Synthesis of multicyclic 2-pyridones from a formyl and

chlorometylene substituted precursor using a strategy of

directed diversity-oriented synthesis

Golla Krishna Prasad

Student Degree Thesis in Chemistry 45 ECTS Master’s Level Under Supervision of: Prof Fredrik Almqvist & Dr Magnus Sellstedt Examiner: Prof Mikael Elofsson Department of Chemistry Umeå University

ABSTRACT

Multi ring-fused 2-pyridones have shown interesting activity in a variety of biological systems. It

would be of great interest to explore the biological application of even more multi heterocyclic

ring fused 2-pyridones. ‘Diversity oriented synthesis’ is an excellent concept emerged in recent

years to access compounds with structurally and stereochemically diverse skeletons. This

served as an efficient avenue for synthesizing various ring fused 2-pyridones using formyl,

chloromethylene substituted 2-pyridone (6) as a starting compound. By treating the starting

material (6) with various nucleopliles, different sized heterocyclic ring fused 2-pyridones has

been synthesized. Additionally, an improved methodology for the synthesis of naphthyridones

was presented.

TABLE OF CONTENTS

1. INTRODUCTION........................................................................................................................1

2. RESULTS AND DISCUSSIONS................................................................................................3

2.1 Synthesizing multi hetero cyclic ring fused 2-pyridone compounds by using directed diversity

oriented synthesis………………………………………………………………………………………………………………….……….….3

2.2 Synthesis of chloromethylene substituted 2-pyridone………………………………………………………………….4

2.3 Synthesis of formyl and chloromethylene substituted 2-pyridone ……………………………….……………..4

2.4 Synthesis of 5-membered hetero cyclic ring fused 2-pyridones…………………………………………………….5

2.4.1 N-Substituted pyrroles fused to 2-pyridones at 6th and 7th positions………………………………………….5

2.4.2 Synthesis of thiophene and furan fused 2-pyridones at 6,7 positions……….……………….…………….…..5

2.4.3 Synthesis of γ-lactam fused 2-pyridone at 6th and 7th positions……………………………….…………………….6

2.5 Synthesis of six -membered heterocyclic ring-fused 2-pyridones…….……….…………….……………………7

2.5.1 Previous methodology to synthesize Napthyridones……………………………………………………………………7

2.5 .2 An improved methodology to synthesize Naphthyridones……………………………………………………………8

2.6 Synthesis of seven- membered heterocyclic ring-fused 2-pyridones .…………….…………..……………….10

2.7 Synthesis of eight- member heterocyclic fused 2-pyridones ………….…………………………..………….…..11

3. CONCLUSION……………….……………………………………………………………………………….……………………12

4. ACKNOWLEDGEMENT………………….………………………………………………………….………………….…13

5. EXPERIMENTS …….…………………………………………………………………….………………………………...14

5.1 General ………………………………………………………………………………………………………….………………………..14 5.2 Synthesis ……………………………………………………………………………………………………………………………………14

6. REFERENCES…………………………..........................................................................................23

ABBREVIATIONS

AcOH acetic acid

calcd calculated

DMF N,N’-dimethylformamide

DMSO dimethylsulfoxide

DOS diversity oriented synthesis

EtOAc ethyl acetate

EtOH ethanol

eq. equivalents

HPLC high performance liquid chromatography

HRMS high resolution mass spectroscopy

IR infrared

LC-MS liquid chromatography mass spectrometry

MeCN acetonitrile

MeOH methanol

mol. molecular

MWI microwave irradiation

NMR nuclear magnetic resonance

Ac acetyl

ON overnight

Ph phenyl

RT room temperature

TEA triethylamine

TFA trifluoro aceticacid

UV ultraviolet

1

1. INTRODUCTION

Heterocyclic compounds are widely present in biologically active, natural and synthetic

compounds. The majority of pharmaceutical products, pesticides, insecticides, cosmetics and

dyes e.t.c are heterocyclic compounds. In fact, in the Comprehensive Medicinal Chemistry

(CMC) data base, more than 67% of the compounds contain heterocyclic rings1. New strategies

to synthesize various heterocyclic compounds are significant for the lead identification and lead

optimization processes. In this context, it is certainly a more interesting and worthwhile

strategy to access skeletally different compounds rather than the single central fragment, for it

widens the scope to find lead compounds in the drug discovery process2.

2-pyridones are the significant core structures in synthetic as well as in natural products having

diverse medicinal properties like antifungal3, angiotensin converting enzyme inhibition4-6, Aβ

peptide aggregation7,8 , antibacterial9,10 and anticancer11,12 (Fig 1).

N

OH

COOHO

HOOC

N

O

MeO

ONHS

Et2N

1 2

A58365A, ACE Inhibitor (nM)5 RO 65 – 8835/001 Inhibitor of amyloid formation8

Figure 1. Example of biologically active molecules containing 2-pyridones.

The thiaozolo ring-fused 2-pyridones 3 and 4 possess various biological properties like

antibacterial13, inhibition of aggregation of an Alzheimer’s peptide into amyloids14 and

inhibition of curli protein aggregation15. By making new multiring fused systems based on 3 and

4 we envision it will result in substance classes with interesting activity in variety of biologiacal

systems. In order to prepare large number of mulitiring fused 2-pyridones that are diverse but

with maintained peptidomimetic backbone a directed diversity oriented synthesis has been

developed.

2

N

S

O COOLi

N

S

CF3

O COOLi

3 4

Figure 2 Biologically active thiazolo ring-fused 2-pyridones with peptidomimetic backbones13,15.

The aim of this project was to prepare the formyl and chloromethylene substituted 2-pyridone

(6) from the compound chloromethylene substituted 2-pyridone (5). Using this as starting

compound, a small series of multi ring-fused 2-pyridones were to be synthesized.

N

S

O COOMe

Cl

N

S

O COOMe

Cl

O

H

5 6

Figure 3. Structure of chloromethylene and formyl chloromethylene substituted 2-pyridone compounds.

3

2. RESULTS AND DISCUSSIONS

2.1. Synthesizing multi heterocyclic ring fused 2-pyridone compounds by using a directed diversity

oriented synthesis

Inspired by the concepts of diversity oriented synthesis16 5 to 8 membered heterocyclic

ring fused 2-pyridones were synthesized using the formyl chloro substituted 2-pyridone (6) as

starting material. Compound 6 has two reactive electrophilic sites at left hand side, the

reactions were carried out in this side and gave compounds that have large change in one part

and no change in the other part (Fig 4).

Figure 4. Overview to access multi heterocyclic ring fused 2-pyridones using directed diversity

oriented synthesis strategy.

N

S

O COOMe

S

N

S

O COOMe

Cl

O

H

N

S

O COOMe

N

N

S

NH O

S

COOMeMeOOC

S

O COOMe

N

O

O

+N

S

O COOMe

HN

O

N

S

O COOMe

ON

S

O COOMe

N

N

S

O COOMe

N

N

S

O COOMe

N

S

O COOMe

N

O

O

4

2.2. Synthesis of chloromethylene substituted 2-pyridone

The chloromethylene substituted 2-pyridone 5 was synthesized by using the chlorinated

Meldrum’s acid derivative (8) and ∆2thiazoline (7) according to the previously published

procedure17. One of these two 2-pyridone building blocks, the ∆2thiazoline (7) was synthesized

from nitriles according to the previously published procedure18. The chlorinated Meldrum’s acid

derivative (8) was synthesized from chloroacetic acid using a slight modification of previously

published precedures19.

S

COOMe

O O

O O

MWI 120oC

3 MIN

N

S

O COOMe

Cl+

TFADCE

yield 74%

ClHO

7 8 5

Scheme 1. Synthesis of chloromethylene substituted 2-pyridone (5).

2.3. Synthesis of formyl and chloromethylene substituted 2-pyridone

We made an attempt to introduce a formyl group at the sixth position in

compound 5 by following the previously published procedure20, in which the reaction was

carried out by using 4 eq of Arnold’s reagent and refulxing in acetonitrile. Unfortunately the

compound 6 was obtained in lower yield (22%). However, increasing the amount of Arnold’s

reagent to 10 eq gave the desired compound 6 in 53% yield (scheme 2).

N

S

O COOMe

Cl

N

Cl

N

S

O COOMe

Cl

O

H

yield 53%

+

MeCN,80oC

3.5 h

10 eq

5 6

Scheme 2. Synthesis of formyl chloromethylene substituted 2-pyridone.

5

2.4. Synthesis of 5-membered heterocyclic ring-fused 2-pyridones

Several 5-membered heterocyclic fused 2-pyridones at 6th and 7th positions were

synthesized using different nuclophiles.

2.4.1. N-Substituted pyrroles fused to 2-pyridones at 6th and 7th positions

In order to synthesize N-substituted pyrrole fused 2-pyridones, the starting material 6 was

treated with amines in the presence of K2CO3 using acetonitrile as a solvent at room

temperature. However, when the aniline was used as reaction component the reaction mixture

was refluxed at 70oC overnight (Scheme 3).

N

S

O COOMe

Cl

O

H

RNH2K2CO3

MeCNN

S

O COOMe

NR

6 9

9a R = Me 85%

9b R = Ph 77%

Scheme 3. Synthesis of N- substituted pyrrole fused 2-pyridones.

2.4.2. Synthesis of thiophene and furan fused 2-pyridones at 6,7 positions.

The preparation of thiophene fused 2-pyridones at 6th and 7th positions is a three

step one pot reaction. The first step is the SN2 reaction. Subsequent hydrolysis of the thioester

and then addition of AcOH accelerated the dehydration reaction, which resulted in 10a. The

same procedure was followed for the preparation of furane fused 2-pyridone (10b). But in this

reaction the dehydration step was not accelerated by AcOH. The reason for low yield was its

low stability (Scheme 4).

6

N

S

O COOMe

Cl

O

HN

S

O COOMe

X

X= O,S

2. CH3OH, RT

3. CH3COOH, RT

1.AcX-K

+, MeCN, RT

6 10

10a X = S 70%

10b X= O 23%

Scheme 4. Synthesis of thiophene and furan fused 2-pyridones

2.4.3. Synthesis of a γ-lactam fused 2-pyridone

When the starting material 6 was treated with sodium azide in MeCN under

microwave irradiation at 80oC to obtain the azide-substituted 2-pyridone compound 11

interestingly, a small amount of byproduct 12 was observed. As this product has two

peptidomimetic amide bonds, it enhanced our curiosity to increase the yield of 12. Fortunately,

using the same reaction conditions with an additional increase in temperature from 80oC to

100oC and reaction time from 10 to 30 min gave 62% yield of 12 (Scheme 5).

N

S

O COOMe

Cl

O

H

NaN3

MWI 80oC N

S

O COOMe

N3

O

H

MeCN, MeOH

30 min

6 11

N

S

O COOMe

Cl

O

H

MeCN, MeOH

MWI 100oC N

S

O COOMe

HN

O30 min

NaN3

6 12

Yield 62%

Scheme 5. Synthesis of γ-lactam fused 2-pyridones.

7

Mechanism

Although the mechanism of the above reaction is not clear, a possible mechanism is a Schmidt type of

reaction.

N

S

O COOMe

Cl

O

H

NaN3, MeCN

MWI 100oC

N

S

O COOMeO

H

N

N

S

O COOMe

NN

+

N

N

S

O COOMe

NNN

H

..

.._

HO

+

O

.._

:

Figure 5: Outline of tentative mechanism.

2.5. Synthesis of six-membered heterocyclic ring fused 2-pyridones

In order to synthesize six-membered heterocyclic rings fused to 2-pyridones we

focused on a previously published three component reaction21, which access naphthyridones.

The central fragment of these compounds shows a structural similarity to the natural product

lophocladine A, which has δ-opiod receptor antagonist activity22. Here, we present an improved

methodology to synthesize naphthyridones by changing the starting compound.

2.5.1. Previous methodology to synthesize naphthyridones

The three component reaction described before, used compound 13 as the starting

material21. Compound 13 was stirred with various aldehydes and amines at which resulted in

dihydro naphthyridones. In this method oxidation was nessesary and this was done by using

chloronil or oxygen (Fig 6).

8

N

S

O COOMe

, R2CHO1.R

1NH2

AcOH,

N

S

O COOMe

NR

1

R2

+

MeOH, MeCN

O

H2.Oxidation

13 14

Figure 6. Previous method to synthesize naphthyridones21.

2.5.2. An improved methodology to synthesize naphthyridones

Here, to synthesize naphthyridones compound 6 was used as starting material instead

of compound 13 (Scheme6). The naphthyridones were isolated in high yields in low reaction

times and as there is a methylene chloride at seventh position, the intermediate

dihydronaphthridone spontaneously becomes aromatic as it eleminates the chloride and this is

confirmed by mechanism. Hence, there is no need to use any oxidizing agent in this method as

it was in the previous procedure21 (Scheme 6).

N

S

O COOMe

Cl PhCHO,

MeNH2, AcOH

MWI 80oC,10 min

N

S

O COOMe

N

Ph

+MeOH, MeCN

O

H

Cl-

6 15

Yield 74%

Scheme 6. Overview of naphthyridone synthesis from compound 6.

9

The below mechanism supports the spontaneous aromatization of dihydronaphthyridones.

Mechanism

N

S

O COOMe

Cl

O

H

N

S

O COOMe

N

R2

R1

R1NH2

-HCl

N

S

O COOMeNR

1

Cl

N

S

O COOMe

N

R2

Cl

R1

R2CHO

N

S

O COOMe

Cl

HNR

1

N

S

O COOMe

Cl

NR

1

R2

+

+

+

Figure 7. Outline of tentative mechanism.

Besides the synthesis of charged naphthyridonium salts, the uncharged napthyridone

16 was also prepared. As with the previous method21, it was found that excess of ammonium

acetate (4 eq) was necessary to produce uncharged naphthyridones (Scheme 7).

N

S

O COOMe

Cl PhCHO

MeCOO-NH4

+

, AcOH

MWI 80oC,10 min

N

S

O COOMe

N

Ph

MeOH, MeCN

yield 62%

O

H

6 16

Scheme 7. Synthesis of uncharged naphthyridones.

10

2.6. Synthesis of seven-membered heterocyclic ring-fused 2-pyridones

For the synthesis of seven-membered heterocyclic fused 2-pyridones, first the starting

material 6 was reduced to alcohol compound 17. Next, the alcohol compound 17 was treated

with different amines to give amino alcohols. Finally, the amino alcohols were reacted with

triphosgene to give 7-membered carbamate compounds (Scheme 8). It was possible to purify

the resulting compounds in each step for most amines but with aniline it was difficult to purify

the resulted carbamate and this compound was excluded.

N

S

O COOMe

Cl

O

H

NaBH4

N

S

O COOMe

Cl

MeOH,MeCN

yield 80%

OH

6 17

N

S

O COOMe

Cl

OH

N

S

O COOMe

NH

RNH2,K2CO3

MeCN, rt

R

OH

17 18

18a R=Me 58%

18b R=Bn 63%

N

S

O COOMe

HN

OH

N

S

O COOMe

triphosgene

DCM, rt

N

O

O

RR

18 19

18a 19a R=Me 63%

18b 19b R=Bn 61%

Scheme 8. Overview of carbamate synthesis.

11

2.7. Synthesis of an eight-membered heterocyclic ring-fused 2-pyridones

In order to make an 8-membered heterocyclic ring fused 2-pyridone, compound 6

was first treated with BOC protected cysteine which resulted in a thio ether compound. Next,

the BOC deprotection was carried out by adding TFA in CH2Cl2 (1:1), which also promoted the

formation of the imine. If basic work-up was performed, thiophene fused 2-pyridone (10a)

was observed. In order to avoid unwanted thiophene fused 2-pyridone formation, the TFA was

removed by evoporation without proceeding with any basic work-up. The triacetoxy

borohydride was unable to reduce the imine. Sodium borohydride was used as an alternative

for the reductive amination (Scheme 9).

N

S

O

Cl

COOMeO

H N

S

O COOMe

S

NH

MeOOC

yield 22%

1. BOC cysteinemethylester

K2CO3, MeOH, MeCN

2.TFA, DCM

3.NaBH4, MeOH

6 20

Scheme 9. Synthesis of 8-membered heterocyclic fused 2-pyridone.

12

3. CONCLUSION

Suitable reaction conditions to synthesize the starting compound, a formyl and chloro-

methylene substituted 2-pyridone were developed. Directed diversity-oriented synthesis was

used as a platform to synthesize multi heterocyclic ring-fused 2-pyridones in which one part of

the compounds was constant but with large change in the other part. In order to obtain

different heterocycles and rings, various nucleophiles were used. It was also shown that it is

possible to make new seven and eight-membered ring-fused 2-pyridones with this strategy.

Additionally, an improved methodology to synthesize charged and uncharged naphthyridones

has been developed.

These compounds will be hydrolyzed and screened on various biological systems and will

hopefully show significant biological activity on a particular biochemical process or processes.

13

4. ACKNOWLEDGEMENT

I would like to thank my supervisor, Fredrik Almqvist for allowing and encouraging me to do

this project. I am profoundly grateful to Magnus Sellstedt who helped me immensely for what I

have learned from him is invaluable. I would also like to thank other members of the group;

Christoffer Bengtsson, Syam Krishnnan, Munawar Hussain, The Hung Dang, Karl Gustafson and

wu lian pao for their encouragement and assistance. Lastly, I also wish to thank all the members

involved in general club meeting for I have attained valuable information from their work and

article discussions.

14

5. EXPERIMENT

5.1 General

All reactions were carried out under nitrogen atmosphere, unless otherwise stated. CH2Cl2 and DMF were passed through a column of Al2O3 and MeCN was distilled from CaH2. These were stored over 3 Å molecular sieves. The amberlite® IR-120(plus) ion exchange resin was washed with methanol prior to use. Optical rotations were mesured on a perkin Elmer polarimeter at 20oC and 589 nm. TLC was performed om silica gel 60 F254 (Merck) with detection by UV light. Flash coloumn chromatography was performed on silica gel (Merk, 60 AO, 40.63 µm). 1H NMR and 13C NMR spectra were recorded on a Bruker DRX-400 spectrometer. NMR experiments were conducted at 298K and spectra were calibrated against solvent residue to 7.26 (1H) 77.0 (13C) for CDCl3, 3.30 (

1H) 49.0 (13C) for MeOD d4. IR spectra were recorded on ATI Mattson Infinity Series FTIRTM. HRMS was conducted using a BrukermicrOTOF II mass spectrometer with electrospray ionization. LC-MS was conducted on a Micromass ZQ mass spectrometer. One of the NMR spectras is quite complex (compound 20) due to diastereomeric mixture.

Fractional proton integrals indicate presence of isomers. (maj) indicate peak from major isomer

and (min) indficate peak from minor isomer in both 1H and 13C spectra.

5.2 Synthesis

5-Chloromethyl-2,2-dimethyl-[1,3]dioxane-4,6-dione (8)

Chloroacetic acid 4725 mg (50.00 mmol), Meldrum’s acid 7209 mg (50.02 mmol), and 4-

dimethylaminopyridine, 7326 mg (59.97 mmol) were dissolved in 250 ml CH2Cl2 and cooled at -

10oC. Dicyclohexylcarbodimide, 1350 mg (55.01 mmol) in 50 ml CH2Cl2 was added slowly over

one hour and the resulting mixture was stirred overnight at room temperature. The reaction

mixture was filtered off and dichloromethane phase was washed three times with 2% aqueous

KHSO4 solution and then brine. The organic phase was concentrated under reduced pressure

gave 8, 5787 mg (53%) as solid.

IR λ 3012, 1732, 1667, 1538, 1380, 1264, 1380, 1264, 1199, 1155, 906; 1H NMR (400 MHz,

CDCl3) 15.54 (m, 1H), 4.88 (s, 2H), 1.76 (s, 6H); 13C NMR (400 Mz,CDCl3) 188.5, 170.4 (broad), 159.6

(broad), 106.0, 91.7, 41.8, 27.1 (2C)

15

5-Cyclopropylmethyl-2,3-dihydro-thiophene-3-carboxylic acid methyl ester (7)

Thiazoline 7 was prepared according to published procedure18.

The data is in agreement with published data18.

7-Chloromethyl-8-cyclopropyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic acid meth

yl ester (5)

Thiazoline 7 (1990 mg, 10 mmol) and Meldrum’s acid (10) (5500mg, 25 mmol), were dissolved in 15 ml 1,2 dichloroethane. Trifluoroacetic acid (1.54 ml, 20 mmol) was added. The solution was heated with microwave irradiation for 3 min then quenched by addition of saturated NaHCO3 and then extracted with CH2Cl2. The organic extract was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure and resulting residue was purified with silica gel chromatography using heptane ; ethyl acetate 1:2 as elutent to give 2236 mg (74%) of compound 5. [α]D -255 (c 1.0, CHCl3); IR λ 2952, 1747, 1648, 1578, 1484, 1430, 1202, 1170, 842, 734;

1H NMR (400 MHz, CDCl3) 6.40 (s, 1H), 5.58 (dd, J = 8.6, 2.4 Hz, 1H), 4.62 (d, J = 12.5 Hz, 1H), 4.50 (d, J = 12.5 Hz, 1H), 3.79 (s, 3H), 3.66 (dd, J = 11.7, 8.6 Hz, 1H), 3.50 (dd, J = 11.7, 2.4 Hz, 1H), 1.72-1.63 (m, 1H), 1.00-0.87 (m, 2H), 0.69-0.60 (m, 2H); 13C NMR (400 Mz,CDCl3) 168.5, 161.1, 152.1, 148.4, 115.6, 112.3, 62.9, 53.3, 42.7, 31.8, 10.5, 7.4, 7.2

7-Chloromethyl-8-cyclopropyl-6-formyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3-carboxylic

acid methyl ester (6)

5319 mg (53 mmol) DMF in 30 ml MeCN, was cooled at 0oC. Oxalyl chloride, 6.06 ml (53

m mol), was slowly added and the resulting slurry was then stirred at room temperature for 10

minutes. Compound 5, 1600 mg (5.3 mmol) in 20 ml MeCN was added and the mixture was

heated at 75oC in oil bath for 4 hours. The mixture was quenched with saturated aqueous

NaHCO3 and extracted with CH2Cl2. The organic extract was dried over anhydrous Na2SO4 ,

filtered and concentrated under reduced pressure. The residue was purified by

chromatography on silica gel using heptane : ethyl acetate 1:1 as eluent to give 925 mg (53%) of

compound 6 as yellow foam.

[α]D -417 (c 0.5,CHCl3); IR λ 1747, 1671, 1635, 1473, 1214, 1147, 737; 1H NMR (400 MHz, CDCl3)

10.35 (s, 1H), 5.67 (dd, J=9.20,2.35 Hz, 1H), 5.34 (d, J=9.68Hz, 1H), 5.09 (d, J=9.68 Hz, 1H), 3.82

(s,3H), 3.74 (dd, J=9.2, 11.87 Hz,1H), 3.56 (dd, J=11.74, 2.35 Hz, 1H), 1.82-1.68 (m, 1H), 1.12-

0.92 (m, 2H), 0.81-0.7 (m,2H); 13C NMR (400 Mz,CDCl3) 190.1, 167.8, 162.1, 158.2, 155.5, 116.9,

114.3, 63.4, 53.6, 36.9, 31.5, 10.5, 8.0, 7.3

16

8-Cyclopropyl-6-methyl-4-oxo-2,3,4,6-tetrahydro-1-thia-3a,6-diaza-s-indacene-3-carboxylic acid

methyl ester (9a) Compound 6 (50 mg, 0.15 mmol) and K2CO3 (32 mg, 0.22 mmol) were taken up in 2ml

acetonitrile. 0.14 ml (0.22 mmol) methylamine (1.6M in MeOH) was added and the reaction

mixture was stirred at room temperature over night. Saturated aqueous NaHCO3 was added

and the mixture was extracted with CH2Cl2. The organic extract was dried over anhydrous

Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column

chromatography on silica gel using heptane : ethyl acetate 1:3 as eluent to give 40 mg (85%) of

compound 9a as yellow foam.

[α]D -206 (c 0.5,CHCl3); IR λ 1739, 1649, 1587, 1317, 1175, 1206, 762; 1H NMR (400 MHz,

CDCl3); 7.35 (d, J=1.99 Hz, 1H), 6.74 (d, J=2.0 Hz, 1H), 5.55 (dd, J=7.7, 1.20 Hz, 1H), 3.81 (s,3H),

3.75(s,3H), 3.59 (dd, J=11.4, 7.6 Hz,1H), 3.46 (dd, J=11.4, 2.0 Hz, 1H), 1.72-1.64 (m, 1H), 0.87-

0.79 (m,2H), 0.71-0.64 (m, 2H); 13C NMR (400 Mz,CDCl3) 167.7, 158.9, 133.4, 127.5, 121.1,

114.3, 112.4, 106.9, 61.2,52.8, 37.4, 32.1, 10.8, 5.8, 5.5

8-Cyclopropyl-4-oxo-6-phenyl-2,3,4,6-tetrahydro-1-thia-3a,6-diaza-s-indacene-3-carboxylic acid

methyl ester (9b)

Compound 6 (110 mg, 0.33 mmol) and K2CO3 (67.4 mg, 0.48 mmol) were taken up in 2ml

acetonitrile. Aniline (77.6 mg, 0.84 mmol) was added and the reaction mixture was refluxed at

70oC overnight. Cooled the reaction mixture at room temperature and saturated aqueous

NaHCO3 was added and the mixture was extracted with DCM. The organic extract was dried

over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was

purified by column chromatography on silica gel using heptane: ethyl acetate 1:2→1:3 as eluent

to give 95 mg (77%) of compound 9b as yellow foam.

[α]D -86 (c 0.5,CHCl3); IR λ 1742, 1651, 1597, 1496, 1243, 1208, 754, 691; 1H NMR (400 MHz,

CDCl3) 7.80 (d, J=2.2 Hz,1H), 7.51-7.45 (m, 4H), 7.39-7.33 (m, 1H), 7.18 (d, J=2.2 Hz, 1H), 5.59

(dd, J=7.7, 1.8 Hz, 1H), 3.78 (s, 3H), 3.63 (dd, J=7.7, 11.5 Hz, 1H), 3.49 (dd, J=11.5, 1.9 Hz), 1.79-

1.70 (m,1H), 0.90-0.84 (m,2H), 0.78-0.69 (m, 2H); 13C NMR (400 Mz,CDCl3) 169.7, 159.1, 140.1,

134.4, 129.2 (2C), 128.2, 127.3, 121.7 (2C), 118.9, 115.8, 110.2, 106.7, 61.3, 53.0, 32.1, 10.9,

6.0, 5.6.

17

8-Cyclopropyl-4-oxo-2,3-dihydro-4H-1,6-dithia-3a-aza-s-indacene-3-carboxylic acid

methyl ester (10a)

Compound 6 (50 mg, 0.15 mmol) and potassium thio acetate (17 mg, 0.15 mmol) were

taken up in 2ml acetonitrile and stirred at room temperature for 2 hours. 2 ml MeOH was

added to the above reaction mixture and stirred at room temperature for 2 hours. 180 mg (3

mmol) AcOH was added and stirred at room temperature for 1 hour. The saturated aqueous

NaHCO3 was added to the reaction mixture and extracted with CH2Cl2 three times. The organic

extract was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure.

The residue was purified by coloumn chromatography on silica gel using heptane: ethylacetate

1:1 as eluent to give 31 mg (70%) of compound 10a as yellow foam.

[α]D -95 (c 0.5, CHCl3); IR λ 1739, 1643, 1586, 1197, 1153, 854, 762; 1H NMR (400 MHz, CDCl3)

8.29 (d, J=3.2 Hz,1H), 7.37 (d, J=3.2 Hz,1H), 5.55 (dd, J=7.8, 2.0 Hz, 1H), 3.78 (s, 3H), 3.63 (dd,

J=11.7, 7.9 Hz,1H), 3.39 (dd, J=11.7, 2.0 Hz,1H), 1.80-1.72 (m, 1H), 0.98-0.88 (m, 2H), 0.75-0.65

(m,2H); 13C NMR (400 Mz,CDCl3) 169.2, 157.4, 140.9, 137.2, 130.1, 129.2, 114.4, 107.6, 61.1,

53.1, 32.0, 10.9, 6.5, 6.2

8-Cyclopropyl-4-oxo-2,3-dihydro-4H-6-oxa-1-thia-3a-aza-s-indacene-3-carboxylic acid

methyl ester (10b)

Compound 6 (100 mg, 0.3 mmol) and 29.4 mg (0.3 mmol) potassium acetate were taken up in

2ml DMF and stirred at room temperature for 2 hours. 2ml MeOH was added to the above

mixture and stirred at room temperature for 2 hours. 360 mg (6 mmol) AcOH was added and

stirred at room temperature for 1 hour. The saturated aqueous NaHCO3 was added and

extracted with ethyl acetate three times. The organic extract was dried over anhydrous Na2SO4,

filtered and concentrated under reduced pressure. The residue was purified by coloumn

chromatography on silica gel using heptane: ethylacetate 1:1 as eluent to give 17 mg (22%) of

compound 10b as yellow foam.

1H NMR (400 MHz, CDCl3) 8.20 (d, J=1.3, 1H), 7.66 (d, J=1.3), 5.51 (dd, J=7.5, 1.5 Hz,1H), 3.78

(s,3H), 3.6 (dd, J=7.5, 11.5, 1H), 3.48 (dd, J=11.5, 1.5 Hz, 1H), 1.78-1.60 (m, 1H), 0.91-0.79 (m,

2H), 0.76-0.62 (m, 2H); 13C NMR (400 Mz,CDCl3) 169.3, 157.8, 143.9, 136.3, 134.0, 125.4, 117.5,

104.4, 60.9, 53.1, 31.9, 10.7, 5.98, 5.6

18

8-Cyclopropyl-4,5-dioxo-2,3,4,5,6,7-hexahydro-1-thia-3a,6-diaza-s-indacene-3-carboxylic acid

methylester (12)

Compound 6 (32.7 mg, 0.1 mmol) of, and NaN3 (7.1 mg, 0.11 mmol) were dissolved in 1 ml 1:1

mixture of MeOH and MeCN. The reaction mixture was heated with microwave irradiation at

100oC for 30 min and then concentrated under reduced pressure. The residue was absorbed

onto silica and purified with silica gel chromatography using CH2Cl2 : MeOH 95:5 as eluent to

give 19 mg (62%)of compound 12 as yellow color solid.

[α]D -120 (c 0.5,CHCl3+CH3OH); IR λ 1694, 1664, 1594, 1504, 1265, 1168, 771, 695; 1H NMR (400

MHz, CDCl3+MeOD-d4) 5.37 (d, J=8.8,1H), 4.14-3.99 (m, 2H), 3.58-3.49 (m, 1H), 3.47 (s, 3H), 3.36

(d, 11.5 Hz, 1H), 1.37-1.27 (m,1H), 0.65-0.51 (m,2H), 0.39-0.22 (m, 2H); 13C NMR (400 MHz,

CDCl3+MeOD-d4) 174.5, 172.3, 167.4,160.9, 159.2, 118.4, 114.2, 66.7, 57.0, 48.5, 35.9, 13.0, 9.6,

9.4

9-Cyclopropyl-6-methyl-4-oxo-7-phenyl-2,3-dihydro-4H-1-thia-3a,6-diaza-cyclopenta[b]naphthalene-

3-carboxylic acid methyl ester (15)

Compound 6 (30 mg, 0.092 mmol) and) benzaldeyhde (15 mg, 0.14 mmol) were dissloved in a

1:1 mixture of 1.5 ml acetonitrile and methanol. Then methanolic methylamine (1.6 M) (0.14

ml, 0.22 mmol) was added followed by acetic acid (15 eq) and the solution was heated with

microwave irradiation at 80oC for 10 minutes and then concentrated under reduced pressure.

The residue was absorbed onto silica and purified with silicagel chromatography using CH2Cl2:

MeOH 95:5 as eluent to give 29 mg (74%) of compound 15 as yellow foam.

[α]D -481 (c 0.5,CHCl3); IR λ 1739, 1649, 1586, 1354, 1319, 854, 761; 1H NMR (400 MHz,

CDCl3+MeOD) 9.15 (s, 1H), 7.74 (s,1H), 7.41-7.32 (m, 3H), 7.30-7.24 (m, 2H), 5.53 (dd, J=8.7, 1.6

Hz,1H), 3.08 (s, 3H), 3.66 (dd, J=8.6, 12.0 Hz, 1H), 3.44 (dd, J=1.6, 12.0 Hz,1H), 1.52-1.44 (m,1H),

0.89-0.75 (m, 2H), 0.47-0.35 (m,2H); 13C NMR (400 MHz, CDCl3+MeOD-d4) 169.2, 162.0, 159.1,

155.9, 149.6, 149.3, 133.0, 132.9, 130.9 (2C), 130.2 (2C), 122.8, 120.4, 110.0, 64.6, 54.8, 47.6,

33.2, 10.5, 8.8, 8.7

9-Cyclopropyl-4-oxo-7-phenyl-2,3-dihydro-4H-1-thia-3a,6-diaza-cyclopenta[b]naphthalene-

3-carboxylic acid methyl ester (16)

Compound 6 (50 mg, 0.15 mmol), benzaldehyde 40 mg (0.38 mmol), and ammonium acetate 47

mg (0.61 mmol) were dissloved in a 1.1 ml acetonitrile and 1.1 ml methanol. Acetic acid 0.13

ml (2.3 mmol) was added and the reaction mixture was heated with microwave irradiation at

100oC for 20 minutes. The mixture was diluted with dichloromethane and washed with

19

saturated aqueous NaHCO3. The aqueous phase was extracted with CH2Cl2 and the organic

extract was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue

was purified coloumn chromatography on silica gel using heptane : ethyl acetate 2:1 as eluent

to give 35 mg (61%) of compound 16.

[α]D -220 (c 0.5,CHCl3); IR λ 1755, 1655, 1591, 1262, 1171, 769, 695. 1H NMR (400 MHz, CDCl3)

9.52 (s, 1H), 8.15-8.12 (m, 1H), 8.11-8.05 (m, 2H), 7.54-7.43 (m, 3H), 5.96 (dd, J=2.0, 11.6 Hz,

1H), 3.8 (s, 3H), 3.71 (dd, J=11.6, 8.4 Hz, 1H), 3.55 (dd, J=2.08, 11.63), 1.80-1.70 (m, 1H), 1.17-

1.04 (m,2H), 0.78-0.68 (m,2H); 13C NMR (400 Mz,CDCl3) 168.6, 160.5, 159.2, 151.4, 146.5, 144.9,

139.1, 129.6, 128.8 (2C), 127.5 (2C), 117.4, 112.5, 109.0, 62.3, 53.5, 31.6, 9.6, 7.3 (2C)

7-Chloromethyl-8-cyclopropyl-6-hydroxymethyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-

3-carboxylic acid methyl ester (17)

Compound 6, 98 mg (0.3 mmol) was dissolved in 4ml, 1:1 acetonitrile and methanol. Sodiumborohydride, 13 mg (0.35 mmol) was added and the mixture was stirred at room temperature for 15 minutes and then quenched by addition of 1 M aqueous hydrochloric acid. the mixture was extracted with CH2Cl2 and the organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica gel using heptane : ethyl acetate 1:2→1:4 as eluent to give 78 mg (80%) of compound 17. [α]D -244 (c 0.5,CHCl3); IR λ 3418, 1744, 1625, 1569, 1477, 1226, 1147, 742, 602;

1H NMR (400 MHz, CDCl3) 5.57 (dd, J = 8.7, 2.2 Hz, 1H), 4.80-4.69 (m, 2H), 4.70-4.59 (m, 2H), 3.78 (s, 3H), 3.73-3.65 (m, 1H), 3.65 (dd, J = 11.8, 8.7 Hz, 1H), 3.48 (dd, J = 11.8, 2.2 Hz, 1H), 1.75-1.64 (m, 1H), 1.03-0.88 (m, 2H), 0.77-0.67 (m, 2H); 13C-NMR (400MHz,CDCl3) 168.3, 161.7, 148.6, 147.5, 125.8, 113.3, 63.3, 57.8, 53.4, 38.3, 31.6, 11.1, 7.6, 7.2

General procedure for SN2 reaction between 17 and primary amines

Compound 17 (1.0 eq) and K2CO3 (1.5 eq) was taken up in acetonitrile (30ml/ mmol of 17). The

amino compound (3.0 eq) was added and the reaction was stirred at room temperature over

night. Saturated aqueous NaHCO3 was added and the mixture was extracted with CH2Cl2. The

organic extract was dried over anhydrous Na2SO4, filtered and concentrated under reduced

pressure. The residue was purified by coloumn chromatography on silica gel.

8-Cyclopropyl-6-hydroxymethyl-7-methylaminomethyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyridine-3

-carboxylic acid methyl ester (18a)

Compound 17, 66 mg (0.20 mmol) was converted to 38 mg (59%) of compound 18a by

following the above general procedure, in this reaction methylamine was used as an amino

20

reagent. To purify the compound 18a, CH2Cl2: MeOH 95: 5 was used as eluent in the

chromatography.

[α]D -310 (c 0.5,CHCl3); IR λ 3295, 1733, 1635, 1490, 1477, 1351, 1218, 792, 649; 1H NMR (400

MHz, CDCl3) 5.61 (dd, J=8.5,2.5 Hz,1H), 4.71 (d, J=12.2 Hz, 1H), 4.55 (d,J=12.2 Hz, 1H),4.02 (d,

J=11.5 Hz,1H), 3.79 (s,3H), 3.65 (dd, J=8.5,11.5 Hz,1H), 3.47 (dd, J=11.5, 2.5Hz,1H), 3,29 (bs,2H),

1.66 (m,1H), 0.95 (m,1H), 0.63(m,1H); 13C-NMR (400MHz, CDCl3) 169.1, 161.8, 152.5, 147.2,

126.6, 114.3, 63.9, 57.7, 53.9, 50.2, 36.8, 31.9, 12.1, 8.3, 7.9.

7-(Benzylamino-methyl)-8-cyclopropyl-6-hydroxymethyl-5-oxo-2,3-dihydro-5H-thiazolo[3,2-a]

pyridine-3-carboxylic acid methyl ester (18b)

Compound 17, 70 mg (0.21 mmol) was converted to 54 mg (63%) of compound 18b by

following the above general procedure, in this reaction benzyl amine was used as an amino

reagent. To purify the compound 18b, CH2Cl2: MeOH 95:5 was used as eluent in the

chromatography.

[α]D -193 (c 0.5,CHCl3); IR λ 3306,1745, 1627, 1497, 1452, 1207, 1173, 739, 697; 1H NMR (400

MHz, CDCl3) 7.39-7.32 (m, 4H), 7.31-7.26 (m,1H), 5.60 (dd, J=2.5, 8.6 Hz, 1H), 4.72 (d, J=12.4,

1H), 4.58 (d, J=12.4, 1H), 4.01 (d, J=11.5 Hz, 1H), 3.94-3.86 (m, 3H), 3.80 (s, 3H), 3.64 (dd,

J=11.6, 8.7 Hz, 1H), 3.47 (dd, J=11.6, 2.5 Hz, 1H), 1.61-1.53 (m, 1H), 0.84-0.76 (m, 2H), 0.62-0.52

(m, 2H); 13C-NMR (400MHz, CDCl3) 168.6, 161.2, 151.9, 146.7, 138.9, 128.6(2C), 128.4 (2C),

127.4, 127.0, 133.8, 63.4, 57.2, 54.3, 53.2, 46.9, 31.5, 11.5, 7.7, 7.3

General procedure for carbamate formation

The aminoalcohol (1.0 eq ) and triethylamine (6.0 eq) were dissolved in CH2Cl2 (50ml / mmol of

amino alcohol). Triphosgene (0.5) was added and the solution was stirred at room temperature

for one hour. Saturated aqueous NaHCO3 was added, the reaction mixture was extracted with

CH2Cl2. The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under

reduced pressure. The residue was purified with silica gel chromatography.

21

10-Cyclopropyl-8-methyl-4,7-dioxo-2,3,5,7,8,9-hexahydro-4H-6-oxa-1-thia-3a,8-diaza-cyclohepta[f]

indene-3-carboxylic acid methyl ester (19a)

Amino alcohol 19a (49 mg, 0.15 mmol) was converted to 30 mg (57%) of compound 20a by

using the above general procedure. Inorder to purify the compound 19a Heptane: ethyl acetate

1:3 was used as eluent in the chromatography.

[α]D -255 (c 0.5,CHCl3); IR λ 1710, 1639, 1588, 1512, 1351, 1214, 1094, 746, 701; 1H NMR (400

MHz, CDCl3) 5.50 (dd, J=8.5, 2.3 Hz, 1H), 5.07-4.95 (m, 2H), 4.58-4.44 (m, 2H), 3.74 (s, 3H), 3.61

(dd, J=8.6, 11.5 Hz, 1H), 3.44 (dd, J=11.5, 2.4 Hz), 3.00 (s, 3H), 1.56-1.47 (m, 1H), 1.00-0.87 (m,

2H), 0.60-0.51 (m, 2H); 13C-NMR (400 MHz, CDCl3) 168.3, 159.1, 158.8, 147.3, 146.4, 120.7,

111.8, 66.5, 62.9, 53.3, 48.8, 38.0, 31.6,10.8, 8.1, 8.0

8-Benzyl-10-cyclopropyl-4,7-dioxo-2,3,5,7,8,9-hexahydro-4H-6-oxa-1-thia-3a,8-diaza-cyclohepta[f]

indene-3-carboxylic acid methyl ester (19b)

Amino alcohol 19b, 46 mg (0.11 mmol) was converted to 30 mg (61%) of compound 20b by

using the above general procedure. Inorder to purify the compound 19b CH2Cl2: MeOH 95 : 5

was used as eluent in the chromatography.

[α]D -196 (c 0.5,CHCl3); IR λ 1744, 1711, 1632, 1586, 1507, 1211, 743; 1H NMR (400 MHz, CDCl3)

7.37-7.22 (m, 5H), 5.58 (dd, J=8.5,2.4 Hz,1H), 5.25-5.12 (m, 2H), 4.72-4.43 (m, 4H), 3.83 (s, 3H), 3.66 (dd, J=11.7, 8.6 Hz, 1H), 3.50 (dd, J=2.4, 11.7 Hz, 1H), 1.13- 1.05 (m, 1H), 0.83-0.73 (m, 2H), 0.48-0.33 (m,2H); 13C-NMR (400 MHz, CDCl3) 168.3, 159.3, 158.9, 147.7, 146.2, 136.3, 126.7(2C), 128.2 (2C), 128.0, 120.5, 111.8, 66.7, 62.9, 53.5, 53.3, 45.8, 31.6, 10.4, 7.9, 7.6.

8-Cyclopropyl-2-oxo-6,11-dithia-3,14-diaza-tricyclo[7.6.0.03,7

]pentadeca-1(9),7-diene-4,

13-dicarboxylic acid dimethyl ester (20)

Compound 6 (65mg, 0.2 mmol) and K2CO3 (27 mg, 0.2 mmol) were taken up in 1:2 mixture of

1.8 ml methanol and acetonitrile. To this mixture N-boc protected cysteinmethyl ester (0.2

mmol) was added and stirred at room temperature for 2 hours. Then the reaction was washed

with NaHCO3 and extracted with CH2Cl2. The extract was dried over anhydrous Na2SO4, filtered

and concentrated under reduced pressure. The residue was dissolved in 1 ml CH2Cl2 and 1 ml of

trifluroaceticacid was added and stirred at room temperature for 10 min. the solvent was

removed under reduced pressure. The residue was dissolved in 2ml methanol and 110 mg (15

mmol) of sodium borohydride was added, stirred at room temperature over night. Diluted with

CH2Cl2 and washed with saturated aqueous NaHCO3 and extracted with CH2Cl2. The organic

phase was dried with anhydrous Na2SO4 the compound was purified by using reverse phase

HPLC (30%H20, 70%MeCN, 0.1%TFA, 25 min). The purified compound dissolved in 5 ml

22

methanol and treated 1ml The amberlite® IR-120(plus) ion exchange resin for 5 min and filtered and evaporated the solvent under reduced pressure gave 18 mg (22%) of 2:1

diastereomeric mixture of compound 20.

[α]D -40 (c 0.5,CHCl3); IR λ 1733, 1629, 1576, 1576, 1501, 1206, 1173, 823, 739; 1H NMR (400

MHz, CDCl3) 5.62 (maj) &5.59 (min) (dd, j=8.6,2.5 Hz, 1H), 4.20-3.85 (m,4H), 3.80 (min) & 3.79

(maj) (s, 3H), 3.77-3.73 (m,1H), 3.71 (s,3H),3.68-3.60 (m, 1H),3.51-3.45 (m, 1H),3.09 (min) &

3.06 (maj) (dd, J=15.8, 2.2 Hz,1H), 2.87 (min) &2.79 (maj) (dd, J=15.8, 5.6 Hz, 1H),2.47

(s,1H),1.70-1.58 (m, 1H), 1.00-0.86 (m, 2H),0.85-0.76 (m, 1H), 0.71-0.62 (m,1H); 13C-NMR (400

MHz, CDCl3) 172.8, 168.7 (min), 168.6 (maj), 160.7, 150.9 (min), 149.9 (maj), 146.3 (min), 146.2

(maj), 123.3 (maj)122.6 (min),112.5, 63.9 (maj), 63.6 (min), 63.3 (min), 63.2 (maj), 53.3, 52.4,

42.4 (maj), 42.1 (min), 32.9 (min), 32.8 (maj), 31.5 (maj),31.4 (min), 28.9 (min), 28.6 (maj), 11.3,

7.9,7.8(min), 7.5 (maj)

23

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