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Osmotic processes as targets for drug design: Part I. The polyol pathway; design and synthesis of inhibitors of the aldose reductase enzyme. Part II. The bacterial osmotolerance; a study of the structural requirements for the glycine betaine-choline transport system
Abdel-Ghany, Yasser Saad, Ph.D.
The Ohio State University, 1992
300 N. ZeebRd. Ann Arbor, MI 48106 OSMOTIC PROCESSES AS TARGETS FOR DRUG DESIGN
Part I. The Polyol Pathway Design and Synthesis of Inhibitors of the Aldose Reductase Enzyme Part II. The Bacterial Osmotolerance A Study of the Structural Requirements for the Glycine Betaine-Choline Transport System
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
YasserS. Abdel-Ghany, B.S., M.S.
The Ohio State University 1992
Dissertation Committee: Approved by
Duane D. Miller, Ph.D. Robert W. Brueggemeier, Ph.D. Dennis R. Feller, Ph.D. Duane D. Miller, Adviser Larry W. Robertson, Ph.D. College of Pharmacy DEDICATION
®© %t>©$® m w M % m m $ m i f
My Family, My Educators, My Advisors and My Friends ACKNOWLEDGMENTS
I would like to express my everlasting thanks and gratitude to Dr. Duane D. Miller for his sincere guidance, constructive supervision and unending enthusiasm throughout the course of my graduate career. The unconditional friendship and support that Dr. Miller and his family have offered is beyond description, and was a big factor in the completion of the work described here.
I would also like to express my indebtedness to the members of my graduate committee, especially Dr. Dennis Feller and Dr. Robert Brueggemeier for their continuous help and advice.
It is my duty to acknowledge, with appreciation, the efforts of Dr. Peter F. Kador, Dr. Tadashi Mizogouchi and Dr. Anita Malik for their help in the biological evaluation of the new aldose reductase inhibitors. The same goes for Dr. Calvin Kunin and Dr. Hua Tong for performing the salt tolerance and the mutation studies for the glycine betaine analogs. My gratitude is also addressed to Dr. LeRudulier and Dr. Poggi for carrying out the radiolabeled uptake studies of glycine betaine , choline and proline.
My cordial thanks is also addressed to Dr. Mike Darby for his help in the radioactive synthesis and to John Miller for his help in obtaining the spectral analysis.
My thanks and appreciation also goes to my good friends Dr. Robert Curley, Dr. Jeff Herndon and Dr. Pat Cyr for their unsolicited but much appreciated advice and for their never ending support. Finally I will remain in debted to the following friends and colleagues that stood by me throughout my graduate career
Helen A. Whelan, without her friendship, love and support, I wouldn't have been able to make it through the graduate years.
Dr. Hassan Salama, my friend and former advisor, that taught me what chemistry is all about.
Mustapha Beleh, my student, friend and colleague who was always there and ready to help.
My friends and lab-mates, Kim, Jeff, Meri, Ron, Hong, Kazu, Vimon and Yoshi.
Joan Dendera and Kathy Brooks for their help and assistance throughout my stay at The Ohio State University.
Finally, my friends over the years Ali, Adam, Sinout, Setsuko, Farida, Shamim, Saroj, Nader, Michelle, Steve, Levon and Allen.
iv VITA
August 26, 1959 ...... Born - Alexandria, EGYPT
June, 1981 ...... B. S. Pharmaceutical Sciences, University of Alexandria, College of Pharmacy, Alexandria, EGYPT
Sept. 1981 - April 1985 ...... Graduate Teaching Associate, University of Alexandria, College of Pharmacy, Alexandria, EGYPT
April 1985 ...... M.S. Pharmaceutical Sciences, University of Alexandria, College of Pharmacy, Alexandria, EGYPT
April 1985 - Sept. 1987 ...... Associate Lecturer, University of Alexandria College of Pharmacy, Alexandria, EGYPT
Sept 1987 - Sept. 1989 ...... Egyptian - American Peace Fellowship The Ohio State University Columbus, Ohio
Sept 1989 - Sept. 1991 ...... Egyptian Scientific Mission Fellowship The Ohio State University Columbus, Ohio
Sept. 1991 - Sept. 1992 ...... Graduate Teaching Assistant The Ohio State University Columbus, Ohio
Sept. 1992- present ...... Graduate Research Associate The Ohio State University Columbus, Ohio
PUBLICATIONS AND ABSTRACTS
1. Rida, S. M .; Salama, H. M .; Labouta, I. M .; A.-Ghany, Y. S. Synthesis of Some 3-(Benzimidazol-2-ylmethyl)Thiazolidinone Derivatives as Potential Antimicrobial Agents Pharmazie 1985, 4 0 , 727-728.
v 2. Rida, S. M .; Salama, H. M .; Labouta, I. M. ; A.-Ghany, Y. S. Synthesis and In Vitro Antimicrobial Activites of Thiazolo[3,2-a]benzimidazol-3(2H)- ones Pharmazie 1986, 4 1 , 324-326.
3. Rida, S. M .; Labouta, I. M .; Salama, H. M .; A.-Ghany, Y. S .; El-Ghazzaui, E. ; Kader, O. Synthesis and In Vitro Antimicrobial Evaluation of Some Benzimidazol-2-ylmethylthioureas, Benzimidazol-2-ylacetylthio semicarbazides, and Products of Their Condensation with Monochloroacetic Acid Pharmazie 1986, 41 , 475-478.
4. Miller, D. D .; Abdel-Ghany, Y. S. ; Donkor, I . ; Kador, P. F .; Mizoguchi, T. Chemoreactive Analogs as Possible Irreversible Inhibitors of Aldose Reductase US-Japan Aldose Reductase Workshop Kona, Hawaii, February 1991.
5. Abdel-Ghany, Y. S .; Donkor, I . ; Kador, P .; Mizoguchi, T . ; Malik, A .; Miller, D. Novel Alrestatin Analogs with Potent Reversible and Irreversible Inhibitory Activity for the Enzyme aldose Reductase 203rd American Chemical Society National Meeting, San Francisco, California, April 1992, Abst. No. 135.
6. Abdel-Ghany, Y. S .; Miller, D. D .; Inhat, M. ; Kunin, C. ; Tong, H. Glycine Betaine and Choline Transport Systems as Possible Routes of Delivering Cytotoxic Analogs into Enteric Bacteria 25th Annual Graduate Student Meeting in Medicinal Chemistry, Ann Arbor, M l, June 1992.
Fields of Study
Major Field: Pharmacy
Specific Field: Synthetic Medicinal Chemistry TABLE OF CONTENTS
PAGE Dedication ...... ii
Acknowledgement i i i
VITA v
List of Figures...... x
List of Tables...... xii
List of Schemes ...... xiii
PART 1: THE POLYOL PATHWAY Design and Synthesis of Inhibitors of the Aldose Enzyme CHAPTER INTRODUCTION 1.1. Overview ...... 2 1.2. Diabetes Mellitus ...... 5 1.3. Types and Treatment of Diabetes Mellitus ...... 5 1.4. Diabetic Complications ...... 8 1.5. Sorbitol Metabolism ...... 9 1.6. The Role of the Polyol Pathway in Diabetic Complications ...... 11 1.7. The Aldose Reductase Enzyme ...... 13 1.8. Aldose Reductase Inhibitors ...... 14 1.9. Aldose Reductase Binding Sites ...... 19 STATEMENT OF PROBLEMS AND OBJECTIVES 2.1. Irreversible Inhibitors of Aldose Reductase ...... 23 2.2. Alrestatin Analogs as Reversible and Irreversible Inhibitors of Aldose reductase ...... 26 2.3. Alconil Analogs as Reversible and Irreversible Inhibitors of Aldose Reductase ...... 30 2.4. Radio-labeled Analog of 5-lodoacetamido Alrestatin... 33 RESULTS AND DISCUSSION 3.1. Chemistry ...... 34 3.1.1. Synthesis of 5-Substituted Alrestatin ...... 34 3.1.2. Synthesis of Benzenesulfonamide Analogs of Alrestatin ...... 38 3.1.3. Synthesis of Nitrophenol Analogs of Alrestatin 40 3.1.4. Synthesis of Alrestatin Carboxylic Surrogates as Affinity Labels for Aldose Reductase ...... 40 3.1.5. Synthesis of 2-Substituted Alconil Analogs ...... 43 3.1.6. Synthesis of Alconil Analogs Modified at the Hydantoin Ring ...... 45 3.1.7. Synthesis of 14C-Labeled Analog of 5-lodo acetamido Alrestatin ...... 48 3.2. Biological Results ...... 49 3.2.1. Irreversible Inhibitors ...... 51 3.2.2. Reversible Inhibitors ...... 54 3.3. Summary...... 62 EXPERIMENTAL...... 63
PART 2: THE BACTERIAL OSMOTOLERANCE A Study of the Structural Requirements of the Glycine Betaine-Choline Transport System. INTRODUCTION 5.1. Overview ...... 98 5.2. Background ...... 99 5.3. Compatible Solutes ...... 99 5.3.1. Potassium Ions ...... 101 5.3.2. Trehalose ...... 102 5.4. Osmoprotectants ...... 103 5.5. The Choline-Glycine Betaine Pathway ...... 105 5.6. Glycine Betaine Analogs ...... 107 5.7. Miscellaneous Osmoprotectant Molecules ...... 109 5.8. Relation Between Osmoprotection and Urinary Tract Infections ...... 110 STATEMENT OF PROBLEMS AND OBJECTIVES...... 111
RESULTS AND DISCUSSION 7.1. Chemistry ...... 115 7.1.1. Synthesis of Sulfobetaine ...... 115 7.1.2. Synthesis of Phosphobetaine ...... 116 7.1.3. Synthesis of N-Betainylbenznesulfonamide 117 7.1.4. Synthesis of Sulfonamide Analogs ...... 118 7.1.5. Synthesis of Sulfobetaine Analogs...... 120 7.2. Biological Studies ...... 121 7.2.1. Salt Tolerance Studies ...... 122 7.2.2. Effect of MPMS on the Osmoprotective Activity of GB, Choi and Proline in Mutant Strains of E. coli.. 131 7.2.3. Uptake Studies ...... 133 7.3. Summary...... 137
EXPERIMENTAL...... 138
BIBLIOGRAPHY 153 LIST OF FIGURES
FIGURE PAGE
1. Compatible Organic Osmolytes ...... 4
2. Clinically Used Sulfonylureas ...... 7
3. Insulin Sensitizer ...... 8
4. The Polyol Pathway ...... 10
5. The Crystal Structure of the Human Placenta Aldose Reductase ...... 14
6. Clinically Tested Carboxylic Acids ARIs ...... 15
7. Clinically Tested Acidic Cyclic Amides ARIs ...... 16
8. Kador-Sharpless Pharmacophor Model for ARIs ...... 21
9. Proposed Affinity Labels on Alrestatin Backbone ...... 27
10. Proposed Affinity Labels from Carboxylic Surrogates of Alrestatin ...... 30
11. Proposed Affinity Labels on Alconil Backbone ...... 31
12. Proposed Alconil Analogs Modified at the Hydantoin Ring 32
13. The 14C-Labeled Analog 5-lodoacetamido Alrestatin ...... 33
14. The Metabolically Linked Choline and Glycine Betaine T ransport Systems...... 107
15. Proposed Analogs of Compound 105 ...... 113
16. The Proposed Sulfobetaine Analogs ...... 114
17. The Effect of the Tested Compounds on the Maximum Salt Tolerance of the Salt Resistant Strain E coli 31 ...... 125 18. The Effect of the Tested Compounds on the Maximum Salt Tolerance of the Standard Strain E coli K10...... 126
19. The Effect of the Tested Compounds on the Maximum Salt Tolerance of the Salt Sensitive Strain E coli ATCC 25922...... 127
20. The Effect of Various Concentrations of Choi Alone, or in Combination with MPMS on Growth of E coli ATCC 25922.... 130
21. Uptake of [14C]-GB by Three Strains of E coli...... 134
22. Uptake of [14C]-Chol by Three Strains of E coli...... 135
23. Uptake of [14C]-Proline by Three Strains of E coli...... 136
xi LIST OF TABLES
TABLES PAGE
1. Reversible and Irreversible Inhibition of Rat Lens AR by Affinity andPhotoaffinity Labels Analogs of Alrestatin ...... 24
2 . Reversible and Irreversible Inhibition of Rat Lens AR by Affinity Labels Analogs of Alconil ...... 25
3. Proposed Benzenesulfonamide Analogs of Alrestatin ...... 28
4. Proposed Nitrophenol Analogs of Alrestatin...... 29
5. IC50 Values and % Irreversible Inhibition Activities of Compounds (45-49) ...... 52
6 . IC50 Values and % Irreversible Inhibition Activities of the Alconil Analogs (63-66) ...... 53 7. IC50 Values and % Irreversible Activities of the Benzenesulfo namide Carboxylic Surrogates of Alrestatin (51-56) ...... 55
8 . IC50 and % Irreversible Inhibitory Activities of the Nitrophenol Carboxylic Surrogates of Alrestatin (57-59) ...... 55
9. IC 50 Values and % Irreversible Inhibition Activities of the Alconil Analogs (67-68) ...... 57
10. IC50 and % Irreversible Inhibitory Activities of Affinity Labels from the Bezenesulfonamide Analogs of Alrestatin (60-62)...... 59
11. The Inhibitory Activities of the Tested Compounds Against RLAR and RKALR...... 60
12. Osmoregulated Systems in E coli...... 101
13. The Effect of the Tested Compounds on Bacterial Maximum Salt Tolerance ...... 123
14. Effect of MPMS on Salt-Tolerance of Mutant Strains of E. coli Grown in MM Alone or MM Containing GB, Choi or Proline 132
xii LIST OF SCHEMES
SCHEME PAGE
1. Synthesis of 5-Amino Alrestatin ...... 35
2. Synthesis and Decomposition of Bicyclo[2.2.2]octa-2,5- diene-2-carboxylic Acid ...... 36
3. Synthesis of 5-Substituted Alrestatins ...... 37
4. Attempts for the Synthesis of Compound 50 ...... 38
5. Synthesis of Benzenesulfonamide Analogs of Alrestatin ...... 39
6. Synthesis of Nitrophenol Analogs of Alrestatin ...... 41
7. Synthesis of Carboxylic Surrogates Affinity Labels ...... 42
8. Synthesis of 2-Substituted Alconil ...... 44
9. Synthesis of 2-Betainyl Alconil Analog ...... 45
10. Synthesis of 9-Flourene Carboxyl-benzenesulfonamide ...... 46
11. Synthesis of 1 -Benzensulfonyl-3-[9-(9H)-Flourenyl]Urea ...... 47
12. Synthesis of the Radiolabeled Analog of 34 ...... 48
13. Enzymatic Conversion of Choline to Glycine Betaine ...... 105
14. Synthesis of Sulfobetaine ...... 115
15. Synthesis of Phosphobetaine ...... 116
16. Synthesis of N-Betainyl Benzenesulfonamide ...... 117
17. Synthesis of 105 Analogs ...... 119
18. Synthesis of 105 Analogs ...... 120
19. Synthesis of Sulfobetaine Analogs ...... 121
xiii PART 1
THE POLYOL PATHWAY
DESIGN AND SYNTHESIS OF INHIBITORS OF THE ALDOSE REDUCTASE ENZYME CHAPTER I
INTRODUCTION
1.1. Overview:
Mammalian cells lack a mechanically rigid-cell wall and, consequently, they are incapable of sustaining significant osmotic pressure difference. Thus, the cells need to live in an isosmotic environment in order to survive. The great nineteenth century French physiologist, Claude Bernard, has established the fundamental principle of mammalian physiology stating that the extracellular fluid constitutes the "internal environment" for cells and that the constancy of this internal environment is essential to the normal functioning of the cells and ultimately their survival .1’3
In accord with this principle, the volume, composition, and, hence the osmotic make up of the internal environment of almost all human tissue cells are very tightly regulated .3>4 The striking exception is the kidney medulla in which the blood and interstitial solutes concentration are much higher than the rest of 3
the body and vary considerably with the operation of the renal concentrating
mechanisms.4-5 These unique cells adapt by a controlled accumulation of
"compatible organic osmolytes." Thus, cellular osmotic balance is maintained while still keeping relatively constant intracellular electrolyte concentrations which, if altered might perturb the structure and function of intracellular biological macro-molecules.3-4-6
Four predominant compatible organic osmolytes have been identified in renal cells namely glycerophosphoryl-choline (GPC; 1), myo-inositol (2), betaine (3), and sorbitol (4), (Figure 1).7-8 The sum of the renal intracellular concentrations of these four compatible osmolytes were found to correlate highly with the osmolarity of extracellular fluid, suggesting that they act in combination as compatible osmolytes.9-10 Thus, the unusual rise in the concentration of any of these osmolytes will result in the decrease of the concentrations of the others.4 In contrast to the beneficial effect of accumulating appropriate amounts of compatible osmolytes during normal cell osmoregulation, too much of the osmolytes may be detrimental. That could be due to excessive swelling of the cell or due to sharp drop in the intracellular ionic strength.4 4
CH2 OH OH O H j HO — CH
CH2-0—P —0 HO myo-lnositol (2) HO — CH2 I HC — OH
I 0M H O - C * H m + JJ Betaine I Sorbitol / 'sS S ^ ' K 0 m (3) H C -O H (4)
HC — OH I CH2OH
Figure 1: Compatible Organic Osmolytes
An outstanding example of the harmful effect of organic osmolytes over accumulation is the cellular pathology associated with sorbitol accumulation which occurs in tissues that normally do not contain high levels of this sugar alcohol. This abnormal accumulation of sorbitol happens in cases of hyperglycemia associated with Diabetes Mellitus. Mounting experimental evidence suggests the involvement of sorbitol accumulation in the development of diabetic complications.11’14 Therefore the focus of this research will be on ways to interfere with sorbitol metabolism as means of A preventing the development of Diabetic Complications. 5
1.2. Diabetes Mellitus:
Diabetes mellitus (DM), as we know it today, has been recognized as an illness as early as 1500 B C .13 By the end of the eighteenth century, the relationship between the pancreas and DM was established. Although traditionally thought of as a singular disease state, DM is actually a series of complex chronic metabolic disorders characterized by symptomatic glucose intolerance and an abnormal increase in blood glucose levels (a condition known as hyperglycemia). According to the American Diabetes Association there are about 14 million diabetics in the United States alone . In the mid 1980s, DM was considered the seventh leading underlying cause of death in the U.S. 15
The U.S. market for drugs to control blood sugar level alone was estimated as
$940 million in 1990 with an expected increase to $1.2 billion by 1995, indicating a large financial burden on health care services.16
1.3. Types and Treatment of Diabetes Melleitus:
Two major types of DM have been defined by the National Diabetes Data
Group.17 Type I, or insulin dependent DM (IDDM), is characterized by low levels or the complete absence of circulating insulin. IDDM usually occurs in childhood or early adulthood (formerly known as Juvenile Diabetes) and it 6
accounts for approximately 10% of all diabetic cases. Heredity, viral infections, and autoimmune conditions have been linked to the development of this type of diabetes. IODM is controlled by daily injections of insulin, and patients are prone to develop the ketoacidosis if insulin is withheld. Type II, or non-insulin dependent DM (NIDDM) is by far the most common type of diabetes, accounting for approximately 90% of the total diabetic population.16 Patients of this type still retain the ability to secrete some insulin, are usually over 40, and are often obese. In this case circulating insulin falls to inadequate levels or the cellular responses to insulin for glucose uptake or utilization fail.
The treatment of DM varies considerably between IDDM and NIDDM. As mentioned above, Type I patients require insulin injection in addition to weight loss and diet control. In diabetics whose pancreas still produces insulin, oral hypoglycemics such as sulfonylureas (Figure 2) are often used in conjunction with weight loss, diet, and insulin administration if necessary.18 The exact mechanism of sulfonylurea action is unknown. They do not cause p-cells to make more insulin. Rather, they sensitize (3-cells to respond to glucose by releasing insulin secretory granules already there. 7
H H H k s /
Chlorpropamide Tolazamide
Glimepride
Figure 2: Clinically Used Sulfonylureas
Efforts to deal with insulin fluctuation have led to the development of orally administered insulin sensitizers such as pioglitazone and CP-72467 (Figure
3). These compounds are now in phase II clinical trials. They were shown to increase glucose uptake by peripheral tissues which results in decreasing the need of circulating insulin in blood.15 Pioglitazone
H CP-72467
Figure 3: Insulin Sensitizers
1.4. Diabetic Complications:
Currently available treatments of diabetes can correct acute life-threatening symptoms but do not prevent the development of the tissue damaging complications which are largely responsible for the morbidity and mortality of
DM . 19 In general, diabetic complications affect tissues that are not dependent on insulin for glucose transport and metabolism. 20 Examples of diabetic complications are: neuropathy (peripheral nerve dysfunction), nephropathy
(intracapillary glomerulo sclerosis), vascular complications (capillary basement 9
membrane thickness, precyte loss in capillaries, aneurysm), retinopathy,
cataracts, alteration of some immunological reactions, and skin and bone
disorders.11-21'2S
In general, diabetic patients have twice the normal incidence of cardiovascular
disease such as macroangiopathy and stroke, 25 times the normal incidence of
blindness, 20 times the normal incidence of gangrene and an increased risk of developing neurological and renal disease.18 Overall, the net effect of diabetic
complications is that the average lifespan of a diabetic is only two-thirds that of a non diabetic.19
1.5. Sorbitol Metabolism (the Polyol Pathway):
Sorbitol, the common name given to D-glucitol, is the polyol (polyhydric alcohol or alditol) of D-glucose. In mammalian tissues, sorbitol is produced as an intermediate in the conversion of glucose to fructose in a two-step metabolic process known as the Polyol Pathway (Figure 4). Two enzymes are involved in the polyol pathway: Aldose Reductase [low Michaelis-Menten constant (Km) aldehyde reductase, EC1.1.121, ALR2] which catalyzes the reduction of glucose to sorbitol, 26-27 and Sorbitol Dehydrogenase (L-iditol dehydrogenase, EC 1.1.1.14) which catalyzes the oxidation of sorbitol to fructose.28 10
HC = 0 HO — CH2 I I HC — OH NADPH + H + NADP * HC — OH I I HO — CH HO — CH I ^ . I HC — OH Aldose Reductase HC — OH I I HC — OH (AR) HC — OH I I ch 2oh CH2OH L-Sorbitol D-Glucose (D-Glucitol)
ho — ch 2 I NAD* NADH + H + c = o I HO — CH D-Fructose I HC — OH Sorbitol I Dehydrogenase HC — OH I CH2OH
Figure 4: The Polyol Pathway
The first physiological role attributed to sorbitol was in seminal vesicles and placentas of sheep, where it serves as the source of fructose, a nutrient for sperms and conceptus 4>29 Recently, its role as a compatible osmotically active organic solute in mammalian renal medulla has been p r o p o s e d .8 -1 2 ,2 9 ,3 0
Under normal physiological conditions, very little sorbitol was found in any tissues except the renal medulla. This is because of the low affinity of the first 11
enzyme in the polyol pathway (aldose reductase ; AR) toward glucose. Any excess intracellular glucose will be used up by the hexokinase in glycolysis.
However, in the case of DM, there is a large increase in blood glucose levels even in well-controlled diabetic patients. Glucose can freely pass in and out of the cells to achieve equilibrium, especially in tissues that are not dependent on insulin for their glucose transport. In these cases, the glycolytic pathways are saturated and there is an increased flux through the polyol pathway towards sorbitol formation. The rate of sorbitol formation exceeds the rate of its oxidation by sorbitol dehydrogenase into fructose. That, together with the fact that sorbitol lacks permeability through cellular plasma membranes, leads to the intracellular build up of sorbitol concentrations.14-20
1.6. The Role of the Polyol Pathway in Diabetic Complications:
The suspected association between the polyol pathway and diabetic complications was strengthened by the work of Kinoshita, who demonstrated the adverse effects of AR activation in the rat lens.31 This research has led to the development of the "Osmotic Hypothesis" of cellular damage leading to diabetic complications. Briefly, the osmotic hypothesis states that increased flux through the polyol pathway leads to the intracellular accumulation of sorbitol. Increased intracellular concentration of sorbitol leads to intracellular osmotic pressure build up which causes accumulation of water, cell swelling and changes in cell membrane permeability. K32-34 The change in the 12
membrane permeability causes the cell to lose essential metabolites and enzymes particularly myoinositol, reduction in glutathione, ATP, and Na-K-
ATPase activities. This results in a loss of metabolic function and ultimately cell destruction .14«32 It has been suggested that polyol induced pathology may also result from changes in cellular redox potentials resulting from the rapid depletion of NADPH and from the inverse relationship between the cellular concentration of myoinositol and aldose reductase activity.11
The glycation theory presents a quite different view of diabetic complications.
In this theory, nonenzymatic modification of protein by glucose is considered the critical event in the development of complications. 35*36 Very recently,
Vander Jagt proposed a new integrative model for the etiology of diabetic complications. The essential features of this theory are as follows:
1) hyperglycemia results in increased production of methylglyoxal in extrahepatic tissues; 2) ketoacidosis leads to production of acetol and methylglyoxal, primarily from hepatic metabolism of acetone; 3) aldose reductase reduces methylgloxal to acetol; 4) accumulation of acetol and glucose produces covalent modification of proteins leading to altered physiological functions. 36 From the above it could be concluded that Aldose
Reductase is the key enzyme in the development of diabetic complications.
The inhibition of this enzyme will help delay the onset of such complications. 13
1.7. The Aldose Reductase Enzyme:
Aldose reductase (AR) is the rate-limiting enzyme in the polyol pathway. 14 AR
is a member of a aldo-keto reductases superfamily.37 These cytosolic
enzymes are monomeric, NADPH-dependent reductases that preferentially
reduce aromatic aldehydes over nonaromatic aldehydes and aldoses. AR has
been found in numerous mammalian organs and tissues. 4-37 The enzymes
isolated from various tissues are monomers with apparent molecular weights
ranging from 28 to 45 KD. 4<38 The amino acid sequences of aldose
reductases from different sources have been determined. 4.37,39*41 u was
found to contain 315 amino acids with high degree of sequence identity (>84%)
among the reported protein sequences.4
The crystal structure of the pig lens AR and human placenta AR has been
recently reported. 42-43 The study revealed that AR folds into a three dimensional structure similar to that of the triose phosphate isomerase p/a- barrel (Figure 5). 44 As observed in all other p/a-barrel enzymes, the active site of AR is located at the COOH-terminal end of the p barrel. 45-46 The study also reveals an unusual NADPH-binding domain in AR where residues from both the N-terminal and the C-terminal regions are brought together to participate in the formation of the coenzyme binding site. 44 Kinetic studies of isolated AR revealed the presence of two forms of the enzyme which vary in their Km values for the substrate. Factors such as temperature, the presence of substrates, osmotic environment and exposure to oxidizing agents govern the ratio between the two forms of the enzyme.47'49 14
Figure 5: Ribbon Drawing of the Peptide Backbone from the Crystal Structure
of Human Placenta Aldose Reductase
1.8. Aldose Reductase Inhibitors:
Inhibition of AR has been the major goal in the treatment of late-onset diabetic complications. Massive work has been done in the development of inhibitors to this enzyme and a wide array of structurally diverse compounds have been found to inhibit AR.18-19*50'53 Although aldose reductase inhibitors (ARIs)
span a remarkable diversity of structures, almost all of them are acidic to some
degree and can generally be grouped into certain functional classes. ARIs
could be grouped into two major structural classes the carboxylic acids 15
and the acidic cyclic amides, with members of both classes currently in clinical studies. Figures 6 and 7 show representative examples of the clinically tested
ARIs from these two structural classes . 11.16,19,50,52,54
H H O'
CF3 (5) Alre8tatin (6) Tolrestat
0
ci Br Br
O
(7) FR 74366 (8) Ponalrostat (Statil)
\J
r - e O uJ>0 - H CF3
(9) Epalrestat (10) Zopolrestat
Figure 6: Clinically Tested Carboxylic Acids ARIs H — N
(11)Sorbinil (12) M 79175
H — N = o
ch 3o och 3 (13) Alconil (14) HOE-252 0 H
(15) CT112 (16) ADN138
Figure 7: Clinically Tested Acidic Cyclic Amides ARIs 17
Of these clinically tested ARIs, Alrestatin (5) was the first orally active ARI. 55
Tolrestat (6) and Zopolrestat (10) are among the most potent carboxylic
containing ARIs. 54 Epalrestat (9) is close to being marketed in Japan for diabetic complications. 33 Alconil (13) and HOE-252 (14) are among the most potent of ARIs known. 16,56,57 Sorbinil (11) is now being used as a standard against which new ARIs are tested. In addition, several natural products were also found to exhibit AR inhibitory activity. Among the naturally occurring ARIs are Questrin (17) and Gossypol (1 8 ).58-59
OH OHC OH OH HO HO CHO OH OH HO OH OH HO
Questrin Gossypol (17) (18 )
Recent literature is showing a continuous interest in developing more potent and less toxic ARIs 60'79. Several new structural classes and hybrids of the known ARIs have been introduced as potent inhibitors and are progressing into clinical trials; examples are: 1,2,4-triazolidine-3,5-dione derivatives (19,20),
3,4-isoxazolidinedione analog (21) ,21 spiro[imidazoline-4,4'(1'H)-quinazoline]-
2,2',5(3'H)-trione derivatives (22,23) ,80 ethyl 3-hydroxy-2(5H)-oxopyrrole-4- carboxylate derivatives (24,25), 81 and (2,6-dimethylphenylsulphonyl) nitromethane (2 6 ).82 18
V ' H Ri r \ , - 1 = / y (19), R = n -CsHuCO (21) (2 0 ),R 1 = CCI 3CO
N
o H-N Xtc ch 3 H (22), X = F (24) (23), X = Cl
(26)
Due to the toxicity associated with the potent spirohydantoin moiety containing
ARIs such as Sorbinil (11) and Alconil (13), 83-84 several bioisosteric modification of these ARIs were synthesized and were proven to be at least as active as their parent compounds. They were also found to lack toxicity associated with the hydantoin ring (compounds 24,27 and 28) .58-85 19
COOH HN
Cl
(27) (28)
Most of the reported ARIs are non-specific and they were also found to inhibit
the closely related enzyme Aldehyde Reductase [ALR, EC .1.1.1.20]. 51 >73
ALR and AR possess approximately 51% sequence homology, both are
monomeric NADPH-dependent oxidoreductases, and share some substrate
specificity. 82 The physiological role of ALR has not been established.
However, due to its high specificity for aromatic aldehydes, a general function
in detoxification of carbonyl compounds has been proposed.4
1.9. Aldose Reductase Binding Sites:
All the above mentioned ARIs display either uncompetitive or noncompetitive
inhibition, indicating that these compounds do not compete with either the
substrate or the nucleotide cofactor for their binding sites. Moreover,
competition studies suggests that all of these inhibitors interact reversibly at a common region on the AR enzyme. 2 0
The active site pocket of AR is reported to be very large and extremely hydrophobic, which explain the better affinity of the enzyme for aromatic than aliphatic aldehydes ,43-86 As mentioned earlier the substrate binding site of AR lies near the C-terminal end of the p barrel and the unique NADPH-binding domain involves residues from both the C- and N-terminals. 43-44 The inhibitor binding domain, however, has not yet been well identified. Liu and coworkers reported the involvement of sulfhydryl containing residues in the inhibitor binding sites. 87 They also reported that carboxymethylation of bovine lens
AR altered the sensitivity of the enzyme towards Sorbinil (11) and not Tolrestat
(6) indicating different distinct binding sites for different ARIs at the inhibitor binding domain. 88 A similar finding has been reported utilizing site directed mutagenesis studies. These studies suggested the involvement of Lys263 in the catalytic function of the enzyme, changing it to neutral or acidic residue dramatically changed the kinetic parameters of AR. In addition, mutation at the
Lys263 altered the enzyme sensitivity toward Sorbonil (11) and Alconil (13) type ARIs, however it has little effect on the /C/s of Tolrestat (6) or Statil (8) type inhibitors. Again, these findings illustrate a significant difference in the inhibitor association sites for ARIs depending on their structure. 89
A model for the inhibitor binding site has been proposed by Kador and
Sharpless ,90 based on the known structure activity relationships of ARIs, computational chemistry, and biochemical observations. This model (Figure 8) includes: 1) a primary lipophilic (aromatic) region separated (center to center) by approximately 2.8-3.8 A (di) from an electrophilic group; 2) a secondary 21
lipophilic region located about 2.8-6.1 A ( 0(2 ) from the electrophilic group and coplanar with the primary lipophilic region; 3) two hydroxy groups located 2.8-
3.8 A (c/ 3 ) and 8.0-9.3 A (c/ 4 ) from the center of the primary lipophilic region.
0 II
HO
d-t = 2.8-3.8 A c /2 - 2.8-6.1 A d3 = 2.8-3.8 A d4 = 8.0-9.3 A
Figure 8: Kador-Sharpless Pharmacophor Model for ARIs
According to this model, a three point interaction-site attachment should result from a combination of hydrophobic bonding and a reversible charge transfer reaction. Compounds with higher affinity for the proposed receptor model will be more potent ARI. Potency is expected to increase with either addition of selective lipophilic substituents or by addition of groups that could increase charge transfer capabilities .38 The inhibition of the enzyme could either be due to reversible conformational changes, before or after the binding of the substrate or the cofactor, or due to steric interference of the catalytic site produced by partial overlapping of bound inhibitor and the catalytic site.52 CHAPTER II
STATEMENTS OF PROBLEMS AND OBJECTIVES
As mentioned in the introduction, the enzyme aldose reductase remains a subject of great interest in chemical, biological, and clinical research. It is now well established that inhibiting aldose reductase will aid in preventing or at least delaying the development of diabetic complications. A large number of relatively potent reversible inhibitors of AR have been developed and the approximate sites to which they bind at the enzyme were proposed. However, the detailed interactions between the enzyme and the inhibitor at the inhibitor binding site, and its relation to the catalytic function of enzyme, is not clearly understood. A clear and concise understanding of the intimate interactions between the enzyme and the inhibitor, and the exact make up of the inhibitor binding site, will lead to the development of more potent and highly specific inhibitors for AR. That could partially be achieved by the use of affinity and photoaffinity ligands that can irreversibly bind to the enzyme.
Generally, affinity or photoaffinity ligands are compounds that have high affinity for a particular enzyme or receptor protein, and they have within their structure a chemoreactive or photoreactive moiety that can form a covalent bond with
-2 2 - 23
reactive groups in their binding site on a specific protein .91-93 Tracing these
covalently bound ligands will provide a clearer understanding of their binding
site. In addition, affinity ligands could provide a prolonged inactivation of the
enzyme and consequently reduce the need for frequent administration of
potential drugs from this type.
2.1. Irreversible Inhibitors of Aldose Reductase:
Protein modification and protection studies, and the crystal structure of AR,
revealed a score of nucleophilic amino acid residues near or close to the
enzyme binding sites: a sulfhydryl group and a tyrosine residue near the
inhibitor binding site, basic residues (lysine, arginine and histidine) at the
cofactor binding site, and a lysine and a tyrosine residues at the substrate
binding site. 42,43,88,89,94-96
To gain more information about the nature of enzyme inhibitor interaction, the
location of the amino acids at the inhibitor binding site, and the structural and
spatial limitations of the binding site, several affinity and photoaffinity analogs of the known ARIs are being utilized as specific probes. These probes, with an appropriately placed electrophilic functional groups, can react irreversibly with any of the nucleophilic residues near or around their binding site at the enzyme. Electrophiles such as isothiocyanate, haloacetamides, and sulfonyl flouride have been placed in different positions on three different ARIs:
Alrestatin (5), Sorbinil (11), and Alconil (13). 52,96-101 These same inhibitors 24
carrying an azide moiety were used as photoaffinity labels for AR . 94,ioi jh e
reversible and irreversible activities of the reported affinity and photoaffinity analogs of Alrestatin and Alconil are shown in Tables 1 and 2.
Table 1: Reversible and Irreversible Inhibition of Rat Lens AR by
Affinity and Photoaffinity Labels Analogs of Alrestatin.
0
Inh bition
Compound # R Reversible IC 50 (pM) Irreversible % at 10*4 M
5 H 0.11 0.0
29 NCS 0.30 56 j
30 n 3 3.60 34*
31 n h c o c h 2ci 0.55 0.0
32 NHCOCH2Br 0.60 46.0
34 n h c o c h 2i 0.40 89.0
* After 1 h photolysis 25
Table 2: Reversible and Irreversible Inhibition of Rat Lens AR by
Affinity Labels Analogs of Alconil.
H — N
x
4
Inhi Dition
Compound # XR Reversible IC5 0 (pM) Irreversible % at 10*4 m
35 HH 0.86 0.0
36 H 2-NCS 0.23 56.0
37 H 3-NCS 0.09 54.0
38 H 4-NCS 0.28 21.0
39 F 2-NCS 0.50 56.0
40 F 2-S02F 0.07 12.0
41 H 2- NHCOCH2 CI ND* 0.0
42 H 2- NHCOCH2Br ND* 0.0
43 H 2- NHCOCH2I ND* 25.6
44 H 4-NHCOCH2l ND* 41.2
* Not determined. 26
The data presented in Tables 1 and 2 show that the concept of affinity labeling
is clearly applicable to the AR enzyme. It also reveals the presence of a
reactive nucleophilic residue at or near the inhibitor binding site. The same eiectrophilic moieties produced better irreversible inactivation of the enzyme when placed on the Alrestatin molecule than when placed on the Alconil backbone. Up to 89 % irreversible inhibition was achieved by Alrestatin substituted at position 5 with iodoacetamido group (compound 34). However,
34 has only one fourth of the reversible activity (as indicated by the IC 50 values) of Alrestatin itself.
2.2. Alrestatin Analogs as Reversible and Irreversible Inhibitors of AR:
In an attempt to reach an irreversible AR inhibitor with higher potency and better affinity for the enzyme, we designed a series of affinity labels with enhanced lipid solubility (compounds 45,47*49 ; Figure 9). This is in agreement with the notion that increasing the lipid solubility will enhance the potency of ARIs. 38>102 The chemoreactive moieties in the designed inhibitors (45-49) are a reactive " Michael-acceptor" type. This type of affinity labeling has been successfully utilized before in irreversibly labeling a-|- adrenoceptors.103 27
Figure 9 : Proposed Affinity Labels on Alrestatin Backbone
All the affinity labels for AR reported to date were aimed at the nucleophile located near the enzyme domain that binds the aromatic portions of Alrestatin
(position-5), Alconil (positions-2,3 and 4), and Sorbinil (position-8). In order to explore the presence of different nucleophiles at or near the binding domain of 28
the carboxylic group of Alrestatin, we designed alrestatin analogs with benzenesulfonamides and nitrophenols as carboxylic acid surrogates
(Tables 3 and 4). To these carboxylic surrogates, different chemoreactive groups could be attached to probe for nucleophiles at the carboxylic acid binding domain (Figure 10).
Table 3: Proposed Benzenesulfonamide Analogs of Alrestatin
Compound Number R R1
50 H H
51 H N 0 2
52 4-N 02 H
53 3-NO2 H
54 2-NO2 H
55 4-NH2 H
56 4-NH2 N 0 2 29
Table 4: Proposed Nitrophenol Analogs of Alrestatin
Compound Number R Ri
57 HH
58 H n o 2
59 n o 2 n o 2 30
(60) R = C l; (61) R = I ; (62) R = +N(CH3)3
Figure 10: Proposed Affinity Labels From Carboxylic Surrogates of Alrestatin
The rationale behind the the design of compound 62 was to examine the effect that a permanently charged moiety will have on the enzyme inhibitor binding
interaction. In addition, it might shed some light on the nature of the nucleophilic residue at the inhibitor binding site.
2.3. Alconil Analogs as Reversible and Irreversible Inhibitors of AR:
As could be seen in Table 2, similar irreversible inhibition of AR was obtained with Alconil analogs substituted at position 2 or 3 with electrophilic moieties.
As a continuation of the above mentioned rationale we designed a series of
Alconil analogs substituted at position 2 with the lipophilic "Micheal-acceptor" 31
type electrophiles (Figure 11). Compound 66 was designed for the same
reason as compound 62. In addition, we design Alconil analogs modified at the
hydantoin ring part of the molecule, to investigate the possibility of applying the affinity labeling technique at this part of the molecule (Figure 12).
O—
o
R = R =
(63) (64)
R = l H (66)
Figure 11: Proposed Affinity Labels on Alconil Backbone 32
Figure 12: Proposed Alconil Analogs Modified at the Hydantoin Ring 33
2.4. Proposed Radiolabeled Analog of 5-lodoacetamldo-1,3 -dioxo-1 H-
benz[de]isoquinoline-2(3H)-acetic acid (69):
As shown in Table 1 compound 34 was the most active irreversible inhibitor of
AR with 89% irreversible inactivation of the enzyme. That prompted us to synthesize the 14C-labe!ed analog of 34 (compound 69; Figure 13) to identify the nucleophilic residue that covalently binds to this inhibitor.
o
c N ^ 0
H
Figure 13: The 14C-Labeled Analog Of 5-lodoacetamido -1,3 -dioxo-1 H-
benz[de]isoquinoline-2(3H)-acetic acid CHAPTER III
RESULTS AND DISCUSSION
3.1. CHEMISTRY:
3.1.1. Synthesis of 5-Substituted Alrestatins (Compounds 45-49):
Synthesis of the key intermediate 5-amino-1,3-dioxo-1H-benz[de]isoquinoline-
2(3H)-acetic acid (5-aminoalrestatin) (75), was achieved by modification of the reported procedures 55,92,93 which reduced the reaction time, produced a cleaner product, and gave better yields. The procedure utilized for the synthesis of 5-aminoalrestatin in comparison to the reported procedure is outlined in Scheme 1.
The bicyclo acid (75) was synthesized according to the reported procedure
103,104 by reacting cyclohexadiene and propiolic acid. However, the fractional distillation step was skipped because heating initiated the reverse Diels Alder reaction to produce benzoic acid (Scheme 2).
-34- 35
HCI*H 2NCH2CO2CH3
(7 ° ) ' TEA,Toluene, A
w^ L s J s N02 sh h 2n c h 2co o h ' DMF.A ^H2,P d /C ,3 h 2 h kEtOAc
H2, Pd/C 30% KOH MeOH, MeCN.RT r || 2 h
(72)
Scheme 1: Synthesis of 5-Aminoalrestatin (74) 36
Reaction of the key intermediate 5-aminoalrestatin (74) with maleic anhydride, or the appropriate acid chloride yielded the target compounds 45-49 (Scheme
3). However, heating at elevated temperature was avoided in the synthesis of compound 45, to avoid the decomposition of the bicyclo acid.
0
Scheme 2: Synthesis and Decomposition of Bicyclo[2.2.2]octa-2,5-diene-2-
carboxylic Acid (75) 37
CH 2C i2,Py Toluene, 48 hr ^ water trap AH O.v N. x.O THF.Py 4h, RT
CH2CI2,Py A THF, DMAP, A 2 hr
Scheme 3 : Synthesis of 5-Substituted Alrestatins (45-49) 38
3.1.2. Synthesis of Benzenesulfonamide Analogs of Alrestatin
(Compounds 50-56):
Attempts to synthesize the lead compound in this series, 1,3-dioxo-1H- benzen[de]isoquinoline-2(3H)-acetyl benzenesulfonamide (50) by reacting the potassium salt of 1,8-naphthalimide (79) with chloroacetamidobenzenesulfon- amide ( 78, Scheme 4) failed. This was probably due to the acidity of the sulfonimide hydrogen which exceeds that of the naphthalimide and results in its precipitation.
o H o = s - nh 2 O O N DMAP N'S = 0 . c ' V 6 CH2CI2 6 (76) (78)
■J o Mo 0 ^°6 1 = 0 (79) (50)
Scheme 4: Attempts for the Synthesis of Compound 50 39
The synthesis was achieved by applying the facile procedure reported by
Matassa and coworkers 105 for the conversion of carboxylic acids to A/-sulfonyl
amides using a (water soluble) carbodiimide in methylene chloride. As outlined
in Scheme 5, the desired products (50-54) were obtained in very high yields;
and the reactions required minimum work up. The only modification to
Matassa's procedure is that the water soluble 1-[3-(dimethylamino)propyl]-3-
ethylcarbodiimde hydrochloride was added to an ice-cooled suspension of the
acid [alrestatin(5), or 5-nitroalrestatin (73)] , the appropriate substituted
benzenesulfonamide, and dimethylaminopyridine (DMAP) in methylene
chloride under argon. When the ingredients were mixed at room temperature, or in a different order, the reaction did not proceed as clean, and tarry products were obtained. The only work up required for this reaction was vigorous shaking with 1 N HCI. The products either fall out of solution or remain in the organic layer, while the unreacted materials are washed out in the aqueous layer.
o
Scheme 5: Synthesis of Benzenesulfonamide Analogs of Alrestatin (50-56) 40
For the synthesis of compounds 55 and 56, 4-aminobenzenesulfonamide
(sulfanilamide) was protected as trifylate before applying Matassa's procedure.
The protecting group was conveniently cleaved during the acidic work up to
give the target compounds 55 and 56.
3.1.3. Synthesis of Nitrophenol Analogs of Alrestatin
(Compounds 57-59):
The synthesis of the target compounds was achieved by the condensation of
2-amino-4-nitro-phenol (80) with 1,8-naphthalic anhydride (81) or 3-nitro-1,8- naphthalic anhydride (70) in refluxing DMF to give compounds 57 and 58, respectively. Treatment of 57 with 10 % nitric acid in concentrated sulfuric acid gave the dinitro analog (59, Scheme 6).
3.1.4. Synthesis of Alrestatin Carboxylic Surrogates as Affinity
Labels for AR (compounds 60-62):
The synthesis of the target compounds 60-62 is outlined in Scheme 7.
Sulfanilamide (82) was chloroacetylated by treatment with chloroacetic anhydride (77) in methylene chloride using pyridine as the base to give
/V4-chloroacetamido benzenesulfonamide (83). The reaction of 83 with alrestatin (5) in Matassa's conversion procedure 105 yielded the target compound 60 in very good yield. The 4-iodoacetamido benzenesulfonamide 41
analog of alrestatin (61) was prepared by the treatment of the chloroacetamido analog (60) with excess Nat in acetone under Finkelstein halide exchange reaction conditions .106 Replacement of the chlorine atom in 60 with trimethylamine provided the quaternary ammonium analog (62; Scheme 7).
OoN
OH H
DMF
(80)
NO< (57) R= H (58) R= NO 2
(59)
NO:
Scheme 6 : Synthesis of Nitrophenol Analogs of Alrestatin (57-59) 42
S 02NH2 s o 2n h 2
Py.
c h 2c i 2
n h 2 -•V'- (82) (83) o
Cl O = \
carbodiimide □MAP, CH2CI2
(CH3)3N
s / W, I N o o = \ ' W ^= \ \ XW Cl +
° ^ N ^ 0
(61)
Scheme 7 : Synthesis of Carboxylic Surrogates Affinity Labels 43
3.1.5. Synthesis of 2-Substituted Alconil Analogs (63-66):
The synthesis of the target compounds is outlined in Scheme 8. The key intermediate 2-aminospiro(9H)-fluorene-914,-imidazoline-2\5,-dione (85) was prepared according to the reported procedures 57,99,1 oo by reacting 2-amino-
9-fluorenone (84) with ammonium carbonate and potassium cyanide under
Bucherer-Berg’s reaction conditions. 107 Treatment of 85 with maleic anhydride in toluene under water trap, or the appropriate acid chloride in acetonitrile yielded the target compounds 63-65 in good yields.
The 2-betainyl-spiro(9H)-fluorene-9,4'-imidazoline-2,,4,-dione (66) was obtained by the treatment of the reported 2-chloroacetamido analog (4 1 )" with trimethylamine in acetone (Scheme 9) 44
0 = r N •
(84)
(85)
0 = *
0 —
(64)
(65) 0=<
(63)
Scheme 8: Synthesis of 2-Substituted Aiconil (63-65) 45
O O
+ c ls ^ o ^ cl
(85) (77)
(CH3)3N A s ^ C I A n N N ^ ^ + S
Scheme 9: Synthesis of 2-Betainyl Alconil Analog (66)
3.1.6. Synthesis of Alconil Analogs Modified at the Hydantoin
Ring (67,68):
The synthesis of 9-fluorene-carboxyl-benzenesulfonamide (68) was fairly simple. We were able to convert the commercially available 9-fluorene carboxylic (86) acid into the benzenesulfonimide analog by applying Matassa's procedure 105 to give 68 in fairly high yield (Scheme 10). On the other hand, the synthesis of 1-benzensulfonyl-3-[9-(9H)-fluorenyl]urea (67) was somewhat problematic. We were able to convert 9-aminofluorene (87) to the corresponding urea analog (88); however, coupling of (88) with benzenesulfonyl chloride (89) using different bases (pyridine, DMAP or sodium 46
methoxide) and in different solvents (acetone, pyridine or DMF) under different reaction conditions did not give the desired product (Scheme 11). In contrast, treatment of the amine ( 8 8 ) with benzenesulfonyl isothiocyanate (90) in dry acetonitrile gave the desired product (67) in a single step.
OH 0 0 = S -N H 2 DMAP,UI CH2CI2
X N:C:N (86 ) 6 HCI (76)
N-S
(68 )
Scheme 10: Synthesis of 9-Fluorene Carboxyl-benzenesulfonamide (68) 47
NH VNH, H - N KNCO
(87) (88 )
(90) | (89)
SOoNCO O SOoCI II o
o H-N H
Scheme 11: Synthesis of 1-Benzenesulfonyl-3-[9-(9H)-Fluorenyl]Urea (67) 48
3.1.7. Synthesis o f 14C-Labeled Analog of 5-lododacetamido-1,3 -
dioxo-1H-benz[de]isoquinoline-2(3H)-acetic acid (69)
The radioactive atom was chosen to be placed in the iodoacetamido part of the inhibitor molecule (34). That will allow for the tracing of the covalently bound radioactivity even after the acid digestion of the enzyme protein. The cold synthesis (Scheme 12) was done by a slight modification of the reported procedures. 52,98,1 oo The molar equivalence of iodoacetic anhydride was reduced from 5 to 3 molar equivalent and the reaction temperature was increased from 0 °C to 120 °C. [1- 14C] Iodoacetic anhydride was prepared by
American Radiolabeled Chemicals, Inc., from [1- 14C] iodoacetic acid and iodoacetyl chloride. 108 The low radiochemical yield obtained was due to the fact that, the radioactive reactant was not the rate limiting material of the reaction, and the maximum theoretical radiochemical yield is only 25 %.
Scheme 12: Synthesis of the Radiolabeled Analog 69 49
3.2. Biological Results:
The inhibitory activities of the new compounds were tested against cloned rat lens aldose reductase enzyme (RLAR ).109 Biological tests were performed at the National Eye Institute, National Institutes of Health, by Drs. Peter Kador,
Anita Malik and Tadadshi Mizogouchi.
Two types of experiments were used. First, the tested compound was added directly to the enzyme mixture and the inhibition of RLAR expressed as molar concentration that cause 50% inhibition (IC 50 values) was compared to that of a reference standard. In the second set of experiments, the tested compound was preincubated with the enzyme and the enzyme was isolated by gel filtration on NAP-5 desalting column using phosphate buffer containing 2- mercaptoethanol as an eluent and assayed for residual activity. This treatment was reported to break the enzyme-inhibitor adduct of the known reversible
ARIs regardless of their affinity to the enzyme and 100% recovery of the enzyme activity is usually obtained. 96 If the tested compound can act as an alkylating agent, then the % inactivation of the enzyme after gel filtration is considered as irreversible inhibition. 50
For the determination of the inhibitory activity, 1 mM of the tested compound was dissolved in 1-5 ml of NaOH (0.004 N) diluted with water to form 10 mL total volume of a stock solution. Different dilutions (1 X 10’4 -1 X 10'7 M) of the compound were prepared from the stock solution to determine the IC 50 value. The activity of the enzyme was determined by spectrophotometrically following the decrease in NADPH concentration at 340 nm, using DL- glyceraldehyde as the substrate. 96 The concentrations of the inhibitors needed to cause 50% inhibition of the enzyme activity (IC 50) were estimated from least square regression of the Marquart-Leveberg iterative curve fitting algorithm of the dose response curves. 100 The IC 50 values of the tested compounds were compared to their parent unsubstituted inhibitor (alrestatin or alconil) as the reference standard. For the determination of the irreversible inhibitory activity, the enzyme was passed through a NAP-5 desalting column, then incubated for 15 minutes at room temperature with 0.1 mM of the tested compound. Unreacted reagents were then removed by gel filtration, and the remaining aldose reductase activity was determined as above .108 These procedures have been previously described in detail. 52,96-98 51
3.2.1. Irreversible Inhibitors
IC50 values and the percentage of irreversible inhibition of rat lens aldose reductase (RLAR) by 5-substituted alrestatin analogs (45-49), and 2- substituted alconil analogs (63-65) are recorded in Tables 5 and 6 respectively.
As could be seen in Table 5, conjugated carbonyl amides substituents at position 5 of alrestatin molecule dramatically enhance the inhibitory activity of the parent compound, the IC 50 values went down to 0.08 pM (47) as compared to 1.6 pM for alrestatin. The irreversible inhibition activity seems to be a function of the lipid solubility, up to 40 % irreversible inhibition was achieved with the lipophilic analogs 45 and 48. In contrast, the same type analogs on the alconil backbone showed marked depression of the inhibitory activity (IC 50 values) with no significant irreversible inhibition (Table 6). This indicates that alrestatin type inhibitors probably bind at a different site than that of the alconil type inhibitors. This finding supports the previously reported notion that different ARIs bind at distinct sites on the enzyme depending on their structure type .88,89 52
Table 5: IC50 Values and % Irreversible Inhibitory Activities of
Compounds (45-49)
Compound # R ICso(uM) %lrreversible Inhibition at 0.1 mM
5 H 1.6 0.0 %
45 0.11 4 0 %
H
46 1 0.13 13.3%
H 0 47 0.08 2.7%
0
0 48 1.16 0.0 % H Sd 49 'H 0.80 37.85 % Table 6: IC50 Values and % Irreversible Inhibitory Activities of the
Alconil Analogs (63-66)
Compound # R IC50 (uM) %lrreversible Inhibition at 0.1 mM
91 H 0.86 0.0 %
63 14.4 5 .8 %
1 64 ° ^ y ° 2.15 7 .3 %
H O 65 3.88 7 .0 %
0
H 66 9.73 0.0 % 0 • 54
3.2.2. Reversible Inhibitors
Tables 7 and 8 show the RLAR inhibitory activities of the carboxylic acid surrogates of alrestatin (compounds 50-56). As could be seen in Table 7 bioisosteric replacement of the carboxylic moiety of alrestatin with benzenesulfonamide moiety yielded inferior inhibitors to the parent molecule.
However, substitution with nitro groups at different positions of these analogs generally produced inhibitors with better activities and are comparable to that of alrestatin (compounds 5 1 ,5 3 and 56). Similar results are obtained with the nitrophenol analogs (Table 8), indicating that the carboxylic binding domain can accommodate bulky substituents as long as acidic functionality is maintained.
Substitution of the hydantoin ring of Alconil with acidic functionality have been tried before with limited success. 100 The benzenesulfonamide analogs of
Alconil that we proposed 67 and 68 showed better activities than the reported ones, however they were still inferior inhibitors in comparison to their parent compound (Table 9). However this finding indicates that the hydantoin binding domain can recognize benzenesulfonamide as a bioisostere of the hydantoin ring. 55
Table 7: IC 50 Values and % Irreversible Inhibitory Activities of the
Benzenesulfonamide Carboxylic Surrogates of Alrestatin
(51-56)
Compound # R Rl IC50 (pM) % Irreversible
Inhibition at 0.1 mM
Alrestatin (5) - H 1.6 0.0%
50 H H 43.7 3.3 %
51 H N 02 2.31 0.0 %
52 4-NO2 H >100 2.3 %
53 3-NO2 H 10.7 0.0 %
54 2-NO2 H >100 0.0 %
55 4-NH2 H 100.7 0.0 %
56 4-NH2 N 02 1.94 7.7 % 56
Table 8: IC 50 Values and % Irreversible Inhibitory Activities of the
Nitrophenol Carboxylic Surrogates of Alrestatin (57-59)
Compound # R Ri IC5 0 O1M) % Irreversible Inhibition at 0.1 mM
Alrestatin (5) - H 1.6 0.0 %
57 HH >100 0.0 %
58 H N 0 2 >100 0.0 %
59 NO2 N 0 2 8.9 0 .0 % 57
Table 9: IC50 Values and % Irreversible Inhibitory Activities of
the Alconil Analogs (67-68)
Compound # Structure IC 5 0 (|xM) % Irreversible Inhibition at 0.1 mM
H 0 91 0.86 0 .0 % 0 ^ 3
92a100 183 0 .0 % o f e
N' N\\ 92b100 1360 0.0 %
67 15.3 0.0 % o - ju - v IJ H C M 3
68 8.4 - C ^O 58
Table 10 shows the comparative inhibitory activities of the affinity labels from the benzenesulfonamide carboxylic surrogates of alrestatin (60-62). The data indicate the presence of a nucleophilic residue at or near the carboxylic binding domain. The fact that the less reactive electrophile chloroacetamido analog
(60) showed better irreversible inhibitory activity than the more reactive iodoacetamido analog (61) indicates that alkylation was not a function of the reactivity of the electrophile at the inhibitor molecule. Another possible explanation is that steric factors interfere with the proper alignment of 61 to be assessable to the nucleophile.
The permanently charged compounds, 4-betanylamidobenzenesulfonamide analog of alrestatin (62) and 2 -betanylamido analog of alconil (66) showed no enhancement of the inhibitory activities of their parent compounds (Tables 6 and 10). This suggests that the nucleophiles at the inhibitor binding site are probably not a charged residue (such as glutamte or aspartate), rather they could be nucleophilic groups of tyrosine, arginine, lysine, histidine or cysteine. 59
Table 10: IC50 Values and Irreversible Inhibitory Activities of
Affinity Labels from the Benzenesulfonamide Analogs
of Alrestatin (60-62)
Compound # R IC 5 0 (pM) % Irreversible Inhibition
at 0.1 mM
60 Cl 58.6 22.20%
61 1 22.0 13.52%
62 +N(CH3)3 45.0 0.0 %
To test the specificity and selectivity of the new compounds for AR, their inhibitory activities were examined on RLAR and the closely related rat kidney aldehyde reductase (RKALR). The results are shown in Table 11. 60
Table 11: The Inhibitory Activities of the Tested Compounds Against
RLAR and RKALR
Compound # Structure RLAR RKALR
ICsofuM) ICso GiM) ------*r ■ " ------r*0 " ° . N . O 48 1.16 2.55
49 0.80 46.40 ' & . v
52 >100 37.00 O ^ N ^ O O 6o
55 66 100.7 8.83
" T > .
58 >100 16.00 61
Table 11: (Continued)
rAr,' ' 8I- 0 ‘ N^ c' 60 65 58.6 49.2
f S ° i ~ r y X * .° O h 61 65 22.00 >100
The data shows that several compounds show selective inhibitory activity for either RLAR or RKALR. However, several of the new compounds inhibit both of the enzymes almost with the same inhibitory potency ( I C 5 0 values). In a very broad generalization, it could be concluded that benzenesulfonamido and nitrophenol analogs of alrestatin are better inhibitors for RKALR than RLAR
(compounds 52, 55 and 58). On the other hand, 5-substituted alrestatin analogs appear to have more selectivity for AR over ALR (compounds 48 and
49). Enhanced lipid solubility appears to favor AR inhibition over ALR inhibition. 62
3.3. Summary
1. Substitution at the 5-position of alrestatin with bulky conjugated carbonyl
compounds increases its inhibitory activity against RLAR.
The irreversible inhibitory activities of these analogs appears to be
proportional to the lipophilic character of the inhibitor.
2. The carboxylic binding site of alrestatin on the RLAR enzyme can
tolerate bulky substituents as long as the acidic functionality is
maintained. Irreversible inhibition of AR was achieved by attaching
chemoreactive moieties on the carboxylic group surrogate, thus
suggesting the presence of a nucleophilic residue in the vicinity of its
binding site on the enzyme.
3. The ARIs, alrestatin and alconil are probably binding at two different
distinct sites at RLAR. The hydantoin ring of alconil is
bioisosterically replaceable with benzenesulfonamido group.
4. Structural analogs of alrestatin and alconil exhibited preferential,
selective inhibitory activities for aldose reductse (AR) and aldehyde
reductase (ALR). Chapter IV
Experimental
Melting points were determined using Thomas-Hoover melting point apparatus and are uncorrected. Structures of all the new compounds were confirmed by
1H-NMR, elemental analysis and/or MS spectra. 1H-NMR spectra were obtained at the Ohio State University College of Pharmacy, with an IBM
NR/250 FTNMR (250 MHz) with D20 , methanol-d 4 , or DMSO-d6 as solvent and internal reference. Mass spectra were obtained with either a Kratos MS 25
RFA mass spectrometer at the Ohio State College of Pharmacy or with a
Kratos MS-30 mass spectrometer at the Ohio State University Chemical
Instrumentation Center. Anhydrous THF was produced by distillation over sodium using benzophenone as indicator for dryness. Methylene chloride was dried over CaCl 2 then distilled. Acetonitrile was dried over molecular sieves and distilled before use. Elemental analyses were performed by Galbraith
Laboratories, Inc., Knoxville, TN; or by Oneida Research Services, Inc. ,
Whitesboro, NY. All analytical results for the indicated elements were ± 0.4% of the theoretical values.
-63- 64
5-Nitro-1.3-dioxo-1H-benzrde1isoaiiinoline-2(3m-acetlc acid (73)
O
3-Nitro-1,8-naphthalic anhydride (2.4 g, 9.9 mmol) and glycine (0.85 g, 11.3 mmol) in dry DMF (20 mL) were heated under reflux for 2 h. The reaction mixture was treated with activated charcoal, filtered and poured while hot over crushed ice. The yellowish heavy product that separated out was collected by filtration and dried. It was pure enough without any further recrystallization (2.5 g, 84%). mp 267-270 °C (lit. 55 mp 273-275 °C).
1H-NMR 8 9.51 (d,1H,J = 2.2 Hz, ArH ortho to NO2 ), 8.97 (d,1H, J = 2.2 Hz,
ArH ortho to N02), 8.80 (d,1H, J = 8.1 Hz, ArH), 8.70 (d,1H, J = 8.1 Hz, ArH),
8.07 (t, 1H, J = 8.1 Hz, ArH) and 4.75 (s,2H, CH 2COOH). 65
5-Amino-1.3-dioxo-1 H-benzrdelisoquinoline-2(3Hl-acetic acid (741
O
The nitro acid 73 (2 g, 6.7 mmol) was dissolved in DMF (30 mL) and added to a
Parr bottle containing 10% Pd/C (0.2 g). The mixture was hydrogenated at 50 psi for 5 h. Treated with activated charcoal, filtered and concentrated under vacuum. Upon addition of water a heavy precipitate was separated out, collected by filtration, dried and recrystallized from MeOH to give 74 (1.75 g, quantitative), mp 290 °C (lit. 55,92 mp 292 °C).
1H-NMR 5 8.09-8.05 (dd, 2H, ArH), 7.97 (d, 1H, J = 2.2 Hz, ArH ortho to
CO), 7.63 (t, 1H, J = 8.3 Hz, ArH), 7.31 (d, 1H, J = 2.2 Hz, ArH ortho to NH2),
6.03 (broad s, 2H, NH 2) and 4.69 (s, 2H, CH 2COOH). 6 6
Bicvclor2.2.21octa-2.5-diene-2-carboxvlic acid (75) • M y
A mixture of 1,3-cyclohexadiene (3.9 g, 48.6 mmol) and propiolic acid (2.35 g,
33.5 mmol) was stirred at room temperature for one week. The product was isolated by a short gravity column chromatography (-2 0 0 g silica gel) using
CHCI3 / MeOH (98:2) as eluent. Fractions with Rf values of 0.3 were combined and evaporated under vacuum. The residue was recrystallized from hexane to give 75 (2.0 g, 40%). mp 86-88 °C (lit. 95-96 mp 84-85 °C).
1H-NMR (CDCI3) 5 11.08 (broad s, 1H, COOH), 7.47-7.43 (dd, J = 5 Hz, J =
1.8 Hz, 1H, gem. to COOH), 6.38-6.26 (m, 2H, olifenie H), 4.18 (m, 1H, tertiary
H), 3.78 (m, 1H, tertiary H) and 1.39-1.27 (m, 4H, bridge H). 67
5-(Bicvclof2.2.21octa-2.5-diene-2-carboxamido}-1.3-dioxo-1 H- benzfde1isoquinoline-2(3H)-acetic acid (45)
H O'
N I H
The bicyclo acid 75 (0.21 g, 1.4 mmol) was heated under reflux with thionyl chloride (4 mL) for 2 h. Excess thionyl chloride was taken off under vacuum and the residue was coevaporated with toluene (2x10 mL). The resulting residue was dissolved in dry CH 2CI2 (10 mL) and added to a suspension of the amino acid 74 ( 0.36 g, 1.4 mmol) and dry pyridine (0.1 g, 1.4 mmol) in dry
CH2CI2 (20 mL). The reaction mixture was heated under reflux for 4 h.
Solvent was taken off under vacuum and the residue was washed with 10%
HCI (2x10 mL) and recrystallized from aqueous EtOH to give 45 as yellow crystals (0.35 g, 62%). mp 179-181 °C.
1H-NMR S 8.92 (d, J = 1.8 Hz, 1H, ArH ortho to NHCO), 8.79 (d, J = 1.8 Hz,
1H, ArH ortho to NHCO), 8.41-8.37 (m, 2H, ArH), 7.83 (t, J = 7.6 Hz, 1H, ArH),
7.38 (dd, 1H, gem. to CO), 6.45-6.37 (m, 2H, olifenic H), 4.7 (s, 2 H,
CH2 COOH), 4.3 (m, 1H, tertiary H), 3.8 (m, 1H, tertiary H) and 1.25(m, 4H, bridge H). 68
Analysis for C23HisN2Q5#H20
Calculated: C 65.70, H 4.79, N 6.66
Found: C 65.84, H 4.57, N 6.56
5-Maleiamido-1 .3-dioxo-1 H-benzrde1isoquinoline-2(3HFacetic acid f46)
O
A suspension of the amino acid 74 (0.15 g, 0.6 mmol) and maleic anhydride
(0.08 g, 0.8 mmol) in dry toluene (100 mL) was heated under a water trap for
48 h. The product that separated out was collected by filtration, dried and recrystallized from MeOH to give 46 (0.15 g, 79%). mp 201-204 °C.
1H-NMR 8 13.05 (broad s, 1H, COOH), 8.87 (s, 1H, ArH, ortho to N), 8.65
(d, J = 1.9 Hz, 1H, ArH ortho to N), 8.47-8.39 (m, 2H, ArH), 7.85 (t, J = 7.7 Hz,
1H, ArH), 6.50 (d, J = 11.8 Hz, 1H, olifenic H), 6.38 (d, J = 12.0 Hz, 1H, olifenic
H) and 4.72 (s, 2 H, CH2COOH).
MS (m/e) El 350 M+ 69
Analysis for C 18 H10N2O6 • 2 H2O Calculated: C 55.96, H 3.65, N 7.25
Found: C 56.21 , H 3.62, N 7.23
5-Ethvlf umaramido-1.3-dioxo-1 H-benzrde1isoquinoline-2(3H)-acetic acid m
O
H O
Fumaric acid mono ethyl ester (0.48 g, 3.3 mmol) was heated under reflux with thionyl chloride ( 5 mL) for 2 h. Excess thionyl chloride was evaporated under vacuum and the residue was coevaporated with toluene (2x10 mL). The resulting acid chloride was dissolved in dry THF (20 mL) and was added to a suspension of the amino acid 74 (0.3 g, 1.1 mmol) and pyridine (0.2 g, 3 mmol) in THF (100 mL). The mixture was heated under gentle reflux for 2 h. Solvent 70
was evaporated under vacuum and the residue was washed with water and recrystallized from acetone to give 47 as white solid (0.3 g, 6 8 %). mp >300 °C.
1H-NMR 8 13.12 (broad s, 1H, COOH), 11.15 (s, 1H, NH), 8 .8 8 (s, 1H, ArH ortho to N), 8.64 (s, 1H, ArH ortho to N), 8.45-8.37 (m, 2H, ArH), 7.84 (t, J =
7.6 Hz, 1H, ArH), 7.24 (d, J = 15.4 Hz, 1H, olifenic H), 6.78 (d, J = 15.4 Hz, 1H, olifenic H), 4.71 (s, 2H, CH 2COOH), 4.27-4.18 (q, J = 6.98 Hz, 2H, CH 3CH2O) and 1.30-1.24 (t, J = 6.96 Hz, 3H, CM 3CH2O).
Analysis for C 20H16N2O7 • 0.5 H2O
Calculated: C 59.26, H 4.22, N 6.91
Found: C 59.39, H 4.07, N 6.96
5-(franfrCinnamvlamido)-1.3-dioxo-1H-benzrde)isoquinoline-2(3m-acetic acid (48) 71
The same procedure as for 45 was used except that frans-cinnamic acid (0.21 g, 1.4 mmol) was used as starting material. Recrystallization from aqueous acetone gave 48 (0.53 g, 96%). mp 242-244 °C.
1H-NMR 8 10.85 (s, 1H, NH), 8.92 (d, J = 1.8 Hz, 1H, ArH ortho to NHCO),
8.74 (d, J = 1.8 Hz, 1H, ArH ortho to NHCO), 8.46-8.38 (dd,2H, ArH), 7.84 (t, J
= 7.7 Hz, 1H, ArH meta to CON), 7.72-7.66 (m, 3H, CH=CHCO and ortho
ArH), 7.47-7.44 (m, 3H, ArH), 6.88 (d, J = 15.7 Hz, 1H, CH=CHCO) and 4.74
(s, 2 H, CH2COOH).
Analysis for C 23H16N2O5 • 0.5 H2O
Calculated: C 67.47, H 4.18, N 6.84
Found: C 67.49, H 4.19, N 6.81
5-(2-Chloro-frans-cinnamvlamido)-1.3-dioxo-1 H-benz(de] isoquinoline-
2(3H)-acetic acid (49)
u H r " ' O'
N ■ H 72
The same procedure as for 45 was used except that 2-chloro-frans-cinnamic acid (0.26 g, 1.4 mmol) was used as starting material. Recrystallization from acetone gave 49 (0.40 g, 95%). mp 298-300 °C.
1H-NMR 5 11.0 (s, 1H, NH), 8.88 (d, J = 1.9 Hz, 1H, ArH ortho to N), 8.74 (d,
J - 1.9 Hz, 1H, ArH ortho to N), 8.50-8.39 (m, 2H, ArH), 8.0 (d, J = 15.3 Hz,
1H, CH=CHCO), 7.95-7.75 (m, 2H, ArH), 7.6 (m, 1H, ArH), 7.52-7.41 (m, 2H,
ArH), 6.95 (d, J = 15.3 Hz, CH=CHCO) and 4.75 (s, 2H, CH 2COOH).
Analysis for C 23H15N2O5CI • 0.25 H2O
Calculated: C 62.88, H 3.56, N 6.38
Found: C 63.07, H 3.41 , N 6.24
1.3-Dioxo-1H-benz[de]isoquinoline-2(3H)-acetic acid (5)
O H O'
A suspension of 1,8 -naphthalic anhydride (5.0 g, 25 mmol) and glycine (1.89 g,
25 mmol) in DMF (50 mL) was heated under reflux for 2 h. The reaction mixture was treated with activated charcoal, filtered and poured while hot over crushed ice. The heavy off-white product that separated out was collected by 73
filtration and dried. The product was pure enough for subsequent reactions without further purification steps (6.1 g, 95%). mp 268 °C (lit. 55 mp 270-271 °C).
1.3-Dioxo-1 H-benzrde1isoauinoline-2f3H1-acetvl benzenesulfonamide (50)
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.4 g, 2 mmol) was added to a stirred ice-cooled suspension of the acid 5 (0.5 g, 1.95 mmol), benzensulfonamide (0.33 g, 2 mmol) and DMAP (0.26 g, 2 mmol) in dry
CH2CI2 (200 mL) .under argon. The reaction mixture was allowed to warm to room temperature and stirring was continued under argon for 18 h. The reaction mixture was then poured into 1N HCI (100 mL), the separated aqueous layer was extracted with CH 2CI2 (2 x 50 mL), and the combined extracts were washed with water, dried and evaporated. The residue was recrystallized from DMF/H 2O to give 50 (0.65 g, 84%). mp 284-285 °C. 74
1H-NMR 8 8.51-8.42 (m, 4H, ArH), 7.97-7.89 (m, 4H, ArH), 7.75-7.6 (m, 3H,
ArH) and 4.72 (s, 2 H, Ct&COOH).
MS (m/e) El 394 M+
Analysis for C 20H14N2SO5
Calculated: C 60.90, H 3.57, N 7.10
Found: C 61.19, H 3.72, N 7.24
5-Nitro-1.3-dioxo-1 H-benzrde1isoauinoline-2(3H)-acetvl
benzenesulfonamide (51)
The same procedure as for 50 was used except that the nitro acid 73 (0.58 g,
1.95 mmol) was used as starting material. Recrystallization from DMF/H 2O gave 51 (0.7 g, 84%). mp 296-298 °C.
1H-NMR 8 9.51 (d, J = 2.1 Hz, 1H, ArH ortho to N 02), 8.93 (d, J = 2.1 Hz,
1H, ArH ortho to N02), 8.8 (d, J = 7.5 Hz, 1H, ArH), 8.6 (d, J = 7 Hz, 1H, ArH),
8.06 (t, J = 7.6 Hz, 1H, ArH), 7.9 (d, J = 7 Hz, 2H, ArH ortho to S 02), 7.72 (m, 75
1H, ArH para to S0 2 ), 7.62 (t, J = 7.6 Hz, 2 H, ArH meta to SO2) and 4.75 (s,
2H, CH2COOH).
Analysis for C 20H13N3SO7
Calculated: C 54.67, H 2.98, N 9.56
Found: C 54.70, H 3.21 , N 9.70
1.3-Dioxo-1 H-benzrde]isoquinoline-2(3H)-acetvl-(4-nitro- benzenetsulfonamide (52)
The same procedure as for 50 was used except that 4-nitrobenzene sulfonamide (0.4 g, 2 mmol) was used instead of benzenesulfonamide as starting material. Recrystallization from MeOH gave 52 (0.7 g, quantitative), mp 275 °C.
1H-NMR 8 8.50-8.41 (m, 6 H, ArH), 8.17 (d, J = 8.2 Hz, 2H, ArH), 7.86 (t, J =
8 Hz, 2H, ArH), 4.76 (s, 2H, CJ&COOH).
Analysis for C 20H13N3SO7
Calculated: C 54.66, H 2.98, N 9.57
Found: C 54.39 , H 3.20, N 9.60 76
1.3-Ploxo-1 H-benzfdelisoQuinoline-2(3m-acetvl-(3-nitro- benzenelsulfonamide (53)
The same procedure as for 50 was used except that 3-nitrobenzene sulfonamide (0.4 g, 2 mmol) was used instead of benzenesulfonamide.as starting material. Recrystallization from acetone gave 53 (0.72 g, quantitative), mp 275-277 °C.
1H-NMR 8 8.59-8.53 (m, 2H, ArH), 8.49-8.42 (m, 4H, ArH), 8.32 (d, J = 7.8
Hz, 1H, ArH ortho to NO2), 7.94 (t, J = 8 Hz, 1H, ArH meta to NO2), 7.86 (t, J
= 7.5 Hz, 1H ArH) and 4.75 (s, 2H, CH 2COOH).
MS (m/e) El 439 M+.
Analysis for C 20H13N3SO7
Calculated: C 54.66, H 2.98, N 9.57
Found: C 54.87, H 3.18, N 9.64 77
1.3-Pioxo-1 H-benzrde1isoquinoline-2f3Hl-acetvl-(2-nitro- benzeneteulfonamide (541
The same procedure as for 50 was used except that 2-nitrobenzene sulfonamide (0.4 g, 2 mmol) was used instead of benzenesulfonamide.as starting material. Recrystallization from acetone gave 54 (0.72 g, quantitative), mp 285 °C.
1H-NMR d 8.48-8.44 (m, 4H, ArH), 8.14 (d, J = 7.4 Hz, 1H, ArH), 8.06 (d, J
= 7.5 Hz,1H, ArH), 7.97-7.82 (m, 4H, ArH) and 4.80 (s, 2H, CH 2COOH).
Analysis for C 20H13N3SO7 • 0.75 H2O
Calculated: C 53.04, H 2.89, N 9.27
Found: C 53.02 , H 3.06, N 9.38 1.3-Dioxo-1 H-benzrde1isoauinoline-2f3H)-acetvl-(4-amino- benzeneteulfonamide (55)
A mixture of sulfanilamide (0.85 g, 4.9 mmol) tritylchloride (1.5 g, 5.4 mmol) and triethylamine (1 g, 9.8 mmol)in dry CH 2CI2 (20 mL) was stirred overnight at room temperature. The solvent was evaporated under vacuum and the residue was washed with 2% acetic acid, H 2O and dried to give the /V4-protected sulfanilamide. The /V4-protected sulfanilamide (1.78 g, 4.3 mmol) was mixed with the acid 5 (1 g, 3.9 mmol) and DMAP (0.52 g, 4.3 mmol) in dry CH 2CI2
(200 mL) and cooled in an ice-bath under argon. 1-[3-(dimethyl amino)propyl]-
3-ethylcarbodiimide hydrochloride (0.82 g, 4.3 mmol) was added and the reaction mixture was stirred overnight under argon at room temperature. The resulting solution was shaken with 1N HCI (100 mL) and the separated aqueous layer was extracted with CH 2CI2 (2 x 100 mL). The combined non- aqueous layers were washed with water and evaporated under vacuum. The resulting residue was washed with MeOH and recrystallized from DMF/H 2O to give 55 (1.3 g, 81%). mp 268-270 °C. 79
1H-NMR 8 12.17 (s, 1H, S02NHC0), 8.51-8.46 (m, 4H, ArH), 7.88 (t, J = 7.7
Hz, 2H, ArH), 7.51 (d, J = 8.7 Hz, 2H, ArH ortho to S02), 6.58 (d, J = 8.7, 2H,
ArH ortho to NH2), 6.18 (s, 2H, NH2) and 4.67 (s, 2H, ChbCOOH).
Analysis for Q 20H15N3SO5
Calculated: C 58.67, H 3.69, N 10.26
Found: C 58.68, H 3.59, N 10.24
5-Nitro-1.3-dioxo-1H-benzrde1isoauinoline-2f3H)-acetvl-f4-amino- benzene)sulfonamide (56)
The same procedure as for 55 was used except that the 5-nitro acid 73 (1.2 g,
3.9 mmol) was used instead of the acid 5. The final product was washed with ether, MeOH and recrystallized from DMF/H 2 O to give 56 (0.5 g, 30 %). mp
255-256 °C.
1H-NMR 8 12.19 (s, 1H, S0 2NHC0), 9.51 (d, J = 2.2 Hz, 1H, ArH ortho to
N 0 2), 8.94 (d, J = 2.1 Hz, 1H, ArH ortho to N02), 8.81 (d, J = 8.1 Hz, 1H,
ArH), 8.67 (d, J = 7 Hz, 1H, ArH), 8.06 (t, J = 7.8 Hz, 1H, ArH), 7.51 (d, J = 8.6 80
Hz, 2H, ArH ortho to S02), 6.58 (d, J = 8.7 Hz, 2H, ortho to NH2), 6.18 (s, 2H,
NH^ and 4.7 (s, 2 H, CikCOOH).
Analysis for C 2oHi4N4S0 7 • 0.25 H20
Calculated: C 52.34, H 3.18, N 12.21
Found: C 52.39, H 3.16, N 11.92
Af*-fChloroacetyh sulfanilamide (831
O n o = s - n h 2
o
A solution of chloroacetic anhydride (77; 5.6 g, 32.7 mmol) in dry CH 2CI2 (10 mL) was added dropwise to a suspension of sulfanilamide (82 ; 5 g, 29.0 mmol) and pyridine (5.0 g, 63.0 mmol) in dry CH 2CI2 (100 mL). The reaction mixture was heated under reflux for 4 h. The product that separated out was collected by filtration, washed with 1N HCI and recrystallized from MeOH to give colorless crystals of 83 (6.7 g, quantitative), mp 218-220 °C (lit. 166,167 mp
217 °C). 81
1.3-Pioxo-1 H-benzrdelisoauinoline-2(3H)-acetvl-f4-chloro- acetamidolbenzenesulfonamide (60)
The same procedure as for 50 was used except that 83 (0.46 g, 2 mmol) was used instead of bezenesulfonamide as starting material. Recrystallization from
MeOH gave 60 (0.76 g, 80 %). mp 276-278 °C.
1H-NMR 6 12.59 (broad s, 1H, CONHSO 2), 10.75 (s, 1H, NHCO), 8.52-8.42
(m, 4H, ArH), 7.91-7.75 (m, 6H, ArH), 4.71 (,s, 2H, NCH 2CO) and 4.30 (s,2H,
CICH2CO).
Analysis for C 22H16N3SO6CI • 0.25 H2O
Calculated: C 53.88, H 3.39, N 8.57
Found: C 53.93, H 3.51 , N 8.50 82
1.3-Pioxo-1 H-benzrde1isoauinoline-2(3H)-acetvl-(4-iodo- acetamido)benzenesulfonamide (61)
TT-' o
A suspension of 60 (0.2 g, 0.4 mmol) and potassium iodide (0.4 g, 2.4 mmol) in acetone (20 mL) was stirred at room temperature for 48 h. Solvent was evaporated under vacuum and the residue was washed with H 2 0 , dried and recrystallized from DMF/H 2O to give 61 (0.2 g, 84 %). mp 258-260 °C.
1H-NMR S 12.59 (broad s, 1H, CONHSO2), 10.75 (s, 1H, NHCO), 8.56-8.45
(m, 4H, ArH), 7.96-7.80 (m, 4H, ArH), 7.77-7.74 (m, 2H, ArH), 4.71 (s, 2H,
NCH2CO) and 3.84 (s,2H, ICH 2CO).
Analysis for C 22H16N3SO6I
Calculated: C 45.77, H 2.79, N 7.28
Found: C 46.17, H 2.90, N 7.14 83
1.3-Dioxo-1 H-benz[de1isoauinoline-2(3H)-acetvl-(4-betainvl- amidolbenzenesulfonamide hydrochloride (62)
Trimethylamine ( 0.12 g, 2 mmol) was added to a suspension of 60 (0.24 g, 0.5 mmol) in acetone (20 mL) and the reaction mixture was heated under reflux for
3 h. The product that separated out was collected by filtration, washed with acetone and dried. The product was purified by dissolving it into 1N NaOH , treatment with charcoal, filtration and reprecipitation by 1N HCI to give 62 (0.14 g, 56 %). mp 278-282 °C.
1H-NMR (DMSO-de + NaOD) 5 7.88 (d, J = 7.9 Hz, 1H, ArH), 7.79 (d, J = 7.9
Hz, 1H, ArH), 7.59-7.46 (m, 4H, ArH) 7.40 (t, J = 7.6 Hz, 2H, ArH), 7.23 (d, J =
8.5 Hz, 2H, ArH), 3.88 (s, 4H, NCH 2CON and (CH 3)3 N+CH2CO) and 3.17 (s,
9H, (CH 3)3N+).
Analysis for C 25H25N4SO6CI
Calculated: C 55.10, H 4.62, N 10.28
Found: C 55.08, H 4.88, N 10.38 84
1.3-Dioxo-2(3HH2-hvdroxv-5-nitroDhenvn-1 H-benzfdel isoauinoline (57)
OH
0 N O
A mixture of 1,8-naphthalic anhydride (1 g, 5 mmol) and 2-amino-4-nitro-phenol
(0.85 g, 5.5 mmol) in DMF (10 mL) was heated under reflux for 4 h. The reaction mixture was treated with activated charcoal, filtered and poured while hot over crushed ice. The product that separated out was collected by filtration, dried and recrystallized from aqueous MeOH to give 57 (1.68 g, quantitative), mp >300 °C.
1H-NMR 8 11.36 (broad s, 1H, OH), 8.54-8.49 (m, 4H, ArH), 8.37 (d, J = 2.8
Hz, 1H, ArH ortho to N02), 8.27-8.22 (dd, J = 2.8, 9.1 Hz, 1H, ArH ortho to
N02), 7.94-7.88 (t, J = 7.9 Hz, 2H, ArH) and 7.19-7.15 (d, J = 9.1 Hz, 1H, ArH ortho to OH).
Analysis for C i 8 HioN20 5
Calculated: C 64.67, H 3.02, N 8.38
Found: C 64.41 , H 3.01 , N 8.31 85
5-Nitro-1.3-dioxo-2(3HH2-hvdroxv-5-nitrophenvl)-1 H-benzrdel
JfipqylnollTO.ffg)
OH
OON
NO
The same procedure as for 57 was used except that 3-nitro-1,8-naphthalic
anhydride (1.2 g, 5 mmol) was used as starting material. Recrystallization from
acetone/MeOH gave 58 (1.36 g, 73 %).
mp >300 °C.
1H-NMR 8 11.44 (broad s, 1H, OH), 9.56 (s, 1H, ArH), 8.97 (s, 1H, ArH),
8.85 (d, J = 8.2 Hz, 1H, ArH), 8.71 (d, J = 7.2 Hz, 1H, ArH), 8.40 (d, J =2.3 Hz,
1H, ArH ortho to N02), 8.29-8.24 (dd, J = 2.5, 9.1 Hz, 1H ortho to N02), 8.13-
8.07 (t, J = 7.8 Hz, 1H, ArH) and 7.20-7.16 (d, J = 9.1 Hz, 1H, ArH ortho to
OH).
Analysis for C-isHgNaOz
Calculated: C 57.00, H 2.39, N 11.08
Found: C 56.70, H 2.42, N 10.90 86
5-Nitro-1.3-dioxo-2(3HH2-hvdroxv-3.5-dinitrophenyl)-1 H- benzrdelisoauinoline (59)
NO
OH
0 N O
NO
A solution of 57 (0.5 g, 1.5 mmol) in conc. H 2SO4 (5 mL) was cooled in an ice- bath. A solution of HNO 3 (0.18 g, 3.1 mmol) in conc. H 2 SO4 (2 mL) was dropped slowly to the original solution with stirring such that the reaction temperature did not exceed 20 °C. After addition was complete the ice bath was removed and the reaction mixture was allowed to stir at room temperature for 90 min, then poured over crushed ice. The heavy off-white product that separated out was collected by filtration, dried and recrystallized from MeOH to give 59 (0.6 g, 95 %). mp 280-281 °C.
1H-NMR S 9.57 (d,J = 1.4 Hz, 1H,ArH), 8.98 (d, J = 1.3 Hz, 1H, ArH), 8 .8 8 -
8.83 (m, 2H, ArH), 8.73-8.70 (d, J = 7.2 Hz, 1H, ArH), 8.62-8.59 (m, 1H, ArH) and 8.14-8.08 (t, J = 8.0 Hz,1H, ArH).
Analysis for C 18 H8 N4O9
Calculated: C 50.94, H 1.90, N 13.21
Found: C 50.85, H 1.94, N 13.12 87
2-Aminospiro(9HVfluoren-9.4,-imidazoline-2l-5,-dione (85)
NH2
A mixture of 2-amino-9-fluorenone (1.95 g, 1 mmol), potassium cyanide (1.3 g,
20 mmoi) and ammonium carbonate (3.84 g, 40 mmol) in EtOH (90 %, 50 mL) was placed in a 300 mL Parr stainless steel general purpose bomb and heated in an oil-bath at 110 °C for 48 h. The bomb was cooled, opened and the contents were acidified with 10 % HCI to -pH 3 and the solid that separated out was collected by filtration. The product was purified by dissolution in 10 %
NaOH , treatment with activated charcoal, filtration and reprecipitation with 10
% HCI to give 85 (2 g, 75 %). mp 300 °C (lit 57 mp 310-314 °C). 88
2-(Bicvclor2.2.21octa-2.5-diene-2-carboxamidol-spiro-(9H)-fluoren-9.4l- imidazoline-g.S'-dione (63)
The bicyclo acid 75 (0.56 g, 3.6 mmol) was heated under mild reflux with thionyl chloride (5 mL) for 2 h. Excess thionyl chloride was evaporated under vacuum and the residue was coevaporated with toluene (2 x 20 mL). The acid chloride was then dissolved in dry CH 3CN (20 mL)and added to a suspension of 85 (0.5 g, 1.8 mmol) and pyridine (0.3 g, 4 mmol) in dry CH 3CN (50 mL).
The reaction mixture was stirred at room temperature for 24 h. The product that separated out was collected by filtration, dried and recrystallized from
DMF/H2O to give 63 (0.67 g, 89 %). mp >300 °C.
1H-NMR 8 11.26 (broad s, 1H, CONJHCO), 9.78 (s, 1H, amide NH), 8.61 (s,
1H, imidazoline NH), 7.83-7.79 (m, 4H.ArH), 7.47-7.41 (m, 2H, ArH), 7.32-7.23
(m, 2H, ArH and CH=CCO), 6.41-6.32 (m, 2H, olifinic H), 4.22 (m, 1H, 3°H), 3.8
(m, 1H, 3°H) and 1.35-1.25 (m, 4H, bridge H).
MS (m/e) El 397 M+.
Analysis for C 24H19N3O3 • H2O
Calculated: C 69.38, H 5.09, N 10.12
Found: C 69.12, H 5.27, N 10.57 89
2-Mal8amido-spiro-(9H)-fluoren-9.4l-imidazoline-2l.5l-dione (64)
A mixture of 85 (0.24 g, 0.9 mmol) and maleic anhydride (0.1 g, 1 mmol) in dry toluene (100 mL) was heated under a water trap for 48 h. The product that separated out was collected by filtration, dried and recrystallized from
DMF/H20 to give 64 (0.3 g, 95 %). mp >300 °C.
1H-NMR 8 11.29 (broad s, 1H, CONHCO), 8.63 (s, 1H, NH), 7.84-7.78 (m,
3H, ArH), 7.64-7.61 (m, 1H, ArH), 7.48-7.43 (m, 2H,ArH), 7.34-7.27 (t, J = 7.6
Hz, 1H,ArH), 6.47-6.42 (d, J = 12 Hz, 1H, olifenic H) and 6.33-6.28 (d, J = 12
Hz, 1H, olifenic H).
MS (m/e) El 345 M+.
Analysis for CigHnN 30 4 • 1.5 H2O
Calculated: C 61.29, H 3.79, N 11.28
Found: C 61.36, H 3.87, N 11.31 90
2-Ethylfumaramido-spiro-(9HVfluoren-9.4l-imidazoline-2,.5l-dione (65)
H O
0
Fumaric acid monoethyl ester (0.54 g, 3.7 mmol) was heated under reflux with thionyl chloride (4 mL) for 2 h. Excess thionyl chloride was distilled under vacuum and the residue was coevaporated with toluene (2x10 mL). The acid chloride was dissolved in CH 3CN (10 mL) and added to a suspension of 85
(0.5 g, 1.8 mmol) and pyridine (0.3 g, 4 mmol) in CH 3CN (20 mL). The reaction mixture was heated under reflux for 4 h. The product that separated out was collected by filtration, dried and recrystallized from DMF/H 2O to give 65 (0.55 g, 75 %). mp >300 °C.
1H-NMR 8 11.29 (broad s, 1H, CONJJCO), 10.75 (s, 1H, Amide NH), 8.63 (s,
1H, imidazoline NH), 7.92 (d, J = 1.6 Hz, 1H, ArH), 7.87-7.80 (t, J = 8.3 Hz, 2H,
ArH), 7.67-7.63 (dd, J = 1.7 Hz, 8.2 Hz, 1H, ArH), 7.49-7.44 (t, J = 6.7 Hz, 2H,
ArH), 7.34-7.28 (t, J = 7.3 Hz, 1H, ArH), 7.21-7.15 (d, J = 15 Hz, 1H, olifenic H),
6.74-6.67 (d, J = 15.4 Hz, 1H, olifenic H), 4.27-4.18 (q, J = 6.98 Hz, 2H,
CH3CH2O) and 1.31-1.22 (t, J = 6.96 Hz, 3H, CH 3CH2O) 91
Analysis for C 2 1 H 1 7 N 3 O 5
Calculated: C 64.44, H 4.37, N 10.73
Found: C 64.33, H 4.60, N 10.49
2-Chloroacetamido-spiro(9HHIuoren-9.4,-imidazoline-2,.5,-dione(41)
O
A mixture of X+23 (0.5 g, 1.8 mmol) chloroacetic anhydride (0.4 g, 2.1 mmol) and DMAP (0.02 g, 0.1 mmol) in dry CH 3CN (20 mL) and the reaction mixture was stirred overnight at room temperature. The product that separated out was collected by filtration and recrystallized from DMF/H 2O to give 41 (0.6 g, 90 %). mp >305 °C (lit. 99 mp >285 °C )
MS (m/e) El 343 M++ 2 , 342 M++1, 341 M+.
1H-NMR 8 11.29 (broad s, 1H, CONHCO), 10.5 (s, 1H, amide NH), 8.62 (s,
1H, imidazoline NH), 7.83-7.72 (m, 3H, ArH), 7.67-7.55 (dd, J = 1.7 Hz, 8.2 Hz,
1H, ArH), 7.5-7.4 (m, 2H, ArH), 7.33 (t, J = 7.3 Hz, 1H, ArH) and 4.25 (s, 2H,
CICH2CO). 92
2-Betainvlamido-SPiro-(9H)-fluoren-9.4'-imidazoline-2l.5l-clione (66)
H i cr N y \ + 'y ^N(CH3)3
Trimethylamine (0.14 g, 2 mmol) was added to a suspension of 86 (0.2 g, 0.6 mmol) in acetone (20 mL). The reaction mixture was heated under reflux for 4 h. The product that separated out was collected by filtration, dried and recrystallized from H 2 0 /acetone to give 66 (0.21 g, 89 %). mp 270-274 °C.
1H - N M R (D20) 8 7.73-7.68 (m, 2H, ArH), 7.59 (s, 1H, ArH), 7.41-7.38 (m,
3H, ArH), 7.31-7.25 (m, 1H. ArH), 4.13 (s, 2 H, NCH 2 C O ) and 3.21 (s, 9H,
( C H 3 ) 3 N + ).
MS (m/e) El 365 M+.
Analysis for C 20 H21N4O3CI • 1.75 H20
Calculated: C 55.56, H 5.82, N 12.95
Found: C 55.79, H 5.85 , N 12.66 93
1-Benzenesulfonvl-3-r9-(9HMIuorenvnurea (67)
O .______. V»1H II v i H-N
A solution of benzenesulfonyl isocyanate (0.6 g, 3.2 mmol) in dry CH 3CN (25 mL) was added dropwise with stirring to a suspension of 9-amino-fluorene (0.5 g, 2.7 mmol) in dry CH 3CN (25 mL). After the addition was complete the reaction mixture was heated gently under reflux for 2 h, allowed to cool and filtered. The filtrate was evaporated under vacuum and the residue was recrystallized from MeOH to give 67 (0.5 g, 50 %). mp 218-220 °C.
1H-NMR 8 7.97-7.93 (d, J = 6.9 Hz, 2H, ArH), 7.82-7.79 (d, J = 7.4 Hz, 2H,
ArH), 7.73-7.61 (m, 3H, ArH), 7.42-7.23 (m, 6 H, ArH) 7.08-7.04 (d, J = 8 Hz,
1H, NHCO) and 5.69-5.65 (d, J = 8 Hz, 1H, benzylic H).
MS (m/e) El 364 M+.
Analysis for C 20H16N2SO3
Calculated: C 65.91 , H 4.42, N 7.68
Found: C 65.52 , H 4.52, N 7.51 94
N-(9-Fluoren-carboxyl)"benzensulfonamide (68)
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.99 g, 5.2 mmol) was added to an ice-cooled mixture of 9-fluorenecarboxylic acid (1 g,
4.7 mmol), DMAP (0.63 g, 5.2 mmol) and benzenesulfonamide (0.81 g, 5.2 mmol) in dry CH 2CI2 (200 mL) under argon. The mixture was allowed to stir at room temperature for 18, then poured into 1N HCI (200 mL) and the separated aqueous layer was extracted with CH 2CI2 (2 x100 mL). The combined extracts were washed with H 2O, dried and evaporated under vacuum and the residue was recrystallized from CH 2Cl2/petroleum ether to give 68 (1.05 g, 63 %). mp
162-164 °C.
1H-NMR 8 12.85 ( broad s, 1H, S0 2NMCO), 7.89-7.86 (d, J = 7.2 Hz, 2H,
ArH), 7.83-7.81 (m, 1H, ArH), 7.65-7.62(d, J = 7.2 Hz, 2H, ArH), 7.57-7.53 (m,
2 H, ArH), 7.45-7.30 (m, 6 H, ArH) and 4.95 (s, 1H, benzylic H).
MS (m/e) El 349 M+-
Analysis for C 20H15NSO3 • H2O
Calculated: C 65.38, H 4.66, N 3.81
Found: C 65.38 , H 4.60, N 3.80 95
5-lodoacetamido-1.3-dioxo-1 H-benzrde1isoquinoline-2(3H)-acetic acid (34)
O
H
lodoacetic anhydride (35 mg, 0.1 mmol) was added to a suspension of the amino acid 74 (10 mg, 0.037 mmol) in dry THF (5 mL) and the reaction mixture was heated under reflux for 1 h. THF was then distilled off under vacuum and the residue was dissolved in little acetone then precipitated with H 2 O to give 34
(15 mg, 95%). m.p. 230-234 °C. (lit. 98-100 mp 222-223 °C). 96
r14C1-5-lodoacetamido-1.3-dioxo-1H-benzrde1isoauinoline-2(3HVacetic acid (69):
O
H
14C-lodoacetic anhydride (66.63 mg, 0.188 mmol, 1.316 mCi, specific activity =
7 mCi / mmol) was added to a suspension of the amino acid 74 (20.18 mg,
0.075 mmol) in dry THF (15 mL) and the reaction mixture was heated under reflux for 3 h and stirred at room temperature overnight. Solvent was evaporated under vacuum, the residue was dissolved in acetone (~ 5 mL). The product was precipitated with H 2O, collected by filtration and dried to give 69
(25.35 mg, 77 % chemical yield; 0.132 mCi, 10 % radiochemical yield; specific activity = 2.29 mCi / mmol), mp 230 °C. PART 2
THE BACTERIAL OSMOTOLERANCE
A STUDY OF THE STRUCTURAL REQUIREMENTS FOR THE GLYCINE
BET AINE-CHOLINE TRANSPORT
SYSTEMS
-97- CHAPTER V
INTRODUCTION
5.1. Overview:
The ability of living cells to adapt to fluctuation in the osmolarity of their
surroundings is fundamental to their survival within a given habitat. Cellular
adaptation to osmotic stress is considered to be a "cardinal biological process" that protects the cell against the lethal effects of dehydration. The concept of osmoadaptation has recently become a subject of considerable interest . 110-114
An oversimplified definition of osmoadaptation could be given as the cellular accumulation of material that keeps the cytoplasmic osmotic strength slightly higher than that of the environment. While many eukaryotic cells possess systems for osmoadaptation (or osmoprotection),i ' 3 the focus here will be on the prokaryotic osmoadaptation mechanisms, specifically in enteric bacteria.
-98- 99
5.2. Background:
Because the bacterial cytoplasmic membrane is permeable to water but not to
most other metabolites, hyperosmotic or hypoosmotic shock causes a rapid
efflux or influx of water which results in a change in the cytoplasmic volume . 111
Since bacterial cell walls are rigid and can withstand pressures up to 1 0 0
atm, 115 hypoosmotic shock generally causes only minor increase in the
cytoplasmic volume . 116 On the other hand, hyperosmotic shock results in a
considerable shrinkage in the cell volume, a process known as plasmolysis . 117
Plasmolysis results in the inhibition of a variety of physiological processes
ranging from nutrient uptake to DNA replication, a condition th a t, if not rapidly corrected, will lead to cell destruction .118,119 |n order to survive, bacterial cells must maintain a positive turgor, an outward pressure that results from the maintenance of cytoplasmic osmolarity above that of the extracellular milieu.11°-111
5.3. Compatible Solutes:
To achieve a positive turgor, bacteria respond to osmotic stress by increasing the intracellular concentration of a limited number of solutes (osmolytes) that will help increase the cytoplasmic osmotic pressure. These osmolytes are termed "compatible solutes" because they are accumulated by the cell in high 100
intracellular concentrations without notable toxicity to the normal cellular
functions .110,111,114,120 Several types of bacteria utilize the same strategy of
osmotic adaptation, but the most studied organisms have been various
Halobacteria, Escherichia coli, Klebsiella pneumonia and members of
Salmonella species , 121 The bacterial osmoadaptation mechanisms are
controlled by a small number of genes that are known as osmotic tolerance
genes (osm genes) .113 Their activities are exquisitely regulated at the
transcription level, or by modulation of their protein products (Table 1 2 ),
providing an integrated response which enables cells to grow at a wide range
of extracellular osmolarities .122
The primary bacterial response to osmotic challenge is the accumulation of
potassium ions, with a concomitant increase in the cytoplasmic concentration
of glutamate which serves as a counter ion .123-125 Several other compatible solutes are also accumulated as a result of osmotic stress, these include carbohydrates such as trehalose ( 1 , 1-oc-D-glucopyranosyl-a-D-gluco- pyranose), and glucosylglycerol; 123,125,127,128 amino acids such as alanine, glutamine and proline; i29 and also quaternary ammonium compounds such as proline betaine (/S/./V-dimethylproline) and glycine betaine (GB ; N,N,N- trimethylglycine). 110-113,120,122,125,126,130 Osmotic challenge also causes alteration in the outer membrane porin proteins and in membrane-derived oligosaccharides. 123,131 101
Table 12. Osmoregulated Systems in E. coli 113
System Function Activation 3 Gene / Protein^
K d p High-affinity K + transport High Gene
T r e Trehalose synthesis High ?
P r o P Low-affinity G B / proline transport High Protein
P ro U High-affinity G B transport High Gene & protein
B e t Choline transport / G B synthesis High ?
MDO Membrane derived oligosaccharides Low Protein
O m p C Outer membrane porin High Gene
O m p F Outer membrane porin Low Gene a Activity is induced at high or low osmolarity b Stimulation is at the transcriptional (gene) or enzymatic (protein) level.
5.3.1 Potassium Ions:
Potassium ions are the most prevalent cations in the cytoplasm of bacteria and consequently they serve as one of the major intracellular osmolytes that maintain positive turgor. The cytoplasmic concentration of K + in a wide variety of bacterial species has been found to be nearly proportional to the osmolarity of the growth medium .132 Two major selective K + transport systems have been extensively characterized in E. coli : T r k which is the steady state K + 102
transporter having a relatively low affinity for K + {Km = 1 .5 mM) and K d p
which has a much higher affinity ( Km = 2 jiM) and is a subject to osmotic
transcriptional regulation. 124 Both Trk and Kdp belong to the o s m genes
and their activities are greatly stimulated by hyperosmotic shock. 133 The
osmotic-triggered increase in the cytoplasmic concentration of g lu t a m a t e and
g lu t a m in e is attributed to the enhancement of their rate of synthesis, since
bacteria grown in hyperosmotic media devoid of exogenous amino acids still
possess high levels of these two amino acids . 134
5.3.2. Trehalose:
T r e h a l o s e is probably the most important carbohydrate with respect to
osmoregulation. 123,125.127,128 jh e structure ( 9 3 ) of this disaccharide is very
unusual in having the glycosidic oxygen atom axial to the two glucose rings,
both of which have the C 1 a-conformation .112
c h 2o h
HO OH HO OH OH OH
(9 3 )
Trehalose has been found to be synthesized in a number of bacteria including
E. coli as a result of osmotic stress. The o s m gene controlling trehalose synthesis is the Tre gene. The synthesis starts by the condensation of glucose 103
6 -phosphate and uridine diphosphate-glucose to yield trehalose 6 -phosphate
which is subsequently hydrolyzed to trehalose . 135 Trehalose accumulation in
E. coli might occur as a result of other signals beside osmotic stress, for
example mutations in E coli that resulted in tyrosine overproduction also
caused trehalose accumulation. 136 It has also been found that E. coli can
grow with trehalose as a sole carbon source . 137
5.4. Osmoprotectants:
Proline (94), proline betaine (95) and glycine betaine (3) are considered to
be the most important osmolytes in the the bacterial osmoadaptation
mechanisms . 110-113,120-122 They are known as osmoprotectant molecules because, in addition to their ability to provide a positive turgor to the cytoplasm, they can protect crucial cellular enzymes and macromolecules against high ionic strength and in some cases even against heat inactivation T 38
(94) (95) (3) 104
The mechanism by which these molecules protect the cells against hyperosmotic shock has not yet been clarified. It has been proposed that because of their dipolar character osmoprotectants form a "protective shell" around proteins excluding ions that may otherwise induce adverse conformational changes .113 Thus, osmoprotectants can stabilize the structure and consequently the metabolic functions of cellular proteins under high osmotic pressure environments.
The accumulation of these osmoprotectant molecules is also controlled by the o s m genes. E. coli and $. typhimurium have three independent proline transport systems: PutP, ProP and P r o U .139-142 o f these P r o P and P r o U were found to be involved in the accumulation of proline during osmotic stress, and both are active transport systems that exhibit Michaelis-Menten kinetics and are energized by ATP .113 P r o P is a low affinity ( Km = 0 .3 mM) proline transporter whose expression is stimulated several-fold during osmotic shock;
P r o U is a binding-protein-dependent transport system, with relatively higher affinity ( Km = 0 .2 mM), whose transcription is induced by elevated osmolarity or by high potassium glutamate concentrations .110,111,114,143 An interesting observation is that glycine betaine transport was also found to be mediated by the P r o P and P r o U systems T 11 What is even more interesting is the fact that these transport systems have much higher affinity ( Km of 4 4 pM for P r o P and
1 pM for P r o U ) for glycine betaine than for proline, which indicates that glycine betaine is a superior osmoprotectant than proline .m .H 4 105
It has been reported that solutes that carry a net electric charge are generally more deleterious to protein stability than non polar or zwitterionic solutes .144
In addition, the in vivo preference of glycine betaine over K + and glutamate for the maintenance of the cellular osmolarity suggests that glycine betaine is more compatible than either of them .114 From the above, it can be concluded that glycine betaine is the most important solute in the bacterial osmo adaptation mechanisms.
5.5. The Choline-Glycine Betaine Pathway:
Cyanobacteria and a few other CC>2-fixing prokaryotes are able to carry out the de novo synthesis of glycine betaine ( e.g. from basic carbon source such as glucose), i 45. ^ but most bacteria synthesize it by oxidation of choline ( 9 6 )
(Scheme 13 ).147’149
\ + OH N Choline Oxidase
(96) (97)
Betaine Aldehyde Dehydrogenase S \ || ' o (3) Scheme 13: Enzymatic Conversion of Choline to Glycine Betaine 106
The metabolic conversion of choline to glycine betaine consists of two distinct enzymatic steps. The first is the oxidation of choline into betaine aldehyde
(97), and the second is the oxidation of this aldehyde to the end product glycine betaine. Both reactions in this pathway are catalyzed by a membrane associated, 0 2 -dependent, electron-transfer-linked enzyme (choline oxidase), whereas a soluble NADP-dependent dehydrogenase catalyzes the second step only ,122 This pathway is also osmotically regulated, i.e. both enzyme activities appear simultaneously when the osmotic pressure of a medium containing choline is increased by an addition of an osmolyte (salts or sugars ).111 E coli has a high-affinity and a low-affinity transport systems for choline with Km values of 8 (iM and 1.5 mM, respectively. These systems are only expressed under osmotic stress . 111 The osm genes that govern choline transport and the Choline-Glycine betaine pathway (Figure 14) are the Bet genes . 1so-152 y^us, choline itself is not considered as an osmoprotectant, and bacteria that lack the B et genes {e.g. Salmonella typhimurium ) can not produce glycine betaine from choline .111
From the above it can be concluded that bacterial cells have developed a number of mechanisms by which they can withstand fluctuations in the osmotic strength of their environments. Among these mechanisms the glycine betaine transport and synthesis are the best characterized and the most efficient osmoadaptation mechanisms. / ^ 'OH S ' ^ q Choline Glycine Betaine
Choline Betaine. Glycine Aldehyde Betaine
Figure 14: The Metabolically Linked Choline and Glycine Betaine Transport Systems.
5.6. Glycine Betaine analogs:
Because of the importance of glycine betaine as an osmoprotectant, several researchers have studied the osmoprotectant activities of some of its analogs.114*122.151,153,154 The trimethylamino group of glycine betaine was reported to be important for activity because the dimethylamino glycine was found to be significantly less active than glycine betaine and both methyl glycine and glycine itself were totally devoid of osmoprotectant activity .122
However, it was possible to replace betaine's quaternary amine with the permanently charged dimethylsulfonium group and retain most of the 108
osmoprotectant activity . 154 Dimethylthetin (98) had been shown to be an acceptable substitute for choline as a methyl donor in mammals .155
Dimethylthetin was tested against the standard, glycine betaine, as well as two dimethylthetin analogs, 3-dimethylsulfoniopropionate (99) and 3-dimethyl sulfonio- 2-methyl-propionate ( 100).
0 \\ A
(9 8 ) (99) (100)
Dimethylthetin (98) was confirmed to be as active as glycine betaine both in intracellular uptake and in supporting E coli growth under osmotic stress. The other two analogs possess negligible activity in comparison to glycine betaine.
From this study, it was concluded that the quaternary amine of glycine betaine is bioisosterically replaceable with permanently charged moieties without significant loss of activity or recognition by glycine betaine transport systems. It was also concluded that the optimum distance between the two functional groups of glycine betaine is one unsubstituted methylene group ,154 109
5.7. Miscellaneous Osmoprotectant Molecules:
Several natural and synthetic compounds were tested for their ability to be accumulated and/or to protect bacterial cells under hyperosmotic conditions.
3-(N -Morpholino)propanesulfonate (MOPS; 101) was found to be actively accumulated by E. coli and not by S. typhimurium under osmotic stress. The active transport system for MOPS was found to be different from that of glycine betaine, because S. typhimurium that has PorP and ProU was not able to accumulate MOPS. The charge difference between MOPS and choline ruled out the possibility of the involvement of the choline transporters ( B et -gene products) in the accumulation of MOPS. It was concluded that MOPS is accumulated by E. coli via an uncharacterized osmotically regulated transport system. However, MOPS is considered more of an osmolyte than an osmoprotectant .153
O (101) (102)
Recently, it was reported that the naturally occurring cyclic nonproteinaceous amino acid Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid;
1 0 2 ) could act as an efficient osmoprotectant in different types of bacteria 110
including E. c o li. It was also reported that both glycine betaine transporters
ProP and ProU are involved in the uptake and accumulation of ecotine and that ProP is the main system for its transport .114
5.8. Relationship between Osmoprotection and Urinary Tract Infections:
Urine is a variable, but generally good, culture medium for enteric gram- negative bacteria, enterococci, and other organisms that commonly cause infections in the urinary tract .156 The most common causative organism is E. coli, which is responsible for about 80-85 % of all uncomplicated urinary tract infections. Klebsiella species, Proteous species, Enterococcus species and to a lesser extent Pseudomonas species account for a majority of the remainder of the urinary tract infections .1 5 7 ,1 5 8 Conditions that tend to inhibit bacterial growth in the urinary tract are high osmolarity and low pH, therefore bacteria rely on their osmoadaptive mechanisms in order to survive the extremely diverse osmotic environment within the urinary tract .156,159
Human urine was found to contain glycine betaine and even greater levels of its biosynthetic precursor choline .4,156,159 jhat, together with the fact that bacterial cells have a well-developed transport systems for glycine betaine and for choline, explains the ability of urinary tract pathogens to survive urinary tract osmolarity. From the above, it could be concluded that the bacterial Glycine
Betaine-Choline transport system is potentially a very fruitful target for antimicrobial drug design. CHAPTER VI
STATEMENT OF PROBLEMS AND OBJECTIVE
As noted in Chapter V bacterial cells have well developed mechanisms by which they can adapt to osmotic stress. The most efficient among the bacterial osmoadaptive mechanisms is the choline / glycine betaine system. The presence of such system in bacteria facilitates bacterial infections of organs with high osmolarity such as the urinary tract.
Compounds that can interfere with glycine betaine uptake or its synthesis from choline will be expected to have antibacterial activity in fluids of high osmolarity such as urine (which was found to contain both choline and glycine betaine).
The transport system for glycine betaine and choline could be used to deliver toxic analogs into bacterial cells under conditions of osmotic stress.
The therapeutic potential of such compounds prompted us to study the structural requirements for the uptake and the osmoprotectant activity of glycine betaine. As discussed in Chapter V the quaternary ammonium end of glycine betaine could be replaced, with complete retention of the osmoprotectant activity, by other permanently charged cationic moieties such
-111- 112
as dimethylsulfonium group. The goal of the present study is to further
investigate the structural requirement of the choline / glycine betaine transport
system by the replacement of the carboxylic acid end of glycine betaine with
different carboxylic bioisosteres. The bioisosteres that we chose to test were
the sulfonic acid analog (sulfobetaine; 103), the phosphonic acid analog
(phosphobeaine, 104) and the A/-arylsulfonylamide analogs (105).
O O
(103) (104) (105)
The aromatic portion of 105 provided us with a vehicle to which different cytotoxic alkylating groups such as a-haloacetamido group (Figure 15), could be linked. This allows for the investigation of the possibility of utilizing the choline-glycine betaine transport system to deliver such cytotoxic moieties into bacterial cells. This will be a reasonable way to interfere with the osmoadaptive mechanisms in bacteria and may lead to a development of a new class of antibacterial agents. 113
(106) R = H (109) R = Cl
(107) R = CH3 (110) R = Br
(108)R = (CH3)3N+ (111) R = I
Figure 15: Proposed Analogs of Compound 105
The fact that MOPS (101) was found to be actively taken in by £ coli under osmotic stress 153 prompted us to try replacing the quaternary ammonium end of glycine betaine with quaternary cyclic ammonium moieties. However, because MOPS did not show good osmoprotectant activity we limited the distance between the sulfonic group and the the cyclic amine to one methylene 114
unit, which will agree with the reported structure requirements for glycine betaine recognition .154 These analogs provided us with a set of compounds
(Figure 16) that have both ends of glycine betaine bioisosterically replaced.
I L . o * - S O ," N ^ S O , .N j^ S O g
(1 1 2 a) (112b) (112c)
Figure 16: The Proposed Sulfobetaine Analogs CHAPTER VII
RESULTS AND DISCUSSION
7.1. CHEMISTRY:
7.1.1. Synthesis of (A/,/V,AMrimethylammonio)methanesulfonate
(Sulfobetaine; 103)
Sulfobetaine was synthesized with a slight modification of the procedure reported by King 160 as outlined in Scheme 14. The pH was kept at - 6 and
Amberlite MB-1 deionizing resin was used instead of the Rexyn-300 resin as described in the original procedure.
HCOH (CH3)2NH NaHSO3 ► HO. ^ ^ ^ l l o (113) o o II 1. (CH3)2S 0 4 II - pH~6 S—O 2. Amberlite MB-1 O (114) (103)
Scheme 14: Synthesis of Sulfobetaine (103)
-115- 116
7.1.2. Synthesis of (A/,A/,/V-trimethylammonio)methane
phosphonate (phosphobetaine; 104)
The desired compound was obtained by the acid hydrolysis of the diethyl ester precursor (116), which in turn was obtained by reacting diethylphosphite (115) with formaldehyde and dimethylamine followed by quaternarization with methyliodide (Scheme 15 )161.
o r o y 1. (CH3)2N,CH20 + p_n'^v,s 0 I 0 ^ ■ - ■ ’ - »- (CH3)3N -'I -P 0 H 2- 3 O (115) (116)
O i. hci + y _ 0- 2.Amberlite MB1 ^ Scheme 15: Synthesis of Phosphobetaine 104 117 7.1.3. Synthesis of N-Betainylbenzenesulfonamide (105) The synthesis of compound 105 is outlined in Scheme 16. The synthesis was initiated by chloroacetylation of benzenesulfonamide (76) with chloroaceticanhydride and DMAP to give /V-chloroacetylbenzene-sulfonamide (78) 162. Treatment of 78 with trimethylamine in boiling acetone yielded the target compound 105. c h 2c i 2 (76) (77) (78) O _ (CH3)3N Y ^ N (105) Scheme 16: Synthesis of N-Betainyl benzenesulfonamide (105) 118 7.1.4. Synthesis of Sulfonaimde Analogs (Compounds 106-111) The synthesis of the target compounds (106-111) started with sulfanilamide (82; Scheme 17). Treatment of 82 with chloroacetic anhydride and pyridine following the standard sulfanilamide acylation procedures 163-165 yielded only the /V4-chloroacetylated compound (83). Using DMAP instead of pyridine in this reaction we were able to get the /V^/V^-dichloroacetylated analog compound 117. This was found to be a cleaner reaction, required less molar equivalence of chloroacetic anhydride and provided better yield than the reported procedure for the synthesis of 117 .166 Reaction of 83 or 117 with trimethylamine in refluxing acetone gave the corresponding betainyl derivatives 118 and 108. Treatment of 108 with 1 eq. of NaOH followed by neutralization with HCI cleaved the /V4-betainyl group to give the /Vr-betainylsulfanilamide dihydrochloride (119). Treatment of 119 with the appropriate acid anhydride and pyridine in refluxing CH 2CI2 gave the corresponding analogs 106,107 and 109 in good yields (Scheme 18). Compound 110 was obtained by reacting 119 with bromoacetic anhydride and pyridine in CH 2CI2 at room temperature because heating allows pyridine to replace the bromine atom of the product to give the pyridinium analog. Compound 111 was prepared by reacting 119 with iodoacetic anhydride in CH 2CI2 at room temperature in absence of the base. 119 n h 2 + (82 ) (77) Pyridine DMAP ♦ H 0 O ■ II * 0 = S 0 = S “ NH2 Y ^ CI (83) [j (117) H' n Y ^ c | o (CH3)3N I I(CH3)3N o - *■ N, o = s - n h 2 0=S'NY^S(C H 3)3 o (118) | (108 ) I Cl 1 Cl h ' N ' ^ ' N (CH 3)3 H y^N(CH3)3 Scheme 17: Synthesis of 105 Analogs 120 + N(CH3)3 Cl = f ' NY ~ 0 = S ' N|J ^ N ( C H 3)3 1. NaOH t f * 2. HCI Cl h ' NY ^ niN(CH3)3 nh2 *hci o (108) (119) o o NCH33 »A 0 A„ c h 3 or R CH2CH3 c h 2c i Y‘ CH2Br.HBr 0 CH2I Scheme 18: Synthesis of 105 Analogs 7.1.5. Synthesis of Sulfobetaine Analogs (Compounds 112a-c) The synthesis of the sulfobetaine analogs was accomplished by the treatment of the commercially available sodium hydroxy methanesulfonate ( 1 2 0 ) with appropriate cyclic amine to give the intermediates (121a-c). These compounds were then quaternarized to the target compounds by the treatment with methyl iodide (Scheme: 19). 121 r \ -* NH + H 0 ^ S 0 3 Na -► X N ^ s°3 Na (120) (121 a-c) c h 3i 112 t ch 3 a (C H 2)4 b (CH2)s r \ c c (CH2CH2)20 K ^ S 0 3 (112 a-c) Scheme 19: Synthesis of Sulfobetaine Analogs 7.2. Biological Studies: Biological evaluation of the new compounds was performed at The Ohio State University College of Medicine, Department of Internal Medicine by Dr. Hua H. Tong under the supervision of Dr. Calvin Kunin. The effect of the new compounds on enteric bacterial osmotolerance was tested in three strains of E coli ,with different salt tolerance, grown in different osmotic environments. 122 7.2.1. Salt-Tolerance Studies The bacterial salt tolerance is defined as the maximum molar NaCI concentration that the bacterial cells can withstand. The cells were grown in minimal media (MM) containing increasing concentrations of NaCI (0.1-1.0 M in 0.1 M increments) and the ability of the compounds to enhance or inhibit the bacterial salt tolerance was tested. In addition, the effect of the tested compounds on the osmoprotectant ability of glycine betaine (GB), and its biosynthetic precursor choline (Choi), was tested to determine if we could interfere with the effect or the uptake of either. The composition of the media used and the growth conditions are reported elsewhere .168 The strains of E. coli used are: I. a salt resistant strain E coli 31 which was obtained from clinical isolates from young women with urinary tract infections; ii. a standard strain E. coli K10 which was provided by LT.Smith (University of California, Davis); and iii. a salt sensitive strain E. coli ATCC25922 which was obtained from the American Type Culture Collection. Target compounds were tested at a concentration of 0.1 mM. Maximum salt- tolerance of the different strains of E. coli grown in minimal media (MM), or in MM containing GB or Choi (0.1 mM) are recorded in Table 13. Maximum salt- tolerance of bacteria grown in MM containing the test compounds alone or in 123 combination with GB or Choi are also recorded in Table 13. The effect of representative members of the novel compounds on the maximum salt- tolerance of E. coli 31, K10 or ATCC25922 is graphically presented in Figures 17,18 and 19 respectively. Table 13. The Effect of the Tested Compounds on Bacterial Maximum Salt Tolerance. Maximum Salt -Tolerance (M NaCI)a Compound# Media 6 E.coH31 E.coli K10 Ecoli ATCC25922 MM 0.65 ±0.05 0.49 ±0.03 0.42 ±0.04 MM+GB 0.92 ±0.04 0.90 ±0.04 0.83 ± 0.05 MM+Chol 0.88 ±0.04 0.81 ± 0.04 0.82 ± 0.02 MM 0.83 ±0.06 0.83 ±0.06 0.27 ±0.06 103 MM+GB 0.90 ±0.00 0.93 ± 0.06 0.86 ±0.06 MM+Chol 0.90 ±0.00 0.90 ±0.10 0.33 ±0.10 MM 0.60 ± 0.00 0.40 ± 0.00 0.33 ± 0.06 104 MM+GB 0.90 ±0.00 0.87 ± 0.06 0.80 ± 0.00 MM+Chol 0.83 ±0.00 0.77 ± 0.06 0.77 ±0.06 MM 0.73 ±0.06 0.56 ± 0.06 0.50 ±0.00 105c MM+GB 0.90 ±0.00 0.90 ± 0.00 0.83 ±0.06 MM+Chol 0.90 ±0.00 0.86 ± 0.06 0.80 ± 0.00 MM 0.70 ± 0.00 0.57 ± 0.07 0.40 ± 0.00 106d MM+GB 0.90 ±0.00 0.90 ± 0.00 0.86 ± 0.07 MM+Chol 0.80 ± 0.00 0.83 ±0.07 0.80 ± 0.00 124 Table 13 (Continued): MM 0.63 ±0.06 0.30 ±0.00 0.10 ± 0.00 109® MM+GB 0.90 ±0.00 0.86 ±0.06 0.86 ±0.06 MM+Chol 0.80 ± 0.00 0.80 ± 0.00 0.80 ± 0.00 MM 0.70 ±0.00 0.50 ±0.00 0.13 ±0.06 112a MM+GB 0.93 ±0.06 0.97 ±0.06 0.87 ±0.06 MM+Chol 0.86 ±0.06 0.60 ± 0.00 0.20 ± 0.00 MM 0.43 ±0.05 0.35 ±0.06 0.13 ±0.06 112b MM+GB 0.93 ±0.05 0.90 ±0.00 0.86 ±0.06 MM+Chol 0.88 ±0.05 0.50 ±0.00 0.20 ± 0.00 MM 0.77 ±0.06 0.63 ±0.06 0.20 ± 0.10 112c MM+GB 0.97 ±0.06 0.97 ±0.06 0.83 ± 0.06 MM+Chol 0.90 ±0.00 0.87 ±0.06 0.23 ± 0.06 a- Maximum salt-tolerance was measured as the maximum NaCI molar concentration at which there was 50% or more growth compared with tubes in which there was full growth (average of three observations ± S.D), b• Media consists of minimum media(MM), MM+ 0.1 mmol GB or MM+ 0.1 mmol Choi with and without 0.1 mmol of the tested compound, c■ Compounds 108,118 and 119 showed similar activity to that of 105, d Compound 107 showed similar activity to that of 106, e■ Compounds 110,111 showed similar activity to that of 109. Figure 17: The effect of the tested compounds on the maximum salt maximum onthe compounds the tested of effect The 17: Figure Maximum Salt Tolerance 0.0 0.2 0.4 0.6 0.8 1.0 1.2 - oto 0 105 103 Control ** Significant from its control at p < 0.05 (Paired student T-test) student (Paired <0.05 atp its control from Significant ** * Significant from its control at p < 0.01 (Paired student T-test) student (Paired 0.01 3) Bars a'CHA V g < , O S ^ C ( MM+GB nt h E. coli 31 coli E. 109c MM+Chol □ T ci 112 b 125 Figure 18: Figure Maximum Salt Tolerance . - 0.4 0.0 0.2 0.6 .8 0 1.0 1.2 - Control * Significant from its control at p < 0.01 (Paired student T-test) student (Paired < 0.01p at its control from Significant * tolerance of the Standard Strain Standard the of tolerance salt maximum the on compounds tested the of effect The Bars represent SDM (n3)SDM > represent Bars Standared 103 V'Viw, 0^YS( - * H (C S Y ^ 0 , w i V ' V ' O S " “ o c e 0 109c 105 IF iq k E. coli K10 coli E. irci r hNi ) K 0 MM+Chol □ 112b 126 Figure 19: Figure Maximum Salt Tolerance 0.0 0.2 0.4 0.6 0.8 1.0 1.2 - - - oto 103 Control The effect of the tested compounds on the maximum salt maximum the on compounds tested the of effect The oeac fteSl estv tanEE Strain Sensitive Salt the of tolerance ** Significant from its control at p < 0.01 (Paired student T-test) student (Paired 0.01 3) SDM represent Bars Salt Sensitive Sensitive Salt (CH. v ‘ n so - MG MM+Chol □ MM+GB l f sN CHs)l H (C R N^ ,s o w v y o £ 0 109c 105 iAC 52 ) 25922 ATCC li o c -tT ^ h 0 coli ATCC 25922 ATCC coli 01 112b ch . , 127 128 The results indicated that bioisosteric replacement of the carboxylic group of GB with a sulfonic group (sulfobetaine; compound 103) produces an osmoprotectant with equipotent activity to GB in both the standard and the salt resistant strains used (Figures 16 and 17). It showed some toxicity towards the salt sensitive strain, which was completely reversed by GB and to a lesser extent by Choi (Figure 18). The phosphonic acid analog (compound 104) did not show any osmoprotectant activity or toxicity towards any of the tested strains (Table 13). This indicates that the sulfonic, and not the phosphonic, group can replace the carboxylic group part of GB with complete retention of its osmoprotectant activity. The N -benzenesulfonyl amide analog of GB (compound 105) showed slight osmoprotectant activity at the concentration used (Table 13, Figure 18). That indicates that the GB/Chol transport system can recognize the A/-arylsulfonyl amide group as bioisostere of the carboxylic group of GB. This type of bioisosteric replacement of the carboxylic group has been utilized before in different systems 105. Addition of 4-amino or 4-betainyl amide groups on the aromatic ring of 105 (compounds 119 and 108 respectively) did not enhance its osmoprotectant activity (Table 13). The N 4 regioisomer of compound 108 (compound 118) showed similar activity to that of 105 (Table 13). When the alkylating arm (haloacetamido moiety) was attached to N4 of compound 119 (compounds 109-111) they showed strong toxicity to both the standard ( K 1 0 ) and the salt sensitive ( ATCC25922 ) E. coli strains (Table 13). This toxicity was completely reversed by both GB and Choi (Figures 17 and 18). The fact 129 that compounds 109-111 did not show any toxicity at zero molar salt concentration indicates that these compounds are probably utilizing the osmotically triggered GB/Chol transport systems to get into the bacterial cells and cause cytotoxicity. The presence of the natural substrates for these transport systems can prevent such toxicity. To prove that the toxicity is due to the electrophilic character and not due to the size of compounds 109-111, we tested compounds 106 and 107 in which the halogen atom is replaced by a hydrogen atom or a methyl group respectively. The results indicated that neither 106 nor 107 possess any toxicity; in fact, they showed slight osmoprotectant activity (Table 13). When we replaced the trimethylammonium portion of the sulfobetaine (compound 103) with other cyclic ammonium moieties, such as methylpyrrolidinium (compound 112a), methylpiperidinium (compound 112b) or methylmorpholinium (compound 112c), we observed marked inhibition of the bacterial maximum salt-tolerance of the standard and the salt sensitive strains (Figures 17 and 18) and to a lesser extent in the salt resistant strain (Figure 16). When GB was supplied, the salt-tolerance was fully restored. In contrast, salt-tolerance was not restored with addition of Choi. This observation indicates that compounds 112a-c may be interfering with the Choi and not with GB transport system. Methyl piperidinio-methanesulfonate (MPMS; 112b) was chosen to further investigate the effect of this type of compounds on the osmoprotectant effect of Choi. The salt sensitive strain E coli ATCC 25922 was grown in various combination of Choi and MPMS, in minimal medium containing 0.6 M NaCI (Figure.20). The osmoprotectant effect of Choi was 130 almost completely blocked with equimolar concentrations of MPMS. In addition, concentrations as low as 0.03 mM of MPMS were enough to partially reduce the osmoprotective activity of Choi. ZcoJi ATCC 25922 1.0 0.8 O O Choline (O 0.6 UJ o 0.03 z < 00 a: 0.4 m 0.06 o cn oo 0.13 < 0.2 ■ 0.25 I > .1 0.5 0.25 0.12 0.06 0.03 CHOLINE (mM) Figure 20: The effect of various concentrations of Choi alone, or in combination with MPMS on growth of E. coli ATCC 25922. Cells were grown in the presence of 0.6 M NaCI the growth was assessed by measuring the absorbance after 48 h. Concentrations of MPMS (mM) are indicated by arrows. 131 7.2.2. Effect of MPMS on the Osmoprotective Activity of GB, Choi and Proline in Mutant Strains Of E. coli Salt-tolerance studies were performed in two mutant strains with specific genetic deletions in the GB and/or Choi transport system to study the effect of MPMS. The parent strain E. coli MC4100 lacks the Bet genes so it can not accumulate Choi or oxidize it to GB. The mutant strain E. coli EF047 also lacks the Bet genes. In addition it lacks GB and proline transport systems (PutP, ProP and ProU). As expected, E. coli MC4100 responded to the osmoprotective effect of GB and proline, but not to Choi (Table 14). The addition of MPMS totally blocked the activity of proline, but did not suppress the osmoprotective effect of GB. The salt-tolerance of E. coli EF047 was not enhanced by GB, Choi or proline, and it was not inhibited by MPMS. 132 Table 14. Effect of MPMS on salt-tolerance of mutant strains of E. coli grown In MM alone or MM containing GB, Choi or proline Salt Tolerance (M NaCI)a E. coll MC4100b E. coll EF047 c Compound** Alone + MPMS Alone + MPMS Control 0.4 0.3 0.4 0.4 GB 0.8 0.8 0.4 0.4 Choi 0.4 0.3 0.4 0.4 Proline 0.5 0.3 0.4 0.4 a-. Values are mean of three experiments (variation is < 0.1M NaCI). b• Parent strain that can not utilize Choline. c- Mutant strain that is unable to utilize GB, Choi or proline. d Identical results were obtained when the osmoprotectants and MPMS were tested alone or together at 0.1 and 1.0 mM. 133 7.2.3. Uptake Studies Effect of MPMS on the Uptake of [14C]-GB, [14C]-Chol, and [14C]-Proline by E. c o ll : The effect of MPMS on the ability of different strains of E coli to accumulate 14C-labeled GB, Choi and proline was tested. Tests were performed by Marie- Christine Poggi under the supervision of Dr. LeRudulier at Laboratorie de Biologie vegetale et Microbiologie, Universite de Nice-Sophia Antapolis, Nice, France. The detailed experimental procedures are reported elsewhere . 168 Results are shown in Figures 21, 22 and 23. For each substrate (GB.Chol or proline), uptake was measured with cells grown in minimal medium in the absence or the presence of NaCI (0.8 M), following pretreatment with MPMS for 5 or 60 min. All strains showed strong stimulation of the radiolabeled substrates' uptake after being challenged with high osmolarity. 5 Min preincubation with MPMS strongly inhibit the uptake of Choi and proline but did not affect GB uptake. After 60 min preincubation with MPMS, Choi uptake was almost completely blocked in all strains, about 90% inhibition was observed in the case of proline, and 70 to 85% inhibition of GB uptake was observed depending on the salt sensitivity of the cells. 134 SALT RESISTANT STANDARD SALT SENSITIVF I50 r 150 r 150 100 100 too 50 50 5 10 Time (min) Figure 21: Uptake of [ 14C]-GB by three strains of E. coli. Cells were grown in MM in the presence or absence of 0.8 M NaCI or 1mM MPMS. Transport was assayed at a final concentration of 25 pM. (A) MM alone, (A) MM preincubated for 5 min with MPMS, (■ ) preincubated for 60 min; (o) MM+ 0.8 M NaCI without MPMS, (•) MM+ 0.8 M NaCI preincubated for 5 min with MPMS, (□) MM+ 0.8 M NaCI preincubated with MPMS for 60 min. 135 SALT RESISTANT STANDARD SALT SENSITIVE 10 T "I I 5 10 Time (min) Figure 22: Uptake of [ 14C]-Chol by three strains of £ coli. Cells were grown in MM in absence(A) or presence of 0.8 M NaCI (B). Transport was assayed at a final concentration of 25 pM; (A) MM alone, (A) MM preincubated for 5 min with MPMS; (o) MM + 0.8 M NaCI without MPMS, (•) MM + 0.8 M NaCI preincubated for 5 min with MPMS, (□) MM + 0.8 M NaCI preincubated with MPMS for 60 min. 136 SALT RESISTANT STANDARD SALT SENSITIVE 3 0 0 3 0 0 3 0 0 r c o CL 200 200 o E c ® o 100 100 100 a. 3 a> c o CL T im e ( m i n ) Figure 23: Uptake of [ 14C]-proline by three strains of E. coli. Cells were grown in MM in the presence or absence of 0.8 M NaCI or 1mM MPMS. Transport was assayed at a final concentration of 50 pM. (A) MM alone, (A) MM preincubated for 5 min with MPMS, (■) preincubated for 60 min; (o) MM + 0.8 M NaCI without M PM S, (•) MM + 0.8 M NaCI preincubated for 5 min with MPMS, (□) MM + 0.8 M NaCI preincubated with MPMS for 60 min. 137 7.3. Summary: Based on the above data it is possible to conclude that the carboxylic group of GB is bioisosterically replaceable with sulfonic but not phosphonic groups (compounds 103 and 104). The /V-benzenesulfonyl amide analog of GB (compound 105) was not as good as an osmoprotectant as GB or sulfobetaine. However, it did provide slight osmoprotection which suggests recognition by the GB/Chol transport system (Table 13). This system can be utilized to deliver cytotoxic analogs of GB (compounds 109-111) into bacterial cells under osmotic stress. The Choi transport system is inhibited by the methyl cyclicammonio methanesulfonate (compounds 112a-c; Table 13). MPMS (compound 112b) was found to be a strong inhibitor of the Choi transport system {B e t -gene products) which was significantly blocked after short preincubation with MPMS. The transport system for proline (ProP system) was also inhibited by short preincubation with MPMS. The GB transport system (ProU system), however, was not inhibited by short preincubation with MPMS, whereas prolonged preincubation with MPMS reduced GB uptake by up to 85 % (Figure 21). 138 CHAPTER VIII EXPERIMENTAL The general information about instrumentation, elemental analysis and solvents used provided in the introduction of Chapter IV is also applicable to this chapter. A/.MAf-Trimethvlamoniomethanesulfonate(103) O A/,/V,/V-Trimethylamoniomethanesulfonate was synthesized according to the reported procedure of King except that the pH was kept at ~6 and Amberlite MB-1 deionizing column was used to obtain the zwitterion 1 0 3 (72%). mp 318 °C (Lit . 160 mp 320 °C). 139 N.N.N-T rimeth vlammoniomethvlDhosphonate (1041 O + »■ ° *H Diethyl trimethylammoniomethylphosphonate iodide (116) was prepared according to the reported procedure (62%) mp 120-122 °C (lit. 25 mp 126 °C). The diethyl ester 116 (2.1 g, 6.4 mmol) was refluxed with concentrated HCI (10 mL) for 4 h and the solvent was removed under vacuum. The resulting solid was dissolved in H20 (150 mL), brought to -pH 8 with 10% aqueous NaOH and passed through a column of Amberlite MB-1 desalting resin. Solvent was removed under vacuum to give 104 (0.74 g, 72%). mp 285 °C (lit . 161 mp270 °C). 1H-NMR 8 3.23 (d, 2H, CH2) and 3.1 (s, 9H, 3 x CH 3). MS (m/e) FAB 153M+ Analysis for c 4h 12n o 3p Calculated: C 31.38, H 7.90, N 9.15. Found: C 31.25, H 7.72, N 8.99. 140 M-Chloroacetvlbenzenesulfonamide (78) Y " a A suspension of benzenesulfonamide (76 ; 1.8 g, 11.4 mmol), chloroacetic anhydride (77 ; 2.1 g, 12.3 mmol) and DMAP (1.4 g, 11.4 mmol) in dry CH 2CI2 (20 mL) was heated at reflux for 5 h. The reaction mixture was cooled and washed with 1N HCI (2 X 20 mL). The non aqueous layer was concentrated at reduced pressure. Upon addition of hexane the product precipitated out of solution and was collected by filtration. Recrystallization from toluene gave 78 (2.4 g, 90%). mp 94-96 °C (lit . 162 mp 104-105 °C). /V-Betainvlbenzenesulfonamide (105) O - N (C H 3)3 141 Trimethylamine (4 m L, 25% aqueous solution, 17 mmol) was added to 78 (1 g, 5.0 mmol) in acetone (30 mL) and the reaction mixture was heated at reflux for 5 h. The product that separated out was collected and recrystalllzed from H20 to give colorless crystals of 105 (0.61 g, 56%). mp 280-283 °C. 1H-NMR 8 7.73 (d, J = 7.8 Hz, 2H, ArH, ortho), 7.46 (m, 3H, ArH), 3.79 (s, 2H, CH2) and 3.04 (s, 9H, 3 x CH 3). MS (m/e) El 256 M+ . Analysis for CnH^NOaS Calculated: C 51.54, H 6.29, N 10.92. Found: C 51.57, H 6.20, N 10.78 A/L/V*-Dichloroacetvlsulfanilamide (117) H o H Nvlfx^ cl o A solution of chloroacetic anhydride (77; 30 g, 175.45 mmol) in dry CH 2CI2 (100 mL) was added over a period of 2 h to a cooled suspension of sulfanilamide (82 ; ice bath ; 10 g, 58.0 mmol) and DMAP (15 g, 122.7 mol) in 142 dry CH2CI2 (200 m L). The reaction mixture was heated at reflux for 4 h , upon which a heavy precipitate separated out that was collected by filtration and washed with 1 N HCI. The solid was dissolved in 10% aqueous NaOH solution, treated with activated charcoal and reprecipitated with 10% aqueous HCI. Recrystallization from MeOH gave yellowish crystals of 117 in almost quantitative yield, mp 246 °C (lit . 166 mp 215 °C). 1H-NMR (Methanol-d4) 5 7.95 (d, J = 8 Hz, 2H, ArH), 7.8 (d, J = 8 Hz, 2H, ArH), 4.2 (s, 2 H, Chfc) and 4.05 (s, 2 H, Chfc). Analysis for C i 0H i0CI2N2O4S Calculated: C 36.93, H 3.10, N 8.61 Found: C 37.12 , H 3.19, N 8.64 Aft-Betainvlsulfanilamide Hydrocholride (118) O II 0 = S — NH2 N(CH3)3 o 143 The same procedure as for 105 was applied using 83 (1 g, 4.02 mmol) as starting material. Recrystallization from H 20/acetone gave 118 (0.66 g, 50%) as colorless crystals, mp 268 °C. 1H-NMR 8 7.78 (d, J = 7 Hz, 2H, ArH), 7.58 (d, J = 7 Hz, 2H, ArH), 4.2 (s, 2H, C H 2 ) and 3.25 (S, 9H , 3 x C H 3 ) . MS (m/e) El 271 M+ . Analysis for C^H^NsOaS^HCI Calculated: C 42.92, H 5.89, N 13.65 Found: C 42.94, H 5.78, N 13.37 AW-Pibetainvlsulfanilamide Hydrochloride (108) 0 = S " >s[|X ^ N(CH3)3 N(CH3)3 Trimethylamine (5.6 mL, 25%w/v , 23.2 mmol) was added to a suspension of 117 (1 g, 3.0 mmol) and the resulting solution was heated at reflux for 2 h. The product that separated out was collected and recrystallized from H20/acetone to give white clusters of 108 (0.9 g, 81%). mp 302 °C (dec). 144 1H-NMR S 7.73 (d, J = 8.6 Hz, 2H, ArH), 7.52 (d, J = 8.6 Hz, 2H, ArH), 4.15 (s, 2 H, N1C0 CH2), 3.77 (s, 2 H, NHCOCtb), 3.21 (s, 9H, 3 x CH 3) and 3.03 (s, 9H, 3 x CH3). MS (m/e) El 370 M+. Analysis for C 16H26N40 4S«HCI Calculated: C 47.22, H 6 .6 8 , N 13.76 Found: C 47.15, H 6.77, N 13.75 Af*-Betainvlsulfanilamide Dihvdrochloride (1191 H . + CI N(CH3)3 n h 2 *h c i A mixture of 108 (0.8 g, 2.0 mmol) and NaOH (0.1 g, 2.5 mmol) in H20 (10 mL) was heated at reflux for 15 min and acidified to -pH 4 with 10% aqueous HCI. The solvent was removed at reduced pressure and the organic material was extracted from the residue with MeOH (2 x 25 mL). The product was precipitated with ether to give 119 (0.5 g, 74%). mp 248 °C (lit. 169 mp 254 °C). 145 General Procedure for the Synthesis of /V4-Acvl-AP-betainvl Sulfanilamides (106.107.109-111) Aft-Acetvl-AT-betainvlsulfanilamide (1061 N(CH3)3 0 A mixture of 119 (0.35 g, 1.0 mmol) acetic anhydride (0.122 g 1.2 mmol) and dry pyridine (0.2 g, 2.5 mmol) in dry CH 2CI2 (10 mL) was heated at reflux for 4 h during which the product separated out. It was collected by filtration and recrystallized from MeOH/ether to give 106 (0.3 g, quantitative), mp 263-265 °C. 1H-NMR 8 7.7 (d, J = 8 Hz, 2H, ArH), 7.48 (d, J = 8 Hz, 2H, ArH), 3.78 (s, 2H, CH2), 3.04 (s, 9H, 3 x CH 3) and 2.03 (s, 3H, COCH 3). Analysis for Ci3Hi9N 30 4S*H20 Calculated: C 47.12, H 6.39, N 12.68 Found: C 47.29, H 6.34, N 12.38 146 Aft-ProDionvl-A/7-betainvlsulfanilamide hydrochloride (1071 O Cl II . N - + 0 = s x N(CH3)3 The same procedure as for 106 was used except that propionic anhydride (0.15 g , 1.2 mmol) was used instead of acetic anhydride to give 107 (0.35 g, 92%). mp 240-241 °C. 1H-NMR 8 7.7 (d, J = 8.6 Hz, 2H, ArH), 7.48 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 2H, COCH2) 3.05 (s, 9H, 3 x CH 3 ), 2.3 (q, J = 7.6 Hz, 2 H, CH2CH3) and 1.03 (t, J = 7.6 Hz, 3H, CH2CH3). Analysis for C^^il^C^S'HCI Calculated: C 46.21 , H 6.09, N 11.54 Found: C 46.31 , H 6.35, N 11.76 147 yyft-Chloroacetyl-Ar-betalnylsiilfanilamide (1091 0 " + o = s ' Y ' N(CH3)3 o h y - c i The same general procedure as for 106 was applied except that the anhydride used was 77 ( 0.2 g, 1.2 mmol) to give 109 (0.3 g, 84%). Crystallization solvent was H20/acetone. mp 225-227 °C. 1H-NMR 8 7.75 (d, J = 8 Hz, 2H, ArH), 7.55 (d, J = 8 Hz, 2H, ArH), 4.18 (s, 2H, N1COCH2), 3.8 (s, 2 H, COCh^CI) and 3.05 (s, 9H, 3 x Cth). MS (m/e) El 347 M+. Analysis for C ^ H -^ C I^ C ^ S ^ O Calculated: C 42.68, H 5.51 , N 11.48 Found: C 42.75, H 5.53, N 11.68 148 Aft-BromoacetyW-betainvlsulfanilamide Hvdrobromide (110) O« Hi . Br 0 = S n |< ^ N ( C H 3)3 o "'V“ 0 The same general procedure as for 106 was applied except that bromoacetylbromide (0.24 g, 1.2 mmol) was used instead of acetic anhydride and the reaction was carried out at room temperature to give 110 (0.45 g, 93%). Crystallization solvent was MeOH/ether. mp 212-214 °C. 1H-NMR 8 7.74 (d, J = 8.5 Hz, 2H, ArH), 7.53 (d, J = 8.5 Hz, 2H, ArH), 3.95 (s, 2H, NICOCH2), 3.8 (s, 2H, COCh^Br) and 3.06 (s, 9H, 3 x CH 3 ). Analysis for C 13H18 BrN30 4S*HBr Calculated: C 32.99, H 4.04, N 8.88 Found: C 33.29, H 4.24, N 8.91 149 /V4-lodoacetvl-/V7-betainylsulfanilamide (111 ) 0 " + II .N y v - ' +11 o = s Y N(CH3)3 The same general procedure as for 106 was applied except that pyridine was omitted, iodoacetic anhydride (0.42 g, 1.2 mmol) was used instead of acetic anhydride and the reaction was carried out at room temperature overnight to give 111 (0.56 g, quantitative). Crystallization solvent was H 20/acetone. mp 198-200 °C. 1H-NMR 8 7.72 (d, J = 6.7 Hz, 2H, ArH), 7.50 (d, J = 6.7 Hz, 2 H, ArH), 3.79 (s, 4H, 2 x CH2 ) and 3.05 ( s, 9H, 3 x CH 3). Analysis for c 13h 18 in 3o 4s Calculated: C 35.54, H 4.13, N 9.56 Found: C 35.16, H 4.52, N 9.32 150 MMethvlpyrrolidiniomethanesulfonate (1 12al c h 3 0 N\ ^ s° 3 Pyrrolidine (0.79 g, 11.1 mmol) was added to a solution of sodium hydroxymethanesulfonate(120 ; 1.0 g, 7.4 mmol) in H20 (20 mL) and the reaction mixture was stirred at room temperature for 3 h. The H20 was then distilled off under reduced pressure to give A/-pyrrolidinylmethanesulfonate (121a; 1.3 g, 90%). Quaternarization was carried out by stirring 121a (0.5 g, 2.6 mmol) with iodomethane (0.4 g, 2.8 mmol) in aqueous methanol solution overnight. The zwitterion was obtained by passing an aqueous solution of the quaternary ammonium salt through a column of Amberlite MB-1 desalting resin. Distillation of H20 under reduced pressure gave 112a(0.3 g, 62%). mp 220-222 °C. 1H-NMR 8 4.4 (s, 2H), 3.7-3.5 (m, 4H), 3,18 (s, 3H) and 2.2-2.0 (m, 4H). Analysis for C 6H13N03S Calculated: C 40.20, H 7.31 , N 7.81 Found: C 39.93, H 7.47, N 7.70 151 AFMethvlpiperidiniomethanesulfonate M 12b1 c h 3 N + .SO , The same procedure as for 112a was used except that piperidine (0.94 g, 11.0 mmol) was used instead of pyrrolidine to give 112b (0.25 g, 53%). mp 224-226 °C. 1H-NMR 6 4.38 (s, 2H), 3.62-3.55 (m, 2H), 3.42-3.32 (m, 2H), 3.22 (s, 3H), 1.80 (m, 4H) and 1.57-1.54 (m, 2H). MS (m/e) FAB 194M+ Analysis for c 7 h 15n o 3s Calculated: C 43.52, H 7.82, N 7.25 Found: C 43.38, H 7.82, N 7.03 152 M-Methylmorpholiniomethanesulfonate (112c) c h 3 The same procedure as for 112a was used except that morpholine (0.97 g, 11.0 mmol) was used to give 112c (0.22 g, 45%). mp 272-274 °C. 1H-NMR 8 4.55 (s, 2H), 3.99 (t, J = 7 Hz, 4H), 3.79-3.68 (m, 2H), 3.6-3.5 (m, 2H) and 3.39 (s, 3H). Analysis for C 6H13N 04S Calculated: C 36.91 , H 6.71, N 7.17 Found: C 36.56 , H 6.77, N 7.00 BIBLIOGRAPHY 1. Rankin, J.C.; Davenport, J. Animal Osmoregulation ; Blackie & Son Ltd.: London 1981; p2. 2 . Abramow, M. Control Mechanisms in Mammals in Mechanisms of Osmoregulation in Animals; Gilles, R., Ed.; John Wiley & Sons:New York 1979; p 349-403. 3. Gilles,R.; Hoffman, E., K .; Bolis, L. Volume and Osmolality Control in Animal Cells Advances in Comparative & Environmental Physiology 1991, Volume 9. 4. Garcia-Perez, A .; Burg, M.B. 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