Synthesis, in vitro structure-activity relationship, and in vivo studies of 2-arylthiazolidine-4-carboxylic acid amides as anticancer agents

Bioorganic & Medicinal Chemistry 18 (2010) 477–495

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis, in vitro structure–activity relationship, and in vivo studies of

2-arylthiazolidine-4-carboxylic acid amides as anticancer agents

Yan Lu ^a, Zhao Wang ^a, Chien-Ming Li ^b, Jianjun Chen ^a, James T. Dalton ^b, Wei Li ^a, Duane D. Miller ^a,

*

^aDepartment of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, United States

^bDivision of Pharmaceutics, College of Pharmacy, The Ohio State University, Columbus, OH 43210, United States

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of (2RS,4R)-2-arylthiazolidine-4-carboxylic acid amide (ATCAA) was synthesized. Antiprolifera-

tive activity against melanoma and prostate cancer cells compared with control cells (ﬁbroblast and

RH7777, respectively) was evaluated. Compound 3id showed the best selectivity and growth-inhibition

activity against three melanoma cell lines (B16-F1, A375, and WM-164). Compounds 15b and 3ac had

good selectivity and potency against four prostate cancer cell lines (DU 145, PC-3, LNCaP, and PPC-1).

The structure–activity relationship (SAR) of the side chain, the thiazolidine ring, and phenyl substituents

is discussed. Cell cycle analysis showed that the percentage of cancer cells undergoing apoptosis (sub-G1

phase) increased after treatment with 1b and 3ad, which also strongly inhibited melanoma colony for-

mation. In vivo studies on nude mice bearing A375 melanoma tumors showed that compound 1b inhib-

ited tumor growth in a dose-dependent manner. At a dose of 10 mg/kg, 1b signiﬁcantly inhibited

melanoma tumor growth and showed higher efﬁcacy than did dacarbazine at 60 mg/kg.

Ó 2009 Published by Elsevier Ltd.

Received 23 October 2009

Revised 2 December 2009

Accepted 5 December 2009

Available online 11 December 2009

Keywords:

Melanoma

Prostate cancer

Thiazolidine

Antiproliferative activity

Structure–activity relationship

Colony formation

In vivo evaluation

1. Introduction

In previous contributions from our laboratory, we discovered

that, by replacing the glycerol backbone in lysophosphatidic acid

Cancer ranks second in diseases leading to mortality, following

only cardiovascular diseases. One-quarter of all deaths in the Uni-

ted States are caused by cancers.¹Out of the many cancer diseases,

the incidence of cutaneous malignant melanoma is increasing rap-

idly throughout the world. Melanoma metastasizing to major or-

gans (stage IV) is virtually incurable. Currently, dacarbazine

(DTIC) is the only FDA-approved drug to treat advanced mela-

noma; it provides complete remission in only 2% of patients.^2–4

Prostate cancer is the most common noncutaneous malignancy

in men in Western countries. One out of nine men over 65 years

of age is diagnosed with prostate cancer in the United States.⁵It ac-

counts for one-third of all male cancer diagnoses and 9% of male

deaths as a result of cancer.⁶

(LPA, 1-acyl-sn-glycerol-3-phosphate), the resulting 2-arylthiazoli-

dine-4-carboxylic acid amides (ATCAAs) were potent cytotoxic

agents for prostate cancer and melanoma.^7–12One of earlier deriv-

atives (2RS,4 R)-2-phenyl-thiazolidine-4-carboxylic acid octade-

cylamide 1a and (2RS,4R)-2-(4-acetamidophenyl)-thiazolidine-4-

carboxylic acid hexadecylamide 1b (Fig. 1) were sent to the U.S.

National Cancer Institute 60 human tumor cell line anticancer drug

screen (NCI-60). The NCI-60 screening data indicated that com-

pounds 1a and 1b strongly inhibited the growth of all nine types

of cancer cells with GI₅₀values ranging from 0.12 lM (leukemia,

CCRF-CEM cell line) to 10.9

l

M (colon cancer, HCC-15 cell line).

CONH(CH₂)_nCH₃

NH

S

Abbreviations: ATCAA, 2-arylthiazolidine-4-carboxylic acid amide; ATCAs, 2-

arylthiazolidine-4-carboxylic acids; CDDP, cisplatin; DTIC, dacarbazine; DCC, N,N⁰-

dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; DMEM, Dulbecco’s

modiﬁed Eagle medium; EDCI, 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide;

FBS, fetal bovine serum; FLIPR, ﬂuorescence imaging plate reader; GPCR, G-protein-

coupled receptors; HBSS, Hank’s buffered salt solution; HOBT, 1-hydroxybenzotri-

azole; LPA, lysophosphatidic acid; NOE, nuclear Overhauser effect; Oleoyl-LPA,

1-oleoyl lysophosphatidic acid; SAR, structure–activity relationship; SRB, sulforho-

damine B.

O

HO P O

OH

O

(CH₂)_nCH₃

OH

R

ATCAA

1a n=17, R=H

LPA

1b n=15, R=NHCOCH₃

* Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448 3446.

E-mail address: dmiller@uthsc.edu (D.D. Miller).

Figure 1. Structures of LPA and ATCAA.

0968-0896/$ - see front matter Ó 2009 Published by Elsevier Ltd.

doi:10.1016/j.bmc.2009.12.020

478

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

1b was very potent against melanoma (GI₅₀= 0.13–1.48

against all eight tumor cell lines) and prostate cancer (GI₅₀

0.17–0.27 M against two tumor cell lines). Based on these preli-

lM

=

2. Chemistry

l

The general synthesis of (2RS,4R)-2-arylthiazolidine-4-carbox-

minary cytotoxic screenings, extensive structure–activity rela-

tionship (SAR) and in vivo studies were carried out. In this

article, we report the synthesis, biological evaluation, and SAR

studies of ATCAA analogues based on 1a and 1b for both mela-

noma and prostate cancer. These analogs were further assessed

for their ability to inhibit melanoma colony formation. The

in vivo efﬁcacy of compound 1b was tested in a xenograft model

using human A375 melanoma tumors. This class of compounds

showed potent antitumor activity and selectivity, which could

represent the basis for their development into novel chemothera-

peutic drugs.

amides is shown in Scheme 1. L-Cysteine reacted with appropriate

benzaldehydes in ethanol and water at ambient temperature to

give cyclized (2RS,4 R)-2-arylthiazolidine-4-carboxylic acids (AT-

CAs),¹³which were converted to the corresponding Boc-protected

derivatives 2a–2j. Reaction of 2a–2j with appropriate amines using

EDCI/HOBt gave corresponding amides, which were subsequently

treated with triﬂuoroacetic acid (TFA) to form the target com-

pounds 1a, 1b, and 3aa–3jc. Acetylation of the thiazolidine amino

group in compound 1b with acetyl chloride gave acylation deriva-

tive 4. Ester 5 was obtained by esterifying 2i with DCC/DMAP cat-

alyzed by (1S)-(+)-10-camphorsulfonic acid followed by TFA

COOH 2a R¹=3,4,5-trimethoxy

2b R¹=3,4-dimethoxy

COOH

O

2c R¹=2-methoxy

O

S

NBoc

S

NH

2d R¹=3-methoxy

2e R¹=4-methoxy

b

a

HS

OH

2f R¹=4-dimethylamino

2g R¹=2-acetylamino

NH₂

L-cysteine

R¹

Aldehyde

2h

R¹=3-acetylamino

2i R¹=4-acetylamino

2j R¹=H

R¹

2a-2j

R¹

ATCA

CONR²R³

NH

CONHC₁₆H₃₃

NH

CONHC₁₆H₃₃

S

NCOCH₃

e

S

c, d

R¹

NHCOCH₃

4

NHCOCH3

1b

For

, R³=H,

1a, 1b, 3aa-3jc

R³=CH , 3ai is 4S isomer of 3ae

3ia

R¹and R²see ³below

j

COOR²

NH

CONHC₁₆H₃₃

NH

C₁₆H₃₃HNOC

HN

COOH

NBoc

S

f, d

2a-2j

R¹

NHCOCH₃

H₃COCHN

R¹=4-NHCOCH , R²=n-C₁₆H₃₃

5

3

9

CH₂NHR²

NH

CON

O

CHO

S

NBoc

S

NBoc

S

i ,d

g

h

R¹

8a R¹=H, R²=n-C₁₆H₃₃

6

7

8b R¹=4-NHCOCH₃, R²=n-C₁₆H₃₃

3ib R¹=4-acetylamino, R²=1-adamantanyl;

3ic R¹=4-acetylamino, R²=2-adamantanyl;

3id R¹=4-acetylamino, R²=9H-fluoren-2-yl;

3ie R¹=4-acetylamino, R²=anthracen-2-yl;

3if R¹=4-acetylamino, R²=4-bipheny:

1a R¹=H, R²=n-C₁₈H₃₇

1b R¹=4-acetylamino, R²=n-C₁₆H₃₃

;

3ba R¹=3,4-dimethoxy, R²=n-C₁₈H₃₇

;

3bb R¹=3,4-dimethoxy, R²=(Z)-Octadec-8-enyl;

;

3aa R¹=3,4,5-trimethoxy, R²=n-C₁₈

H ;

3bc R¹=3,4-dimethoxy, R²=(E)-Octadec-8-enyl;

37

3ab R¹=3,4,5-trimethoxy, R²=(Z)-Octadec-8-enyl;

3ac R¹=3,4,5-trimethoxy, R²=(E)-Octadec-8-enyl;

3ca R¹=2-methoxy, R²=n-C₁₆H₃₃

;

3da R¹=3-methoxy, R²=n-C₁₆H₃₃

3ea R¹=4-methoxy, R²=n-C₁₆H₃₃

;

3ig R¹=4-acetylamino, R²=2-benzothiazolyl;

3ih R¹=4-acetylamino, R²=(Z)-Hexadec-9-enyl;

3ii R¹=4-acetylamino, R²=Nonadec-10-ynyl;

3ad R¹=3,4,5-trimethoxy, R²=n-C₁₆H₃₃

;

3ae R¹=3,4,5-trimethoxy, R²=n-C₁₄H₂₉

3af R¹=3,4,5-trimethoxy, R²=n-C₁₂H₂₅

3ag R¹=3,4,5-trimethoxy, R²=n-C₁₀H₂₁

3ah R¹=3,4,5-trimethoxy, R²=n-C₈H₁₇

3fa R¹=4-dimethylamino, R²=n-C₁₆H₃₃

;

3ga R¹=2-acetylamino, R²=n-C₁₆H₃₃

;

3ja R¹=H, R²=n-C₁₆H₃₃

;

3ha R¹=3-acetylamino, R²=n-C₁₆H₃₃

;

3jb R¹=H, R²=(Z)-Octadec-8-enyl;

3jc R¹=H, R²=(E)-Octadec-8-enyl

3ia R¹=4-acetylamino, R²=n-C₁₈

H

, R³=CH₃;

;

37

Scheme 1. Synthesis of ATCAA. Reagents and conditions: (a) C₂H₅OH, H₂O (70–99%); (b) Boc₂O, 1 N NaOH, 1,4-dioxane, H₂O (89–97%); (c) EDCI, HOBt, Et₃N, R²R³NH, CH₂Cl₂;

(d) TFA, CH₂Cl₂; (e) CH₃COCl, pyridine, CH₂Cl₂(74%); (f) DCC, DMAP, 0.3 equiv (1S)-(+)-10-camphorsulfonic acid, C₁₆H₃₃OH (94%); (g) EDCI, HOBt, NMM, HNCH₃OCH₃, CH₂Cl₂

(71%); (h) LAH, THF (84%); (i) R²NH₂, NaBH₃CN, HOAc; (j) LAH, THF, 0 °C to rt or B₂H₆, THF, rt to reﬂux.

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

479

treatment.¹⁴Reducing amide 1b with LAH or B₂H₆did not provide

the desired amine 8b; instead, disulfanyl dimer 9 was obtained. 8a

and 8b were prepared from (2RS,4R)-2-arylthiazolidine-4-carbal-

dehyde 7, which was obtained from reducing Weinreb amide 6.¹⁵

The reductive amination of aldehyde 7 with hexadecylamine and

NaBH₃CN¹⁶followed by Boc deprotection gave amines 8a and 8b.

The Fmoc-protected (2RS,4R)-(4-acetylamino-phenyl)-thiazoli-

the major isomer appeared at 5.32 ppm. The C4–H signal of the

major isomer appeared at 4.33 ppm, while the minor isomer ap-

peared at 3.90 ppm. The signals from the two unequivalent protons

of C5–H_aand C5–H_bshowed four sets of peaks; each set appeared

as a doublet of doublet (dd) as a result of geminal coupling and

with additional vicinal coupling to C4–H. These peaks were cen-

tered at 3.69 ppm, 3.41 ppm, 3.39 ppm, and 3.28 ppm, respectively,

with upﬁeld signals partially overlapping with CH₂signals (3.26–

3.21 ppm) from the long fatty chain near the carboxylic amide

CONHCH₂C₁₅H₃₁. The absolute structures of the two diastereomers

can be readily assigned using 1D nuclear Overhauser effect (NOE)

experiments (Fig. 2). When the peak at 4.33 ppm (C4–H of the ma-

jor isomer) was irradiated, the peak at 5.32 ppm (C2–H of the ma-

jor isomer) did not show any detectable NOE (Fig. 2B); when the

peak at 3.90 ppm (C4–H of the minor isomer) was irradiated, the

peak at 5.58 ppm (C2–H of the minor isomer) showed a strong

NOE (Fig. 2C). Because the chirality at C4 is ﬁxed (R-conﬁguration

dine-4-carboxylic acid 10 was prepared by reacting L-cysteine with

4-acetylaminobenzaldehyde, followed by reaction with Fmoc-Cl in

the presence of triethanolamine (TEA) with a 99% yield. Subse-

quent EDCI/HOBt coupling reaction yielded carboxamide 11. 4-

Aminophenyl intermediate 12 was obtained by acid hydrolysis in

methanol. Compound 12 was further reacted with different acid

chlorides or sodium cyanate¹⁷to afford derivatives 14a–14c. Com-

pounds 13a–13b and 15a–15c were obtained by deprotection of

the Fmoc group from 12 and 14a–14c, respectively (Scheme 2).

The open-ring compounds 16a–16d were prepared as shown in

Scheme 3. Compounds 16a–16c were easily obtained from (R)-2-

amino-3-(methylthio)propanoic acid or (R)-3-(benzylthio)-2-

(tert-butoxycarbonylamino)propanoic acid. Compound 16d was

readily prepared in a ﬁve-step procedure from 2-aminoacetic acid

at 38.4% overall yield.

We characterized each compound with nuclear magnetic reso-

nance (NMR), mass spectroscopy, and elemental analysis. Because

of the presence of two chiral centers at the thiazolidine ring (C2

and C4 positions, the chirality at C4 is ﬁxed), the ¹H NMR spectra

of ATCAAs indicated two diastereomers with a ratio of 7:3. The

chemical shifts for C2–H, C4–H, and C5–H_a/bon the thiazolidine

ring were greatly different between the two diastereomers. For

example, in the case of 1b (Fig. 2A), the C2–H signal of the minor

isomer appeared downﬁeld (5.58 ppm in CDCl₃), while that of

from starting material L-cysteine), the major isomer was clearly

(2S,4R), and the minor isomer was (2R, 4R) as indicated in Figure

2. The relative distribution of these two conﬁgurations can also

be expected based on their molecular structures, because it is ste-

rically more favorable when the two bulky groups are attached to

opposite sides of the ﬁve-membered ring.

3. Biological results and discussion

3.1. Antiproliferative effects of ATCAA against melanoma and

prostate cancer

The diastereomeric mixtures of compounds 3aa–15c were used

to evaluate their in vitro inhibitory activity and toxicity against

COOH

NFmoc

CONHR²

NFmoc

O

S

c

a, b

HS

OH

NH2

L-cysteine

HN

O

HN

O

HN

11

O

4-acetylamino-

benzaldehyde

10

CONHR²

e

S

NH

13a R²=n-C₁₂H₂₅

CONHR²

NFmoc

2

13b

R =n-C₁₆H₃₃

S

NH₂

13a-13b

d

CONHR²

NFmoc

CONHR²

NH

NH₂

12

S

R²=n-C₁₆H₃₃

S

15a R⁵= SO₂CH₃

15b R⁵= COCH₂Cl

15c R⁵= CONH₂

f

e

NHR⁵

14a-14c

NHR⁵

15a-15c

Scheme 2. Reagents: (a) C₂H₅OH, H₂O (78%); (b) Fmoc-Cl, Et₃N, CH₂Cl₂(99%); (c) EDCI, HOBt, Et₃N, R²NH₂, CH₂Cl₂(71%); (d) CH₃COCl, CH₃OH (48%); (e) DBU, CH₂Cl₂(46–66%)

(f) 15a CH₃SO₂Cl, pyridine (80%); 15b ClCH₂COCl, pyridine (51%); 15c NaOCN, HOAc, CH₃OH, H₂O (88%).

480

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

CONHR

NH

S

CONHR

NH₂

CONHR

H₃CS

16a

NH

S

16b

16d

NH

S

NHAc

CONHR

NHCOCH₃

1b

NH

3ja

NH₂

S

16c

R=C₁₆H₃₃

NHCOCH₃

O

S

NHR

O

NH

d, e

a, b, c

S

NHR

S

OH

NH₂

16b

16a

NHCOCH₃

COOH

NHBoc

CONHR

NH₂

NHBoc

S

c

b

16c

O

NHR

NH

d, e

a, b, c

NH₂CH₂CONHR

NH₂CH₂COOH

16d

NHCOCH₃

Scheme 3. Reagents: (a) Boc₂O, 1 N NaOH; (b) EDCI, HOBt, C₁₆H₃₃NH₂; (c) CF₃COOH; (d) N-(4-formyl-phenyl)-acetamide, MeOH, (e) NaBH₃CN.

two human melanoma cell lines (A375 and WM-164), one mouse

melanoma cell line (B16-F1), four human prostate cancer cell lines

(DU 145, PC-3, LNCaP, and PPC-1), ﬁbroblast cells (control cell line

for melanoma), and RH7777 (control cell line for prostate cancer).

Because preparing pure diastereomers was not easy to achieve, the

IC₅₀values were obtained on diastereomeric mixtures to select the

most promising compounds. The standard sulforhodamine B (SRB)

assay was used to evaluate the antiproliferative activity of different

compounds in melanoma and prostate cancer cells. The results are

summarized in Tables 1 and 2. Compounds 1a and 1b were chosen

as control compounds for both tumor cell lines in these in vitro

studies. Cisplatin (CDDP), DTIC, and Sorafenib (Nexavar, Bay43-

9006,¹⁸which had been granted Fast Track designation by FDA to

treat advanced melanoma) were used as reference compounds

for melanoma. The antiproliferative data showed that all three

melanoma cell lines were resistant to cisplatin. DTIC was inactive

because of the lack of bioactivation in vitro.^19,20The average IC₅₀

cells is threefold. Our purpose in modifying chemical structure was

to enhance the selectivity between tumor cells and control cells

and to increase or maintain antitumor potency. Thus, we used

the average IC₅₀value of all melanoma and prostate cancer cells

for comparison. Selectivity is deﬁned as the ratio of IC₅₀values in

the control cell line and the average in tumor cell lines. From Ta-

bles 1 and 2, the most selective compound (3id) had a selectivity

of 11.3-fold in melanoma cells against ﬁbroblast cells; compounds

3ac and 15b showed an improved selectivity of 9.1-fold and 9.4-

fold for prostate cancer cells, respectively.

3.2. Effects of different amide chains, C4 chirality, and chain

length of ATCAA

ATCAA molecules were designed from LPA structure, which

contains a long aliphatic chain. To examine whether the fatty chain

played an essential role in cytotoxicity, we explored the possibili-

ties of mimic fatty chain with bulky ring or aromatic systems such

as adamantanyl, ﬂuorenyl, and anthrancenyl. Although the anti-

value of Sorafenib on three melanoma cell lines is 5.1

lM, and

the selectivity of Sorafenib between ﬁbroblast cells and melanoma

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

481

H_a

O

H

H_b

5

H

5

H_b

N

(CH₂)₁₅CH₃

N

(CH₂)₁₅CH₃

S

H

Ar

4

N

2

H

2

H

Ar

H

NOE effect

Minor: 2R, 4R

Major: 2S, 4R

NHCOCH₃

Ar =

C2-H

major

C4-H

major

C2-H

minor

C4-H

minor

A. ¹H NMR

B. No NOE between C4-H and C2-H (major)

C. Strong NOE between C4-H and C2-H (minor)

5.5

5.0

4.5

4.0

3.5

3.0

Chemical Shift (ppm)

Figure 2. (A) Expanded ¹H NMR spectrum of two diastereomers of 1b (ratio 7:3) in CDCl₃; (B) 1D NOE from irradiation of C4–H of the major isomer at 4.33 ppm, no NOE

observed at 5.32 ppm; (C) 1D NOE from irradiation of C4–H of minor isomer at 3.90 ppm, shows strong NOE at 5.58 ppm. Thus, protons at C2–H and C4–H are ‘cis-position’ in

the minor isomer.

proliferative data showed that 1- or 2-adamantanyl amides and 2-

benzothiazolylamide (3ib, 3ic, and 3ig) decreased activity against

most melanoma and prostate cancer cells, introducing certain aro-

matic bulky groups such as 9H-ﬂuoren-2-yl (3id), anthrancen-2-yl

(3ie), and 4-biphenyl (3if) retained their proliferative activity for

both types of cancers. Of particular interest, while introducing 9-

Short chain lengths with 8–10 carbon atoms in 4-carboxamide

fatty chains (3ah, 3ag) displayed low potency. As chain length in-

creased, potency increased, but toxicity also increased as measured

on the RH7777 cell line (Fig. 3). Hexadecyl chains (3ad) displayed

both the highest potency and selectivity against cancer cells among

the examined chains, with an IC₅₀= 0.4 lM for PPC-1 cells. How-

ﬂuoren-2-yl (3id) showed a comparable IC₅₀value of 2.6

l

M with

ever, further increasing chain length (18 carbons, 3aa) reduced po-

tency and toxicity. Interestingly, adding either a cis- or trans-

double bond in the C18 side chain restored potency dramatically

(cis and trans-octadec-8-enyl), demonstrating that both length

and composition of the side chain are critical for their activity.

trans-Octadec-8-enyl chain (3ac, 3bc, 3jc) showed slightly better

activity than did the cis-isomers (3ab, 3bb, 3jb) against prostate

cancer cells. Introducing a cis-double bond in the C16 fatty chain

(3ih) or an alkyne in the C18 chain (3ii) did not increase overall po-

tency on either cancer cell line. Introducing a branched aliphatic

chain (3ia) decreased the potency in both melanoma cells (average

1b (1.8 M) on melanoma cells, the selectivity of 3id increased to

l

11.3-fold from 8.9-fold in 1b. This structural modiﬁcation provided

us a new approach to optimize the drug-like properties of ATCAA

molecules, in which the hydrophobic fatty lipid chain could be

modiﬁed with other groups.²¹

To investigate whether the chiral center at the 4-thiazolidine

position had an effect on the potency of ATCAA, the 4S isomer

(3ai) was prepared using

D-cysteine as starting material. After

comparing activity of 4R (3ae) with 4S (3ai) isomers in both pros-

tate cancer cell lines and RH7777 cells (Table 4), no signiﬁcant ste-

reoselectivity at the thiazolidine 4-position was found. Therefore,

changing the chirality at C4 on the thiazolidine ring did not sub-

stantially affect either activity or selectivity.

IC₅₀= 16.7

age IC₅₀= 2.6

IC₅₀= 1.8 M for melanoma, 1.1

l

M, 9.3-fold decrease) and prostate cancer cells (aver-

M, 2.4-fold decrease) compared with 1b (average

M for prostate cancer).

l

482

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

Table 1

Antiproliferative effects of ATCAA in three melanoma cell lines and ﬁbroblast cell line

S

R¹

XR²R³

N

R⁴

a

Average IC₅₀of three melanoma cells IC₅₀s.

Ratio = (IC₅₀of ﬁbroblast cell)/(the average IC₅₀of melanoma cell lines).

b

3.3. Effects of substitutions on 2-phenyl of ATCAA

sulted in loss of selectivity against melanoma cells (1.8-fold for

15c vs 8.9-fold for 1b). A similar trend with 15a and 15c was found

in prostate cancer, except that 15b showed improved growth inhi-

The antiproliferative effects on both melanoma and prostate

cancer indicated that when methoxy, 3,4-dimethoxy, and 3,4,5-tri-

methoxy were introduced to 2-phenyl (3aa–3ea), potent activity

was preserved or increased compared with that of 1a. For example,

(2RS,4R)-2-(3,4,5-trimethoxy-phenyl)thiazolidine-4-carboxylic acid

bition of prostate cancer cells (average IC₅₀of 15b is 1.0

pared with 6.9 M for 1a and 1.1 M for 1b) and a 9.4-fold

selectivity against prostate cancer cells.

lM com-

l

We also investigated the importance of o-, m-, and p-substitu-

tions in the 2-phenyl ring relative to acetylamino and methoxy

groups. Interestingly, o-, m-, and p-isomers showed different activ-

ities. p-Isomers (3ea, 1b) had the best selectivity and activity for

both cancer cell lines. m-Isomers (3da, 3ha) had similar average

IC₅₀s against melanoma compared with p-isomers, but their toxic-

octadecylamide (3aa) (average IC₅₀= 2.9

lM) was 2.4-fold more

potent than was 1a (average IC₅₀= 6.9 M) against prostate cancer

l

cells. Subsequently, different groups were introduced to the para-

phenyl position. Replacements of 4-NHCOCH₃(1b) with 4-

N(CH₃)₂(3fa) showed comparable activity on A375 (IC₅₀= 1.8 vs

2.1

l

M) and WM-164 cells (IC₅₀= 1.5 vs 1.1

l

M) but slightly lower

M).

ity also increased on both ﬁbroblast (2.5

lM and 6.3 lM) and

selectivity on ﬁbroblast cells (IC₅₀= 21.0 vs 16.0

l

RH7777 cells (3.6 M and 4.9 M). o-MeO and o-NHAc analogues

l

Although both 4-N(CH₃)₂(3fa) and 4-NH₂(13b) substituted AT-

(3ca, 3ga) showed slightly less potency and selectivity on both

CAAs were more potent than was 1a against prostate cancer cells

melanoma and prostate cancer cells.

(average IC₅₀= 2.8

1a), they were less potent than was 1b (average IC₅₀= 1.1

and selective ratio = 6.3 for 1b). Methanesulfonamide (15a) or 2-

chloroacetamide (15b) were less potent (IC₅₀= 5.7–113.6 M)

and less toxic (IC₅₀= 25.4–>100 M) on melanoma cell lines com-

pared with 1b. Introducing a ureido, 4-NHCONH₂(15c) instead of a

4-NHCOCH₃kept partial potency (average IC₅₀= 3.9 M) but re-

lM for 3fa, 1.8 lM for 13b, and 6.9 lM for

lM

3.4. Effects of 4-linkage of ATCAA and thiazolidine ring

l

To investigate the importance of amide linkage on C4 of ATCAA,

the carboxamide was replaced with an ester and amine. The isos-

teric replacement of the amide by an ester resulted in 5 with mark-

edly decreased activity against all cancer cell lines. The amine

l

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

483

Table 2

Antiproliferative effects of ATCAA in four prostate cancer cell lines and RH7777 cell line

S

R¹

XR²R³

N

R⁴

a

Average IC₅₀of four prostate cells IC₅₀s.

Ratio = (IC₅₀of RH7777 cell)/(average IC₅₀of prostate cell lines).

b

derivatives 8a and 8b also failed to show any increased potency,

although they did show lower toxicity (60–84.5 M) than did 1a

(29.8 M) and 1b (16 M) in ﬁbroblast cells.

l

Thiazolidine-4-carboxylic acid has been reported to be effective

for treating advanced cancers.^22,23To examine the importance of a

central thiazolidine ring, we synthesized 3-acetyl-thiazolidine

compound 4 and a series of thiazolidine ring-opened compounds

16a–16d as shown in Schemes 1 and 3. Antiproliferative activity of

16a–16d against melanoma is summarized in Table 3. When acetyl

was introduced to 3-NH of thiazolidine, activity decreased in both

cancer cell lines (average IC₅₀= 16 and 10.3 lM against melanoma

Table 3

Antiproliferative effects of thiazolidine ring-open compounds against two melanoma

cell lines and ﬁbroblast cell line

ID

IC₅₀SEM (lM)

A375

WM-164

Fibroblast

16a

16b

16c

16d

3ja

1b

12.9 1.3

108.5 4.8

15.1 1.1

11.9 0.6

ND^a

14.4 0.8

61.2 3.6

>100

16.4 1.2

24.0 1.3

2.2 0.2

16.0 2.5

144.9 5.2

12.8 2.1

10.2 0.5

0.7 0.1

2.1 0.2

1.1 0.1

Figure 3. Amide carbon chain length relative to IC₅₀in prostate cancer cell lines

(average IC₅₀of DU 145, PC-3, LNCaP, and PPC-1 cells) and RH7777 cell line.

a

ND = not determined.

484

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

Table 4

Antiproliferative effects of different chain length amides against prostate cells and R7777 cells

Compounds

Conﬁguration at 4-position

R

IC₅₀SEM (

LNCaP

lM)

DU 145

PC-3

PPC-1

RH7777

3ah (4R)

3ag (4R)

n-C₈H₁₇

n-C₁₀H₂₁

n-C₁₂H₂₅

n-C₁₄H₂₉

n-C₁₆H₃₃

n-C₁₈H₃₇

(Z)-Octadec-8-enyl

(E)-Octadec-8-enyl

17.3 1.4

4.1 0.3

>20

4.4 0.3

2.7 0.7

2.2 0.3

14.0 0.5

2.1 0.1

>20

6.0 0.8

CONHR

*

3af (4R)

3ae (4R)

3ai (4S)

3ad (4R)

3aa (4R)

3ab (4R)

3ac (4R)

2.8 0.1

2.2 0.1

2.1 0.1

1.7 0.1

4.1 0.7

1.2 0.1

0.9 0.1

2.9 0.1

2.6 0.1

2.9 0.1

1.8 0.1

4.9 0.4

1.5 0.1

1.2 0.1

1.4 0.1

2.0 0.3

1.9 0.1

0.8 0.1

1.6 0.2

0.7 0.1

0.6 0.1

1.0 0.1

0.6 0.1

0.7 0.1

0.4 0.1

1.0 0.1

0.5 0.1

0.3 0.1

6.5 0.1

6.1 0.6

5.8 0.8

7.2 0.1

14.0 0.5

8.4 0.8

6.8 0.7

S

NH

*

O

Figure 4. Flow cytometric analysis of selected ATCAA compounds 3ad and 1b. Effect of selected compounds on cell cycle progression of prostate cancer LNCaP and melanoma

A375 cells was examined according to the procedure described in Section 5. Sub-G1 phase accumulation was induced by ATCAA compounds.

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

485

and prostate cancer, respectively). Cleavage of both the C–S and C–

N bond (16a) led to substantial decrease in both potency (12.9–

regulated breakdown of membrane phospholipids that are known

to stimulate G-protein-coupled receptors (GPCR) signaling. GPCR

activation could be detected through changes in intracellular cal-

cium concentration. To examine whether ATCAA compounds inhi-

bit cancer cell growth via LPA-GPCR pathway, a ﬂuorescence

imaging plate reader FLIPR assay (Ca²⁺ﬂux response) was con-

ducted to screen 3ad for dose-dependent agonist and antagonist

activities on the LPA1-, LPA3-, and LPA5-transfected Chem-1 cells.

Oleoyl-LPA and a known LPA antagonist compound Ki16425²⁴

were used as a positive and negative control, respectively. In ago-

nist assay mode, oleoyl-LPA provided EC₅₀values of 31 nM,

120 nM, and 12 nM for LPA1, LPA3, and LPA5, respectively. When

referencing E_maxof oleoyl-LPA, 15% activation was considered as

an agonist. Compound 3ad did not exhibit any dose-dependent

agonist activity on LPA1, LPA3, or LPA5 (Fig. 5). We also tested

antagonism of 3ad, and 50% inhibition was considered as an antag-

onist when referencing EC₈₀. The negative control, Ki16425, had an

IC₅₀value of 46 nM on LPA1. However, 3ad did not exhibit any

dose-dependent antagonist activity on the LPA1, LPA3, or LPA5

(Fig. 6). The FLIPR assay results suggested that 3ad did not display

cytotoxicity as a result of antagonism or agonism of the LPA

receptors. ATCAA compounds exhibit cytotoxicity through other

mechanisms.

14.4

trol cells) compared with 1b and 3ja (0.7–2.1

2.0–16.0 M on control cells). Opening the thiazolidine ring from

the C–S bond (16b) caused a loss of potency on both cancer and

control cells (>100 M). Cleavage of the C–N bond in the thiazoli-

dine ring (16c) also resulted in decreased activity (12.8–15.1 M)

lM on melanoma cell lines) and toxicity (61.2

lM on the con-

lM on cancer cells,

l

and no improvement on selectivity compared with 3ja. Removing

the C–S bond (16d) reduced the activity of 1b from IC₅₀= 1.1–

2.1 lM to 10.2–11.9 lM. These results suggested that the presence

of the thiazolidine ring is critical for activity. Attempts to reduce

the amide with LAH or B₂H₆resulted in the C–S bond cleavage in

thiazolidine. A disulfanyl dimer (9), which completely lost antican-

cer activity, was obtained, and this result is consistent with the

above ﬁnding that the thiazolidine ring played an essential role

in ATCAA potency.

3.5. Flow cytometric analysis

Flow cytometric analysis was performed on human melanoma

A375 cells and human prostate cancer LNCaP cells to examine

the effect of the new synthetics on cell cycle progression. Analysis

of the DNA content of control cells (Fig. 4A–D, top graphs) showed

typical distribution of peaks corresponding to cells in G1/G0 phase

(65.67–66.97%), S-phase (24.16–28.36%), and G2/M-phase (5.97–

8.87%). Upon treatment with compounds 3ad and 1b at 1-, 5-,

3.7. ATCAA inhibits human melanoma colony formation

Cells grown in 96-well plates do not reﬂect many properties

associated with three-dimensional tumors. Human tumor colony

formation assay (ex vivo soft agar colony assay) has been sug-

gested as an in vitro method to predict the response of an individ-

ual patient’s tumor to chemotherapeutic agents.^25,26As a prelude

to in vivo animal experiments, we investigated the ability of 1b

and 3ad to inhibit melanoma colony formation by using a well-

established method.^27–29We performed studies with A375 human

melanoma cells. Compounds 1b and 3ad could effectively inhibit

10-, and 20-lM concentrations for 48 h, ﬂow cytometric analysis

showed that these compounds signiﬁcantly induced apoptosis

(sub-G1 phase) in cancer cells in a dose-dependent manner. In

both cell lines, the accumulation of cells in sub-G1 phase became

apparent at 5 lM (Fig. 4).

3.6. Intracellular calcium mobilization assays of ATCAA

The initial design of ATCAA was derived from contriving mimics

of lysophosphatidic acid (LPA), a lipid mediator generated via the

A375 colony formation at 2

(Fig. 7). At a concentration of 20

l

M, the lowest tested concentration

l

M, compound 3ad almost

Figure 5. FLIPR intracellular calcium mobilization assay in agonist mode. Calcium ﬂux in LPA1-, LPA3-, and LPA5-expressing Chem-1 cell lines induced by oleoyl-LPA. Oleoyl-

LPA increased [Ca²⁺]_iin a dose-dependent manner; 3ad and LPA antagonist Ki15425 did not show agonist effect.

486

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

Figure 6. (A) Response of 3ad and oleoyl-LPA on LPA1, LPA3, and LPA5 kinetic data. Based upon these kinetic traces exhibited by 3ad, the relatively small Ca²⁺ﬂux is nonLPA

receptor mediated. (B) Antagonist data of 3ad, which did not show antagonism on LPA1, LPA3, or LPA5. Ki16425 had an IC₅₀= 46 nM on LPA1.

completely inhibited colony formation, while compound 1b inhib-

ited colony formation by 75%. At the highest tested concentration,

100 lM, melanoma colony formation was completely inhibited by

both 1b and 3ad.

mined to be 42 mg/kg. Two nontoxic dosages (5 mg/kg and

10 mg/kg) were used. We also included DTIC (60 mg/kg),³¹the gold

standard for melanoma treatment, as a positive control to assess

the efﬁcacy of compound 1b. Brieﬂy, male athymic nude mice were

injected subcutaneously with 2.5 ꢀ 10⁶A375 cells. Treatment be-

gan on day 7 after tumor inoculation, and 1b was injected once a

day. Each group was composed of eight mice. The results are

shown in Figure 8. Compound 1b at 5 mg/kg showed moderate

melanoma tumor growth inhibition. After 22 days of treatment,

the percentage of tumor reduction was 25%. At a higher dose of

10 mg/kg, compound 1b showed signiﬁcant melanoma tumor

growth inhibition with 62% of tumor reduction. Compared with

DTIC at a dose of 60 mg/kg, which inhibited tumor growth by

42% at the end of treatment, compound 1b clearly showed superior

activity in this human melanoma xenograft model. All mice dis-

played normal activities, and no signiﬁcant body weight loss was

observed during the experiment.

3.8. Antitumor efﬁcacy of ATCAA on human melanoma cancer

A375 xenografted athymic nude mice

We chose human melanoma A375 cells to test efﬁcacy in vivo

because ATCAA exhibited potency both in vitro assay and colony

formation assay against A375 cells. A375 xenograft tumors also

exhibited faster tumor growth rate. While compound 3ad was

more potent than was 1b in inhibiting melanoma colony forma-

tion, it had much poorer selectivity between melanoma cancer

cells and ﬁbroblast cells (2.0-fold for 3ad vs 8.9-fold for 1b) as

shown in Table 1. Therefore, we chose 1b for our in vivo studies

against A375 melanoma tumors. ATCAA compound 1b was exam-

ined for in vivo evaluation and was formulated with 80% Tween 80

and 20% Captex 200 because of its limited water solubility.³⁰We

ﬁrst determined the maximally tolerated dose (MTD) in ICR mice.

These mice are less expensive than nude mice and are commonly

used for toxicity testing. The MTD for compound 1b was deter-

4. Conclusions

We synthesized a series of ATCAA compounds and thiazolidine

ring-opened analogues. Chemical modiﬁcation and structure–

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

487

Figure 7. Compounds 3ad and 1b inhibit melanoma colony formation.

Figure 8. Growth of A-375 tumor treated with compound 1b or DTIC. (A) in vivo results of treatment of 1b and DTIC on nude mice bearing A375 tumors compared with

control group. (B) Average body weight change in control and treatment groups.

activity relationship of ATCAA compounds were investigated with

different substituted 2-phenyl, thiazolidine ring, 4-position link-

age, and 4-carboxamide groups (Fig. 9) based on biological evalua-

tion against melanoma and prostate cancer cells in vitro. Two

compounds, 3id and 3ac, and another, 15b, are promising agents

against melanoma and prostate cancer cells, respectively. Com-

pounds 1b and 3ad effectively inhibited A375 melanoma colony

formation and induced apoptosis. Compound 1b at 10 mg/kg sig-

niﬁcantly inhibited melanoma tumor growth in vivo and showed

higher efﬁcacy than did DTIC at 60 mg/kg. These compounds did

not seem to work via the LPA-GPCR pathway. Additional work is

under way to investigate the mechanisms of action of ATCAA and

to perform further in vivo studies with other active ATCAA com-

pounds and in prostate cancer xenografted models.

5. Experimental section

5.1. General

All reagents were purchased and used without further puriﬁca-

tion from Sigma–Aldrich Chemical Co. (St. Louis, MO), Fisher Scien-

a. Free NH and thiazolidine ring contribute to anticancer

activity; 4R and 4S isomers have similar potency and activity

b. Fatty chains (C-16 optimum);9H-fluoren-2-

yl is the best for melanoma; (E)-octadec-8-

enyl unsaturated chains is best for prostate

cancer.

S

*

R¹

N

R⁴

*

X

R²R³

c

. amide is better than ester and amine

d

. Phenyl substitutions: EDG are better than EWG; para-andmeta-

substituents are comparable, with ortho-substituents being slightly worse

Figure 9. SAR relationship of ATCAA.

488

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

tiﬁc (Pittsburgh, PA) and AK Scientiﬁc, Inc (Mountain View, CA).

Moisture-sensitive reactions were conducted in an argon atmo-

sphere. Argon gas was purchased from NexAir Medical Gas, Inc.,

TN. Routine thin layer chromatography (TLC) was performed on

aluminum-backed uniplates (Analtech, Newark, DE). NMR spectra

were obtained on a Bruker AX 300 (Billerica, MA) spectrometer.

Chemical shifts were reported as parts per million (ppm) relative

to TMS in CDCl₃or DMSO-d₆. Mass spectral data were collected

on a Bruker ESQUIRE electrospray/ion trap instrument in positive

and negative ion modes. Elemental analyses were performed by

Atlantic Microlab Inc. (Norcross, GA).

[MꢂH]^ꢂ. Anal. Calcd for C₂₈H₄₇N₃O₂S: C, 68.51; H, 9.61; N, 8.48.

Found: C, 68.67; H, 9.67; N, 8.58.

5.3.3. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid octadecylamide (3aa)

Yield: 65.3%. ¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.31 (t, 0.7H,

J = 6.6 Hz and br, 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and

5.28 (s, s, 0.3H and 0.7H), 4.34 and 3.93 (dd, J = 7.8 Hz, 4.2 Hz, m,

1H), 3.88 and 3.87 (s, s, 6H), 3.80 and 3.79 (s, s, 3H), 3.68 and

3.42 (dd, J = 11.1 Hz, 4.2 Hz, m, 2H), 3.31–3.19 (m, 2H), 2.52 (br,

1H), 1.50 (m, 2H), 1.25 (s, 30H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI)

m/z 551.5 [M+H]⁺, 573.5 [M+Na]⁺, 549.2 [MꢂH]^ꢂ. Anal. Calcd for

C₃₁H₅₄N₂O₄S: C, 67.59; H, 9.88; N, 5.09. Found: C, 67.38; H, 9.95;

N, 5.09.

5.2. General procedure for preparing (2RS,4R)-2-arylthiazolidine-

3, 4-dicarboxylic acid 3-tert-butyl ester 2a–2j

A mixture of

L

-cysteine (3.16 g, 26.11 mmol) and appropriate

5.3.4. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid octadec-8-cis-enylamide (3ab)

aldehyde (26.15 mmol) in ethanol (300 mL) and water (30 mL)

was stirred at room temperature for 6–15 h, and the separated so-

lid was collected, washed with diethyl ether, and dried to obtain

(2RS,4 R)-2-arylthiazolidine-4-carboxylic acid (ATCA) with yields

of 70–99%. At 0 °C, ATCA (5.95 mmol) was dissolved in 1 N NaOH

(6 mL) and 1,4-dioxane (15 mL); then di-tert-butyldicarbonate

(2.80 g, 12.80 mmol) was added slowly and stirred at room tem-

perature for 1 h. The reaction mixture was concentrated in a vac-

uum and washed with ethyl acetate (20 mL). The aqueous phase

was adjusted to pH 4 by adding 1 N HCl or 5% KHSO₄, then ex-

tracted with ethyl acetate, dried with magnesium sulfate, ﬁltered,

and concentrated in a vacuum to give corresponding compounds

2a–2j as white foam-solids with yields of 89–97%. 2a–2j were used

further in the next step of synthesis without puriﬁcation.

Yield: 58.8%.¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.31 (t, 0.7H,

J = 6.0 Hz and br, 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and

5.28 (s, s, 0.3H and 0.7H), 5.37–5.32 (m, 2H), 4.34 and 3.94 (dd,

J = 7.2 Hz, 4.2 Hz, m, 1H), 3.88 and 3.87 (s, s, 6H), 3.84 and 3.84

(s, s, 3H), 3.68 and 3.43 (dd, J = 11.1 Hz, 4.2 Hz, m, 2H), 3.33–3.21

(m, 2H), 2.01–1.97 (br, 4H), 1.97 (br, 1H), 1.55–1.44 (m, 2H), 1.26

(s, 22H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 549.5 [M+H]⁺, 547.2

[MꢂH]^ꢂ. Anal. Calcd for C₃₁H₅₂N₂O₄S: C, 67.84; H, 9.55; N, 5.10.

Found: C, 67.49; H, 9.55; N, 5.12.

5.3.5. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid octadec-8-trans-enylamide (3ac)

Yield: 42.1%. ¹H NMR (300 MHz, CDCl₃) d 7.19 and 6.29 (t, 0.7H,

J = 6.3 Hz and br, 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and

5.28 (s, s, 0.3H and 0.7H), 5.37 (br, 2H), 4.34 and 3.95 (dd,

J = 7.8 Hz, 4.2 Hz, m, 1H), 3.88 and 3.87 (s, s, 6H), 3.84 (s, 3H),

3.68 and 3.43 (dd, J = 11.1 Hz, 4.2 Hz, m, 2H), 3.31–3.19 (m, 2H),

1.97 (br, 4H), 1.55–1.46 (m, 2H), 1.26 (s, 22H), 0.87 (t, 3H,

J = 6.6 Hz). MS (ESI) m/z 583.2 [M+Na]⁺, 549.5 [M+H]⁺, 547.2

[MꢂH]^ꢂ. Anal. Calcd for C₃₁H₅₂N₂O₄S: C, 67.84; H, 9.55; N, 5.10.

Found: C, 68.10; H, 9.62; N, 5.11.

5.3. General procedure for preparing (2RS,4R)-2-arylthiazolidine-

4-carboxylic acid amide 1a, 1b, and 3aa–3jc

A mixture of appropriate Boc-protected carboxylic acids (2a–2j,

0.3–0.5 g), EDCI (1.2 equiv), and HOBT (1.05 equiv) in CH₂Cl₂

(20 mL) was stirred at room temperature for 10 min. To this solu-

tion, appropriate amine (1.05 equiv) and Et₃N (1.2 equiv) were

added, and stirring continued at room temperature for 6–15 h.

The reaction mixture was diluted with CH₂Cl₂(30 mL) and sequen-

tially washed with water, satd NaHCO₃, and brine and dried over

MgSO₄. The solvent was removed under reduced pressure to yield

a crude oil, which was stirred with TFA (0.6–1 mL) in 20 mL CH₂Cl₂

at room temperature for 1–8 h to cleave the Boc group. The reac-

tion mixture was concentrated, washed with satd NaHCO₃, and

dried over MgSO₄. The solvent was removed to yield a crude solid;

ATCAAs 1a–3jc were puriﬁed by silica column ﬂash chromatogra-

phy using hexane/ethyl acetate gradient elution system. Yield

was reported as 2-step yield.

5.3.6. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3ad)

Yield: 46.7%. ¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.30 (t, 0.7H,

J = 6.3 Hz and br, 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and

5.28 (d, d, 0.3H, J = 10.2 Hz and 0.7H, J = 10.8 Hz), 4.34 and 3.93 (dd,

br, m, J = 4.2 Hz, 1H), 3.89 and 3.88 (s, s, 6H), 3.85 and 3.84 (s, s,

3H), 3.68 and 3.45–3.39 (dd, m, 2H), 3.38–3.19 (m, 2H), 2.63 and

2.52 (br, br, 1H), 1.53 (m, 2H), 1.25 (s, 26H), 0.87 (t, 3H,

J = 6.3 Hz). MS (ESI) m/z 524.1 [M+H]⁺, 523.8 [MꢂH]^ꢂ. Anal. Calcd

for C₂₉H₅₀N₂O₄S: C, 66.63; H, 9.64; N, 5.36. Found: C, 66.41; H,

9.63; N, 5.30.

5.3.1. (2RS,4R)-2-Phenyl-thiazolidine-4-carboxylic acid

octadecylamide (1a)

5.3.7. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid tetradecylamide (3ae)

Yield: 76.1%. ¹H NMR (300 MHz, CDCl₃) d 7.63–7.37 (m, 6H),

5.70 (br, 2H), 5.67 and 5.49 (s, s, 1H, 0.3H and 0.7H), 4.90 and

4.63 (br, t, J = 4.5 Hz, 1H), 3.66–3.46 (m, 2H), 3.30–3.26 (m, 2H),

1.54–1.50 (m, 2H), 1.25 (s, 30H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI)

m/z 461.4 [M+H]⁺. Anal. Calcd for C₂₈H₄₈N₂OSꢁCF₃COOH: C, 62.69;

H, 8.59; N, 4.87. Found: C, 62.94; H, 8.84; N, 4.95.

Yield: 51.0%. ¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.27 (t, 0.7H,

J = 6.3 Hz and br, 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.56 and

5.28 (d, d, J = 10.2 Hz and 0.7H, J = 10.5 Hz), 4.34 and 3.93 (dd,

J = 4.2 Hz, m, 1H), 3.89 and 3.88 (s, s, 6H), 3.85 and 3.84 (s, s,

3H), 3.68 and 3.45–3.36 (dd, m, 2H), 3.34–3.19 (m, 2H), 2.51 (br,

1H), 1.50 (m, 2H), 1.25 (s, 22H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI)

m/z 495.6 [M+H]⁺. Anal. Calcd for C₂₇H₄₆N₂O₄S: C, 65.55; H, 9.37;

N, 5.66. Found: C, 65.49; H, 9.41; N, 5.59.

5.3.2. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (1b)

Yield: 61.3%. ¹H NMR (300 MHz, CDCl₃) d 7.54–7.43 (d,d, 4H,

J = 8.4 Hz), 7.25 (br, 1H), 5.62 and 5.33 (s, s, 0.3H and 0.7H), 4.36

and 4.05 (br, 1H), 3.69 and 3.43 (dd, m, 2H, J = 4.2 Hz), 3.31–3.24

(m, 2H), 2.19 (s, 3H), 1.53–1.47 (m, 4H), 1.26 (s, 26H), 0.88 (t,

3H, J = 6.9 Hz). MS (ESI) m/z 490.5 [M+H]⁺, 512.5 [M+Na]⁺, 488.1

5.3.8. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid dodecylamide (3af)

Yield: 63.9%. ¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.31 (t, br,

0.7H and 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and 5.29

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

489

(d, d, br, 0.3H, J = 7.8 Hz and 0.7H, J = 7.5 Hz), 4.34 and 3.93 (br, m,

1H), 3.89 and 3.88 (s, s, 6H), 3.85 and 3.84 (s, s, 3H), 3.68 and 3.47–

3.36 (m, m, 2H), 3.33–3.19 (m, 2H), 2.53 (br, 1H), 1.53 (m, 2H), 1.25

(s, 18H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 490.3 [M+Na]⁺. Anal.

Calcd for C₂₅H₄₂N₂O₄S: C, 64.34; H, 9.07; N, 6.00. Found: C,

64.29; H, 9.06; N, 5.92.

3.88 (s, s, s, s, 6H), 3.69 and 3.43 (dd, J = 11.1 Hz and 4.2 Hz, m, 2H),

3.33–3.17 (m, 2H), 1.96 (br, 4H), 1.52–1.43 (m, 2H), 1.26 (s, 22H),

0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 519.5 [M+H]⁺, 541.3 [M+Na]⁺,

517.2 [MꢂH]^ꢂ. Anal. Calcd for C₃₁H₅₀N₂O₄S: C, 69.45; H, 9.71; N,

5.40. Found: C, 69.57; H, 9.86; N, 5.20.

5.3.15. (2RS,4R)-2-(2-Methoxy-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3ca)

5.3.9. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid decylamide (3ag)

Yield: 38.8%. ¹H NMR (300 MHz, CDCl₃) d 7.52–7.27 and 7.04–

6.92 (m, 4H), 7.35 (br, 1H), 5.93 and 5.63 (s, s, 1H), 4.37 and 4.23

(dd, J = 7.8 Hz and 4.2 Hz, br, 1H), 3.92 and 3.89 (s, s, 3H), 3.67,

3.42–3.35 (dd, m, 2H), 3.33–3.20 (m, 2H), 1.53 (m, 2H), 1.25 (s,

26H), 0.88 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 463.4 [M+H]⁺. Anal. Calcd

for C₂₇H₄₆N₂O₂S: C, 70.08; H, 10.02; N, 6.05. Found: C, 69.97; H,

9.96; N, 6.02.

Yield: 50.0%. ¹H NMR (300 MHz, CDCl₃) d 7.21 and 6.32 (br, br,

0.7H and 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and 5.28 (s,

s, 0.3H and 0.7H), 4.34 and 3.94 (dd, J = 7.8 Hz and 4.2 Hz, m, 1H),

3.89 and 3.88 (s, s, 6H), 3.85 and 3.84 (s, s, 3H), 3.68 and 3.45–3.36

(dd, m, 2H), 3.34–3.19 (m, 2H), 1.53 (m, 2H), 1.25 (s, 14H), 0.87 (t,

3H, J = 6.9 Hz). MS (ESI) m/z 461.9 [M+Na]⁺. Anal. Calcd for

C₂₃H₃₈N₂O₄S: C, 62.98; H, 8.73; N, 6.39. Found: C, 62.98; H, 8.71;

N, 6.28.

5.3.16. (2RS,4R)-2-(3-Methoxy-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3da)

5.3.10. (2RS,4R)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid octylamide (3ah)

Yield: 33.6%. ¹H NMR (300 MHz, CDCl₃) d 7.47–6.87 (four dou-

blets, 4H, J = 8.7 Hz), 7.30 (br, 1H), 5.64 and 5.33 (s, s, 1H), 4.36

and 4.25 (dd, J = 4.2 Hz, br, 1H), 3.88 and 3.83 (s, s, 3H), 3.69,

3.46–3.35 (dd, J = 10.2 Hz and 4.2 Hz, m, 2H), 3.30–3.24 (m, 2H),

1.53–1.48 (m, 2H), 1.25 (s, 26H), 0.88 (t, 3H, J = 7.2 Hz). MS (ESI)

m/z 485.5 [M+Na]⁺. Anal. Calcd for C₂₇H₄₆N₂O₂S: C, 70.08; H,

10.02; N, 6.05. Found: C, 69.88; H, 9.93; N, 5.97.

Yield: 27.7%. ¹H NMR (300 MHz, CDCl₃) d 7.21 and 6.34 (br, br,

0.7H and 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.55 and 5.28 (s,

s, 0.3H and 0.7H), 4.34 and 3.93 (dd, J = 7.8 Hz and 4.2 Hz, m, 1H),

3.89 and 3.88 (s, s, 6H), 3.85 and 3.84 (s, s, 3H), 3.68 and 3.45–3.33

(dd, J = 10.8 Hz and 7.8 Hz, m, 2H), 3.31–3.19 (m, 2H), 2.61 (br, 1H),

1.53 (m, 2H), 1.30–1.26 (br, 30H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI)

m/z 411.8 [M+H]⁺, 433.5 [M+Na]⁺. Anal. Calcd for C₂₁H₃₄N₂O₄S: C,

60.11; H, 8.41; N, 6.68. Found: C, 60.36; H, 8.24; N, 6.56.

5.3.17. (2RS,4R)-2-(4-Methoxy-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3ea)

Yield: 28.8%. ¹H NMR (300 MHz, CDCl₃) d 7.86–6.88 (m, 4H),

7.36 (br, 1H), 5.69 and 5.32 (s, s, 1H), 4.39 and 4.46 (dd, br, 1H),

3.90 and 3.82 (s, s, 3H), 3.68, 3.50–3.40 (dd, m, 2H), 3.30–3.24

(m, 2H), 1.53–1.45 (m, 2H), 1.26 (s, 26H), 0.88 (t, 3H, J = 6.9 Hz).

MS (ESI) m/z 463.1 [M+H]⁺. Anal. Calcd for C₂₇H₄₆N₂O₂S: C,

70.08; H, 10.02; N, 6.05. Found: C, 70.13; H, 10.12; N, 6.00.

5.3.11. (2RS,4S)-2-(3,4,5-Trimethoxy-phenyl)-thiazolidine-4-

carboxylic acid tetradecylamide (3ai)

Yield: 49.4%. ¹H NMR (300 MHz, CDCl₃) d 7.20 and 6.30 (t, br,

0.7H and 0.3H), 6.77 and 6.71 (s, s, 0.7H and 1.3H), 5.56 and 5.28

(s, s, 0.3H and 0.7H), 4.34 and 3.93 (br, m, 1H), 3.89 and 3.88 (s,

s, 6H), 3.85 and 3.84 (s, s, 3H), 3.68 and 3.45–3.35 (dd, J = 3.9 Hz,

m, 2H), 3.34–3.19 (m, 2H), 2.51 (br, 1H), 1.50 (m, 2H), 1.25 (s,

22H), 0.87 (t, 3H, J = 6.6 Hz). MS (ESI) m/z 495.6 [M+H]⁺. Anal. Calcd

for C₂₇H₄₆N₂O₄S: C, 65.55; H, 9.37; N, 5.66. Found: C, 65.29; H,

9.42; N, 5.66.

5.3.18. (2RS,4R)-2-(4-Dimethylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3fa)

Yield: 62.5%. ¹H NMR (300 MHz, CDCl₃) d 7.33 and 6.70 (dd,

J = 8.7 Hz, 4H), 7.39 and 6.43 (br, 1H), 5.55 and 5.25 (s, s, 1H),

4.33 and 3.87 (dd, J = 4.2 Hz, t, 1H), 3.69 and 3.45–3.39 (dd,

J = 4.2 Hz, m, 2H), 3.32–3.12 (m, 2H), 2.96 and 2.95 (s, s, 3H),

1.55–1.46 (m, 2H), 1.25 (s, 26H), 0.87 (t, 3H). MS (ESI) m/z 476.4

[M+H]⁺. Anal. Calcd for C₂₈H₄₉N₃OS: C, 70.68; H, 10.38; N, 8.83.

Found: C, 70.61; H, 10.49; N, 8.70.

5.3.12. (2RS,4R)-2-(3,4-Dimethoxy-phenyl)-thiazolidine-4-

carboxylic acid octadecylamide (3ba)

Yield: 29.0%. ¹H NMR (300 MHz, CDCl₃) d 7.51–6.97 (m, 3H),

6.85 (t, 1H), 5.64 and 5.37 (s, s, 1H), 4.69 and 4.51 (dd, br, m,

1H), 3.91, 3.90, 3.89 and 3.88 (s, s, s, s, 6H), 3.64–3.42 (m, 2H),

3.35–3.23 (m, 2H), 2.52 (br, 1H), 1.51–1.43 (m, 2H), 1.25 (s, 30H),

0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 521.5 [M+H]⁺. Anal. Calcd for

C₃₀H₅₂N₂O₃S: C, 69.18; H, 10.06; N, 5.38. Found: C, 69.36; H,

10.01; N, 5.36.

5.3.19. (2RS,4R)-2-(2-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3ga)

Yield: 52.7%. ¹H NMR (300 MHz, CDCl₃) d 8.65 (s, 1H), 7.87 (d,

J = 7.8 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.16

(t, J = 7.8 Hz, 1H), 6.78 (br, 1H), 5.52 and 5.48 (s, s, 1H), 4.16 (br,

1H), 3.61 and 3.39–3.32 (dd, m, 2H), 3.30–3.20 (m, 2H), 2.90 (br,

1H), 2.25 (s, 3H), 1.54–1.50 (m, 2H), 1.25 (s, 26H), 0.87 (t, 3H,

J = 6.9 Hz). MS (ESI) m/z 512.3 [M+Na]⁺, 488.1 [MꢂH]^ꢂ. Anal. Calcd

for C₂₈H₄₇N₃O₂S: C, 68.51; H, 9.61; N, 8.48. Found: C, 68.57; H,

9.55; N, 8.47.

5.3.13. (2RS,4R)-2-(3,4-Dimethoxy-phenyl)-thiazolidine-4-

carboxylic acid (Z)-octadec-8-enylamide (3bb)

Yield: 29.1%. ¹H NMR (300 MHz, CDCl₃) d 7.27 and 6.38 (br, 0.7H

and 0.3H), 7.11–6.80 (m, 3H), 5.58 and 5.31 (s, s, 0.3H and 0.7H),

5.39–5.34 (m, 2H), 4.35 and 4.00 (dd, J = 7.8 Hz and 4.2 Hz, m,

1H), 3.93, 3.92, 3.90 and 3.89 (s, s, s, s, 6H), 3.70 and 3.44 (dd,

J = 10.8 Hz and 4.2 Hz, m, 2H), 3.34–3.19 (m, 2H), 2.03–1.99 (br,

4H), 1.56–1.44 (m, 2H), 1.28 (s, 22H), 0.89 (t, 3H, J = 6.9 Hz). MS

(ESI) m/z 519.3 [M+H]⁺. Anal. Calcd for C₃₁H₅₀N₂O₄S: C, 69.45; H,

9.71; N, 5.40. Found: C, 69.17; H, 9.61; N, 5.18.

5.3.20. (2RS,4R)-2-(3-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadecylamide (3ha)

Yield: 70.1%. ¹H NMR (300 MHz, CDCl₃) d 7.75 and 7.65 (s, s,

0.7H, 0.3H), 7.51–7.22 (m, 4H), 7.19 and 6.48 (br, 1H), 5.59 and

5.34 (s, s, 0.3H, 0.7H), 4.32 and 3.93 (dd, J = 3.9 Hz, t, J = 6.9 Hz,

0.7H, 0.3H), 3.68 and 3.42–3.34 (dd, J = 3.9 Hz, m, 2H), 3.31–3.16

(m, 2H), 2.60 (br, 1H), 2.17 (s, 3H, J = 6.9 Hz), 1.52 (m, 2H), 1.25

(s, 26H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 512.3 [M+Na]⁺. Anal.

Calcd for C₂₈H₄₇N₃O₂S: C, 68.51; H, 9.61; N, 8.48. Found: C, 68.54;

H, 9.65; N, 8.49.

5.3.14. (2RS,4R)-2-(3,4-Dimethoxy-phenyl)-thiazolidine-4-

carboxylic acid (E)-octadec-8-enylamide (3bc)

Yield: 13.3%. ¹H NMR (300 MHz, CDCl₃) d 7.26 and 6.33 (br, 1H),

7.09–6.82 (m, 3H), 5.57 and 5.29 (s, s, 0.3H and 0.7H), 5.37 (br, 2H),

4.34 and 3.95 (dd, J = 8.1 Hz and 4.2 Hz, m, 1H), 3.91, 3.90, 3.89 and

490

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

5.3.21. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid (1-methyl-octadecyl)-amide (3ia)

5.3.28. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadec-9-cis-enylamide (3ih)

Yield: 68.7%. ¹H NMR (300 MHz, CDCl₃) d 9.91 (s, 1H) 7.56 (s, br,

4H), 7.49 (s, br, 1H), 5.77 and 5.76 (s, s, 1H), 4.89 and 4.79 (dd,

J = 3.9 Hz, t, br, 0.6H, 0.4H), 3.67 and 3.44 (m, t, 2H), 3.37–3.19

(m, 2H), 3.08 and 2.97 (s, s, 3H), 2.18 (s, 3H), 1.56 (m, 2H), 1.25

(s, 30H), 0.87 (t, 3H, J = 7.2 Hz). MS (ESI) m/z 532.3 [M+Na]⁺. Anal.

Calcd for C₃₁H₅₃N₃O₂SꢁCF₃COOHꢁH₂O: C, 59.70; H, 8.50; N, 6.33.

Found: C, 60.15; H, 8.71; N, 6.27.

Yield: 18.1%. ¹H NMR (300 MHz, CDCl₃) d 7.86–7.41 (m, 4H),

7.30 (br, 1H), 7.09 and 6.42 (br, t, 1H), 5.57 and 5.39 (s, s, 1H),

5.37–5.32 (m, 2H), 4.32 and 3.89 (dd, J = 4.2 Hz, t, J = 7.8 Hz, 1H),

3.68 and 3.43–3.36 (dd, J = 4.2 Hz, m, 2H), 3.31–3.20 (m, 2H),

2.51 (br, 1H), 2.18 (s, 3H), 2.01–1.97 (br, 4H), 1.54–1.46 (m, 2H),

1.29 and 1,27 (s, s, 18H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z

510.4 [M+Na]⁺, 486.1 [MꢂH]^ꢂ. Anal. Calcd for C₂₅H₄₈N₃O₂S: C,

68.95; H, 9.30; N, 8.62.

5.3.22. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid adamantan-1-ylamide (3ib)

5.3.29. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid nonadec-10-ynylamide (3ii)

Yield: 73.0%. ¹H NMR (300 MHz, DMSO-d₆) d 10.04 (s, 1H), 7.79

(br, 1H), 7.50 (dd, J = 8.7 Hz, 4H), 5.74 and 5.58 (s, s, 1H), 4.27 and

3.96 (br, 1H), 3.45–3.35 and 3.16–2.99 (m, m, 2H), 2.04 (s, 3H), 1.94

(s, 6H), 1.62 (s, 6H). MS (ESI) m/z 400.3 [M+H]⁺. Anal. Calcd for

C₂₂H₂₉N₃O₂SꢁCF₃COOH: C, 56.13; H, 5.89; N, 8.18. Found: C,

56.39; H, 5.89; N, 8.18.

Yield: 40.5%. ¹H NMR (300 MHz, CDCl₃) d 7.83–7.41 (m, 4H),

7.26 (br, 1H), 7.23 and 6.43 (br, br, 1H), 5.57 and 5.31 (s, s, 1H),

4.32 and 3.90 (dd, J = 4.2 Hz, t, J = 7.2 Hz, 1H), 3.68 and 3.43–3.36

(dd, J = 4.2 Hz, m, 2H), 3.32–3.20 (m, 2H), 2.51 (br, 1H), 2.18 (s,

3H), 2.13 (t, 4H), 1.54–1.44 (m, 2H), 1.31 and 1,27 (s, s, 22H),

0.87 (t, 3H, J = 6.6 Hz). MS (ESI) m/z 514.3 [M+H]⁺, 512.1 [MꢂH]^ꢂ.

Anal. Calcd for C₃₀H₄₇N₃O₂S: C, 70.13; H, 9.22; N, 8.18. Found: C,

70.21; H, 9.14; N, 8.05.

5.3.23. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid adamantan-2-ylamide (3ic)

Yield: 45.3%. ¹H NMR (300 MHz, DMSO-d₆) d 9.99 (s, 0.5H), 8.08

and 8.02 (dd, J = 7.2 Hz, 1H), 7.58–7.38 (m, 4H), 5.56 and 5.53 (s, s,

1H), 4.30 and 3.88 (t, J = 6.0 Hz, br, 1H), 3.36 and 3.16–2.93 (dd, m,

2H), 2.03 (s, 3H), 1.82–1.49 (m, 15H). MS (ESI) m/z 400.3 [M+H]⁺,

398.0 [MꢂH]^ꢂ. Anal. Calcd for C₂₂H₂₉N₃O₂Sꢁ1/2 CF₃COOH: C,

60.51; H, 6.51; N, 9.20. Found: C, 60.62; H, 6.83; N, 8.93.

5.3.30. (2RS,4R)-2-Phenyl-thiazolidine-4-carboxylic acid

hexadecylamide (3ja)

Yield: 39.3%. ¹H NMR (300 MHz, CDCl₃) d 7.55–7.31 (m, 5H),

7.26 and 6.41 (br, br, 1H), 5.62 and 5.34 (d, J = 9.9 Hz, d,

J = 11.4 Hz, 1H), 4.35 and 3.93 (dt, dd, , J = 7.5 Hz and 4.2 Hz, 1H),

3.71 and 3.45–3.30 (dd, J = 4.2 Hz m, 2H), 3.30–3.19 (m, 2H), 2.66

and 2.53 (br, 1H), 1.52 (m, 2H), 1.25 (s, 26H), 0.88 (t, 3H,

J = 6.9 Hz). MS (ESI) m/z 455.4 [M+Na]⁺, 431.1 [MꢂH]^ꢂ. Anal. Calcd

for C₂₆H₄₄N₂OS: C, 72.17; H, 10.25; N, 6.47. Found: C, 71.98; H,

10.02; N, 6.34.

5.3.24. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid-(9H-ﬂuoren-2-yl)-amide (3id)

Yield: 45.7%. ¹H NMR (300 MHz, DMSO-d₆) d 10.33 and 10.13 (s,

s, 1H), 10.00 and 9.97 (s, s, 1H), 7.97 and 7.94 (s, br, 1H), 7.84–7.24

(m, 7H), 7.63–7.24 (m, 4H), 5.69 and 5.59 (s, s, 1H), 4.39 and 4.04 (t,

J = 5.7 Hz, m, 1H), 3.91 (s, 2H), 3.47–3.29 and 3.13 (m, 2H), 2.05 (s,

3H). MS (ESI) m/z 430.1 [M+H]⁺. Anal. Calcd for C₂₅H₂₃N₃O₂SꢁCF_3-

COOH: C, 56.13; H, 5.89; N, 8.18. Found: C, 56.01; H, 5.87; N, 8.01.

5.3.31. (2RS,4R)-2-Phenyl-thiazolidine-4-carboxylic acid (Z)-

octadec-8-enylamide (3jb)

Yield: 46.1%. ¹H NMR (300 MHz, CDCl₃) d 7.53–7.31 (m, 5H),

7.26 and 6.42 (br, br, 1H), 5.61 and 5.34 (s, br, 1H), 5.34 (br, 2H),

4.35 and 3.93 (dd, J = 4.2 Hz, t, J = 7.5 Hz, 0.7H and 0.3H), 3.71

and 3.45–3.39 (dd, J = 4.2 Hz, m, 2H), 3.37–3.19 (m, 2H), 2.54 (br,

1H), 2.01–1.99 (m, 4H), 1.52–1.45 (m, 2H), 1.29 and 1.26 (s,

22H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 459.4 [M+H]⁺. Anal. Calcd

for C₂₈H₄₆N₂OS: C, 73.31; H, 10.11; N, 6.11. Found: C, 73.15; H,

10.09; N, 6.14.

5.3.25. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid anthracen-2-yl)-amide (3ie)

Yield: 45.7%. ¹H NMR (300 MHz, DMSO-d₆) d 10.49 and 10.27 (s,

s, 1H), 10.00 and 9.97 (s, s, 1H), 8.55–8.46 (m, 3H), 8.06–8.03 (m,

3H), 7.68–7.44 (m, 4H), 7.58–7.44 (m, 4H), 5.70 and 5.60 (s, s,

1H), 4.43 and 4.07 (t, br, 1H), 3.49–3.35 and 3.17 (m, t, 2H), 2.04

(s, 3H). MS (ESI) m/z 442.1 [M+H]⁺. Anal. Calcd for

C₂₆H₂₃N₃O₂SꢁCF₃COOH: C, 60.53; H, 4.35; N, 7.56. Found: C,

60.31; H, 4.37; N, 7.71.

5.3.32. (2RS,4R)-2-Phenyl-thiazolidine-4-carboxylic acid (E)-

octadec-8-enylamide (3jc)

Yield: 35.7%. ¹H NMR (300 MHz, CDCl₃) d 7.47–7.31 (m, 5H),

7.26 and 6.42 (br, br, 1H), 5.61 and 5.37 (br, br, 1H), 5.34–5.33

(br, J = 13.8 Hz, 2H), 4.35 and 3.91 (br, br, 0.7H and 0.3H), 3.71

and 3.45–3.37 (dd, J = 4.2 Hz, m, 2H), 3.34–3.19 (m, 2H), 2.66 and

2.54 (br, 1H), 1.96–1.95 (m, 4H), 1.54–1.42 (m, 2H), 1.26 (s, 22H),

0.87 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 459.4 [M+H]⁺. 457.1 [MꢂH]^ꢂ.

Anal. Calcd for C₂₈H₄₆N₂OS: C, 73.31; H, 10.11; N, 6.11. Found: C,

73.36; H, 10.16; N, 6.12.

5.3.26. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid biphenyl-4-ylamide (3if)

Yield: 35.1%. ¹H NMR (300 MHz, CDCl₃) d 9.27 and 8.74 (s, s,

1H), 7.83–7.30 (m, 13H), 7.17 and 6.90 (s, br, 1H), 5.68 and 5.40

(s, 0.3H and s, 0.7H), 4.53 and 4.21 (dd, J = 3.9 Hz, t, 1H), 3.84–

3.79 and 3.59–3.43 (dd, , J = 3.9 Hz, m, 2H), 2.16 and 2.19 (s, s,

3H). MS (ESI) m/z 440.1 [M+Na]⁺, 416.0 [MꢂH]^ꢂ. Anal. Calcd for

C₂₄H₂₃N₃O₂S: C, 69.04; H, 5.55; N, 10.06. Found: C, 68.87; H,

5.32; N, 10.01.

5.4. (2RS,4R)-3-Acetyl-2-(4-acetylamino-phenyl)-thiazolidine-

4-carboxylic acid hexadecylamide (4)

5.3.27. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid benzothiazol-2-ylamide (3ig)

Under 0 °C, to a mixture of 1b (0.30 g, 0.61 mmol) and pyridine

(0.10 g, 1.26 mmol) in 10 mL CH₂Cl₂, was added acetyl chloride

(0.19 g, 2.42 mmol) and stirred at room temperature for 8 h. The

reaction mixture was washed with satd NaHCO₃and dried over

MgSO₄. The solvent was concentrated and puriﬁed through column

chromatography to yield 4 (0.24 g, 73.6%) as a white solid. ¹H NMR

(300 MHz, CDCl₃) d 7.50 (br, 1H), 7.50–7.34 (d, J = 8.4 Hz, 4H), 7.02

and 6.55 (br, br, 1H), 5.97 (s, 1H), 5.07 and 4.70 (br, br, 1H), 3.67

Yield: 37.9%. ¹H NMR (300 MHz, CDCl₃) d 10.56 (br, 1H), 7.85–

7.78 (dd, J = 14.4 Hz and 7.8 Hz, 2H), 7.55–7.41 (m, 5H), 7.36–

7.29 (m, 2H), 5.68 and 5.41 (s, 0.15H and d, J = 9.6 Hz, 0.85H),

4.64 and 4.32 (br, t, 1H), 3.80 and 3.60–3.44 (m, 2H), 2.79 (br,

1H), 2.19 (s, 3H). MS (ESI) m/z 421.1 [M+Na]⁺. Anal. Calcd for

C₁₉H₁₈N₄O₂S₂: C, 57.26; H, 4.55; N, 14.06. Found: C, 57.17; H,

4.32; N, 14.01.

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

491

and 3.14–3.08 (dd, J = 4.2 Hz, br, 2H), 3.31–3.29 (m, 2H), 2.17 (s, 3H),

1.98 (s, 3H), 1.54(m, 2H), 1.25 (s, 26H), 0.87 (t, 3H, J = 6.9 Hz). MS

(ESI) m/z554.5 [M+Na]⁺, 530.2[MꢂH]^ꢂ. Anal. Calcdfor C₃₀H₄₉N₃O₃S:

C, 67.76; H, 9.29; N, 7.90. Found: C, 67.49; H, 9.40; N, 7.75.

3.31–3.08 (m, 2H), 2.92 (m, 2H), 1.64 (m, 2H), 1.24 (s, 26H), 0.85 (t,

3H, J = 6.6 Hz). MS (ESI) m/z 419.1 [M+H]⁺). Anal. Calcd for

C₂₆H₄₆N₂S ꢁ 2HCl ꢁ 1/2H₂O: C, 62.37; H, 9.86; N, 5.60. Found: C,

62.29; H, 9.91; N, 5.80.

5.5. (2RS,4R)-2-(4-Acetylamino-phenyl)-thiazolidine-4-

carboxylic acid hexadecyl ester (5)

5.6.2. (2RS,4R)-Hexadecyl-(2-(4-acetyl-phenyl-thiazolidin-4-

ylmethyl)-amine hydrochloride (8b)

Yield: 48.4%. ¹H NMR (300 Hz, CDCl₃) d 7.35–7.57 (m, 4H), 5.52

(s, 0.55H), 5.47 (s, 0.45H), 4.02, 3.52 (br, 1H), 3.00–3.24 (m, 2H),

2.61–2.92 (m, 4H), 2.17 (s, 3H), 1.48–1.61 (m, 2H), 1.25 (s, 26H),

0.87 (t, 3H, J = 6.9 Hz); MS (ESI) m/z 476.4 [M+H]⁺. Anal. Calcd for

C₂₈H₄₉N₃OSꢁHCl: C, 65.65; H, 9.84; N, 8.20. Found: C, 65.55; H,

9.73; N, 8.04.

A

mixture of Boc-protected carboxylic acid 2i (0.549 g,

1.50 mmol), DCC (0.372 g, 1.8 mmol), DMAP (0.055 g, 0.45 mmol),

(1S)-(+)-10-camphorsulfonic acid (0.105 g, 0.45 mmol), and hexa-

decanol (0.726 g, 3.0 mmol) in CH₂Cl₂(15 mL) was stirred at room

temperature for 24 h. The reaction mixture was ﬁltered, then

washed with satd NaHCO₃and brine (10 mL), and dried over

MgSO₄. The pure Boc-protected ester was obtained after column

chromatography (0.83 g, 93.8%). To a solution of Boc-protected es-

ter (0.83 g, 1.41 mmol) in CH₂Cl₂(10 mL) was added TFA (1 mL)

and stirred for two days. The reaction mixture was concentrated,

washed with satd NaHCO₃, and dried over MgSO₄. The solvent

was removed, and the crude compound was puriﬁed by column

chromatography using hexane/ethyl acetate gradient elution sys-

tem to yield 5 as a white solid (0.41 g, 59.5%). ¹H NMR (CDCl₃) d

7.52–7.45 (m, 4H), 7.21 and 7.17 (br, br, 1H), 5.79 and 5.52 (s,

1H), 4.19 (t, 2H, J = 6.6 Hz), 4.18–4.14 and 3.96 (m, t, 1H,), 3.49–

3.35 and 3.20–3.07 (m, m, 2H), 2.63 (br, 1H), 2.18 and 2.17 (s, s,

3H), 1.69–1.62 (m, 2H), 1.25 (s, 26H), 0.88 (t, 3H); MS (ESI) m/z

513.3 [M+Na]⁺, 489.1 [MꢂH]^ꢂ. Anal. Calcd for C₂₈H₄₆N₂O₃S: C,

68.53; H, 9.45; N, 5.71. Found: C, 65.47; H, 9.40; N, 5.75.

5.7. Synthesis of disulfanyl dimer (9)

Under 0 °C, to a solution of 1b (330 mg, 0.67 mmol) in THF

(10 mL) was charged B₂H₆(8.0 mL, 1 M in THF) and reﬂuxed for

24 h. Then the reaction mixture was quenched by satd NH₄Cl and

dried over MgSO₄. Disulfanyl dimer 9b was separated from a silica

gel column using hexane/ethyl acetate gradient elution system

(127 mg, 41%). ¹H NMR (500 MHz, CDCl₃) d 7.44 and 7.21 (d, d,

8H), 7.40 (t, 2H, J = 6.0 Hz), 3.69 (q, 4H), 3.37 (q, 2H, J = 4.0 Hz),

3.29–3.18 (m, 4H), 3.13 (dd, 2H, J = 4.0 Hz, J = 14.0 Hz), 2.71 (dd,

2H, J = 14.0 Hz, J = 8.5 Hz), 2.17 (s, 6H), 1.51–1.48 (m, 4H), 1.25

and 1.29 (s, 52H), 0.87 (t, 6H). MS (ESI) m/z 1003.9 [M+Na]⁺. Anal.

Calcd for C₅₆H₉₆N₆O₄S₂: C, 68.53; H, 9.86; N, 8.56. Found: C, 68.54;

H, 9.74; N, 8.31.

5.6. General procedure for preparing (2RS,4R)-alkyl-2-aryl-

thiazolidin-4-ylmethyl)-amine 8a–8b

5.8. Synthesis of (2RS,4R)-2-(4-acetylamino-phenyl)-

thiazolidine-3,4-dicarboxylic acid 3-(9H-ﬂuoren-9-ylmethyl)

ester (10)

To a mixture of Boc-protected carboxylic acid 2j (5.6 mmol),

EDCI (6.7 mmol), HOBt (5.6 mmol), and NMM (13.4 mmol) in

CH₂Cl₂(20 mL) was added O,N-dimethyl-hydroxylamine hydro-

chloride (5.9 mmol); the mixture was stirred continuously at room

temperature for 10 h. The reaction mixture was diluted with

CH₂Cl₂(30 mL) and sequentially washed with water, 5% HCl, satd

NaHCO₃, and brine and dried over MgSO₄. The solvent was re-

moved under reduced pressure to yield a crude product meth-

oxy-methyl-amide 6, which was recrystallized from ethyl ether/

hexane as white crystals and used directly in the next step without

puriﬁcation (71%). Under ꢂ40 °C, LAH (1.0 mL, 2 M in THF) was

added to a solution of 4-(methoxy-methyl-carbamoyl)-2-phenyl-

thiazolidine-3-carboxylic acid tert-butyl ester (0.20 g, 0.57 mmol)

in THF (10 mL) and stirred for 30 min. The reaction mixture was

quenched with ethyl acetate (10 mL) and satd NH₄Cl (10 mL). Then

5% H₂SO₄(20 mL) was added under 0 °C. The mixture was ex-

tracted with ethyl acetate and dried with MgSO₄. The solvent

was removed and pure aldehyde 7 (0.14 g, 84.3%) was puriﬁed as

a colorless oil after column chromatography. To a solution of 7

(0.14 g) in MeOH (3 mL) was charged hexadecylamine (1 equiv)

in MeOH (7 mL). After stirring for 10 min, HOAc (1.3 equiv) and

NaBH₃CN (2.5 equiv) were added and stirred for 2 h. The reaction

mixture was quenched with satd NaHCO₃(10 mL), extracted with

ethyl acetate, and dried with MgSO₄. The solvent was removed un-

der reduced pressure to yield a crude 4-hexadecylaminomethyl-2-

phenyl-thiazolidine-3-carboxylic acid tert-butyl ester. Deprotec-

tion with TFA yielded product 8a. Then 8a was converted to a cor-

responding hydrochloride by using 2 M HCl/Et₂O. Compound 8b

was prepared using the same method.

At 0 °C, to a suspension of (2RS,4R)-2-(4-acetylaminophenyl)-

thiazolidine-4-carboxylic acid (1.00 g, 3.76 mmol) in 15 mL CH₂Cl₂

was added triethylamine (0.38 g, 3.80 mmol) and 9-ﬂuorenylmeth-

yl chloroformate (0.98 g, 3.80 mmol). After stirring for 30 min, the

mixture was washed with 5% HCl and dried with MgSO₄. The sol-

vent was removed, and crude compound was puriﬁed by column

chromatography using hexane/ethyl acetate gradient elution sys-

tem to yield Fmoc-protected acid as a white foam 10 (1.82 g,

99%). Yield: 99%. ¹H NMR (300 MHz, CDCl₃) d 10.24 (br, 1H),

7.73–7.05 (m, 13H), 6.14–5.85 (m, 1H), 5.05–4.38 (m, 3H), 3.99

(br, 1H), 3.36–3.23 (m, 2H), 2.22 (s, 3H). MS (ESI) m/z 511.1

[M+Na]⁺, 486.9 [MꢂH]^ꢂ.

5.9. Preparation of (2RS,4R)-2-(4-aminophenyl)-thiazolidine-4-

carboxylic acid amide 13a–13b

The Fmoc-protected acid 10 (3.97 g, 8.135 mmol), EDCI

(1.2 equiv) and HOBT (1.0 equiv) in CH₂Cl₂(80 mL) was stirred at

room temperature for 10 min. To this solution, dodecylamine or

hexadecylamine (1.0 equiv) and Et₃N (1.2 equiv) were added and

stirred continuously at room temperature for 3.5 h. The reaction

mixture was sequentially washed with water, satd NaHCO₃, and

brine, and then dried over MgSO₄. The solvent was removed under

reduced pressure to yield a crude oil 11 (4.11 g, 70.98%), which was

used in the next step without puriﬁcation. 11 (2.0 g) was added to

a solution of CH₃COCl (4.5 mL) in MeOH (10 mL) under 0 °C and

stirred at room temperature for four days. The solvent was evapo-

rated on vacuum and diluted with CH₂Cl₂(30 mL), then washed

with satd NaHCO₃, and dried over MgSO₄to obtain the crude com-

pound 12 as a yellow solid (0.9 g, 47.8%). To a solution of 12

(1 equiv) in CH₂Cl₂(10 mL) was added 1,8-diazabicycloundec-7-

ene (1.5 equiv) and stirred for 30 min to 1 h. The mixture was

washed with 2.5% HCl, extracted with CH₂Cl₂, and dried with

5.6.1. (2RS,4R)-Hexadecyl-(2-phenyl-thiazolidin-4-ylmethyl)-

amine hydrochloride (8a)

Yield: 42.9%. ¹H NMR (300 Hz, CDCl₃) d 7.66–7.38 (m, 5H), 5.87

(s, 0.55H), 5.79 (s, 0.45H), 4.34 and 3.96 (br, 1H), 3.34–3.32 (m, 2H),

492

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

MgSO₄. The solvent was removed on vacuum and puriﬁed by col-

umn chromatography using hexane/ethyl acetate gradient elution

system to give 13a and 13b as a light yellow solid.

using hexane/ethyl acetate gradient elution system to give 15b

as a white solid. Yield: 53.2%. ¹H NMR (300 MHz, CDCl₃) d 8.26

(s, 1H), 7.58–7.46 (m, 4H), 7.19 and 6.38 (br, br, 1H), 5.59 and

5.33 (s, s, 1H), 4.34 and 3.91 (br, br, 0.7H and 0.3H), 4.20 (s,

2H), 3.69 and 3.39 (dd, J = 3.9 Hz, br, 2H), 3.33–3.26 (m, 2H),

2.52 (br, 1H), 1.56 (br, 2H), 1.25 (s, 26H), 0.87 (t, 3H, J = 6.0 Hz).

MS (ESI) m/z 541.5 [M+Na]⁺, 522.3 [MꢂH]^ꢂ. Anal. Calcd for

C₂₈H₄₆ClN₃O₃S: C, 64.15; H, 8.84; N, 8.02. Found: C, 64.06; H,

8.88; N, 7.91.

5.9.1. (2RS,4R)-2-(4-Amino-phenyl)-thiazolidine-4-carboxylic

acid dodecylamide (13a)

Yield: 65.7%. ¹H NMR (300 MHz, CDCl₃) d 7.31–7.24 (m, 4H),

7.29 and 6.39 (br, br, 1H), 5.51 and 5.24 (br, 0.3H, d, J = 9.6 Hz,

0.7H), 4.32 and 3.86 (br, br, 1H), 3.74 (br, 2H), 3.67 and 3.40 (dd,

dd, J = 4.2 Hz, J = 8.1 Hz, 2H), 3.29–3.20 (m, 2H), 2.44 (br, 1H),

1.53–1.47 (m, 2H), 1.25 (s, 18H), 0.87 (t, 3H, J = 6.9 Hz). MS (ESI)

m/z 392.4 [M+H]⁺, 390.1 [MꢂH]^ꢂ. Anal. Calcd for C₂₂H₃₇N₃OS: C,

67.47; H, 9.52; N, 10.73. Found: C, 67.34; H, 9.52; N, 10.65.

5.10.3. (2RS,4R)-2-(4-Ureido-phenyl)-thiazolidine-4-carboxylic

acid hexadecylamide (15c)

At 0 °C, to a solution of 12 (240 mg, 0.32 mmol) in water (5 mL)

were added sodium cyanate (64 mg, 0.98 mmol) and acetic acid

(0.15 mL). The mixture was stirred at 50 °C for 24 h. It was ex-

tracted with ethyl acetate and dried over MgSO₄. The solvents

were removed to yield the crude Fmoc-protected product 14c,

which was used in the next step without further puriﬁcation

(87.7%). To the solution of 14c (200 mg, 0.28 mmol) in CH₂Cl₂

(10 mL) was added 1,8-diazabicycloundec-7-ene (99 mg, 0.65

mmol) and stirred for 45 min. The mixture was washed with 1 N

HCl, extracted with CH₂Cl₂, and dried with MgSO₄. The solvent

was removed on vacuum, and the resulting crude material was

puriﬁed by column chromatography using hexane/ethyl acetate

gradient elution system to give 15c. Yield: 51.1%. ¹H NMR

(300 MHz, CDCl₃) d 7.56–7.33 (m, 4H), 7.28 and 6.46 (br, 1H),

6.82 (br, 1H), 5.57 and 5.33 (s, 0.3H, s, 0.7H), 4.78 (s, 2H), 4.33

and 3.91 (dd, J = 4.2 Hz, t, J = 7.5 Hz, 0.7H and 0.3H), 3.68 and

3.41 (dd, J = 11.1 Hz and 4.2, m, 2H), 3.32–3.25 (m, 2H), 2.60 (br,

1H), 1.56–1.45 (m, 2H), 1.27 (s, 26H), 0.89 (t, 3H, J = 6.9 Hz). MS

(ESI) m/z 513.5 [M+Na]⁺, 491.4 [M+H]⁺, 489.1 [MꢂH]^ꢂ. Anal. Calcd

for C₂₇H₄₈N₄O₃S₂ꢁH₂O: C, 63.74; H, 9.51; N, 11.01. Found: C, 63.89;

H, 9.01; N, 10.91.

5.9.2. (2RS,4R)-2-(4-Amino-phenyl)-thiazolidine-4-carboxylic

acid hexadecylamide (13b)

Yield: 53.2%. ¹H NMR (300 MHz, CDCl₃) d 7.31–7.25 (m, 4H),

7.26 and 6.39 (br, br, 1H), 5.52 and 5.23 (br, 0.3H, br, 0.7H), 4.32

and 3.86 (br, m, 1H), 3.72 (br, 2H), 3.68 and 3.41 (dd, J = 3.9 Hz,

dd, J = 8.1 Hz, 2H), 3.29–3.20 (m, 2H), 2.44 (br, 1H), 1.51–1.47 (m,

2H), 1.25 (s, 26H), 0.88 (t, 3H, J = 6.9 Hz). MS (ESI) m/z 448.5

[M+H]⁺, 446.1 [MꢂH]^ꢂ. Anal. Calcd for C₂₆H₄₅N₃OS: C, 69.75; H,

10.13; N, 9.39. Found: C, 69.80; H, 10.21; N, 9.16.

5.10. General procedure for preparing (2RS,4R)-2-(4-amido-

phenyl)-thiazolidine-4-carboxylic acid hexadecylamide 15a–

15c

5.10.1. (2RS,4R)-2-(4-Methanesulfonylaminophenyl)-thiazolidine-

4-carboxylic acid hexadecylamide (15a)

At 0 °C, to a solution of 12 (450 mg, 0.672 mmol) in CH₂Cl₂

(10 mL) were added pyridine (88 mg, 1.11 mmol) and methanesul-

fonyl chloride (144 mg, 1.25 mmol) and stirred at room tempera-

ture for 10 h. The reaction mixture was washed with satd

NaHCO₃and dried over MgSO₄. The solvent was removed to yield

the crude Fmoc-protected product 14a, which was used in the next

step without further puriﬁcation (79.7%). To the solution of 14a

(310 mg, 0.414 mmol) in CH₂Cl₂(10 mL) was added 1, 8-diazabicy-

cloundec-7-ene (94.5 mg, 0.622 mmol) and stirred for 1 h. The

mixture was washed with 2.5% HCl, extracted with CH₂Cl₂, and

dried with MgSO₄. The solvent was removed on vacuum and puri-

ﬁed by column chromatography using hexane/ethyl acetate gradi-

ent elution system to give 15a as a white solid. Yield: 45.9%. ¹H

NMR (300 MHz, CDCl₃) d 7.52–7.46 and 7.24–7.19 (m, 4H), 7.15

and 6.33 (br, 0.3H, br, 0.7H), 6.52 (br, 1H), 5.57 and 5.33 (d,

J = 10.8 Hz, 0.3H, d, J = 11.4 Hz, 0.7H), 4.33 and 3.89 (br, br, 0.7H

and 0.3H), 3.69 and 3.42 (dd, J = 4.2 Hz, m, 2H), 3.32–3.22 (m,

2H), 3.02 and 3.01 (s, s, 3H), 2.65 and 2.50 (br, 1H), 1.52–1.48

(m, 2H), 1.25 (s, 26H), 0.87 (t, 3H, J = 6.6 Hz). MS (ESI) m/z 548.4

[M+Na]⁺, 524.2 [MꢂH]^ꢂ. Anal. Calcd for C₂₇H₄₇N₃O₃S₂: C, 61.67;

H, 9.01; N, 7.99. Found: C, 61.91; H, 9.03; N, 7.91.

5.11. (2R)-2-Amino-N-hexadecyl-3-methylsulfanyl-propion-

amide (16a)

At 0 °C, (2R)-2-amino-3-methylsulfanyl-propionic acid (2.5 g,

18.5 mmol) was dissolved in 1 N NaOH (20 mL) and 1,4-dioxane

(40 mL); then di-tert-butyldicarbonate (8.1 g, 37 mmol) was added

slowly and stirred at room temperature overnight. The reaction

mixture was concentrated in vacuum and washed with ethyl ace-

tate (20 mL). The aqueous phase was adjusted to a pH 3 by adding

1 N HCl, then extracted with ethyl acetate, dried with magnesium

sulfate, ﬁltered, and concentrated on vacuum to gave Boc-pro-

tected (2R)-2-amino-3-methylsulfanyl-propionic acid as a white

solid (90.96%). A mixture of Boc-protected (2R)-2-amino-3-meth-

ylsulfanyl-propionic acid (1.76 g, 7.5 mmol), EDCI (1.2 equiv), and

HOBT (1.0 equiv) in CH₂Cl₂(35 mL) was stirred at room tempera-

ture for 10 min. To this solution, hexadecylamine (1.0 equiv) and

Et₃N (1.2 equiv) were added and stirring continuously at room

temperature for 10 h. The reaction mixture was diluted with

CH₂Cl₂(50 mL) and sequentially washed with water, satd NaHCO₃,

and brine, and dried over MgSO₄. The solvent was removed under

reduced pressure to yield a crude oil, which was stirred with TFA

(3 mL) in 20 mL CH₂Cl₂at room temperature for 1 h to cleave the

Boc group. The reaction mixture was concentrated, diluted with

ethyl acetate, washed with satd NaHCO₃, and dried over MgSO₄.

The solvent was removed to yield a crude solid, which was puriﬁed

by column chromatography using hexane/ethyl acetate gradient

elution system as a white solid. Yield: 63.4%. Mp (hexane) 55–

56 °C. ¹H NMR (300 MHz, CDCl₃) d 7.42 (br, 1H), 3.95 and 3.50

(br, dd, 1H, J = J = 3.9 Hz, 9.0 Hz), 3.24 (q, 2H), 3.02 and 2.66 (dd,

dd, 2H, J = 3.9 Hz, J = 9.0 Hz, J = 13.8 Hz), 2.10 (s, 3H), 1.52–1.48

(m, 2H), 1.25 (s, 26H), 0.88 (t, 3H). MS (ESI) m/z 359.9 [M+H]⁺,

357.1 [MꢂH]^ꢂ.

5.10.2. (2RS,4R)-2-[4-(2-Chloro-acetylamino)-phenyl]-thiazoli-

dine-4-carboxylic acid hexadecylamide (15b)

At 0 °C, to a solution of 12 (390 mg, 0.582 mmol) in CH₂Cl₂

(10 mL) were added pyridine (72 mg, 9.11 mmol) and chloro-acet-

yl chloride (122 mg, 1.08 mmol) and stirred at room temperature

for 10 h. The reaction mixture was washed with satd NaHCO₃

and dried over MgSO₄. The solvents were removed to yield the

crude Fmoc-protected product 14b, which was used in the next

step without further puriﬁcation (50.7%). To the solution of 14b

(220 mg, 0.295 mmol) in CH₂Cl₂(10 mL) was added 1, 8-diazabicy-

cloundec-7-ene (103 mg, 0.678 mmol) and stirred for 20 min. The

mixture was washed with 1 N HCl, extracted with CH₂Cl₂, and

dried with MgSO₄. The solvent was removed on vacuum and the

resulting crude material was puriﬁed by column chromatography

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

493

5.12. (2R)-2-(4-Acetylamino-benzylamino)-N-hexadecyl-3-

methylsulfanyl-propionamide (16b)

trated, diluted with EtOAc (50 mL), and washed with satd NaHCO₃.

The white precipitate 2-amino-N-hexadecylacetamide was ob-

tained by treatment with 1 N HCl (10 mL) (77.7%). To a suspension

of 2-amino-N-hexadecylacetamide (200 mg, 0.60 mmol) in MeOH

(15 mL) was added N-(4-formylphenyl)-acetamide (100 mg,

0.61 mmol). After stirring for 5 min, HOAc (75 mg) was charged

and stirred for 20 min. Then NaBH₃CN (93 mg, 1.48 mmol) was

added and stirred for 1 h. The reaction mixture was quenched with

satd NaHCO₃, extracted with ethyl acetate, and dried on MgSO₄.

The solvent was removed, and 16d was puriﬁed by column chro-

matography using hexane/ethyl acetate gradient elution system

as a white solid. Yield: 53.1%. Mp (hexane) 81–83 °C. ¹H NMR

(300 MHz, CDCl₃) d 7.48 (d, 2H, J = 8.4 Hz), 7.24 (d, 2H, J = 8.4 Hz),

7.15 (br, 1H), 7.15 (br, 1H), 3.72 (s, 2H), 3.27 (s, 2H), 3.26–3.22

(m, 2H), 2.17 (s, 3H), 1.51–1.47 (m, 2H), 1.25 (s, 26H), 0.87 (t,

3H, J= 6.6 Hz). MS (ESI) m/z 468.4 [M+Na]⁺, 444.1 [MꢂH]^ꢂ. Anal.

Calcd for C₂₉H₅₁N₃O₂S: C, 72.76; H, 10.63; N, 9.43. Found: C,

73.00; H, 10.77; N, 9.35.

To a suspension of 16a (244 mg, 0.728 mmol) in MeOH (10 mL)

was added N-(4-formylphenyl)-acetamide (119 mg, 0.728 mmol).

After stirring for 5 min, HOAc (75 mg) was charged to the mixture

and stirred for 20 min. Then NaBH₃CN (115 mg, 1.82 mmol) was

added and stirred for 2 h. The reaction mixture was quenched with

satd NaHCO₃, extracted with ethyl acetate, and dried on MgSO₄.

The solvent was removed to yield a crude solid, which was puriﬁed

by column chromatography using hexane/ethyl acetate gradient

elution system. Yield: 61.1%. Mp (hexane) 83–84 °C. ¹H NMR

(300 MHz, CDCl₃) d 7.48–7.24 (m, 4H), 7.40 (br, 1H), 7.19 (br,

1H), 3.70 (q, J = 9.0 Hz, 2H), 3.28–3.18 (m, br, 3H), 2.98 (dd,

J = 3.6 Hz, 1H), 2.58 (dd, J = 9.6 Hz, J = 13.8 Hz, 1H), 1.49–1.47 (m,

2H), 1.25 (s, 26H), 0.88 (t, 3H, J = 6.3 Hz). MS (ESI) m/z 506.4

[M+H]⁺, 504.1 [MꢂH]^ꢂ. Anal. Calcd for C₂₉H₅₁N₃O₂S: C, 68.86; H,

10.16; N, 8.31 Found: C, 69.98; H, 10.12; N, 8.47.

6. Biology

5.13. (2R)-2-Amino-3-benzylsulfanyl-N-hexadecyl-propionamide

(16c)

6.1. Cell culture and cytotoxicity assay of melanoma

A mixture of (2R)-3-benzylsulfanyl-2-tert-butoxycarbonylami-

no-propionic acid (0.5 g, 1.61 mmol), EDCI (0.371 g 1.93 mmol),

and HOBT (0.228 g, 1.69 mmol) in CH₂Cl₂(20 mL) was stirred at

room temperature for 10 min. To this solution, hexadecylamine

(0.409 g, 1.69 mmol) and Et₃N (0.195 g, 1.93 mmol) were added

and stirred continuously at room temperature for 10 h. The reac-

tion mixture was sequentially washed with water, satd NaHCO₃,

and brine, and then dried over MgSO₄. The solvent was removed

under reduced pressure to yield a crude product, which was stirred

with TFA (3 mL) in 20 mL CH₂Cl₂at room temperature for 2–3 h to

cleave the Boc group. The reaction mixture was concentrated, di-

luted with ethyl acetate, washed with satd NaHCO₃, and dried over

MgSO₄. The solvent was removed to yield a crude solid, which was

puriﬁed by column chromatography using hexane/ethyl acetate

gradient elution system as a white solid. Yield: 53.7%. Mp (hexane)

66–67 °C. ¹H NMR (300 MHz, CDCl₃+ D₂O) d 7.51 (br, 1H), 7.35–

7.20 (m, 5H), 3.75 (s, 2H), 3.70 (br, 1H), 3.28–3.13 (m, 2H), 2.99

(br, 1H), 2.90–2.68 (m, 1H), 1.48–1.46 (m, 2H), 1.25 (s, 26H), 0.88

(t, 3H, J = 6.9 Hz). MS (ESI) m/z 435.4 [M+H)⁺, 433.1 [MꢂH]^ꢂ. Anal.

Calcd for C₂₆H₄₆N₂OS: C, 71.83; H, 10.67; N, 6.44. Found: C, 71.82;

H, 10.51; N, 6.51.

We examined the antiproliferative activity of ATCAA analogues

in two human melanoma cell lines (A375 and WM-164) and one

mouse melanoma cell line (B16-F1). We used activity on ﬁbroblast

cells as a control to determine the selectivity of these compounds

against melanoma. A375 cells and B16-F1 cells were purchased

from ATCC (American Type Culture Collection, Manassas, VA,

USA). WM-164 cells were derived from metastatic melanoma tu-

mors and were a gift from Dr. Meenhard Herlyn (Wistar Institute,

Philadelphia, PA, USA). Human dermal ﬁbroblast cells were pur-

chased from Cascade Biologics, Inc., Portland, OR, USA. All cell lines

were cultured in DMEM (Cellgro Mediatech, Inc., Herndon, VA,

USA), supplemented with 5% FBS (Cellgro Mediatech), 1% antibi-

otic/antimycotic mixture (Sigma–Aldrich, Inc., St. Louis, MO,

USA), and bovine insulin (5 lg/mL; Sigma–Aldrich). Cultures were

maintained at 37 °C in a humidiﬁed atmosphere containing 5% CO₂.

Standard sulforhodamine B assay was used. Cells were exposed to

a wide range of concentrations for 48 h in round-bottomed 96-well

plates. Cells were ﬁxed with 10% trichloroacetic acid and washed

ﬁve times with water. After cells were air-dried overnight and

stained with SRB solution, total proteins were measured at

560 nm with a plate reader. IC₅₀(i.e., concentration that inhibited

cell growth by 50% of no treatment controls) values were obtained

by nonlinear regression analysis with GraphPad Prism (GraphPad

Software, San Diego, CA).

5.14. 2-(4-Acetylamino-benzylamino)-N-hexadecyl-acetamide

(16d)

At 0 °C, aminoacetic acid (2.08 g, 27.7 mmol) was dissolved in

1 N NaOH (28 mL) and 1,4-dioxane (25 mL); then di-tert-butyldi-

carbonate (12.1 g, 55.4 mmol) was added slowly and stirred at

room temperature overnight. The reaction mixture was concen-

trated in vacuum and washed with ethyl acetate (20 mL). The

aqueous phase was adjusted to a pH 3 by adding 1 N HCl, then ex-

tracted with ethyl acetate, dried with magnesium sulfate, ﬁltered,

and concentrated on vacuum to gave Boc-protected aminoacetic

acid as a white crystalline solid (92.3%). A mixture of Boc-protected

aminoacetic acid (0.87 g, 5 mmol), EDCI (1.152 g, 6 mmol), and

HOBT (0.675 g, 5 mmol) in CH₂Cl₂(25 mL) was stirred at room

temperature for 10 min. To this solution, hexadecylamine (1.21 g,

5 mmol) and Et₃N (0.606 g, 6 mmol) were added and stirred con-

tinuously at room temperature for 10 h. The reaction mixture

was diluted with CH₂Cl₂(50 mL) and sequentially washed with

water, satd NaHCO₃, and brine, and dried over MgSO₄. The solvent

was removed under reduced pressure to yield a crude oil, which

was stirred with TFA (3 mL) in 20 mL CH₂Cl₂at room temperature

for 1 h to cleave the Boc group. The reaction mixture was concen-

6.2. Cell culture and cytotoxicity assay of prostate cancer

We examined the antiproliferative activity of ATCAA analogues

in four human prostate cancer cell lines (LNCaP, DU 145, PC-3, and

PPC-1). We used LPL receptor-negative RH7777 cells as a control to

determine the selectivity of these compounds. LNCaP, PC-3, DU

145, and RH7777 cells were purchased from ATCC. Dr. Mitchell

Steiner at the University of Tennessee Health Science Center kindly

provided PPC-1cells. All prostate cancer cell lines were cultured in

RPMI 1640 (Cellgro Mediatech), supplemented with 10% FBS (Cell-

gro Mediatech). RH7777 cells were cultured in DMEM medium

with 10% FBS. Cultures were maintained at 37 °C in a humidiﬁed

atmosphere containing 5% CO₂. 1000 to 5000 cells were plated into

each well of 96-well plates depending on growth rate and exposed

to different concentrations of a test compound for 96 h in three to

ﬁve replicates. Cell numbers at the end of the drug treatment were

measured by the SRB assay. Brieﬂy, the cells were ﬁxed with 10% of

trichloroacetic acid and stained with 0.4% SRB, and the absorbances

494

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

at 540 nm were measured using a plate reader (DYNEX Technolo-

gies, Chantilly, VA). Percentages of cell survival versus drug con-

centrations were plotted, and the IC₅₀values were obtained by

nonlinear regression analysis using WinNonlin (Pharsight Corpora-

tion, Mountain View, CA).

6.6. Formulation for in vivo studies

Compound 1b was dissolved into a co-solvent system that was

composed of 80% Tween 80 (Sigma–Aldrich, St. Louis, MO) and 20%

Captex 200 (Abitec Corporation, Columbus, OH). Dacarbazine

(DTIC) (Sigma–Aldrich, St. Louis, MO) was dissolved in saline

solution.

6.3. Cell cycle analysis

Flow cytometry analysis was performed as described else-

where²⁵to study cell cycle phase distribution. Brieﬂy, A375 and

LNCaP cells (4 ꢀ 10⁶) were seeded in each 10-cm dish; A375 and

LNCaP cells were synchronized using 0.5% charcoal-stripped

DMEM and RPMI medium for 72 h, respectively. After that, the

medium was changed to the media containing 10% FBS with differ-

6.7. Animals

Male athymic nude mice age 4–5 weeks were purchased from

Harlan Laboratories (Harlan Laboratories, Indianapolis, IN, USA).

The laboratory housing the animals met all Association for Assess-

ment and Accreditation and Laboratory Animal Care speciﬁcations.

All of the procedures were conducted in accordance with the

guidelines of our Institutional Animal Care and Use Committee.

ent concentrations (0, 1, 5, 10 and 20 lM) of testing compounds.

Cells were incubated for additional 24 h. All cells were collected

by trypsinization. Cell pellets were washed by PBS. The superna-

tant was discarded, and the pellets were ﬁxed in ice-cold 70% eth-

anol at 4 °C overnight. Ethanol was removed by centrifugation and

cell pallets were washed with PBS twice. 1 mL of PBS containing

6.8. In vivo evaluation of antitumor efﬁcacy

Logarithmic growth phase A375 cells were prepared in FBS-free

100

bated for 1 h. Cells were centrifuged and re-suspended in 1 mL of

PBS containing propidium iodide (50 g/mL). Cell cycle analysis

l

g/mL RNAase A was then added and cell pallets were incu-

DMEM medium (Cellgro Mediatech) at a concentration of

5 ꢀ 10⁷viable cells/mL and placed on ice. The cell suspension

l

was mixed with BD Martigel (BD Biosciences, Waltham, MA,

was conducted with a FACS Calibur Autometer (Beckton Dickinson,

San Diego, CA, USA). Data were analyzed and graphs were prepared

using the ^MODFIT2.0 program (Verity Software House, Topsham, ME,

USA).

USA) at a 1:1 ratio. This cell suspension (100 lL) was injected sub-

cutaneously in the right dorsal ﬂank of each mouse. When tumor

sizes reached about 150 mm³, about seven days after cell inocula-

tion, all mice bearing tumors were divided into control and treat-

ment groups based on tumor size (n = 8 per group). Each group

had similar average tumor size. Mice in control groups were in-

6.4. FLIPR intracellular calcium mobilization assay

jected intraperitoneally with 50 lL vehicle solution only (negative

Chem-1 cells (Chemicon Int., Inc., Billerica, MA) were stable-

transfected with LPA₁, LPA₃, and LPA₅, respectively. Cells are main-

tained in DMEM/10% FBS/100 U/mL penicillin. Cells are passaged

by washing with Ca²⁺and Mg2⁺-free HBSS (10 mL/T75) and incu-

bated with 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) for 5–10 min

at 37 °C. Cells are loaded with Fluo-4 NW, and the assay was read

control) or DTIC at 60 mg/kg (positive control) once daily. Tumor

volume was measured twice weekly with a Traceable^Òelectronic

digital caliper (Fisher Scientiﬁc, Inc., Pittsburgh, PA) and calculated

using the formula a ꢀ b²ꢀ 0.5, where a and b represented the lar-

ger and smaller diameters, respectively.³²Tumor volume was ex-

pressed as cubic millimeters. Data were expressed as mean SE

for each group and plotted as a function of time. Percentage tumor

reduction at the conclusion of the experiment (22 days after initi-

ating treatment) was calculated from the formula 100–

100 ꢀ [(T ꢂ T₀)/(C ꢂ C₀)], where T represents mean tumor volume

of a treated group on a speciﬁc day, T₀represents mean tumor vol-

ume of the same group on the ﬁrst day of treatment, C represents

mean tumor volume of a control on a speciﬁc day, and C₀repre-

sents mean tumor volume of the same group on the ﬁrst day of

treatment.³³Animal activity and average body weight of each

group were monitored during the entire experiment period to as-

sess compound toxicity. At the end of treatment, all mice were

euthanized by CO₂followed by cervical dislocation, and tumors

were harvested for further studies.

for 180 s using the FLIPR^TETRA. The signal is proportional to [Ca²⁺

]

i

and thus can be used to quantify differences in calcium mobiliza-

tion in ATCAA-treated and control cells. Percentage activations

were determined upon initial addition of 3ad followed by 10-

min incubation at 25 °C. To study antagonism of ATCAA on LPAs,

cells were incubated with 3ad for 10 min at 25 °C. Following com-

pound incubation, reference agonists (oleoyl-LPA) were added at

EC₈₀to determine percentage inhibition. Compound 3ad was

plated in an eight-point, fourfold serial dilution series with a top

concentration of 10 lM. The concentration described here reﬂects

the ﬁnal concentration of the compound during the antagonist

assay.

6.5. Colony-formation assay

NCI-60 cell line cytotoxicity data of 1a and 1b are available via

the internet at http://pubs.acs.org.

To measure colony formation and growth of melanoma, A375

cells were plated at a colony-forming density (2000 cells per well

on 6-well plates). Cells were grown in DMEM supplemented with

FBS (Atlanta Biologicals, Lawrenceville, GA) and an antibiotic–

antimycotic solution (Sigma, St. Louis, MO) at 37 °C in an atmo-

sphere of 95% air and 5% CO2.²⁷Cells were treated with com-

pounds 1b and 3ad at different concentrations (2, 20, and

Acknowledgment

This research was supported by funds from Department of

Pharmaceutical Sciences, College of Pharmacy, University of Ten-

nessee Health Science Center; the Van Vleet Endowed Professor-

ship; Division of Pharmaceutics, The Ohio State University;

USPHS grant 1R15CA125623, and University of Tennessee Technol-

ogy Maturation Grant. Zhao Wang acknowledges the support from

the Alma & Hal Reagan Fellowship. We thank Dr. George Wood’s

group in the College of Pharmacy, University of Tennessee Health

Science Center, for their initial help with formulations for com-

pound 1b; Dr. Dave Dickason and Dr. Tai Ahn at GTx, Inc., for their

help in developing drug formulations in this study; Dr. David Arm-

bruster at the University of Tennessee Health Science Center for

100 lM). Compounds were added to the medium from 1 mM

DMSO stock solutions, and corresponding dilution of DMSO was

used as negative (vehicle) control. Cells were grown for 14 days,

and colonies were stained with 0.1% crystalline blue for 30 min

and rinsed with distilled water to remove excess dye. Plates were

photographed, and the number of colonies was measured by Ar-

tek 880 Automated Colony Counter (Artek Systems Corporation,

Farmingdale, NY).

Y. Lu et al. / Bioorg. Med. Chem. 18 (2010) 477–495

495

13. Schubert, M. P. J. Biol. Chem. 1936, 114, 341.

14. Zhao, L.; Huang, W.; Liu, H.; Wang, L.; Zhong, W.; Xiao, J.; Hu, Y.; Li, S. J. Med.

Chem. 2006, 49, 4059.

15. Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.

16. Burns, C. J.; Guitton, J.-D.; Baudoin, B.; Lelievre, Y.; Duchesne, M.; Parker, F.;

Fromage, N.; Commercon, A. J. Med. Chem. 1997, 40, 1763.

editorial assistance; and Dr. David Hamilton at the University of

Tennessee Health Science Center animal facility for assistance in

developing the approved animal protocols.

Supplementary data

17. Kurzer, F. Org. Synth. 1951, 31, 8.

18. Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R. A.;

Schwartz, B.; Simantov, R.; Kelley, S. Nat. Rev. Drug Disc. 2006, 5, 835.

19. McFadyen, M. C. E.; Melvin, W. T.; Murray, G. I. Mol. Cancer Ther. 2004, 3, 363.

20. Rooseboom, M.; Commandeur, J. N. M.; Vermeulen, N. P. E. Pharmacol. Rev.

2004, 56, 53.

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.bmc.2009.12.020.

21. Lu, Y.; Li, C. M.; Wang, Z.; Ross, C. R., 2nd; Chen, J.; Dalton, J. T.; Li, W.; Miller, D.

D. J. Med. Chem. 2009, 52, 1701.

References and notes

22. Brugarolas, A.; Gosalvez, M. Lancet 1980, 1, 68.

23. Liu, J.; Qu, S.; Li, B.; Li, D. Yiyao Gongye 1988, 19, 203.

1. Cancer Facts & Figures; American Cancer Society: Atlanta, 2007.

2. Carlson, J. A.; Ross Jeffery, S.; Slominski, A.; Linette, G.; Mysliborski, J.; Hill, J.;

Mihm, M., Jr. J. Am. Acad. Dermatol. 2005, 52, 743.

3. Gogas, H. J.; Kirkwood, J. M.; Sondak, V. K. Cancer (Hoboken, NJ, U.S.A.) 2007, 109,

455.

24. Ohta, H.; Sato, K.; Murata, N.; Damirin, A.; Malchinkhuu, E.; Kon, J.; Kimura, T.;

Tobo, M.; Yamazaki, Y.; Watanabe, T.; Yagi, M.; Sato, M.; Suzuki, R.; Murooka,

H.; Sakai, T.; Nishitoba, T.; Im, D. S.; Nochi, H.; Tamoto, K.; Tomura, H.; Okajima,

F. Mol. Pharmacol. 2003, 64, 994.

25. Meyskens, F. L., Jr.; Moon, T. E.; Dana, B.; Gilmartin, E.; Casey, W. J.; Chen, H. S.;

Franks, D. H.; Young, L.; Salmon, S. E. Br. J. Cancer 1981, 44, 787.

26. Von Hoff, D. D.; Casper, J.; Bradley, E.; Sandbach, J.; Jones, D.; Makuch, R. Am. J.

Med. 1981, 70, 1027.

27. Zmijewski, M. A.; Li, W.; Zjawiony, J. K.; Sweatman, T. W.; Chen, J.; Miller, D. D.;

Slominski, A. T. Steroids 2009, 74, 218.

28. Zmijewski, M. A.; Li, W.; Zjawiony, J. K.; Sweatman, T. W.; Chen, J.; Miller, D. D.;

Slominski, A. T. Photochem. Photobiol. Sci. 2008, 7, 1570.

29. Zbytek, B.; Janjetovic, Z.; Tuckey, R. C.; Zmijewski, M. A.; Sweatman, T. W.;

Jones, E.; Nguyen, M. N.; Slominski, A. J. Invest. Dermatol. 2008, 128, 2271.

30. Panayiotis, P.; Constantinides, C. M. L. J. Controlled Release 1995, 34, 109.

31. Povlsen, C. O.; Jacobsen, G. K. Cancer Res. 1975, 35, 2790.

32. Guo, W.; Wu, S.; Liu, J.; Fang, B. Cancer Res. 2008, 68, 7403.

33. Chen, J.; Smith, M.; Kolinsky, K.; Adames, V.; Mehta, N.; Fritzky, L.; Rashed, M.;

Wheeldon, E.; Linn, M.; Higgins, B. Cancer Chemother. Pharmacol. 2007, 59, 651.

4. Jemal, A.; Tiwari Ram, C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer

Eric, J. CA Cancer J. Clin. 2004, 54, 8.

5. Bosland, M. C.; McCormick, D. L.; Melamed, J.; Walden, P. D.; Zeleniuch-

Jacquotte, A.; Lumey, L. H. Eur. J. Cancer Prev. 2002, 11, S18.

6. Karan, D.; Holzbeierlein, J.; Thrasher, J. B. Cancer Res. 2009, 69, 2.

7. Gududuru, V.; Hurh, E.; Dalton, J. T.; Miller, D. D. J. Med. Chem. 2005, 48, 2584.

8. Li, W.; Lu, Y.; Wang, Z.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem. Lett. 2007,

17, 4113.

9. Chen, J.; Wang, Z.; Lu, Y.; Dalton, J. T.; Miller, D. D.; Li, W. Bioorg. Med. Chem.

Lett. 2008, 18, 3183.

10. Li, W.; Wang, Z.; Gududuru, V.; Zbytek, B.; Slominski, A. T.; Dalton, J. T.; Miller,

D. D. Anticancer Res. 2007, 27, 883.

11. Gududuru, V.; Zeng, K.; Tsukahara, R.; Makarova, N.; Fujiwara, Y.; Pigg, K. R.;

Baker, D. L.; Tigyi, G.; Miller, D. D. Bioorg. Med. Chem. Lett. 2006, 16, 451.

12. Gududuru, V.; Hurh, E.; Sullivan, J.; Dalton, J. T.; Miller, D. D. Bioorg. Med. Chem.

Lett. 2005, 15, 4010.

Article Doi

Synthesis, in vitro structure-activity relationship, and in vivo studies of 2-arylthiazolidine-4-carboxylic acid amides as anticancer agents

DOI: 10.1016/j.bmc.2009.12.020

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2009.12.020

Full text of DOI:10.1016/j.bmc.2009.12.020

Products guided by the article

R&D Labs maybe for 1208224-26-3

Relevant to this article

Hot Product