265,266,267,268] have been employed for the study of these systems. 6.3.1 Retrosvnthetic Strategy of lodinated Affinity and Photoaffinitv Labels Several guidelines have been followed during the design of the irreversibly binding derivatives 251 -252 and their retrosynthetic strategy: 1. The irreversible label must have a high affinity for the receptor [269]. Simple modifications of iodo- and diiodo-TMQ may allow the incorporation of an irreversibly binding functionality without significant loss of receptor affinity. Closely related reversible derivatives will be synthesized to evaluate reversible receptor affinity. 2. In an effort to limit the amount of nonspecific binding, molecules which would have a weaker affinity for the receptor should be eliminated. Because of the stereodependence of binding to 8-adrenergic (S »R ) and TXA2 (R»S) receptors (Tables 8 and 9), the irreversible labels will be stereospecifically synthesized. This rationale will eliminate the "enantiomeric impurity" which has a lesser affinity for the specific receptor. In an effort to conserve the chiral intermediates, the synthetic methodology will be perfected on racemic compounds before application to the asymmetric methodology. The irreversibly binding group must be placed in either the 3- or 4-position of the aromatic ring. The portion of the receptor which interacts with the trimethoxybenzyl ring is unknown. For irreversible alkylation of to occur, the reactive group must be properly positioned (R)-enantiomer (S)-enantiomer Photoaffinity Labels a.) R — -N 3 Affinity Labels o b.) R = -NCS c.) R = -NHCOCH2 X X = Ci,Br,l o d.) R = -NHCOCH=CHC02Et within the receptor to alkylate the area of interest. This will require a number of reactive groups to be placed in either the 3- or 4-position to probe for the optimal interaction with the benzyl ring. These groups will vary in size, shape, and reactivity (photolabile or electrophillic) in order to irreversibly bind to the receptor. Irreversible labels require a marker to trace the receptor bound ligand during protein mapping and amino acid sequencing [269], This marker is frequently a radiolabel. This radiolabel must be incorporated late in the synthetic scheme in order to minimize the cost, disposal, and handling of radioactive materials. lodo-TMQ derivatives can have the stable isotope of iodine (127l) replaced with the short-lived radioisotope (125|, t,/2 = 60 days) to provide the necessary marker. Unfortunately, the synthesis of iodo- 246 and diiodo-TMQ 248 introduced the iodine atom very early in the synthetic pathway [241,254]. Therefore, an original synthetic approach is required for the synthesis of the iodinated irreversible labels. Whenever possible, the radiolabel will be introduced after the irreversible group. In some cases (steric interference or chemical reactivity), it will be necessary to introduce the radiolabel prior to the introduction of the irreversible group. The synthetic methodology will be performed utilizing the stable isotope of iodine (127l) before application to the radiochemical synthesis. 187 5. The final prerequisite for the design of the synthetic pathway is to limit intermolecular alkylation of trimetoquinol’s secondary amine. By isolating trimetoquinol labels as their amine salts, significant intermolecular alkylation of these analogues can be prevented. Several affinity and photoaffinity labels which contain a secondary amine have been studied [270,271,272], The retrosynthetic strategies which encompasses all of these guidelines are shown in Schemes XXIII and XXIV. The retrosynthetic strategy begins with the asymmetric synthesis of the tetrahydroisoquinoline (THIQ) nucleus (see Chapter IX for the approaches to the asymmetric synthesis of these THIQs). Scheme XXIII. Retrosynthesis of 4-Substituted Diiodotrimetoquinol Irreversible Labels. HO Protect-0 NH N - Protect HO Protect-O N-label N-label Protect-0 Protect-0 N - Protect I* = I1 2 7 or I1 2 5 N-label = affinity or photoaffinity label NH Scheme XXIV. 188 Retrosynthesis of 3-Substituted lodotrimetoquinol Irreversible Labels. Protect-0 HO NH Protect-O N - Protect HO N-label N-label OCH OCH Protect-O Protect-O N - Protect Protect-O Protect-O N - Protect NO. NO OCH OH f = I127 or I125 N-label = affinity or photoaffinity label Aromatic iodination occurs much later in the synthetic pathway when compared to iodo- and diiodo-TMQ, but there are a several steps which will eventually involve radiochemical synthesis. Iodination (127l or 125l) is required at this point in the scheme because the benzyl substituent must be appropriately activated with minimal steric bulk to allow selective iodination of this trisubstituted ring. The iodinated ring will then be converted to an iodinated aniline ring for subsequent irreversible functionalization. At this point, whenever possible, the stable isotope (127l) will be converted to the radioisotope (125l) through a trimethyltin derivative [268] to limit the number of radiosynthetic steps. In order to limit intermolecular alkylation, the final step will be simultaneous deprotection 189 of the secondary amine and catechol functionalities. In order to conserve the chiral intermediates, this entire approach will be explored with racemically synth esized tetrahyd ro isoquinolines. 6.3.2. Precursors of Irreversible Iodinated Derivatives as Lead Compounds Several racemic intermediates 253-261 will be prepared to evaluate the synthetic methodology during the preparation of the irreversibly binding analogues. Many of these intermediates will be converted to catecholamines 262-270 in one step by simultaneous deprotection of the catechol and amine functionalities. These deprotected compounds will be evaluated for (3-adrenergic and TXA2 functional and binding properties. This random screening process may yield valuable information on the structural activity relationships of related trimetoquinol analogues and potentially produce a novel lead compound for further study. 190 RO r3 R Ri RL, Ea R Bi R* Ea 253 Bn H n o 2 H 262 HH n o 2 H 254 Bn H n h 2 H 263 HH n h 2 H 255 Bn H NHAc H 264 HH NHAc H 256 Bn 1 n h 2 1 265 H 1 n h 2 1 257 Bn n o 2 OH H 266 H n o 2 OHH 258 Bn n o 2 OH 1 267 H n o 2 OH 1 259 Bn n o 2 o c h 3 1 268 H n o 2 o c h 3 1 260 Bn n h 2 o c h 3 1 269 H n h 2 o c h 3 1 261 Bn NHAc o c h 3 1 270 H NHAc o c h 3 1 CHAPTER VII RESULTS AND DISCUSSION 7.1 Chemistry 7.1.1 N-Phenvlethvl-Trimetoquinol The synthesis of 1,2,3,4-tetrahydroisoquinolines is generally accomplished by two routes: a Pictet-Spengler cyclization or a Bischler- Napieralski cyclization [273]. Both the Pictet-Spengler method [215] and Bischler-Napieralski method [226] have been used to produce trimetoquinol 176, and the Bischler-Napieralski method has been described for the synthesis trimetoquinol precursor 271 [221,223,226]. This trimetoquinol precursor is a functional intermediate for the production of N-substituted trimetoquinol HO BnO NH NH HO BnO OCH3 176 271 OCH 191 192 derivatives [221,223] and was utilized in the synthesis of N-phenylethyl trimetoquinol 249 (Scheme XXV). Commercially available amine hydrochloride 98 was converted to its free base and heated with 3,4,5-trimethoxyphenylacetic acid in toluene for 72 h with azeotropic removal of water via a Dean-Stark trap to form amide 272 [221] (Scheme XXV). This amide was cyclized under Bischler-Napieralski conditions with phosphorous oxychloride in anhydrous acetonitrile to form an air-sensitive imine [221,223] which was immediately reduced with sodium borohydride in ethanol [221] to form tetrahydroisoquinoline 273 which was isolated as its oxalate salt. The free base of this amine was acylated with phenylacetyl chloride to form amide 274. This amide has restricted rotation about the amide bond which produces a complicated 1H NMR spectrum (Figure 29). Elevated temperature studies in deuterated chloroform at 323 K or dimethylsulfoxide at 357 K did not allow for a rapid interconversion between these conformations and did not clarify the 1H NMR spectrum. Reduction of the amide with borane- tetrahydrofuran complex to form tetrahydroisoquinoline 275 which was isolated as its oxalate salt, restored free rotation about the nitrogen and provided a simpler 1H NMR spectrum (Figure 30). The benzyloxy ethers were removed from the amine free base with a 1:1 mixture of concentrated hydrochloric acid/methanol to form a mixture of products. This mixture was purified by silica gel chromatography with methanol/diethyl ether/ammonium hydroxide/chloroform Scheme XXV. 193 Synthesis of N-Phenylethyi-Trimetoquinol 249. 1. NaO H/CH 2 CI2 BnO BnO 2 CH2COOH BnO " A A N H 2 HCI BnO X jO 272 OCH, 98 c h 3o o c h 3 o c h 3 OCH, toluene reflux c h 3o BnO 1. POCI3 /CH 3 CN * B n 0 (COOH, 2 1 -N .° H /C H 2« 2 & 2. NaBH 4 2. PhCH2COCI O C H 3 Na 2C 0 3 /CH 2CI2 3. oxalic acid / IpOH 273 OCH, (COOH)2 CH30 ’ 1. NaOH / CH 2CI2 2. LAH / THF 3. oxalic acid / Et20 c h 3o C H ,0 \ HCI r i N — 1. NaOH / CH 2CI2 i 2. HCI / MeOH ^,och3 249 rTr so c h 3 c h 3o (10/10/1/79) as the eluent. The fractions were acidified with dilute hydrochloric acid, evaporated and recrystallized from ethanol/diethyl ether to yield N-phenylethyl-trimetoquinol 249 as a hydrochloride salt. 194 7. 5 7.0 6.5 6.0 Figure 29. Partial 1H NMR Spectrum of N-Phenylacetamide 274 in CDCI3. 6 . SI S . S Figure 30. Partial 1H NMR Spectrum of N-Phenylethylamine 275 in CDCI3. 7.1.2 Amidine-Trimetoquinol Several preparations have appeared in the literature for synthesizing benzamidine systems via imino chlorides [274], ureas [275], and thioureas [276,277,278]. Because of similarities of the Bischler- Napieralski cyclization of ureas [275] and thioureas [276] to the synthesis of trimetoquinol, this method was chosen as the route to synthesize the amidine derivative of trimetoquinol 250. The retrosynthesis of urea 276 and thiourea Scheme XXVI. Retrosynthetic Approach to Urea 276 and Thiourea 277. N=C=X OCH3 OCH OCH3 X = O or S 276 = o 277 = s BnO 196 277 for Bischler-Napieralski cyclization is shown in Scheme XXVI. These compounds can be synthesized by two possible routes which involve the reaction of an amine with an acylating reagent. In each route either the phenylethylamine or the trimethoxyphenyl group can be functionalized for use as the acylating reagent. The initial investigation began by reacting trimethoxyaniline 278 with methyl chloroformate to form aryl urethane 279 (Scheme XXVII). This urethane was added to a solution of the anion of phenylethylamine 98 in toluene to form urea 276 in only 26% yield. A second product was also isolated from the Scheme XXVII. Synthesis of Urea 276 from 3,4-Dibenzyloxyphenylethylamine and Urethane 278. o HN A.o c h 3 methyl chloroformate *■ c h 3o OCH3 Na 2CQ3 /toluene CH30 OCH3 NaH / toluene o c h 3 o c h 3 278 279 OCH 197 reaction mixture and identified by 1H NMR as urea 280. Thus, some of the desired amide was lost by the addition of a second anion of phenylethylamine 98 to form the symmetrical urea. No reaction was observed in the absence of anion formation, and decomposition was observed under sulfuric acid catalysis. The second route investigated reversed the urethane and anion formation. Arylalkylurethane 281 was formed from the free base of amine 98 and methyl chloroformate (Scheme XXVIII). This urethane was added to a solution of the anion of trimethoxyaniline 279 in toluene to form urea 276 in only 20% yield. A second product was also isolated and identified by 1H NMR as urea 282. With low yields and the formation of the symmetrical urea by-products, the reaction of an amine with a urethane is not a feasible route to urea 276. Scheme XXVIII. Synthesis of Urea 276 from Trimethoxyaniline and Urethane 281. n h 2 CH30 o c h 3 BnO methyl chloroformate OCH3 *■ KOH / water BnO NaH toluene ■HCI 98 281 o c h 3 o c h 3 Scheme XXIX. 198 Synthesis of Amidine 283 via Thiourea 277. BnO N=C=S BnO CH3O —0CH;l water 5 °c CH3O — iJV^'OCH3 EtOH OCH 3 284 BnO 1. POCI3 /CH 3CN > 1 2. HCI/CHCI3 BnO N HCI HN OCH 283 OCH CH3O Reaction of phenylethylamine 98 with an isocyanate derivative was chosen as an alternate method to form urea 276. The shortest route to the protected amidine 283 from a compound which can undergo a Bischler- Napieralski cyclization would involve the preparation of thiourea 277. Isothiocyanate 284 was formed in one step from trimethoxyaniline 279 and thiophosgene [279] (Scheme XXIX). The crude isothiocyanate was reacted with the free base of amine 98 to produce thiourea 277. Cyclization with phosphorous oxychloride in anhydrous acetonitrile followed by salt formation with hydrogen chloride (g) in chloroform to produce the protected amidine 282. However, this synthetic route did not provide an acceptable elemental analysis for this compound. The product from this pathway was not further studied because of the successful results from the pathway described below. 199 Scheme XXX. Synthesis of Isocyanate 285. OH toluene reflux OCH OCH OCH OCH3 OCH3 OCH3 286 287 r = OEt 285 7 ~ l n h 2nh 2 288 r = n h n h 2 soci. 289 r = n*33 NaN(V HCI t 1 NaN3/toluene OCH 290 The key intermediate in the preparation of urea 276 is isocyanate 285 which can be prepared by two routes (Scheme XXX). 3,4,5-Trimethoxybenzoic acid 286 was heated with ethanol and sulfuric acid to form ethyl ester 287 [280]. This ester was heated with hydrazine to form the benzoyl hydrazide 288 [281]. The hydrazide was converted to the benzoyl azide 289 with hydrochloric acid and sodium nitrite at 0 °C [282], A second route for the formation of this benzoyl azide is the conversion of benzoic acid 286 to its acid chloride 290 with thionyl chloride in benzene [283] followed by reaction with sodium azide in toluene to form benzoyl azide 289 [279], Isocyanate 285 is formed by heating the benzoyl azide in toluene and collected by vacuum distillation [279] or generated in situ for subsequent use. 200 Scheme XXXI. Synthesis of Amidine-TMQ 250 from Urea 276. BnO N=C=Q BnO NH, BnO CH30 toluene OCH3 276 T ' OCH3 285 c h3o BnO 1. POCI3/CH3CN BnO - N HCI 1 2. HCI / MeOH HN OCH 283 OCH. HO cone HCI / MeOH N HCI HO HN OCH 250 OCH A toluene solution of the free base of phenylethylamine 98 was mixed with a toluene solution of isocyanate 285 at room temperature and subsequently heated to reflux for 1.5 h to produce urea 276 (Scheme XXXI). Cyclization with phosphorous oxychloride in anhydrous acetonitrile produced a mixture of compounds. The desired amidine 283 was purified by silica gel chromatography with methanol/benzene/triethylamine (5/95/0.5) as the eluent 201 and subsequent salt formation in hydrogen chloride (g)/methanol. The benzyloxy ethers were removed with (1:1) concentrated hydrochloric acid/methanol to produce amidine-TMQ 250. 7.1.3 Modified Trimetoquinol Derivatives Several intermediates for the synthesis of iodinated analogues of trimetoquinol have been converted to catecholamines to study the structural activity relationships of modified benzyl substituents. Each compound only required a single synthetic reaction to prepare a catecholamine. These racemic analogues successfully completed include 4-nitro-, 4-amino, and 4-hydroxy-3-nitro derivatives 262, 263. and 266. The approach to synthesizing these compounds is identical to that employed for the synthesis of N-phenylethyl-trimetoquinol as described above. The general preparation of these compounds begins with the conversion of protected amine 98 to its free base which is heated with the appropriate phenylacetic acid 291-293 to form amides 294-296 (Scheme XXXII). Both 4-nitro- and 4-aminophenylacetic acid are commercially available, but 4-hydroxy-3-nitrophenylacetic acid 293 was prepared by the nitration of 4-hydroxyphenylacetic acid 297 with nitric acid in glacial acetic acid. These amides were cyclized under Bischler-Napieralski conditions with phosphorous oxychloride in anhydrous acetonitrile and subsequently reduced with sodium borohydride in ethanol to form the protected tetrahydroisoquinolines 253. 254. Scheme XXXII. 202 General Synthesis of Catecholamines 262, 263, and 266. BnO CH,COOH BnO X T 1-, 1. POCI3 /CH 3 CN „ toluene 2 . NaBH 4 / EtOH 3. HCI / MeOH 291 R = NOz R, = H 292 R = NH2 Ri = H 294 R = NOz R, = H 293 R = OH R, = N 02 H N 0 3 / H 0 A c 295 r = n h 2 r , = h 297 R = OH R, = H — 2.96 R = OH R, = N 0 2 BnO HO cone HCI / MeOH BnO NH HCI reflux HO 2 253 R = n o R, = H __ H2 50psi 262 R = N02 R, = H 254 r = nh 2 r , = h Raney Ni 263 r = nh 2 R-) = h MeOH 257 R = OH Rn = n o 2 266 R = OH R, = n o 2 and 257. The formation and cyclization of amide 295 both proceeded in very low yield. An alternate route for synthesizing tetrahvdroisoquinoline 254 was to reduce nitro derivative 253 by catalytic hydrogenation with Raney nickel (Scheme XXXII). This efficient procedure does not reduce the benzyloxy ethers [284] and was much easier than reduction with acidic tin(ll) chloride. Reduction with hydrazine and Raney nickel in refluxing methanol can also be used [285]. The benzyloxy ethers were removed by heating in 1:1 203 concentrated hydrochloric acid/methanol to afford catecholamines 262, 263. and 266. Several iodinations were attempted on tetrahydroisoquinolines 254 and 257 to provided benzyloxy protected, iodinated trimetoquinol derivatives 256 and 258 (Scheme XXXIII). For 4-amino derivative 254, standard iodinating procedures for anilines such as sodium iodide/Chloramine T [286], sodium iodide/thallium chloride [257], and iodine monochloride [287,288,289] all failed to give an iodinated product. Likewise, nitrophenol derivative 257 also failed to give an iodinated product with sodium iodide/thallium chloride [290] or iodine monochloride [291]. The result is rather perplexing because both 4-aminophenylacetic acid 292 and 4-hydroxy-3-nitrophenylacetic acid 293 were easily iodinated with iodine monochloride to provide acids 298 and 299 in high yield (Scheme XXXIV) [292,293]. Therefore, the original route via amide formation and subsequent cyclization was investigated to provide iodo-derivatives 256 and 258. The appropriately substituted acetic acid was heated with the free base of amine 98 for 72 h with azeotropic removal of water via a Dean-Stark trap to afford amides 300 and 301 (Scheme XXXV). Cyclization of the amides with phosphorous oxychloride in anhydrous acetonitrile followed by reduction with sodium borohydride in ethanol produced a mixture of compounds from which the desired derivatives 255 and 258 could not be purified by either silica gel chromatography or crystallization. One of the possible unwanted side reactions Scheme XXXIII. 204 Attempted Synthesis of Iodinated Analogues 255 and 258. BnO BnO iodination N H H C I NH HCI BnO BnO 254 R i = H R 2 = N H 2 R 3 = H 255 r , = i r 2 = nh 2 r 3 = i 257 R 1 = N 0 2 R 2 = O H R 3 = H 258 r , = n o 2 r 2 = oh r 3 = i Scheme XXXIV. Synthesis of lodoacids 298 and 299. ch2cooh C H o C O O H i c i I . aq. HCI nh2 292 298 ch2cooh C H o C O O H ICI DMF NO. OH 299 for the formation of the tetrahydroisoquinoline system is simultaneous dehalogenation by sodium borohydride. In order to avoid dehalogenation of amino derivative 255. the reducing hydride was changed to the deactivated Scheme XXXV. 205 Attempted Synthesis of lodo-THIQs 255 and 258 from the Corresponding Amides. BnO c h 2 c o o h BnO NH. toluene 298 R -) = I R 2 = N H 2 R 3 = I 300 r - | = i r 2 = n h 2 r 3 = i 299 r - i = n o 2 r 2 = o h r 3 = i 301 R-I = n o 2 r 2 = o h r 3 = I BnO 1. POCI3 /C H 3CN NH HCI BnO 2 . NaBH 4 / EtOH 3. HCI I MeOH 255 R f = I R 2 = N H 2 R 3 = I 258 R -j = n o 2 r 2 == o h r 3 = I reducing agent, sodium cyanoborohydride, for a second attempt to reduce the unstable intermediate imine. However, sodium cyanoborohydride was unable to reduce the imine intermediate. Several of the amides already described also have potential use to provide the starting material necessary for asymmetric catalytic hydrogenation (see Chapter XI). Two other amides were prepared for potential use via a racemic or asymmetric route. Nitration of 4-methoxyphenylacetic acid 302 with fuming nitric acid in glacial acetic acid provided phenylacetic acid 303 Scheme XXXVI. Attempted Synthesis of Phenylacetic Acid 304 by lodination of Phenylacetic Acid 303. CH2COOH CH2COOH CH2COOH 90% H N 0 3 iodination HOAc (Scheme XXXVI) in moderate yield. Several iodinations including iodine monochloride [291] and iodine/silver trifluoroacetate [294] failed to produce an iodinated derivative. Therefore, 4-hvdoxv-3-iodo-5-nitrophenvlacetic acid 299 was methylated with dimethylsulfate and potassium carbonate in acetone to produced acid 304 in moderate yield (Scheme XXXVII). The success of this reaction was limited by the solubility of the acid salt of this compound in acetone and required two recrystallization to provide a pure product. These liabilities required a more efficient means of synthesizing the respective amides. Therefore, the nitrophenolic amides 296 and 301 were methylated with iodomethane or dimethylsulfate to provide amides 305 and 306 (Scheme XXXVIII). These compounds were retained for potential use in the asymmetric synthesis of trimetoquinol analogues. Scheme XXXVII. 207 Synthesis of 3-lodo-4-methoxy-5-nitrophenylacetic Acid 304. C H o C O O H c h 2c o o h Dimethyl Sulfate K 2 C 0 3 NO. acetone OH 299 Scheme XXXVIII. Synthesis of Methoxyiodoamides 305 and 306. ^ Bno methyl iodide or dimethylsulfate ------► BnO k 2c o 3 B i acetone V r 3 296 Rt = n o 2 r 2 = o h r 3 = h 305 r ■, = n o 2 r 2 = o c h 3 r 3 = h 301 Rt = n o 2 r 2 = o h r 3 = i 306 r 1 = n o 2 r 2 = o c h 3 r 3 = i 7.1.4 N-Protected Trimetoquinol Derivatives The retrosynthetic approach for the synthesis of the irreversible iodinated trimetoquinol derivatives 251-252 requires protection of the secondary amine. By protecting this amine early in the synthetic strategy, selective formation of the irreversible label on the aromatic amine is unnecessary required. Early protection of this amine was also utilized in an approach to produce chiral intermediates from chiral HPLC (see Chapter XI). The major criteria for 208 (CH3)3Sil O — Si(CH3) 3 OCH3 HCI 00N Y OSi(CH3)3 O co2 CH3OSi(CH3) 3 Figure 31. Mechanism for Trimethylsilyl Iodide Cleavage of Methylurethanes [295]. selecting the appropriate protecting group are stability during subsequent synthetic steps and an intrinsic lability for simultaneous removal with the benzyloxy ethers. The protecting group selected with these qualities was the methyl urethane group. This functionality can be removed with trimethylsilyl iodide (TMSI) [295,296] by intermediate conversion to a trimethylsilyl urethane which is subsequently cleaved by acidic methanol (Figure 31). The by-products of this reaction, methoxytrimethylsilane and methyl iodide, are volatile and can be removed under reduced pressure during purification of the hydrophilic product [295], Some irreversible trimetoquinol derivatives may contain an amide, but the proposed mechanism for urethane cleavage by trimethylsilyl iodide excludes the cleavage of amides [295]. For amides, the unstable trimethylsilyl urethane intermediate would revert back to the amide because iodide attack on silicon is much more favorable than attack at the 209 (CH3)3Sil ^ 0 - S i ( C H 3) 3 I Figure 32. Rationale for the Stability of Amides in the Presence of Trimethylsilyl Iodide. a-carbon to release the positive charge of the intermediate (Figure 32). The synthesis of urethane 307 was accomplished by heating isoquinoline 253 with methyl chloroformate, potassium carbonate, and the phase transfer catalyst Adogen 464 in acetone [297] (Scheme XXXIX). This urethane derivative has restricted rotation about the amide bond resulting in a complex 1H NMR spectrum of two conformations. Elevated temperature studies in deuterated chloroform at 325 K showed a partial collapse of the 1H NMR spectrum to aid in the identification of particular proton resonances (Figure 33). This 1H NMR sample could not be heated much higher in deuterated chloroform without the loss of solvent. Catalytic hydrogenation of the nitro group with Raney nickel in methanol/dimethylformamide provides amino derivative 308 without the need for selective alkylation between a secondary and an aromatic amino group (Scheme XXXIX). This procedure also provides the necessary protection of the secondary amine for while acetylating the aromatic amine with acetic anhydride to provide acetamide derivative 309. Several attempts to iodinate either Scheme XXXIX. 210 Synthesis of Methyl Urethanes 307-309. BnO BnO H2 60psi methyl chloroformate Raney Ni NH -HCI OCH ► BnO k2c o 3 BnO MeOH/DM F Adogen 464 acetone 253 307 NO NO BnO BnO OCH BnO BnO OCH 308 309 NH NHAc N-protected amine 308 or acetamide 309 with iodine monochloride [287,288,289] or iodine/silver trifluoroacetate [294] failed provide significant amounts of an iodinated derivative. A small scale reaction of amine 308 with 2.2 equivalents of benzyltrimethylammonium dichloroiodate (BTMA ICI2) and calcium carbonate in methanol/dichloromethane [298] for 24 h at room temperature provided a mixture of monoiodo derivative 310 and diiodo derivative 311 in 39% and 20% yield respectively (Scheme XL). Reaction of acetamide derivative 309 under identical conditions failed to produce an iodinated compound. The 1H NMR spectra of each of these urethanes is a mixture of two conformations at room temperature. The assignments of 211 JLA. 3 2 5 K 3 1 3 K 2 9 3 K i i i i 'i—-i i | i -i i i j 1 1 l i | i r i i 3.0 ?. 5 7.0 6.5 Figure 33. Partial 1H NMR Spectrum of Nitro Urethane 307 in CDCI3 at 298, 313 and 325 K. Scheme XL. 212 Synthesis of lodourethanes 310 and 311. BnO BnO BTMA ICI BnO OCH BnO OCH 308 NH NH 310 R = I, = H 311 R = R-, = I particular proton resonances were determined by analogy to the temperature study of the 4-nitro derivative 307. Isoquinoline 257 was converted to its urethane derivative 312 with methyl chloroformate and triethylamine in acetone followed by saponification of the aryl carbonate with 20% aqueous potassium hydroxide in methanol at room temperature to yield nitrophenolic urethane 312 (Scheme XLI). The 1H NMR spectrum of this nitrophenolic derivative showed three conformations in a ratio of 10:9:1 at room temperature. Temperature elevation studies in deuterated chloroform at 323 K also showed a partial collapse of the 1H NMR spectrum to aid in the identification of specific proton resonances (Figure 34). Further 1H NMR studies in deuterated dimethylsulfoxide to 357 K were less useful than the chloroform study, lodination of this derivative followed a similar pattern to the amino derivative 308. lodination with iodine monochloride failed [293], but iodination with benzyltrimethylammonium dichloroiodate and sodium bicarbonate Scheme XLI. 213 Synthesis of 4-Hydroxy-3-iodo-5-nitro- Urethane 313. BnO BnO 1. methyl chloroformate ...... OCH BnO Et3N acetone BnO NO 2. KOH / MeOH n o 2 257 312 OH OH BnO BTMA ICI2 BnO OCH CH2CI2/MeOH n o 2 NaHC0 3 313 OH in methanol/dichioromethane [299] for 24 h at room temperature provided the iodinated derivative 313 in 37% yield (Scheme XLI). The 1H NMR spectrum of this derivative showed only two conformations at room temperature and was solved by analogy to 4-nitro derivative 307 temperature studies. 214 3 2 3 K 2 9 8 K 7.5 7.0 6.5 Figure 34. Partial 1H NMR Spectrum of 4-Hydroxy- 3-nitro Urethane 312 in CDCI3 at 298 and 323 K. 215 7.2 Biological Evaluation Each of the catecholamine derivatives described above has been evaluated for (3-adrenergic and thromboxane A2 receptor activities. B-Adrenergic evaluation was performed in guinea pig atria (BJ and tracheal (B2) strips to evaluate increased heart rate and smooth muscle relaxant properties respectively. Thromboxane A2 antagonist properties were determined from the ability of these catecholamines to inhibit agonist (U46619) induced platelet aggregation and serotonin secretion. The N-phenylethyl derivative 249 was a weak agonist in both ^ and 32 adrenergic systems (Table 10). This derivative was slightly more potent than the N-benzyl derivative 214 on B1 tissue, but less potent on (32 tissue [221]. Unlike the N-benzyl derivative which has 13-receptor subtype selectivity [221], the N-phenylethyl derivative is devoid of any receptor subtype selectivity. Table 10. Comparative Adrenergic Agonist Activities of Trimetoquinol and N-Phenylethyl-Trimetoquinol on Guinea Pig Right Atrial (3,) and Tracheal (32) Tissues. Compd # Description B, (n-4) pD,a B, (n=3) pD,a 176 (±)-TMQ 7.91 ±0.09 7.09 ±0.11 249 N-PhEt-TMQ 4.62 ± 0.36 4.64 ± 0.23 a pD2 = - log ECso, data represents the mean pD2 value ± S.E. 216 Thus, the extension of the alkyl chain provides no significant benefit in the 6-adrenergic profile. A similar result occurs for the N-phenylethyl derivative 249 in thromboxane A2 receptor systems (Table 11). This derivative is slightly less potent in preventing U46619-induced platelet aggregation in comparison to the N-benzyl derivative, but it is slightly more potent in preventing serotonin secretion. However, the extension of the alkyl chain also provides no significant benefit in the thromboxane A2 profile. Table 11. Comparative Potencies of Trimetoquinol Derivatives for the Inhibition of U46619 (1 (xM) Induced Platelet Aggregation and Serotonin Secretion. U46619 induced platelet U46619 induced Compd # Description aggregation Serotonin secretion filCso! filC ^ 176 (±)-TMQb 5.94 ± 0.21 5.88 ± 0.13 249 N-PhEt-TMQ 4.42 ± 0.12 4.46 ± 0.36 250 Amidine-TMQ NAe NDd 262 4-N02-TMQ 4.35 ± 0.05 4.22 ± 0.07 263 4-NH2-TMQ 3.89 ± 0.04 3.84 ± 0.04 266 4-0H-3-N02-TMQ 5.90 ± 0.10 5.95 ± 0.19 a PlCgo = - log ICgQ, data represents the mean pIC^ value ± S.E. b value determined simultaneously with derivatives 262.263. and 266 ClNA = No antagonist activity at 400 jxM dND = not determined Table 12. 217 Comparative Adrenergic Agonist Activities of Trimetoquinol and Amidine Trimetoquinol on Guinea Pig Tracheal (32) Tissue. Compd # Description ikEDa! 176 (±)-TMQ (n = 3) 7.46 ± 0.11 250 Amidine-TMQ (n = 5) 3.95 ± 0.12 a pD2 = - log ECgo, date represents the mean pD2 value ± S.E. The biological profile of the planar amidine derivative 250 supports the stereoselective interactions of trimetoquinol with each receptor system. This planar analog is a very weak B2 agonist with a substantial loss in potency as compared to trimetoquinol (Table 12). Since this compound was such a weak 62 agonist, the B1 properties were not evaluated. This compound also failed to show thromboxane A2 antagonism at a 400 |iM dose (Table 11). The modified benzyl derivatives 262, 263, and 266 were all active as B-agonists (Table 13). The rank order of potency of increased heart rate and for tracheal smooth muscle relaxation in comparison to the trimetoquinol isomers is (S)-(-)-TMQ > 4-N02-TMQ > 4-NH2-TMQ > 4-0H-3-N02-TMQ > (R)-(+)-TMQ. In comparison to (S)-(-)-TMQ, each of these derivatives failed to show receptor subtype selectivity. These compounds are of comparable potency to the previously synthesized modified 1-benzyl derivatives 244-248 [242]. The modified benzyl derivatives 262, 263, and 266 were also concentration-dependent thromboxane A2 antagonists (Table 11). The rank Table 13. 218 Comparative Adrenergic Agonist Activities of Trimetoquinol Analogues on Guinea Pig Right Atrial (8^ and Tracheal (32) Tissues. Compd # Description <"=3) B, (n=4-9) pD?a P0?a 209 (SM-)-TMQ 8.35 ± 0.13 8.80 ± 0.14 210 (R)-(+)-TMQ 5.84 ± 0.18 5.76 ± 0.05 262 4-N 02-TMQ 7.26 ± 0.26 7.49 ± 0.12 263 4-NH2-TMQ 6.21 ± 0.04 6.75 ± 0.10 266 4-OH-3-N02-TMQ 5.91 ± 0.31 6.55 ± 0.15 = - log EC, so, data represents the mean pD2 value ± S.E. order potency of for thromboxane A2 antagonist properties for the inhibition of platelet aggregation and serotonin release was (±)-TMQ = 4-0H-3-N02-TMQ » 4-N02-TMQ > 4-NH2-TMQ. The modification of the 1-benzyl substituent to the 4-0H-3-N02 derivative 266 provided significant potency for the inhibition of platelet aggregation (a-subtype) and serotonin secretion that is comparable to previously synthesized iodo-TMQ derivatives 246 and 248 [242]. The nitrophenolic ring can be ionized as physiological pH providing a potential ionic interaction with a cationic group on the receptor. The lack of significant potency for 4-NH2-TMQ rules out potential hydrogen bonding interactions of 4-0H-3-N02-TMQ for this increased potency. Further studies of with rat vascular aorta (x-subtype) are planned to investigate potential receptor subtype selectivity. 219 7.3 Summary 1. The synthesis of N-phenylethyl-TMQ 249 has been completed. This compound has similar activity to the previously reported N-benzyl derivative and provides no improvement of the biological profile in either 6-adrenergic or thromboxane A2 receptor systems. 2. The synthesis of amidine-TMQ 250 has been achieved. This planar analog has a dramatic loss of activity in both 13-adrenergic and thromboxane A2 receptor systems. The conversion of the chiral center of trimetoquinol into a planar amidine provides additional support for the importance of the chiral center for the stereoselective interactions of the trimetoquinol isomers with the appropriate receptor systems. 3. Several intermediates toward the synthesis of irreversible iodinated trimetoquinol analogues have been converted to catecholamines for biological evaluation in both 13-adrenergic and thromboxane A2 receptor systems. The increased potency of 4-0H-3-N02-TMQ 266 in a thromboxane A2 receptor system with a concurrent loss of activity in 6-adrenergic receptor systems constitutes this compound as a potential new lead compound to optimize thromboxane A2 antagonist properties. This compound appears to have increased potency due to an ionic interaction via the nitrophenolic ring with a complementary group on the receptor. Progress toward the synthesis of irreversible iodinated trimetoquinol analogues has been achieved. Racemic N-methyl iodourethanes 312 and 314 have been recently synthesized as a model for the synthesis of chiral irreversible iodinated derivatives 251 and 252. The success of these synthetic pathways should be applicable to the synthesis of chiral radioiodinated derivatives from the asymmetric synthesis of chiral intermediates (Chapter XI). Several amides 301. 302, 306. and 307 have been prepared for potential use in the asymmetric synthesis of trimetoquinol derivatives. The cyclization of amides 301 and 302 to the corresponding tetrahydroisoquinolines was unsuccessful. CHAPTER VIII EXPERIMENTAL The general information concerning instrumentation, elemental analysis, and solvent preparation provided in Chapter 4 is also applicable to this chapter. Additionally, dry toluene was stored over anhydrous magnesium sulfate and filtered prior to use. Anhydrous toluene was stored over sodium and distilled prior to use. Anhydrous acetonitrile was heated to reflux for at least 3 h with phosphorous pentoxide, distilled and stored over 4A molecular sieves. Brine was diluted with distilled water to prepare V* strength and V 2 strength brine. BnO BnO OCH OCH C H ,0 N-(2-(3,4-Bis(benzyloxv))phenvlethvl)-(3A5-trimethoxvphenyl)acetamide, (272) : 3,4-Dibenzyloxyphenethylamine hydrochloride (10.0 g, 27 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 ml_) and 221 222 dichloromethane (100 mL). The layers were separated, and the organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in toluene (200 mL) and charged with 3,4,5-trimethoxyphenylacetic acid (7.95 g, 35.1 mmol). The mixture was heated to reflux for 72 h with azeotropic removal of water via a Dean-Stark trap. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (200 mL) and washed successively with 1.2 N hydrochloric acid (200 mL), 1/4 saturated brine (200 mL), 10% aqueous sodium hydroxide (200 mL), and 1'A saturated brine (200 mL). The organic layer was then dried with anhydrous magnesium sulfate and evaporated in vacuo to a solid. This solid was recrystallized from hot toluene to yield 10.7 g (73%) of a white solid, mp 111-112 °C (lit. [221] mp 109-111 °C). ’H NMR (CDCI/TMS) : 6 7.43-7.30 (m, 10H, ArH), 6.80 (d, J = 8.2 Hz, 1H, ArH), 6.73 (d, J = 1.9 Hz, 1H, ArH), 6.50 (dd, J = 8.2 and 1.9 Hz, 1H, ArH), 6.36 (s, 2H, ArH), 5.40 (bs, 1H, NH), 5.13 (s, 2H, ArCH20), 5.11 (s, 2H, ArCH20), 3.83 (s, 3H, CH 30), 3.79 (s, 6 H, 2 x CH30), 3.43-3.37 (m, 2H, NCH2), 3.42 (s, 2H, CH2CO), 2.64 (t, J = 6.8 Hz, 2H, ArCH2). I 223 BnO BnO OCH OCH 6.7-Bis(benzvloxv)-1-(3A5-trimethoxybenzvl)-1.2,3,4-tetrahvdroisoquinoline oxalate salt (273) : Amide 272 (6.94 g, 12.8 mmol) was dissolved in anhydrous acetonitrile (150 mL) under argon and charged with phosphorous oxychloride (6.0 mL, 64.1 mmol). The resulting solution was heated to reflux for 3.5 h. After cooling to room temperature under argon, solvent was evaporated in vacuo. The residue was rinsed with anhydrous acetonitrile (30 mL) and evaporated in vacuo. The resulting oil was dissolved in absolute ethanol (100 mL) and cooled to 0 °C with an ice bath. Sodium borohydride (4.85 g, 128 mmol) was added portionwise to prevent excessive foaming. The flask was fitted with an anhydrous calcium sulfate drying tube, and the suspension was allowed to come to room temperature with stirring overnight. Ethanol was removed in vacuo, and the residue was dissolved in a 3:1 mixture of benzene:diethyl ether (200 mL). This solution was successively washed with 10% aqueous sodium hydroxide (2 x 200 mL), brine (200 mL), and dried over anhydrous magnesium sulfate. Solvent was evaporated in vacuo, and the residue was dissolved in methanol (30 mL), and added dropwise to a solution of oxalic acid dihydrate (1.62 g, 12.82 mmol) in isopropanol. After standing for 1 h, the mixture was carefully diluted with diethyl ether (150 mL) to form a bilayer system. After standing overnight, the precipitate was collected to yield 3.98 g (51 %) of a white solid, mp 214-216 °C. 1H NMR (free base, CDCI/TMS) : 6 7.46-7.30 (m, 10H, ArH), 6.73 (s, 1H, ArH), 6.69 (s, 1H, ArH), 6.44 (s, 2H, ArH), 5.12 (s, 2H, ArCH20), 5.07 (s, 2H, ArCH 20), 4.16-4.09 (m, 1H, ArCHN), 3.83 (s, 3H, CH 30), 3.82 (s, 6H, 2 x CH30), 3.15-3.05 (m, 2H, CH2), 2.87-2.68 (m, 4H, 2 x CH2), 2.03 (bs, 1H, NH). 1H NMR is in agreement with that reported as the free base from the hydrochloride salt [221]. FAB MS m/z 526.3 (m + 1, 88 %), 344.1 (m + 1 - C14H14, base). Analysis for CagH^NTOg : calc. C, 68.28; H, 6.06; N, 2.28; found C, 67.92; H, 5.86; N, 2.38. 6.7-Bis(benzvloxv)-2-phenvlacetvM-(3.4.5-trimethoxvbenzvl)-1.2.3.4- tetrahvdroisoquinoline. (274) : Amine oxalate 273 (500 mg, 0.81 mmol) was converted to its free base with 10% aqueous sodium hydroxide (50 mL) and dichloromethane (50 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (50 mL). The organic extracts were 225 combined and washed with 10% aqueous sodium hydroxide (100 mL), 1'A strength brine (100 mL), and dried over anhydrous magnesium sulfate. The organic solution was charged with anhydrous magnesium sulfate (5.0 g) and anhydrous sodium carbonate (5.0 g). The suspension was vigorously stirred for 15 min before a solution of phenylacetyl chloride (0.2 mL, 1.51 mmol) in dichloromethane (20 mL) was added dropwise over 5 min at room temperature. The suspension was stirred for 30 min and filtered. The organic layer was washed successively with 1.2 N hydrochloric acid (100 mL), 1/4 strength brine (100 mL), 5% aqueous sodium hydroxide (2 x 100 mL), 1/< strength brine (100 mL), and dried over anhydrous magnesium sulfate. The solvent was evaporated in vacuo, The clear white oil was dissolved in a minimal amount of hot methanol. After cooling to room temperature, the flask was placed in the freezer. The deposited solid was collected after 72 h to yield 374 mg (72%) of a white solid, mp 122-123 °C. The compound is a mixture of two conformations in solution providing a complex 1H NMR spectrum. Temperature elevation studies in CDCI 3 to 323 K and d6-DMSO to 357 K did not simplify the 1H NMR spectrum in order to assign proton resonances. FAB MS m/z 644.3 (m + 1, 22%), 462.3 (m + 1 - C14H14, base). Analysis for C 41H41N 10 6 : calc. C, 76.49; H, 6.42; N, 2.18; found C, 76.38; H, 6.42; N, 2.11. 226 BnO BnO (COOH)2 c h3o’ 6.7-Bis(benzvloxv)-2-(2-phenvlethv8)-1-(3A5-trimethoxvbenzvl)-1,2,3.4- tetrahvdroisoquinoline oxalate salt, (275) : To a solution of amide 274 (900 mg, 1.40 mmol) in anhydrous tetrahydrofuran (25 mL) in a 3-neck flask fitted with a stopper, septum, and condenser with an argon port was added a 1.0 M solution of borane/tetrahydrofuran complex (5.0 mL, 5.0 mmol). The solution was heated to reflux for 2.5 h, and subsequently cooled to room temperature. Excess reagent was quenched by the addition of ethanol (5 mL), and solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (50 mL) and 5% aqueous sodium hydroxide (100 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (50 mL). The organic extracts were combined and successively washed with 5% aqueous sodium hydroxide (100 mL), 1/4 strength brine (2 x 100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in diethyl ether (25 mL) and added dropwise to a solution of oxalic acid dihydrate (197 mg, 1.40 mmol) in diethyl ether (25 mL). After standing overnight, the precipitate was collected to yield 227 845 mg (84%) of a white solid, mp 214-216 °C. 1H NMR (free base, CDCI/TMS) : 6 7.42-7.15 (m’ 15H, ArH), 6.66 (s, 1H, ArH), 6.29 (s, 2H, ArH), 6.19 (s, 1H, ArH), 5.10 (s, 2H, ArCH20), 4.87 (ABq, J= 13.7 Hz, Au = 20.5 Hz, 2H, ArCH20), 3.82 (s, 3H, CH 30), 3.77 (s, 6 H, 2 x CH30), 3.25-3.15 (m, 1H, ArCHN), 3.09-2.66 (m, 8 H, 4 x CH2), 2.52-2.43 (m, 2H, CH2). FAB MS m/z 630.3 (m + 1, base), 448.1 (m + 1 - C 14H14, 59%) Analysis for C^H^I^Og : calc. C, 71.75; H, 6.30; N, 1.95; found C, 71.48; H, 6.28; N, 1.88. •HCI CH30 6.7-Dihvdroxv-2-(2-phenvlethvl)-1-(3.4.5-trimethoxvbenzvn-1.2,3.4- tetrahydroisoquinoline hydrochloride. (249) : Amine 275 (200 mg, 0.28 mmol) was converted to its free base with 5% aqueous sodium hydroxide (50 mL) and dichloromethane (50 mL). The layers were separated, and the organic layer was washed with % strength brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in a mixture of methanol (10 mL) and concentrated hydrochloric acid (10 mL) and subsequently heated to reflux for 24 h under an argon atmosphere. After cooling to room temperature, solvent was evaporated in vacuo. The resulting 228 oil was rinsed with ethanol (10 mL) and evaporated in vacuo. This was repeated for a total of three rinses. The resulting oil was recrystallized from ethanol/diethyl ether to yield 91 mg (67%) of a light brown solid, dp 118-120 °C (with darkening). TLC on silica gel with methanol/diethyl ether/ammonium hydroxide/chloroform (10/10/1/79) as the eluent revealed an impurity that could not be removed by recrystallization. The product was purified by silica gel chromatography with the same eluent as TLC. The pooled fractions were acidified with 1.2 N hydrochloric acid and evaporated in vacuo. The residue was heated in absolute ethanol (10 mL), filtered and evaporated in vacuo. Recrystallization from minimal hot absolute ethanol/diethyl ether yielded 55 mg (60%) of a tan solid, dp 122-125 (with darkening). ’H NMR (D 20) : 6 7.20-7.08 (m, 5H, ArH), 6.64 (s, 1H, ArH), 6.24 (s, 2H, ArH), 5.87 (s, 1H, ArH), 4.40-4.38 (m, 1H, ArCHN), 3.60 (s, 9H, 3 x CH30), 3.38-3.31 (m, 4H, 2 x CHZ), 3.30-2.92 (m, 6H, 3 x CH2). FAB MS m/z 450.2 (m + 1, base), 268.1 (m + 1 - C 10H14O3, 86 %). Analysis for C^HggN^OgCI^HgO : calc C, 62.12; H, 6.95; N, 2.68; found C, 62.35; H, 6.51; N, 2.99. 229 N-(3A5-Trimethoxyphenvl) methyl urethane, (279) : 3,4,5-trimethoxyaniline 278 (2.0 g, 10.9 mmol) was suspended in dry toluene (75 mL) with anhydrous sodium carbonate (2.0 g). The suspension was charged with methyl chloroformate (2.0 ml, 25.8 mmol) and stirred at room temperature for 72 h. The solids were removed by gravity filtration, and the filtrate was washed successively with 5% aqueous sodium hydroxide (100 mL), Vz saturated brine (100 mL), 1.2 N hydrochloric acid (100 mL), Vz saturated brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The solid residue was recrystallized from a minimal amount of hot toluene to yield 2.07 g (79%) of a white solid, mp 102-104 °C. 1H NMR (CDCI/TMS) : 6 6.68 (s, 2H, ArH), 6.58 (bs, 1H, NH), 3.84 (s, 6H, 2 x CH3OAr), 3.80 (s, 3H, CH 3OAr), 3.77 (s, 3H, CH3OCOR). FAB MS m/z 242.1 (m + 1, 80%), 241.1 (m, base), 226.0 (m - CH3). Analysis for C 11H15N1Os : calc. C, 54.77; H, 6.27; N, 5.81; found C, 54.92; H, 6.09; N, 5.79. 230 N-(3.4-Bis(benzvloxv)phenvlethvl methyl urethane. (281) : According to the procedure of Brossi [300], 3,4-dibenzyloxyphenethylamine hydrochloride (2.0 g, 5.41 mmol) was suspended in water (50 mL), and the solution was rendered basic with 20% aqueous potassium hydroxide (2 drops). Methyl chloroformate (2.0 mL, 25.8 mmol) was added, and the mixture was stirred for 15 min while maintaining the pH > 10 with 20% aqueous potassium hydroxide. The solid was isolated by vacuum filtration, rinsed liberally with water, and dried in a desiccator to yield 2.1 g (99%) of a light brown solid, mp 73-74.5 °C. 1H NMR (CDCI/TMS) : 6 7.46-7.30 (m, 10H, ArH), 6.87 (d, J = 8.1 Hz, 1H, ArH), 6.77 (d, J = 1.9 Hz, 1H, ArH), 6.69 (dd, J = 8.1 and 1.9 Hz, 1H, ArH), 5.14 (s, 2H, ArCH20), 5.13 (s, 2H, ArCH20), 4.60 (bs, 1H, NH), 3.67 (s, 3H, CH 30), 3.40-3.32 (m, 2H, NCH2), 2.69 (t, J = 6.8 Hz, 2H, ArCH2). FAB MS m/z 392.1 (m + 1, 42%), 391.1 (m, base). Analysis for C 24H25N10 41/4H20 : calc. C, 72.80; H, 6.49; N, 3.54; found C, 72.97; H, 6.33; N, 3.55. 231 o OCHoCH OCH3 Ethyl 3,4,5-trimethoxvbenzoate. (287) : According to the procedure of Maxwell [280], the mixture of 3,4,5-trimethoxybenzoic acid 286 (5.0 g, 23.6 mmol), concentrated sulfuric acid (5 mL), and ethanol (100 mL), were heated to reflux for 14 h. Solvent was evaporated in vacuo, and the residue was dissolved in diethyl ether (125 mL). The ether layer was washed with water (125 mL), aqueous saturated sodium bicarbonate (125 mL), dried over anhydrous magnesium sulfate and evaporated in vacuo. The resulting solid was recrystallized from 95% ethanol to yield 5.19 g (92%) of white needles, mp 51-53 °C (lit. [301] mp 52.5-54.5 °C). 1H NMR (CDCI/TMS) : 6 7.31 (s, 2H, ArH), 4.37 (q, J = 7.1 Hz, 2H, OCH2), 3.92 (s, 6 H, 2 x CH30), 3.91(s, 3H, CH3O), 1.40 (t, J = 7.1 Hz, 3H, CH,CH 20). o NHNH- OCH3 3.4.5-Trimethoxvbenzovl hvdrazide. (288) : Ethyl ester 287 (5.0 g, 21 mmol) was dissolved in a mixture of 95% ethanol (25 mL) and toluene (25 mL). 232 Anhydrous hydrazine (10.0 mL, 810 mmol) was added, and the solution was heated to reflux for 23 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was triturated in diethyl ether to produce a solid. The solid was collected by vacuum filtration and recrystallized from 95% ethanol to yield 3.16 g (67%) of white needles, mp 155-157 °C (lit. [302] mp 162 °C). 1H NMR (CDCI/TMS) : 6 7.31 (bs, 1H, CONH), 6.97 (s, 2H, ArH), 4.10 (bs, 2H, NHg), 3.90 (s, 6H, 2 x CH30), 3.89 (s, 3H, CH 30). o o c h 3 3.4-5-Trimethoxvbenzovl azide, (289) : According to the procedure of Mohunta [282], benzoyl hydrazide 288 (1.0 g, 4.42 mmol), was suspended in 1.2 N hydrochloric acid (50 mL) in a 125 mL Erlenmeyer flask at 0 °C. A solution of sodium nitrite (600 mg, 8.7 mmol) in water (10 mL) was added, and the mixture was stirred for 1.5 h at 0 °C. The solid was collected by vacuum filtration, washed with water, and dried in a desiccator to yield 812 mg (77%) of a white solid, mp 83-85 °C (lit. [303] mp 85.5-86.5 °C). 1H NMR (CDCI/TMS) : 6 7.29 (s, 2H, ArH), 3.93 (s, 3H, CH 30), 3.91 (s, 6 H, 2 x CH30). IR (CHCI3) 2146 (N3) cm'1. 233 o o c h 3 3.4.5-Trimethoxvbenzo vl chloride, (290) : According to the procedure of Reeve [283], 3.4.5-trimethoxvbenzoic acid 286 (25.0 g, 0.12 mol) was dissolved in a mixture of benzene (150 mL) and a catalytic amount of dimethylformamide (2 drops). Thionyl chloride (20 mL, 0.27 mol) was added, and the mixture was slowly heated to reflux over 30 min to avoid excessive foaming. The mixture was monitored by IR spectroscopy for the disappearance of the carboxyl OH. After heating for 4 h, solvent was removed by short path distillation at atmospheric pressure. The product was purified by vacuum distillation (bp 119- 136 °C / 0.5-2.2 mm Hg), and solidified on standing to yield 24.0 g (84%) of a white solid, mp 78-79 °C (lit. [283] mp 77-78 °C). 1H NMR (CDCI/TMS) : 6 7.38 (s, 2H, ArH), 3.96 (s, 3H, CH30), 3.93 (s, 6 H, 2 x CH30). IR (neat) 1751 (COCI) cm'1. 234 NCO OCH3 3,4,5-Trimethoxyphenvl isocyanate, (285) : According to the method of Stogryn [279], a solution of benzoyl chloride 290 (10.0 g, 43 mmol) in anhydrous toluene (75 mL) was added dropwise to a slurry of sodium azide (8.45 g, 123 mmol) in anhydrous toluene (50 mL) over 30 min. The reaction was monitored by IR spectroscopy for the disappearance of the acyl halide absorption (1751 cm'1). After stirring at room temperature for 21.5 h, the mixture was filtered to remove the solids. The filtrate was heated to reflux. The reaction was monitored by evolution of N 2 through a bubbler and IR spectroscopy for the disappearance of the acyl azide absorption (2142 cm'1). Solvent was removed by distillation at atmospheric pressure. The product was purified by vacuum distillation (bp 95-99 °C / 0.25 mm Hg) and solidified on standing to yield 7.19 g (79%) of a white solid, mp 42-43 °C (lit. [279] 43-43.5 °C). 1H NMR (CDCI3/TMS) : 6 6.32 (s, 2H, ArH), 3.83 (s, 6H, 2 x CH30), 3.81 (s, 3H, CH3O). IR (neat) 2267 (NCO) cm'1. 1 235 N,-(2-(3.4-Bis(benzvloxv))phenvlethvl)-N-(3.4,5-trimethoxvphenvl)urea. (276) : 3,4-Dibenzyloxyphenethylamine hydrochloride (678 mg, 1.83 mmol) was converted to its free base with 10% aqueous sodium hydroxide (50 mL) and dichloromethane (50 mL). The organic layer was washed with V* strength brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in dry toluene (25 mL). Benzoyl azide 289 (500 mg, 2.1 mmol) was suspended in dry toluene (25 mL) and heated to reflux for 1.5 h. After cooling to room temperature, the amine solution was added. The resulting mixture was heated to reflux for 15 min and allowed to cool. The deposited crystals were collected by vacuum filtration to yield 805 mg (81%) of a white solid, mp 162-162.5 °C. ’H NMR (CDCI/TMS) : 6 7.44-7.28 (m, 10 H, ArH), 6.85 (d, J = 8.1 Hz, 1H, ArH), 6.80 (d, J = 2.0 Hz, 1H, ArH), 6.68 (dd, J = 8.1 and 2.0 Hz, 1H, ArH) 6.48 (s, 2H, ArH), 6.12 (s, 1H, ArNH), 5.11 (s, 4H, 2 x ArCH20), 4.63 (t, J= 5.8 Hz, 1H, NHCH2), 3.79 (s, 3H, CH 30), 3.76 (s, 3H, CH30), 3.47-3.39 (m, 2H, NHCH,), 2.23 (t, J = 6.7 Hz, 2H, ArCH2). FAB MS m/z 543.3 (m + 1, 85%), 542.3 (m, base). Analysis for Ca^^N/Dj. : calc. C, 70.83; H, 6.32; N, 5.16; found C, 71.19; H, 6.45; N, 5.13. 236 alternate procedure: 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (500 mg, 1.35 mmol) was converted to its free base with 5% aqueous sodium hydroxide (25 mL) and dichloromethane (25 mL). The aqueous layer was extracted with dichloromethane (25 mL), and the organics were combined. The combined extracts were washed with 5% aqueous sodium hydroxide (50 mL), Va strength brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in dry toluene (100 mL) and charged with a 50% dispersion of sodium hydride in mineral oil (65 mg, 1.35 mmol). The mixture was heated to reflux for 2 h. After cooling to room temperature, a solution of aryl urethane 279 (326 mg, 1.35 mmol) in dry toluene (100 mL) was added over 1 h via an addition funnel. Upon complete addition, the mixture was heated to reflux overnight. After cooling to room temperature, the solution was quenched with methanol (20 mL) and evaporated in vacuo. The residue was dissolved in dichloromethane (50 mL) and 5% aqueous sodium hydroxide (50 mL). The aqueous layer was extracted with dichloromethane (50 mL), and the extracts were combined. The organic extracts were washed successively with 5% aqueous sodium hydroxide (100 mL), V a strength brine (100 mL), 1.2 N hydrochloric acid (2 x 100 mL), Va strength brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was recrystallized from minimal hot toluene to yield 187 mg (26%) of a white solid, mp 159-161 °C. Spectroscopic data is in agreement with spectra from the formation 237 of urea 276 described above. A second product was identified by 1H NMR after column chromatography of the mother liquor as N,N’-bis(phenylethyl)urea 280. ’H NMR (CDCI/TMS) : 6 7.45-7.25 (m, 20H, ArH), 6.85 (d, J = 8.1 Hz, 2H, ArH), 6.75 (d, J = 2.0 Hz, 2H, ArH), 6.66 (dd, J = 8.1 and 2.0 Hz, 2H, ArH), 5.14 (s, 4H, ArCH20), 5.12 (s, 4H, ArCH20), 3.95 (bt, J = 5.8 Hz, 2H, NH), f 3.35-3.27 (m, 4H, CH 2N), 2.65 (t, J= 6.7 Hz, 4H, ArCH2). alternate procedure: 3,4,5-Trimethoxyaniline 279 (350 mg, 1.91 mmol) and a 50% dispersion of sodium hydride in mineral oil (92 mg, 1.91 mmol) were dissolved in dry toluene (40 mL) and heated to reflux for 2.5 h. The solution was charged with a solution of arylalkylurethane 281 (748 mg, 1.91 mmol) in hot, dry toluene, and the mixture was heated to reflux overnight. After cooling to room temperature, the mixture was quenched by the addition of methanol (10 mL). Solvent was evaporated in vacuo, and the residue was dissolved in dichloromethane (50 mL) and 1.2 N hydrochloric acid (50 ml). The aqueous layer was extracted with dichloromethane (50 ml), and the organic extracts were combined. The organic extracts were washed with 1.2 N hydrochloric acid (100 mL), 1A strength brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was recrystallized twice from a minimal amount of hot toluene to yield 205 mg ( 20%) of a white solid, mp 157-159 °C. Spectroscopic data is in agreement with spectra from the formation of urea 276 described above. 238 A second product was identified by 1H NMR after column chromatography of the mother liquor as N,N’-bis(aryl)urea 282. 1H NMR (CDCI/TMS) : 6 6.71 (bs, 2H, NH), 6.64 (s, 4H, ArH), 3.83 (bs, 18H, CH30). alternate procedure: 3,4-Dibenzyloxyphenethylamine hydrochloride (3.0 g, 8.1 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 mL) and dichloromethane (100 mL). The organic layer was washed with 1/4 strength brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in dry toluene (75 mL). Isocyanate 285 (1.8 g, 8.6 mmol) was dissolved in dry toluene (25 mL) and added to the amine solution. The resulting mixture was heated to reflux for 30 min. The condenser was removed and the mixture was concentrated to ca. 50 mL and allowed to cool. The deposited crystals were collected by vacuum filtration to yield 3.05 g (69%) of a white solid, mp 160-162 °C. Spectroscopic data is in agreement with spectra from the formation of urea 276 described above. 239 OCH. CH30 N’-(2-(314-Bis(benzvloxv))phenv!ethyl)-N-(31415-trimethoxvphenvl)thiourea1 (277): Thiophosgene (1.0 mL, 13.12 mmol) was suspended in an ice slurry (50 mL) in a 250 mL 2-neck flask fitted with an addition funnel and a thermometer. A solution of 3,4,5-trimethoxyaniline 279 (2.0 g, 10.92 mmol) in chloroform (50 mL) was added dropwise while maintaining an internal temperature of about 5 °C. Upon complete addition the mixture was allowed to stir for an additional 15 min. The layers were separated, and the chloroform layer was dried over anhydrous sodium sulfate. Chloroform and excess thiophosgene were removed by short path distillation. The crude, dark red isothiocyanate 284 (2.08 g, 85%) was used without further purification. 3,4-Dibenzyloxyphenethylamine hydrochloride was converted to its free base with dichloromethane (50 mL) and 5% aqueous sodium hydroxide (50 mL). The aqueous layer was extracted with dichloromethane (25 mL) and the organic extracts were combined. The extracts were washed with 5% aqueous sodium hydroxide (50 mL), 1/4 strength brine, dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in 95% ethanol (25 mL) and added dropwise to a solution of the crude isothiocyanate in 95% ethanol (25 mL) over 5 min. The mixture was stirred at room temperature for 30 min, and the deposited solid was collected by vacuum filtration. Recrystallization from 95% ethanol yielded 2.63 g (58%) of a white solid, mp 95-98 °C. 1H NMR (CDCI/TMS) : 6 7.45-7.29 (m, 11H, 10 x ArH and ArNH), 6.81-6.78 (m, 2H, ArH), 6.58 (dd, J = 8.1 and 2.1 Hz, 1H, ArH), 6.24 (s, 2H, ArH), 5.96 (bt, J= 4.7 Hz, 1H, NHCH2), 5.11 (s, 4H, 2 x ArCH20), 3.85-3.78 (m, 2H, NHCH,), 3.82 (s, 3H, CH30), 3.69 (s, 6H, 2 x CH30), 2.82 (t, J = 6.7 Hz, 2H, ArCH2). FAB MS m/z 559.3 (m + 1, base), 558.3 (m + 1 - H, 40%), 242.0 (m + 1 - C22H210 2, 48%). Analysis for C ^ H ^ ^ O ^ : calc. C, 68.80; H, 6.13; N, 5.01; found C, 68.53; H, 6.01; N, 5.02. BnO N HCI BnO HN OCH OCH 6.7-Bis(benzvloxv)-1-(3.4.5-trimethoxvphenvlamino)-3.4-dihvdroisoquinolme hydrochloride. (2831 : Urea 276 (500 mg, 0.92 mmol) was dissolved in phosphorous oxychloride (5 mL) and anhydrous acetonitrile (50 mL) under an argon atmosphere and heated to reflux for 4.5 h. After cooling to room temperature, solvent was removed in vacuo. The residue was dissolved in chloroform (50 mL), washed with 5% aqueous sodium hydroxide (50 mL), Vfc strength brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was subjected to silica gel chromatography with methanol/benzene/triethylamine (5/95/0.5) as the eluent. Appropriate fractions were pooled and evaporated in vacuo. Anhydrous hydrogen chloride (g) was bubbled into a solution of the residue in chloroform. The addition of ether resulted in precipitation of the product. The solid was collected by vacuum filtration to yield 179 mg (35%) of a white solid, mp 192-194 °C. This compound did provide acceptable elemental analysis. An analytical sample was recrystallized from saturated hydrogen chloride (g)/methanol to yield a white solid, mp 154-155 °C. 1H NMR (CDCI/TMS) : 6 7.96 (s, 1H,=N+HR), 7.51-7.27 (m, 11H, ArH), 6.74 (s, 1H, ArH), 6.22 (s, 2H, ArH), 5.21 (s, 2H, ArCH2Q), 5.20 (s, 2H, ArCH20), 5.02 (bs, 1H,ArNHR), 3.84 (s, 9H, 3 x CH30), 3.37 (t, J = 6.3 Hz, 2H, N+CH2), 2.85 (t, J = 6.3 Hz, 2H, ArCH2). FAB MS m/z 525.4 (m + 1 - HCI, base), 524.4 (m - HCI, 55%). Analysis for CggHggNgOgCI, V a H 2 0 : calc. C, 67.96; H, 5.97; N, 4.95; found C, 67.86; H, 5.87; N, 5.06. alternate procedure: In a manner similar to Roushdi [276], thiourea 277 (500 mg, 0.89 mmol) was dissolved in phosphorous oxychloride (5 mL) under an argon atmosphere and heated to reflux for 3 h. The solution was poured into a slurry of ice-water (150 mL) and extracted with chloroform (125 mL). The extract was washed successively with 1% strength brine (100 mL), 5% aqueous sodium hydroxide (100 mL), 1/4 strength brine (100 mL), dried over anhydrous magnesium sulfate, and concentrated in vacuo to 25 mL. Anhydrous hydrogen chloride (g) was bubbled into the solution for 30 min. The solution was diluted with diethyl ether until it became cloudy. After standing overnight, the precipitate was collected by vacuum filtration to yield 171 mg (34%) of a bright orange solid, mp 192-193.5 °C. Spectroscopic data is in agreement with spectra from the cylcization of urea 276 described above. HO •HCI HO HN OCH OCH 6.7-Dihvdroxv-1-(3.4.5-trimethoxvphenvlamino)“3,4-dihvdroisoquinolme hydrochloride, (250) : Amidine 283 (500 mg, 0.89 mmol) was dissolved in a mixture of methanol (20 mL) and concentrated hydrochloric acid (20 mL) under argon. The mixture was heated to reflux for 5 h, and cooled to room temperature. Solvent was evaporated in vacuo. The residue was rinsed with isopropanol (20 mL) and evaporated in vacuo. The resulting oil was recrystallized from methanol/diethyl ether to yield 300 mg (89%) of an off white solid, dp 169-171 °C (with darkening). 1H NMR (D20 ) : 6 7.28 (s, 1H, ArH), 6.76 243 (s, 1H, ArH), 6.63 (s, 2H, ArH), 3.70 (s, 6H, 2 x CH30), 3.67 (s, 3H, CH30), 3.33 (t, J = 6.7 Hz, 2H, N+CH2), 2.74 (t, J= 6.7 Hz, 2H, ArCH^. FAB MS m/z 345.1 (m + 1 - HCL, base), 344.1 (m - HCI, 24%). Analysis for C18H21N20 5CI,-H20 : calc. C, 54.21; H, 5.81; N, 7.02; found C, 54.01; H, 5.92; N, 6.96. N-(3.4-Bis(benzvloxv)phenvlethv0-4-nitrophenvlacetamide. (294): 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (10.0 g, 27.0 mmol) was converted to its free base with 5% aqueous sodium hydroxide (250 mL) and dichloromethane (250 mL). The layers were separated and the organic layer was washed with brine (250 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The light brown oil was dissolved in toluene (150 mL) and charged with 4-nitrophenylacetic acid 291 (7.2 g, 39.8 mmol). The suspension was heated to reflux under argon for 72 h with azeotropic removal of water by a Dean-Stark trap. After cooling to room temperature, solvent was removed in vacuo. The residue was dissolved in dichloromethane (250 mL), extracted with saturated sodium bicarbonate (2 x 125 mL), washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The product was recrystallized in two crops from a minimal amount of hot toluene to yield 10.06 g (75%) of a pale yellow solid, mp 132-133.5 °C. 1H NMR (CDCI3): 6 8.13 (AA’XX’, J= 6.8 and 2.0 Hz, ArH ortho to N02), 7.48-7.27 (m, 12H, ArH and m etato N 0 2), 6.80 (d, J= 8.1 Hz, 1H, ArH), 6.72 (d, J= 2.0 Hz, 1H, ArH), 6.53 (dd, J = 8.1 and 2.0 Hz, 1H, ArH), 5.32 (bt, 1H, NH), 5.14 (s, 2H, ArCH20), 5.12 (s, 2H, ArCH20), 3.51 (s, 2H, ArCH2CO), 3.44 (m, 2H, NHCH2), 2.66 (t, 2H, A rC H jQ -y. El MS m/z 496 (m+, 7.5%), 91 (C7H/, base). Analysis for C3oH28N20 5: calc. C, 72.56; H, 5.68; N, 5.64; found C, 72.17; H, 5.40; N, 5.51. BnO BnO NH HCI NO 6.7-Bis(benzvloxv)-1-(4-nitrobenzvn-1.2,3,4-tetrahvdroisoquinoline hydrochloride. (253) : To a suspension of amide 294 (5.0 g, 10.0 mmol) in anhydrous acetonitrile (20 mL) under argon at room temperature was added phosphorus oxychloride (5 mL) and the resulting solution was heated to reflux for 4.5 h. The solution was cooled to room temperature, and solvent was removed in vacuo. The oily residue was dissolved in chloroform (75 mL) and diluted with an ice slurry (40 mL). The biphasic solution was vigorously stirred while a 5% solution of sodium hydroxide was added dropwise until the aqueous layer was basic to pH paper. The layers were separated, and the organic layer was washed with 5% aqueous sodium hydroxide (50 mL), brine (30 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was suspended in ethanol (50 mL) and cooled to 0°C. Sodium borohydride (0.95 g, 25 mmol) was added portionwise with vigorous stirring over 10 min to prevent excessive foaming. The mixture was allowed to warm to room temperature with stirring overnight. Solvent was removed in vacuo, and the residue was partitioned between ethyl acetate (100 mL) and water (100 ml). The organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in methanol (30 mL) and stirred overnight with a solution of saturated hydrogen chloride(g)/methanol (10 mL). Solvent was removed in vacuo. The residue was dissolved in hot ethanol, filtered and concentrated by evaporation on a hot plate. After cooling, the deposited product was collected by vacuum filtration and washed with diethyl ether to yield 2.37 g (46%) of a light yellow solid, mp 213-214.5 °C. The mother liquor was evaporated and a second crop of crystals was produced from hot ethanol to yield an additional 1.36 g (total yield 72%) of a pale yellow solid, mp 210-213°C. 1H NMR (CD3OD) 6 8.23 (d, J= 8.7 Hz, 2H, ArH ortho to N 0 2), 7.52 (d, J = 8.7 Hz, 2H, ArH meta to N 0 2), 7.46-7.26 (m, 10H, ArH), 6.91 (s, 1H, ArH), 6.61 (s, 1H, ArH), 5.14 (s, 2H, ArCH20), 4.95 (s, 2H, ArCH20), 4.80 (t, J= 7.2 Hz, 1H, ArCHRN), 3.58-3.49 (m, 2H, 4-N 0 2ArCH2), 3.39-3.22 (m, 2H, NCH2), 3.06-2.98 (m, 2H, ArCh^CH,). FAB MS m/z481.4 (m + 1 - HCI, base), 246 344.2 (m - HCI - C7H6N0 2). Analysis for CgoHggNgO^HgO : calc. C, 67.35; H, 5.84; N, 5.24; found C, 67.31; H, 5.55; N, 5.18. HO NH -HCI HO NO 6.7-Dihvdroxv-1 -(4-nitro benzyl)-1,2.3.4-tetrahvdroisoquinoline hydrochloride, (262) : Amine 253 (500 mg, 0.97 mmol) was dissolved in a mixture of concentrated hydrochloric acid (4 mL) and methanol (4 mL). The solution was heated to reflux overnight under argon. After cooling to room temperature, solvent was evaporated in vacuo. The residue was rinsed with methanol (10 mL) and evaporated in vacuo twice. The resulting solid was recrystallized from ethanol/diethyl ether to yield 264 mg (81%) of a golden solid, mp 178-179 °C. 1H NMR (CD3OD) : 6 8.26 (d, 8.7 Hz, 2H, ArH ortho to N 0 2), 7.56 (d, J= 8.7 Hz, 2H, ArH meta to N02), 6.64 (s, 1H, ArH), 6.44 (s, 1H, ArH), 4.74 (t, J = 7.4 Hz, 1H, ArCHRN), 3.60-3.47 (m, 2H, N02ArCH2), 3.34-3.21 (m, 2H, CH2N), 3.02-2.94 (m, 2H, ArCH2). FAB MS m/z 301.2 (m + 1 - HCI, base). Analysis for C^H^N.O^I, 1/2H20 : calc. C, 55.58; H, 5.25; N, 8.10; found C, 55.79; H, 5.40; N, 7.75. 247 N-(3,4-Bis(benzvloxv)phenvlethv0-4-aminophenvlacetamide, (295): 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (2.0 g, 5.4 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 mL) and dichloromethane (100 mL). The layers were separated and the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The light brown oil was dissolved in toluene (75 mL) and charged with 4-aminophenylacetic acid 292 (1.0 g, 6.6 mmol). The suspension was heated to reflux under argon for 69 h with azeotropic removal of water by a Dean-Stark trap. After cooling to room temperature, solvent was removed in vacuo. The residue was dissolved in dichloromethane (125 mL) and saturated sodium bicarbonate (125 mL). The layers were separated, and the aqueous layer was extracted with dichloromethane (125 mL). The organic extracts were combined, washed with brine (250 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The product was recrystallized in three crops from a minimal amount of hot toluene to yield 1.223 g (49%) of an off white solid, mp 99-100 °C. 1H NMR (CDCI/TMS): 6 7.46-7.27 (m, 10H, ArH), 6.88 (AA’XX’, J = 6.4 and 2.0 Hz, 2H, ArH meta to NH2), 6.81 (d, J = 8.2 Hz, 1H, 248 ArH), 6.68 (d, J = 2.0 Hz, 1H, ArH), 6.55 (AA’XX1, J= 6.4 and 2.0 Hz, 2H, ArH ortho to NHg), 6.51 (dd, J = 8.2 and 2.0 Hz, 1H, ArH), 5.34 (bm, 1H, CONH), 5.14 (s, 2H, ArCH20), 5.10 (s, 2H, ArCH20), 3.57 (bs, 2H, NH2), 3.41-3.33 (m, 2H, NCH2), 3.39 (s, 2H, ArCH2CO), 2.61 (t, J= 6.8 Hz, 2H, CH2). El MS m/z 466 (m+, 4.8%), 91 (C7H7+, base). Analysis for C30H30N2O3 : calc. C, 77.23; H, 6.48; N, 6.00; found C, 76.94; H, 6.46; N, 5.82. BnO NH -2HCI BnO NH 1-(4-aminobenzvn-6.7-Bis(benzvloxv)-1.2.3,4-tetrahvdroisoquinoline dihvdrochloride. (2541: p-Nitrobenzyl derivative 253 (500 mg, 0.97 mmol) was dissolved in methanol (20 mL) in a Parr bottle. The solution was charged with a slurry of Raney Nickel (0.25 mL) and hydrogenated at 50 psi for 2 h. The mixture was double filtered and evaporated in vacuo. The oily residue was dissolved in methanol (5 mL) and charged with a solution of 3 N hydrochloric acid/methanol (1 mL). After stirring for 4 h, solvent was evaporated in vacuo. The solid residue was dissolved in minimal hot methanol and diluted with diethyl ether until the mixture became cloudy. The solid was collected by vacuum filtration to yield 408 mg (81%) of an off white solid, mp 211-213 °C. 1H NMR 249 (CD3OD) : 6 7.48-7.25 (m, 14H, ArH), 6.91 (s, 1H, ArH), 6.65 (s, 1H, ArH), 5.13 (s, 2H, ArCH20), 4.97 (s, 2H, ArCH20), 4.75 (t, J= 7.5 Hz, 1H, ArCHRN), 3.54- 3.16 (m, 4H, 2 x CH2), 3.06-2.98 (m, 2H, CH2). FAB MS m/z 451.4 (m + 1 - 2HCI, base), 344.2 (m - 2HCI - C ^ W , 68%). Analysis for C30H32N2O2CI2^ VaH20 : calc. C, 68.24; H, 6.20; N, 5.31; found C, 68.10; H, 6.17; N, 5.05. alternate procedure: Amide 295 (1.0 g, 2.14 mmol) was suspended in anhydrous acetonitrile (15 mL) and charged with phosphorous oxychloride (4 mL). The resulting solution was heated to reflux for 3.5 h under an argon atmosphere. After cooling to room temperature, solvent was evaporated in vacuo. The resulting oil was rinsed with anhydrous acetonitrile (15 mL), and evaporated in vacuo. The residue was dissolved in ethanol (25 mL) and cooled to 0 °C. Sodium borohydride (810 mg, 21.4 mmol) was added portionwise to the solution to avoid excessive foaming. The mixture was stirred overnight while warming to room temperature. Solvent was evaporated in vacuo, and the residue was dissolved in a mixture of 5% aqueous sodium hydroxide (50 mL) and dichloromethane (50 mL). The organic layer was washed with Vz strength brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The mixture was subjected to silica gel chromatography with methanol/ethyl acetate/triethylamine (8/91/1) as the eluent. Appropriate fractions were pooled and evaporated in vacuo. The residue was dissolved in ethanol (8 mL) and 250 charged with a solution of 3 N hydrochloric acid/ethanol (2 mL). The solution was diluted with ether to induce crystallization. The solid was collected by vacuum filtration to yield 244 mg, (22%) of a pale yellow solid, mp 205-209 °C. 1H NMR is in agreement with the spectra from the procedure on page 248. HO NH -2 HCI HO NH 1-(4-ammobenzvl)-6,7-dihvdroxv-1.2.3,4-tetrahvdroisoquinoline dihvdrochloride. (263): Amine 254 (200 mg, 0.38 mmol) was dissolved in a mixture of concentrated hydrochloric acid (4 mL) and methanol (4 mL).. The solution was heated to reflux overnight under argon. After cooling to room temperature, solvent was evaporated in vacuo. The residue was rinsed with methanol (10 mL) and evaporated in vacuo. The methanol rinse was repeated, and the resulting solid was recrystallized from ethanol/diethyl ether. The deposited solid was collected by vacuum filtration, rinsed with diethyl ether, and dried under vacuum for 36 h at 56 °C to yield 88 mg (66%) of a tan solid, mp 260-262 °C. 1H NMR (CD3OD) : 6 7.52-7.40 (AA’BB’, 4H, ArH ortho and meta to NHg) 6.63 (s, 1H, ArH), 6.47 (s, 1H, ArH), 4.70 (t, J= 7.3 Hz, 1H, ArCHRN), 3.53-3.43 (m, 2H, CH2), 3.33-3.28 (m, 1H, CH2), 3.26-3.17 (m, 1H, CH2), 251 3.02-2.93 (m, 2H, CH^. FAB MS m/z271.1 (m + 1 - 2HCI, base). Analysis for C16H20N2O2CI21/2H2O : calc. C, 54.56; H, 6.01; N, 7.95; found C, 54.18; H, 6.15; N. 7.70. c h 2c o o h OH 4-Hvdroxv-3-nitrophenvlacetic acid, (293) : In a manner similar to the procedure of Hiigel [304], a suspension of 4-hydroxyphenylacetic acid 297 (5.0 g, 33 mmol) in glacial acetic acid (12.5 mL) was cooled to about 10 °C with an ice bath. A solution of 70% nitric acid (2.5 mL) in glacial acetic acid (4.0 mL) was added dropwise over 15 min while maintaining the temperature at 10-20 °C. Upon complete addition, the suspension was stirred in an ice bath for 1 h and then at room temperature for 2 h. The mixture was poured into 1.2 N hydrochloric acid/ice slurry (50 mL) and placed in the refrigerator. The product was collected by vacuum filtration, washed with water and air dried to yield 5.34 g (82%) of a bright yellow solid, mp 144-145 °C (lit. [304] 145-146 °C). 1H NMR (CDCI/TMS/DMSO) ; 11.14 (bs, 1H, COOH), 10.57 (s, 1H, ArOH), 7.80 (s, 1H, ArH), 7.39 (d, J= 8.6 Hz, 1H, ArH), 7.03 (d, J= 8.6 Hz, 1H, ArH), 3.52 (s, 2H, CH2). 252 BnO BnO 500 a : N-(3.4-Bis(benzvloxv)phenvlethvl)-4-hvdroxv-3-nitrophenvlacetamide, (296): 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (10.0 g, 27.0 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 ml_) and dichloromethane (100 mL). The layers were separated and the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The light brown oil was dissolved in toluene (125 mL) and charged with 4-hydroxy-3-nitrophenylacetic acid 293 (5.86 g, 29.7 mmol). The suspension was heated to reflux under argon for 72 h with azeotropic removal of water by a Dean-Stark trap. After cooling to room temperature, solvent was removed in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed with a solution of ammonium bicarbonate (5 g/100 mL), saturated ammonium chloride (100 mL) brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The product was recrystallized in two crops from a minimal amount of hot toluene to yield 9.3 g (67%) of a bright yellow solid, mp 135-137 °C. 1H NMR (CDCI3): 10.51 (bs, 1H, OH), 7.91 (d, J = 2.2 Hz, 1H, ArH ortho to N 0 2), 7.48-7.29 (m, 11H, 10 x ArH and ArH para to NO^, 7.08 (d, J= 8.7 Hz, 1H, ArH meta to N 0 2), 6.82 (d, J= 8.2 Hz, 1H, ArH), 253 6.71 (d, J= 2.0 Hz, 1H, ArH), 6.58 (dd, J= 8.2 and 2.0 Hz, 1H, ArH), 5.33 (bt, 1H, NH), 5.13 (s, 2H, ArCH20), 5.12 (s, 2H, ArCH20), 3.47-3.39 (m, 2H, NCH2), 3.39 (s, 2H, ArCH2CO), 2.67 (t, J = 6.7 Hz, 2H, ArCH2). FAB MS m /z513.4 (m + 1 , base), 512.4 (m, 78%), 316.2 (C22H20O2, 31%), 181.1 (ChH7N,04i 39%). Analysis for C-joH^Np,, : calc. C, 70.30; H, 5.51; N, 5.47; found C, 70.29; H, 5.50; N, 5.41. BnO NH -HCI BnO NO OH 6.7-Bis(benzvloxv)-1-(4-hvdroxv-3-nitrobenzvh-1.2.3.4- tetrahvdroisoquinoline hydrochloride. (257): Amide 296 (3.3 g, 6.4 mmol), was suspended in anhydrous acetonitrile (30 mL) under an argon atmosphere. The suspension was charged with phosphorous oxychloride (15 mL) and heated to reflux for 3.5 h. After cooling to room temperature, solvent was removed in vacuo. The residue was rinsed with anhydrous acetonitrile (30 mL) and evaporated in vacuo. The dark green oil was suspended in ethanol (40 mL) and cooled to 0 °C. Sodium borohydride (1.21 g, 32 mmol) was added portionwise to avoid excessive foaming. The suspension was stirred overnight warming to room temperature. Solvent was evaporated in vacuo, and the residue was dissolved in a mixture of ethyl acetate (100 mL) and 5% ammonium hydroxide (100 mL). The layers were separated, and the organic layer was washed with brine (100 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The residue was dissolved in methanol (30 mL) and charged with 3 N hydrochloric acid/methanol. After stirring at room temperature overnight, the solid was collected by vacuum filtration to yield 2.21 g (64%) of an off white solid, mp 204-207 °C. 1H NMR (CD3OD/acetone) : 8.03 (d, J = 2.0 Hz, 1H, ArH ortho to N 0 2), 7.57 (dd, J = 8.6 and 2.0 Hz, 1H, ArH para to NCy, 7.44-7.24 (m, 10H, ArH), 7.14 (d, J= 8.6 Hz, ArH meta to N 0 2), 6.93 (s, 1H, ArH), 6.79 (s, 1H, ArH), 5.11 (s, 2H, ArCH20), 4.97 (s, 2H, ArCH20), 4.96 (t, J= 6.0 Hz, 1H, ArCHRN), 3.62-3.34 (m, 4H, CH2), 3.08-3.03 (m, 2H, CH2). El MS m/z 496.3205 (m+ - HCI calc. 496.1998, 1.3%), 344 (m+ - HCI - CyHgN^g, 62%), 91 (C7H/, base). Analysis for CgoH^N^sCI^/aH^ : calc. C, 66.48; H, 5.58; N, 5.17; found C, 66.43; H, 5.44; N, 5.05. 255 HO NH HCI HO NO OH 6.7-dihvdroxv-1-(4-hvdroxv-3-nitrobenzvl)-1.2,3,4-tetrahvdroisoquinoiine hydrochloride, (266) : Isoquinoline 257 (200 mg, 0.37 mmol) was dissolved in a mixture of concentrated hydrochloric acid (4 mL) and methanol (4 mL) under an argon atmosphere. The mixture was heated to reflux overnight and cooled to room temperature. Solvent was evaporated in vacuo. The residue was rinsed in methanol (10 mL) and evaporated in vacuo. The methanol rinse was repeated, and the resulting solid was dissolved in excess ethanol, filtered, and concentrated by evaporation on a hot plate to about 5 mL. Diethyl ether was added until the mixture became cloudy. The deposited solid was collected by vacuum filtration, rinsed with diethyl ether, and dried under vacuum at 56 °C for 36 h to yield 106 mg (82%) of a rust colored solid, dp 253-255 °C (with bubbling). 1H NMR (CD3OD) : 8.05 (d, J= 2.1 Hz, 1H, ArH ortho to NCy, 7.56 (dd, J = 8.6 and 2.1 Hz, 1H, ArH para to N 0 2), 7.18 (d, J = 8.6 Hz, 1H, ArH meta to N02), 6.63 (s, 1H, ArH), 6.54 (s, 1H, ArH), 4.66 (t, J = 7.2 Hz, 1H, ArCHRN), 3.51-3.41 (m, 2H, CH2), 3.31-3.23 (m, 1H, CH2), 3.14-3.05 (m, 1H, CH2), 3.01-2.92 (m, 2H, CH2). FAB MS m/z 317.1 (m + 1 - HCI, base). 256 Analysis for C ^H ^N ^C I, : calc. C, 54.48; H, 4.86; N, 7.94; found C, 54.19; H, 4.97; N, 7.69. 4-Amino-3,5-diiodophenvlacetic acid, (298) : To a suspension of 4-aminophenylacetic acid 292 (5.0 g, 33 mmol) in 1.2 N hydrochloric acid (250 mL) was added a solution of iodine monochloride (11.5 g, 71 mmol) in 1.2 N hydrochloric acid (50 mL) over a 10 min period. Upon complete addition, the solution was heated to 70 °C for 2 h. After cooling to room temperature, the flask was placed in the refrigerator overnight. The precipitate was collected by vacuum filtration, washed with water, and air dried for 3 d to yield 12.3 g (92%) of an off white solid, dp 222-224 °C (with darkening). An analytical sample was prepared by dissolving the product in dilute sodium hydroxide, filtering the solution, and acidifying the filtrate with dilute with 1.2 N hydrochloric acid to precipitate the product. The solid was collected as before to yield a white solid, dp 215°C (purple vapor while decomposing). 1H NMR (CDCI/TMS/DMSO) : 6 12.18 (bs, 1H, COOH), 7.53 (s, 2H, ArH), 4.91 (bs, 2H, NH2), 3.37 (s, 2H, CH2). El MS 402 (m+, 99%), 357 (m+ - C02H, base). Analysis for C8H7N102l2 : calc. C, 23.85; H, 1.75; N, 3.48; found C, 23.56; H, 1.85; N 3.33. 257 NO. OH 4-Hvdroxv-3-iodo-5-nitrophenvlacetic acid. (299) : To a solution of 4-hydroxy- 3-nitrophenylacetic acid 293 (1.5 g, 7.6 mmol) in dimethylformamide (5 mL) was added a solution of iodine monochloride (1.36 g, 8.4 mmol) in dimethylformamide (5 mL). The reaction mixture was stirred at room temperature for 2 h before being poured into 1.2 N hydrochloric acid (40 mL) and placed in the refrigerator overnight. The product was collected by vacuum filtration, washed with water, and air dried for 3 d to yield 2.13 g (87%) of a bright yellow solid. An analytical sample was prepared by dissolving the product in dilute sodium hydroxide, filtering the solution, and precipitating the product by the addition of dilute acid. The compound was collected by vacuum filtration, washed liberally with water, and air dried for 3 d to yield a golden solid, dp 207-209 °C (with darkening, lit. [293] dp 213 °C). 1H NMR (CDCI/TMS/DMSO) : 6 12.42 (bs, 1H, COOH), 11.01 (bs, 1H, ArOH), 8.04 (d, J= 1.4 Hz, 1H, ArH), 7.97 (d, J= 1.4 Hz, 1H, ArH), 3.60 (s, 2H, CH2). 258 i N-(2-(3,4-Bis(benzvloxv)phenvlethvl)-(4-amino-3,5-diiodophenyl)acetamide, (3 0 0 ): 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (1.0 g, 2.7 mmol) was converted to its free base with 5% aqueous sodium hydroxide (50 mL) and dichloromethane (50 mL). The layers were separated, and the organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in toluene (50 mL) and charged with 4-amino-3,5-diiodophenylacetic acid 298 (1.4 g, 3.5 mmol). The mixture was heated to reflux for 96 h with azeotropic removal of water via a Dean-Stark trap. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in a mixture of methanol (40 mL) and chloroform (160 mL) and washed with 5% aqueous sodium hydroxide (2 x 200 mL), brine (200 mL), dried with anhydrous magnesium sulfate, and evaporated in vacuo to a solid. This solid was recrystallized from a large volume of toluene to yield 1.7 g (88%) of a white solid, mp 187-188 °C. 1H NMR (CDCI/TMS) : 6 7.47-7.29 (m, 12H, 10 x ArH and 2 x ArH ortho to I), 6.85 (d, J= 8.1 Hz, 1H, ArH), 6.71 (d, J= 2.0 Hz, 1H, ArH), 6.54 (dd, 8.1 and 2.0 Hz, 1H, ArH), 5.30 (bt, 1H, NHCO), 5.15 (s, 2H, ArCH20), 5.13 (s, 2H, 259 ArCH20), 4.59 (bs, 2H, ArNH2), 3.43-3.36 (m, 2H, CH2N), 3.27 (s, 2H, ArCH2CO), 2.65 (t, J= 6.7 Hz, 2H, A rO -y. FAB MS m/z719.2 (m + 1, 52%), 176.1 (C10H10N1O2, base). Analysis for C30H28N2O3l2 : calc. C, 50.16; H, 3.93; N, 3.90; found C, 50.49; H, 3.80; N, 3.81. BnO BnO NO- OH N-(2-(3.4-Bis(benzvloxv)phenvlethvlM4-hvdroxv-3-iodo-5- nitrophenvhacetamide. (301) : 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (3.12 g, 8.4 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 mL) and dichloromethane (100 mL). The layers were separated, and the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in toluene (100 mL) and charged with 4-hydroxy-3-iodo-5-nitrophenylacetic acid 299 (3.0 g, 9.3 mmol). The mixture was heated to reflux for 72 h with azeotropic removal of water via a Dean-Stark trap. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (100 mL) and washed with 5% aqueous sodium hydroxide (100 mL), brine (100 mL), dried with anhydrous sodium sulfate, and evaporated in vacuo to a solid. This solid was recrystallized from toluene to yield 2.81 g (52%) of a yellow solid, mp 145-147 °C. 1H NMR (CDCI/TMS) : 6 11.22 (bs, 1H, OH), 7.94 (d, J= 1.7 Hz, 2H, ArH ortho and para to N 0 2), 7.47-7.29 (m, 10H, ArH), 6.85 (d, J= 8.0 Hz, 1H, ArH), 6.72 (d, J 1.8 = Hz, 1H, ArH), 6.60 (dd, J= 8.0 and 1.8 Hz, 1H, ArH), 5.40 (bt, 1H, NH), 5.14 (s, 2H, ArCH20), 5.13 (s, 2H, ArCH20), 3.48-3.40 (m, 2H, CH2N), 3.33 (s, 2H, ArCH2CO), 2.68 (t, J = 6.7 Hz, 2H, ArCH2). FAB MS m/z 639.2 (m + 1, 95%), 638.2 (m, base). Analysis for O^H^NgC^ : calc. C, 56.44; H, 4.26; N, 4.39; found C, 56.55; H, 3.97; N, 4.62. c h 2c o o h o c h 3 4-Methoxv-3-nitrophen vlacetic acid. (303) To a solution of 4-methoxyphenylacetic acid 302 (10.0 g, 60 mmol) in glacial acetic acid (20 mL) was added a solution of fuming nitric acid (20 mL) in glacial acetic acid (30 mL) dropwise over 45 min while cooling the reaction mixture as needed with an ice bath. Upon complete addition, the mixture was stirred with the ice bath for an additional hour, then at room temperature for 2 h. The reaction mixture was poured into a solution of 1.2 N hydrochloric acid (125 mL) and placed in the refrigerator overnight. The crystals were collected, washed with water, and air dried for 3 d to yield 6.23 g (49%) of a light brown solid, mp 122-123.5 °C. An analytical sample was prepared by dissolving the product in dilute sodium hydroxide, filtering the solution, and precipitating the product by the addition of dilute acid. The compound was collected by vacuum filtration, washed liberally with water, and air dried for 3 d to yield a white solid, mp 125.5-126.5 °C (lit. [305] mp 132-133 °C). 1H NMR (CDCI/TMS/DMSO) : 6 12.34 bs, 1H, COOH), 7.76 (d, J = 2.0 Hz, 1H, ArH ortho to N 0 2), 7.52 (dd, J = 8.7 and 2.0 Hz, 1H, ArH para to N 0 2), 7.22 (d, J - 8.7 Hz, 1H, ArH meta to N 0 2), 3.93 (s, 3H, CH30), 3.61 (s, 2H, CH2). CHpCOOH NO OCH3 3-lodo-4-methoxv-5-nitrophenvlacetic acid, (304) : To a suspension of anhydrous potassium carbonate (2.0 g) in acetone (20 mL) was added 4-hydroxy-3-iodo-5-nitrophenylacetic acid 299 (600 mg, 1.86 mmol) and dimethyl sulfate (2.0 mL). The mixture was heated to reflux overnight, and cooled to room temperature. Solvent was evaporated in vacuo, and the residue was triturated in a solution of saturated sodium bicarbonate. The mixture was filtered, acidified with 1.2 N hydrochloric acid, and placed in the refrigerator. The precipitate was collected by vacuum filtration to yield 319 mg (51%) of a light brown solid. An analytical sample was prepared by dissolving the product 262 in dilute sodium hydroxide, filtering the solution, and precipitating the product by the addition of dilute acid. The compound was collected by vacuum filtration, washed liberally with water, and air dried for 3 d to yield a white solid, mp 130.5-132 °C. 1H NMR(CDCl3/TMS/DMSO) : 6 12.31 (bs, 1H, COOH), 8.04 (d, J = 1.9 Hz, 1H, ArH), 7.82 (d, J= 1.9 Hz, 1H, ArH), 3.89 (s, 3H, CH30), 3.65 (s, 2H, CH2). El MS m/z 336.9447 (m+ calc. 336.9447, base). Analysis for CgHgN^Ogli : calc. C, 32.07; H, 2.39; N, 4.16; found C, 32.08; H, 2.34; N, 4.24. N-(2-(3.4-Bis(benzvloxv)phenvlethvl)-(4-miethoxv-3-nitrophenvl)acetamide, (305) : Amide 296 (2.0 g, 3.9 mmol) was suspended in acetone (50 mL) and charged with anhydrous potassium carbonate (550 mg, 3.9 mmol) and iodomethane (1.5 ml, 23.4 mmol). The mixture was heated to reflux for 6 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate (100 mL) and 5% aqueous sodium hydroxide (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL), and the organic extracts were combined. The organic layer was washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting solid 263 was recrystallized from a minimal amount of hot toluene to yield 1.98 g, (96%) of a light brown solid, mp 124-125 °C. 1H NMR (CDCI/TMS) 6 7.66 (d, J = 2.2 Hz, 1H, ArH ortho to N 0 2), 7.47-7.27 (m, 11H, 10 x ArH and ArH para to N 0 2), 6.97 (d, J= 8.7 Hz, 1H, ArH meta to NOz), 6.83 (d, J= 8.1 Hz, 1H, ArH) 6.71 (d, 2.0 Hz, 1H, ArH), 6.57 (dd, J= 8.1 and 2.0 Hz, 1H, ArH), 5.37 (bt, 1H, NH), 5.12 (s, 2H, ArCH20), 5.10 (s, 2H, ArCH20), 3.88 (s, 3H, CH30), 3.46-3.39 (m, 2H, CH2N), 3.39 (s, 2H, ArCH2CO), 2.66 (t, J = 6.7 Hz, ArCH2). FAB MS m/z565.2 (m + K, 65%), 527.3 (m + 1, base), 526.3 (m, 93%), 316.1 (C22H20O2, 39%). Analysis for C31H30N2O6 : calc. C, 70.71; H, 5.74; N, 5.32; found C, 70.73; H, 5.65; N, 5.21. i N-(2-(3.4-Bis(benzvloxv)phenvlethv0-(3-iodo-4-methoxv-5- nitrophenvOacetamide, (3061 : Amide 301 (150 mg, 0.24 mmol) was suspended in acetone (50 mL) and charged with anhydrous potassium carbonate (650 mg, 0.47 mmol) and dimethylsulfate (0.05 ml, 0.47 mmol). The mixture was heated to reflux overnight. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (20 mL) and water (20 mL). The organic layer was washed with brine (10 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting solid was recrystallized twice from a minimal amount of hot toluene to yield 105 mg, (69%) of a light brown solid, mp 148-149 °C. 1H NMR (CDCI/TMS) 6 7.88 (d, J = 2.1 Hz, 1H, ArH ortho to N 0 2), 7.63 (d, J = 2.1 Hz, 1H, ArH ortho to I), 7.47-7.29 (m, 10H, ArH), 6.86 (d, J = 8.2 Hz, 1H, ArH) 6.74 (d, J= 1.9 Hz, 1H, ArH), 6.60 (dd, J= 8.2 and 1.9 Hz, 1H, ArH), 5.44 (bt, 1H, NH), 5.14 (s, 4H, ArCH20), 3.95 (s, 3H, CH30), 3.47-3.39 (m, 2H, CH2N), 3.35 (s, 2H, ArCH2CO), 2.68 (t, J = 6.8 Hz, ArCH2). FAB MS 653.1 (m +1, 93%), 652.1 (m, base). Analysis for : calc. C, 57.07; H, 4.48; N, 4.29; found C, 56.77; H, 4.34; N, 4.40. BnO BnO OCH NO- 6.7-Bis(benzvloxv)-2-carbomethoxv-1 -(4-nitrobenzvl)-1,2,3,4- tetrahvdroisoquinoline, (307) ; To a suspension of isoquinoline 253 (3.5 g, 6.8 mmol) in acetone (50 mL) was added potassium carbonate (5.61 g, 40.6 mmol), Adogen 464 (125 mg), and methyl chloroformate (2.1 mL, 27.1 mmol). The mixture was stirred at room temperature overnight and evaporated in vacuo. The residue was dissolved in water (250 mL) and dichloromethane (250 mL). The layers were separated, the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and solvent was removed in vacuo. The resulting oil was crystallized from methanol to yield 3.125 g (86%) of bright yellow needles, mp 133-135 °C. 1H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. A temperature elevation study reveals an increasing interconversion between the two conformations with increasing temperature and a progression toward a single spectrum. A complete collapse to a single spectrum was not possible due to solvent limitations. 1H NMR (CDCI3): 6 8.10-8.03 (m, 2H, ArH ortho to NCy, 7.46-7.31 (m, 10H, ArH), 7.16-7.09 (m, 2H, ArH meta to N 02), 6.69 and 6.66 (s, 1H, ArH), 5.27-5.10 (m, 1H, ArCHRN), 5.13 (s, 2H, ArCH20), 5.07-5.00 (m, 2H, ArCH20), 4.12-4.08 and 3.77-3.72 (m, 1H, CH2N), 3.67 and 3.46 (s, 3H, CH30), 3.25-3.15 (m, 1H, CH2N), 3.11-3.04 (m, 2H, N 0 2ArCH2), 2.78-2.61 and 2.53-2.37 (m, 2H, ArCH2). FAB MS m/z 539.4 (m + 1, 4.7%), 402.3 (m - CyH6N-|02, base). Analysis for C32H30N2O6: calc. C, 71.36; H, 5.61; N, 5.20; found C, 71.32; H, 5.61; N, 5.17. 266 BnO BnO OCH NH 1 -(4-Aminobenzvl)"6,7-bis(benzvloxv)-2-carbomethoxv-1,2,3,4- tetrahydroisoquinoline, (308) : In a Parr bottle, compound 307 (2.0 g, 3.7 mmol) was dissolved in dimethylformamide (30 mL) and diluted with methanol (30 mL). Raney nickel (2 ml slurry, washed 3 x 4 mL methanol) was added to the solution. The bottle was evacuated with a water aspirator, filled with hydrogen (60 psi) and shaken on a Parr hydrogenator for 24 h at room temperature. The resulting suspension was gravity filtered to remove catalyst and concentrated in vacuo. The oil was dissolved in ethyl acetate (50 mL) and washed with water (50 mL), brine (50 mL), dried over anhydrous sodium sulfate and concentrated in vacuo. The resulting solid was triturated in methanol to yield 1.56 g (83%) of a tan solid, mp 85-88 °C. ’H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. 1H NMR (CDCI3/TMS) : 6 7.45-7.27 (m, 10H, ArH), 6.81-6.77 (m, 2H, ArH meta to NH2), 6.67 and 6.63 (s, 1H, ArH), 6.60-6.54 (m, 2H, ArH ortho to NH2), 6.44 and 6.33 (s, 1H, ArH), 5.13-4.95 (m 1H, ArCHRN), 5.11 (s, 2H, ArCH20), 4.99 and 4.92 (s, 2H, ArCH20), 4.14-4.08 and 3.82-3.75 (m, 1H, CH2N), 3.69 and 3.52 (s, 3H, CH30), 3.28-3.13 (m, 1H, CH2N), 2.97-2.59 (m, 3H, 2 x N H ^rC H , and 1 x ArCH2), 267 2.55-2.40 (m, 1H, ArCH2). FAB MS m/z 509.4 (m + 1, 3.1%), 402.2 (m + 1 - C7H8N„ base). Analysis for C32H32N20 43/4H20 : calc. C, 73.61; H, 6.47; N, 5.37; found C, 73.80; H, 6.22; N, 5.29. BnO BnO OCH HN CH 1-(4-Acetamldobenzvn-6.7-Bis(benzvloxv)-2-carbomethoxv-1.2.3.4- tetrahvdroisoaumoline. (309) : Amino urethane 308 (500 mg, 0.98 mmol) was dissolved in acetic anhydride (8 mL) and heated to reflux for 8 h. After cooling to room temperature, the solution was poured into an ice slurry (50 mL) and extracted with ethyl acetate (2 x 50 mL). The organic extracts were combined and washed with water (2 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was triturated in methanol (5 mL) to produce a solid. The product was collected by vacuum filtration to yield 440 mg (81%) of a pale yellow solid, mp 144-46 °C. 1H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. 1H NMR (CDCI/TMS) : 6 7.45-7.23 (m, 13H, NHAc and 10 x ArH and 2 x ArH meta to NHAc), 6.99-6.93 (m, 2H, ArH ortho to NHAc), 6.68 and 6.64 (s, 1H, ArH), 6.46 and 6.36 (1H, ArH), 5.21-5.01 (1H, ArCHRN), 5.06 (s, 2H, ArCH20), 4.99 and 4.92 (s, 2H, ArCHzO), 4.12-4.08 and 3.82-3.72 (m, 1H, CH2N), 3.68 268 and 3.49 (s, 3H, CH30), 3.25-3.15 (1H, CH2N), 3.10-2.92 (m, 2H, AcNHArCH2), 2.90-2.43 (m, 2Hr ArCH2), 2.13 (s. 3H, CH3COR). FAB MS m/z 551.4 (m + 1, 6.6%), 402.2 (m + 1 - CgH^IN^O,, base). Analysis for C^H^NgOg 1/4H20 : calc. C, 73.56; H, 6.26; N, 5.05; found C, 73.61; H, 6.28; N, 4.80. BnO BnO OCH NH. 1-(4-Amino-3-iodobenzvD-6.7-bis(benzvloxv)-2-carbomethoxv-1.2.3.4- tetrahvdroisoquinoline, (310) : In a manner similar to the methodology of Kajigaeshi [298], amino urethane 308 (180 mg, 0.35 mmol) was dissolved in a mixture of methanol (2 mL) and dichloromethane (5 mL). The solution was charged with calcium carbonate (500 mg) and benzyltrimethylammonium dichloroiodate (BTMA ICI2, 270 mg, 0.77 mmol) and stirred for 24 h at room temperature. The suspension was gravity filtered, and the filtrate was diluted with dichloromethane (15 mL) and water (20 mL). The layers were separated, and the organic layer was washed with 5% aqueous sodium thiosulfate (20 mL), brine (20 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was subjected to silica gel chromatography with ethyl acetate/hexanes/dichloromethane (10/40/50) as the eluent. The appropriate fractions of the second compound to elute were pooled to yield 87 mg, (39%) 269 of a brown oil. 1H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. 1H NMR (CDCI/TMS) : 6 7.46-7.27 (m, 11H, 10 x ArH and ArH ortho to I), 6.82-6.76 (m, 1H, ArH para to I), 6.88-6.61 (m, 2H, ArH and ArH metato I), 6.42 and 6.28 (s, 1H, ArH), 5.12 (s, 2H, ArCH20), 5.08-4.85 (m, 3H, ArCH20 and ArCHRN), 4.12-4.06 and 3.79-3.75 (m, 1H, CH2N), 3.71 and 3.55 (s, 3H, CH3OCO), 3.35-3.15 (m, 1H, CH2N), 2.97-2.63 (m, 3H, ArCH2), 2.56-2.43 (m, 1H, ArCH2). FAB MS m/z 635.2 (m + 1, 1.7%), 634.2 (m, 2.8%), 633.1 (m - 1, 5.2%), 402.2 (m - C ^ N ^ , base). Elemental analysis was not submitted. BnO BnO OCH NH 1-(4-Amino-3,5-diiodobenzvl)-6.7-bis(benzvloxv)-2-carbomethoxv-1,2,3,4- tetrahvdroisoquinoline, (311) : From the above procedure, the appropriate fractions of the first compound to elute from the column were pooled to yield 55 mg, (20%) of a brown foam. 1H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. 1H NMR (CDCI/TMS) : 6 7.47-7.27 (m, 12H, 10 x ArH and 2 x ArH ortho to I), 6.69 and 6.67 (s, 1H, ArH), 6.39 and 6.24 (s, 1H, ArH), 5.13 (s, 2H, ArCH20), 5.06-4.97 (m, 2H, ArCH20), 4.94-4.84 (m, 1H, ArCHRN), 4.07-4.03 and 3.79-3.66 (m, 1H, CH2N), 3.72 and 3.60 (s, 3H, CH3OCO), 3.38-3.15 (m, 1H, CH2N), 2.92-2.65 (m, 3H, ArCH2), 2.56-2.45 (m, 1H, ArCH2). FAB MS m /z 761.1 (m + 1, 2.5%), 760.2 (m, 1.6%), 759.2 (m - 1, 2.9%), 402.2 (m - C^HgN^, base). An acceptable elemental analysis was not obtained. BnO BnO OCH NO. OH 6,7-Bis(benzvioxv)-2-carbomethoxv-1-(4-hvdroxv-3-nitrobenzvh-1,2,3.4- tetrahvdroisoquinoline, (312) : To a suspension of isoquinoline 257 (1.0 g, 1.9 mmol) in dichloromethane (10 mL) was added triethylamine (6 mL) and methyl chloroformate (4 mL). The mixture was stirred at room temperature overnight and evaporated in vacuo. The residue was dissolved in water (50 mL) and dichloromethane (50 mL). The organic layer was washed with 1.2 N hydrochloric acid (2 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and solvent was removed in vacuo. The resulting oil was dissolved in a mixture of 20% aqueous potassium hydroxide (2 mL) and methanol (10 mL). After stirring overnight at room temperature, the mixture was cooled in an ice bath and acidified with concentrated hydrochloric acid until acidic to pH paper. The mixture was concentrated in vacuo, and the residue was dissolved in 1.2 N hydrochloric acid (50 mL) and dichloromethane (50 mL). The organic layer was washed with 1.2 N hydrochloric acid (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was triturated in diethyl ether (4 mL). The solid was collected by vacuum filtration to yield 769 mg (74%) of a bright yellow solid, mp 124-125 °C. 1H NMR is a complex spectrum of three conformations in a 10:9:1 ratio at room temperature. 1H NMR (CDCI3): 6 10.49 (s, 1H, OH), 7.88, 7.80 and 7.71 (s, 1H, ArH ortho to N02), 7.46-7.19 (m, 11H, 10 x ArH and ArH para to N02), 7.05-6.98 (m, 1H, ArH meta to N 02), 6.70 and 6.67 (s, 1H, ArH), 6.54, 6.48 and 6.41 (s, 1H, ArH), 5.20-4.95 (m, 5H, 4 x ArCH20 and ArCHRN), 4.15-3.94 and 3.80-3.65 (m, 1H, CH2N), 3.66, 3.51 and 3.46 (s, 3H, CH30), 3.32-3.11 (m, 1H, CH2N), 3.10-2.88 (m, 2H, ArCH>CHRN), 2.88-2.43 (m, 2H, ArCH2). FAB MS m/z 555.4 (m + 1, 4.4%), 402.2 (m + 1 - CjH^Og, base). Analysis for C32H30N2O71/2H2O: calc. C, 68.20; H, 5.54; N, 4.97; found C, 68.40; H, 5.41; N, 4.92. BnO BnO OCH NO. OH 6.7-Bis(benzvloxv)-2-carbomethoxv-1-(4-hvdroxv-3-iodo-5-nitrobenzvn- 1.2.3.4-tetrahvdroisoquinoline. (313) : In a manner similar to the methodology of Kajigaeshi [299], hydroxy urethane 312 (50 mg, 0.090 mmol) was dissolved 272 in a mixture of methanol (2 mL) and dichloromethane (5 mL). The solution was charged with sodium bicarbonate (250 mg) and benzyltrimethylammonium dichloroiodate (BTMA ICI2, 35 mg, 0.099 mmol) and stirred for 24 h at room temperature. The suspension was gravity filtered, and the filtrate was diluted with dichloromethane (15 mL) and 1.2 N hydrochloric acid (20 mL). The layers were separated, and the organic layer was washed with 5% aqueous sodium thiosulfate (20 mL), brine (20 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was subjected to silica gel chromatography with ethyl acetate/hexanes/dichloromethane (5/45/50) as the eluent. The appropriate fractions were pooled and evaporated in vacuo to a yellow oil (23 mg, 37% yield). The oil was triturated in diethyl ether to yield 16 mg (26%) of a bright yellow solid, mp 160-161 °C. 1H NMR is a complex spectrum of two conformations in a 1:1 ratio at room temperature. 1H NMR (CDCI/TMS) : 5 11.23 (s, 1H, OH), 7.79-7.67 (m, 2H, ArH ortho and para to N 0 2) 7.47-7.26 (m, 10H, ArH), 6.69 (bs, 1H, ArH), 6.52 and 6.44 (bs, 1H, ArH), 5.20-4.98 (m, 5H, 2 x CH20 and ArCHRN), 4.18-4.00 and 3.72-3.60 (m, 1H, CH2N), 3.68 and 3.57 (s, 3H, CH30), 3.40-3.12 (m, 1H, CH2N), 2.91 (d, 2H, J = 6.4 Hz, ArCH2CHRN), 2.84-2.60 (m, 1H, ArCH2), 2.60-2.38 (m, 1H, ArCH2). FAB MS m /z 681.0 (m + 1, 4.7%), 402.1 (m + 1 - CyH^Ogl,, base). Analysis for CagHagNgO^ : calc. C, 56.48; H, 4.30; N, 4.12; found, C, 56.84; H, 4.15; N, 4.20. i Part 2 Section B Approaches to the Asymmetric Synthesis of Irreversibly Binding lodinated Derivatives of Trimetoquinol , 273 CHAPTER IX INTRODUCTION 9.1 Synthesis of Chiral 1-(Benzyl)-1,2,3,4-Tetrahydroisoquinolines Typically, asymmetric synthesis is the synthetic conversion of a prochiral group into a chiral group in such a manner that the stereoisomeric products are produced in unequal amounts [306]. However, this definition is frequently extended to encompass all methodologies of converting racemic compounds into chiral compounds. This extended definition includes methods such as achiral chromatographic separation of diastereomers, fractional crystallization of diastereomeric salts, enzymatic resolution, and chiral separation of racemates [306,307]. In each of these cases, chemical or physical procedures are required for the separation of the racemic mixtures. Thus, these methodologies are not classical asymmetric syntheses even though they produce enantiomerically enriched compounds [306]. 9.1.1 Separation of Diastereomers The separation of diastereomeric derivatives of a racemic compound utilizes differences in physical properties to separate the diastereomers. 274 275 Generally this involves separation of the diastereomers based on differences in solubility, boiling point, or chromatographic properties such as adsorption and partitioning [306,308]. This methodology requires an optically pure agent for the formation of the diasteromers (Figure 35). This technique has been used quite extensively and is the most general method for the isolation of optically active compounds. The most common techniques are achiral chromatography of diastereomeric derivatives and fractional recrystallization of diastereomeric salts [306,308]. Racemic compound (R and S) ...... Optically pure reagent (D) ir Diastereomeric mixture (RD and SD) Physical Seperation (achiral chromatorgapy, or fractional recrystallization) Diastereomer RD Diastereomer SD Regeneration ’ ' , Y " Enantiomer (R) Optically pure reagent (D) Enantiomer (S) Optically pure reagent (D) Figure 35. Resolution of Diasteromeric Derivatives. . 276 9.1.1.1 Achiral Chromatography of Diastereomeric Derivatives Chromatographic separations of diastereomeric compounds involves the chemical formation of the diastereomers, achiral chromatography based on physical properties such as adsorption or partitioning, and conversion of the purified derivative to the chiral substrate and chiral reagent [306] (Figure 35). The chemical reactions involved in the formation and disassociation of the diastereomeric derivatives must be mild enough to prevent racemization of the desired ligand’s chiral center. It is also beneficial to be able to recycle the chiral derivatizing reagent. Some examples of chiral functionalizing reagents that are useful for this methodology include camphor sulfonic acid, mandelic acid, menthol, ephedrine, 1-phenylethylamine, tartaric acid derivatives, and amino acid derivatives (Figure 36) [308]. The major drawback to this methodology is the appropriate selection of both the chiral functionalizing reagent and achiral chromatographic system for successful application [308]. 9.1.1.2 Fractional Recrvstallization of Diastereomeric Salts Fractional recrystailization of diastereomeric salts involves the selective crystallization of a diastereomeric salt from a racemic mixture with a chiral reagent [306,308]. Typically for racemic bases, camphor sulfonic acids or tartaric acid derivatives are used as the chiral reagent [308], For racemic acids, ephedrine or 1-phenylethylamine can be used [308] (Figure 36). This technique is based on differing solubilities of the diastereomeric salts. Usually, a number (1S)-(+)-10-Camphorsulfonic Acid (R)-Mandelic Acid (1 R,2S,5R)-(-)-Menthol ■ O ^ O H H -C -N H C H 3 H 1 H-C-OH H-C-OR NH2 1 R (1 R,2 S)-(-)-Ephedrine (R)-I-Phenylethylamine ( 2 R, 3R)-(+)-Tartaric Acid L-Amino Acid Derivatives Derivatives Figure 36. Chiral Derivatizing Reagents for Diasteromeric Separations. of recrystallizations are required for the successful purification of a single enantiomer from a racemic mixture [306,308], 9.1.2 Enzymatic Resolution of Racemic Amines A great deal of work has been published on the enzymatic resolution of racemic alcohols, and carboxylic acids. There has also been considerable amount success in the resolution of racemic amino acids. However, other compounds which contain amino groups have received little attention [309], The use of lipases and proteases to catalyze the amidation between a carboxylic ester and a racemic primary amine has been recently investigated [310,311]. The racemic primary amine, an active ester 278 o H3C ^ ^ O ^ C F 3 314 (2,2,2-trifluoroethyl butyrate 314), and the protease, subtilisin Carlsberg, in the hydrophobic solvent 3-methyl-3-pentanol provided the conditions necessary for selective conversion of one enantiomer of the amine to the amide [309,311]. By controlling the incubation length according to general theory of enzyme- catalyzed kinetic resolution [312], high enantioselective conversion to the amide was observed [309,311], The derivatized amide is simply separated from the unreacted amine by column chromatography and cleaved to the enantiomerically enriched amine. By extending the length of the incubation, the free amine portion becomes concentrated with the enantiomer of the opposite configuration as compared to that converted to the amide [309,311]. Again, the amine can be purified from the amide by column chromatography. Thus, enzymatic resolution can furnish either enantiomer based on the length of the incubation [309,311]. 9.1.3 Chiral HPLC of Racemic Mixtures The direct resolution of racemic mixtures by chiral stationary phases is a rapidly evolving discipline. This technique relies on the formation of transient diastereomeric complexes between the sample and the stationary phase. The 279 enantiomer forming the more stable diastereomer is retained on the column allowing the enantiomer forming the less stable diastereomer to elute first [313], A number of chiral stationary phases (CSP) have received considerable attention. Cellulose is a natural chiral polymer used for paper chromatography 1 resolution of amino acids but lacks the mechanical strength for use in high-performance liquid chromatography (HPLC) [314]. Modified cellulose ester and carbamate derivatives provide the required mechanical strength for successful chiral HPLC. These stationary phases, such as ChiralcelR OD (Figure 37), are able to interact with the solutes by hydrogen bonding, dipole-dipole interactions, and charge transfer interactions [314], In addition to these interactions, the cellulose stationary phases produce a chiral cavity that is very important for steric interactions with the solute. The magnitude of chiral resolution is related to the ability of the solute to interact with the stationary phase by this inclusion phenomena [314]. This stationary phase can be useful for the separation of 1,2,3,4-tetrahydroisoquinolines [232,315], The Pirkle-type chiral stationary phases consists of a short modified amino acid chain bound by a linker to silica [313], The most common stationary phase is a 3,5-dinitrobenzoylphenylglycine covalently bound to aminopropylsilanized silica (Figure 37) [316]. This chiral stationary phase is capable of attractive interactions such as hydrogen bonding, dipole-dipole interactions, and charge-transfer complexes as well as steric interactions to 280 Chiralcel OD CH, OR O R = JL E -0 RO OR Coated on silica gel Pirkle Phase Si— O OEt NH n o 2 Figure 37. Chiral Stationary Phases. discriminate between enantiomers [317]. Unfortunately, derivatization of many amines to naphthamides is required to facilitate the chiral recognition process [316,317]. This type of chiral stationary phase has been used to separate the a-naphthamides of 1,2,3,4-tetrahydroisoquinolines [318] and has been utilized for the determination of the enantiomeric excess from the asymmetric synthesis of 1,2,3,4-tetrahydroisoquinolines [319,320]. 281 9.1.4 Asymmetric Synthesis of 1-Substituted THIQs 1-Substituted 1,2,3,4-tetrahydroisoquinolines and related systems are important biologically active molecules from natural and synthetic sources [321,322]. Trimetoquinol 176 is an important example for the need to develop drugs as a single enantiomer because the enantiomers of trimetoquinol 209 and 210 have stereoselective biological activities [206,217,218,220], Biological systems are chiral. Therefore, it is reasonable to assume that there will be a preferential interaction of one stereoisomer with the system in comparison to its antipode [323]. These interactions can occur during absorption, protein binding, metabolism, renal clearance, and at the receptor [323]. Therefore, it is beneficial to produce drugs in an optically pure form. For drugs like trimetoquinol, recent advances in asymmetric synthesis provide the opportunity for the stereospecific synthesis of the enantiomers. These asymmetric methods are the alkylation of chiral formamidines and asymmetric catalytic hydrogenation [319,324], 9.1.4.1 Alkvlation of Chiral Formamidines Initial work on the C-C bond formation with secondary amines provided a new route to the formation of 1 -substituted-1,2,3,4-tetrahydroisoquinolines with formamidines [325]. For this technique, a secondary amine is converted to a formamidine derivative. Metalation of the formamidine results in a stabilized a-amino carbanion which can be readily alkylated by an electrophile. 282 Subsequent removal of the formamidine results in an a-alkylated secondary amine (Figure 38) [325,326]. By changing the substitution on the formamidine to a chiral substituent, the environment of the carbanion can be stereochemically induced to provide stereospecific alkylation [319,326]. This methodology has produced numerous tetrahydroisoquinolines and related systems [320,325,326]. rI RNCH(OMe )2 mf H *-BuLi .L f u .HH ------. ^ Y " Nv L i- N H R R R i - X H O H ' ► _ NH Figure 38. Methodology for cx-Lithiation of Formamidines. 9.1.4.2 Asymmetric Catalytic Hydrogenation Asymmetric catalytic hydrogenation can be divided into two groups: reduction of a chiral alkene mediated by preferential reduction of the double bond from a single diastereotopic face and reduction of an achiral alkene with a homogeneous chiral catalyst from a single enantiotopic face [327]. Catalytic hydrogenations with a homogenous chiral catalysts have been the most successful (Scheme XLII) [327]. Some of the earliest work involved the 283 use of soluble chiral rhodium-phosphine catalysts for the reduction of alkenes [327]. Recently, chiral ruthenium-phosphine catalysts has been used for the reduction of dehydro precursors of 1 -substituted-1,2,3,4-tetrahydroisoquinolines [324,328], including the protected dehydro trimetoquinol derivative 315 with ruthenium(ll)-BINAP catalyst 316 (Scheme XLIII) [324]. In addition to producing chiral tetrahydroisoquinolines, BINAP-ruthenium(ll) catalysts have great synthetic utility in the reduction of enamides, a, 6- and B.y- unsaturated acids, allylic alcohols, and f3-keto esters [329,330,331,332,333], Scheme XL.il. Reduction of an Achiral Alkene with a Chiral Catalyst. chiral catalyst where 4 R2 Scheme XLIII. Chiral Catalytic Hydrogenation of Dehydro Trimetoquinol Derivative 315. BnO BnO BnO H2 (4 atm) RT BnO OCH3 Ru(S-BINAP)(OAc)2 OCH3 316 OCH3 EtOH/CH2CI2 OCH3 CH 3 O (5:1) CH 3 O CHAPTER X STATEMENT OF THE PROBLEM AND OBJECTIVES In order to synthesize the chiral irreversible trimetoquinol derivatives 251 and 252, a number of potential chiral intermediates are required. This section will discuss the approaches investigated for the production of the necessary chiral intermediates. These approaches are chiral high-performance liquid chromatography (chiral-HPLC), enzymatic resolution, and asymmetric synthesis. Several of these approaches investigated the production of trimetoquinol enantiomers 209 and 210 for comparison to authentic samples from the pharmaceutical manufacturer [334]. The products from asymmetric synthesis were analyzed by HPLC to determine enantiomeric excess (Pirkle column and TAGIT method) [335,336], 10.1 Chiral HPLC Chiral HPLC utilizing a ChiralcelR OD column was investigated as a potential route for the production of desired chiral intermediates. Chiralcel columns have a general application for the separation of compounds containing 284 285 aromatic, carbonyl, nitro, sulfinyl, cyano, or hydroxyl groups [315]. This technique has been successfully used for the preparative separation of dibenzyloxy 8-fluoro-TMQ 318 to its enantiomers [232]. In order to evaluate the potential of a preparative column to supply the necessary intermediates, amines 253 and 254 and methyl urethanes 307 and 308 were analyzed. These derivatives were examined on an analytical ChiralcelR OD column with various mobile phases for possible preparative separation. BnO NH BnO OCH 318 10.2 Enzymatic Resolution Several primary amines have been successfully resolved by enzymatic resolution [309-311]. A successful application of this methodology can resolve large quantities of material rather inexpensively in a relatively short time. Based on the successful resolution of several arylalkylprimary amines 319-321 [309], the dibenzyloxy trimetoquinol precursor 271 was evaluated for potential enzymatic resolution. Reaction times and temperatures were varied in an attempt to resolve the enantiomers. A successful resolution of the trimetoquinol 286 n h 2 n h 2 n h 2 ch3 ch3 319 320 321 precursor 271 may then be extended to provide the necessary chiral precursors for the irreversible derivatives 251 and 252. 10.3 Chiral Formamidine Synthesis Meyer’s chiral formamidine methodology has been successfully applied to the synthesis of several tetrahydroisoquinolines [320,325,326], During the development of this methodology, most studies either lacked functionalization or contained inert methyl ethers [320,325,326], More recently, a few publications demonstrated this methodology with additional functional groups such as benzyloxy ethers, amides, and hindered esters [337,346], A successful application of this technique to the synthesis of functionalized trimetoquinol derivatives requires an application of this technique to protected aniline 322 and phenol 323 benzyl bromides for the asymmetric synthesis of tetrahydroisoquinolines 324 and 325, This technique was also applied to the synthesis of trimetoquinol enantiomers 209 and 210 for comparison to authentic samples. 3 2 2 R = -NH-Protect 3 2 4 R = -NH-Protect 3 2 3 R = -O-Protect 3 2 5 R = -O-Protect 10.4 Asymmetric Catalytic Hydrogenation Noyori’s BINAP-ruthenium(ll) catalysts have been reported to asymmetrically reduce (Z)-1 -benzylidene-1,2,3,4-tetrahydroisoquinolines [324,338] and has been applied to the synthesis of trimetoquinol enantiomers [324]. However, other researchers reported difficulties in the preparation and successful use of this catalyst [330], This technique was applied to the synthesis of trimetoquinol enantiomers in our lab for a potential application in the synthesis of the irreversibly binding derivatives 251 and 252. Difficulties with this technique in our lab required an analogous investigation of the asymmetric reduction of enamides 326 and 327 in an effort to circumvent inherent solubility problems of the related trimetoquinol precursor 328. Upon the successful reduction of both (Z)-enamides 326 and 328. other (Z)-enamides 329-331 and an enurethane 332 were investigated. 2 8 8 RO CH30 CH N - ^ C H RO C H 3 O C H 3 O OCH; C H 3 O OCH. C H 3 O 326r = c h 3 327 328R r B n 329 R-t = no2 r2 = h 332R-) = no2 r 2 = h 330 Rl = OAc R2 = N 02 331 R1 = OH R2 = no2 CHAPTER X! RESULTS AND DISCUSSION 11.1 Chiral HPLC Preparative chiral HPLC has been used to separate the isomers of 6.7-dibenzyloxy-8-fluorotrimetoquinol 318 as its free base utilizing ChiralcelR OD as the chiral stationary phase [232]. With this chiral column in hand, the initial approach for the production of chiral intermediates for the synthesis of chiral irreversible iodinated trimetoquinol derivatives 251 and 252 involved the evaluation of various modified trimetoquinol derivatives for separation by a Chiralcel ODR analytical column. This inquiry evaluated several solvent systems compatible with the chiral stationary phase for the separation of the tetrahydroisoquinoline free bases 253 and 254 and urethanes 307, and 308. The free base of 6,7-dibenzvloxv-8-fluorotrimetoquinol 318 was evaluated as a prototype compound and displayed good separation (t, = 47.2 min, t, = 54.5 min, a = 1.20, Rs = 1.39, Figure 39). The free bases of 253 and 254 were selected for evaluation by chiral HPLC because aniline derivative 254 was a potential penultimate intermediate for iodination. Since the desired irreversible labels may eventually be radioiodinated, it would be desirable to perform the 290 Figure 39. Chiral HPLC Chromatogram for 8-Fluoro-TMQ Derivative 318. preparative chromatography prior to radioiodination to avoid duplicating equipment needs for both "cold" and "hot" chromatography. Nitro derivative 253 was used to evaluate the general applicability of the chromatography system to other trimetoquinol derivatives. These amines were analyzed with varying concentrations of isopropanol in hexanes as the mobile phase. In sharp contrast to the 8F-TMQ derivative 318 which provided near baseline separation, these amines showed very little inclination for separation with an isopropanol/hexanes mobile phase. In an effort to optimize analyte properties, urethanes 307 and 308 were evaluated. Acylation of the secondary amine provides an additional functional group to influence analyte/stationary phase diastereomeric complex formation. These urethanes also produced unremarkable chromatograms with an isopropanol/hexanes or a 100% isopropanol mobile phase. Since this stationary phase is compatible with pure Figure 40. HPLC Chromatogram of Aminourethane 308 on an Analytical ChiralcelR OD Column. alcohols as the mobile phase [315], urethanes 307 and 308 were evaluated with methanol as the mobile phase. This system resolved aminourethane 308 enantiomers with near baseline separation (t, = 36.0 min, t2 = 40.2 min, a = 1.15, Rs = 1.87, Figure 40). However, the successful resolution of the urethane 308 by analytic chiral HPLC still provides formidable obstacles: 1. The general application of this technique to other derivatives is questionable since nitrourethane 308 or amines 253 and 254 were not adequately resolved under any chromatographic conditions. Therefore, the applicability of this technique to other trimetoquinol derivatives suggests that each trimetoquinol derivative be independently evaluated. 292 2. Several synthetic steps to form the target compounds are required following separation of the urethane enantiomers. This means that gram quantities of chiral materials may be required to synthesize a sufficient quantity of the target compounds for biological evaluation. This would require multiple injections to separate a sufficient quantity of material. Both of these obstacles can demand a significant amount of labor and materials. Therefore, other methods for providing large quantities of chiral intermediates were investigated. 11.2 Enzymatic Resolution Dibenzyloxy protected trimetoquinol derivative 271 was utilized a prototype secondary amine for enzymatic resolution. The use of a protease has been recently described for the resolution of arylalkyl primary amines 319-321 [309] which contain a chiral center similar to the chiral center of trimetoquinol. Although the enzymatic resolution of a secondary amine has not been described, the potential application for providing large quantities of enantiomerically pure trimetoquinol derivatives is of interest. The procedure involves the enzyme-catalyzed enantioselective amidation of one enantiomer with an active ester. By controlling the length of the reaction, either the amide (shorter reaction times) or the unreacted amine (longer reaction time) becomes enantiomerically enriched [309]. Butyric anhydride 333 was 293 Scheme XLIV. Synthesis of 2,2,2-Trifluoroethyl Butyrate 314. 0 0 o n n cf 3ch2oh n ------► / ' v x ^ o cf 3 333 314 mixed with 2,2,2-trifluoroethanol, heated to reflux for 24 h, and distilled to provide 2,2,2-trifluoroethyl butyrate 314 (Scheme XLIV). This active ester (0.5 M) was shaken at 250 rpm with the free base of 271 (0.5 M) and subtilisin Carlsberg (2 mg/mL) at 30, 37, or 45 °C for 72 h. The progress of the reaction was monitored at various times by thin layer chromatography and reverse-phase HPLC for the formation of the butyramide derivative. No reaction was observed at any temperature during the 72 h reaction. Under these conditions, this procedure [309] is not directly applicable to trimetoquinol derivative 271. The optimal conditions for the enantioselective resolution of this trimetoquinol derivative were not further investigated. 294 11.3 Asymmetric Synthesis 11.3.1 Chiral Formamidine Synthesis The application of this strategy to the asymmetric synthesis of chiral trimetoquinol derivatives began with the actual asymmetric synthesis of trimetoquinol stereoisomers 209 and 210 for comparison to authentic samples. The retrosynthetic strategy begins with the formation of a common chiral tetrahydroisoquinoline intermediate which could be alkylated with several benzyl halides to provide 1-benzyl substituted tetrahydroisoquinolines (Scheme XLV). After alkylation, hydrazinolysis of the formamidine portion followed by deprotection of the catechol ring provides the chiral trimetoquinol derivatives Scheme XLV. Retrosynthetic Strategy for Synthesis of 1-Benzyl Substituted THIQs via Chiral Formamidines. BnO BnO tBuO OCH3 tBuO 295 The chiral tetrahydroisoquinoline intermediate is formed from two parts, a tetrahydroisoquinoline and a chiral formamidine derived from L- or D-valine (Scheme XLV). Pictet-Spengler cyclization of 3,4-dibenzyloxyphenethylamine hydrochloride 98 with formaldehyde in formic acid provided isoquinoline 334 which was isolated as its oxalate salt (Scheme XLVI). Several steps were required to prepare the second chiral formamidine fragment [337,341]. Reduction of L-valine 335 with sodium borohydride and boron trifluoride etherate in tetrahydrofuran provide (S)-valinol 336 (Scheme XLVII) [337]. This aminoalcohol was converted to a formamide with ethyl formate and O-alkylated with isobutene 337 and boron trifluoride etherate in dioxane to provide ether 338. The formamide was cleaved with potassium hydroxide and converted to formamidine 339with N,N-dimethylformamidedimethyl acetal. Transamidination with the free base of isoquinoline 334 provides (S)-isomer 340 which could be isolated as its oxalate salt. The same sequence can be utilized to convert D-valine 341 to (R)-isomer 344 (Scheme XLVIII). Scheme XLVI. Synthesis of Tetrahydroisoquinoline 334. 1. h c o h /h c o 2h 2. oxalic acid NH (COOH) 2 98 334 Scheme XLVII. 296 Synthesis of (S)-Chiral Formamidine 340. 1. ethyl formate 2- CH3 COOH CH2OH CH2 CH3 h2n - 4« h NaBH4 H2N - f - H 337 BF3 Et20 c h 3 * ^ s C H 3 ch3 ^ ch, —,------> . THF BF3'Et20 335 336 dioxane CH2OtBu NMe2 CH2OtBu 1. NaOH H^HN " H H 2. Me2NCH(OMe2) CH3 ^ CH-i C H , ^ CH, 338 339 BnO BnO (COOH)2 BnO X X > BnO p-TsOH toluene 340 2. oxalic acid tBuO EtOH / Et20 In order to synthesize the enantiomers of trimetoquinol, the appropriate benzyl halide was required. Reduction of 3,4,5-trimethoxybenzaldehyde 345 with sodium borohydride provided benzyl alcohol 346 (Scheme XLIX). A solution of this alcohol was halogenated with hydrogen bromide (g) in chloroform to provide benzyl halide 347. This compound readily decomposes and was purified by silica gel chromatography prior to use. Scheme XLVIII. 297 Synthesis of (R)-Chiral Formamidine 344. 1. NaBH 4 COOH BF3 Et20 /T H F CH2OtBu CH2OtBu 1. NaOH H -4 -N H j, 2 . ethyl formate -4— NH H H 1 I t f 3 . CH3 A o 2. Me 2 NCH(OMe2) ■*VH CH, 'CH, CH, NMe, c h 2 ^ c h 3 CH3 CH3 341 337 CH3 * 342 343 BF 3-Et20 dioxane 1 . BnO BnO YYl (COOH)2 BnO IX 1-" BnO ll I N\.o " J---- p-TsOH toluene 2 . oxalic acid 344 tBuO EtOH / EtzO Scheme XLIX. Synthesis of 3,4,5-Trimethoxybenzyl Bromide 347. NaBH, HBr (g) THF/lpOH CHCI OCH OCH OCH OCH3 345 346 347 Alkylation of the free base of tetrahydroisoquinoline 340 was performed in two steps by forming the corresponding cx-lithio anion at -78 °C followed by alkylation with benzyl halide 347 at -100 °C (Scheme L). Following workup, the 298 Scheme L. Chiral Formamidine Synthesis of (S)-Trimetoquinol 209. BnO BnO 1 . t-BuLi BnO BnO XXX. 2 . CH2Br 340 3u ~OCH3 tBuO o c h 3 tBuO 347 OCH3 348 BnO HO 1.NH2 NH2 /H 20 NH HCI NH-HCI HO HOAc / IpOH Bn° conc. HCI OCH 2. HCI MeOH 349 209 OCH CH,0 crude formamidine was cleaved with a mixture of hydrazine, water, and glacial acetic acid in isopropanol. The desired compound was purified by silica gel chromatography and converted to its hydrochloride salt with hydrogen chloride (g) in methanol to provide (S)-dibenzyloxy-TMQ 349 in only 22% yield. The benzyl ethers were removed with concentrated hydrochloric acid and methanol to provide (S)-trimetoquinol 209. The same sequence was performed on (R)-isomer 344 to provide (R)-dibenzyloxy-TMQ 351 in only 13% yield (Scheme LI). Initially, the reason for the poor yields was unknown. Eventually, it was determined that the chiral formamidine intermediates 340 and 344, which were required as their free bases, contained sufficient water to limit the success 299 Scheme LI. Chiral Formamidine Synthesis of (R)-Dibenzyloxy Trimetoquinol Derivative 351. BnO ^ Bn° 1. t-BuLi ► BnO ? CHoBr II I A c h3o 344 NV ch3o J ^ o c h 3 ” Y l i tBuO ' ° C H 3 CH30 tBuO 347 °CH3 350 BnO 1. NH2 NH2 /H 20 HOAc / IpOH BnO xx>~ 2. HCI ' 351 L i Y ^ o c h 3 ch3o of this technique even though these compounds were dried in vacuo with phosphorous pentoxide prior to use. Both the free base and oxalate salts of (S)-chiral formamidine 340 were shown to be partial hydrates upon elemental analysis. The chiral formamidine methodology is successful for producing compounds of high enantiomeric purity. The (S)-dibenzyloxy derivative 349 was initially analyzed as its a-naphthamide derivative by chiral HPLC on a Pirkle column to determine enantiomeric excess [319]. Even though this compound showed a single peak, the determination of the enantiomeric excess is difficult 300 because the separation of racemic derivative 352 (Scheme LI I) was not very impressive (t, = 23.7 min, ^ = 26.6 min, a = 1.13, Rs = 0.84, Figure 41). Noyori [324], reported a second method for analyzing 1-substituted tetrahydroisoquinolines for enantiomeric excess. Analysis by in situ conversion of racemic dibenzyloxy derivative 271 to a diastereomeric pair with 2,3,4,6-tetra-0-acetyl-l3-D-glucopyranosyl isothiocyanate (TAGIT) (Scheme LI 11) and separation on a reverse phase column provided much better resolution (tn = 13.6 min, t2 = 15.4 min, a = 1.16, Rs = 2.44, Figure 42). In addition, the TAGIT method is applicable to catechol derivatives such as trimetoquinol [336], (S)-Dibenzyloxy derivative 349 (94% ee), (R)-dibenzyloxy derivative 351 (97% ee), and (S)-trimetoquinol 209 (99% ee) synthesized from the chiral formamidine route each showed high enantiomeric excess by the TAGIT method. Several other halides were also prepared for potential alkylation of the chiral tetrahydroisoquinoline intermediates 340 and 344. Both esters and amides have been utilized in a successful application of this strategy [337], Therefore, the first protecting groups employed were an acetamide and an acetate. Reduction of 4-acetamidobenzaldehyde 354 with sodium borohydride provide benzyl alcohol 355 which was halogenated with hydrogen bromide (g) in chloroform to produce benzyl bromide 356 (Scheme LIV). Acetylation of 4-hvdroxvbenzaldehvde 357 with acetic anhydride provided benzaldehvde 358 (Scheme LV). This aldehyde was reduced with sodium borohydride to benzyl Scheme Lll. 301 Synthesis of Trimetoquinol Naphthamide 352. BnO BnO 1. POCI3 /CH 3CN NH HCI BnO BnO 2. NaBH4 / EtOH OCH OCH 3. HCI (g) / MeOH 272 271 OCH OCH BnO N BnO 352 OCH 1 I 1 / Figure 41. Chiral HPLC of (±) Trimetoquinol Naphthamide 352. Scheme LIU. 302 In s itu Generation of TMQ-TAGIT Diastereomers. CH2OA c AeO BnO AcO NHHCI BnO OCH TAGIT 271 Et3N / DMFI CH3CN OCH OCH3 CHoOAc OBn AcO NH N AcO OAc 353 OCH Figure 42. TAGIT HPLC Chromatogram for (±)-Dibenzyloxytrimetoquinol Derivative 271(1 = solvent, 2 = quenched TAGIT, 3 = (S)-isomer, 4 = (R)-isomer). Scheme LIV. 303 Synthesis of 4-Acetamidobenzyl Bromide 356. C H o O H NaBH, HBr (g) THF CHCI NHAc NHAc NHAc 355 356 Scheme LV. Synthesis of 4-Acetoxybenzyl Bromide 360. C H o O H PBr THF OAc OAc OAc 358 359 360 alcohol 359 and converted to benzyl bromide 360 with phosphorous tribromide. 4-Acetoxybenzyl bromide readily decomposes and was purified by silica gel chromatography prior to use. Each of these benzyl halides has relatively acidic protons as compared to the lithiated tetrahydroisoquinoline which is formed in situ. Therefore, these compounds were mixed with potassium hydride prior to use for alkylation in an attempt to avoid quenching the lithio anion [337]. Since these benzyl halides are para-substituted aromatic rings, the alkylated products should contain aromatic protons which are readily identifiable by 1H NMR spectroscopy. However, the only identifiable compound following the 304 reaction and workup was tetrahydroisoquinoline 334. Therefore other protecting group which lack potentially acidic hydrogens were investigated. Since amides were successfully used with this methodology [337], the phthalimide protecting group was chosen to protect the aniline. Phthalic anhydride 361 was heated with p-toluidine 362 in glacial acetic acid to form phthalimide 363 (Scheme LVI). This compound was a-brominated with N-bromosuccinimide (NBS) and the radical initiator 2,2’-azobisisobutyronitrile (AIBN) in carbon tetrachloride to provide benzyl bromide 364. Several protecting groups for phenols are stable to lithiation conditions, but the mildly acidic removal of methoxymethoxy ethers from phenols led to its selection as the desired protecting group [106], 4-Hydroxybenzaldehyde 357 Scheme LVI. Synthesis of N-(4-Bromomethylphenyl)phthalimide 364. HOAc O O 361 3 6 3 o NBS /AIBN O 364 Scheme LVIL 305 Synthesis of 4-Methoxymethoxybenzyl Bromide 367. H CH2OH CH2Br 1. NaH NaBH4 CBr4/PPh3 THF iiu o OH OCH2OCH3 OCH2OCH3 357 365 366 367 was converted to its anion with sodium hydride and alkylated with chloromethyl methyl ether to provide benzaldehyde 365 (Scheme LVI I). This aldehyde was reduced with sodium borohydride to alcohol 366 and brominated with carbon tetrabromide and triphenylphosphine to form benzyl bromide 367. This compound readily decomposes and was purified by column chromatography prior to use. Both benzyl bromide 364 and 367 were reacted with the lithio anion of chiral formamidine 340 or 344. Since these benzyl halides are para-substituted aromatic rings, the alkylated products should contain aromatic protons which are readily identifiable by 1H NMR spectroscopy. Following workup, several products were seen by TLC, but the only identifiable compound was tetrahydroisoquinoline 334. Since these compounds do not contain relatively acid hydrogens as compared to the lithio anion, the source of the quenching material must be coming from chiral formamidine 340 or 344. The elemental analysis of the free base of (S)-formamidine 340 did show hydration and may 306 be sufficient to prevent formation of the desired product. The eventual success of chiral catalytic hydrogenation halted further research with chiral formamidines. 11.3.2 Asymmetric Catalytic Hydrogenation Chiral catalytic hydrogenation of dehydro trimetoquinol derivative 315 with a ruthenium(ll) catalyst has been reported (Scheme XLIII) [324], The required dehydro derivative is prepared by the Bischler-Napieralski cyclization of amide 272 with phosphorous oxychloride to the unstable dihydroisoquinoline intermediate 368 followed by acylation of the imine with concurrent migration of the double bond to enamide 315 with acetic anhydride and triethylamine (Scheme LVIII). Two preparations of the required air-sensitive catalyst have been described [328,339]. Commercially available ruthenium compounds 369 or 370 undergo high temperature ligand exchange with (S)- or (R)-2,2’-bis(diphenylphosphino)-1,1’-binaphthyl to form intermediate chloride catalysts 371 and 372 (Scheme LIX). The second step of this one-pot reaction involves the displacement of the chlorides with sodium acetate to form the ruthenium diacetate catalyst 373 and 374. The diacetate catalyst is extremely air-sensitive and quickly turns from bright yellow to green when exposed to air. Therefore, catalyst preparation requires the use of Schlenk techniques. Several attempts to prepare this catalyst and reduce enamide 315 with crude or crystalline catalyst according to the described procedures failed. Catalyst formation proceeded as described except for crystal formation. Scheme LVIII. 307 Synthesis of Dibenzyloxy Enamide 315. BnO BnO BnO IX 1- BnO OCH, OCH 272 368 OCH, OCH c h 3o BnO BnO CH Ac2Q /E t3N OCH 315 OCH. Scheme LIX. Reported Literature Syntheses of Ru(BINAP) Catalysts. S-BINAP NaOAc [RuCI2 (cod)]n ------► Ru(S-BINSAP)CI2 Et3N Ru(S-BINAP)(OAc)2 t-BuOH toluene Et,N 373 369 reflux 371 reflux R-BINAP NaOAc [RuCI2 (benzene ) ]2 ...... - > Ru(R-BINAP)CI2 ■■■■■■■ > Ru(R-BINAP)(OAc)2 D M F 100 ° C MeOH 370 372 RT 374 The crystals initially deposited in the Schlenk tubes as yellow needles which eventually turned green within a short period of time. In addition, the poor solubility of enamide 315 in the 5:1 ethanol/dichloromethane solvent system 308 Table 14. Reaction Conditions for Catalytic Hydrogenation Entry Compd # Description Catalyst" S o lven t O utcom e 1 315 (Z)-BnO-enamide Ru(S-BINAP)(OAc)jb EtOH/CH2CI2 no rxn (5:1) 2 315 (Z)-BnO-enamide Ru(S-BINAP)(OAc)2 EtOH/CH2CI2 no rxn (1:1) 3 376 (Z)-MeO-enamide R u (S-BINAP)(OA c )2 EtOH/CH2CI2 fain t crude (2:1) 4 377 (E)-MeO-enamide R u (S-BINAP)(OA c )2 EtOH/CH2CI2 no rxn crude (2:1) 5 315 (Z)-BnO-enamide R u (S-BINAP)(OA c )2 EtOH/CH2CI2 no rxn crude (2:1) 6 376 (Z)-MeO-enamide R u (R-BINAP)CI2 EtOH/CH2CI2 fain t crude (2:1) 7 315 (Z)-BnO-enamide R u (R-BINAP)CI2 EtOH/CH2CI2 no rxn crude (2:1) 8 315 (Z)-BnO-enamide R u (R-BINAP)(OA c )2 EtOH/CH2CI2 partial crude" (2:1) 9 315 (Z)-BnO-enamide Ru(R-BINAP)(OAc)2 H O Ac no rxn crude" 10 315 (Z)-BnO-enamide R u (S-BINAP)(OA c )2 H O Ac no rxn crude" 11 315 (Z)-BnO-enamide R u (S-BINAP)(OA c )2 HOAC/CH2CI2 no rxn crude (5:2) 12 315 (Z)-BnO-enamide R u (S-BINAP)CI2 EtO H 60 °C full rxn crude 13 376 (Z)-MeO-enamide R u (S-BINAP)CI2 EtO H 6 0 °C full rxn crude 14 315 (Z)-BnO-enamide R u (R-BINAP)CI2 EtO H 6 0 °C full rxn crude a = all catalysts prepared from [RuCI2(benzene)]2 unless otherwise noted b = prepared from [RuCI2(cod)]n c = utilized 2.1 equiv NaOAc instead of 20 equiv d = catalyst prepared with HOAc as solvent instead of DMF catalyst may be Ru(R-BINAP)C!2 e = utilized 2.1 equiv NaOAC in HOAc as solvent 309 described [324] posed further problems. Several modifications of the described methodology were investigated to successfully reduce enamide 315 (Table 14). Readily available reagents were used to prepare an alternate compound for experimentation. 3,4-Dimethoxyphenylethylamine 120 was heated with 3,4,5-trimethoxyphenylacetic acid in toluene to form amide 375 (Scheme LX). 4 Bischler-Napieralski cyclization with phosphorous oxychloride in anhydrous acetonitrile followed by acylation with acetic anhydride and triethylamine provided a mixture of isomers 376 and 377. These isomers could be readily separated by selective crystallization from ethanol. By cooling the reaction Scheme LX. Synthesis of Methoxy Enamides 376 and 377. CH2COOH toluene reflux c h 3o CH30 c h 3o ■ f c h 3o c h 3o 1 . p o c i3 / c h 3c n o c h 3 c h 3o 2. Ac20 / E t 3N o c h 3 c h 3o 376 c h 3o c h 3o 377 310 mixture to 0 °C for the acylation step, (E)-isomer 377 is predominantly produced. However, only (Z)-isomer 376 is able to be reduced by asymmetric catalytic hydrogenation, so the mixture can be converted exclusively to (Z)-isomer 376 by thermal isomerization in refluxing toluene (Scheme LXI). These enamide derivatives also suffer from poor solubility in the described solvent system [324], but they were also utilized to determine optimal conditions for successful hydrogenation. Scheme LXI. Thermal Isomerization of (E)-Enamide 377 to (Z)-Enamide 376. CH toluene CH. reflux OCH 377 376 OCH Each of the enamides were investigated (Table 14) under various conditions with several modifications for catalyst preparation. The initial effort (Entry 1) attempted to reproduce the literature method for catalytic hydrogenation for 72 h at room temperature [324], but no reaction was observed. The reaction mixture was actually a suspension in 5:1 ethanol/dichloromethane, so dichloromethane was added until (Z)-enamide 315 311 dissolved (Entry 2). Again, no reaction was observed. This lead to the investigation of methoxy protected (Z)-enamide 376 (Entry 3) which produced two very faint spots on TLC which were different from starting material after 72 h. These spots were not of sufficient intensity to warrant further investigation. (E)-Enamide 377 was evaluated (Entry 4) to verify the literature citation that only the (Z)-isomer would reduce under these conditions [324], and as expected no reaction was observed. A final attempt to reduce Z-enamide 315 in 2:1 ethanol/dichloromethane also failed (Entry 5). The crude intermediate dichloride catalyst 372 was reported to be an efficient hydrogenation catalyst for enamides [328] and was evaluated with both enamides 315 and 376 (Entry 6 and 7). Partial reduction of methoxy enamide 376 was observed, but could not be improved by extending the reaction time nor could it be repeated. For this particular reaction, contamination with another catalyst from the equipment was possible. In addition, reduction of benzyloxy enamide 315 was not successful, so it was felt that the reduction of methoxy enamide 376 was the result of a contaminating catalyst from the equipment. The next attempt altered catalyst preparation. Most of the catalysts used were crude mixtures since several attempts to crystallize the catalyst resulted in catalyst oxidation. This crude mixture contained several equivalents of unreacted acetate which may have hindered the reaction. Therefore, only 2.1 equivalents of acetate were used (Entry 8) for the successful partial reduction of benzyloxy enamide 315 Unfortunately, this result could not be duplicated. 312 To this point, it appeared that successful catalyst preparation was still the major hinderance. Since ruthenium catalyst ligands are easily displaced with acids [332], the acetate catalysts 373 and 374 were prepared and hydrogenated in glacial acetic acid with varying amounts of sodium acetate (Entry 8,9,and 10). In each case no reaction was observed. Since catalyst preparation and enamide solubility were problematic, one final set of reaction conditions were investigated. Enamides 315 and 376 were both much more soluble in hot ethanol, so the hydrogenation was performed at an elevated temperature. The proper selection of the appropriate catalyst was still required. Since several researchers describe the successful preparation and use of dichloride catalysts 371 and 372 [328,330,332], they were selected as the catalysts for elevated temperature hydrogenation (Entry 12, 13 and 14). In each case, the enamide was successfully reduced with 60 psi of hydrogen at 60 °C in ethanol for 72 h (Scheme LXI I). These acetamide derivatives have restricted rotation about the amide bond and produce two conformations in solution resulting in complex 1H NMR spectra. Temperature elevation studies in CDCIg at 323 K or d6-DMSO at 357 K were unable to clarify the 1H NMR spectra (methoxy derivative 379. Figure 43). The structures of amides 317. 378. and 379 were verified by comparing their 1H NMR spectra to those of the unambiguously synthesized racemic amides 380 and 382 (Schemes LXI 11 and LXIV). Scheme LXII. 313 Synthesis of Trimetoquinol Derivatives by Chiral Catalytic Hydrogenation. RO H2 60 psi RO CH N ^CH, Ru(S- or R-BINAP)CI2 ------OCH OCH, EtOH 60 °C OCH OCH, c h 3o 315 R = Bn 317 R = Bn (S) 376r = c h 3 378 R = Bn (R) 379 R = CH3 (S) T—I------1------1------1------1------1------1 T f ' T" ' 1..... 7.0 6.5 6.0 Figure 43. Partial 1H NMR Spectra of Methoxy Acetamide 379. In order to determine the enantiomeric excess from the chiral reduction, the acetamide group must be cleaved. Several attempts to cleave the acetamide group according the literature procedure with hydrazine and Scheme LXIII. 314 Synthesis of (±) Dibenzyloxy Acetamide 380. BnO BnO NH HCI -o- BnO BnO CH OCH OCH 271 352 OCH OCH Scheme LXIV. Synthesis of (±)-Methoxy Trimetoquinol Derivative 382. CH3° 1. POCI3 /C H 3CN ► NH HCI CH30 2. NaBH 4 / EtOH k ^ . O C H , OCH 3. HCI (g) / MeOH 375 381 V ™ , OCH c h 3o Ac20 /E t 3N CH OCH CH2CI2 382 OCH CH30 potassium hydroxide in ethylene glycol were only moderately successful (Scheme LXV) [324]. Benzyloxy (S)-acetamide 317 was cleaved in only 24% yield, and methoxy acetamide decomposed under these conditions. In addition, hydrolysis with 70% sulfuric acid caused decomposition and no reaction was observed with 10% sulfuric acid. A more efficient means of removing the Scheme LXV. 315 Hydrazinolysis of Benzyloxy Trimetoquinol Derivative 349. BnO ^ ^ BnO BnO 1.NH2NH2 /KOH ; ► BnO 5 OCH 170 °C ethylene glycol 2. HCI (g) / MeOH 317 S ^ o c h 3 c h3o acetamide group is currently under investigation. Deacetylated (S)-dibenzyloxy amine derivative 349 was shown to have an enantiomeric excess of 95% by the TAGIT method as described above. The importance of this methodology is the extension to dehydro trimetoquinol derivatives 329-332 which would provide the chiral intermediates necessary for the synthesis of irreversibly binding iodinated trimetoquinol derivatives 251 and 252. Bischler-Napieralski cyclization of amide 294 with phosphorous oxychloride in anhydrous acetonitrile with subsequent acetylation with acetic anhydride and triethylamine provided a mixture of compounds. These compounds were separated by silica gel chromatography to provide (Z)-enamide 329 (Scheme LXVI). Catalytic reduction of this enamide with ruthenium(ll) dichloride catalyst 383 in ethanol at 60 °C provided acetamide 384 which also has restricted rotation about the amide bond and a complex 1H NMR spectra (Figure 44). The downfield aromatic resonances from 8.17-8.01 and 7.22-7.07 ppm are characteristic of a para-substituted nitro ring showing that the Scheme LXVI. 316 Synthesis of (S)-4-Nitro Trimetoquinol Derivative 384 by Chiral Catalytic Hydrogenation. BnO 1. POCI3 /CH 3CN -► BnO N -^ C H 2. A c20 / Et3N BnO H2 60 psi Ru (S-BINAP)CI2 BnO CH 383 EtOH 60 °C 384 NO. nitro group is stable to these hydrogenation conditions. The successful reduction of nitroenamide 329 has provided the impetus for Bischler-Napieralski cyclization of amide 296 with phosphorous oxychloride in anhydrous acetonitrile followed by acetylation with acetic anhydride and triethylamine to provide (Z)- enamide 330 (Scheme LXVI I). Likewise, amide 294 may be cyclized with phosphorous oxychloride and acylated with methylchloroformate and triethylamine to form urethane 332 (Scheme LXVIII). This work is currently in progress. 317 ea & in ID lO CS 8 .58.0 7.5 7 .0 6.5 6 Figure 44. Partial 1H NMR Spectrum of (S)-4-Nitro Acetamide 384. 318 Scheme LXVII. Attempted Synthesis of 4-Acetoxy-3-nitro Enamide 330. BnO BnO J l BnO :CO" BnO N 0 2 296 330 OAc Scheme LXVIII. Attempted Synthesis of 4-Nitro Enurethane 332. BnO BnO 2 . methyl chloroformate Et3N 11.4 Summary 1. The enantiomers of aminourethane derivative 308 were successfully resolved on an analytical ChiralcelR OD column with methanol as the mobile phase. The generally applicability of this technique to successfully resolve other trimetoquinol derivatives is suspect since the enantiomers of free bases 253 and 254 as well as urethane 307 were unable to be separated. 2. The enzymatic resolution of dibenzyloxy derivative 271 with the protease subtilisin Carlsberg and an active ester was unsuccessful at several temperatures. 3. Dibenzyloxy trimetoquinol derivatives 349 and 351 were asymmetrically synthesized in high enantiomeric purity with chiral formamidines. However, the required isoquinoline intermediates were found to be partial hydrates which severely hindered the success of this methodology. Several functionalized benzyl bromides were prepared to supply functionalized trimetoquinol derivatives. Multiple attempts to produce these functionalized derivatives were unsuccessful. 4. The determination of enantiomeric purity was accomplished by the in situ preparation of TAGIT-diastereomers with subsequent separation on a reverse phase column. This technique is suitable for the analysis of both dibenzyloxy and catechol derivatives. Asymmetric catalytic hydrogenation of enamide derivatives was successfully accomplished with ruthenium-BINAP dichloride catalysts at elevated temperatures and pressures in a Parr hydrogenator. The catalytic reduction of nitroenamide 329 provides a technique for the successful asymmetric synthesis of functionalized trimetoquinol derivatives. CHAPTER XII EXPERIMENTAL The general information concerning instrumentation, elemental analysis, and solvent preparation provided in Chapters 4 and 8 is also applicable to this chapter. Additionally, UV spectra were obtained at The Ohio State University College of Pharmacy with a Kontron Uvikon 860 Spectrophotometer. Optical rotations were determined at The Ohio State University College of Pharmacy with a Perkin Elmer 241 Polarimeter. Anhydrous toluene was stored over sodium/benzophenone and distilled prior to use. Anhydrous dioxane was stored i over lithium aluminum hydride and distilled prior to use. General Approach for the Analytical Resolution of Racemic Amines and Urethanes by Chiral-HPLC on a Chiralcel-OD Column : Racemic amine free bases or urethanes were dissolved in dichloromethane for sample injection. The HPLC system specifications : Beckman System Gold HPLC utilizing two model 11 OB pumps, a model 406 analog interface, and a model 167 scanning uv detector (utilized at 254 nm). The system was fitted with a Chiralcel-OD 321 322 column (Beckman, 10 microns, 25 cm x 4.6 mm) with isopropanol/hexanes, water/methanol, or methanol as the mobile phase and flow rates from 0.3 -1.0 mL/min. General Procedure for Enantiomeric Purity Determination by Chiral-HPLC on a Pirkle Column : In a manner similar to Meyers [335], the tetrahydroisoquinoline was dissolved in dichloromethane (2 mL/mmol) and charged with a-naphthoyl chloride (1.5 equiv) and triethylamine (0.25 mL/mmol). The mixture was stirred at room temperature for 30 min, and evaporated in vacuo. The residue was subjected to silica gel chromatography with ethyl acetate/hexanes/dichloromethane (10/40/50) as the eluent to remove excess a-naphthoyl chloride and triethylamine hydrochloride. The appropriate fractions were pooled and solvent was evaporated in vacuo. The solid residue was dissolved in hot isopropanol for sample injection. The HPLC system specifications : Beckman System Gold HPLC utilizing two model 110B pumps, a model 406 analog interface, and a model 167 scanning uv detector (utilized at 254 nm). The system was fitted with a Pirkle Covalent PhenylglycineR column (Regis, Morton Grove, IL, 5 microns, Spherisorb, 25 cm x 4.6 mm) with 10% isopropanol/hexanes as the mobile phase at a flow rate of 5 mL/min. 323 General Procedure for Enantiomeric Purity Determination with TAGIT by RP-HPLC on an ODS-8 Column : In a manner similar to the procedure of Nishi [336], the tetrahydroisoquinoline (1 mg) was dissolved in dimethylformamide (1 mL). A 100 \.iL aliquot of analyte solution was vortexed with 100 \iL of a 0.5% (w/v) solution of TAGIT (2,3,4,6-tetra-0-acetyl-l3-D-glucopyranosyl isothiocyanate) in acetonitrile and 20 ^iL of a 10% (v/v) solution of triethylamine in acetonitrile. After standing for 15 min, The sample was quenched with 20 fxL of a 10% (v/v) solution of ethanolamine in acetonitrile. After an additional 5 min, the sample was diluted with 200 of 10% (v/v) acetic acid, and this acidic mixture is ready for injection. The HPLC system specifications : Beckman Model 420 or 421 System Controller utilizing two model 112 pumps and a model 153 fixed wavelength uv detector (254 nm). The system was fitted with an IBM EC-C8 column (5 microns, 15 cm x 4.6 mm) with acetonitrile/0.05 M phosphate buffer (pH 3.0) as the mobile phase. Dibenzyloxy derivatives were analyzed with 40% acetonitrile at a flow rate of 2 mL/min while catechol derivatives were analyzed with 28% acetonitrile with a flow rate of 1 mL/min. 324 BnO NH HCI BnO OCH3 OCH (±)-6.7-Bis(benzvlox v)-1 - (3.4,5-trimethoxvbenz vl)-1.2,3,4- tetrahvdroisoquinoline hydrochloride. (271) : Amide 272 (15.0 g, 27.7 mmol) was dissolved in anhydrous acetonitrile (30 ml_) under argon and charged with phosphorous oxychloride (15 mL). The resulting solution was heated to reflux for 4.5 h, and allowed to cool to room temperature under argon. Solvent was evaporated in vacuo. The resulting oil was dissolved in chloroform (100 mL) and poured in to an ice slurry (200 mL). The layers were separated, and the aqueous layer was extracted with chloroform (100 mL). The organic extracts were combined and washed successively with 5% aqueous sodium hydroxide (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in absolute ethanol (100 mL) and cooled to 0 °C with an ice bath. Sodium borohydride (4.19 g, 111 mmol) was added portionwise to prevent excessive foaming. The flask was fitted with an anhydrous calcium sulfate drying tube, and the suspension was allowed to stir overnight warming to room temperature. Ethanol was removed in vacuo, and the residue was dissolved in chloroform (200 mL). This solution was 325 successively washed with 5% aqueous sodium hydroxide (200 mL), water (200 mL), brine (200 mL), and dried over anhydrous magnesium sulfate. Solvent was evaporated in vacuo, and the residue was dissolved in methanol (30 mL), and charged with a saturated solution of hydrogen chloride (g) in methanol (10 mL). After stirring overnight, the solid was collected by vacuum filtration. The solid was dissolved in hot ethanol, filtered and diluted with diethyl ether until the solution became cloudy. After standing overnight, the precipitate was collected to yield 11.02 g (71%) of a white solid, mp 209-210 °C (lit. [221] mp 205-207 °C). 1H NMR (CD3OD) : 6 7.45-7.27 (m, 10H, ArH), 6.89 (s, 1H, ArH), 6.63 (s, 1H, ArH), 6.58 (s, 2H, ArH), 5.12 (s, 2H, ArCH20), 4.95 (ABq, J = 12.1 Hz, Au = 15.2 Hz, 2H, ArCH20), 4.68 (t, J= 7.2 Hz, 1H, ArCHRN), 3.80 (s, 6H, 2 x CH30), 3.74 (s, 3H, CH30), 3.51-3.43 (m, 1H, CH2), 3.35-3.24 (m, 2H, CH2), 3.10-2.96 (m, 3H, CH2). o 2.2.2-Trifluoroethvl butyrate, (314) : Butyric anhydride 333 (22.5 mL, 0.14mol) was placed in a 100 mL rb flask and cooled to 0 °C with an ice bath. 2.2.2-trifluoroethanol (10 mL, 0.14 mol) was added dropwise over 30 min via an addition funnel. The ice bath was removed, and the mixture was stirred at room temperature overnight followed by heating the mixture to reflux for 24 h. The 326 product was collected by short path distillation to yield 20.42 g (88%) of clear liquid, bp 110-115 °C (lit. [340] bp 111-112 °C) with a noxious odor. 1H NMR (CDCI/TMS) : 6 4.47 (q, JHF = 8.5 Hz, 2H, CH2CF3), 2.40 (t, J = 7.4 Hz, 2H, RCH2C0 2), 1.69 (m, J= 7.4 Hz, 2H, CK>CH3), 0.97 (t, J= 7.4 Hz, 3H, CH3). General Approach to the Enzymatic Resolution of a Dibenzyloxy Trimetoquinol Derivative : Tetrahydroisoquinoline 271 (5.0 q. 0.01 mmol) was converted to its free base with 5% aqueous sodium hydroxide (100 mL) and chloroform (100 mL). The layers were separated, the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo to an oil. The oil was dried under high vacuum over phosphorous pentoxide for 24 h before being dissolved in 3-methyl-3-pentanol (20 mL) to provide a 0.5 M solution. The mixture was charged with ester 314 (1.5 mL, 0.01 mmol, 0.5 M) and subtilisin Carlsberg (40 mg, 2 mg/ml). The suspension was shaken on a Lab-Line Orbit Environ Shaker at 250 rpm for 72 h at 30, 37, and 45 °C. The progress of the reaction was monitored by TLC and RP-HPLC at various times to monitor enzymatic conversion of the free base amine to an amide. No reaction was observed at any temperature during the experiment. 327 BnO -'tk ^ v s /NH (cooh)2 6.7-Bis(benzvloxv)-1,2,3,4-tetrahvdroisoquinoline oxalate salt, (334) : 3,4-Dibenzyloxyphenethylamine hydrochloride 98 (10.0 g, 27 mmol) was dissolved in a formic acid (20 ml_) and charged with a 37% (w/w) solution of formaldehyde (2.0 mL, 27 mmol). The mixture was heated at 50 °C overnight. After cooling to room temperature, the mixture was concentrated in vacuo to a thick oil. This oil was dissolved in ethanol (50 mL) and added dropwise to a solution of oxalic acid (6.8 g, 54 mmol) in ethanol (50 mL) with vigorous stirring. The precipitate was collected by vacuum filtration to yield 8.69 g (93%) of a white solid. This material is a mixture of 2 compounds. The oxalate salts were converted to their free bases by stirring with chloroform (100 mL) and 10% aqueous sodium hydroxide (100 mL). The layers were separated, the aqueous layer was extracted with chloroform (100 mL), and the organic extracts combined. The chloroform extracts were washed with 10% aqueous sodium hydroxide (100 mL), water (2 x 100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting solid was subjected to silica gel chromatography with ethyl acetate/dichloromethane/triethylamine (10/10/1) as the eluent. After the first compound had eluted, the eluent was changed to ethyl acetate/dichloromethane/ methanol/triethylamine (10/10/1/1) 328 to elute the desired product. Appropriate fractions were pooled an evaporated in vacuo to yield 5.6 g (60%) of a light brown solid that was immediately used without further purification. 1H NMR (free base, CDCI/TMS) : 6 7.45-7.25 (m, 10H, ArH), 6.67 (s, 1H, ArH), 6.89 (s, 1H, ArH), 5.11 (s, 2H, ArCH20), 5.10 (s, 2H, ArCH20), 3.88 (s, 2H, ArCH2N), 3.07 (t, J= 5.9 Hz, 2H, ArCH2CbL,N), 2.65 (t, J= 5.9 Hz, 2H, ArCb^CH;,), 1.87 (bs, 1H, NH). A portion of this material was converted to its oxalate salt by dissolving the amine in ethanol and adding this solution to a solution of oxalic acid dihydrate (2 equiv) in ethanol. The product was collected by vacuum filtration to yield a white solid, dp 220-221 °C 1H NMR (CD3OD) : 6 7.44-7.28 (m, 10H, ArH), 6.88 (s, 1H, ArH), 6.84 (s, 1H, ArH), 5.12 (s, 2H, ArCH20), 5.10 (s, 2H, ArCH20), 4.21 (s, 2H, ArCH2N), 3.42 (t, J = 6.3 Hz, 2H, ArCH2CHJ, 2.98 (t, J= 6.3 Hz, 2H, ArCb^CH,). FAB MS m/z346.2 (m + 1, base). Analysis for C^H ^^O g 1/4H20 : calc. C, 68.25; H, 5.84; N, 3.18; found C, 68.40; H, 5.60; N, 3.22. c h2o h h2n - 4 - h c h 3 ''* s c h 3 (S)-2-Amino-3-methvl-butanol, (336) : In a manner similar to Meyers [337], L-valine 335 (22.2 g, 0.19 mol) was suspended in anhydrous tetrahydrofuran (300 mL) at 0 °C, and charged with sodium borohydride (14.4 g, 0.38 mol). 329 Boron trifluoride etherate (94 mL, 0.76 mol) was added dropwise to the mixture over 15 min. The ice bath was removed, and the mixture was stirred for 2 h at room temperature and heated to reflux overnight. After cooling to room temperature, the reaction was quenched by the careful dropwise addition of methanol (120 mL) over 30 min with subsequent stirring at room temperature for an additional 30 min. Solvent was evaporated in vacuo, and the solid residue was dissolved by heating to reflux in 20% aqueous sodium hydroxide (120 mL) for 1 h. After cooling to room temperature the solution was extracted with chloroform (3 x 100 mL) and then diethyl ether (2 x 100 mL). The organics were combined, washed with brine (200 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo at room temperature to yield 18.0 g (92%) of an oil that was used without purification. 1H NMR (CDCI/TMS) : 6 3.64 (dd, J = 10.5 and 3.9 Hz, 1H, CH20), 3.29 (dd, J = 10.5 and 8.8 Hz, 1H, CH20), 2.60-2.52 (m, 1H, RR'CHN), 1.96 (bs, 3H, NH2 and OH), 1.56 (m, J= 6.7 Hz, 1H, CH(CH3)2), 0.93 (d, J=6.7 Hz, 3H, CH3), 0.91 (d, J= 6.7 Hz, 3H, CH3). O CH2OtBu A I H H N -1 — H c h3 ^ c h3 fS)-N-ri-f(1.1-dimethvlethoxv)methvn-2-methvlpropyl1formamide. (338) : According to the procedure of Dickman [341], (S)-valinol 336 (18.0 g, 0.17 mol) and ethyl formate (14.0 g, 0.19 mol) were heated to reflux for 1 h 330 under an argon atmosphere. After cooling to room temperature, solvent was evaporated in vacuo. The resulting oil was triturated in diethyl ether until a solid formed. The solid was collected by vacuum filtration to yield 18.62 g (81%), mp 66-67.5 °C (lit. [341,342] mp not reported). The 1H NMR spectrum is complicated by restricted rotation about the amide bond resulting in 2 conformations in a 2:1 ratio. 1H NMR (CDCI/TMS) : 6 8.24 and 8.00 (d, J = 1.7 Hz, and d, J= 13.5 Hz, 1H, CHO), 6.65-6.55 and 6.25-6.18 (bm, 1H, NH), 3.83- 3.79 (m, 1H, RR’CHN), 3.75-3.60 (m, 2H, CH20), 3.19-3.10 (m, 1H, OH) 1.88 (m, J= 6.9 Hz, 1H, CH(CH3)2), 0.99-0.91 (m, 6H, C H (C hy2). The crude (S)- formamide (10.0 g, 76 mmol) was suspended in anhydrous dioxane (65 mL) in a 500 mL pressure bottle immersed in an ice bath. The bottle was charged with liquid isobutene 337 (65 mL) and boron trifluoride etherate (18 mL). The bottle was sealed with a rubber stopper, removed from the ice bath, and stirred at room temperature for 3 h. The stopper was carefully removed. After evolution of the gas had ceased, the mixture was poured into a 500 mL separatory funnel containing 5% aqueous sodium hydroxide (125 mL). The aqueous layer was extracted with dichloromethane (3 x 100 mL), and the extracts were combined. The organic extracts were washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was purified by vacuum distillation to yield 8.2 g (57%) of a clear colorless oil, bp 114 °C /1.65 mm Hg (lit. [341] 80-85 °C / 0.05 mm Hg). The 1H NMR spectrum is complicated by restricted rotation about the amide bond resulting in 331 2 conformations in a 2:1 ratio. 1H NMR (CDCI/TMS) : 6 8.22 and 8.04 (d, J = 1.5 Hz, and d , J = 12.0 Hz, 1H, CHO), 5.90 (bs, 1H, NH), 3.90-3.80 and 3.20-3.10 (m, 1H, RR’CHN), 3.53-3.30 (m, 2H, CH20), 1.93-1.87 (m, J = 6 .6 Hz, 1H, CH^CHgJg), 1.17 (s, 9H, q C H g )/ 0.97-0.91 (m, 6H, CH(Chy2). NMe2 CH2OtBu H ^ N — f - H C H 3 ' ^ N C H 3 (S)-N'-M -F(1.1 - Pi methyl ethoxy) methv II-2-methyl pro pvII-N.N- dimethvlmethanimidamide, (339) : (S)-formamide 338 (8.2 g, 44 mmol) was dissolved in ethanol (30 mL), charged with 20% aqueous potassium hydroxide (60 mL), and heated to reflux overnight. After cooling to room temperature, the mixture was extracted with diethyl ether (3 x 100 mL), and the extracts were combined. The ether layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo at room temperature. The resulting oil was charged with N,N-dimethylformamide dimethyl acetal (8.6 mL, 65 mmol) and heated to reflux for 1 h under an argon atmosphere. The mixture was concentrated in vacuo and vacuum distilled to yield 7.54 g, (80%) of a clear colorless oil, bp 110-115 °C /1.0 mm Hg (lit [341] bp 55-65 °C / 0.05 mm Hg). 1H NMR (CDCI/TMS) : b 7.23 (s, 1H, HCNN), 3.51 (dd, J= 8.9 and 5.7 Hz, 1H, CH20), 3.17 (dd, J = 8.9 and 7.0 Hz, 1H, CH20), 2.82 (s, 6H, 332 N(CH3)2), 2.74-2.70 (m, 1H, RR’CHN), 1.84 (m, J= 6.5 Hz, 1H, CH(CH3)2), 1.16 (S, 9H, C(CH3)3), 0.86 (d, J = 6.6 Hz, 6H, (C H ^C H ). [a]*5 -16.5 0 (c = 1.0, THF) (lit. [341] [a]*5 -15.9 0 (c = 0.98, THF)). BnO _ (COOH)2 1 BnO tBuO (S)-6.7-B is (benzyl ox v)-2-riT1 -Td.1 -dimethvlethoxv) methyl!-2- methvlpropvlliminolmethvll-1,2.3,4-tetrahvdroisoquinoline oxalate salt (340) : In a manner similar to the procedure of Meyers [343], (S)-dimethylformamidine 339 (2.05 g, 9.55 mmol) was dissolved in anhydrous toluene (50 mL) and charged with the free base of isoquinoline 334 (3.0 g, 8.68 mmol) and a catalytic amount of p-toluenesulfonic acid (2 mg). The mixture was heated to reflux for 48 h, cooled to room temperature, and evaporated in vacuo. The residue was dissolved in ethyl acetate, washed with saturated sodium bicarbonate (100 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The oily residue was submitted to silica gel chromatography with ethyl acetate/ dichloromethane/triethylamine (25/25/1) as the eluent. Appropriate fractions were pooled and evaporated in vacuo to yield 3.92 g (88%) of a thick, viscous, pale yellow oil. 1H NMR (free base, CDCI/TMS) : 6 7.43-7.22 (m, 11H, ArH, and CHNN), 6.68 (s, 1H, ArH), 6.67 (s, 1H, ArH), 5.10 (s, 4H, ArCH20), 4.35 (d, J = 3.9 Hz, 2H, ArCH2N), 3.52 (dd, J = 8.8 and 5.3 Hz, 1H, CH2Ot-Bu), 3.42 (t, J= 5.7 Hz, 2H, ArCH2CH>N), 3.20 (dd, J = 8.8 and 7.2 Hz, 1H, CH2Ot-Bu), 2.77-2.66 (m, 3H, ArCH,CH2N and RR’CHN), 1.83 (m, J = 6.5 Hz, CH(CH3)2), 1.13 (s, 9H, C(CH3)3), 0.87 (d, J = 6.6 Hz, 3H, ( C ]i) 2CH), 0.86 (d, J = 6.6 Hz, 3H, (CHJaCH). Analysis for C ^H ^N A 1/2H20 : calc. C, 75.68; H, 8.28; N, 5.35; found C, 75.38; H, 8.28; N, 5.40. [a]*5 -18.5° (c = 1.5, CHCI3). A portion of this oil was converted to its oxalate salt by dissolving the formamidine (140 mg, 0.27 mmol) in diethyl ether (10 mL) and adding this solution dropwise to a solution of oxalic acid dihydrate (0.54 mmol) in diethyl ether (10 mL). The mixture oiled. Ethanol was added (2 mL), and the oil was triturated until a precipitate formed. After standing overnight, the precipitate was collected by vacuum filtration to yield 82 mg (50%) of a white solid, mp 117-119 °C. The 1H NMR is complicated due to 2 conformations in solution in about a 3:2 mixture. 1H NMR (CD3OD) : 6 8.10 (s, 1H, CHNN), 7.44-7.27 (m, 10H, ArH), 6.93 and 6.89 (s, 1H, ArH), 6.88 and 6.79 (s, 1H, ArH), 5.12 and 5.11 (s, 4H, ArCH20), 4.65 and 4.58 (s, 2H, ArCH2N), 3.82-3.79 (m, 1H, CH2Ot-Bu), 3.72-3.58 (m, 2H, ArCH.CH/sl), 3.41-3.31 (m, 1H, CH2Ot-Bu), 3.19-3.12 (m, 1H, RR’CHN), 2.98-2.88 (m, 2H, A rC H A -y, 1.95 (m, 1H, CH(CH3)2), 1.14 and 1.09 (s, 9H, C(CH3)3), 1.02-0.97 (m, 6H, (C H ^C H ). FAB MS m/z 515.5 (m + 1 - C2H20 4, base). Analysis for 334 C35H44N2°7'1/4H20 : calc. C, 69.00; H, 7.36; N, 4.60; found C, 68.60; H, 7.17; N, 4.68. CH2OtBu H —|— NH H 1 CH3 ''"jis'CH30 (R)-N-M-ff1,1 -dimethvlethoxv)methvn-2-methvlpropvl1formamide, (342) : In a manner similar to Meyers [337], D-valine 341 (25 g, 0.21 mol) was suspended in anhydrous tetrahydrofuran (300 mL) at 0 °C, and charged with sodium borohydride (16.1 g, 0.43 mol). Boron trifluoride etherate (105 mL, 0.85 mol) was added dropwise to the mixture over 30 min. The ice bath was removed, and the mixture was stirred for 2 h at room temperature and heated to reflux overnight. After cooling to room temperature, the reaction was quenched by the careful dropwise addition of methanol (125 mL) over 30 min with subsequent stirring at room temperature for an additional 30 min. Solvent was evaporated in vacuo, and the solid residue was dissolved by heating to reflux in 20% aqueous sodium hydroxide (100 mL) for 1 h. After cooling to room temperature the solution was extracted with chloroform (3 x 100 mL). The organics were combined, washed with brine (200 mL),dried over anhydrous sodium sulfate, and evaporated in vacuo at room temperature to yield 19.5 g (89%) of (R)-valinol as an oil that was used without purification. According to the procedure of Dickman [341], (R)-valinol (19.5 g, 0.19 mol) and ethyl formate 335 (15.4 g, 0.21 mol) were heated to reflux for 1 h under an argon atmosphere. After cooling to room temperature, solvent was evaporated in vacuo. The resulting oil was triturated in diethyl ether until a solid formed. The solid was collected by vacuum filtration to yield 19.1 g (77%) of the (R)-formamide as a white solid, mp 65-66 °C (lit [341,342] mp not reported). The 1H NMR spectrum is in agreement with that described for the (S)-isomer on page 330. The (R)-formamide (19.1 g, 146 mmol) was suspended in anhydrous dioxane (100 mL) in a 500 mL pressure bottle immersed in an ice bath. The bottle was charged with liquid isobutene 337 (150 mL) and boron trifluoride etherate (35 mL). The bottle was sealed with a rubber stopper, removed from the ice bath, and stirred at room temperature for 3 h. The stopper was carefully removed. After evolution of the gas had ceased, the mixture was poured into a 500 mL separatory funnel containing 5% aqueous sodium hydroxide (250 mL). The aqueous layer was extracted with dichloromethane (3 x 150 mL), and the extracts were combined. The organic extracts were washed with brine (200 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was purified by vacuum distillation to yield 17.7 g (65%) of a clear colorless oil, bp 105-110 °C / 1.5 mm Hg (lit. [341] 80-85 °C / 0.05 mm Hg) that was used without further purification. The 1H NMR spectrum is in agreement with that described for the (S)-isomer on page 331. (R)-N*-n -f(1,1 -Pi methyl ethoxy) methyl!-2-methvl prop yll-N.N- dimethvlmethanimidamide, (343) : (R)-formamide 342 (17.7 g, 95 mmol) was dissolved in ethanol (100 mL), charged with 20% aqueous potassium hydroxide (100 mL), and heated to reflux overnight. After cooling to room temperature, the mixture was extracted with diethyl ether (3 x 100 mL), and the extracts were combined. The ether layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo at room temperature. The resulting oil was charged with N,N-dimethylformamide dimethyl acetal (17 mL, 128 mmol) and heated to reflux for 1 h under an argon atmosphere. The mixture was concentrated in vacuo and vacuum distilled to yield 16:62 g, (82%) of a clear colorless oil, bp 106-110 °C /1.0 mm Hg (lit [341] bp 55-65 °C / 0.05 mm Hg). 1H NMR spectrum is in agreement with that described for the (S)-isomer on page 331. H d 5 +16.8 0 (c = 1.0, THF). 337 BnO ___ >|Yl (COOH)2 N» o '-' ------L_ tBuOT ' (R)-6,7-Bis(benzvloxv)-2-rrf1 -f(1,1 -d i methyl ethoxy) methvll-2- methvlpropvniminolmethvn-1.2.3,4-tetrahvdroisoquinoline oxalate salt, *3441 : In a manner similar to the procedure of Meyers [343], (R)-dimethyformamidine 343 (1.59 g, 7.4 mmol) was dissolved in anhydrous toluene (50 mL) and charged with the free base of isoquinoline 334 (2.33 g, 6.7 mmol) and a catalytic amount of p-toluenesulfonic acid (3 mg). The mixture was heated to reflux for 48 h under argon, cooled to room temperature, and evaporated in vacuo. The residue was dissolved in ethyl acetate (50 mL), washed with saturated sodium bicarbonate (50 mL), brine (25 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The oily residue was submitted to silica gel chromatography with ethyl acetate/dichloromethane /triethylamine (25/25/1) as the eluent. Appropriate fractions were pooled and evaporated in vacuo to yield 2.95 g (85%) of a thick, viscous, pale yellow oil. 1H NMR spectrum for the free base is in agreement with that described for the (S)-isomer on page 333. A portion of this oil was converted to its oxalate salt by dissolving the formamidine (250 mg, 0.49 mmol) in diethyl ether (10 mL) and 338 adding this solution dropwise to a solution of oxalic acid dihydrate (122 mg, 0.97 mmol) in diethyl ether (20 mL). After standing, the precipitate was collected by vacuum filtration to yield 293 mg (quantitative) of a white solid, mp 117-118 °C. 1H NMR spectrum for the oxalate salt is in agreement with that described for the (S)-isomer on page 333. [a]p5+21.0 0 (c = 1.5, CHCI3). FAB MS m/z 515.4 (m + 1 -C2H20 4, base). Analysis for C35H44N20 7-1/2H20 : calc. C, 68.50; H, 7.39; N, 4.56; found C, 68.74; H, 7.22; N, 4.54. OCH 3.4.5-Tr8methoxvbenzvl alcohol. (346) A solution of 3.4.5-trimethoxybenzaldehyde 345 (10.0 g, 51 mmol) in a mixture of tetrahydrofuran (45 mL) and isopropanol (5 mL) was charged with sodium borohydride (2.0 g, 53 mmol) and stirred overnight at room temperature. Solvent was evaporated in vacuo, and the residue was dissolved in ethyl acetate (100 mL) and water (100 mL). The layers were separated, and the organic layer was washed with brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The product was purified by vacuum distillation to yield 8.8 g (87%) of a clear colorless liquid, bp 165-172 °C / 1.0 mm Hg (lit. [344] bp 115-120 °C / 0.03 mm Hg). 1H NMR (CDCI/TMS) 339 : 6 6.61 (s, 2H, ArH), 4.64 (s, 2H, CH2), 3.87 (s, 6H, 2 x CH30), 3.84 (s, 3H, CH30), 1.72 (bs, 1H, OH). OCH O C H 3 3,4.5-Trimethoxvbenzvl bromide, (3471 : Hydrogen bromide (g) was bubbled into a solution of 3,4,5-trimethoxybenzyl alcohol 346 (5.0 g, 25.2 mmol) in chloroform (50 mL) over 1.5 h. The solution was dried with anhydrous magnesium sulfate and evaporated in vacuo to produce 6.06 g (92%) of a dark solid, mp 69-72 °C. This material was purified prior to use by passing the material through a short silica gel column with chloroform as the eluent. Evaporation of the appropriate fractions yielded a light yellow solid, mp 74-75.5 °C (lit. [345] mp 75-78 °C). 1H NMR (CDCI/TMS) : 6 6.62 (s, 2H, ArH), 4.47 (s, 2H, CH./ 3.86 (s, 6H, 2 x CH30), 3.84 (s, 3H, CH30). 340 BnO NH HCI BnO OCH OCH (S)-6.7-bis(benzvloxv)-1-(3,4,5-trimethoxvbenzvn-1,2,3.4- tetrahvdroisoquinoline hydrochloride, (349) : In a manner similar to Meyers [346], (S)-formamidine free base 340 (400 mg, 0.78 mmol) was dissolved in anhydrous tetrahydrofuran (6 mL) in an oven dried 3-neck flask fitted with an argon port, an addition funnel, and a septum inlet. Benzyl bromide 347 (264 mg, 1.01 mmol) was dissolved in anhydrous tetrahydrofuran in the addition funnel. The flask was cooled to -78 °C with a C 02/acetone bath. A solution of 1.7 M t-butyl lithium in pentane (0.55 mL, 0.93 mmol) was added to the light yellow solution of formamidine producing a deep red solution. After stirring for 30 min at -78 °C, the temperature was lowered to -100 °C with a C 02/diethyl ether bath. The benzyl bromide solution was added dropwise over a 15 min period turning the deep red solution to a bright yellow. After stirring for an additional 30 min at -100 °C, the reaction was quenched with 20% aqueous ammonium chloride (1 mL) and allowed to warm to room temperature. The mixture was concentrated in vacuo to remove solvent. The residue was dissolved in ethyl acetate (20 mL), washed with 20% aqueous ammonium chloride (20 mL), brine (10 mL) dried over anhydrous sodium sulfate, and evaporated in vacuo. The orange oil (517 mg, 96% yield) was dissolved in a mixture of isopropanol (4 mL), water (2 mL), hydrazine (2 mL), and acetic acid (1 mL). After stirring overnight at room temperature, solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate (20 mL), washed with water (20 mL), brine (10 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was subjected to silica gel chromatography (deactivated with 2% triethylamine) with ethyl acetate/dichloromethane (50/50) as the eluent. Appropriate fractions were pooled and evaporated in vacuo. The oily residue was dissolved in a saturated solution of hydrogen chloride (g) in methanol (10 mL) and stirred overnight. Solvent was evaporated in vacuo, and the residue was dissolved in hot ethanol (4 mL), filtered, and diluted with diethyl ether until cloudy. The deposited solid was collected by vacuum filtration to yield 97 mg (22%) of an off white solid, mp 174-175 °C. Spectroscopic data is consistent with the spectra for the racemic product on page 325. [a]^5 +28.2 0 (c = 0.25, MeOH). Optical purity was determined to be 94% by the TAGIT method (page 323). 342 (R)-6,7-bis(benzvloxv)-1 - (3,4,5-trimethoxvbenzvl)-1,2,3,4- tetrahvdroisoauinoline hydrochloride. (351) : Prepared as for the (S)-isomer above from (R)-formamidine 344 (500 mg, 0.97 mmol) to yield 69 mg (13%) of a white solid, mp 166-168 °C. 1H NMR is in agreement with that described for the racemic product on page 325. [a]^5 -26.1° (c = 0.5, MeOH). Optical purity was determined to be 97% ee by the TAGIT method (page 323). HO NH HCI HO OCH OCH (S)-6.7-dihvdroxv-1-(3,4.5-trimethoxvbenzvl)-1,2,3,4-tetrahvdroisoquinoline hydrochloride. (209) : (S)-lsoquinoline 349 (50 mg, 0.09 mmol) via chiral formamidine synthesis was dissolved in a mixture of methanol (4 mL) and concentrated hydrochloric acid (4 mL) under an argon atmosphere. The mixture was heated to reflux overnight, cooled to room temperature, and evaporated in vacuo. The residue was rinsed with methanol (10 mL) and evaporated in vacuo. This procedure was repeated for a total of three rinses. The residue was dissolved in hot ethanol (4 mL), filtered, and diluted with diethyl ether until the mixture became cloudy. The precipitated product was collected by vacuum filtration to yield 32 mg (94%) of a white solid, dp 155-157 °C (with darkening, lit. [334] dp 151-153.5 °C). 1H NMR (CD3OD) : 6 6.65-6.60 (m, 4H, ArH), 4.65-4.58 (m, 1H, ArCHRN), 3.82 (s, 6H, 2 x CH30), 3.76 (s, 3H, CH30), 3.45-3.35 (m, 2H, CH2N), 3.30-3.20 (m, 1H, ArCH2, obscured by solvent), 3.02-2.90 (m, 3H, ArCH2). [a]*5 -18.0° (c = 0.05, MeOH) [lit. [334] [a]*0 -28.5° (c = 1.16, MeOH)]. Optical purity was determined to be 99% ee by the TAGIT method on page 323. BnO BnO OCH (±)-6.7-Bis(benzvloxv)-2-(1 -naphthovl)-1 -(3 A5-trimethoxvbenzvD-1,2,3.4- tetrahvdroisoquinoline, (352) : To a solution of tetrahydroisoquinoline 271 (500 mg, 0.89 mmol) in dichloromethane (20 mL) was added 1-naphthoyl chloride (254 mg, 1.32 mmol) and triethylamine (2 mL). The mixture was stirred at room temperature for 4 h, and washed with 5% aqueous sodium hydroxide (20 mL), brine (20 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was purified by silica gel chromatography with ethyl acetate/hexanes/dichloromethane (10/40/50) as the eluent. Appropriate fractions were pooled and evaporated in vacuo. The resulting oil < was dissolved in minimal hot ethyl acetate, allowed to cool, and placed in the freezer. The deposited solid was collected by vacuum filtration to yield 456 mg (76%) of a white solid, mp 147-148 °C. The compound is a mixture of multiple conformations in solution providing a complex 1H NMR spectrum. Temperature elevation studies in d6-DMSO to 357 K did not simplify the 1H NMR spectrum sufficiently for the assignment of proton resonances. FAB MS m/z 680.4 (m + 1, 29%), 498.4 (m - C10Hl3O3, base). Analysis for : calc. C, 77.74; H, 6.08; N, 2.06; found C, 77.54; H, 6.10; N, 2.01. c h2oh NHAc 4-Acetamidobenzvl alcohol, (355) : 4-Acetamidobenzaldehyde 354 (5.0 g, 30.6 mmol) was suspended in tetrahydrofuran (100 mL) at 0 °C with an ice bath. The suspension was charged with sodium borohydride (1.16 g, 30.6 mmol) and stirred at room temperature for 6 hours. The mixture was diluted with 5% aqueous sodium hydroxide (50 mL) and the layers were separated. The aqueous layer was extracted with diethyl ether (2 x 100 mL) and the organic extracts were combined. The extracts were washed with 5% aqueous sodium hydroxide (50 mL), brine 950 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo to yield 2.38 g (54%) of a yellow-orange solid, mp 116-119 °C. An analytical sample was recrystallized from nitromethane to yield a yellow-orange solid, mp 118-120 °C (lit. [350] mp 120-121 °C). 1H NMR (DMSO/CDCI/TMS) : 6 10.5 (bs, 1H, NH), 8.35 (d, J = 8.4 Hz, 2H, ArH ortho to NHAc), 7.32 (d, J = 8.4 Hz, 2H, ArH ortho to CH2OH), 5.76 (t, J = 5.6 Hz, 1H, OH), 5.31 (d, J = 5.6 Hz, 2H, CH2Br), 2.90 (s, 3H, CH3CON). CH2Br NHAc 4-Acetamidobenzvl bromide. (356) : 4-Acetamidobenzyl alcohol 355 (2.0 g, 12.1 mmol), was suspended in chloroform (200 mL). The mixture was charged with crushed anhydrous calcium chloride (10 g) and saturated with hydrogen bromide. The mixture was stirred at room temperature for 4 h, filtered and evaporated in vacuo. The resulting oil was triturated in acetone to yield 280 mg (10%) of a yellow-orange solid, mp 144-148 °C. An analytical sample was 346 recrystallized from ethyl acetate to yield a yellow-orange solid, mp 159-162 °C (lit. [347] mp 163-165 °C). 1H NMR (DMSO/CDCI/TMS) : 6 7.57 (d, J = 8.6 Hz, 2H, ArH ortho to NHAc), 7.36 (bs, 1H, NH), 7.31 (d, J = 8.6 Hz, 2H, ArH ortho to CH2Br), 4.58 (s, 2H, CH2Br), 2.07 (s, 3H, CH3CON). CHO OAc 4-Acetoxvbenzaldehvde. (358) : 4-Hydroxybenzaldehyde 357 (10.0 g, 82 mmol) was dissolved in acetic anhydride (20 mL) and heated to reflux overnight. After cooling to room temperature, the mixture was evaporated in vacuo. The resulting liquid was distilled to yield 9.7 g (72%) of a pale yellow liquid, bp 104 °C / 1.2 mm Hg (lit. [348] bp 266-268 °C). ’ H NMR (CDCI/TMS) : 6 9.99 (s, 1H, CHO), 7.92 (AA'XX’, J= 6.5 and 2.0 Hz, 2H, ArH ortho to CHO), 7.28 (AA’XX’, J = 6.5 and 2.0 Hz, 2H, ArH ortho to oAc), 2.34 (s, 3H, CH3C0 2). 347 ch2oh OAc 4-Acetoxvbenzvl alcohol. (359) : 4-Acetoxybenzaldehyde 358 (5.0 g, 30.4 mmol) was suspended in tetrahydrofuran and charged with sodium borohydride (1.15 g, 30.4 mmol). The mixture was stirred for 6 h at room temperature and diluted with 5% aqueous sodium hydroxide (50 mL). The layers were separated, and the aqueous layer was extracted with diethyl ether (2 x 100 mL). The organic portions were combined, washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting liquid was purified by silica gel chromatography with ethyl acetate/hexanes as the eluent. Appropriate fractions were pooled and evaporated in vacuo to yield 2.83 g (56%) of a clear liquid (lit. [349] oil, bp not reported). 1H NMR (CDCI/TMS) : 6 7.38 (d, J = 8.6 Hz, 2H, ArH ortho to CH20), 7.07 (d, J = 8.6 Hz, 2H, ArH ortho to OAc), 4.68 (bs, 2H, CH20), 2.30 (s, 3H, CH3C0 2), 1.72 (bs, 1H, OH). 348 CH2Br OAc 4-Acetoxvbenzvl bromide, (360) : 4-Acetoxybenzyl alcohol 359 (2.0 g, 12 mmol) was dissolved in dichloromethane (20 mL) and cooled to 0 °C with an ice bath. The solution was charged with phosphorous tribromide (1.26 mL, 13.2 mmol) and stirred at 0 °C for 2 h. The mixture was quenched with an ice slurry (20 mL) and saturated sodium bicarbonate (20 mL). The layers were separated, and the organic layer was washed with water (2 x 20 mL), brine (20 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting brown solid was purified by silica gel chromatography with chloroform as the eluent. Appropriate fractions were pooled and evaporated in vacuo to yield 1.23 g of a white solid, mp 49.5-51 °C (lit. [350] mp 54-55 °C. 1H NMR (CDCI/TMS) : 6 7.40 (AA'XX’, J = 6.6 and 1.8 Hz, 2H, ArH ortho to CH2Br), 7.06 (AA'XX’, J= 6.6 and 1.8 Hz, 2H, ArH ortho to OAc), 4.49 (s, 2H, CH2Br), 2.30 (s, 3H, CH3C02). 349 O o N-(4-Methvlphenvl)phthaSimide, (363) : A mixture of p-toluidine 362 (5.0 g, 47 mmol), phthalic anhydride 361 (7.6 g, 51 mmol), and glacial acetic acid were heated to reflux for 2 h. After cooling to room temperature, the solid was collected by vacuum filtration, washed liberally with water (200 mL), and air dried to yield 10.54 g (95%) of a tan solid, mp 201-204 °C (lit. [351] mp 204 °C). 1H NMR (CDCI/TMS) : 6 7.95-7.92 (m, 2H, ArH ortho to ring), 7.78- 7.75 (m, 2H, ArH meta to ring), 7.30 (s, 4H, ArH), 2.40 (s, 3H, CH3). N-(4-BromomethvlphenvDphthalimide. (364) : To a suspension of N-(4-methylphenyl)phthalimide 363 (3.0 g, 12.6 mmol) in degassed carbon tetrachloride (40 mL) under argon was added N-bromosuccinimide (2.25 g, 12.6 mmol) and a catalytic amount of 2,2’-azobisisobutyronitrile (AIBN, 5 mg). The mixture was heated to reflux for 4 h. The solution was allowed to cool to room temperature with stirring overnight. The suspension was diluted with chloroform (360 mL), washed with a mixture of saturated sodium bicarbonate 350 (50 mL) and water (100 mL), water (3 x 100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting solid was recrystallized by dissolving the material in a mixture of ethanol (300 mL) and chloroform (100 mL) and concentrating the solution to about 200 mL. The solid was collected by vacuum filtration to yield 3.85 g (96%) of a white solid, mp 212-214.5 °C which contains small amounts of dibromo compound (ca. 5%) and starting material (ca. 5%) from the NMR spectrum. The product was purified as needed by silica gel chromatography with chloroform as the eluent to yield a white solid, mp 215-216 °C (lit. [352] mp 210-212 °C). ’ H NMR (CDCI/TMS) : 6 7.99-7.95 (m, 2H, ArH ortho to ring), 7.82-7.79 (m, 2H, ArH meta to ring), 7.56-7.43 (AA’BB’, 4H, ArH), 4.53 (s, 2H, CH2Br). El MS m/z 316.9848 (m+(81Br), calc. 316.9874, 3.6%), 314.9881 (m+(79Br), calc. 314.9895, 3.5%), 236 (base). CHO och2ch3 4-Methoxvmethoxvbenzaldehvde. f365) . In a manner similar to the procedure of Winkle [128], sodium hydride (1.44 g, 36 mmol, washed 3 x 5 mL petroleum ether) was suspended in a mixture of anhydrous tetrahydrofuran (20 mL) and dimethylformamide (5 mL) in a 3-neck flask fitted with an addition funnel, a 351 condenser with an argon port, and a stopper. To this suspension was added a solution of 4-hydroxybenzaldehyde 357 (4.0 g, 32.8 mmol) in anhydrous tetrahydrofuran over 15 min (exothermic reaction). Following an additional 30 min of stirring at room temperature, a solution of chloromethyl methyl ether (2.99 mL, 39.3 mmol) in anhydrous tetrahydrofuran (5 mL) was added dropwise over 10 min, and subsequently stirred at room temperature for an additional 2 h. The mixture was diluted with water (50 mL) and diethyl ether (50 mL), the layers were separated, and the organic layer was washed with water (50 mL), 5% aqueous sodium hydroxide (2 x 50 mL), brine (50 mL), dried over anhydrous sodium sulfate, and evaporated in vacuo. The resulting oil was purified by Kugelrohr distillation (bath temp. 100-110 °C / 0.5 mm Hg) to yield 4.81 g (88%) of a clear colorless oil (lit. [353] bp 121-125 °C / 6.0 mm Hg). 1H NMR (CDCI/TMS) : 6 9.90 (s, 1H, CHO), 7.84 (AA’XX1, J= 6.8 and 2.0 Hz, 2H, ArH ortho to carbonyl), 7.15 (AA’XX’, J = 6.8 and 2.0 Hz, 2H, ArH ortho to OMOM), 5.26 (s, 2H, OCH20), 3.50 (s, 3H, CH30). CH2OH OCH2CH3 4-Methoxvmethoxvbenzvl alcohol, (366) : Benzaldehyde 365 (4.3 g, 26 mmol) was dissolved in ethanol (40 mL) and cooled to 0 °C with an ice bath. Sodium 352 borohydride (9.8 g, 0.26 mol) was added portionwise to the mixture over 15 min to avoid an excessive reaction. The mixture was allowed to warm to room temperature with stirring overnight. Solvent was evaporated in vacuo, and the residue was dissolved in a mixture of water (100 ml_) and diethyl ether (100 mL). The aqueous layer was extracted with diethyl ether (100 ml_), and the extracts were Combined. The extracts were washed with water (100 mL), brine (100 ml), dried over anhydrous sodium sulfate, and evaporated in vacuo. The liquid was purified by Kugelrohr distillation (bath temp. 100-105 °C/0.5 mm Hg) to yield 3.36 g (77%) of a clear, colorless oil (lit. [353] bp 122 °C / 2 mm Hg). 1H NMR (CDCI/TMS) : 6 7.28 (m, 2H, ArH ortho to CH20), 7.02 (m, 2H, ArH ortho to OMOM), 5.17 (s, 2H, OCH20), 4.62 (bs, 2H, ArC I-y, 3.47 (3H, CH30), 1.75 (bs, 1H, OH). CH2Br OCH2CH3 4-Methoxvmethoxvbenzvl bromide. (367) . According to the methodology of Lan [354], benzyl alcohol 366 (1.8 g, 10.7 mmol) was dissolved in diethyl ether (50 mL) and charged with triphenylphosphine (3.0 g, 11.4 mmol) and carbon tetrabromide (3.8 g, 11.4 mmol). The resulting suspension was stirred for 3 h at room temperature, evaporated in vacuo, and subjected to silica gel 353 chromatography with 5% ethyl acetate/hexanes as the eluent. Appropriate fractions were pooled and evaporated in vacuo to yield a pale yellow oil that was used immediately. The compound has significant decomposition within a few hours even when stored in a desiccator. 1H NMR (CDCI/TMS) : 6 7.32 (d, J = 8.6 Hz, 2H, ArH ortho to CH2Br), 7.00 (d, J = 8.6 Hz, 2H, ArH ortho to OMOM), 5.18 (s, 2H, 0 C H 20), 4.49 (s, 2H, CH2Br), 3.47 (s, 3H, CH30). BnO J i BnO ch3 OCH OCH (Z)-2-Acetvl-6.7-bis(benzvloxv)-1-(3.4,5-trimethoxvbenzvlidene)-3.4-dihvdro- 2(1H)-isoquinoline, (315) : Amide 272 (3.0 g, 5.5 mmol) was suspended in anhydrous acetonitrile (20 mL) under an argon atmosphere. The suspension was charged with phosphorous oxychloride (10 mL) and heated to reflux for 3 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was diluted with dichloromethane (50 mL), ice (50 g), and diluted with a solution of saturated sodium bicarbonate portionwise (evolution of gas) until the mixture was basic to pH paper. The layers were separated, and the aqueous layer was extracted with dichloromethane (50 ml). The organic extracts were combined, washed with saturated sodium bicarbonate (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in a mixture of acetic anhydride (10 mL) and triethylamine (10 mL) and stirred at room temperature overnight. Solvent was evaporated in vacuo, and the residue was dissolved in dichloromethane (50 mL) and water (50 mL). The organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was recrystallized twice from ethanol to yield 1.29 g (41%) of a pale yellow solid, mp 196-197.5 °C. 1H NMR (CDCI/TMS) : 6 7.51-7.30 (m, 10H, ArH), 7.25 (s, 1H, =CHR), 6.71 (bs, 3H, ArH), 6.58 (s, 1H, ArH), 5.21 (ABq, J = 11.8 Hz, Au = 29.3 Hz, ArCH20), 5.16 (s, 2H, ArCH20), 5.03-4.99 (m, 1H, CH2N), 3.86 (bs, 9H, 3 x CH30), 3.17-3.10 (m, 2H, ArCH2), 2.68-2.62 (m, 1H, CH2N), 1.80 (s, 3H, CH3CO). FAB MS m/z 566.4 (m + 1, 98%), 565.4 (m, base). UV 330 (e 26 900, methanol). Analysis for CgsHasNTOgV^HgO : calc. C, 73.73; H, 6.28; N, 2.46; found C, 73.83; H, 6.14; N, 2.36. 355 (R)-2,2’-Bis(diphenvlphosphino)-1 -1 ’-binaphthvUruthenium dichloride, Ru(R-BINAF)CI„, (372) : According to the procedure of Kitamura [355], benzeneruthenium(ll) chloride dimer ([RuCI2(benzene)]2, 370, 42 mg, 0.084 mmol) and (R)-(-)-2,2’-bis(diphenylphosphino)-1,1 ’-binaphthyl (R-BINAP, 100 mg, 0.16 mmol) were quickly weighed in the air and transferred to a 10 mL argon filled conical flask. The mixture of solids is charged with degassed anhydrous dimethylformamide (1 mL) via a syringe under a constant flow of argon. The dark suspension is immersed in an oil bath at 100 °C for 10 min. The clear, dark reddish-brown solution was cooled to room temperature and evaporated by high vacuum for 10 h with vigorous stirring. The resulting solid was quickly weighed in air to yield 143 mg (112%) of a dark, reddish-brown solid that was used without further purification. Large scale reactions ( > 200 mg) require 0.5-1 mol% of catalyst, small scale reactions ( < 200 mg) require 1-2 mol%, reactions utilizing older batches (over 2 weeks) of catalyst require up to 6 mol% of catalyst. The solid was stored under argon, and is probably a mixture of dimethylformamide complexes generalized as 356 RuCI2(R-BINAP)(DMF)n [355]. This compound is air sensitive in solution and changes from a bright reddish-brown to a blackish-green when exposed to air for a short period of time. The catalyst remained active for about 4 weeks when stored in a ground glass flask under argon. It is however, much less sensitive to air than Ru(R-BINAP)(OAc)2. (S ^ ^ ’-BisfdiphenvlphosphinoM -1 ’-binaphthvHruthenium dichloride, Ru(S-BINAP)CL. (383) : Prepared as for the (R)-isomer described above with S-BINAP to yield 135 mg (106%) of a dark, reddish-brown solid that was used without further purification. The solid was stored under argon, and is probably a mixture of dimethylformamide complexes generalized as RuCI2(S-BINAP)(DMF)n [355], This compound is air sensitive in solution and changes from a bright reddish-brown to a blackish-green when exposed to air for a short period of time. The catalyst remained active for about 4 weeks when stored in a ground glass flask under argon. It is however, much less sensitive to air than Ru(S-BINAP)(OAc)2. 357 N-(2-(3.4-Dimethoxyphenvl)ethvl-(3,4.5-trimethoxvphenvl)acetamideT375) : To a solution of 2-(3,4-dimethoxyphenyl)ethylamine 120 (10 g, 55 mmol) in toluene (100 mL) was added 3,4,5-trimethoxyphenylacetic acid (13.7 g, 61 mmol). The suspension was heated to reflux for 72 h with azeotropic removal of water via a Dean-Stark trap. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (200 mL), washed with 5% aqueous sodium hydroxide (2 x 100 ml), water (100 mL), 1.2 N hydrochloric acid (2 x 100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting solid was recrystallized from hot toluene to yield 19.4 g (90%) of a pale yellow solid, mp 97-98 °C (lit. [356] mp 100-101 °C). 1H NMR (CDCI/TMS) : 6 6.72 (d, J = 8.1 Hz, 1H, ArH), 6.65 (d, J = 2.0 Hz, 1H, ArH), 6.50 (dd, J = 8.1 and 2.0 Hz, 1H, ArH), 6.34 (s, 2H, ArH), 5.51 (bt, 1H, NH), 3.86 (s, 3H, CH30), 3.843 (s, 3H, CH30), 3.837 (s, 3H, CH30). 3.80 (s, 6H, 2 x CH30), 3.49- 3.42 (m, 4H, CH2N and ArCH2CON), 2.70 (t, J =6.9 Hz, 2H, ArCH2). 358 CH30 CH CH30 N— CH CH3 O OCH CH3 O OCH CH 3 O (Z)- and (E)-2-Acetvl-6,7-dimethoxv-1 -(3A5-trimethoxybenzvlidene)-3,4- dihvdro-2(1 H)-isoquinoline. (376 and 377) : Amide 375 (4.0 g, 10.3 mmol) was suspended in anhydrous acetonitrile (20 mL) under an argon atmosphere. The solution was charged with phosphorous oxychloride (10 mL) and heated to reflux for 3 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (50 mL) and charged with an ice slurry (50 mL). The mixture was rapidly stirred while saturated sodium bicarbonate (vigorous evolution of gas) was added dropwise until the mixture became basic to pH paper. The layers were separated, and the organic layer was washed with saturated sodium bicarbonate (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in acetic anhydride (10 mL) and triethylamine (10 mL). The mixture was stirred overnight at room temperature, and subsequently diluted with dichloromethane (50 mL). The solution was washed with water (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, 359 and evaporated in vacuo. The resulting oil was recrystallized from ethanol to yield 878 mg (21 %) of a pale yellow solid, mp 208-210 °C. 1H NMR reveals that this material is (Z)-isomer 376 from the upfield shift of the acetyl resonance [357]. 1H NMR (CDCI/TMS) : 6 7.13 (s, 1H, =CHR), 6.74 (s, 2H, ArH), 6.69 (s, 1H, ArH), 6.63 (s, 1H, ArH), 5.09-5.03 (m, 1H, CH2N), 3.99 (s, 3H, CH30), 3.90 (s, 3H, CH30), 3.87 (s, 9H, 3 x CH30), 3.21-3.10 (m, 2H, ArCH2), 2.73 (m, 1H, CH2N), 1.81 (s, 3H, CH3CON). El MS m/z 413.1834 (m+ calc. 413.1838, base). UV 331 (e 32,900; MeOH). Analysis for C^H^^O /AH/]) : calc. C, 66.09; H, 6.63; N, 3.35; found C, 66.00; H, 6.18; N, 3.21. The mother liquor was concentrated yielding 802 mg (19%) of a pale yellow solid, mp 145-146 °C. 1H NMR reveals that this material is (E)-isomer 377 from the downfield shift of the acetyl resonance [357], 1H NMR (CDCI/TMS) : 6 6.79 (s, 1H, ArH), 6.64 (s, 1H, ArH), 6.52 (s, 2H, ArH), 6.45 (bs, 1H, =CHR), 4.01 (t, J = 6.5 Hz, 2H, CH2N), 3.87 (s, 3H, CH30), 3.83 (s, 3H, CH30), 3.76 (s, 6H, 2 x CH30), 3.47 (s, 3H, CH30), 2.92 (t, J = 6.5 Hz, 2H, ArCH2), 2.31 (s, 3H, CH3CON). El MS m/z 413.1836 (m+ calc. 413.1838, base). UV 305 (s 15,100; MeOH). Analysis for C ^H ^^O g-y^O : calc. C, 66.09; H, 6.63; N, 3.35; found C, 66.35; H, 6.31; N, 3.15. 360 CH OCH (Z)-2-Acetvl-6,7-Dimethoxv-1 -(3A5-trimethoxybenzvlidene)-3,4-dihvdro- 2(1 HHsoquinoline, (376) : Amide 375 (5.0 g, 12.8 mmol) was suspended in anhydrous acetonitrile (20 mL) under an argon atmosphere. The solution was charged with phosphorous oxychloride (10 mL) and heated to reflux for 4 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (50 mL) and charged with an ice slurry (50 mL). The mixture was rapidly stirred while adding 50% aqueous sodium hydroxide dropwise until the mixture became basic to pH paper. The layers were separated, and the organic layer was washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), and dried over anhydrous sodium sulfate. The resulting solution was cooled to 0 °C, and charged with acetic anhydride (10 mL) and triethylamine (10 mL). The mixture was stirred overnight while warming to room temperature. The solution was washed with 1.2 N hydrochloric acid (2 x 100 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil contained both the (E)- and (Z)-isomers in a 9:1 E:Z ratio (TLC with methanol/diethyl ether/chloroform 361 (1/25/25) as the eluent). The (E)-isomer was converted to the (Z)-isomer by dissolving the mother liquor in toluene (25 mL) and heating to reflux for 2 h. After cooling to room temperature, solvent was removed in vacuo. The resulting oil was recrystallized from ethanol to yield 2.46 g (46%) of a pale yellow solid, mp 210-211 °C. This compound was identified as the (Z)-isomer since its 1H NMR spectrum is in agreement with that described for the (Z)-isomer on page 359. BnO BnO CH3 OCH3 OCH (S)-2-Acetvl-6.7-bis(benzvloxv)-1-(3.4.5-trimethoxvbenzvl)-1,2.3,4- tetrahvdroisoquinoline hydrochloride. (317) : (Z)-Enamide 315 (500 mg, 0.88 mmol) was suspended in ethanol (20 mL) in a Parr bottle. The system was evacuated by high vacuum pump to less than 1 mm Hg and filled with argon. This degassing procedure was repeated for a total of five cycles. The catalyst (Ru(S-BINAP)CI2, 383. 7 mg, 8.8 fxmol) was quickly weighed in the air and immediately added to the bottle. The system was evacuated and filled five times as described above. The bottle was connected to the hydrogenator quickly while the flask was under the reduced pressure of a water aspirator. 362 The system was filled to 30 psi with hydrogen, evacuated with the water aspirator, and refilled to 30 psi with hydrogen. After heating to 60 °C under regulation of a thermocouple, hydrogen content was increased to 60 psi, and the mixture was shaken from 50-60 psi for 72 h. Upon cooling to room temperature, the apparatus was disassembled, the contents were transferred to a rb flask, and solvent was evaporated in vacuo. The dark oil was subjected to silica gel chromatography with methanol/diethyl ether/chloroform (1/50/50) to separate the product from the homogeneous catalyst. Appropriate fractions were pooled and evaporated in vacuo to yield 362 mg (72%) of a tacky foam that was used without recrystallization. The 1H NMR spectrum is in agreement with that described for the racemic acetamide on page 366. OCH c h 3o (F0-2-Acetvl-6.7-bis(benzvloxv)-1-(3,4.5-trimethoxvbenzvn-1,2,3,4- tetrahvdroisoquinoline hydrochloride. (378) : (Z)-Enamide 315 (500 mg, 0.88 mmol) was suspended in ethanol (20 mL) in a Parr bottle. The system was evacuated by high vacuum pump to less than 1 mm Hg and filled with argon. This degassing procedure was repeated for a total of five cycles. 363 The catalyst (Ru(R-BINAP)CI2, 372, 7 mg, 8.8 nmol) was quickly weighed in the air and immediately added to the bottle. The system was evacuated and filled five times as described above. The bottle was connected to the hydrogenator quickly while the flask was under the reduced pressure of a water aspirator. The system was filled to 30 psi with hydrogen, evacuated with the water aspirator, and refilled to 30 psi with hydrogen. After heating to 60 °C under regulation of a thermocouple, hydrogen content was increased to 60 psi, and the mixture was shaken from 50-60 psi for 72 h. Upon cooling to room temperature, the apparatus was disassembled, the contents were transferred to a rb flask, and solvent was evaporated in vacuo. The dark oil was subjected to silica gel chromatography with methanol/diethyl ether/chloroform (1/50/50) to separate the product from the homogeneous catalyst. Appropriate fractions were pooled and evaporated in vacuo to yield 362 mg (72%) of a tacky foam that was used without recrystallization. The 1H NMR spectrum is in agreement with that described for the racemic acetamide on page 366. 364 CH OCH3 OCH (S)-2-Acetvl-6,7-dimethoxv-1 -(3.4,5-trim ethoxvbenzvh-1,2,3,4- tetrahvdroisoquinoline. (379) : (Z)-Enamide 376 (1.5 g, 3.6 mmol) was suspended in ethanol (20 mL) in a Parr bottle. The system was evacuated by high vacuum pump to less than 1 mm Hg and filled with argon. This degassing procedure was repeated for a total of five cycles. The catalyst (Ru(S-BINAP)CI2l 383, 29 mg, 36 j.imol) was quickly weighed in the air and immediately added to the bottle. The system was evacuated and filled five times as described above. The bottle was connected to the hydrogenator quickly while the flask was under the reduced pressure of a water aspirator. The system was filled to 30 psi with hydrogen, evacuated with the water aspirator, and refilled to 30 psi with hydrogen. After heating to 60 °C under regulation of a thermocouple, hydrogen content was increased to 60 psi, and the mixture was shaken from 50-60 psi for 72 h. Upon cooling to room temperature, the apparatus was disassembled, the contents were transferred to a rb flask, and solvent was evaporated in vacuo. The dark oil was subjected to silica gel chromatography with methanol/diethyl ether/chloroform (1/50/50) as the eluent to separate the product from the homogeneous catalyst. Appropriate fractions were pooled and evaporated in vacuo to yield 1.5 g (quantitative) of a tacky foam that was used without recrystallization. The 1H NMR spectrum is in agreement with that of the racemic acetamide on page 368. El MS m/z 415.1999 (m+ calc. 415.1995, 0.16%), 234.1 (m - C 10H13O3, base), 192.1 (m - C12H150 4, 60%). BnO BnO OCH OCH (±)-2-Acetvl-6.7-bisf benzvloxvM -(3.4.5-tr imethoxvbenzvl)-1.2.3,4- tetrahvdroisoquinoline. (380) : To a solution of tetrahydroisoquinoline 271 (100 mg, 0.18 mmol) in dichloromethane (5 mL) was added acetic anhydride (2 mL) and triethylamine (2 mL). The mixture was stirred at room temperature for 72 h and poured into a 1.2 N hydrochloric acid ice slurry (15 mL). The mixture was extracted with dichloromethane (2x15 mL) and, the extracts were combined. The organic extracts were washed with brine (25 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield 71 mg (70%) of a yellow solid, mp 128-129 °C. The 1H NMR is complicated by restricted rotation about the amide bond to produce two conformations in a 1:1 ratio at room temperature. The compound is a mixture of two conformations in solution providing a complex 1H NMR spectrum. Temperature elevation studies in d6-DMSO to 357 K did not simplify the 1H NMR spectrum sufficiently for the assignment of proton resonances. FAB MS m/z 568.5 (m + 1, 20%), 386.2 (m - CioH130 3> base). Analysis for C35H37N1061/4H20 : calc C, 73.47; H, 6.61; N, 2.45; found C, 73.31; H, 6.63; N, 2.93. NH HCI OCH OCH (±)-6.7-Dimethoxv-1-(3,4,5-trimethoxvbenzvl)-1,2,3,4-tetrahvdroisoqumoline hydrochloride, (381) : Amide 375 (3.0 g, 7.7 mmol) was suspended in anhydrous acetonitrile (10 mL) under an argon atmosphere. The solution was charged with phosphorous oxychloride (5 mL) and heated to reflux for 3.5 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (20 mL) and charged with an ice slurry (50 mL). The mixture was rapidly stirred while 5% aqueous sodium hydroxide was added dropwise until basic to pH paper. The layers were 367 separated, and the organic layer was washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in ethanol and cooled to 0 °C with an ice bath. Sodium borohydride (584 mg, 15.4 mmol) was added, and the mixture was stirred overnight with warming to room temperature. Solvent was evaporated in vacuo. The residue was dissolved in ethyl acetate (100 mL), washed with 5% aqueous sodium hydroxide (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The resulting oil was dissolved in a saturated solution of hydrogen chloride (g) in methanol (10 mL) and evaporated in vacuo. The residue was recrystallized from ethanol/diethyl ether to yield 1.06 g (34%) of a colorless solid, mp 140-141 °C (lit. [356] mp 131-132 °Cfrom methanol). 1H NMR (CD3O D ): 6 6.81 (s, 1H, ArH). 6.65 (s, 2H, ArH), 6.62 (s, 1H, ArH), 4.76 (bt, J = 8.0 Hz, 1H, ArCHRN), 3.821 (s, 6H, 2 x CH30), 3.816 (s, 3H, CH30), 3.75 (s, 3H, CH30), 3.69 (s, 3H, CH30), 3.60-3.38 (m, 3H, CH2), 3.14-3.05 (m, 3H, CH2). 368 c h3o CH OCH OCH (±1-2-Acetyl-6,7-dimethoxv-1 -(3,4,5-trim ethoxvbenzvl)-1,2,3,4- tetrahvdroisoquinoline, (382) : Isoquinoline 381 (500 mg, 1.23 mmol) was dissolved in a mixture of dichloromethane (10 mL), triethylamine (2 mL), and acetic anhydride (2 mL). The mixture was stirred at room temperature overnight and then diluted with dichloromethane (40 mL). This solution was washed with 1.2 N hydrochloric acid (2 x 50 mL), water (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo to yield 273 mg (53%) of a yellow solid, mp 138-142 °C.' The compound is a mixture of two conformations in solution providing a complex 1H NMR spectrum. Temperature elevation studies in CDCI3 to 323 K and de-DMSO to 357 K did not simplify the 1H NMR spectrum sufficiently for the assignment of proton resonances. El MS m /z 415.1996 (m+ calc. 415.1995, 0.15%), 234.1 (m - C l0H13O3, base), 192.1 (m - C12H1504, 55%). Analysis for C ^H ^N peH p : calc. C, 63.73; H, 7.21; N, 3.23; found C, 63.81; H, 6.81; N, 3.35. 369 BnO NH HCI BnO OCH OCH (S)-6,7-Dibenzvloxv-1-(3,4,5-trimethoxvbenzvn-1,2,3,4- tetrahvdroisoquinoline hydrochloride. (349) : (S)-Acetamide 317 (310 mg, 0.55 mmol) was dissolved in a mixture of 20% aqueous potassium hydroxide (4 mL), hydrazine (4 mL), and ethylene glycol (10 mL). The mixture was heated to 160 °C reflux for 24 h and then cooled to room temperature. The mixture was diluted with dichloromethane (50 mL) and water (50 mL) and the layers were separated. The aqueous layer was extracted with dichloromethane (25 mL), and the organic extracts were combined. The organic portion was washed with 5% aqueous sodium hydroxide (50 mL), brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in a saturated solution of hydrogen chloride (g) in methanol (2 mL) and diluted with diethyl ether to precipitate 75 mg (24%) of an off white solid, mp 148-151 °C. Spectroscopic data is consistent with the spectra for the racemic derivative reported on page 325. [«]q5 +23.5 0 (c = 0.5, MeOH). Optical purity was determined to be 95% by the TAGIT method on page 323. 370 BnO BnO CH (Z)-2-Acetvl-6.7-bis(benzvloxv)-1-(4-nitrobenzvlidene)-3,4-dihvdro-2(1H)- isoquinollne, (329) : Amide 294 (5.0 g, 10.1 mmol) was suspended in anhydrous acetonitrile (20 mL) under an argon atmosphere. The suspension was charged with phosphorous oxychloride (10 mL) and heated to reflux for 3 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was diluted with dichloromethane (50 mL) and an ice slurry (50 mL). A solution of 50% aqueous sodium hydroxide was added dropwise until the mixture was basic to pH paper. The layers were separated, and the aqueous layer was extracted with dichloromethane (50 ml). The organic extracts were combined, washed with 5% aqueous sodium hydroxide (100 mL), brine (100 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was dissolved in a mixture of acetic anhydride (10 mL), triethylamine (10 mL) and toluene (100 mL) and heated to reflux for 4 h. After cooling to room temperature, solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (50 mL) and 1.2 N hydrochloric acid (50 mL). The organic layer was washed with brine (50 mL), dried over anhydrous magnesium sulfate, and evaporated in vacuo. The residue was subjected to silica gel chromatography with diethyl ether/chloroform (1/1) as the eluent. Appropriate fractions were pooled and evaporated in vacuo. The resulting yellow oil was recrystallized from isopropanol to yield 847 mg (16%) of a yellow solid, mp 125-127 °C. The mother liquor (600 mg, 28% total yield) was used without isolation as a solid. 1H NMR (CDCI/TMS) : 6 8.02 (d, J = 8.8 Hz, 1H, ArH ortho to N02), 7.47-7.21 (m, 12H, 10 x ArH and ArH meta to N 02), 6.79 (s, 1H, ArH), 6.64 (s, 1H, ArH), 6.51 (bs, 1H, =CHR), 5.19 (s, 2H, ArCH20), 4.76 (s, 2H, ArCH20), 3.89 (t, J = 6.5 Hz, 2H, CH2N), 2.88 (t, J = 6.5 Hz, 2H, ArCH2), 2.27 (s, 3H, CH3CO). FAB MS m/z 521.4 (m + 1, base), 520.4 (m, 66%), 429.2 (m + 1 - C7H7, 48%). UV 366 (e 21 865, methanol). Analysis for C32H28N20 5 : calc. C, 73.83; H, 5.42; N, 5.38; found C, 73.87; H, 5.34; N, 5.45. BnO BnO CH NO. (S)-2-Acetvl-6.7-bis(benzvloxv)-1-(4-nitrobenzvn-1.2.3,4- tetrahvdroisoquinoline hydrochloride, (384) : (Z)-Enamide 329 (600 mg, 1.15 mmol) was suspended in ethanol (20 mL) in a Parr bottle. The system was evacuated by high vacuum pump to less than 1 mm Hg and filled with argon. This degassing procedure was repeated for a total of five cycles. The catalyst (Ru(S-BINAP)CI2, 383. 54 mg, 68 nmol) was quickly weighed in the air and immediately added to the bottle. The system was evacuated and filled five times as described above. The bottle was connected to the hydrogenator quickly while the flask was under the reduced pressure of a water aspirator. The system was filled to 30 psi with hydrogen, evacuated with the water aspirator, and refilled to 30 psi with hydrogen. After heating to 60 °C under regulation of a thermocouple, hydrogen content was increased to 60 psi, and the mixture was shaken from 50-60 psi for 72 h. Upon cooling to room temperature, the apparatus was disassembled, the contents were transferred to a rb flask, and solvent was evaporated in vacuo. The dark oil was subjected to silica gel chromatography with diethyl ether/dichloromethane (5/95) as the eluent to separate the product from the homogeneous catalyst. Appropriate fractions were pooled and evaporated in vacuo. The resulting oil was recrystallized from a minimal amount of ethyl acetate to yield 120 mg (20%) of a yellow solid, mp 152-153 °C. The mother liquor was evaporated to yield an additional 304 mg (424 mg, 70% total yield) that was used without further purification. The compound is a mixture of two conformations in solution providing a complex 1H NMR spectrum. Temperature elevation studies of related compounds in CDCI3 to 323 K and de-DMSO to 357 K did not simplify the 1H NMR spectrum sufficiently for the assignment of proton resonances. FAB MS m/z 523.4 (m + 1, 49%), 386.2 (m - Cj.Hg^Oe, base). 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