Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors

Bioorganic & Medicinal Chemistry 14 (2006) 2162–2177

Bicyclic and tricyclic thiophenes as protein tyrosine

phosphatase 1B inhibitors

A. F. Moretto,^aS. J. Kirincich,^aW. X. Xu,^aM. J. Smith,^aZ.-K. Wan,^aD. P. Wilson,^a

B. C. Follows,^aE. Binnun,^aD. Joseph-McCarthy,^aK. Foreman,^a,ꢀD. V. Erbe,^b

Y. L. Zhang,^bS. K. Tam,^bS. Y. Tam^aand J. Lee^a,*

^aChemical and Screening Science, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA

^bCardiovascular and Metabolic Diseases, Wyeth Research, 200 Cambridge Park Drive, Cambridge, MA 02140, USA

Received 13 September 2005; revised 31 October 2005; accepted 1 November 2005

Available online 21 November 2005

Abstract—A novel pyridothiophene inhibitor of PTP1B was discovered by rational screening of phosphotyrosine mimics at high

micromolar concentrations. The potency of this lead compound has been improved signiﬁcantly by medicinal chemistry guided

by X-ray crystallography and molecular modeling. Excellent consistency has been observed between structure–activity relationships

and structural information from PTP1B-inhibitor complexes.

1. Introduction

PTP1B upon cross-validation with detailed enzymology,

NMR-binding studies, and X-ray crystallography. Such

a high frequency of false positives could be due to the

low pK_aand thus high reactivity of Cys215 at the en-

zyme active site.^15,16Inhibition of PTP1B enzymatic

activity via oxidation of the cysteine residue has been

reported before.^17–19

Protein tyrosine phosphatase 1B (PTP1B) has been

shown to be a negative regulator in the insulin and leptin

receptor pathways.^1–3Two independent studies of

PTP1B-deﬁcient mice have revealed phenotypes of en-

hanced insulin sensitivity, improved glycemic control,

and resistance to high-fat diet induced obesity.^4,5Fur-

thermore, treatment of diabetic mice with PTP1B anti-

sense oligonucleotides reduced the expression level of

the enzyme and subsequently normalized blood glucose

and improved insulin sensitivity.^6,7Thus, PTP1B has be-

come an attractive therapeutic target for the treatment

of type 2 diabetes and obesity. A number of companies

and academic institutions have small molecule programs

targeting this enzyme.^8–14

The lead compound 1 was discovered by screening sev-

eral hundred compounds at high micromolar concentra-

tion for novel phosphotyrosine mimetics (Fig. 1).

Compound 1 was a reversible and competitive inhibitor

of PTP1B with a K_iof 230 lM at pH 7.4. Inhibition of

the enzyme by this compound was independent of pH

(K_i= 200 lM at pH 5.5).^20,21

Binding of 1 to the enzyme active site was further con-

ﬁrmed by X-ray crystallography (Fig. 2). Upon binding

of 1, the WPD loop of PTP1B adopted a closed confor-

Our PTP1B small molecule inhibitor program started

with a high-throughput screening (HTS) campaign.

About 6000 hits were identiﬁed with more than 40%

inhibition at a concentration of 20 lg/mL. However,

none of the selected HTS hits were real inhibitors of

Keywords: Protein tyrosine phosphatase 1B; Thiophene; Structure-

based design.

*

Corresponding author. Tel.: +1 617 665 5674; fax: +1 617 665

5682; e-mail: jklee@wyeth.com

ꢀ

Present address: OSI Pharmaceuticals, Inc. 1 Bioscience Park Drive,

Farmingdale, NY 11735, USA.

Figure 1. Compound 1, a reversible inhibitor of PTP1B.

doi:10.1016/j.bmc.2005.11.005

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2163

Figure 3. Variation of the bicyclic scaﬀolds on compound 1.

Figure 2. X-ray crystal structure of inhibitor 1 bound to PTP1B.

hydrolysis with LiOH provided compound 3 (Fig. 3).

Compounds 4–7 were prepared in a similar manner.

The ester precursor of 2 was prepared by alkylation of

methyl 3-hydroxy-2-naphthoate with ethyl bromoace-

tate, followed by hydrolysis to give compound 2.

mation. The thiophene ring was sandwiched between

Phe182 and Tyr46, which provided p-interactions and

mimicked the phenyl group of phosphotyrosine (p-

Tyr). The acidic side chain at the 3-position was buried

deep down in the enzyme active site, mimicking the

phosphate group of p-Tyr. The carboxyl group formed

a salt bridge with Arg221 and multiple hydrogen bonds

with the backbone amides of Gly218, Ile219, and

Gly220 at the bottom of the active site. The ether oxy-

gen formed water mediated hydrogen bonds with

Ala217 and Arg221. The carboxyl group at the 2-posi-

tion interacted with Lys120 via a salt bridge. The 5-po-

sition of 1 packed tightly against the side chain of Ile219.

Because of these multiple interactions, the inhibitor

positioned itself nicely at the enzyme active site.

Further derivatization of benzothiophenes (compounds

8–17 in Table 1) was carried out according to the steps

in Scheme 1, followed by straightforward functional

group manipulations such as nitro reduction, acylation,

reductive amination, amide formation, and cross-

coupling reactions.^25–27

Synthesis of thienobenzothiophene analogs was carried

out following the steps in Scheme 2. 4-(Methoxycarbon-

yl)-3-nitrobenzoic acid or 2-nitro-isophthalic acid was

treated with allyl bromide to protect the carboxylic acid

moiety. The resulting allyl ester was treated with methyl

2-mercaptoacetate to eﬀect formation of the second ring.

Converting this bicycle to the corresponding triﬂate and

Though the potency of 1 was weak, the availability of

structural information provided guidance for further

optimization. We report here our medicinal chemistry

eﬀort in signiﬁcantly improving the potency of 1. The

structural–activity relationship will be discussed in con-

junction with X-ray structural information and molecu-

lar modeling.

Table 1. Structure–activity relationship of the 3, 6, and 7 positions of

benzothiophenes

2. Chemistry

Compound

X

R₁

R₂

PTP1B K_i(lM)

Synthesis of various bicyclic scaﬀolds was carried out

following the general synthetic steps in Scheme 1.^22,23

As an example, reaction of ethyl 2-chloronicotinate

and sodium methoxy-carbonylmethoxide aﬀorded 3-

hydroxyfuro[2,3-b]pyridine-2-carboxylate.²⁴Subsequent

alkylation with tert-butylbromoacetate followed by

8

9

H

Cl

Br

H

61

>2500

52

Me

F

10

11

H

42

12

13

14

15

H

128

26

30

20

Scheme 1. Reagents: (i) NaH or K₂CO₃, methyl 2-mercaptoacetate or

methyl 2-hydroxyacetate, DMF; (ii) ethyl bromoacetate or tert-butyl

2-bromoacetate, K₂CO₃, DMF (iii) LiOH, THF/H₂O.

16

17

H

Cl

Me

119

37

2164

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

PTP1B active site (Fig. 2). Modeling suggests that a

di-ﬂuoro methylene or the addition of a methyl alcohol

to the methylene carbon could be tolerated at this posi-

tion; in the latter case, a bound water would be

displaced.

Substitution at the 6-position oﬀered the ﬁrst hint at

potency improvement. A simple chloro (8) or bromo

(11) group provided a 3- to 4-fold increase in inhibitory

activity. Electron-rich aromatic groups were favorable

at this position (12 vs 13 or 14), as well as a carbonyl

group (i.e., 15). Though all these substitutions oﬀered

modest improvement in potency ranging from 2- to 8-

fold versus that of 4, the structure–activity relationship

was relatively ﬂat with K_ivalues ranging from 20 to

150 lM.

Scheme 2. Reagents: (i) allyl bromide, K₂CO₃, DMF; (ii) methyl 2-

mercaptoacetate, LiOH, DMF; (iii) Tf₂O, TEA, CDCl₃; (iv) methyl 2-

mercaptoacetate, NaH, DMF; (v) ethyl bromoacetate, K₂CO₃, DMF;

(vi) Pd(PPh₃)₄, morpholine, THF; (vii) DPPA, TEA, toluene, t-buOH;

(viii) HCl, EtOAc, MeOH; (ix) aldehyde/ketone, NaB(OAc)₃H, DCE;

(x) LiOH, THF/H₂O.

To our surprise, a simple methyl group at the 7-position

of the benzothiophene yielded a compound (17) with a

K_iof 37 lM, a 4-fold improvement over that of 4. A

model of 17 docked into the PTP1B active site indicates

that the methyl group has some van der Waals interac-

tion with the side chain of Tyr46 (Fig. 4). Since substitu-

tions at either the 6- or 7-position provided modest

improvement in potency, combining 6,7-di-substitution

in the form of a benzo-fusion provided a tricyclic thio-

phene compound 18 (Fig. 5). The eﬀect of this di-substi-

tution turned out to be additive and compound 18 had a

K_iof 11 lM, a 15-fold improvement over that of 4. Two

other tricyclic thiophene analogs (19 and 20) were pre-

pared varying the middle ring. Consistent with the

repeating treatment with methyl 2-mercaptoacetate

formed the second thiophene ring. This was followed

by alkylation with ethyl bromoacetate and deprotection

of the allyl ester. Subsequent Curtius rearrangement of

the free acid gave the entire thienobenzothiophene back-

bone, with an aniline nitrogen available for subsequent

functionalization such as reductive amination.

3. Results and discussion

Optimization of 1 started with varying the pyridothi-

ophene scaﬀold. Thiophene as the ﬁrst ring of the bicy-

clic scaﬀold was crucial for better inhibitory activity

against PTP1B (Fig. 3). A simple replacement of the sul-

fur atom with an oxygen atom resulted in signiﬁcant loss

of potency (1 vs 3). Expansion of the 5-membered thio-

phene ring to a 6-membered benzene ring also reduced

activity signiﬁcantly (2 vs 4).^28–30On the contrary, the

second ring was less sensitive to modiﬁcations. Moving

the nitrogen from the 7-position (1) to the 4-position

(6) or replacing of the nitrogen with a carbon (4) was

allowed, but nitrogen at the 5-position (5) was not toler-

ated. Contraction of the 6-membered ring to a 5-mem-

bered thiophene ring was also tolerated.

The incompatibility of a polar nitrogen atom at the 5-

position was consistent with the structural information

in Figure 2, because this position interacted closely with

the hydrophobic side chain of Ile219. Furthermore,

molecular modeling suggested that this position would

not allow further substitution.

Figure 4. A model of compound 17 docked into the PTP1B active site.

Compound 17 and Tyr46 are shown in a van der Waals sphere

representation.

Benzothiophenes were selected for further derivatization

at the 3, 6, and 7-positions, since benzothiophene (4) of-

fered a slightly better activity than pyridothiophene (1)

and more ﬂexibility in synthesis. The carboxymethoxy

side chain at the 3-position appeared to be optimal. A

ﬂuorine was tolerated at the methylene carbon of the

side chain (8 vs 10), but a simple methyl group at the

same position resulted in signiﬁcant loss of activity (8

vs 9). This likely reﬂects the energy cost of placing a

hydrophobic group into the polar environment of the

Figure 5. Tricyclic thiophene PTP1B inhibitors.

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2165

SAR of the bicyclic thiophenes, a thiophene ring was

tolerated (20), while a pyridine nitrogen at the 5-position

(19) resulted in signiﬁcant loss of potency. The thi-

enobenzothiophene template of 20 was chosen over that

of 18 to be the new backbone of our chemical series due

to greater novelty and synthetic ﬂexibility.

resulted in slight loss in activity. Nevertheless, a piper-

dine group was introduced as a functional handle to

gain further access to the second phosphotyrosine-bind-

ing site. A variety of sulfonamide derivatives were syn-

thesized (e.g., 28 and 29). Compound 29 turned out to

be one of the most potent analogs in this chemical series,

with a K_iof 370 nm. The X-ray co-crystal structures of

29 and PTP1B gave insight into the interactions that

resulted in this dramatic increase in activity, as well as

into ways that these interactions can be further opti-

mized. As shown in Figure 6, the acid moieties of 29

bind to the active site in a similar binding mode as the

bicyclic thiophene compounds (Fig. 2). In addition,

one of the sulfonamide oxygens hydrogen bonds to the

backbone nitrogen of Gly 259 and the other enters into

interactions with Arg24 and Arg254 through bridging

water molecules. These interactions overcome any unfa-

vorable eﬀect of the proximity of the piperidine nitrogen

to the hydrophobic Met258 side chain. However, the

benzyl moiety is pointing out into solvent. This shows

that, presumably, further optimization of the side chain

and/or the main scaﬀold, and therefore increased poten-

cy, is possible.

To further improve the potency and selectivity of 20, our

approach was to extend the inhibitor toward the second

phosphotyrosine-binding site.^31,32Molecular modeling

suggested that substitution at the 5- or the 6-position

could lead to the desired secondary interactions with

the enzyme. Therefore, a number of compounds with

each substitution pattern were synthesized and assayed.

Table 2 shows representative examples of 5- or 6-substi-

tuted thienobenzothiophene compounds and their activ-

ity in our in vitro enzymatic assay. As predicted from

molecular modeling, both the 5- and 6-positions oﬀered

opportunity for potency improvement. A cyclohexylm-

ethylamino group at either position yielded compounds

with sub-micromolar potency (23 and 24), which was an

order of magnitude better than that of 20. In general,

substitution at the 6-position was more favorable than

that at the 5-position (22 vs 21, 24 vs 23, and 26 vs

25), suggesting that the former position might provide

a better trajectory toward the second phosphotyrosine-

binding site. Thus, further extension of the molecule

was derived from the 6-position.

In general, the thienobenzothiophene analogs were very

selective against other protein tyrosine phosphatases

such as CD45 and LAR (ꢀ1000-fold), except the highly

homologous TCPTP (Table 3).^33,34Nevertheless,

extending the inhibitor from the active site into the sec-

ond phosphotyrosine-binding site did shift the relative

selectivity of PTP1B vs TCPTP (20 vs 29) slightly. Com-

pound 20, only binding to the active site, favored

TCPTP by about 2-fold. Compound 29, spanning the

active and the second phosphotyrosine-binding site,

was equally potent against both enzymes.

Modeling suggested that the potency improvement from

the cyclohexyl (26) or cyclohexylmethyl (24) group was

mainly due to their van der Waals interaction with the

Met258 side chain. Thus, it was not surprising that polar

functionality such as oxygen (27) on the cyclohexyl ring

Table 2. In vitro activity of 5- and 6-substituted thienobenzothioph-

enes

Compound

R₁

R₂

PTP1B K_i(lM)

21

22

23

Cl

H

Cl

H

10

3.5

0.92

24

25

26

27

H

0.68

1.7

H

0.74

2.4

Figure 6. Co-crystal structure of 29 and PTP1B.

Table 3. Inhibitory activity of compounds 20 and 29 against various

protein tyrosine phosphatases

28

29

H

1.6

Compound

K_i(lM)

PTP1B

CD45

LAR

TCPTP

0.37

20

29

9.2

0.37

>1250

325

1100

>500

4.1

0.38

2166

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4. Conclusion

5.2.2. 3-(Carboxymethoxy)-2-naphthoic acid (2). Methyl

3-hydroxy-2-naphthoate (500 mg, 2.47 mmol), ethyl

bromoacetate (619 mg, 3.71 mmol), and potassium car-

bonate (682 mg, 4.94 mmol) were suspended in 5 mL

DMF. The mixture was stirred at 60 ꢁC for 16 h. EtOAc

(100 mL) was then added and the organic layer was

washed with water, brine, and dried over anhydrous

Na₂SO₄. Solvent was removed under reduced pressure

to aﬀord methyl 3-(2-ethoxy-2-oxoethoxy)-2-naphthoate

(2a; 783 mg, >95%) as a light yellow oil. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.30 (t, J = 7.07 Hz, 3 H)

3.97 (s, 3H) 4.29 (q, J = 7.07 Hz, 2H) 4.81 (s, 2H) 7.14

(s, 1H) 7.37–7.45 (m, 1H) 7.49–7.56 (m, 1H) 7.72 (d,

J = 8.08 Hz, 1H) 7.84 (d, J = 8.08 Hz, 1H) 8.37 (s,

1H). MS (ESI, pos.) calcd for C₁₆H₁₆O₅m/z [M+H]=

288.10, found 288.09.

In summary, a novel pyridothiophene lead was identi-

ﬁed via screening of phosphotyrosine mimetics at high

micromolar concentrations. Starting with this weak

but well-characterized lead, potency has been im-

proved almost three orders of magnitude. This

improvement was achieved via medicinal chemistry

guided by X-ray crystallography and molecular model-

ing. More importantly, the consistency of structure–

activity relationship data with crystallographic infor-

mation conﬁrms that we can utilize structure-based

drug design for eﬃciently eﬀecting further optimiza-

tion. This serves as a good starting point for develop-

ing low nanomolar PTP1B inhibitors by further

optimizing the occupancy of the active site and the

second phosphotyrosine-binding site. Eﬀort in that re-

gard will be reported in due course.

Compound 2a (780 mg, 2.70 mmol) was dissolved in

2 mL THF and 6 mL of 1 N LiOH. The mixture was

stirred at room temperature for 16 h. THF was removed

under reduced pressure and the solution was acidiﬁed

with 1 N HCl slowly. The white precipitate was ﬁltered,

washed with water, and dried to aﬀord compound 2

5. Experimental

5.1. General

1

(514 mg, 77%). H NMR (400 MHz, DMSO-d₆) d ppm

Commercial reagents and solvents were used as re-

ceived without further puriﬁcation. ¹H NMR spectra

were recorded on a Bruker 400 MHz spectrometer.

LC–MS data were collected using a Micromass LCT

mass spectrometer with electrospray ionization in con-

junction with a Waters 2795 LC system. Liquid chro-

4.86 (s, 2H) 7.37 (s, 1H) 7.42 (t, J = 7.45 Hz, 2H) 7.55

(t, J = 7.33 Hz, 1H) 7.83 (d, J = 8.08 Hz, 1H) 7.96 (d,

J = 7.83 Hz, 1H) 8.25 (s, 1H). HRMS: calcd for

C₁₃H₁₀O₅+ H+, 247.06010; found (ESI-FTMS,

[M+H]¹⁺), 247.0596.

matography (LC–MS) was performed using

a

5.2.3. 3-(Carboxymethoxy)furo[2,3-b]pyridine-2-carbox-

ylic acid (3). Following the literature procedure,²⁴ethyl

2-chloronicotinate (1.0 g, 5.4 mmol) was converted to

methyl 3-hydroxyfuro[2,3-b]pyridine-2-carboxylate (3a;

1.07 g, >95%) as a crude, oﬀ-white solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 4.03 (s, 3H) 7.33 (dd,

J = 7.83, 4.80 Hz, 1H) 8.13 (dd, J = 7.83, 1.77 Hz, 1H)

8.55 (dd, J = 4.80, 1.77 Hz, 1H).

Phenomenex C18 column (Mercury MS Luna 5l

C18(2), 20 · 2 mm) with mobile phase of 0.1% formic

acid in H₂O (A) and 0.1% formic acid in CH₃CN (B)

and a gradient of 15–100% B in 3 min followed by

1.5 min at 100% B. HRMS data was recorded on a

Bruker APEXIII-7T FTMS spectrometer with electro-

spray ionization.

5.2. Synthesis

A solution of 3a (200 mg, 1 mmol), K₂CO₃(143 mg,

1 mmol), and tert-butyl bromoacetate (153 mL, 1 mmol)

in acetone was heated at reﬂux for 16 h. The cooled solu-

tion was evaporated, the residue was dissolved in ethyl

acetate and water, and the organic phase was dried and

evaporated. The crude produce was puriﬁed by ﬂash

chromatography using 20% ethyl acetate/hexane as eluent

to give methyl 3-(2-tert-butoxy-2-oxoethoxy)furo[2,3-

b]pyridine-2-carboxylate (3b; 116 mg, 38%) as a white sol-

id. ¹H NMR (400 MHz, CDCl₃) d ppm 1.46 (s, 9H) 3.98

(s, 3H) 4.99 (s, 2H) 7.31 (dd, J = 7.83, 4.80 Hz, 1 H)

8.19 (dd, J = 7.83, 1.77 Hz, 1H) 8.52 (dd, J = 4.80,

1.77 Hz, 1H).

5.2.1.

3-(Carboxymethoxy)thieno[2,3-b]pyridine-2-car-

boxylic acid (1). Methyl 3-hydroxythieno[2,3-b]pyri-

dine-2-carboxylate (1a) was prepared following

literature procedures.³⁵To a 5 mL DMF solution of

1a (100 mg, 0.48 mmol) was added potassium carbonate

(132 mg, 0.96 mmol) followed by ethyl bromoacetate

(96 mg, 0.58 mmol). The reaction mixture was stirred

at 60 ꢁC for 4 h, then cooled to room temperature and

poured into water (50 mL). A white precipitate emerged,

and the solid was ﬁltered, washed with water, and dried

to aﬀord methyl 3-(2-ethoxy-2-oxoethoxy)thieno[2,3-

b]pyridine-2-carboxylate (1b; 115 mg, 81%). Compound

1b (110 mg, 0.37 mmol) was dissolved in 1 mL THF and

2 mL of 1 N LiOH. The mixture was stirred at room

temperature for 16 h. The solution was acidiﬁed with

1 N HCl slowly and a white precipitate was formed.

The solid was ﬁltered, washed with water, and dried to

A solution of 3b (155 mg, 0.4 mmol), LiOH hydrate

(63 mg, 4 equiv) in THF (1 mL), MeOH (1 mL), and

water (1 mL) was stirred at room temperature for 3 h.

The volatiles were evaporated, the white residue was dis-

solved in water (10 mL), and 10% HCl was added drop-

wise until the pH ꢀ4. The resulting precipitate was

ﬁltered and dried to provide 3 (39 mg, 44%) as a white

1

give compound 1 (73 mg, 78%). H NMR (400 MHz,

DMSO-d₆) d ppm 5.03 (s, 2H) 7.55 (dd, J = 8.34,

4.80 Hz, 1H) 8.36 (d, J = 8.34 Hz, 1H) 8.75 (d,

J = 4.80 Hz, 1H); HRMS: calcd for C₁₀H₇NO₅S + H+,

254.01177; found (ESI-FTMS, [M+H]¹⁺), 254.0116.

1

solid. H NMR (400 MHz, DMSO-d₆) d ppm 5.12 (s,

2H) 7.47 (dd, J = 7.83, 4.80 Hz, 1H) 8.31 (dd, J = 7.83,

1.52 Hz, 1H) 8.52 (dd, J = 4.80, 1.52 Hz, 1H); HRMS:

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2167

calcd for C₁₀H₇NO₆+ H+, 238.03462; found (ESI-

FTMS, [M+H]¹⁺), 238.0344.

slowly. A white precipitate was collected, washed with

water, and dried to give compound 5 (28 mg, 35%). H

1

NMR (400 MHz, DMSO-d₆) d ppm 2.58 (s, 3H) 5.04

(s, 1H) 7.84 (s, 1H) 9.07 (s, 1H); HRMS: calcd for

C₁₁H₉NO₅S + H+, 268.02742; found (ESI-FTMS,

[M+H]¹⁺), 268.0273.

5.2.4. 3-(Carboxymethoxy)benzo[b]thiophene-2-carboxyl-

ic acid (4). To a solution of 3-methoxycarbonylmethoxy-

benzo[b]thiophene-2-carboxylic acid methyl ester

(100 mg, 0.36 mmol) in THF (2.0 mL) was added

2.5 M LiOH in H₂O (0.3 mL). The reaction mixture

was stirred vigorously for 24 h before adding 1 N HCl

(1.5 mL). The solution was diluted with H₂O (50 mL)

and extracted with ethyl acetate (50 mL). The organic

layer was dried over MgSO₄and ﬁltered. The solvent

was removed under reduced pressure and then triturated

with diethyl ether to give 4 as a white solid (59 mg, 66%).

¹H NMR (400 MHz, MeOD) d ppm 5.23 (s, 2H) 7.64–

7.70 (m, 1H) 7.72–7.79 (m, 1H) 8.05 (d, J = 8.08 Hz,

1H) 8.23–8.30 (m, 1H); MS (ESI, neg.) Calcd for

C₁₁H₈O₅S m/z [MꢁH] = 251.01, found 251.09.

5.2.6.

3-(Carboxymethoxy)thieno[3,2-b]pyridine-2-car-

boxylic acid (6). A solution of 3-mercaptopicolinic acid

(750 mg, 3.9 mmol) and hydrochloric acid (3 drops) in

methanol (100 mL) was heated at reﬂux for 48 h. The

cooled solution was neutralized with aqueous sodium

hydroxide and sodium bicarbonate, and evaporated to

provide crude methyl 3-mercaptopicolinate (6a; 662 mg,

1

>95%). H NMR (400 MHz, CDCl₃) d ppm 4.02–4.03

(m, 3H) 7.29 (dd, J = 8.08, 4.55 Hz, 1H) 7.69 (dd,

J = 8.34, 1.52 Hz, 1 H) 8.50 (dd, J = 4.29, 1.52 Hz, 1H).

A solution of 6a (149 mg, 0.88 mmol), ethyl bromoace-

tate (367 mg, 2.5 equiv), and potassium carbonate

(520 mg) in DMF was heated at 90 ꢁC for 48 h. The

cooled solution was acidiﬁed with aqueous hydrochloric

acid, diluted with water (20 mL), and extracted with eth-

yl acetate (3· 30 mL). The combined organic layers were

washed with saturated aqueous sodium chloride and

dried with MgSO₄. The crude product was puriﬁed by

ﬂash chromatography (10–75% ethyl acetate/hexane)

to provide ethyl 3-(2-ethoxy-2-oxoethoxy)thieno[3,2-

b]pyridine-2-carboxylate (6b; 66 mg, 24%) as a red solid.

5.2.5. 3-(Carboxymethoxy)-6-methylthieno[3,2-c]pyridine-

2-carboxylic acid (5). Mercaptoacetic acid methyl ester

(0.19 mL, 2.14 mmole) and sodium methoxide (289 mg,

5.35 mmole) were dissolved in 40 mL DMF. The mixture

was stirred at room temperature for 5 min. 2,4-Dichloro-

6-methyl-nicotinic acid ethyl ester (0.5 g, 2.14 mmole) in

10 mL DMF was then added. The mixture was stirred at

room temperature for 2 h. tert-Butyl bromoacetate

(0.43 mL, 3.21 mmole) was then added and the mixture

was stirred at 70 ꢁC for 16 h. DMF was removed under re-

duced pressure. DCM (20 mL) was added and the organic

layer was washed with water, saturated NaHCO₃, brine,

andthendriedoveranhydrousNa₂SO₄. Thecrude product

was puriﬁed by ﬂash chromatography using ethyl acetate/

hexane (0–20%) as eluent. Pure fractions were combined

and evaporated to give methyl 3-(2-tert-butoxy-2-oxoeth-

oxy)-4-chloro-6-methylthieno[3,2-c]pyridine-2-carboxylate

(5a; 404 mg, 51% two steps) as a yellowish green solid. ¹H

NMR (400 MHz, CDCl₃) d ppm 1.47 (s, 9H) 2.63 (s, 3H)

3.92 (s, 3H) 4.86 (s, 2H) 7.44 (s, 1H); MS (ESI, pos.) calcd

for C₁₆H₁₈ClNO₅S m/z [M+H] = 372.06, found 372.18.

¹H NMR (400 MHz, CDCl₃)

d

ppm 1.24 (t,

J = 7.20 Hz, 3H) 1.41 (t, J = 7.07 Hz, 3H) 4.23 (q,

J = 7.07 Hz, 2H) 4.41 (q, J = 7.07 Hz, 2H) 5.47 (s,

2H) 8.08 (dd, J = 8.34, 1.52 Hz, 1H) 8.66 (dd,

J = 4.55, 1.52 Hz, 1H).

A solution of 6b (55 mg, 0.18 mmol) and LiOH hydrate

(37 mg, 5 equiv) in THF (2 mL) and water (2 mL) was

stirred at room temperature for 18 h. The solution was

evaporated, acidiﬁed to pH 4, and cooled to 0 ꢁC. The

resulting precipitate was collected by ﬁltration and dried

under vacuum to provide 7 (4 mg, 9%) as an oﬀ-white

1

Methyl 4-chloro-3-(2-ethoxy-2-oxoethoxy)-6-methyl-thi-

eno[3,2-c]pyridine-2-carboxylate (5a⁰) was synthesized

following the procedure in the preparation of 5a.

solid. H NMR (400 MHz, DMSO-d₆) d ppm 5.29 (s,

2H) 7.34 (dd, J = 8.21, 4.42 Hz, 1H) 7.50 (dd, J = 8.34,

4.55 Hz, 1H) 8.46 (dd, J = 8.34, 1.52 Hz, 1H) 8.69 (dd,

J = 4.55, 1.52 Hz, 1H).

A solution of 5a⁰(640 mg, 1.9 mmol) in 10 mL MeOH/

EtOAc (1:1) was shaken under a hydrogen atmosphere

at 45 psi in the presence of 10% Pd/C (0.5 g) for 16 h.

The mixture was ﬁltered and the solvent was removed

under reduced pressure. The crude product was puriﬁed

by ﬂash chromatography eluting with a gradient from

10% to 75% ethyl acetate in hexane. Pure fractions were

combined and evaporated to give methyl 3-(2-ethoxy-2-

oxoethoxy)-6-methylthieno[3,2-c]pyridine-2-carboxylate

(5b; 134 mg, 23%) as a yellow solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.27 (t, J = 7.07 Hz, 3H)

2.68 (s, 3H) 3.91 (s, 3H) 4.24 (q, J = 7.07 Hz, 2H) 5.09

(s, 2H) 7.49 (s, 1H) 9.22 (s, 1H).

5.2.7. 3-(Carboxymethoxy)thieno[3,2-b]thiophene-2-car-

boxylic acid (7). A solution of methyl 3-chlorothioph-

ene-2-carboxylate

(2.75 g,

15.6 mmol)

methyl

thioglycolate (1.42 mL, 15.6 mmol) and potassium car-

bonate (4.74 g, 31.2 mmol) in DMF (60 mL) was heated

at 60 ꢁC for 18 h. The cooled solution was diluted with

water (100 mL) and extracted with ethyl acetate (3·

50 mL). The combined organic layers were dried over

MgSO₄, ﬁltered, evaporated, and puriﬁed by ﬂash chro-

matography (2–35% ethyl acetate/hexane) to provide

methyl 3-(2-methoxy-2-oxoethylthio)thiophene-2-car-

1

boxylate (7a; 81 mg, 5%). H NMR (400 MHz, CDCl₃)

d ppm 3.75 (s, 3H) 3.76 (s, 2H) 3.89 (s, 3H) 7.09 (d,

J = 5.31 Hz, 1H) 7.26 (s, 1H) 7.51 (d, J = 5.31 Hz, 1H).

Compound 5b (92 mg, 0.3 mmol) and LiOH (56 mg,

2.34 mmol) were suspended in 2 mL THF and 3 mL

water. The mixture was stirred at room temperature

overnight. The reaction was acidiﬁed with 1 N HCl

A solution of 7a (240 mg, 0.97 mmol) and sodium tert-

butoxide (230 mg, 2.5 equiv) in DMF (7 mL) was heated

2168

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

at 60 ꢁC for 18 h. The cooled solution was acidiﬁed with

aqueous hydrochloric acid and extracted with ethyl ace-

tate (3· 20 mL). The combined organic layers were dried

over MgSO₄, ﬁltered, and evaporated to provide methyl

rated under reduced pressure followed by addition of

15 mL DCM. The organic layer was washed with water,

brine, and dried over anhydrous Na₂SO₄. The crude

product was puriﬁed by ﬂash chromatography eluting

with a gradient of ethyl acetate in hexane. Pure fractions

were combined and evaporation of solvents gave 6-chlo-

ro-3-(1-methoxycarbonyl-ethoxy)-benzo[b]thiophene-2-

carboxylic acid methyl ester (9a; 254 mg, 94%) as a

3-hydroxythieno[3,2-b]thiophene-2-carboxylate

(7b;

163 mg, 76%) as a red solid. ¹H NMR (400 MHz,

CDCl₃) d ppm 3.93 (s, 3 H) 7.21 (d, J = 5.31 Hz, 1H)

7.63 (d, J = 5.31 Hz, 1 H).

1

white solid. H NMR (400 MHz, CDCl₃) d ppm 1.71

A solution of 7b (140 mg, 0.85 mmol) ethyl bromoace-

tate (100 lL, 0.94 mmol) and sodium tert-butoxide

(75 mg, 0.78 mmol) in DMF (3 mL) was heated at

40 ꢁC for 2 h. The cooled solution was acidiﬁed with

aqueous hydrochloric acid and extracted with ethyl ace-

tate. The combined organic layers were dried over

MgSO₄, ﬁltered, and evaporated to provide methyl 3-

(2-ethoxy-2-oxoethoxy)thieno[3,2-b]thiophene-2-car-

(d, J = 6.82 Hz, 3H) 3.70 (s, 3H) 3.90 (s, 3H) 5.30 (q,

J = 6.82 Hz, 1H) 7.37 (dd, J = 8.59, 1.77 Hz, 1H) 7.70

(d, J = 1.77 Hz, 1H) 7.98 (d, J = 8.59 Hz, 1H).

Compound 9a (250 mg, 0.76 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give

compound 9 (211 mg, 92%) as a white solid. H NMR

1

(400 MHz, DMSO-d₆) d ppm 1.56 (d, J = 6.82 Hz, 3H)

5.26 (q, J = 6.82 Hz, 1H) 7.50 (dd, J = 8.59, 1.77 Hz,

1H) 7.95 (d, J = 8.59 Hz, 1H) 8.13 (d, J = 1.77 Hz,

1H). HRMS: calcd for C₁₂H₉ClO₅S + H+, 300.99320;

found (ESI-FTMS, [M+H]¹⁺), 300.9934.

1

boxylate (7c; 195 mg, 64%) as a pale-brown liquid. H

NMR (400 MHz, CDCl₃) d ppm 1.28 (t, J = 7.07 Hz,

3H) 3.89 (s, 3H) 4.26 (q, J = 7.07 Hz, 2H) 5.03 (s, 2H)

7.19 (d, J = 5.31 Hz, 1H) 7.58 (d, J = 5.31 Hz, 1H).

A solution of 7c (71 mg, 0.24 mmol) and LiOH hydrate

(37 mg, 0.88 mmol) in THF (2 mL) and water (2 mL)

was heated at 40 ꢁC for 4 h. The cooled solution was

evaporated, and the resulting aqueous mixture was acid-

iﬁed with hydrochloric acid. The resulting yellow precip-

itate, compound 7 (60 mg, 98%), was collected by

5.2.10. 3-(Carboxy-ﬂuoro-methoxy)-6-chloro-benzo[b]thi-

ophene-2-carboxylic acid (10). 6-Chloro-3-hydroxy-

benzo[b]thiophene-2-carboxylic acid methyl ester

(200 mg, 0.824 mmol) was dissolved in 10 mL DMF, fol-

lowed by addition of K₂CO₃(342 mg, 2.47 mmol) and

bromo-ﬂuoro-acetic

acid

ethyl

ester

(117 lL,

1

ﬁltration. H NMR (400 MHz, DMSO-d₆) d ppm 4.54

0.99 mmol). The mixture was stirred at 70 ꢁC for 16 h.

DMF was evaporated under reduced pressure, followed

by addition of 15 mL DCM. The organic layer was

washed with water, brine, and dried over anhydrous

Na₂SO₄. The crude product was puriﬁed by ﬂash chro-

matography eluting with a gradient of ethyl acetate in

hexane. Pure fractions were combined and evaporation

of solvents gave 6-chloro-3-(ethoxycarbonyl-ﬂuoro-

methoxy)-benzo[b]thiophene-2-carboxylic acid methyl

ester (10a; 161 mg, 56%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.43 (t, J = 7.07 Hz, 3H)

3.93 (s, 3H) 4.44 (q, J = 7.07 Hz, 2H) 6.17 (d,

J = 59.37 Hz, 1H) 7.42 (dd, J = 8.59, 1.77 Hz, 1H) 7.76

(d, J = 1.77 Hz, 1H) 7.91 (d, J = 8.59 Hz, 1H).

(s, 2 H) 7.45 (d, J = 5.31 Hz, 1H) 7.85 (d, J = 5.31 Hz,

1 H); HRMS: calcd for C9H6O5S2 + H+, 258.97294;

found (ESI-FTMS, [M+H]1+), 258.9726.

5.2.8. 3-Carboxymethoxy-6-chloro-benzo[b]-thiophene-2-

carboxylic acid (8). A solution of 6-chloro-3-hydroxy-

benzo[b]thiophene-2-carboxylic acid methyl ester

(5.28 g, 21.8 mmol), K₂CO₃(4.51 g, 32.7 mmol), and

tert-butyl bromoacetate (4.2 mL 28.3 mmol) in 160 mL

DMF was stirred at 50 ꢁC for 18 h. Water was added

and the mixture was extracted with ether (3· 75 mL).

The organic layers were combined, washed with brine,

dried over anhydrous MgSO₄, and ﬁltered. Solvent

was removed under reduced pressure to give 3-tert-but-

oxycarbonylmethoxy-6-chloro-benzo[b]thiophene-2-car-

boxylic acid methyl ester (8a; 7.70 g, 99%) as slightly

Compound 10a (81 mg, 0.23 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give

compound 10 (67 mg, 95%) as a pink solid. H NMR

1

pink solids. H NMR (400 MHz, CDCl₃) d ppm 1.46

(s, 9H) 3.91 (s, 3H) 4.92 (s, 2H) 7.38 (dd, J = 8.59,

2.02 Hz, 1H) 7.71 (d, J = 2.02 Hz, 1H) 8.03 (d,

J = 8.59 Hz, 1H).

(400 MHz, DMSO-d₆) d ppm 6.23 (d, J = 57.35 Hz,

1H) 7.55 (d, J = 8.34 Hz, 2 H) 7.81 (d, J = 8.34 Hz,

2H) 8.20 (s, 1H). HRMS: calcd for C₁₁H₆ClFO₅S + H+,

304.96813; found (ESI-FTMS, [M+H]¹⁺), 304.9684.

Compound 8a (100 mg, 0.28 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give

compound 8 (72 mg, 90%) as a white solid. H NMR

(400 MHz, DMSO-d₆) d ppm 5.00 (s, 2H) 7.52 (d,

J = 8.59 Hz, 1H) 7.97 (d, J = 8.59 Hz, 1H) 8.14 (s, 1H).

5.2.11. 6-Bromo-3-carboxymethoxy-benzo[b]-thiophene-2-

carboxylic acid (11). LiOH monohydrate (2.0 g, 48 mmol)

was added to a solution of 4-bromo-2-ﬂuoro-benzoic acid

methyl ester (5.5 g, 24 mmol) and methyl thioglycolate

(2.1 mL, 24 mmol) in 30 mL DMF at 0 ꢁC. The mixture

was stirred at 0 ꢁC for 30 min, warmed to room tempera-

ture, added to water, and acidiﬁed with 1 N HCl. The

precipitate was ﬁltered, washed with water, and dried to

give 6-bromo-3-hydroxy-benzo[b]thiophene-2-carboxylic

acid methyl ester (11a; 4.89 g, 72%). ¹H NMR (400 MHz,

DMSO-d₆) d ppm 3.81 (s, 3H) 7.55 (d, J = 8.34, 1.26 Hz,

1H) 7.82 (d, J = 8.34 Hz, 1H) 8.19 (s, 1H).

1

5.2.9. 3-(1-Carboxy-ethoxy)-6-chloro-benzo[b]-thiophene-

2-carboxylic acid (9). 6-Chloro-3-hydroxy-benzo[b]thio-

phene-2-carboxylic acid methyl ester (200 mg,

0.82 mmol) was dissolved in 10 mL DMF, followed by

addition of K₂CO₃(342 mg, 2.47 mmol) and 2-bromo-

propionic acid methyl ester (110 lL, 0.99 mmol). The

mixture was stirred at 70 ꢁC for 16 h. DMF was evapo-

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2169

Ethyl bromoacetate (3.0 mL, 27 mmol) was added to a

mixture of 11a (5.1 g, 18 mmol) and sodium tert-butox-

ide (1.9 g, 20 mmol) in 75 mL DMF. The mixture was

stirred at 80 ꢁC overnight. Water was then added and

a precipitate emerged. The solid was ﬁltered, washed

with water, and recrystallized in MeOH to give 6-bro-

mo-3-ethoxycarbonylmethoxy-benzo[b]thiophene-2-car-

boxylic acid methyl ester (11b; 4.5 g, 67%) as a slightly

red at 60 ꢁC until TLC showed the absence of 12a. The

crude product was puriﬁed by ﬂash chromatography to

give 3-tert-butoxycarbonylmethoxy-6-(4-hydroxy-phen-

yl)-benzo[b]thiophene-2-carboxylic acid methyl ester

(13a; 55 mg, 47%) as

a

white solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.48 (s, 9H) 3.92 (s, 3H)

4.89 (s, 1H) 4.93 (s, 2H) 6.93 (d, J = 8.84 Hz, 2H) 7.54

(d, J = 8.84 Hz, 2H) 7.60 (dd, J = 8.34, 1.52 Hz, 1H)

7.85 (d, J = 1.52 Hz, 1H) 8.12 (d, J = 8.34 Hz, 1H).

1

pink solid. H NMR (400 MHz, CDCl₃) d ppm 1.27

(t, J = 7.07 Hz, 3H) 3.91 (s, 3H) 4.24 (q, J = 7.07 Hz, 2

H) 5.02 (s, 2H) 7.53 (dd, J = 8.59, 1.77 Hz, 1H) 7.89

(dd, J = 1.77, 0.51 Hz, 1H) 7.96 (dd, J = 8.59, 0.51 Hz,

1 H).

Compound 13a (50 mg, 0.12 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give

compound 13 (29 mg, 69%) as a light-yellow solid. H

1

NMR (400 MHz, DMSO-d₆) d ppm 5.00 (s, 2H) 6.88

(d, J = 8.84 Hz, 2H) 7.61 (d, J = 8.84 Hz, 2H) 7.71 (d,

J = 8.59, 1H) 7.99 (d, J = 8.59 Hz, 1H) 8.14 (s, 1H)

9.67 (s, 1H). HRMS: calcd for C₁₇H₁₂O₆S + H+,

345.04274; found (ESI-FTMS, [M+H]¹⁺), 345.0428.

Compound 11b (100 mg, 0.27 mmol) was hydrolyzed

following the procedure in the preparation of 1 to give

compound 11 (73 mg, 82%) as a white solid. H NMR

(400 MHz, DMSO-d₆) d ppm 4.99 (s, 2H) 7.64 (dd,

J = 8.59, 1.77 Hz, 1H) 7.90 (dd, J = 8.59, 0.51 Hz, 1H)

8.29 (dd, J = 1.77, 0.51 Hz, 1H). HRMS: calcd for

C₁₁H₇BrO₅S + H+, 330.92703; found (ESI-FTMS,

[M+H]¹⁺), 330.9272.

1

5.2.14. 3-Carboxymethoxy-6-thiophen-2-yl-benzo[b]-thio-

phene-2-carboxylic acid (14). Following the procedure in

the preparation of 12b, 12a (90 mg, 0.25 mmol, 1 equiv),

2-thiophene boronic acid (65 mg, 0.5 mmol), Pd[P(t-

Bu)₃]₂(20 mg, 0.039 mmol), and KF (50 mg, 0.86 mmol)

were used and the reaction mixture was stirred at 60 ꢁC

for 48 h. The crude product was puriﬁed by ﬂash chroma-

tography to yield 3-tert-butoxycarbonylmethoxy-6-thio-

phen-2-yl-benzo[b]thiophene-2-carboxylic acid methyl

5.2.12. 3-Carboxymethoxy-6-phenylbenzo[b]-thiophene-2-

carboxylic acid (12). A solution of 6-chloro-3-hydroxy-

benzo[b]thiophene-2-carboxylic acid methyl ester

(5.28 g, 21.8 mmol), K₂CO₃(4.51 g, 32.7 mmol), and

tert-butyl bromoacetate (4.2 mL, 28.3 mmol) in 160 mL

DMF was stirred at 50 ꢁC for 18 h. Water was added

and the mixture was extracted with ether (3· 75 mL).

The organic layers were combined, washed with brine,

dried over anhydrous MgSO₄, and ﬁltered. Solvent was

removed under reduced pressure to give 3-tert-butoxycar-

bonylmethoxy-6-chloro-benzo[b]thiophene-2-carboxylic

acid methyl ester (12a; 7.70, 99%) as slightly pink solids.

¹H NMR (400 MHz, CDCl₃) d ppm 1.46 (s, 9H) 3.91 (s,

3H) 4.92 (s, 2H) 7.38 (dd, J = 8.59, 2.02 Hz, 1H) 7.71 (d,

J = 2.02 Hz, 1H) 8.03 (d, J = 8.59 Hz, 1H).

1

ester (14a; 86 mg, 62%) as an oﬀ-white solid. H NMR

(400 MHz, CDCl₃) d ppm 1.47 (s, 9H) 3.92 (s, 3H) 4.93

(s, 2H) 7.12 (dd, J = 5.05, 3.54 Hz, 1H) 7.35 (dd,

J = 5.05, 1.14 Hz, 1H) 7.42 (dd, J = 3.54, 1.14 Hz, 1H)

7.68 (dd, J = 8.34, 1.52 Hz, 1H) 7.94 (dd, J = 1.52,

0.63 Hz, 1H) 8.09 (dd, J = 8.34, 0.63 Hz, 1H).

Compound 14a (71 mg, 0.18 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give

compound 14 (4 mg, 7%) as a yellow solid. H NMR

1

(400 MHz, DMSO-d₆) d ppm 5.00 (s, 2H) 7.19 (dd,

J = 5.05, 3.54 Hz, 1H) 7.64 (dd, J = 5.05, 1.14 Hz, 1H)

7.68 (dd, J = 3.54, 1.14 Hz, 1 H) 7.78 (dd, J = 8.34,

1.52 Hz, 1H) 7.99 (d, J = 8.34 Hz, 1H) 8.27 (d,

J = 1.52 Hz, 1H).

Compound 12a (90 mg, 0.25 mmol), Pd₂(dba)₃(4.4 mg,

0.0048 mmol), HP(t-Bu)₃BF₄(2.9 mg, 0.01 mmol), KF

(43 mg, 0.75 mmol), and phenyl boronic acid (34 mg,

0.28 mmol) in 2 mL THF under nitrogen were stirred

at 60 ꢁC for 5 h. The crude mixture was absorbed onto

silica and puriﬁed by ﬂash chromatography to give

3-tert-butoxycarbonylmethoxy-6-phenylbenzo[b]-thio-

phene-2-carboxylic acid methyl ester (12b: 20 mg, 20%)

as a white solid. Compound 12b (20 mg, 0.05 mmol) was

then hydrolyzed following the procedure in the prepara-

tion of 1 to give compound 12 (6 mg, 38%) as a white

5.2.15. 3-Carboxymethoxy-6-(5-methyl-1-phenyl-1H-pyr-

azol-3-ylcarbamoyl)-benzo[b]thiophene-2-carboxylic acid

(15). A solution of 4-(methoxycarbonyl)-3-nitrobenzoic

acid (5.0 g, 22 mmol), isobutylene (20 mL), and concen-

trated sulfuric acid (1 mL) in dioxane (25 mL) was

sealed in a Parr bottle and shaken at room temperature

overnight. A needle was inserted through the silicone

stopper to release excessive pressure. The vessel was

carefully removed from the shaker apparatus and the

reaction solution was stirred open to the atmosphere

for 1 h. The solution was diluted with ethyl acetate

(300 mL) and water (50 mL), neutralized with aqueous

sodium hydroxide, washed with water and brine, and

dried over anhydrous MgSO₄. Evaporation of solvent

yielded tert-butyl 4-(methoxycarbonyl)-3-nitrobenzoate

(15a; 3.38 g, 55%). ¹H NMR (400 MHz, CDCl₃) d

ppm 1.62 (s, 9 H) 3.95 (s, 3H) 7.79 (d, J = 7.83 Hz,

1H) 8.27 (dd, J = 7.83, 1.52 Hz, 1H) 8.48 (d,

J = 1.52 Hz, 1H).

1

solid. H NMR (400 MHz, DMSO-d₆) d ppm 5.00 (s,

2H) 7.42 (m, 1H) 7.51 (t, J = 7.58 Hz, 2H) 7.79 (m,

3H) 8.05 (d, J = 8.34 Hz, 1H) 8.26 (d, J = 0.76 Hz,

1H). HRMS: calcd for C₁₇H₁₂O₅S + H+, 329.04782;

found (ESI-FTMS, [M+H]¹⁺), 329.048.

5.2.13. 3-Carboxymethoxy-6-(4-hydroxyphenyl)-benzo[b]thio-

phene-2-carboxylic acid (13). Following the procedure in

the preparation of 12b, 12a (102 mg, 0.29 mmol), 4-

hydroxyphenylboronic acid (51 mg, 0.37 mmol),

Pd(OAc)₂(8 mg, 0.036 mmol), 2-(dicyclohexylphosphi-

no)-biphenyl (24 mg, 0.068 mmol), and KF (50 mg,

0.86 mmol) were used and the reaction mixture was stir-

2170

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

A solution of 15a (3.3 g, 12 mmol) and methyl thioglyco-

late (1.6 mL, 8.0 mmol) in 15 mL DMF was cooled to

0 ꢁC, and LiOH monohydrate (0.99 g, 24 mmol) was add-

ed over a 15 min period. The reaction mixture was stirred

at 0 ꢁC for an additional 30 min, allowed to warm to room

temperature, and stirred an additional 2 h. The solution

was acidiﬁed, diluted with ethyl acetate (300 mL), and

washed with water and brine, dried over anhydrous

MgSO₄, ﬁltered, and concentrated in vacuo to provide

6-tert-butyl 2-methyl 3-hydroxybenzo[b]thiophene-2,6-

5.2.16. 3-Carboxymethoxy-7-chloro-benzo[b]-thiophene-

2-carboxylic acid (16). Following the procedure in the

preparation of 15b, 3-chloro-2-nitrobenzoic acid methyl

ester (1.98 g, 9.2 mmol) was converted to 7-chloro-3-

hydroxybenzo[b]thiophene-2-carboxylic acid methyl es-

ter (16a; 1.12 g, 50%) after recrystallization from etha-

nol. H NMR (400 MHz, CDCl₃) d ppm 3.98 (s, 3H)

7.38 (t, J = 7.83 Hz, 1H) 7.49–7.54 (m, 1H) 7.87 (dd,

J = 8.08, 1.01 Hz, 1H) 10.09 (s, 1H).

1

dicarboxylate (15 b; 3.10 g, 84%) as a yellow solid. H

Following the procedure in the preparation of 12a, 16a

(1.12 g, 4.62 mmol) was converted to 7-chloro-3-meth-

oxycarbonylmethoxy-benzo[b]thiophene-2-carboxylic

acid methyl ester (16b; 1.34 g, 92%). ¹H NMR

(400 MHz, DMSO-d₆) d ppm 3.69 (s, 3H) 3.88 (s, 3H)

5.12 (s, 2H) 7.57 (t, J = 7.83 Hz, 1H) 7.75 (dd,

J = 7.71, 0.88 Hz, 1H) 8.01 (dd, J = 8.08, 0.88 Hz, 1H).

NMR (400 MHz, CDCl₃) d ppm 1.63 (s, 9H) 3.97 (s,

3H) 7.96 (d, J = 8.4 Hz, 1H) 8.00 (d, J = 8.4 Hz, 1H)

8.39 (s, 1 H).

Compound 15b (3.1 g, 10 mmol) was alkylated with

methyl bromoacetate following the procedure in the prep-

aration of 12a to give 6-tert-butyl 2-methyl 3-(2-methoxy-

2-oxoethoxy)benzo[b]thiophene-2,6-dicarboxylate (15c;

Compound 16 b (100 mg, 0.32 mmol) was hydrolyzed

following the procedure in the preparation of 1 to give

compound 16 (65 mg, 71%). ¹H NMR (400 MHz,

DMSO-d₆) d ppm 5.02 (s, 2 H) 7.53 (t, J = 7.83 Hz,

1H) 7.70 (dd, J = 6.82, 1.01 Hz, 1H) 7.98 (dd, J = 8.08,

1.01 Hz, 1H). HRMS (ESI) calcd for C₁₁H₆ClO₅S

284.96299; found 284.96231.

1

3.17 g, 83%). H NMR (400 MHz, CDCl₃) d ppm 1.61

(s, 9H) 3.77 (s, 3H) 3.91 (s, 3H) 5.02 (s, 2H) 7.99 (d,

J = 8.80 Hz, 1H) 8.08 (d, J = 8.80 Hz, 1H) 8.37 (s, 1H).

A solution of 15c (2.80 g, 7.4 mmol) and triﬂuoroacetic

acid (6 mL) in DCM (40 mL) was stirred at room tem-

perature for 4.5 h. Solvents were removed under reduced

pressure to give 3-(2-methoxy-2-oxoethoxy)-2-(meth-

oxycarbonyl)benzo[b]thiophene-6-carboxylic acid (15d;

5.2.17. 3-Carboxymethoxy-7-methyl-benzo[b]-thiophene-2-

carboxylic acid (17). 3-Methyl-2-nitro-benzoic acid methyl

ester (3.2 g, 17 mmol) was converted to 3-hydroxy-7-meth-

yl-benzo[b]thiophene-2-carboxylic acid methyl ester,

which was subsequently converted to 3-methoxycarbonyl-

1

2.60 g, >95%). H NMR (400 MHz, DMSO-d₆) d ppm

3.69 (s, 3H) 3.87 (s, 3H) 5.10 (s, 2H) 8.00 (d,

J = 8.34 Hz, 1H) 8.09 (d, J = 8.34 Hz, 1H) 8.62 (s, 1H).

methoxy-7-methylbenzo[b]thiophene-2-carboxylic

acid

A solution of 15d (0.97 g, 3 mmol) and thionyl chloride

(10 mL) in toluene (50 mL) was heated at reﬂux over-

night. The cooled solution was concentrated in vacuo,

and the residual thionyl chloride was removed by

azeotroping with added toluene. The crude acid chlo-

ride, methyl 6-(chlorocarbonyl)-3-(2-methoxy-2-oxoeth-

oxy)benzo[b]-thiophene-2-carboxylate (15e), was used

without additional puriﬁcation. A solution of 15e

(75 mg, 0.22 mmol), 3-amino-5-methyl-1-phenylpyraz-

ole (57 mg, 0.33 mmol), and pyridine (53 lL, 0.66 mmol)

in DCM (3 mL) was stirred at room temperature for 4 h.

The reaction was quenched with aqueous sodium bicar-

bonate, diluted with ethyl acetate (50 mL) and washed

with brine, dried over MgSO₄, ﬁltered, and concentrated

in vacuo to provide a crude yellow solid. The crude

product was recrystallized from ethanol to give 3-meth-

oxycarbonylmethoxy-6-(5-methyl-1-phenyl-1H-pyrazol-

methyl ester (17a; 1.56 g, 32% 2 steps), following the proce-

dure in the preparation of 15b and 12a. ¹H NMR

(400 MHz, CDCl₃) d ppm 2.51 (s, 3H) 3.80 (s, 3 H) 3.92

(s, 3H) 5.02 (s, 2H) 7.30 (d, J = 7.07 Hz, 1H) 7.28–7.32

(m, 1H) 7.91 (d, J = 8.08 Hz, 1H).

Compound 17a (100 mg, 0.34 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give com-

pound 17 (81 mg, 90%). ¹H NMR (400 MHz, DMSO-d₆)

d ppm 2.45 (s, 3H) 4.68 (s, 2H) 7.30 (d, J = 7.07 Hz, 1H)

7.33–7.39 (m, 1H) 7.72 (d, J = 7.83 Hz, 1H). HRMS:

calcd for C₁₂H₁₀O₅S + H+, 267.03217; found (ESI-

FTMS, [M+H]¹⁺), 267.0322.

5.2.18.

3-Carboxymethoxy-naphtho[1,2-b]thiophene-2-

carboxylic acid (18). To an oven-dried 50 mL round-bot-

tomed ﬂask under a nitrogen atmosphere were added

1-bromo-naphthalene-2-carboxylic acid ethyl ester

(0.244 g, 0.88 mmol), methyl thioglycolate (0.08 mL,

0.88 mmol), 20 mL DMF, and then sodium hydride

(0.075 g of a 60% suspension in mineral oil, 1.84 mmol).

The resulting mixture was allowed to stir at room tem-

perature for 16 h. To the solution, 25 mL of 1.2 N

HCl was added, at which point a precipitate formed.

The solid was ﬁltered and dried to give 3-hydroxy-naph-

tho[1,2-b]thiophene-2-carboxylic acid methyl ester (18a;

0.134 g, 60%) as an oﬀ-white solid. ¹H NMR (400 MHz,

CDCl₃) d ppm 3.99 (s, 3H) 7.61 (m, 2H) 7.75 (d,

J = 8.59 Hz, 1H) 7.87 (d, J = 8.84 Hz, 1H) 7.94 (dd,

J = 6.06, 3.54 Hz, 1H) 8.10 (dd, J = 6.06, 3.28 Hz, 1

H) 10.12 (s, 1H).

3-ylcarbamoyl)-benzo[b]thiophene-2-carboxylic

acid

methyl ester (15f; 53 mg, 50%) as a white solid. ¹H

NMR (400 MHz, DMSO-d₆) d ppm 3.33 (s, 3H) 3.69

(s, 3H) 3.87 (s, 3H) 5.11 (s, 2H) 6.31 (s, 1H) 7.31 (m,

1H) 7.45 (m, 2H) 7.54 (m, 2H) 7.92 (d, J = 8.59 Hz,

1H) 8.10 (d, J = 8.59 Hz, 1H) 8.49 (s, 1H) 10.57 (s, 1H).

Compound 15f (53 mg, 0.11 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give com-

pound 15 (39 mg, 79%). ¹H NMR (400 MHz, DMSO-d₆)

d ppm 2.26 (s, 3 H) 5.02 (s, 2H) 6.31 (s, 1H) 7.31 (m, 1H)

7.45 (m, 5H) 7.54 (m, 2H) 7.89 (m, 1H) 8.07 (d,

J = 8.59 Hz, 1H) 8.45 (s, 1H) 10.54 (s, 1H). HRMS (ESI)

calcd for C₂₂H₁₇N₃O₆S 452.09109; found 452.08967.

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2171

To an oven-dried 50 mL round-bottomed ﬂask under a

nitrogen atmosphere were added 18a (0.134 g,

0.52 mmol), ethyl bromoacetate (0.06 mL, 0.52 mmol),

potassium carbonate (0.072 g, 052 mmol), and 20 mL

DMF. The resulting mixture was allowed to stir for 16 h

at room temperature. To the mixture, 30 mL of water

was added, at which point a precipitate formed. The solid

was ﬁltered and dried to give 3-ethoxycarbonylmethoxy-

naphtho[1,2-b]thiophene-2-carboxylic acid methyl ester

(18b; 0.116 g, 65%) as a white solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.28 (t, J = 7.20 Hz, 3H) 3.95

(s, 3H) 4.26 (q, J = 7.16 Hz, 2H) 5.07 (s, 2H) 7.58 (m,

2H) 7.76 (m, 1H) 7.93 (m, 1H) 8.02 (m, 1H) 8.11 (m, 1H).

boxylate (20a; 162 mg, 61%) as an orange solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 3.96 (s, 3H) 7.51–7.40 (dd,

J = 6.06, 3.28 Hz, 2H) 7.89–7.80 (m, 2H) 10.06 (s, 1H).

tert-Butyl bromoacetate (0.082 mL, 0.56 mmol) was

added to a mixture of sodium tert-butoxide (50 mg,

0.67 mmol) and 20a (113 mg, 0.43 mmol) in DMF

(10 mL). The resulting mixture was stirred at 60 ꢁC for

16 h. Addition of water followed by extraction with eth-

yl acetate and washing with water gave the crude prod-

uct. Puriﬁcation by ﬂash chromatography gave methyl

3-(tert-butoxycarbonylmethoxy)thieno[3,2-b][1]benzo-

thiophene-2-carboxylate (20b; 140 mg, 86%) of as an

1

orange solid. H NMR (400 MHz, CDCl₃) d ppm 1.47

(s, 9H) 3.91 (s, 3H) 4.97 (s, 2H) 7.47–7.41 (m, 2H)

7.90–7.82 (m, 2H).

To a 25 mL round-bottomed ﬂask, 18b (0.116 g,

0.34 mmol) and 5 mL THF were added, followed by addi-

tion of a solution of LiOH hydrate (0.057 g, 1.35 mmol) in

3 mL water. This mixture was allowed to stir at room tem-

perature for 16 h. At this time 15 mL of 1.2 N HCl was

added, and a precipitate formed. The solid was ﬁltered

Compound 20b (76 mg, 0.2 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to give com-

pound 19 (57 mg, 92%). ¹H NMR (400 MHz, DMSO-d₆)

d ppm 4.69 (d, J = 1.01 Hz, 2H) 7.55–7.46 (m, 2H) 8.08–

8.02 (m, 1H) 8.13–8.09(m, 1H). HRMS (ESIꢁ, m/z) calcd

for [MꢁH]^1ꢁ, 306.97403, found, 306.97322.

1

and dried to give 18 (0.096 g, 93%) as a tan solid. H

NMR (400 MHz, DMSO-d₆) d ppm 5.06 (s, 2H) 7.69

(m, 2H) 7.91 (d, J = 8.84 Hz, 1 H) 7.95 (m, 1H) 8.08 (m,

1H) 8.19 (t, J = 4.0 Hz, 1H); HRMS: calcd for

C₁₅H₁₀O₅S + H+, 303.03217; found (ESI-FTMS,

[M+H]¹⁺), 303.0325.

5.2.21. 3-(Carboxymethoxy)-5-chlorothieno[3,2-b][1]benzo-

thiophene-2-carboxylic acid (21). Triﬂuoromethanesulfo-

nyl anhydride (0.49 mL, 2.9 mmol) was added dropwise

to a 0 ꢁC solution of 7-chloro-3-hydroxy-benzo[b]thio-

phene-2-carboxylic acid methyl ester (355 mg, 1.46 mmol)

in Et₃N (8 mL) and DCM (5 mL). After reaching room

temperature, the mixture was adsorbed onto silica gel

and puriﬁed by ﬂash chromatography to yield 7-chloro-

3-triﬂuoromethane-sulfonyloxy-benzo[b]thiophene-2-car-

boxylic acid methyl ester (21a; 110 mg, 20%) as a white sol-

id. ¹HNMR(400 MHz,CDCl₃)dppm4.01(s, 3H), 7.50(t,

J = 8.0 Hz, 1H), 7.56–7.65 (m, 1H), 7.77 (d, J = 8.1 Hz,

1H).

5.2.19.

3-Carboxymethoxy-thieno[3,2-c]quinoline-2-

carboxylic acid (19). Following the procedure in the

preparation of 18a, except that ethyl 4-chloroquino-

line-3-carboxylate was used as starting material, 3-hy-

droxy-thieno[3,2-c]quinoline-2-carboxylic acid methyl

ester (19a) was obtained as a tan solid (84 mg, 38%).

¹H NMR (400 MHz, DMSO-d₆) d ppm 3.89 (s, 3H)

7.75 (m, 1H) 7.87 (m, 1H) 8.17 (d, J = 7.83 Hz, 1H)

8.24 (d, J = 7.33 Hz, 1H) 9.29 (s, 1H) 11.18 (s, 1H).

Following the procedure in the preparation of 18b, 19a

(84 mg, 0.32 mmol) was alkylated with ethyl bromoace-

tate to give 3-ethoxycarbonylmethoxy-thieno[3,2-c]quin-

oline-2-carboxylic acid methyl ester (19b) as a white

Compound 21a (104 mg, 0.28 mmol) in DMF (2 mL)

was added to a mixture of sodium tert-butoxide

(54 mg, 0.56 mmol) and methyl thioglycolate dissolved

in DMF (1 mL). After 16 h at room temperature, water

was added, and the mixture was neutralized with 1 N

HCl. It was then extracted with ethyl acetate, dried over

MgSO₄, ﬁltered, and evaporated. The resulting solid was

then treated with ethyl bromoacetate (120 ll, 1.1 mmol)

in the presence of K₂CO₃(90 mg, 0.65 mmol) in DMF

(2 mL) at 60 ꢁC. After 24 h, water was added and the

mixture was extracted with ethyl acetate. Removal of

solvent and puriﬁcation by reverse-phase HPLC yielded

methyl 3-(ethoxycarbonylmethoxy)-5-chlorothieno[3,2-

b][1]benzothiophene-2-carboxylate (22b; 19 mg, 16% 2

steps). Subsequent hydrolysis following the procedure

in the preparation of 1 yielded compound 21 (11 mg,

1

solid (51 mg, 46%). H NMR (400 MHz, DMSO-d₆) d

ppm 1.18 (t, J = 7.07 Hz, 3H) 3.91 (s, 3 H) 4.15 (q,

J = 7.07 Hz, 2H) 5.22 (s, 2H) 7.77 (m, 1H) 7.90 (m,

1H) 8.20 (d, J = 7.83 Hz, 1H) 8.30 (d, J = 8.08 Hz, 1H)

9.34 (s, 1H).

Compound 19b (51 mg, 0.15 mmol) was hydrolyzed

according to the procedure in the preparation of 18 to

give compound 19 as a white solid (22 mg, 48%). H

1

NMR (400 MHz, DMSO-d₆) d ppm 5.15 (s, 2H) 7.83

(m, 2H) 8.23 (m, 2H) 9.32 (s, 1H).

5.2.20.

3-(Carboxymethoxy)thieno[3,2-b][1]benzo-thio-

1

phene-2-carboxylic acid (20). Sodium tert-butoxide

(360 mg, 3.7 mmol) was added to a solution of methyl thi-

oglycolate (0.110 mL, 1.23 mmol) in DMF (10 mL). 2-

Chloro-benzo[b]thiophene-3-carboxylic acid methyl ester

(235 mg, 1.12 mmol) was then added and the mixture be-

came orange. When no starting material was observed by

TLC, water was added followed by extraction with ethyl

acetate. Puriﬁcation by ﬂash chromatography yielded

methyl 3-(hydroxy)thieno[3,2-b][1]benzo-thiophene-2-car-

65%) as a white solid. H NMR (400 MHz, DMSO-d₆)

d ppm 4.67 (s, 2H) 7.56 (t, J = 7.83 Hz, 1H) 7.63 (dd,

J = 7.83, 1.01 Hz, 1H) 8.07 (dd, J = 7.83, 1.01 Hz, 1H).

HRMS: calcd for C₁₃H₇ClO₅S₂ꢁH+, 340.93507; found

(ESIꢁ, [MꢁH]^1ꢁ), 340.9345.

5.2.22. 3-(Carboxymethoxy)-6-chlorothieno[3,2-b][1]ben-

zothiophene-2-carboxylic acid (22). 6-Chloro-2-hydroxy-

benzo[b]thiophene-3-carboxylic acid methyl ester

2172

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

(2.46 g, 10.1 mmol) was cooled to 0 ꢁC in a solution

of DCM (25 mL) and Et₃N (25 mL). Triﬂuorometh-

anesulfonyl anhydride (2.56 mL, 15.2 mmol) was added

dropwise and the mixture was stirred at room tempera-

ture for 1 h. The resulting solution was washed with

water, saturated NaHCO₃, and dried over MgSO₄. The

crude material was puriﬁed by column chromatography

to give methyl 6-chloro-2-triﬂuoromethane-sulfonyloxy-

benzo[b]thiophene-3-carboxylate (22a; 2.61 g, 68%) as a

white solid. ¹H NMR (400 MHz, CDCl₃) d ppm 4.00 (s,

3H), 7.50 (dd, J = 8.8, 1.8 Hz, 1H), 7.76 (d, J = 8.8 Hz,

1H), 7.85 (d, J = 1.5 Hz, 1H)

with water, dried over MgSO₄, and concentrated in vac-

uo, aﬀording 2-nitro-isophthalic acid diallyl ester (23a;

6.50 g, 95%). H NMR (400 MHz, CDCl₃) d ppm 4.81

(d, J = 5.81 Hz, 4H) 5.33 (dd, J = 10.48, 1.14 Hz, 2H)

5.41 (dd, J = 17.18, 1.26 Hz, 2H) 5.97 (m, 2H) 7.67 (t,

J = 7.83 Hz, 1H) 8.22 (d, J = 8.08 Hz, 2H).

1

A solution of 23a (6.50 g, 22.3 mmol) in DMF (100 mL)

was cooled to ꢁ78 ꢁC. Methyl thioglycolate (2.02 mL,

22.3 mmol) was then added. The solution was allowed

to stir for a few minutes at ꢁ78 ꢁC, after which excess

DBU was added (8.3 mL, 55.3 mmol). After 1 h, the

reaction was judged complete by TLC. The reaction

mixture was diluted with water (500 mL) and poured

into a large Erlenmeyer ﬂask. The desired product was

precipitated out by adding 10% HCl (5–10 mL drop-

wise). The white solid was ﬁltered, rinsed three times

with water, and allowed to air-dry, aﬀording 3-hy-

droxy-benzo[b]thiophene-2,7-carboxylic acid 7-allyl es-

Methyl thioglycolate (0.169 mL, 1.9 mmol) was added

to a solution of 22a (0.644 g, 1.72 mmol) in Et₃N

(10 mL) and DMF (5 mL), and the mixture was stirred

at room temperature for 16 h. Addition of water fol-

lowed by extraction with ethyl acetate, drying over

MgSO₄, and removal of solvent yielded 0.564 g of crude

product. NaH 60% in oil (65 mg) was added to 449 mg

of the crude product in DMF (8 mL) and stirred for

30 min. Water was added and the mixture was acidiﬁed

with 1 N HCl. Extraction with ethyl acetate, drying over

MgSO₄, and removal of solvent gave methyl 3-(hydro-

xy)-6-chlorothieno[3,2-b][1]benzothiophene-2-carboxyl-

ate (22b; 0.173 g, 43%) as a light orange solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 3.96 (s, 3H) 7.42 (dd,

J = 8.59, 1.77 Hz, 1H) 7.79 (d, J = 8.59 Hz, 1H) 7.88

(d, J = 1.77 Hz, 1H) 10.02 (s, 1H). HRMS (ESIꢁ, m/z)

Calcd for [MꢁH]^1ꢁ, 296.94523, found, 296.4479.

1

ter 2-methyl ester (23b) in quantitative yield. H NMR

(400 MHz, CDCl₃) d ppm 3.98 (s, 3H) 4.94 (d,

J = 5.81 Hz, 2H) 5.34 (dd, J = 10.48, 1.14 Hz, 1H) 5.48

(dd, J = 17.18, 1.26 Hz, 1H) 6.10 (m, 1H) 7.51 (t,

J = 7.71 Hz, 1H) 8.16 (dd, J = 7.96, 1.14 Hz, 1H) 8.31

(dd, J = 7.45, 1.14 Hz, 1H) 10.08 (br s, 1H).

A solution of 23b (1.0 g, 3.4 mmol) in DCM (20 mL)

was cooled to 0 ꢁC. Triethylamine (1.2 mL, 8.6 mmol)

was then added, followed by mesyl chloride (0.28 mL,

3.6 mmol). The reaction mixture was allowed to stir at

0 ꢁC for 30 min, after which it was judged complete by

TLC. Excess water was then added, and the desired

product was extracted into DCM (3· 25 mL). The

organic layers were combined, washed with aqueous

sodium bicarbonate, dried over MgSO₄, and concentrat-

ed in vacuo. The crude product was then dissolved in a

minimal amount of DCM and ﬁltered through a thick

pad of silica gel using a 40% ethyl acetate/hexane solu-

tion, ultimately aﬀording 3-methanesulfonyloxy-

benzo[b]thiophene-2,7-carboxylic acid 7-allyl ester 2-

tert-Butyl bromoacetate (0.10 mL, 0.65 mmol) was add-

ed to a mixture of sodium tert-butoxide (60 mg,

0.63 mmol) and 22b (150 mg, 0.5 mmol) in DMF

(6 mL), and the mixture was stirred at 60 ꢁC for 2.5 h

and then at room temperature for 3 days. Water was

added and the mixture was extracted with ethyl acetate.

After removal of solvent, puriﬁcation by ﬂash chroma-

tography gave methyl 3-(tert-butoxycarbonylmethoxy)-

6-chlorothieno-[3,2-b][1]benzothiophene-2-carboxylate

1

(22c; 140 mg, 68%) of as an orange solid. H NMR

methyl ester (23c; 1.0 g, 79%) as a pale yellow oil. H

(400 MHz, CDCl₃) d ppm 1.47 (s, 9 H), 4.95 (s, 2H),

7.41 (dd, J = 8.6, 2.0 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H),

7.83 (d, J = 2.0 Hz, 1H).

NMR (400 MHz, CDCl₃) d ppm 3.53 (s, 3 H) 3.96 (s,

3H) 4.95 (d, J = 5.81 Hz, 2H) 5.35 (dd, J = 10.36,

1.26 Hz, 1H) 5.48 (dd, J = 17.18, 1.52 Hz, 1H) 6.09

(m, 1H) 7.60 (t, 1H) 8.27 (dd, J = 8.08, 1.26 Hz, 1H)

8.32 (dd, J = 7.45, 1.14 Hz, 1H).

Compound 22c (48 mg, 0.12 mmol) was hydrolyzed fol-

lowing the procedure in the preparation of 1 to yield com-

pound 22 (26 mg, 65%) as an oﬀ-white solid. H NMR

1

Following the procedure in the preparation of 23b, 23c

(1.0 g, 2.7 mmol) was converted to 3-hydroxy-thie-

no[3,2-b][1]benzothiophene-2,5-dicarboxylic acid 5-allyl

ester 2-methyl ester (23d) in quantitative yield. ¹H

NMR (400 MHz, CDCl₃) d ppm 3.97 (s, 3H) 4.96 (d,

J = 5.81 Hz, 2H) 5.36 (dd, J = 10.61, 1.26 Hz, 1H) 5.49

(dd, J = 17.18, 1.26 Hz, 1H) 6.06–6.17 (m, 1H) 7.56 (t,

J = 7.71 Hz, 1H) 8.08 (dd, J = 7.96, 1.14 Hz, 1H) 8.26

(dd, J = 7.58, 1.01 Hz, 1H) 10.04 (br s, 1H).

(400 MHz, DMSO-d₆) d ppm 5.06 (s, 2H) 7.55 (dd,

J = 8.59, 2.02 Hz, 1H) 8.12 (d, J = 8.59 Hz, 1H) 8.32 (d,

J = 2.02 Hz, 1H). HRMS: calcd for C₁₃H₇ClO₅S₂ꢁH+,

340.93507; found (ESIꢁ, [MꢁH]^1ꢁ), 340.93465.

5.2.23. 3-(Carboxymethoxy)-5-[(cyclohexylmethyl)-ami-

no]thieno[3,2-b][1]benzothiophene-2-carboxylic acid (23).

To

a solution of 2-nitro-isophthalic acid (5.0 g,

23.7 mmol) in DMF (80 mL) were added allyl bromide

(10.3 mL, 118.5 mmol) and potassium carbonate (30 g,

237 mmol). The mixture was heated to 50 ꢁC and al-

lowed to stir for 3 h, after which the reaction was judged

complete by TLC. The reaction was quenched with

water and the product was extracted into ethyl acetate

(3· 50 mL). The combined organic layers were washed

To a solution of 23d (1.05 g, 3.02 mmol) in DMF

(25 mL) were added excess potassium carbonate and

ethyl bromoacetate (0.5 mL, 4.5 mmol). The reaction

was allowed to stir at ambient temperature for 2 h after

which it was judged complete by TLC. Excess water was

added to precipitate the desired product 3-ethoxycar-

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2173

bonylmethoxy-thieno[3,2-b][1]benzo-thiophene-2,5-di-

carboxylic acid 5-allyl ester 2-methyl ester (23e) as a

white solid in quantitative yield. H NMR (400 MHz,

To a solution of 23h (250 mg, 0.68 mmol) in DCE

(6.5 mL) was added cyclohexane carboxaldehyde

1

(0.082 mL,

0.68 mmol),

NaBH(OAc)₃

(250 mg,

CDCl₃) d ppm 1.31 (t, J = 7.20 Hz, 3H) 3.92 (s, 3H)

4.28 (q, J = 7.07 Hz, 2H) 4.95 (d, J = 5.56 Hz, 2H)

5.11 (s, 1H) 5.35 (dd, J = 10.36, 1.26 Hz, 1H) 5.48 (dd,

J = 17.18, 1.52 Hz, 1H) 5.96–6.24 (m, 1H) 7.55 (t,

J = 7.71 Hz, 1H) 8.07 (dd, J = 7.96, 1.14 Hz, 1H) 8.24

(dd, J = 7.58, 1.01 Hz, 1H).

1.2 mmol). HOAc (0.05 mL, 0.82 mmol) was then added

dropwise and the solution was allowed to stir at ambient

temperature for 2 h after which the reaction was judged

complete by TLC. The solution was ﬁltered through sil-

ica gel, concentrated in vacuo and puriﬁed by ﬂash

chromatography to give 5-(cyclohexylmethyl-amino)-3-

ethoxycarbonylmethoxy-thieno[3,2-b][1]benzothioph-

ene-2-carboxylic acid methyl ester (23i; 180 mg,

57%)CDCl₃) d ppm 0.97–1.11 (m, 2H) 1.23–1.33 (m,

5H) 1.63–1.81 (m, 6H) 1.83–1.90 (m, 2H) 3.15

(d, J = 6.82 Hz, 2H) 3.91 (s, 3H) 4.27 (q,

J = 7.07 Hz, 3H) 5.07 (s, 2H) 6.67 (dd, J = 7.83,

0.76 Hz, 1H) 7.26 (d, J = 7.83, 1.01 Hz, 1H) 7.34 (t,

J = 7.83 1H).

To a solution of 23e (1.21 g, 2.8 mmol) in anhydrous

THF (50 mL) was added Pd(PPh₃)₄(320 mg,

0.28 mmol) under N₂ﬂow. Concentrated acetic acid

was then added (2.5 mL), and the mixture was allowed

to stir at ambient temperature for 2 h, after which the

reaction was judged complete by TLC. A 20% ethyl

acetate/hexane solution was added to the reaction to

precipitate out the desired product, 3-ethoxycarbonyl-

methoxy-thieno[3,2b][1]benzo-thiophene-2,5-dicarboxyl-

ic acid 2-methyl ester (23f; 0.76 g, 68%). ¹H NMR

(400 MHz, DMSO-d₆) d ppm 1.29 (t, J = 7.07 Hz, 3H)

3.90 (s, 3H) 4.25 (q, J = 7.07 Hz, 2H) 5.26 (s, 2H) 7.72

(t, J = 7.71 Hz, 1H) 8.22 (dd, J = 7.58, 1.01 Hz, 1H)

8.43 (dd, J = 7.96, 0.88 Hz, 1H).

Compound 23i (30 mg, 0.065 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to give

compound 23 (8.2 mg, 28%) as a green solid. H NMR

1

(400 MHz, DMSO-d₆) d ppm 0.94–1.10 (m, 2H) 1.17–

1.35 (m, 3H) 1.63–1.82 (m, 4H) 1.85–1.96 (m, 2H) 3.11–

3.18 (m, 2H) 5.11 (s, 2H) 5.87–5.96 (m, 1H) 6.73 (d,

J = 7.83 Hz, 1H) 7.31 (d, J = 7.83 Hz, 1H) 7.38 (t,

J = 7.83 Hz, 1H). HRMS: calcd for C₂₀H₂₁NO₅S₂+ H+,

420.09339; found (ESI-FTMS, [M+H]¹⁺), 420.0931.

To a solution of 23f (450 mg, 1.14 mmol) in toluene

(7.5 mL) were added diphenylphosphoryl azide

(0.37 mL, 1.71 mmol), excess triethyl amine (0.40 mL,

2.86 mmol), and t-butanol (0.75 mL). The solution was

allowed to stir at ambient temperature for 2 h, after

which the starting material was judged consumed by

TLC. An additional 0.75 mL of t-butanol was then add-

ed and the reaction mixture was heated to 100 ꢁC and al-

lowed to stir for an additional 16 h, after which the

reaction was quenched with excess water and extracted

into DCM (3· 10 mL). The organic layers were com-

bined, washed with water (3· 10 mL), dried with

MgSO₄, and concentrated in vacuo. The crude product

was puriﬁed by ﬂash chromatography using ethyl ace-

tate/hexane (5–20% gradient) as eluent to aﬀord

5- tert-butoxycarbonylamino-3-ethoxycarbonylmeth-

oxy-thieno[3,2-b][1]benzo-thiophene-2-carboxylic acid

5.2.24. 3-(Carboxymethoxy)-6-[(cyclohexylmethyl)-ami-

no]thieno[3,2-b][1]benzothiophene-2-carboxylic acid (24).

To a solution of 3-hydroxy-benzo[b]thiophene-2,6-dicar-

boxylic acid 6-allyl ester 2-methyl ester (3.2 g, 11 mmol)

in DCM (50 mL) were added triethylamine (1.83 mL,

13 mmol), triﬂuoromethanesulfonyl anhydride (2.2 mL,

13 mmol), and DMAP (67 mg, 0.55 mmol) at 0 ꢁC.

The temperature was allowed to rise to room temperate

and the mixture was stirred for 4 h, washed with aque-

ous NaHCO₃, and dried over MgSO₄. The crude prod-

uct was puriﬁed by ﬂash chromatography eluted with

ethyl acetate/hexane to give 3-triﬂuoromethanesulfonyl-

oxy-benzo[b]thiophene-2,6-dicarboxylic acid 6-allyl ester

1

2-methyl ester (24a; 3.88 g, 84%) as a white solid. H

1

methyl ester (23g; 535 mg, 62%). H NMR (400 MHz,

NMR (400 MHz, CDCl₃) d ppm 4.02 (s, 3H) 4.86–

4.93 (m, 2H) 5.34 (dd, J = 10.48, 1.14 , 1H) 5.41–5.50

(m, J = 17.18, 1.52 1H) 5.98–6.15 (m, 1 H) 7.90 (d,

J = 8.59 Hz, 1H) 8.19 (dd, J = 8.72, 1.39 Hz, 1H) 8.59

(s, 1H).

CDCl₃) d ppm 1.28 (t, J = 7.20 Hz, 3H) 3.92 (s, 3H)

4.26 (q, J = 7.16 Hz, 2H) 5.06 (s, 2H) 6.41 (br s, 1H)

7.45 (t, J = 7.96 Hz, 1H) 7.61 (dd, J = 7.83, 1.01 Hz,

1H) 7.92 (d, J = 8.08 Hz, 1H).

A solution of 23g (170 mg, 0.37 mmol) in DCM (6 mL)

was cooled to 0 ꢁC. Triﬂuoroacetic acid (1.2 mL) was

then added, and the solution was allowed to warm to

ambient temperature, stirring for a total of 4 h, after

which the reaction was judged complete by TLC. The

reaction mixture was concentrated in vacuo, dissolved

in ethyl acetate, and washed with sodium bicarbonate

(3· 10 mL). The organic layer was then concentrated

in vacuo, aﬀording 5-amino-3-ethoxycarbonylmethoxy-

thieno[3,2-b][1]benzothiophene-2-carboxylic acid methyl

To a solution of 24a (3.8 g, 8.96 mmol) in DCM were

added methyl thioglycolate (0.96 mL, 10.75 mmol) and

triethylamine (3.75 mL, 26.88 mL) at 0 ꢁC. The temper-

ature was allowed to rise to room temperature. The mix-

ture was stirred for 24 h and washed with aqueous

NaHCO₃, dried over MgSO₄, and puriﬁed by ﬂash chro-

matography eluted with ethyl acetate/hexane to give 3-

methoxycarbonylmethyl-sulfanyl-benzo[b]thiophene-2,

6-dicarboxylic acid 6-allyl ester 2-methyl ester (24b;

1.81 g, 53%) as a light yellow solid. ¹H NMR

(400 MHz, CDCl₃) d ppm 3.51 (s, 3H) 4.00 (s, 3H)

4.84–4.96 (m, 2H) 5.28–5.39 (m, 1H) 5.39–5.51 (m,

1H) 5.96–6.21 (m, 1H) 8.15 (dd, J = 8.59, 1.52 Hz,

1H) 8.25 (dd, J = 8.59, 0.76 Hz, 1H) 8.59 (dd,

J = 1.52, 0.76 Hz, 1H).

1

ester (23h) in a quantitative yield. H NMR (400 MHz,

CDCl₃) d ppm 1.30 (t, J = 7.20 Hz, 3H) 3.91 (s, 3H)

4.28 (q, J = 7.24 Hz, 2H) 5.06 (s, 2H) 6.89 (dd,

J = 7.58, 1.01 Hz, 1H) 7.33 (t, J = 7.71 Hz, 1H) 7.42

(dd, J = 7.97, 0.88 Hz, 1H).

2174

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

To a solution of 24b (1.05 g, 2.76 mmol) in DMF was

added DBU (0.83 mL, 5.53 mmol) at room temperature.

The resultant mixture was stirred for 24 h. To this were

added ethyl bromoacetate (0.92 mL, 8.28 mmol) and

K₂CO₃(1.14 g, 8.26 mmoL). The reaction mixture was

stirred for additional 2 h, then aqueous NH₄Cl

(200 mL) and water (300 mL) were added. The white pre-

cipitate was collected by ﬁltration and washed with water

to give 3-ethoxycarbonylmethoxy-thieno[3,2-b][1]benzo-

thiophene-2,6-dicarboxylic acid 6-allyl ester 2-methyl

1,2-dichloroethane were added to a 50 mL round-bot-

tomed ﬂask under nitrogen. NaBH(OAc)₃(0.123 g,

0.58 mmol) was then added, and the mixture was

allowed to stir at room temperature for 12 h. The mix-

ture was then poured into 100 mL methylene chloride

and washed with three 50 mL portions of saturated sodi-

um bicarbonate solution. The organic layer was dried

over MgSO₄, ﬁltered, and the solvent was removed in

vacuo to give the crude product. This was puriﬁed by sil-

ica gel chromatography (10% ethyl acetate/hexane) to

give 6-cyclohexylmethylamino-3-ethoxycarbonylmeth-

oxy-thieno[3,2-b][1]benzo-thiophene-2-carboxylic acid

1

ester (24c; 1.16 g, 97% two steps). H NMR (400 MHz,

CDCl₃) d ppm 1.29 (t, J = 7.07 Hz, 3H) 4.27 (q,

J = 7.24 Hz, 2H) 4.85–4.92 (m, 2H) 5.07 (s, 2H) 5.30–

5.38 (m, 1H) 5.40–5.51 (m, 1H) 5.97–6.17 (m, 1H) 7.91

(d, J = 7.83 Hz, 1H) 8.13 (dd, J = 8.46, 1.39 Hz, 1H)

8.59 (d, J = 1.52 Hz, 1H).

1

methyl ester (24g; 0.053 g, 46%). H NMR (400 MHz,

CDCl₃) d ppm 0.81–1.10 (m, 3H) 1.13–1.36 (m, 3H) 1.47

(s, 9H) 1.53–1.90 (m, 5H) 3.02 (d, J = 6.82 Hz, 2H) 3.88

(s, 3H) 4.92 (s, 2H) 6.69 (dd, J = 8.72, 2.15 Hz, 1H) 6.90

(d, J = 2.02 Hz, 1H) 7.58 (d, J = 8.59 Hz, 1H).

To a suspension of 24c (920 mg, 2.12 mmol) in THF

(50 mL) was added Pd(PPh₃)₄(240 mg, 0.21 mmol) under

N₂, followed by addition of HOAc (5 mL). The resultant

mixture was stirred at room temperature for 2 h, diluted

with hexane/ethyl acetate (4:1), and the precipitate was

collected by ﬁltration to give 3-ethoxycarbonylmethoxy-

thieno[3,2-b][1]benzo-thiophene-2,6-dicarboxylic acid 2-

Compound 24g (0.053g, 0.11 mmol) was added to a

50 mL round-bottomed ﬂask containing 10 mL THF. A

solution of LiOH^ÆH₂O (0.018 g, 0.43 mmol, 4.0 equiv) in

5 mL water was then added, and the resulting mixture

was allowed to stir at room temperature overnight. The

resulting solution was then acidiﬁed by the addition of ex-

cess 1.2 N HCl. This mixture was then extracted with

three 25 mL portions of ethyl acetate. The organic layer

was then washed with three 50 mL portions of water

and one 50 mL portion of saturated sodium chloride solu-

tion. The organic layer was then dried over MgSO₄, ﬁl-

tered, and the solvent was removed in vacuo to give the

crude product. This was then puriﬁed by reverse-phase

HPLC under basic conditions. The desired product was

isolated pure as the bis-triethylamine salt (24; 0.004 g,

1

methyl ester (24d; 779 mg, 93%) as a white solid. H

NMR (400 MHz, DMSO-d₆) d ppm 1.22 (t, J =

7.07 Hz, 4H) 3.87 (s, 3H) 4.19 (q, J = 7.07 Hz, 2H) 5.19

(s, 2H) 8.05 (dd, J = 8.34, 1.52 Hz, 1H) 8.19–8.26 (m,

1H) 8.75 (dd, J = 1.52, 0.76 Hz, 1H) 13.25 (s, 1H).

To a solution of 24d (290 mg, 0.73 mmol) in toluene

(5 mL) and t-BuOH (0.5 mL) were added diphenylphos-

phoryl azide (0.238 mL, 1.1 mmol) and triethylamine

(0.205 mL) at room temperature. The reaction mixture

was stirred for 30 min, extra 0.5 mL t-BuOH was added,

and the reaction mixture was heated to 100 ꢁC. After stir-

ring at this temperature overnight, the mixture was par-

titioned between DCM and water, and extracted with

DCM, dried over MgSO₄. The crude product was puri-

ﬁed by ﬂash chromatography eluted with ethyl acetate/

DCM to give 6- tert-butoxycarbonylamino-3-ethoxycar-

bonylmethoxy-thieno[3,2-b][1]benzothiophene-2-carbo-

1

6%). H NMR (400 MHz, CDCl₃) d ppm 0.95–1.09 (m,

2H) 1.11–1.28 (m, 18H) 1.57– 1.94 (m, 9H) 2.85–2.94

(m, J = 4.55 Hz, 12H) 2.97–3.07 (m, 2H) 4.81 (s, 2H)

6.69 (d, J = 7.83 Hz, 1H) 6.92 (s, 1H) 7.58 (d,

J = 8.59 Hz, 1H). HRMS: calcd for C₂₀H₂₁NO₅S₂+ H+,

420.09339; found (ESI-FTMS, [M+H]¹⁺), 420.094.

5.2.25. 3-(Carboxymethoxy)-5-(cyclohexylamino)-thieno[3,2-

b][1]benzothiophene-2-carboxylic acid (25). To a solution of

23h (250 mg, 0.68 mmol) in DCE (6.5 mL) were added cyclo-

hexanone (0.071 mL, 0.68 mmol) and NaBH(OAc)₃

(250 mg, 1.2 mmol). HOAc (0.05 mL, 0.82 mmol) was then

added dropwise and the solution was allowed to stir at ambi-

ent temperature for 2 h after which the reaction was judged

complete by TLC. The solution was ﬁltered through silica

gel, concentrated in vacuo, and puriﬁed by ﬂash chromatog-

raphy using a 0–15% ethyl acetate/hexane gradient, aﬀording

5-cyclohexylamino-3-ethoxycarbonylmethoxy-thieno[3,2-

b][1]benzo-thiophene-2-carboxylic acid methyl ester (25a;

180 mg, 59%) as a viscous yellow oil. ¹H NMR

(400 MHz, CDCl₃) d ppm 1.21–1.35 (m, 6H) 1.35–1.49

(m, 2H) 1.65–1.74 (m, 1H) 1.77–1.88 (m, 2H) 2.07–2.18

(m, 2H) 3.36–3.56 (m, 1H) 3.91 (s, 3H) 4.27 (q,

J = 7.24 Hz, 2H) 5.07 (s, 2H) 6.70 (dd, J = 7.83, 1.01 Hz,

1H) 7.24 (dd, J = 7.83, 1.01, 1H) 7.32 (t, J = 7.83 Hz, 1H).

1

xylic acid methyl ester (24e; 195 mg, 57%). H NMR

(400 MHz, CDCl₃) d ppm 1.29 (t, J = 7.20 Hz, 3H)

1.54 (s, 9H) 3.90 (s, 3H) 4.27 (q, J = 7.07 Hz, 2H) 5.04

(s, 2H) 6.72 (s, 1H) 7.19 (dd, J = 8.84, 2.02 Hz, 1H)

7.71 (d, J = 8.59 Hz, 1H) 8.17 (s, 1 H).

To 24e (0.433 g, 0.88 mmol) was added a mixture of

4.4 mL of 1.0 N HCl in ethyl acetate (4.4 mmol), and

1.1 mL methanol. The mixture was allowed to stir at room

temperature for 12 h. The resulting suspension was ﬁl-

tered and the solids were dried under vacuum to give 6-

amino-3-ethoxycarbonylmethoxy-thieno[3,2-b][1]benzo-

thiophene-2-carboxylic acid methyl ester HCl salt as an

1

oﬀ-white solid (24f; 0.302 g, 80%). H NMR (400 MHz,

DMSO-d₆)

d ppm 1.41 (s, 9H) 3.78–3.82 (m,

J = 1.77 Hz, 3H) 4.94–5.12 (m, 1H) 5.13 (d, J = 7.33 Hz,

1H) 6.98 (d, J = 8.08 Hz, 1H) 7.37 (s, 1H) 7.85 (d,

J = 9.09 Hz, 1H).

Compound 25a (30 mg, 0.07 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to give

compound 25 (20 mg, 71%). ¹H NMR (400 MHz,

DMSO-d₆) d ppm 1.15–1.30 (m, 1H) 1.32–1.46 (m, 4H)

Compound 24f (0.100 g, 0.23 mmol) and cyclohexane

carboxaldehyde (0.03 mL, 0.23 mmol), and 20 mL of

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

2175

1.64–1.73 (m, 1H) 1.75–1.85 (m, 2H) 1.98–2.07 (m, 2H)

5.07 (br s, 2H) 5.42 (d, J = 8.59 Hz, 1H) 6.78 (d,

J = 7.58 Hz, 1H) 7.30 (dd, J = 7.83, 1.01 Hz, 1H) 7.35 (t,

J = 7.71 Hz, 1H). HRMS: calcd for C₁₉H₁₉NO₅S₂+ H+,

406.07774; found (ESI-FTMS, [M+H]¹⁺), 406.0771.

5.2.28. 3-(Carboxymethoxy)-6-{[1-(ethylsulfonyl)-piperi-

din-4-yl]amino}thieno[3,2-b][1]benzo-thiophene-2-carbox-

ylic acid (28). NaBH(OAc)₃(227 mg, 1.07 mmol) was

added to a mixture of 26a (259 mg, 0.54 mmol), 4-

oxopiperidine-1-carboxylic

acid

tert-butyl

ester

(129 mg, 0.65 mmol), and acetic acid (50 lL) in DCE

(4 mL). The mixture was stirred at 50 ꢁC for 17 h.

DCM (20 mL) was added and the mixture was washed

with saturated NaHCO₃, dried over MgSO₄, ﬁltered,

and evaporated. Puriﬁcation by ﬂash chromatography

yielded 4-(3-ethoxycarbonylmethoxy-2-methoxycarbon-

yl-thieno[3,2-b][1] benzothiophen-6-ylamino)-piperidine-

1-carboxylic acid tert-butyl ester (28a; 237 mg, 80%).

5.2.26.

3-(Carboxymethoxy)-6-(cyclohexylamino)-thie-

no[3,2-b][1]benzothiophene-2-carboxylic acid (26). To a

solution of 24e (466 mg, 1.0 mmol) in DCM (15 mL) was

added triﬂuoroacetic acid (2 mL). The resultant mixture

wasstirredatroomtemperaturefor2 h. Thesolventwasre-

moved under reduced pressure. The crude product (26a)

wasre-dissolvedinDCE(10 mL).Tothiswereaddedcyclo-

hexanone (0.156 mL, 1.5 mmol) and HOAc (0.088 mL, 1.5

mmol), followed by the addition of NaBH(OAc)₃(424 mg,

2.0 mmol). The resultant mixture was stirred at room tem-

peratureovernight.Thecrudeproductwaspuriﬁedbyﬂash

chromatography eluted with hexane/ethyl acetate to give

the desired product, 6-cyclohexylamino-3-ethoxycarbon-

ylmethoxy-thieno[3,2-b][1]benzo-thiophene-2-carboxyl-

ic acid methyl ester (26b; 260 mg, 65%). ¹H NMR

(400 MHz, CDCl₃) d ppm 1.10–1.26 (m, 2H) 1.33–1.48

(m, 2H) 1.61–1.95 (m, 4H) 2.09 (dd, J = 12.88, 3.28 Hz,

2H) 3.26–3.41 (m, 1H) 3.88 (s, 3H) 4.26 (q, J = 7.07 Hz,

2H) 5.02 (s, 2H) 6.66 (dd, J = 8.72, 2.15 Hz, 1H) 6.91 (d,

J = 2.27 Hz, 1H) 7.57 (d, J = 8.34 Hz, 1H).

¹H NMR (400 MHz, CDCl₃)

d

ppm 1.29 (t,

J = 7.2 Hz, 3H), 1.34–1.44 (m, 2H), 1.47 (s, 9H), 1.79–

1.91 (m, 1H), 2.08 (d, J = 12.1 Hz, 2H), 2.87–3.10 (m,

3H), 3.43–3.58 (m, 1H), 3.78–3.94 (m, 5H), 4.00–4.16

(m, 2H), 4.26 (q, J = 7.1 Hz, 2H), 5.02 (s, 2H), 6.69

(dd, J = 8.6, 2.0 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 7.60

(d, J = 8.6 Hz, 1H).

Compound 28a (204 mg, 0.37 mmol) was stirred in a

mixture of triﬂuoroacetic acid (10 mL) and DCM

(10 mL) at room temperature for 1 h. Evaporation

of solvent gave a triﬂuoroacetic acid salt of 3-ethoxy-

carbonylmethoxy-6-(piperidin-4-ylamino)-thieno[3,2-b][1]-

benzothiophene-2-carboxylic acid methyl ester (28b;

206 mg, 99%). ¹H NMR (400 MHz, DMSO-d₆) d

ppm 1.21 (t, J = 7.1 Hz, 2H), 1.50–1.64 (m, 2H),

2.04–2.15 (m, 2H), 2.96–3.09 (m, 2H), 3.11–3.23

(m, 1H), 3.28–3.40 (m, 2H), 3.57–3.68 (m, 1H),

3.79 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 5.11 (s, 2

H), 6.84 (dd, J = 8.8, 2.0 Hz, 1H), 7.17 (d, J = 2.0

Hz, 1H), 7.75 (d, J = 8.6 Hz, 1H), 8.22–8.54 (m,

2H).

Compound 26b (23 mg, 0.05 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to

give compound 26 (13.2 mg, 63%) as a white solid. H

NMR (400 MHz, DMSO-d₆) d ppm 0.84–1.12 (m, 4H)

1.11–1.31 (m, 2H) 1.40–1.50 (m, 1H) 1.51–1.65 (m,

2H) 1.80 (dd, J = 13.14, 3.03 Hz, 2H) 4.18 (s, 2H) 5.67

(d, J = 7.83 Hz, 2H) 6.59 (dd, J = 8.84, 2.02 Hz, 1H)

6.88 (d, J = 2.02 Hz, 1H) 7.37 (d, J = 8.59 Hz, 1H).

1

5.2.27. 3-(Carboxymethoxy)-6-(tetrahydro-2H-pyran-4-

ylamino)thieno[3,2-b][1]benzothiophene-2-carboxylic acid

(27). NaBH(OAc)₃(90 mg, 0.42 mmol) was added to a

mixture of 26a (85 mg, 0.17 mmol), tetrahydropyran-4-

one (24 ll, 0.2 mmol), and acetic acid (5 lL) in DCE

(4 mL). The mixture was stirred at 50 ꢁC for 17 h.

DCM (20 mL) was added and the mixture was washed

with saturated NaHCO₃, dried over MgSO₄, ﬁltered,

and rotovaped. Puriﬁcation by ﬂash chromatography

yielded 3-ethoxycarbonylmethoxy-6-(tetrahydro-pyran-

4-ylamino)-thieno[3,2-b][1]benzothiophene-2-carboxylic

acid methyl ester (27a; 72 mg, 94%). ¹H NMR

(400 MHz, CDCl₃) d ppm 1.29 (t, J = 7.2 Hz, 3H),

1.44–1.61 (m, 3H), 2.08 (d, J = 14.4 Hz, 2H), 3.44–3.70

(m, 3H), 3.88 (s, 3H), 3.98–4.09 (m, 2H), 4.27 (q,

J = 7.2 Hz, 2H), 5.02 (s, 2H), 6.71 (dd, J = 8.6, 1.8 Hz,

1H), 6.96 (s, 1H), 7.61 (d, J = 8.6 Hz, 1H).

Compound 28b (83 mg, 0.15 mmol) was treated with

ethylsulfonyl chloride (14 ll, 0.15 mmol) in a mixture of

DCM (3 mL) and saturated NaHCO₃(2 mL). The reac-

tion was monitored by LCMS to completion, DCM was

then added, and the organic layer was washed with water,

brine, dried over MgSO₄, and ﬁltered. Evaporation of

solvent yielded 6-(1-ethanesulfonyl-piperidin-4-ylami-

no)-3-ethoxycarbonylmethoxy-thieno[3,2-b][1]benzothi-

ophene-2-carboxylic acid methyl ester (28c; 58 mg, 73%

1

two steps) as a green ﬁlm. H NMR (400 MHz, CDCl₃)

d ppm 1.29 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.5 Hz, 3H),

1.56–1.65 (m, 3H), 2.15–2.28 (m, J = 12.3, 2.9 Hz, 2H),

2.94–3.09 (m, 4H), 3.44–3.59 (m, 1H), 3.79–3.86 (m, 2H),

3.88 (s, 3H), 4.26 (q, J = 7.1 Hz, 2H), 5.02 (s, 2H), 6.70

(dd, J = 8.6, 2.0 Hz, 1H), 6.94 (d, J = 2.0 Hz, 1H), 7.62 (d,

J = 8.6 Hz, 1H).

Compound 28c (58 mg, 0.11 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to give

compound 28 (38 mg, 72%) as a golden yellow solid. H

Compound 27a (72 mg, 0.16 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to give

compound 27 (65 mg, 77%). ¹H NMR (400 MHz,

1

NMR (400 MHz, DMSO-d₆) d ppm 1.23 (t, J = 7.33 Hz,

3H) 1.48–1.35 (m, 2H) 2.06–1.97 (m, 2H) 3.05–2.96 (m,

2H) 3.07 (q, J = 7.41 Hz, 2H) 3.60 (d, J = 12.63 Hz,

2H) 4.55–4.31 (m, 2H) 6.20–5.95 (m, 2H) 6.80 (d,

J = 8.84 Hz, 1H) 7.14 (s, 1 H) 7.73–7.56 (m, 1H). HRMS:

calcd for C₂₀H₂₂N₂O₇S₃ꢁH+, 497.05164; found (ESI-

FTMS, [MꢁH]^1ꢁ), 497.0527.

DMSO-d₆)

d ppm 1.46–1.92 (m, 2H) 1.92 (d,

J = 13.39 Hz, 2H) 3.88 (m, J = 11.12 Hz, 1H) 4.83 (s,

1H) 6.18 (d, J = 7.83 Hz, 1H) 6.82 (dd, J = 8.84,

2.02 Hz, 1H) 7.15 (d, J = 2.02 Hz, 1H) 7.68 (d,

J = 8.84 Hz, 1H). HRMS: calcd for C₁₈H₁₇NO₆S₂+ H+,

408.05701; found (ESI+, [M+H]¹⁺), 408.05609.

2176

A. F. Moretto et al. / Bioorg. Med. Chem. 14 (2006) 2162–2177

5.2.29. 6-{[1-(Benzylsulfonyl)piperidin-4-yl]amino}-3-(carb-

oxymethoxy)thieno[3,2-b][1]benzothiophene-2-carboxylic

acid (29). Compound 28b (65 mg, 0.12 mmol) was treated

with a-tolylsulfonyl chloride (23 mg, 0.12 mmol) in a mix-

ture of DCM (2 mL) and saturated NaHCO₃(2 mL). The

reaction was monitored by LCMS to completion, DCM

was then added, and the organic layer was washed with

water, brine, dried over MgSO₄, and ﬁltered. Evaporation

of solvent yielded 3-ethoxycarbonylmethoxy-6-(1-phen-

ylmethanesulfonyl-piperidin-4-ylamino)-thieno[3,2-b][1]ben-

zothiophene-2-carboxylic acid methyl ester (29a; 37 mg, 53%

two steps) as a green solid. ¹H NMR (400 MHz, CDCl₃) d

ppm 1.29 (t, J = 7.2 Hz, 3H), 1.35–1.50 (m, 2H), 1.99–2.12

(m, J = 13.4 Hz, 2H), 2.71–2.86 (m, 2H), 3.33–3.47 (m,

1H), 3.59– 3.69 (m, 2H), 3.74–3.82 (m, J = 8.6 Hz, 1H),

3.88 (s, 3H), 4.21–4.31 (m, 4H), 5.02 (s, 2H), 6.66 (dd,

J = 8.7, 2.1 Hz, 1H), 6.89 (d, J = 2.3 Hz, 1H), 7.37–7.46

(m, 5H), 7.60 (d, J = 8.6 Hz, 1H).

Diﬀraction data of the hPTP1B-inhibitor complex crys-

tals were collected by in-house conventional X-ray gener-

ator using Raxis-IV image detectors. All data were

collected at 100 K and processed with DENZO/

SCALPACK.³⁷

The structure was solved by molecular replacement with

the available hPTP1B catalytic domain structure model.

Cycles of model rebuilding and reﬁnement were carried

out with CNS (Brunger, A.T. and et. al. copyright:

1997–2001, Yale University), a system for X-ray crystal-

lography and NMR and QUANTA (Accelrys Inc.).

Inhibitor was built into the diﬀerence density after the ﬁrst

cycle of reﬁnement. Water molecules were assigned

according to Fo-Fc maps. The ﬁnal model for hPTP1B-

compound 1 contained residues 2–298, 128 waters, and

1 inhibitor. It exhibited good stereochemistry, with an

averaged bond length and bond angle deviation from ide-

˚

al geometry of 0.007 A and 1.367ꢁ, respectively. The over-

all R factor is 22.3%, and R free is 24.9% with 5% test

Compound 29a (37 mg, 0.06 mmol) was hydrolyzed

according to the procedure in the preparation of 1 to give

compound 29 (22 mg, 65%) as a green solid. H NMR

˚

reﬂections, using diﬀraction data between 20 and 2.0 A.

1

The hPTP1B-compound 29 structure contained residues

2–298, 171 waters, and 1 inhibitor. The averaged bond

length and bond angle deviation from ideal geometry

(400 MHz, DMSO-d₆) d ppm 1.24 (s, 1H) 1.43–1.29 (m,

2H) 1.96 (s, 1H) 2.92 (t, J = 10.74 Hz, 2H) 3.55 (d,

J = 12.38 Hz, 1H) 4.42 (s, 2H) 4.56 (s, 2H) 6.14 (d,

J = 8.08 Hz, 1H) 6.80 (dd, J = 8.59, 2.02 Hz, 1H) 7.15

(d, J = 1.77 Hz, 1H) 7.45–7.36 (m, 5H) 7.67 (d,

J = 8.59 Hz, 1H). HRMS: calcd for C₂₅H₂₄N₂O₇

S₃+ H+, 561.08184; found (ESI+, [M+H]¹⁺), 561.08092.

˚

are 0.01 A and 1.65ꢁ, respectively. The overall R factor

is 21.5%, and R free is 24.9% with 5% test reﬂections, using

˚

diﬀraction data between 20 and 2.1 A. No residues were in

disallowed regions of the Ramachandran plot for both

structures.

5.3. Enzymatic assay

5.5. Molecular modeling

The enzymatic assay was carried out at room tempera-

ture in 96-well plates. The assay buﬀer contained

50 mM of 3,3-dimethyl glutarate, 1 mM EDTA, 1 mM

TCEP, and 0.01% Triton (pH 7.0 with an ionic strength

of 0.15 M adjusted by sodium chloride). The reaction

was initiated by addition of the enzyme at a ﬁnal con-

centration of 10 or 100 nm for PTP1B, 20 nm for

TCPTP, 20 nM for CD45, and 270 nM for LAR, respec-

tively. The initial rate of PTPase-catalyzed hydrolysis of

p-nitrophenol phosphate (pNPP) was measured by fol-

lowing the absorbance change at 405 nm. IC₅₀value

was determined under ﬁxed pNPP concentration of

1 mM. All the assays were carried out in duplicate or

triplicate and the average results are presented. K_iis de-

rived from IC₅₀based on competitive inhibition

K_i= IC₅₀· K_m/(K_m+ [substrate]). PTPases used in the

assays were recombinant human PTP1B (hPTP1B cata-

lytic domain, residues 1–299, expressed and puriﬁed

according to literature procedures²¹), recombinant hu-

man TCPTP (residues 1–299, expressed and puriﬁed

in-house), recombinant human CD45 (cytoplasmic do-

main, residues 484–1281, purchased from Biomol), and

recombinant human LAR (soluble catalytic LAR-D1

domain, residues 1275–1613, purchased from Biomol).

Analogs were initially docked into the active site of the

PTP1B protein structure from the X-ray complex with

compound 1. As additional protein–inhibitor complex

structures were determined the binding site model was

reﬁned. Typically 1000 Monte Carlo cycles³⁸were car-

ried out to dock each analog into the site, while also

allowing select protein residues to undergo constrained

movement. Figures 2, 4, and 6 were generated using Py-

MOL (DeLano Scientiﬁc, 2002, San Carlos, CA, USA).

Acknowledgments

We thank Dr. Tarek Mansour for helpful discussion and

reviewing the manuscript. The authors acknowledge Drs.

Nelson Huang and Walter Massefski for their role in the

characterization of the small molecules published herein.

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Article Doi

Bicyclic and tricyclic thiophenes as protein tyrosine phosphatase 1B inhibitors

DOI: 10.1016/j.bmc.2005.11.005

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Authors:

Article abstract of DOI:10.1016/j.bmc.2005.11.005

Full text of DOI:10.1016/j.bmc.2005.11.005

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