Synthesis and Biological Evaluation of new

Synthesis and Biological Evaluation of New Tetrahydrobenzothiophene Derivatives

Lianpao Wu

Student

Degree Thesis in Chemistry 30 ECTS

Master’s Level

Supervisors: Fredrik Almqvist, Dang The Hung

Examiner: Andreas Larsson

Department of Chemistry

Umeå university

Abstract

In a high throughput screening of 17,500 compounds, two hit compounds containing a

tetrahydrobenzothiophene core were identified as interesting biofilm inhibitors of

Escherichia Coli UTI89.Therefore, a small library of this type of compounds has been

synthesized and tested for their activity against the biofilm formation of this bacteria

to explore their structure activity-relationships (SARs) for further study.

The small library of novel class of tetrahydrobenzothiophene compounds included

amide, sulfonamide and sulfonylurea derivatives. Thirteen compounds have been

successfully synthesized from methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]

thiophene-3-carboxylate, which was prepared from Gewald three-component reaction.

From biological data, compounds 4 and 8 were shown to have significant antibiofilm

activity, and preliminary information on the structure activity-relationships of this

class of compounds has been obtained for further investigation.

Keywords

Tetrahydrobenzothiophene, biofilm, Escherichia Coli, structure activity-relationships,

antibacterial, Gewald reaction

II

III

List of abbreviations Å ångström

Ac acetyl

Ar aryl

Boc tert-butoxycarbonyl

Boc2O di (tert-butyl) dicarbonate

DBU 1,8-diazabicyclo[5.4.0]undecene-7

DCC N, N'-Dicyclohexylcarbodiimide

DMF Dimethylformamide

DMSO Dimethyl sulfoxide

e.g. for example

equiv. equivalent(s)

et al. et alii (Latin for “and others”)

etc. et cetera (Latin for “and the rest”)

Et ethyl

h Hours

IR Infrared

LC-MS liquid chromatography-mass spectrometry

Me methyl

MeCN acetonitrile

mL millilitre

mM millmole

MWI Microwave irradiation

NMR Nuclear Magnetic Resonance

Ph phenyl

rt. Room temperature

SAR structure activity-relationship

TBTU O-(Benzotriazol-1-yl)-N, N, N′, N′-tetramethyluronium

tetrafluoroborate

t-Bu tert-butyl

TEA Triethylamine

TFA Trifloroacetic acid

THF Tetrahydrofuran

TLC Thin layer chromatography

UPEC uropathogenic Escherichia Coli

IV

V

Table of contents

1. Introduction ................................................................................................................ 1

1.1 Antibiotic resistance of bacteria and biofilm formation ...................................... 1

1.2. Identification of two hits against biofilm formation ........................................... 2

1.3. Application of Gewald reaction to the synthesis of tetrahydrobenzothiophene

scaffold ............................................................................................................... 2

1.4. Amide bond formation by TBTU ....................................................................... 3

1.5. Objectives ........................................................................................................... 4

2. Results and Discussion .............................................................................................. 5

2.1. Chemistry ............................................................................................................ 5

2.1.1. Modifications of the functional group at the C-2 position ........................... 5

2.2.2. Modifications of the functional group at the C-3 position ........................... 8

2.2. Biological evaluation ........................................................................................ 11

3. Conclusion and future perspectives ......................................................................... 13

5. Acknowledgement ................................................................................................... 14

6. Experimental Section ............................................................................................... 15

7. References ................................................................................................................ 26

VI

1

1. Introduction

1.1 Antibiotic resistance of bacteria and biofilm formation

The war between human and bacteria is still continuing. Nowadays, infectious

diseases are one of the biggest threats to human health.[1,2]

One key problem is the

bacterial evolution of resistance to antibacterial agents. For example when the first

antibiotic sulfonamide was developed in 1930s it saved a large number of people’s

lives. However, the bacteria resistant to sulfonamide appeared about ten years later.

The same thing happened to many antibiotic drugs (i.e. penicillin, streptomycin,

methicillin and linezolid) which were developed decades later.[3]

To win the war,

several approaches for antimicrobial therapy have been studied. It has been mentioned

that targeting bacterial virulence is one of the promising methods. Compared to

traditional antibiotic strategies, which inhibit bacterial growth or kill bacteria, this

approach can slow down the resistance processes, since it decreased the selective

pressure which promotes the growth of antibiotic resistant bacterial strains. In

addition, it has no or little influence on the normal human microbiota.[4-6]

Bacterial adhesion is one of the virulence properties of almost all microbes.[7,8]

Gram

negative bacteria like uropathogenic Escherichia Coli (UPEC) can produce pili and

curli, which help UPEC adhere to host cells and biofilm formation, related to the

recurrent infections and antibiotic resistance.[9-13]

Biofilms are thin layers of microorganisms and can be formed on biotic surfaces (such

as plants and animals) or on abiotic surfaces (such as minerals and surface of dead

organisms). They have their own communication and defense systems.[14,15]

The

biofilm formation is very complex, including five stages as shown in Figure 1.[16]

Many infectious diseases, such as endocarditis and urinary tract infection, are

persistent infection due to the biofilm formation.[5]

Therefore, the quest for novel

biofilm inhibitors is still rapidly growing.

Figure1. Five stages involved in the formation of biofilm. (A) Initial reversible attachment on

solid surface. (B) Other bacteria continued binding to form matrix. (C) Maturation phase:

cells become layered and effects of quorum sensing begin. (D) Clusters reach maximum

thickness. (E) Planktonic bacteria released from matrix dispersion.[16]

2

1.2. Identification of two hits against biofilm formation

Through a high throughput screening of 17,500 compounds, two hits (A and B)

containing a tetrahydrobenzothiophene core were found to have significant activity

against the formation of biofilm of Escherichia Coli UTI89 (Figure 2).

Figure 2. Chemical structures of two hits compounds.

1.3. Application of Gewald reaction to the synthesis of

tetrahydrobenzothiophene scaffold Since discovered in 1961, the Gewald reaction has become an useful method for the

synthesis of the 2-aminothiophene scaffold.[17]

This three-component reaction

involved condensation of a carbonyl compound, an activated nitrile and elemental

sulfur in the present of a base in alcohol or DMF (Scheme 1). Besides the classical

method, several modified approaches (such as solid-supported or microwave

accelerated Gewald reaction) have also been developed.[18,19]

Scheme 1. A general synthesis of 2-aminothiophene scaffold derived from Gewald reaction.

Although the full mechanism of Gewald reaction is not very clear [18]

the most

plausible mechanism is a base-promoted mechanism (Scheme 2).[19]

It involves four

main steps as shown below.

3

Scheme 2. The plausible mechanism of Gewald three-component reaction.

The first step is a Knoevenagel condensation between a carbonyl compound and an

activated nitrile to produce intermediate e. The second step is an addition of elemental

sulfur in the present of a base to give intermediate g. The third step is an intra-

molecular ring closure reaction. Nucleophilic attack by sulfur to the triple bond of the

cyano group give the cyclic imino product i, which in principle has equilibrium with

the tautomeric form 2-aminothiophene j. It was proved that the 2-aminothiophene

occurs exclusively in the amino form.[20-22]

1.4. Amide bond formation by TBTU

Amide functional group plays an important role in medicinal chemistry and biological

chemistry. Generally, it can be directly formed from amines and activated carboxylic

acids. There are myriad coupling reagents (such as DCC, TBTU, etc.) that can be used

to convert the carboxyl group to more reactive functional group such as anhydride,

acyl halide or active esters.[23,24]

TBTU (O-(Benzotriazol-1-yl)-N, N, N', N'-

tetramethyluronium tetrafluoroborate) is one of the less expensive coupling reagents,

which has been widely used in the amide bond formation.[25]

The mechanism of this reaction included several steps: addition of the carboxylate to

TBTU followed by decomposition of the intermediate and addition of the released

anion d to the carbonyl center to give an urea derivative and an activated ester g, and

finally addition of an amine to the activated ester g to form the amide h (Scheme

3).[24]

4

Scheme 3. The proposed mechanism of amide bond formation catalyzed by TBTU.

1.5. Objectives

Regardless of the functional groups at C-2 and C-3 positions, a preliminary study on

analogs of hits A and B showed that derivatives derived from hit A displayed higher

activity than those derived from hit B. Therefore, based on the structure of hit A,

several analogs with C-2 and C-3 modifications have been synthesized and

biologically evaluated to obtain information on the relationship of their structure and

activity against biofilm formation of Escherichia Coli UTI89 for further study.

Scheme 4. Synthesis of methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]thiophene-3-

carboxylate via Gewald reaction. Reagents and conditions: (a) Et2NH, S8, MeOH, room

temperature, 23 h, 82%.

The core scaffold methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo[b]thiophene-3-

carboxylate 3 was synthesized via Gewald three-component reaction (Scheme 4).

From 3, thirteen analogs (A, 4-8, 10-11, 13, 15-18) have been synthesized (Figure 3).

5

Figure 3. Structures of target compounds.

2. Results and Discussion

2.1. Chemistry

2.1.1. Modifications of the functional group at the C-2 position

Five compounds 4, 5, 6, 7 and hit A were successfully synthesized from compound 3

and appropriate acid anhydrides (Scheme 5).[26]

Though the reactivity of the amine

group at C-2 position is quite weak due to its conjugation with the aromatic core

skeleton, the reactions still worked very well with a set of reactive anhydrides. As

shown in table 1, the yields of all compounds are higher than 65%.

6

Scheme 5. Modifications of hit A at the C-2 position. Reagents and conditions: (a) 1.0 equiv.

appropriate acid anhydrides, CH2Cl2, refluxed under N2; (b) NH3 , MeOH, 0 ºC → 110 ºC

(MWI), 1 h, 91%; (c) 2.5 equiv. methanesulfonyl chloride, 2.0 equiv. TEA, CH2Cl2, 0 ºC, 20

min, 96%; (d) 2.0 equiv. sodium methoxide, MeOH, 0 ºC→ 40 ºC overnight, 74%; (e) 1.0

equiv. benzenesulfonyl chloride, 2.0 equiv. TEA, CH2Cl2, 0 ºC→ 50 ºC, 2 days, 47%; (f) 1.0

equiv. 4-methyl benzenesulfonyl cyanide, 1.0 equiv. TEA, toluene, 110 ºC oil bath, 20 h, 75%.

Table 1. Variation of anhydrides in the synthesis of compounds 4-7 and hit A.

Entry Anhydride R2 Product Yield (%)

1

4 69%

2

5 98%

3

6 68%

4

7 65%

5

A 89%

7

Since the anhydride for synthesis of compound 8 is not available at the same reaction

condition as mentioned above, other synthetic pathway was adopted (Scheme 6).

Scheme 6. Synthetic route to compound 8. Reagents and conditions: (a) Ether, 0 ºC to room

temperature, overnight; (b) TEA, CH2Cl2, 0 ºC to room temperature, 5 min, 76%; (c) TFA,

CH2Cl2, 0 ºC to room temperature, 21 h, 60%.

Compound 8 was synthesized via a synthetic sequence shown in scheme 6. First, the

reaction between oxalyl chloride and tert-butanol was done to give intermediate 21.

Then treatment of compound 3 with the intermediate 21 provided compound 3c,

which was treated with CF3COOH to afford compound 8 in 60 % yield after

recrystalization with methanol.[27]

The anhydride for synthesis of compound 9 needs a very hard condition and it is not

stable at room temperature (decomposes to CO2 and ketene),[28,29]

so compound 9 was

attempted to be synthesized from propanedioyl dichloride and tert-butanol. However,

the synthesis of the tert-butyl 3-chloro-3-oxopropanoate intermediate was not

successful due to a possible reason that the propanedioyl dichloride is quite unstable

and very reactive. On the other hand, direct coupling of compound 3 and

propanedioyl dichloride in a base solution also failed. Hence, another synthetic

pathway which was reported by Ju-Yeon Lee et al[30]

was tried (Scheme 7). However,

no product was formed, probably due to the low reactivity of the amine group within

compound 3 compared to aniline in the reported method.

Scheme 7. Synthetic route to compound 9. Reagents and conditions: Meldrum’ acid, Xylene

refluxed overnight.

8

Scheme 5 also shows the pathway for synthesis of target compound 10. The methanol

solution of starting material 4 was saturated with ammonia gas, and this solution was

heated at 110 ºC by microwave for 1 h to give target compound 10 in excellent yield

91%.

To investigate the importance of a base on mesylation reaction of 3, several bases

were tried,[31,32]

but the dimesylated product was observed. Thus, several conditions

were tried (Table 2), but only small amount of the product from entries 3, 4 and 5

were observed.

Table 2. Screening different bases for synthesis of compound 11.

Entry Base Reaction conditions (a) Results

1 TEA CH2Cl2, rt, 30 min dimesylation

2 pyridine CH2Cl2, rt, overnight dimesylation

3 pyridine CH2Cl2, 0 ºC , 9 h mesylation and dimesylation

4 DBU CH2Cl2, -40 ºC →0 ºC, 30 min mesylation and dimesylation

5 TEA CH2Cl2, -40 ºC, 2 h→0 ºC, 0.5 h mesylation and dimesylation

Due to difficulty in purification and low yield of product, another reaction condition

was tried (Scheme 5). To completely convert the starting material to the disubstituted

product, 2.5 equivalent of methanesulfonyl chloride was used for the reaction. Then,

the dimesylated product was mono cleavaged with sodium methoxide and good yield

(74%) of compound 11 was obtained.[33]

The synthesis of compound 12 via treatment of 3 and benzenesulfonyl chloride in the

present of different bases (e.g. TEA, pyridine and sodium carbonate) was also tried.

However, only disubstituted product 3b was obtained (Scheme 5) by using TEA. On

the other hand, a lot of starting material was observed when pyridine or sodium

carbonate was employed in the reaction. This result shows that this reaction is a

thermodynamic control reaction, and the dimer form is the more stable product.

The diarylsulfonylurea 13 was obtained in good yield (75%) by overnight reflux of 4-

methyl benzenesulfonyl cyanide, compound 3 and TEA in toluene.[34]

2.2.2. Modifications of the functional group at the C-3 position

To synthesize compound 14, several reducing agents such as LiAlH4, NaBH4 and

LiBH4 were tried to find out the best one for direct reduction of compound 4[35-38]

(Scheme 8). However, no desired product was obtained and many by-products were

observed. The reduction was also tried through other synthetic pathways [36, 39, 40]

such

as reduction of 3, 3d, 3f and 3g by different conditions, but no desired products were

obtained (Scheme 8).

9

Scheme 8. Synthesis of compound 14. Reagents and conditions: (a) 1.0 equiv. succinic

anhydride, CH2Cl2, refluxed 28 h, 69%; (b) (i) 4.0 eq NaBH4, 4.0 eq MeOH, 0.1 eq

NaB(OAC)3H, THF, 0 ºC → rt, 19 h or (ii) 1.5 eq LiBH4, 1.5 eq MeOH, Et2O, refluxed, 21 h

or (iii) 10.0 eq LiBH4, 1.5 eq MeOH, THF, 65 ºC (MWI), 45 min or (iv) 2.0 eq LiAlH4 (10%

in THF), THF, 0 ºC → rt , 4 h or (v) 2.0 eq LiAlH4 (10% in THF), THF, 65 ºC (MWI), 45

min; (c) 10.0 equiv. 10.0 M NaOH (aq), THF: MeOH: H2O= 5:2:10 refluxed overnight; (d)

4.0 equiv. LiAlH4 in THF (2.0 M), THF, 0 ºC→ 65 ºC (MWI), 2 h; (e) 9.0 equiv. BH3 in THF

(1.0 M), THF, 0 ºC→ 65 ºC (MWI) 1 h; (f) 2.1 equiv. Boc2O, 0.1 equiv. DMAP, dioxane, 80

ºC, 3 h then 3.0 equiv. N2H4.H2O, 40 ºC for 1.5 h, 81%; (g) 2.0 equiv. LiAlH4 in THF (2.0 M),

THF, 0 ºC→ 65 ºC (MWI), 1 h; (h) 3.0 equiv. BH3 in THF (2.0 M), 29 h.

To the best of my knowledge, there is only one patent by Gillen, Kevin James and his

co-workers,[41]

describing synthesis of the primary alcohol compound whose system is

similar to compound 14. The synthetic route used in the patent was adopted with

some modifications (Scheme 9). In order to prepare the aldehyde compound 3l, the

amino group of the starting compound 3 was first protected by an acetyl group,[42]

then followed by hydrolysis 3j, decarboxylation 3k and formylation 3l. However,

when compound 3k was treated with Vilsmeier reagent, an intramolecular ring

closure reaction was observed to give by-product 3m, so the aldehyde 3l could not be

obtained.

10

Scheme 9. Another synthetic pathway for compound 14. Reagents and conditions: (a) 2.5

equiv. acetyl chloride, pyridine, CH2Cl2, 0 ºC → rt, 85%; (b) 10.0 equiv. 10.0 M NaOH (aq),

THF: MeOH: H2O= 5:2:10 refluxed 9 h, 93%; (c) 1.5 equiv. Cu, quinoline, MWI 200 ºC, 10

min, 54%; (d) 1.0 equiv. DMF, 1.0 equiv. POCl3, CH3CH2Cl2, rt → 85 ºC 3 h, then

CH3COONa (aq), 1.5 h, 19%.

The target amide compounds (15-18) were synthesized from compound 3 via a

synthetic sequence including N-Boc protection, hydrolysis, amide bond formation,[43]

N-Boc-deprotection[44]

and acylation (Scheme 10). To ensure complete carbamolyation

of compound 3, two more equivalent of di-tert-butyl dicarbonate are required since

one equivalent of di-tert-butyl dicarbonate resulted in a mixture of starting material,

mono-Boc and di-Boc products. Because compound 3n is sensitivity to nucleophiles,

one of the carbamate groups as well as any excess Boc2O could be converted to tert-

butyl hydrazinecarboxylate by adding excess hydrazine. Moreover, the by-product

tert-butyl hydrazinecarboxylate could be easily removed by silica column, and

compound 3f was achieved in very good yield (81% from 3).[43]

In the coupling reaction either aliphatic or aromatic amines were used in the present

of TEA and the amide coupling reagent TBTU. The yield of some products is quite

low. It is possibly due to the low reactivity of the acid at C-3 position, induced by the

amine group at C-2 position. The amine group is an excellent electron donating group

that decreases the electronpilicity of the carbonyl carbon at C-3 position. The reaction

rate was in the order of morpholine > isopropylamine > aniline > benzyl amine at the

same reaction condition, possibly due to different nucleophilicity.

11

Scheme 10. Synthetic pathway of the target compounds 15-18. Reagents and conditions: (a)

2.1 equiv. Boc2O, 0.1 equiv. DMAP, dioxane, 80 ºC, 3 h; (b) 3.0 equiv. N2H4.H2O, 40 ºC for

1.5 h, 81% (from 3); (c) 10.0 equiv. 10.0 M NaOH (aq), THF: MeOH: H2O= 5:2:10 refluxed

overnight, 85%; (d) 1.0 equiv. TBTU, 1.0 equiv. 2.0 equiv. TEA, ethyl acetate, R1NH2, stirred

at rt; (e) 1.0 ml TFA , 1.0 ml CH2Cl2 stirred at rt; (f) 1.0 equiv. succinic anhydride, CH2Cl2,

refluxed 3 h-7 h 40 min.

Due to stability of 23b-23d, only 23a was purified after treatment with TFA. Finally

these deprotected products were converted to 15-18 by treatment with an equimolar

amount of succinic anhydride (Table 3).

Table 3. Variation of amines in the synthesis of target compounds 15-18.

Entry R1NH2 Product Yield (%)

1

15 27%

2

16 75%

3

17 30%

4

18 65%

2.2. Biological evaluation All the synthesized compounds were evaluated for their activity against biofilm

formation of Escherichia Coli UTI89 using a pili-dependent biofilm assay. In the

biofilm assay, the percentage inhibitory activity of all synthesized compounds was

measured at concentrations of 200 M, 100 M and 50 M. Subsequently,

compounds with good inhibition rate (> 50% at 50 M) were further tested at lower

12

concentrations (25, 12.5, 6.3 and 3.1 M) to determine their ED50 values. In addition,

a growth inhibition assay was also performed to validate that active compounds are

really true inhibitors of biofilm formation and not acting as bactericidal agents. The

results of this evaluation are outlined in table 4.

Table 4. Activity against the growth and biofilm formation of Escherichia Coli UTI89.

Comp.

No.

R1 R2 ED50 of biofilm formation (µM)

growth (µM)

A OMe

25 >50

4 OMe

15 >50

5 OMe

25 >50

6 OMe

25 >50

7 OMe

25 >100

8 OMe

12.5 >100

10 OMe

25 >100

11 OMe

No activity >100

13 OMe

25 >100

15

No activity >100

16

25 >100

17

No activity >100

18

No activity >100

Considering the modification of the cyclic acid group at C-2 position of hit A by other

acid moieties, the data showed that this transformation results in compounds (4-8)

with retained or improved activity. Among them compounds 4 and 8 displayed higher

inhibitory effect on biofilm formation than the hit A, suggesting that an acyclic acid

group is more favorable for the biological activity than the cyclic acid counterpart.

Also, a retained activity in the sulfonylurea analog 13 suggested that modification of

13

the acid moiety at C-2 position by an acid bioisostere strategy may provide potent

biofilm inhibitors.

Regarding the modification of the methyl ester at C-3 position by other amide groups

(15-18) while keeping an acyclic acid moiety constant, the data revealed that only

compound 16 derived from an aryl amine is retained activity compared to the hit A,

suggesting that few amide groups at C-3 position would be tolerated if further

modification on this position is considered.

3. Conclusion and future perspectives

Based on the structure of hit A, several tetrahydrobenzothiophene derivatives (A, 4-8,

10-11, 13, 15-18) have been successfully synthesized to investigate their preliminary

structure-activity relationships. An attempt to transform the ester group into a primary

alcohol was not successful, although several strategies have been employed. A

possible reason is due to a low reactivity of the methyl ester group located in a

conjugate system.

All the synthesized compounds were tested for their antibiofilm activity by evaluation

of their inhibitory effects on biofilm formation of Escherichia Coli UTI89 using a

pili-dependent biofilm assay. As expected, the preliminary information on their SARs

has been observed as a guidance for further investigation. Compared to hit A, several

biofilm inhibitors with improved activity (e.g. 4 and 8) have been investigated by a

simple replacement of the cyclic acid group by an acyclic acid moiety at the C-2

position.

The biological data of the synthesized compounds in the present study suggests that

further modification should focus on other ester groups at C-3 position or acid

bioisostere groups at C-2 position.

14

5. Acknowledgement

Herein I would like to express my deepest gratitude and heartfelt thanks to all the

people who have guided, supported and helped me in various ways during the past

two years.

Firstly, I sincerely thank my supervisor, Professor Fredrik Almqvist, for giving me

this interesting project to work with, for the advices at the beginning of this project

and for the discussions during this work.

Secondly, I would also thank my second supervisor, Dr. Dang The Hung, for all the

helpful advices and discussions and helps in the lab, for your patient guidance during

my thesis writing.

I am also grateful to:

All group members including Christoffer, Magnus, Munawar, Syam, Krishna,

Karl and Lina in the Almqvist’s group, thank you all for being good friends and for

pleasant time. A special thanks to Syam and Magnus for working with me in the lab

during many Saturdays.

The Study Administrator: Barbro and my previous Study Counselor: Bertil. Thank

you for helping me with all sorts of matters when I met during my study.

I wish to thank all my good friends and teachers in China for everlasting care and

support. I also want to thank all my lecturers, classmates and friends in Umeå for

making my days here colorful and memorable. I will keep this memory in my heart

forever.

Last, I would like to thank my parents, who encourage me all the time in my life, and

give me endless love and bless. And I also thank my brother for his supporting and

encouraging me all the time!

15

6. Experimental Section

Unless otherwise stated all the reactions were carried out in oven-dried glassware. All

solvents and reagents were commercially (Sigma-Aldrich, Fluka and Lancaster).The

solvents were dried over 4 Å molecular sieves. Infrared spectra were collected on a

Perkin-Elmer FT-IR Spectrometer. Analytical thin layer chromatography (TLC) was

performed on Merck Silica gel 60 F254 plates. Flash column chromatography was

performed on Sigma-Aldrich silica gel.60. Microwave reactions were carried out in

Biotage Initiator using Smith Process Vial TM

sealed with Teflon septa and an

aluminum crimp top. 1H NMR was recorded at 293 K and 400 MHz and

13C NMR

was recorded at 100 MHz. The spectra were calibrated using the residual peak of

solvent as internal standard [CDCl3 (CHCl3 δH 7.26 ppm, CDCl3 δC 77.1 ppm),

DMSO-d6 (DMSO-d5 δH 2.49 ppm, DMSO-d6 δC 40.0 ppm), MeOH-d4 (CD2HOD δH

3.31 ppm, CD3OD δC 49.0 ppm)].

Methyl 2-amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxylate (3)

To a stirring mixture of phenylcylohexanone (2.0 g, 0.02 mol), methyl cyanoacetate

(1.23 mL, 0.014 mol) and powdered sulfur (0.45 g, 0.014 mol) in methanol (10 mL),

diethylamine (0.72 mL, 0.007 mol) was added dropwise. The mixture was kept

stirring at RT for 22 h. The precipitate product was collected and washed with MeOH.

The mother liquid was recrystalized, a white solid 3 (2.9 g, 82%) was obtained. IR

3445, 3321, 1665 cm-1

; 1H NMR (400 MHz, CDCl3) δ 7.42-7.31 (m, 2H, Ar-H)

7.28-7.21 (m, 3H, Ar-H), 5.98 (bs, 2H, NH2), 3.83 (s, 3H, COOCH3), 3.08-2.93 (m,

2H), 2.85-2.62 (m, 3H), 2.17-2.06 (m, 1H), 1.99-1.85 (m, 1H); 13

C NMR (100 MHz,

CDCl3) δ 166.4, 162.1, 146.0, 132.2, 128.4 (2C), 126.9 (2C), 126.3, 117.0, 105.4,

50.6, 40.8, 32.4, 30.0, 27.2; LC-MS Rf (min) = 5.80, LC-MS m/z (ES+) calculated for

C16H18NO2S [M+H] 288: found 288.

The general procedure for synthesis of compounds A, 4-7

To a solution of compound 3 (208.0 mg, 0.81 mol) in dry CH2Cl2 (10 mL) was added

an equimolar amount of the appropriate acid anhydrides. The mixture was kept at

refluxing for 2~28 h under an atmosphere of nitrogen. The mixture was concentrated

under reduced pressure, and the solid was recrystalized from methanol.

Methyl 2-[[(6-carboxy-3-cyclohexen-1-yl)carbonyl]amino]-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (A)

The light yellow solid was recrystalized from methanol. A white solid A (81.8 mg,

89%) was obtained. IR 3252, 2913, 1691, 1657, 1558, 1243, 1206 cm-1

; 1H NMR

(400 MHz, DMSO-d6) δ 12.3 (bs, 1H, NH), 11.2 (s, 1H, COOH), 7.34-7.28 (m, 4H,

Ar-H), 7.24-7.18 (m, 1H, Ar-H), 5.69 (m, 2H, -CH2-CH=CH-CH2-), 3.84 (s, 3H,

COOCH3), 3.18-3.11 (m, 1H), 3.10-3.04 (m, 1H), 3.00-2.82 (m, 3H), 2.78-2.66 (m,

2H), 2.48-2.30 (m, 4H), 2.05-1.97 (m, 1H), 1.93-1.80 (m, 1H); 13

C NMR (100 MHz,

DMSO-d6) δ 174.7, 171.0, 166.2, 146.1, 130.5, 128.8 (3C), 127.3 (3C), 126.7, 126.2,

125.9, 124.9, 110.9, 52.2, 40.2, 39.5, 39.4, 31.8, 30.0, 26.6, 25.7; LC-MS Rf (min) =

5.87, LC-MS m/z (ES-) calculated for C24H24NO5S [M-H] 438: found 438.

16

Methyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


The light yellow solid was recrystalized from methanol. A white solid 4 (221.0 mg,

69%) was obtained. IR 3193, 3026, 1688, 1665, 1534 cm-1

; 1H NMR (400 MHz,

DMSO-d6) δ 12.25 (bs, 1H, COOH), 10.96 (s, 1H, NH), 7.33-7.18 (m, 5H, Ar-H),

3.82 (s, 3H, COOCH3), 3.00-2.81 (m, 3H), 2.77-2.65 (m, 4H), 2.60-2.53 (m, 2H),

2.04-1.95 (m, 1H), 1.91-1.79 (m, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 173.4,

169.1, 165.2, 146.3, 145.6, 130.1, 128.3 (2C), 126.8 (2C), 126.2, 125.5, 110.7, 51.5,

39.7, 31.3, 30.7, 29.5, 28.6, 26.1; LC-MS Rf (min) = 5.15, LC-MS m/z (ES-)

calculated for C20H20NO5S [M-H] 386: found 386.

Methyl 2-[(4-carboxybutanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


The product was purified by silica gel column chromatography using ethyl DCM:

MeOH= 98:2 (0.1% CH3COOH) → 97:3 (0.1% CH3COOH) as eluent. The solvents

were removed under reduced pressure and the solid was recrystalized from methanol.

Compound 5 (82.6mg, 98%) was obtained as a white solid. IR 3252, 2953, 1713,

1660, 1651 cm-1

; 1H NMR (400 MHz, CDCl3) δ 11.28 (s, 1H, NH), 7.35-7.28 (m, 2H,

Ar-H), 7.25-7.24 (m, 1H, Ar-H), 7.24 -7.19 (m, 2H, Ar-H), 3.86 (s, 3H, COOCH3),

3.04-2.87 (m, 3H), 2.83-2.70 (m, 2H), 2.60 (t, J = 7.40 Hz, 2H), 2.50 (t, J = 7.40 Hz,

2H), 2.16-2.03 (m, 3H), 1.96-1.83 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 178.5,

169.2, 167.0, 147.8, 145.7, 130.4, 128.5 (2C), 126.9 (2C), 126.4, 126.3, 111.1, 51.5,

40.5, 35.4, 32.9, 32.1, 30.1, 26.6, 20.6; LC-MS Rf (min) = 5.67, LC-MS m/z (ES-)


Methyl 2-[(2-carboxybenzoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


The product was purified by silica gel column chromatography using DCM: MeOH=

97:3 as eluent. The solvents were removed under reduced pressure and the yellow oil

was obtained. The yellow oil was recrystalized from methanol. Compound 6 (61.9

mg, 68%) was obtained as a yellow solid. IR 3025, 2917, 1660, 1561 cm-1

; 1H

NMR (400 MHz, CDCl3) δ 11.71 (s, 1H, NH), 8.06 (d, J = 3.76 Hz, 1H, Ar-H), 7.70-

7.62 (m, 2H, Ar-H), 7.62-7.56 (m, 1H, Ar-H), 7.37-7.30 (m, 2H, Ar-H), 7.29-7.27 (m,

1H, Ar-H), 7.25-7.20 (m, 2H, Ar-H), 3.84 (s, 3H, COOCH3), 3.08 -2.93 (m, 3H),

2.87-2.74 (m, 2H), 2.18-2.10 (m, 1H), 2.00-1.87 (m, 1H); 13

C NMR (100 MHz,

CDCl3) δ 170.0, 167.0, 165.6, 147.7, 145.7, 136.0, 132.7, 131.5, 130.8, 129.4, 128.5

(2C), 127.8, 127.0, 126.9 (2C), 126.4, 125.7, 112.0, 51.6, 40.5, 32.1, 30.1, 26.6; LC-

MS Rf (min) = 5.80, LC-MS m/z (ES-) calculated for C24H20NO5S [M-H] 434: found

434.

Methyl 2-[[(1E)-3-carboxy-1-oxo-2-propen-1-yl]amino]-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (7)

The yellow solid was recrystalized from methanol. Compound 7 (52.8 mg, 65%) was

obtained as a yellow solid. IR 3188, 2923, 2492, 1710, 1677, 1591 cm-1

; 1H NMR

(400 MHz, CDCl3) δ 14.5 (bs, 1H, NH), 12.1 (s, 1H, COOH), 7.37-7.30 (m, 2H, Ar-

H), 7.28-7.26 (m, 2H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 6.55-6.42 (d, J = 16.00 Hz, 2H,

17

-CH=CH-COOH), 3.93 (s, 3H, COOCH3), 3.10 -2.96 (m, 3H), 2.89-2.75 (m, 2H),

2.20-2.11 (m, 1H), 2.00-1.87 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 167.1, 163.8,

162.0, 145.2, 144.6, 131.8, 130.3, 129.6, 128.6 (2C), 126.8 (2C), 126.6, 114.5, 77.2,

52.1, 40.3, 32.2, 29.8, 26.5; LC-MS Rf (min) = 5.65, LC-MS m/z (ES-) calculated for

C20H18NO5S [M-H] 384: found 384.

Methyl 2-[(2-tert-butoxy-2-oxo-acetyl)amino]-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3c)

The mixture of butanol (148.2 mg, 2.00 mmol) and dry ether (1.0 mL) was cooled to 0

ºC and then the solution (cooled to 0 ºC) of oxalyl chloride (0.17 mL, 2.00 mmol) in

dry ether (4.0 mL) was added dropwise to the mixture. The resulting mixture was

stirred at 0 ºC for 20 min then stirred at RT overnight. The solvent was removed the

under reduced pressure, and colourless oil 21 was obtained. Then it was dissolved in

dry DCM (8.6 mL) to prepare 0.23M solution. Compound 3 (60.0 mg, 0.21 mmol)

and 58.0 µL TEA were mixed in dry DCM (5.0 mL), and then tert-butyl 2-chloro-2-

oxoacetate 21 (0.23 M, 1.0 mL) was added dropwise to the mixture at 0 ºC under N2.

The mixture was stirred at 0 ºC for 5 min and then washed with saturated NaHCO3

(10 mL) and extracted by DCM (3x20 mL).The organic phase was dried over Na2SO4

(s).The solvent was evaporated, and the residue was purified by silica gel column

chromatography using DCM: MeOH= 99:1 as eluent. The solvents were removed

under reduced pressure to give compound 3c (66.0mg, 76%) as yellow foam. IR

3241, 2915, 1754, 1729, 1693 cm-1

; 1H NMR (400 MHz, CDCl3) δ 12.4 (s, 1H,

NH), 7.39-7.28 (m, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H), 7.25-7.14 (m, 2H, Ar-H),

3.91 (s, 3H, COOCH3), 3.15-2.91 (m, 3H), 2.90-2.72 (m, 2H), 2.21-2.07 (m, 1H),

2.00-1.84 (m, 1H), 1.62 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz, CDCl3) δ 166.1,

158.2, 153.9, 145.6, 145.5, 131.5, 128.5 (2C), 128.1, 126.8 (2C), 126.4, 113.4, 85.3,

51.7, 40.5, 32.3, 29.9, 27.7 (3C), 26.6; LC-MS Rf (min) = 6.50, LC-MS m/z (ES-)


Methyl 2-[(carboxycarbonyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


The solution of compound 3c (63.0 mg, 0.15 mmol) in DCM (6.0 mL) was cooled to

0 ºC, and trifluoroacetic acid (2.0 mL) was added dropwise to the mixture. The

mixture was stirred at RT for 21 h, and then the solvents were evaporated under

reduced pressure and the residue was coevaprorated with Et2O (10x 10 mL) to give a

yellow solid crude which was recrystalized from MeOH. Compound 8 (33 mg, 60%)

was obtained as a yellow solid. IR 3233, 2936, 1765, 1675 cm-1

; 1H NMR (400

MHz, CDCl3) δ 12.5 (s, 1H, NH), 7.35-7.29 (m, 2H, Ar-H), 7.27-7.20 (m, 3H, Ar-H),

3.93 (s, 3H, COOCH3), 3.11-2.93 (m, 3H), 2.87-2.74 (m, 2H), 2.19-2.11 (m, 1H),

1.98-1.87 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 165.9, 158.5, 153.8, 145.3, 144.1,

132.1, 129.3, 128.6 (2C), 126.8 (2C), 126.5, 115.0, 52.0, 40.4, 32.3, 29.8, 26.5; LC-

MS Rf (min) = 5.39, LC-MS m/z (ES-) calculated for C18H16NO5S [M-H] 358: found

358.

Methyl 2-[[(3-aminocarbonyl)propanoyl]amino]-6-phenyl-4, 5, 6, 7-


18

A solution of compound 4 (60.0 mg, 0.21 mmol) in MeOH (5.0 mL) was bubbled

with NH3 (g) in a seal tube for 0.5 h until the solution became clear. The reaction

mixture was heated by microwave irradiation for 1 h at 110 ºC, and the solvent was

evaporated. The concentrated crude was washed with brine and extracted with ethyl

acetate (3x20 mL), dried over Na2SO4 (s), filtered and evaporated. The residue was

purified by silica gel column chromatography using MeOH: DCM= 5:95 (1% HAC)

as eluent. The solvents were evaporated under reduced pressure and compound 10

(54.8 mg, 91%) was obtained as a white solid. IR 3194, 3026, 2923, 1689, 1665,

1533 cm-1

; 1H NMR (400 MHz, CDCl3) δ 11.30 (s, 1H, NH), 7.35-7.29 (m, 2H, Ar-

H), 7.27-7.26 (m, 1H, Ar-H), 7.25-7.20 (m, 2H, Ar-H), 3.87 (s, 3H, COOCH3), 3.05-

2.88 (m, 3H), 2.71-2.85 (m, 6H), 2.17-2.08 (m, 1H), 1.96-1.85 (m, 1H); 13

C NMR

(100 MHz, CDCl3) δ 177.3, 168.3, 166.9, 147.7, 145.7, 130.5, 128.5 (2C), 126.9 (2C),

126.4, 126.3, 111.2, 51.5, 40.5, 32.1, 30.9, 30.0, 28.8, 26.6; LC-MS Rf (min) = 5.37,

LC-MS m/z (ES-) calculated for C20H21N2O4S [M-H] 385: found 385.

Methyl 2-[bis(methylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3a)

Compound 3 (60.0 mg, 0.21 mmol) and TEA (58.0 µL, 0.42 mol) was mixed in dry

DCM (2.5 mL), and the mixture was cooled to 0 ºC then a solution of

methanesulfonyl chloride (16.0 µL in 1.5 mL DCM) was added dropwise thereto.

After 20 min the solution turned slight yellow, and HCl (1.0 mL, 0.5 M) was added.

The mixture was extracted by DCM (3x15 mL) and the solvents were removed under

reduced pressure to give compound 3a (73.6 mg, 96%) as white foam. IR 1717

cm-1

; 1H NMR (400 MHz, CDCl3) δ 7.37-7.31 (m, 2H, Ar-H), 7.27-7.26 (m, 1H, Ar-

H), 7.25-7.22 (m, 2H, Ar-H), 3.88 (s, 3H, COOCH3), 3.50 (s, 3H, -SO2CH3), 3.45 (s,

3H, -SO2CH3), 3.15 -2.98 (m, 3H), 2.92-2.80 (m, 2H), 2.21-2.12 (m, 1H), 1.99-1.87

(m, 1H); 13

C NMR (100 MHz, CDCl3) δ 162.5, 145.1, 138.2, 135.4, 134.0, 131.7,

128.7 (2C), 126.8 (2C), 126.7, 51.8, 42.9, 42.7, 40.3, 33.2, 29.5, 26.7; LC-MS Rf (min)

= 5.57, LC-MS m/z (ES+) calculated C18H19NO6S3 for [M-CH2] 429: found 429.

Methyl 2-[(methylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


A mixture of compound 3a (30.1 mg, 0.06 mmol) and sodium methoxide (3.7 mg,

0.13 mmol) in MeOH (6.0 mL) was stirred at 0 ºC with for 3 h and continued to

stirred at 40 ºC overnight. The mixture was washed with HCl (10.0 mL, 1.0 M) and

extracted with DCM. The organic phase was dried over Na2SO4 (s), filtrated and

concentrated under reduced pressure. The residue was purified by silica gel column

chromatography using ethyl acetate: heptane= 12:88 as eluent. The solvents were

evaporated under reduced pressure to give compound 11 (18.4 mg, 74%) as a white

solid. IR 3135, 1660 cm-1

; 1H NMR (400 MHz, CDCl3) δ 10.19 (s, 1H, NH), 7.36-

7.29 (m, 2H, Ar-H), 7.27-7.25 (m, 1H, Ar-H), 7.25-7.21 (m, 2H, Ar-H), 3.87 (s, 3H,

COOCH3), 3.09 (s, 3H, -SO2CH3), 3.05 -2.86 (m, 3H), 2.82-2.70 (m, 2H), 2.18-2.08

(m, 1H), 1.97-1.84 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 166.3, 148.5, 145.4,

132.1, 128.6 (2C), 126.8 (2C), 126.5, 126.3, 113.0, 51.7, 40.5, 39.7, 33.3, 29.8, 26.8;

LC-MS Rf (min) = 5.67, LC-MS m/z (ES-) calculated for C17H18NO4S2 [M-H] 364:

found 364.

19

Methyl 2-[bis(phenylsulfonyl)amino]-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3b)

To a cooled solution (0 ºC) of compound 3 (60.0 mg, 0.21 mmol) and TEA (58.0 µL,

0.42 mol) in dry DCM (2.5 mL) was added dropwise a solution of benzenesulfonyl

chloride (16.0 µL in 1.5 mL DCM). The solution was stirred at this temperature for 20

min then at RT overnight under an atmosphere of nitrogen. Due to the presence of a

lot of starting material, the mixture was refluxed in 50 ºC oil bath for two days. After

cooling to RT, HCl (1.0 mL, 0.5 M) was added. The mixture was extracted with DCM

(3x15 mL), dried over Na2SO4 (s) and filtered, the solvents were evaporated under

reduced pressure. The residue was purified by silica gel column chromatography

using ethyl acetate: heptane= 1:4 as eluent. Compound 3b (28.1 mg, 47%) was

obtained as a yellow solid. IR 1717 cm-1

; 1H NMR (400 MHz, CDCl3) δ 8.05-7.98

(m, 4H, Ar-H), 7.71-7.66 (m, 2H, Ar-H), 7.60-7.54 (m, 4H, Ar-H), 7.38-7.33 (m, 2H,

Ar-H), 7.29-7.26 (m, 2H, Ar-H), 7.25-7.22 (m, 1H, Ar-H), 3.21 (s, 3H, COOCH3),

3.14-3.00 (m, 3H), 2.92-2.78 (m, 2H), 2.20-2.12 (m, 1H), 2.00-1.88 (m, 1H); 13

C

NMR (100 MHz, CDCl3) δ 161.8, 145.3, 139.5, 139.3, 138.6, 135.7, 134.6, 134.0,

134.0, 131.9, 129.1 (2C), 129.0 (2C), 128.9 (4C), 128.6 (2C), 126.8 (2C), 126.6, 50.9,

40.3, 33.2, 29.6, 26.4; LC-MS Rf (min) = 6.17, LC-MS m/z (ES+) calculated for

C28H26NO6S3 [M+H] 568: found 568.

Methyl 2-[[(4-methylphenyl)sulfonylamino)carbonyl]amino]-6-phenyl-4, 5, 6, 7-


To a stirring solution of compound 3 (30.0 mg, 0.10 mmol) and TEA (29.0 µL, 0.10

mmol) in dry toluene (5.0 mL) was added 4-methylbenzenesulfonyl cyanide (16.0 µL,

0.10 mmol). The reaction mixture was refluxed for 20 h under an atmosphere of

nitrogen. The mixture was concentrated under reduced pressure, and the residue was

recrystalized from methanol to afford a white solid 13 (38.0 mg, 75%). IR 3273,

3231, 1668, 1535 cm-1

; 1H NMR (400 MHz, CDCl3) δ 11.64 (s, 1H, NH), 8.07 (s, 1H,

NH), 7.92 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 7.35-7.28 (m, 5H, Ar-H), 7.25-7.19 (m,

2H, Ar-H), 3.96 (s, 3H, COOCH3), 3.09-2.85 (m, 3H), 2.85-2.69 (m, 2H), 2.42 (s, 3H,

Ar-CH3), 2.18-2.09 (m, 1H), 1.95-1.84 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 165.9,

148.0, 146.9, 145.6, 145.2, 136.1, 131.3, 130.1 (2C), 128.5 (2C), 127.4 (2C), 126.8

(2C), 126.7, 126.4, 112.2, 51.8, 40.4, 32.0, 30.0, 26.6, 21.6; LC-MS Rf (min) = 6.09,

LC-MS m/z (ES+) calculated for C24H25N2O5S2 [M+H] 485: found 485.

General procedure for hydrolysis of the methyl esters

The compound to be hydrolyzed was dissolved in a mixture of V (THF: Methanol: water)

=5:2:10 and aqueous NaOH (10.0 M, 10.0 eq), and the mixture was refluxed

overnight. Solvent was removed under reduced pressure, and the resulting sodium

carboxylate was filtered off and dissolved in water. The free acid was precipitated by

addition of HCl (3.0 M), filtered off, washed with water and then concentrated from

methanol.

2-Amino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxylic acid (3d)

20

By following the general procedure for hydrolysis of the methyl esters, compound 3

(219.7 mg, 0.76 mmol) was converted to compound 3d (189.9 mg, 91%). IR:

3456, 3341, 1628, cm-1

; 1H NMR (400 MHz, MeOH-d4) δ 7.33-7.25 (m, 4H, Ar-

H), 7.23-7.13 (m, 1H, Ar-H), 3.13-3.04 (m, 1H), 3.02-2.93 (m, 1H), 2.83-2.60 (m,

3H), 2.07-1.99 (m, 1H), 1.94-1.82 (m, 1H); 13

C NMR (100 MHz, MeOH-d4) δ 158.7,

146.6, 133.3, 128.0 (2C), 126.5 (2C), 125.7, 115.8, 48.0, 47.8, 41.2, 32.2, 30.3, 26.9;

LC-MS Rf (min) = 5.15, LC-MS m/z (ES-) calculated for C15H14NO2S [M-H] 272:

found 272.

Methyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-

tetrahydrobenzo[b]thiophene-3-carboxylate (3f)

Compound 3 (720.0 mg, 2.50 mmol), Boc2O (1145.0 mg, 5.25 mmol) and DMAP

(30.5 mg, 0.25 mmol) were mixed in dioxane (20 mL), and the mixture was stirred in

an oil bath (80 ºC) for 3h under an atmosphere of nitrogen.Then N2H4.H2O (0.36 ml,

7.50 mmol) was added, and the mixture was stirred at 40 ºC for 1.5 h. The solvents

were removed under reduced pressure and the residue was purified by silica gel

column chromatography with 0-10% ethyl acetate-Heptane to give a white solid 3f

(786.1 mg, 81%). IR 3242, 1715, 1658 cm-1

; 1H NMR (400 MHz, CDCl3) δ 10.28

(bs, 1H, NH), 7.33-7.28 (m, 2H, Ar-H), 7.27-7.25 (m, 1H, Ar-H), 7.24-7.18 (m, 2H,

Ar-H), 3.83 (s, 3H, COOCH3), 3.05-2.84 (m, 3H), 2.81-2.69 (m, 2H), 2.14-2.07 (m,

1H), 1.95-1.85 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz, CDCl3) δ

166.7, 152.2, 150.3, 145.8, 130.7, 128.5 (2C), 126.9 (2C), 126.4, 124.6, 109.6, 81.9,

51.3, 40.6, 32.1, 30.0, 28.2 (3C), 26.7; LC-MS Rf (min) = 7.12, LC-MS m/z (ES+)

calculated for C21H26NO4S [M+H] 388: found 388.

2-(tert-Butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxylic acid (3g)

By following the general procedure for hydrolysis of the methyl esters, compound 3f

(673.0 mg, 1.73 mmol) was converted to compound 3g (551.2 mg, 85%) as a light

brown solid. IR 3281, 2924, 1719, 1636 cm-1

; 1H NMR (400 MHz, DMSO-d6) δ

13.05 (bs, 1H, COOH), 10.4 (s, 1H, NH), 7.33-7.28 (m, 4H, Ar-H), 7.24-7.18 (m, 1H,

Ar-H), 2.99-2.90 (m, 2H), 2.88-2.80 (m, 1H), 2.74-2.63 (m, 2H), 2.02-1.94 (m, 1H),

1.90-1.79 (m, 1H), 1.48 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz, DMSO-d6) δ 167.6,

151.7, 149.0, 146.2, 131.4, 128.8 (2C), 127.3 (2C), 126.7, 124.6, 110.8, 82.2, 40.2,

31.9, 30.0, 28.2(3C), 26.8; LC-MS Rf (min) = 6.05, LC-MS m/z (ES-) calculated for

C20H22NO4S [M-H] 372: found 372.

Methyl 2-acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxylate (3i)

Acetyl chloride (94.0 µL, 1.33 mmol) was added to a solution of compound 3 (151.2

mg, 0.52 mmol) and pyridine (2.3 mL) in DCM (9.2 mL) at 0 ºC. The yellow mixture

was stirred at RT for 45 min and the solution became clear. The solvents were

removed under reduced pressure and the residue was taken up in ethyl acetate and

washed with HCl (1.0 M).The organic phase was dried over Na2SO4 (s), filtered and

the solvent was evaporated under reduced pressure. The residue was purified by silica

gel column chromatography using ethyl acetate: heptane= 1:4 as eluent. Compound 3i

(145.5 mg, 85%) was obtained as white foam. IR 3280, 1672, 1600 cm-1

; 1H NMR

21

(400 MHz, CDCl3) δ 11.23 (s, 1H, NH), 7.35-7.28 (m, 2H, Ar-H), 7.26 -7.20 (m, 3H,

Ar-H), 3.86 (s, 3H, COOCH3), 3.05-2.87 (m, 3H), 2.82-2.70 (m, 2H), 2.25 (m, 3H,

COCH3), 2.15-2.07 (m, 1H), 1.95-1.84 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 167.0

(2C), 148.1, 145.7, 130.3, 128.5 (2C), 126.9 (2C), 126.4, 126.1, 110.8, 51.4, 40.5,

32.1, 30.1, 26.6, 23.7; LC-MS Rf (min) = 5.99, LC-MS m/z (ES+) calculated for

C18H20NO3S [M+H] 330: found 330.

2-Acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxylic acid (3j)

By following the general procedure for hydrolysis of the methyl esters, compound 3i

(145.5 mg, 0.44 mmol) was converted to compound 3j (129.7 mg, 93%) as a light

yellow solid. IR 3281, 2931, 1670, 1638 cm-1

; 1H NMR (400 MHz, DMSO-d6) δ

13.06 (bs, 1H, COOH), 11.16 (s, 1H, NH), 7.34-7.26 (m, 4H, Ar-H), 7.23-7.17 (m,

1H, Ar-H), 3.02-2.79 (m, 3H), 2.76-2.64 (m, 2H), 2.20 (s, 3H, COCH3), 2.03-1.93 (m,

1H), 1.92-1.78 (m, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 167.4, 167.3, 146.9,

146.3, 130.9, 128.8 (2C), 127.3 (2C), 126.6, 125.5, 111.8, 40.3, 31.9, 30.1, 26.7, 23.8;

LC-MS Rf (min) = 5.31, LC-MS m/z (ES-) calculated for C17H16NO3S [M-H] 314:

found 314.

2-Acetylamino-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene (3k)

Compound 3j (102.1 mg, 0.32 mmol) and copper powder (30.7 mg, 0.48 mmol) were

mixed in quinoline (13.4 mL), and the mixture was heated in a Biotage Smith Creator

microwave at 200 ºC for 10 min. The mixture was diluted with H2O (10 mL),

acidified to pH=1 with HCl (5.0 M) and extracted with ethyl acetate (3x15 mL). The

organic phase was dried over Na2SO4 (s), filtered and evaporated under reduced

pressure. The residue was purified by silica gel column chromatography using ethyl

acetate: heptane= 1:4→ 3:7 as eluent. The solvents were evaporated under reduced

pressure and compound 3k (47.4 mg, 54%) was obtained as a white yellow solid. IR

3267, 1635 cm-1

; 1H NMR (400 MHz, CDCl3) δ 7.74 (s, 1H, Ar-H), 7.27-7.22 (m,

2H, Ar-H), 7.21-7.19 (m, 1H, Ar-H), 7.18 -7.12 (m, 1H, Ar-H), 6.30 (s, 1H, NH),

2.99-2.84 (m, 2H), 2.76-2.67 (m, 1H), 2.65-2.54 (m, 2H), 2.10 (s, 3H, COCH3), 2.04-

1.98 (m, 1H), 1.94-1.81 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 166.7, 146.0, 135.7,

131.6, 128.5 (2C), 127.9, 126.9 (2C), 126.3, 113.1, 41.2, 32.2, 30.2, 25.6, 23.3; LC-

MS Rf (min) = 5.20, LC-MS m/z (ES+) calculated for C16H18NOS [M+H] 272: found

272.

2-Chloro-7-phenyl-5, 6, 7, 8-tetrahydro[1]benzothieno[2, 3-b]pyridine (3m)

Phosphorus oxychloride (6.1 µL, 0.6 mmol) was added dropwise to a stirring solution

of DMF (5.1 µL, 0.6 mmol) in 1, 2-dichloroethane (0.5 mL) at 0 ºC, and a solution of

compound 3k (18.1 mg, 0.06 mmol) in dry 1, 2-dichloroethane (1.0 mL) was added

dropwise to the mixture. And the mixture was stirred at RT for 15 min then refluxed

at 85 ºC for 3.5 h. After cooling to RT, a solution of sodium acetate (209.5 mg, 2.55

mmol, in 2.0 mL H2O) was added to the mixture, and the solution was heated at 50 ºC

for 1.5 h. After cooling, the solution was diluted with DCM (10 mL) and extracted

with DCM (3x 10 mL).The organic phase was separated, washed with saturated

sodium bicarbonate solution, dried over Na2SO4 (s), filtered and evaporated under

22

reduced pressure. The residue was purified by silica gel column chromatography

using DCM: heptane= 1:1 as eluent. The solvents were evaporated under reduced

pressure and compound 3m (3.8 mg, 19%) was obtained as a white yellow solid. IR

1582, 1522, 1492, 1426, 1350, 1139, 1121 cm-1

; 1H NMR (400 MHz, CDCl3) δ

7.76 (d, J = 4.12 Hz, 1H, Ar-H), 7.37-7.32 (m, 2H, Ar-H), 7.31-7.23 (m, 4H, Ar-H),

3.22-3.11 (m, 2H), 3.05-2.95 (m, 1H), 2.94-2.75 (m, 2H), 2.30-2.22 (m, 1H), 2.13-

2.01 (m, 1H); 13

C NMR (100 MHz, CDCl3) δ 160.4, 146.8, 145.1, 137.6, 131.7,

130.0, 128.6 (2C), 126.8 (2C), 126.7, 126.6, 119.7, 40.8, 33.3, 29.4, 23.5; LC-MS Rf

(min) = 6.45, LC-MS m/z (ES+) calculated for C17H15ClNS [M+H] 300: found 300.

The general procedure for synthesis of compounds 22a-22d

Compound 3g (80.0 mg, 0.21 mmol), TBTU (69.6 mg, 0.21 mmol) and TEA (70.0

µL, 0.42 mmol) were mixed in ethyl acetate (8.0 mL) .After the mixture was stirred at

RT for 10 min, two equimolar amount of the appropriate amine was added dropwise.

The mixture was stirred for 3 h, 3.5 h, 2 h, and 4.5 h respectively (in the order from

22a to 22d). After dilution with 15 mL ethyl acetate and adding 20 mL H2O, the

organic layer was separated, extracted with ethyl acetate (3x30 mL), dried over

Na2SO4 (s) and evaporated.

N-isopropyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxamide (22 a)

The product was purified by silica gel column chromatography using ethyl acetate:

heptane= 1:10 as eluent. The solvents were evaporated under reduced pressure and

compound 22a (48.2 mg, 54%) was obtained as white solid. IR 3263, 3155, 1702,

1615, 1522 cm-1

; 1H NMR (400 MHz, CDCl3) δ 11.02 (s, 1H, NH), 7.37-7.29 (m, 2H,

Ar-H), 7.28-7.26 (m, 1H, Ar-H), 7.25-7.21 (m, 2H, Ar-H), 5.67 (d, J = 3.6 Hz, 1H, -

NHCH(CH3)2), 4.28-4.17 (m, 1H, -NHCH(CH3)2), 3.07-2.89 (m, 2 H), 2.86-2.71 (m,

3H), 2.24 -2.15 (m, 1H), 2.04-1.92 (m, 1H), 1.50 (s, 9H, CO(CH3)3), 1.24 (d, J= 8 Hz,

3H, -NHCH(CH3)2), 1.25 (d, J= 8 Hz, 3H, -NHCH(CH3)2); 13

C NMR (100 MHz,

CDCl3) δ 165.4, 152.5, 147.4, 145.3, 128.6 (2C), 126.8 (3C), 126.5, 125.6, 112.5,

81.3, 41.4, 40.2, 32.1, 30.1, 28.2 (3C), 26.9, 23.0, 22.9; LC-MS Rf (min) = 6.60, LC-

MS m/z (ES-) calculated for C23H29N2O3S [M-H] 413: found 413.

N-phenyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxamide (22b)



compound 22b (41.6 mg, 43%) was obtained as a white solid. IR 3382, 2984,

1709, 1636, 1518 cm-1

; 1H NMR (400 MHz, CDCl3) δ 10.82 (s, 1H, NH), 7.58-7.47

(m, 3H, Ar-H), 7.41-7.30 (m, 4H, Ar-H), 7.30-7.25 (m, 2 H, Ar-H), 7.19-7.11 (m, 1H,

Ar-H), 3.13-3.03 (m, 1H), 3.03-2.88 (m, 3H), 2.87-2.78 (m, 1H), 2.44-2.17 (m, 1H),

2.16-1.89 (m, 1H), 1.50 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz, CDCl3) δ 164.4,

152.5, 148.7, 145.1, 137.3, 129.1 (2C), 128.6 (2C), 126.9 (2C), 126.6, 126.5, 126.1,

124.7, 120.8 (2C), 112.6, 81.7, 40.2, 32.1, 30.1, 28.2 (3C), 27.0; LC-MS Rf (min) =

6.59, LC-MS m/z (ES-) calculated for C26H27N2O3S [M-H] 447: found 447.

23

[2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thien-3-yl]-4-morpholinylmethanone (22c)


heptane = 1:4 → 1:1 as eluent. The solvents were evaporated under reduced pressure

and compound 22c (44.4 mg, 47%) was obtained as a white solid. IR 3282, 3198,

1714, 1605, 1534 cm-1

; 1H NMR (400 MHz, CDCl3) δ 8.05 (bs, 1H, NH), 7.34-7.29


-NHCH2CH2O-), 3.09-2.91 (m, 2H), 2.84-2.74 (m, 1H), 2.63-2.49 (m, 2H), 2.13-2.05

(m, 1H), 1.96-1.85 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz, CDCl3) δ

166.7, 152.4, 145.5, 139.8, 129.0, 128.5 (2C), 127.4, 126.8 (2C), 126.4, 117.1, 81.7,

67.1 , 67.0, 45.9, 44.9, 40.8, 32.0, 29.8, 28.2 (3C), 24.9; LC-MS Rf (min) = 5.57, LC-

MS m/z (ES+) calculated for C24H31N2O4S [M+H] 443: found 443.

N-benzyl 2-(tert-butoxycarbonylamino)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxamide (22d)



compound 22d (64.4 mg, 65%) was obtained as colourless oil. IR 3029, 2972,

1708, 1618, 1561 cm-1

; 1H NMR (400 MHz, CDCl3) δ 11.06 (s, 1H, NH), 7.39-7.26


NH), 4.61 (d, J = 2.78 Hz, 2H, Ar-CH2NH-), 3.05-2.90 (m, 2H), 2.86-2.69 (m, 3H),

2.20-2.12 (m, 1H), 2.03-1.91 (m, 1H), 1.51 (s, 9H, CO(CH3)3); 13

C NMR (100 MHz,

CDCl3) δ 166.1, 152.5, 148.1, 145.2, 138.1, 128.8 (2C), 128.6 (2C), 127.6, 127.5

(2C), 126.8 (2C), 126.5, 125.7, 112.0, 81.5, 43.5, 40.1, 32.0, 30.0, 28.2 (4C), 26.9;

LC-MS Rf (min) = 6.65, LC-MS m/z (ES+) calculated for C27H31N2O3S [M+H] 463:

found 463.

2-Amino-N-(isopropyl)-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxamide (23a)

Compound 22a (28.0mg, 0.067mmol) was dissolved in dry DCM (1.0 mL), and TFA

(1.0 mL) was added dropwise. The mixture was stirred at RT for 20 min, diluted with

DCM (20 mL), quenched with saturated NaHCO3 solution, washed with brine (15

mL) and extracted with DCM (3x10 mL). The solvent was evaporated under reduced

pressure. The product was purified by silica gel column chromatography using ethyl

DCM: MeOH= 95:5 (0.5% TEA) as eluent. The solvents were evaporated, compound

23a (14.4 mg, 68%) was obtained as yellow oil. IR 3243, 3205, 2924, 1560 cm-1

; 1H NMR (400 MHz, CDCl3) δ 7.37-7.29 (m, 2H, Ar-H), 7.28-7.26 (m, 1H, Ar-H),

7.25-7.18 (m, 2H, Ar-H), 5.50 (d, J = 3.8 Hz, 1H, -NHCH (CH3)2), 4.27-4.16 (m, 1H,

-NHCH(CH3)2), 3.06-2.97 (m, 1H), 2.86 -2.66 (m, 4H), 2.20-2.11 (m, 1H), 2.01-1.89

(m, 1H), 1.21 (d, J= 8 Hz, 3H, -NHCH (CH3)2), 1.23 (d, J= 8 Hz, 3H, -NHCH

(CH3)2); 13

C NMR (100 MHz, CDCl3) δ 165.7, 158.7, 145.5, 128.9, 128.6 (2C),

128.5, 126.8 (2C), 126.4, 118.4, 108.9, 41.0, 40.4, 32.3, 30.1, 27.3, 23.1; LC-MS Rf

(min) = 5.39, LC-MS m/z (ES+) calculated for C18H23N2OS [M+H] 315: found 315.

The general procedure for synthesis of compounds 15-18

24

An equimolar amount of succinic anhydride was added to a stirring mixture of

compounds 23a, 23b, 23c, or 23d in dry CH2Cl2 (3.0 mL).The mixture was refluxed

under an atmosphere of nitrogen for 7.5 h, 5 h, 3 h and 1.5 h respectively. After

dilution with 20 mL DCM, the mixture was washed with brine (20 mL) and extracted

with DCM (3x20 mL). The organic layer was separated, dried over Na2SO4 (s) and

filtered. The solvent was removed under reduced pressure, and purified by silica gel

column chromatography.

N-Isopropyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thiophene-3-carboxamide (15)

The crude was purified by silica gel column chromatography using ethyl DCM:

MeOH= 97.5:2.5 (0.5% CH3COOH) as eluent two times. Compound 23a (14.4 mg,

0.05 mmol) was converted to compound 15 (5.0 mg, 27%) as yellow oil. IR 3297,

3257, 1709, 1681, 1600 cm-1

; 1H NMR (400 MHz, CDCl3) δ 12.01 (s, 1H, NH), 7.30-

7.23 (m, 2H, Ar-H), 7.22-7.19 (m, 1H, Ar-H), 7.18-7.13 (m, 2H, Ar-H), 5.72 (d, J =

3.8 Hz, 1H, -NHCH (CH3)2), 4.20-4.09 (m, 1H, -NHCH(CH3)2), 3.01-2.85 (m, 2H),

2.80-2.65 (m, 7H), 2.18-2.09 (m, 1H), 1.98-1.86 (m, 1H), 1.20 (d, J= 8 Hz, 3H, -

NHCH (CH3)2), 1.18 (d, J= 8 Hz, 3H, -NHCH (CH3)2); 13

C NMR (100 MHz, CDCl3)

δ 176.2, 168.6, 165.4, 145.1, 128.9, 128.6 (2C), 127.4, 126.8 (2C), 126.6, 126.5,

113.9, 41.7, 40.1, 32.0, 31.0, 30.1, 28.9, 26.7, 22.9 (2C); LC-MS Rf (min) = 5.15, LC-

MS m/z (ES-) calculated for C22H25N2O4S [M-H] 413: found 413.

N-Phenyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


Compound 22b (41.6 mg, 0.05 mmol) was dissolved in dry DCM (1.0 mL) at RT, and

TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 20 min, and

diluted with DCM (20 mL), and the solvent was removed under reduced pressure.

Following the general procedure for synthesis of compounds 15-18, and the crude was

purified by silica gel column chromatography using ethyl DCM: MeOH= 99.8 :0.2

(0.1% CH3COOH) → 99 :1 (0.1% CH3COOH) as eluent two times. The solvents were

evaporated under reduced pressure and compound 16 (17.6 mg, 75%) was obtained as

a yellow solid. IR 1719, 1666, 1619 cm-1

; 1H NMR (400 MHz, DMSO-d6) δ 12.14

(bs, 1H, NH), 10.76 (bs, 1H, NH), 9.94 (s, 1H, COOH), 7.72 (s, 1H, Ar-H), 7.70 (s,

1H, Ar-H), 7.37-7.29 (m, 6H, Ar-H), 7.26-7.19 (m, 1H, Ar-H), 7.10-7.04 (t, J = 7.38

Hz, 1H, Ar-H), 3.02-2.70 (m, 5H), 2.68-2.60 (m, 2H), 2.57-2.50 (m, 2H), 2.05-1.97

(m, 1H), 1.93-1.81 (m, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 174.1, 169.6, 163.5,

146.2, 139.6, 129.8, 129.0 (2C), 128.8 (2C), 127.3 (2C), 126.7, 126.3, 123.8, 120.4

(2C), 120.0, 32.1 (2C), 30.7, 30.1, 29.3 (2C), 25.4; LC-MS Rf (min) = 5.42, LC-MS

m/z (ES-) calculated for C25H23N2O4S [M-H] 447: found 447.

[2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo

[b]thien-3-yl]-4-morpholinylmethanone (17)

Compound 22c (44.4 mg, 0.1 mmol) was dissolved in dry DCM (1.0 mL) at RT, and

TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 10 min, and


Following the general procedure for synthesis of compounds 15-18, and the crude was

purified by silica gel column chromatography using ethyl DCM: MeOH= 98 :2 (0.1%

25

CH3COOH) → 97 :3 (0.1% CH3COOH) as eluent two times. The solvents were


colorless oil. IR 3242, 3173, 2921, 1678, 1578, 1542 cm-1

; 1H NMR (400 MHz,

CDCl3) δ 9.51 (s, 1H, COOH), 7.35-7.26 (m, 2H, Ar-H), 7.24-7.18 (m, 3H, Ar-H),

3.74 -3.42 (m, 8H, -NHCH2CH2O-), 3.06-2.91 (m, 2H), 2.82-2.74 (m, 4H), 2.68-2.58

(m, 1H), 2.57-2.46 (m, 2H), 2.15-2.05 (m, 1H), 1.99-1.85 (m, 1H); 13

C NMR (100

MHz, CDCl3) δ 176.8, 168.9, 167.4, 145.4, 136.6, 128.9, 128.5 (2C), 128.2, 126.8

(2C), 126.5, 117.1, 66.9 (2C) , 51.9, 45.0, 40.8, 31.9, 30.7, 29.7, 28.7, 24.8; LC-MS

Rf (min) = 4.64, LC-MS m/z (ES-) calculated for C23H25N2O5S [M-H] 441: found

441.

N-Benzyl 2-[(3-carboxypropanoyl)amino]-6-phenyl-4, 5, 6, 7-tetrahydrobenzo


Compound 18 (64.4 mg, 0.09 mmol) was dissolved in dry DCM (1.0 mL) at RT, and

TFA (1.0 mL) was added dropwise. The mixture was stirred at RT for 10min, and


Following the general procedure for synthesis of compounds 15-18, the crude was

purified by silica gel column chromatography using ethyl DCM: MeOH= 99 :1 (0.1%

CH3COOH) → 97 :3 (0.1% CH3COOH) as eluent two times. The solvents were


a white solid. IR 1725, 1681, 1593 cm-1

; 1H NMR (400 MHz, DMSO-d6) δ 12.21

(bs, 1H, NH), 11.24 (s, 1H, COOH), 8.09 (t, J = 5.88 Hz, 1H, Ar-CH2NH-), 7.37-7.28

(m, 8H, Ar-H), 7.28-7.17 (m, 2H, Ar-H), 4.56-4.39 (m, 2H, Ar-CH2NH-), 3.01-2.70

(m, 5H), 2.66-2.60 (m, 2H), 2.55-2.51 (m, 2H), 2.05-1.97 (m, 1H), 1.95-1.81 (m, 1H); 13

C NMR (100 MHz, DMSO-d6) δ 174.0, 169.3, 165.4, 146.1, 141.2, 139.9, 129.1,

128.8 (2C), 128.7 (2C), 127.6 (2C), 127.3 (2C), 127.1, 126.7, 126.3, 117.4, 43.0, 32.0,

31.0, 30.1, 29.2 (2C), 25.8; LC-MS Rf (min) = 5.34, LC-MS m/z (ES-) calculated for

C26H25N2O4S [M-H] 461: found 461.

26

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