Synthesis of Biologically Important Pyrrole Derivatives in Any 13C and 15N Isotope Enriched Form by Prativa B

Home , Ethyl cyanoacetate

Global Journal of Science Frontier Research Chemistry Volume 12 Issue 2 Version 1.0 February 2012 Type : Double Blind Peer Reviewed International Research Journal Publisher: Global Journals Inc. (USA) Online ISSN: 2249-4626 & Print ISSN: 0975-5896

Synthesis of Biologically Important Pyrrole Derivatives in Any 13C and 15N Isotope Enriched Form By Prativa B. S. Dawadi & Johan Lugtenburg Leiden University, Leiden

Abstract - Recently the synthesis of [3-13C]-, [4-13C]-, and [11-13C]- porphobilinogen, [15N,13C4]-1H - pyrrole-2,3,5 - tricar - boxylic acid, [1-15N]-3-cyano-4-methyl-1H-pyrrole and [2-13C]- and [3-13C]-cyano-4- methyl-3-pyrrolin-2-one have been published. Incorporation of 13C and 15N in these systems at any position and combination of positions has become accessible. Also mild alkylations of active methylene compounds with α-halo carbonyl compounds open up many 3-pyrrolin-2- ones and pyrrole systems based on stable isotope building blocks that have been published. This gives the access to a whole new library of stable isotope enriched pyrroles in any stable isotope enriched form. This is also the case for biliverdin IXα which after enzymatic treatment has been converted into (2R)-phytochromobilin that reacts with its apoprotein to form intact active phytochrome.

Keywords : [1-15N]-3-Cyano-4-methyl-1H-pyrrole, [3-13C], [4-13C]-, and [11-13C]-porphobilinogen, [ 15N,

13C4,] -1H-pyrrole-2,3,5-tricarboxylic acid and biliverdin IXα.

GJSFR -B Classification: FOR Code: 030503, 040203,

Synthesis Of Biologically Important Pyrrole Derivatives In Any 13C And 15N Isotope Enriched Form

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© 2012. Prativa B. S. Dawadi, Johan Lugtenburg.This is a research/review paper, distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License http://creativecommons.org/licenses/by-nc/3.0/), permitting all non commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Synthesis of Biologically Important Pyrrole Derivatives in Any 13C and 15N Isotope Enriched Form

Prativa B. S. Dawadiα & Johan Lugtenburg σ 12 0 2 Abstract - Recently the synthesis of [3-13C]-, [4-13C]-, and of membrane fractions of Heliobacillus mobilis that was 13 15 13 13 [11- C]- porphobilinogen, [ N, C4]-1H -pyrrole -2,3,5 - tricar - grown on media containing [4- C]-aminolevulinic acid 15 13 13 boxylic acid, [1- N]-3-cyano-4-methyl-1H-pyrrole and [2- C]- have been obtained. and [3-13C]-cyano-4-methyl-3-pyrrolin-2-one have been ebruary Besides NMR spectroscopy, vibrational F 13 15 published. Incorporation of C and N in these systems at techniques such as resonance raman spectroscopy any position and combination of positions has become have been applied in heme protein research.14 In this 23 accessible. Also mild alkylations of active methylene compounds case some of the vibrations coupled to an electronic I with -halo carbonyl compounds open up many 3-pyrrolin-2- transition of the chromophore showed enhanced α 6 ones and pyrrole systems based on stable isotope building inelastic scattering up to 10 fold. on blocks that have been published. This gives the access to a Access to pyrroles enriched on each position whole new library of stable isotope enriched pyrroles in any and any combination of positions with stable isotopes V

I stable isotope enriched form. This is also the case for such as 2H, 13C and 15N is essential to study the I biliverdin IXα which after enzymatic treatment has been metabolism of important pyrrole derivatives using non- ersi ue ss converted into (2R)-phytochromobilin that reacts with its invasive isotope sensitive techniques.The chromophores apoprotein to form intact active phytochrome. of heme proteins and photosynthetic reaction centres XII I 15 13 Keywords :[1- N]-3-Cyano-4-methyl-1H-pyrrole, [3- C], have been prepared with stable isotope enriched pyrrole 13 13 15 13 [4- C]-, and [11- C]-porphobilinogen, [ N, C4,] -1H- building blocks. pyrrole-2,3,5-tricarboxylic acid and biliverdin IXα. Recently, we have published a review paper

about the stable isotope enriched systems in heme and )

I. INTRODUCTION ) (bacterio)chlorophyll protein systems that were known at 15 yrroles and their derivatives are one of the most that time. In the meantime a number of important 1 stable isotope enriched pyrrole systems have been important classes of heterocyclic compounds. arch Volume

They exhibit extensive biological and published together with a new method to prepare se P 2 pyrroles and stable isotope enriched building blocks pharmacological properties. Many pyrrole derivatives have shown interesting biological properties such as that allow access to a whole new range of stable isotope 3 4 5 enriched pyrroles. tier Re

antibacterial , antiinflammatory , antioxidant , antitumor, on antifungal6 and immune suppressant activities.7 Highly In this paper we focus on those new functionalized pyrroles are subunits of heme, possibilities that allow access to biliverdin IXα which can chlorophyll, bile pigments, vitamin B12 and pyrrole be converted into (2R)-phytochromobilin, the alkaloids isolated from marine source.8 Atrovastatin chromophore of phytochrome via one enzymatic 16 13 15 (Lipitor) is a drug for lowering cholesterol.9 conversion. We mainly focus on C and N enriched 2 building blocks leading to the labels at all atoms in the Access to stable isotope enriched systems ( H, l of Science Fr

13C and 15N) allows the metabolic conversions of these molecular skeleton of the pyrroles and tetrapyrrole na systems. We have not focused on 2H systems because

systems to be followed with mass spectroscopic Jour 2H occupies the peripheral positions on the molecular techniques when they have at least three stable l isotopes.10 13C-NMR Techniques have been used to system and is more prone to isotope loss and 13 scrambling during the synthetic process. However, the study the conversion of [5- C]-aminolevulinic acid into Globa 13 porphobilinogen in vivo in living Rhodobacter schemes for C incorporation can easily be adjusted to 2 sphaerhoides cells.11 H incorporation as well.

Similarly, the conversion of [2-13C]- and [11-

13C]-porphobilinogen in the body into uroporphyrinogen II. SYNTHESI S AND DISCUSSION 12 III and coproporphyrinogen III has been investigated. 13 13 13 13 a) Synthesis Of [3- C]- , [4- C]- And [11- C]- Very recently the C photo-CIDNAP MAS NMR spectra Porphobilinogen 1. 13 Enzymatic incorporation of [11- C]- and [2,11- α σ 13 Author : Leiden Institute of Chemistry, Leiden University, P.O. Box C2]-porphobilinogen 1 (fig. 1) into uroporphyrinogen I 9502, 2300 RA, Leiden. E-mail : [email protected] and III has been reported.17,18

8 a scheme that allows access to any stable isotopomer CO2H 19 10 9 6 7 and isotopologue has been reported. HO C 2 4 3 Acetic acid 2 is treated with 1 eq of bromine in the presence of trifluoroacetic anhydride to afford a high 5 11 2 yield of the 2-bromoacetic acid which is esterified with N1 ethanol into ethyl bromoacetate 3. The bromine is easily H N H 2 substituted for the cyano group with KCN 4 to give ethyl 1 cyanoacetate 5. The ester function is reduced with NaBH to give an alcohol function in 3- 13 4 1a: [4- C] hydroxypropionitrile 6. Treatment of 6 with aqueous HBr 12

0 13 and subsequent esterification afforded methyl 3- 2 1b: [3- C]

13 bromopropionate 7 in high yield. The SN2 reaction of 1c: [11- C] reagent 7 with nitromethane 8 in the presence of 2 eq

BuLi to obtain methyl 4-nitrobutanoate 9 is somewhat

ebruary Figure 1 : Structure and numbering of porphobilinogen

F difficult. 1 and its highly enriched isotopomers 1a, 1b and 1c. An alternative method to obtain the product 9 is 24 Porphobilinogen 1 is a biosynthetic precursor of to treat reagents 3 and 5 in the presence of NaOEt to tetrapyrrole chromophores in heme proteins, afford diethyl 2-cyanopentanedioate 10.20 Selective I photosynthetic antennae proteins, photosynthetic removal of one of the ester functions in NaCl, DMSO,

on reaction centres and phytochromes. The synthetic H2O gave ethyl 3-cyanopropionate 11. Subsequent 13 15 access to C and N enriched porphobilinogen will reduction of the ester function with NaBH4 afforded 4- V allow access to enrich stable isotopes in the above

I hydroxybutyronitrile 12 which is further converted into 4- I mentioned systems at any possible position. With 13C iodobutyronitrile 13. SN2 substitution of the iodo function ue ersi ue and 15N isotope incorporation in the chromophores of ss with NaNO2 and subsequent conversion of the nitrile these biologically important proteins can be investigated function into ethyl carboxylate is expected to give ethyl

XII I with noninvasive isotope sensitive techniques. 4-nitrobutanoate 9 without problem. In porphobilinogen In figure 1 the structure and numbering of 1 (fig. 1) the carbon atoms 3, 6, 7 and 8 are derived 13 porphobilinogen 1 is depicted. The synthesis of [3- C]-, from the compound 9 and carbon atoms 4, 9 and 10 are [4-13C]- and [11-13C]-porphobilinogen 1a, 1b and 1c via derived from 3-hydroxypropionitrile 6.

)

) a

KCN O O H C OH 4 a 3 a NaBH TFAA, Br2 Br 4a: [13CN] NC 4 NC se arch Volume OEt + OEt OH O EtOH , H 6 2 3 5 13 5a: [3-13C] 6a: [1- C] b CH3NO2 O 8 O 13 NO HBr, MeOH 8b: [ C] 2 6 MeO Br MeO 2 eq BuLi b 7 9 9b: [4-13C] O na lJour of Science Fr on tier Re O

l NC 5 OEt NaCl NaBH4 NC OH 3 NC OEt DMF, H2O Globa CO2Et 10 11 12

TsCl, NaI 12 NC I 13 Scheme 1. The synthesis of ethyl [4-13C]-4-nitrobutyrate 9b. The starting compound acetic acid 2 is commercially 13 13 13 available in the [1- C]-, [2- C]- and [1, 2- C 2] isotopomeric forms.

Synthesis Of Biologically Important Pyrrole Derivatives In Any 13C And 15N Isotope Enriched Form

3-Hydroxypropionitrile 6 is first protected with isocyanoacetonitrile 20. The base (tetramethylguanidine) dihydropyrane via acid catalyzed reaction to afford 3- induced the reaction between compounds 15 and 20 to (tetrahydropyran-2’-yloxy)-propionitrile (scheme 2).19 give 5-cyano-3-(methoxycarbonylethyl)-4 (tetrahydro- DIBAL-H reduction of the nitrile afforded the required 3- pyran-2’-yl-ethyl)-pyrrole 21. The tetrahydropyranyl hydroxypropanal derivative 14. Henry reaction between protective group is removed by acid to obtain the the nitro ester 9 and the aldehyde 14 in the presence of primary alcohol. The alcohol function is converted into a phase transition catalyst and acetylation afforded the the carboxylic acid by Jones oxidation and this acid is nitro derivative 15. subsequently converted into the methyl ester 22. The Isocyanoacetonitrile 20 is the building block that nitrile and methyl ester functions are catalytically provides carbon atoms 2, 5 and 11 and two nitrogen reduced to give an amide function in product 23. Base 12 atoms of porphobilinogen 1. It is shown that in scheme treatment converted the ester and amide functions into 0 2 3 a Strecker reaction of KCN 4, formaldehyde 16 and carboxylic acid and a methylene amine function in

NH4Cl 17 leads to 2-aminoacetonitrile 18. This molecule porphobilinogen 1. The conversion of products 15 and reacted with formic acid 19 in acetic anhydride to give 20 into the pyrrole 21 is a so-called Barton-Zard 21 ebruary the formyl derivative of 2-aminoacetonitrile that upon reacton. F treatment with POCl3 and triethylamine afforded 2- 25 OH DHP a NC THP-O O I DIBAL on 6 14 V

13 I 13 14a:[1- C] I 6a:[1- C] ue ersi ue

ss OAc

b CO2Me NaOH/CT ACl a XII I THP-O O2N CO2Me b 14 + Ac2O/Pyridine NO2 9 14a:[1-13C] 15 13 13 9b:[4- C] 15a:[5- C] ) ) 13 15b:[4- C]

Scheme 2. 3-Hydroxypropionitrile 6 is converted into methyl 5-acetoxy-4-nitro-7-(tetrahydropyran-2’-yloxy)- heptanoate 15, 15a and 15b. se arch Volume

The synthetic routes shown in schemes 1-3 have been developed for the synthesis of [3-13C]-, [4- 13C]- and [11-13C]-porphobilinogen 1. It is clear that based on commercially available 13C and 15N isotope enriched starting materials any carbon and nitrogen with stable isotope enrichment are accessible in porphobilinogen 1. Schemes 1 and 3 can be modified in such a way that a whole series of vicinal acetoxy and nitro derivatives can be made in the required stable na lJour of Science Fr on tier Re

isotope enriched form. l Tertiary butyl isocyanoacetate has been used to

prepare pyrroles with methyl carboxylate groups on the Globa 2-position.22 This building block is accessible in any stable isotope enriched form via reactions analogous to those described in scheme 3. Tosylmethyl isocyanide in all possible stable isotopically labelled forms has been used to prepare pyrroles and other heterocyclic systems via building blocks that can be easily prepared in stable isotope enriched form.23

O c c c HCO2H 19 _ + KCN 2 H H NH Cl H2N CN C N CN + + 4 POCl /NEt 3 3 4 16 17 18 20 4a: [13CN] 18c: [13CN] 20c: [13CN]

_ + c CO2Me CO2Me

12 C N C N THP-O 0 2 MeO C 20 a b 2 a b 15 13 PPTs, MeOH 20c: [1- C] c c

13 15a:[5- C] NC Jones reagent NC ebruary TMG N N F H H

CH N 15b:[4-13C] 2 2 21 22 26 13 21a:[4- C] 22a:[4-13C] 13 13 I 21b:[3- C] 22b:[3- C] 13 on 21c:[ CN] 22c:[13CN]

I I CO H CO2Me 2

ue ersi ue O

ss HO C a b 2 a b PtO2-Pd/H2 aq KOH

XII I HN c c N N H N H 2 H 1 23 13 ) 1a:[4- C] 23a:[4-13C] )

13 13 1b:[3- C]

23b:[3- C] 13 13 1c:[11- C] 23c:[11- C]

se arch Volume Sche me 3. Preparation of 2-aminoacetonitrile 18 and its conversion into isocyanoacetonitrile 20. Synthesis of [4-13C]-porphobilinogen 1a, [3-13C]-porphobilinogen 1b and [11- 13C]-porphobilinogen 1c via base catalyzed condensation of methyl 5-acetoxy-4-nitro-7-(tetrahydropyran-2’-yloxy) -heptanoate 15 and isocyanoacetonitrile 20.

15 This pyrrole is a building block for the b) Synthesis Of [1- N]- Formyl -4-Methyl-1H-Pyrrole 24a biologically important tetrapyrrole systems. In Scheme 4

it is indicated that phthalimide 25 treated with H acrylonitrile 26 to form β-phthalimidopropionitrile 27. 15N- H3C Phthalimide 25a is commercially available. All possible 4 3 O isotopologues of acrylonitrile are accessible via 5 2 na lJour of Science Fr on tier Re isotopically enriched 3-hydroxypropionitrile 6 (scheme

l N1 1).24 H

Globa 24

15 24a: [1- N]

Figure 2. Structure and numbering of [1-15N]-3-formyl-4- methyl-1H-pyrrole 24a.

O O CN CN 1. H2NNH2.H2O NH H2C CN + N N 2. Fumaric acid

O 3. (Ph)2C=NH O 28 25 26 27 29 15 15 15 29a: N 25a: N 27a: N 12

0 OHC CH3 2

NC CH3

NC CH3 ebruary 1. 3 eq LDA N F O a 3 4 H 2. (EtO)2P(O)Cl N O 2.5 N HCl CH3 2 5 24 27 a 15 O H3C 1 N 24a: N 3. H I NC CH3

H3CO on CH 32 3

15 V

OCH 32a: N I

3 I N 30 31 ersi ue CH2C6H5 ss 31a: 15N 33 XII I 15 15 Scheme 4. Reactions to prepare [1- N]-3-formyl-4-methyl -1H-pyrrole 24a starting from[ N]-phthalimide 25a and acrylonitrile 26. The product 3-[(diphenylmethylene) amino] c) Synthesis Of [1-15N, 2, 2’, 3, 3’-13C4]-Pyrrole-2,3,5-

) propionitrile 29 is prepared by first treating - Tricarboxylic Acid 34a. β ) phthalimidopropionitrile 27 with hydrazine hydrate, then O treating the mixture with fumaric acid to obtain the 3' fumaric acid salt of 3-aminopropionitrile followed by 4 3 OH se arch Volume reaction with benzophenone imine 28. Product 29 is 2 O 5 2' OH treated with 3 eq LDA at low temperature and 5' N1 subsequently diethyl chlorophosphate is added followed HO H O by 1,1-dimethoxyacetone 30. After the Wittig-Horner 34 reaction compound 31 is isolated as a mixture of E-and 15 13 34a: [1- N,2,2',3,3'- C ] Z-forms. In a solution of 2.5 N HCl the acetal and the 4 15 nitrogen protection groups are removed and the free Figure 3. Structure and numbering of [1- N, 2, 2’, 3, 3’- 13 amino group and aldehyde group reacted to give a high C4]-pyrrole-2,3,5-tricarboxylic acid 34a. yield of 3-cyano-4-methyl-1H-pyrrole 32. DIBAL-H The synthesis of the product 34a has been reduction of the nitrile function in the product 32 10 13 reported. Commercially available [ C4]-ethyl afforded 3-formyl-4-methyl-1H-pyrrole 24. Repeating this acetoacetate 35a is treated with commercially available 15 reaction with commercially available [ N]-phthalimide [15N]-ammonia in water in the presence of 2- na lJour of Science Fr on tier Re

l 25a afforded [1-15N]-3-formyl-4-methyl-1H-pyrrole 24a. chloroacetaldehyde 37. [1-15N, 2,2’,3,3’-13C4]-2-Methyl- As expected the NH group in the product 32 3-carbethoxypyrrole 38a is obtained via Hantzsch can be easily alkylated under basic condition. In this pyrrole synthesis in 35% yield which is in competition Globa case benzyl bromide has been used to obtain N-benzyl- with a Feist-Benary reaction leading after HCl treatment

3-cyano-4-methylpyrrole 33 in high yield. 13 to [2,2’,3,3’- C4]-2-methyl-3-carbethoxyfuran. In the 1,1-Dimethoxyacetone 30 or its homologous in Hantzsch pyrrole synthesis the enamine of the 3-keto any isotope enriched form is easily accessible via a carboxylate attacks the aldehyde function of the 2- Pummerer reaction of 1-phenyl sulfoxyl acetone.25 Also chloro keto molecule. After ring closure and dehydration many homologous of acrylonitrile are easily accessible. the pyrrole system is obtained. In the Fiest-Benary This means synthetic routes shown in scheme 4 will give reaction the initial reaction is the attack of the anion of access to many isotopically labelled pyrroles directly via the active methylene derivative on the aldehyde function subsequent functional group transformations. of chloroacetaldehyde and subsequent ring closure.25

Product 38a is treated with trichloroacetyl chloride 39 to Treatment with potassium permanganate give a quantitative yield of trichloromethyl keto oxidized the aldehyde function into a carboxylic acid compound 2-methyl-3-carbethoxy pyrrole 40a. function 43a. A final base induced saponification Treatment with ethanol in the presence of potassium afforded [1-15N, 2,2’,3,3’-13C4]-pyrrole-2,3,5-tricarboxylic carbonate afforded the diester 41a, upon cerium acid 34a. ammonium nitrate oxidation the 2-methyl group is converted into an aldehyde function (compound 42a).

O O CO2Et 12 4 2 Cl 4 3 0 3 2 H3C 1 OEt H 37 5 2 Cl3C 39 Cl + NH4OH CH3 OO N1 H ebruary

F 36 35 38 13 15 35a: [1,2,3,4- C ] 36a: [1- N] 38a:[1-15N,2,2',3,3'-13C ] 28 4 4

I CO Et CO Et 2 on 2 CO2Et

V CAN O O

I EtOH O O CH N CH N 3 ue ersi ue N 3 H K2CO3 H OEt H ss H OEt Cl3C 42 XII I 41 40 15 13 15 13 15 13 42a:[1- N,2,2',3,3'- C4] 40a:[1- N,2,2',3,3'- C4] 41a:[1- N,2,2',3,3'- C4]

) CO Et 2 3' )

CO H 2 4 3 KMnO4 KOH O CO H 5 2 se arch Volume N 2 2' EtOH H CO2H OEt HO2C N 1 H 43 34

43a:[1-15N,2,2',3,3'-13C ] 15 13 4 34a:[1- N,2,2',3,3'- C4]

Scheme 5. Synthesis of [1-15N, 2,2’,3,3’-13C ]-pyrrole-2,3,5-tricarboxylic acid 34a. 4

Pyrroles 38a, 40a, 41a, 42a and 43a in scheme Trichloroacetic acid is commercially available in 13C- 5 have isotope incorporation in 15N on position 1 and enriched form; it can be easily converted into the na lJour of Science13 Fr on tier Re 30

l C4 incorporation on positions 2, 2’, 3 and 3’. Via corresponding chloride. scheme 5 many pyrrole systems can be enriched

Globa besides the positions 2, 2’, 3 and 3’. α-Halogenated aldehydes such as 2- chloroacetaldehydes are accessible. Aldehydes that can be easily obtained from stable isotope enriched nitrile esters and alcohol etc.27 A general method to prepare the corresponding α-chloroderivatives has been reported.28 Many α-amino acids are commercially available in stable isotope enriched form. Recently, an efficient method to convert them into 2-chloroaldehydes has been reported.29

The Blaise reaction of acetonitrile 44 with zinc isotope enriched forms. Trimethyl silylation of 35 gives enolate of ethyl iodoacetate 45 has been reported.31 the bis(trimethylsilyloxy)butadiene derivatives 46. Acetonitrile 44 is commercially available in all possible Treatment with 1 mol of bromine afforded methyl 4- isotopomers. Zinc derivative of 45 is accessible via ethyl bromo-3-ketobutyrate.32 This means that besides ethyl 2-bromoacetate 3 (scheme 1). Many other nitriles and α- acetoacetate 35 many 3-ketoesters are accessible. bromoesters are accessible in all possible stable

O O O (H3C)3SiO OMe Zn CH3CN + I OEt H3C OEt CH 12 2 OSi(CH3)3 0 2

35 44 45 46

ebruary

F O O O + H SO Cl Cl 29 2 2 CH 35 H3C OEt 3 I Cl on 48 47 V

I - - I O Br O Br O + ersi ue Ph3P 49 + NaOH NaOH ss Br Ph P Ph3P 3 OEt OEt OEt CH3I O3

XII I 3 50 51

OSi(CH ) O ) 3 3

O )

OEt X OEt OEt 2CH H3C

O se arch Volume O O

52 53 54

Scheme 6. Preparation of ethyl acetoacetate 35 and ethyl 2-chloro-3-oxobutyrate 47 in any isotopomeric form using acetonitrile 44 and ethyl iodoacetate 45.

Ethyl acetoacetate 35 can be easily with the building blocks given in scheme 6 it is clear that monochlorinated by treatment with sulphuryl chloride. a very extended range of pyrroles in all possible stable Subsequent acid catalyzed hydrolysis and carbon isotopomeric forms are now accessible. dioxide elimination results in 1-chloroacetone 48. na lJour of Science Fr on tier Re

Similarly, many monochloroketones will be accessible in l any stable isotope labelled form.

Ethyl bromoacetate 3 treated with triphenyl- Globa phosphine 49 to form triphenyl phosphonium salt 50. After addition of 1eq NaOH and subsequent treatment with CH3I, the propionate phosphonium salt 51 is formed. Further treatment with base and ozonolysis ethyl pyruvate 52 is formed.33 Ethyl pyruvate 52 can be converted in the corresponding trimethyl silyl ether 53.34 Halogenation of the product 53 afforded the 3-halogenopyruvate 54. Using the reactions discussed in scheme 5 together

d) Synthesis Of Ethyl (2Z)-(4-Cyano-5-Oxopyrrolidin-2- 7). This molecule has been described in the Ylidene)Ethanoate 55. 36,37 literature. In this case the SN2 reaction of the anion of CN ethyl cyanoacetate 5 must have been the first step

3 4 followed by a base catalyzed cyclization to the furan derivative 59. 2 5 2' It is to be expected that triethylamine induced 1 O 2'' N alkylations of active methylene derivatives with aldehyde

EtO2C H or keto functions in both the chloride reagent and the active methylene compound will give 1,4-dicarbonyl

12 55 systems in Paal-Knorr pyrrole, Paal-Knorr furan and 0 38 2 Paal-Knorr thiophene syntheses in high yield. Figure 4. Structure and numbering of ethyl (2Z)-(4 - cyano-5-oxopyrrolidin-2-ylidene)ethanoate 55. 2-Cyanoacetamide 60 is expected to react with ethyl 2-chloro-3-oxobutyrate 47 using triethylamine as a

ebruary Ethyl cyanoacetate 5 is treated with ethyl 4- base to give the initial SN2 reaction on the active F chloro-3-oxobutyrate 56 (prepared by chlorination of 46 methylene carbon without reaction on the amide in scheme 6) in the presence of 1 eq of triethylamine in function due to its higher pKa value. 30 refluxing toluene (scheme 7).35 Diethyl 2-cyano-4- The carbonyl group and the amide group cyclised to give compound 61. Acid catalyzed

I oxohexanedioate 57 is the single product in high yield.

This product is the result of an SN2 reaction of the anion dehydration afforded 2-methyl-3-ethoxycarbonyl-4- on of the active methylene compound without competing cyano-5-hydroxypyrrole 62.35 Similarly, ethyl 4-chloro-3- oxoacetate 56 and 2-cyanoacetamide 60 afforded a V Feist-Benary reaction in the Hantzsch pyrrole synthesis I I because the nonnucleophilic base triethylamine cannot high yield of the cyclic derivative 63. Product 55 is

ue ersi ue give the Hantzsch pyrrole system. obtained via acid catalyzed dehydration of product 63.

ss A similar reaction between ethyl cyanoacetate 5 These 2-oxypyrrole derivatives have important and ethyl 2-chloro-3-oxobutyrate 47 afforded 2-amino- pharmaceu tical and biological properties. XII I 3,4-dicarbethoxy-5-methylfuran 59 in high yield (scheme Cl CN 1 5 3 CO Et 6 2 2 ) 4 + O EtO2C )

CO Et EtO2C O CN 2 5 56 57

se arch Volume CO Et EtO2C 2 EtO2C Cl EtO2C CN 4 3 5 + 1 O 2 5 NH2 H3C O 3CH O H3 C O EtO 47

58 59

CN EtO2C CN EtO C EtO C Cl CN EtO2C CN 2 2 + H 3 4 + na lJour of Science Fr on tier Re H3C O 1 l O O O 2 5 OH H3C H3C N H C N O HO 3 H2N H2N H H

Globa 47 60 61 62

CN CN CN 3 4 + H 2 5 O 2' 56 60 1 O + EtO C O 2'' N O H N 2 N 2 HO EtO2C H EtO2C H

Synthesis Of Biologically Important Pyrrole Derivatives In Any 13C And 15N Isotope Enriched Form

e) Synthesis Of [2-13C]-3-Cyano-4-Methyl-3-Pyrrolin-2- Even earlier than the above discussed alkylation One 64a And [3-13C]-3-Cyano-4-Methyl-3-Pyrrolin-2- of active methylene reagents, the Knoevenagel reaction

One 64b. between active methylene compounds, ketones and

aldehydes to form [2-13C]- and [3-13C]-3-cyano-4-

H C CN methyl-3-pyrrolin-3-one 64a and 64b, respectively (fig. 5) 3 4 3 has been reported.39 In scheme 8 it is depicted that 1,1-

dimethoxyacetome 30 and 2-cyanoacetamide 60 in 2 5 refluxing toluene in the presence of ammonium acetate O N1 and acetic acid afforded a high yield of a mixture of (E)-

H and (Z)-2-cyano-3-methyl-4,4-dimethoxybut-2-enamide 12 0 65. Due to the presence of two electron withdrawing 2

64 groups in the molecule the acetal function is relatively 13 64a: [2- C] acid stable. The double bond can easily be reduced by

sodium borohydride in ethanol to form the enantiomeric ebruary 13 F 64b: [3- C] mixtures of 2-cyano-3-methyl-4,4-dimethoxybutanamide

66. 31 Figure 5. Structure and numbering of [2-13C]-3-cyano-4- 13

methyl-3-pyrrolin-2-one 64a and [3- C]-3-cyano-4- I methyl-3-pyrrolin-2-one 64b. on

V 4a 3a CN I

H C CN CH I H C 3 H3C CN 3 3 NC O ersi ue

4 3 ss O O 5 2 O O O 1 + O O H C O H C O NH H C O NH N XII I 3 NH 3 2 3 2 H C 2 H 3 H3C H3C 64 30 60 65 66 13 13 60a: [1- C] 64a: [2- C] )

13 )

60b: [2-13C] 64b: [3- C]

H3C CN NC H3C CN se arch Volume

30 + O O O O OH

CH H3C O H3C H C O H C 3 3 3 H3C H3C 67 68 69

H H CN H C CN 3 O na lJour of Science Fr on tier Re

l O + 67 O O O O H3C O

H3C O 3CH H3C O 3CH Globa H3C 3CH 3CH

70 71 72

Scheme 8. The preparation of [2-13C]-3-cyano-4-methyl -3-pyrrolin-2-one 64a and [3-13C]-3-cyano-4-methyl-3- pyrrolin-2-one 64b from 1,1-dimethoxyacetone 30 and [1-13C]-2-cyanoacetamide 60a and [2-13C]-2-cyanoacetamide 60b, respectively.

Upon mild acid treatment of (E)- and (Z)- 2- As an alternative the Knoevenagel condensation cyano-3-methyl-4,4-dimethoxybutanamide 66 afforded between 1,1-dimethoxyacetaldehyde 70 and 1- 3-cyano-4-methyl-3-pyrrolin-2-one 64 in a high yield. cyanoacetone 67 afforded a high yield of 5,5-dimethoxy- Using [1-13C]-2-cyanoacetamide 60a and [2-13C]-2- 3-cyanopent-3-ene-2-one 71. Treatment of 71 with cyanoacetamide 60b, [2-13C]-64a and [3-13C]-64b have methyl magnesium iodide and cuprous cyanide gave in been prepared, respectively. In order to get to 3-vinyl-4- a high yield of the 1,4-addition product 5,5-dimethoxy-4- methyl-3-pyrrolin-2-one 89 for the ring D of biliverdin IXα methyl-3-cyanopentan-2-one 72. Conversion of product and phytochromobilin (vide infra), 1,1-dimethoxyacetone 72 to the required 3-pyrrolin-2-one was not successful. 30 is treated with 1-cyanoacetone 67 under However, 3-pyrrolin-2-ones are now accessible via

12 Knoevenagel conditions to give an excellent yield of 5,5- alkylation reactions of active methylene compounds with 0 2 dimethoxy-4-methyl-3-cyanopentan-2-one 68 (scheme amide functions. 8). The reduction of the 3,4 double bond and keto Compound 72 ( a protected 1,4-dicarbonyl function occurred simultaneously to give an compound) can easily be converted in the 25

ebruary enantiomeric mixture of 5,5-dimethoxy-4-methyl-3- corresponding pyrrole, thiophene and furan systems. F

cyanopentan-2-ol 69. Pinner reaction of 69 in aqueous The scope of the Knoevenagel reaction has been media to convert the nitrile function into the amide extended.40 At present various 3-pyrrolin-2-ones are 32 function and subsequent ring closure to get 3-(1’- easily available. This is a new approach to pyrrole hydroxyethyl)-4-methyl-3-pyrrolin-2-one could not be synthesis. These systems react with Lawesson’s reagent I realized. to give the corresponding thioamide system 74.25 on

I I Nu ue ersi ue

ss O S SR Nu N N N N

XII I H H H H 76 73 74 75 SnBu3H

) )

se arch Volume N H

Scheme 9. Conversion of 3-pyrrolin-2-one 73 into 2-substituted pyrrole 76 and pyrrole 77.

3-Formyl-4-methyl-1H-pyrrole 24 (scheme 4) The thioamide treated with various alkylhalo - which can be obtained in any stable isotope enriched genides to form the 2-thioalkyl substituted pyrroles. The 2-thioalky is easily substituted for many other 2- form forms the primary building block of both rings B substituents via various nucleophiles.41 The thioalkyl and C of biliverdin IXα 78.

na lJour of Sciencegroup Fr on tier Re has been substituted for hydrogen via radical

l reaction with tributyltin hydride to give the pyrrole without

a substituent on position 2.42

Globa f) Chemoenzymatic Synthesis Of (2R)- Phytochromobilin 80, The Chromophore Of

Phytochromes 78 can be converted into (2R)- Biliverdin IXα 16 phytochromobilin 91 that spontaneously reacts with

the apoprotein of phytochromes to form fully active phytochrome (fig. 6).43,44

COOEt COOEt COOEt H H C H3C O O O H3C H3C 3 EtO P EtO OEt DMF, (COCl)2 O O + N N N N H H H H H H

81 24 79 80 12

0 2 H2/Pd-C H2/Pd-C EtOOC COOEt

ebruary

F CH3 H3C 33 O I N N H H H on

82 I

83 I

ue ersi ue

ss COOEt COOEt XII I H3C H3C 1. CH (CN) H2/Pd-C 2 2 O H 81 O ) 2. DMF, (COCl) N 2 )

N H

H H H NC CN

84 85 se arch Volume

Scheme 10. Conversion of 3-formyl-4-methyl-1H-pyrrole 24 into the necessary building blocks of phytochromobilin 91.

In scheme 10 a synthetic scheme is shown that Vilsmeier formylation afforded in ethyl 3-[2-(2,2- converts 3-formyl-4-methyl-1H-pyrrole 24 into the dicyanoethenyl)-5-formyl-4-methyl-1H-pyrrol-3yl] necessary building blocks of phytochromobilin 91. propanoate 85. Triethyl phosphonoacetate is obtained from the In scheme 11 it is indicated that the reaction of ethyl bromoacetate 3 and triethylphosphite. pyrromethenone building block containing rings A and B Triethyl phosphonoacetate is treated with 3-formyl -4- is accessible in any stable isotope enriched form. methyl-1H-pyrrole 24 (scheme 4) to obtain ethyl 3-(4- na lJour of Science Fr on tier Re l methyl-1H-pyrrol-3-yl)acrylate 79.15 Vilsmeier formylation of product 79 afforded a mixture of the two pyrrole Globa aldehydes which can easily be separated into ethyl 3-(5- formy-4-methyl-1H-pyrrol-3-yl)acrylate 80 and ethyl 3-(2- formyl-4-methyl-1H-pyrrol-3-yl)acrylate 81. Catalytic reductions of the double bond in pyrrole aldehydes 80 and 81 led to 3-[2’-(ethoxycarbonyl)ethyl]-4-methyl-1H- pyrrole-5-aldehyde 83 and 3-[2’-(ethoxycarbonyl)ethyl]- 4-methyl-1H-pyrrole-2-aldehyde 84, respectively. Via the Knoevenagel reaction the 2-formyl group in the product 84 is protected as a dicyanovinyl group. A subsequent

H C O 3CH 3 CH2 H3C O H3C Br CH3 NaBH4 O + Dehydration NH O 2 O N N O H H

86 86 88 89

0 2

CH 3 CH 2 CO2Et ebruary

85 N O 34 H NH H3C H I O on 90 V

I I Scheme 11. Synthesis of pyrromethenone 90 (ring A and ring B) of the phytochrome 91. ue ersi ue

ss 2-Bromopropionamide 86 is condensed with 3- 3-pyrrolin-2-one 89. The condensation of product 89 oxobutanal 87. Subsequent dehydration of the product with product 85 and subsequent deprotection of the XII I 35 45 gave product 88. Reduction of the carbonyl function aldehyde function afforded the product 90. and subsequent dehydration results in 3-vinyl-4-methyl-

HOOC COOH HOOC COOH

) )

10 10 8 12 8 12 9 11 9 11 13 7 CH3 7 CH H3C B C H3C B C 3 23 13 se arch Volume 22 22 23 6 NH N 14 NH N 14 6 15 enzymatic conversion 15 5 5 4 16 4 16 3 3 17 21 17 CH NH HN 24 3 21 NH HN 24 CH3 H2C A D H3C A D 19 18 18 2 1 2 1 19 O O O O H3C H3C

H2C H3C 78 91

HOOC COOH

na lJour of Science Fr on tier Re 10 8 11 12

l 9 7 CH H3C B C 3 22 23 13 NH N Globa 6 14

5 15

CH3 4 16 NH 17 21 NH CH3 3 A 24 D Protein-S 2 18 1 19 O O H3C 91a H3C Figure 6. Structure and numbering of biliverdin IXα 78 and (2R)-phytochromobilin 91. Structure 91a represents the linkage of ring A to the protein of the phytochrome.

In scheme 12 it is indicated that the C and D After introduction of BOC protection of the building blocks are accessible in any stable isotope amide base induced ring closure to the 3-pyrrolin-2-one enriched form. Butyrolactone 92 treated with the sodium with tert- butyl alcoholate is effected.46 Treatment of salt of parachlorobenzene selenol to give the 4- product 96 with tributyl silyl triflate results in the double selenophenyl butyrate that treated with oxalyl chloride to protected pyrrole 97. This building block condensed form the chloride product 81.46 with the pyrrole aldehyde 82 under formation of the Butyrolactone 92 is accessible in any stable pyrromethenone 98.46 A final acid condensation afforded isotope labelled form via a Pinner reaction of 4- in the tetrapyrrole system which after oxidative hydroxybutyronitrile 12 (scheme 1). Product 93 treated desalination gives the vinyl group in the D ring giving the with 2,2-methoxypropylamine 94 to give product 95 after dimethyl ester of biliverdin IXα. A mild saponification 12

0 deacetalization. The protected 1-aminoacetone 94 is afforded in biliverdin IXα 78. 2 easily accessible via nitrosation of ethyl acetoacetate 35 (scheme 5).47

ebruary

Cl Cl F O CH3 H3C 35 Na O CH3 Cl Se (Boc) O

94 2 I NH2 O Se Se O tBuOH on (COCl)2 CH 3 O V

I I

95 ersi ue 92 O 93

Cl NH O ss

XII I Cl

Se Cl Se Cl

Se ) )

H C H C 3 TBSOTf 3 83 CH3

TiCl2/TFA se arch Volume O 3CH CO2Et N N OTBS NH N H Boc Boc O 98 96 97

+ H , perbenzoic acid 90 + 98 78 saponification Scheme 12. Synthesis of biliverdin IXα 78. na lJour of Science Fr on tier Re l

IV. ACKNOW LEDGMENT Globa III. ONCLUSION C This paper is written with great indebtedness to Nowadays there is a strong synthetic effort in investigators who have been involved in pyrrole the pyrrole field. Many new synthetic reactions and new 13 15 synthesis and the preparation of C and N enriched pyrroles are worked out and reported. Many of the building blocks. We dedicate this paper to future building blocks that are used in these processes can be investigators who will use the now accessible made accessible in various stable isotope enriched isotopomers to unravel the role of medically, form. In the near future whole new libraries of stable pharmaceutically and biologically important pyrrole isotope enriched pyrroles will become available. systems without perturbation at the atomic level.

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