X-ray crystal structures and anti-breast cancer property of 3-tert -butoxycarbonyl-2-arylthiazolidine-4-carboxylic acids

Article abstract of DOI:10.1039/c7nj02961f

Diastereomeric '2RS,4R'-2-arylthiazolidine-4-carboxylic acids (ATCAs) were synthesized and their resolution to chiraly pure N-BOC derivatives was attempted by column chromatography. The absolute stereochemistry of the resolved compounds was ascertained by X-ray single crystal structures. Further application of the synthesized compounds was studied for their in vitro anti-breast cancer activity against MCF7 cell line using DOX as a standard by MTT assay method. Cell morphology analysis was carried out by fluorescence microscopy. The compounds containing '2S' absolute configuration in thiazolidine ring and presence of 2-NO₂, 2,6-Cl groups on '2R'-aryl substituent showed significant anti-breast cancer activity where some of the compounds were found to be more active than DOX in terms of induced apoptosis mode of MCF7 cell death.

Full text of DOI:10.1039/c7nj02961f

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PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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ARTICLE
X-ray crystal structures and anti breast cancer property of 3-tert-
butoxycarbonyl-2-arylthiazolidine-4-carboxylic acids
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
b
a
Rohidas M. Jagtap, Shridhar H. Thorat, Rajesh G. Gonnade, Ayesha A. Khan and Satish K.
a
Pardeshi*
DOI: 10.1039/x0xx00000x
The diastereomeric ‘2RS,4R’-2-aryl thiazolidine-4-carboxylic acids (ATCAs) were synthesized and attempted their resolution
to chiraly pure N-BOC derivatives by column chromatography. The absolute stereochemistry of the resolved compounds
was ascertained by x-ray single crystal structures. Further application of synthesized compounds was studied for their in
vitro anti breast cancer activity against MCF7 cell lines using DOX as a standard by MTT assay method. The cell morphology
analysis was carried out by fluorescence microscopy. The compounds containing ‘2S’ absolute configuration in thiazolidine
www.rsc.org/
2
ring and presence of 2-NO , 2,6-Cl groups on ‘2R’-aryl substituent showed significant anti breast cancer activity where
some of the compounds were found to be more active than DOX by induced apoptosis mode of MCF7 cell death.
these compounds against different prostate cancer cell lines,
1
3
melonema and have also explored their structure activity
relationship where 3,4,5-trimethoxyphenyl group linked by
ketone carbonyl to thiazole or thiazolidine ring plays a vital
1
. Introduction
The thiazolidones and thiazolidines are significant scaffolds to
develop the newer and versatile multi-target drugs due to
their broad spectrum of biological activities. Especially
thiazolidine-2,4-dione nucleus has been widely explored by
researchers for their plethora of activities like anti-
hyperglycaemics, aldose reductase inhibitors, anti-cancer, anti-
inflammatory, antiarthritics, anti-microbials, etc. The 1,3-
thiazolidine-4-carboxylic acid (TCA) and its derivatives are
equally noteworthy because of their tremendous biological
potential such as anti acetaminophen hepatotoxicity, HIV
14
role to enhance the anticancer activity. In extension to these
earlier contributions, Miller et. al. have also demonstrated that
the N-BOC and N-fmoc protected ATCAAs exhibits excellent
anticancer activity in vitro against melanoma, prostate cancer
cells as well as in vivo on mice bearing A375 melanoma
1
15
tumors. Li et. al. have also studied ATCAAs as selective and
excellent antiproliferative agents for melanoma where some of
16
the compounds showed activity similar to sorafenib. Lithium
2
17
thiazolidine-4-carboxylate have been studied by Corbi et. al. for
3
4
5
protease in6hibitor, EDG3 7Antagonists, antiproliferative,
Antiasthma, antibacterial, influenza neuraminidase
inhibitors, serotonin metabolite, tyrosinase inhibitors,
preliminary in vitro cytotoxic studies against human HeLa cells. The
recent focus of the anticancer studies revealed a great
attention towards the breast cancer causing MCF7 cells. The
potential of thiazolidine skeleton against MCF7 has been
explored by Gomez-Monterrey et. al. as Indoline-3,2′-thiazolidine
8
9
10
1
1
metallo-β-lactamases inhibitor.
The cancer stood second following cardiovascular diseases in
human mortality. The discovery and development of small
molecule drug (SMD) for cancer treatment has resulted in to
newer additions to the successful cancer chemotherapeutic
drugs. The SMDs with molecular targeting capability have been
always more effective as they exploit the particular genetic
18
based p53 Modulators, Pinho e Melo et. al. as chiral 6,7-
19
bis(hydroxymethyl)-1H,3H-pyrrolo[1,2-c]thiazoles and Serra et. al.
20
as 2-galactosylthiazolidine-4-carboxylic acid amides. The TCA-Cu
(
II) coordination complexes have been found to be significantly
21
cytotoxic towards colon cancer HCT116 cell line. In current
literature, Onen-Bayram et. al
propionyl-thiazolidine-4-carboxylic acid ethyl esters
1
2
additions, dependencies and vulnerabilities of cancer cells.
Considering the SMD application of TCAs, the diastereomeric
-arylthiazolidine-4-carboxylic acids (ATCAs) and their amide
derivatives ATCAAs are focused worldwide by various
22-23
have revealed that the 3-
are
2
significantly active against HUH7 and MV hepatocellular
carcinoma cell lines which are capable to induce caspase-9
dependent apoptosis in HUH7 cells.
(
)
research groups for in vitro anticancer activities against
several human cancer cell lines. Miller et. al. have studied
In order to ascertain the mode of antiproliferative activity exhibited
13
by ATCA amides, Miller et. al. have investigated LPL receptor
mRNA expression using a quantitative sandwich ELISA method
which measures DNA-histone complex released during apoptosis to
demonstrate induced apoptosis in LNCaP, PC-3, and RH7777 cancer
a.
Department of Chemistry, Savitribai Phule Pune University (formerly University of
Pune), Ganeshkhind, Pune-411007, India. Email: skpar@chem.unipune.ac.in;
Tel:+91 020 25601394 Extn. 514; Fax: +91 020 25601394.
Center for Materials Characterization (CMC), National Chemical Laboratory
b.
15
cells. In another significant contributions, Miller et. al. and
Dr. Homi Bhabha Road, Pune 411008, India.
20
Botelho et. al. have proved dose dependant induced apoptosis at
Electronic Supplementary Information (ESI) available. CCDC: 1565464 (2e),
1565470 (3b), 1565479 (3c), 1565478 (3e) and 1565476 (3f). See
DOI: 10.1039/x0xx00000x
sub-G1 phase by ATCA amides using flow cytometric analysis in
LNCaP and MCF-7 cancer cells respectively. Moreover, the docking
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New Journal of Chemistry
1
8
studies by Gomez-Monterrey et. al. has established apoptotic cell propositions of Zhu et. al., on N-BOC protection of 2b, 2c; we could
death mechanism of MCF-7 cells by significantly induced afford and resolve the mixture of ‘2S,4R’ and ‘2R,4R’ diastereomers
accumulation of p53 protein on treatment of spiro thiazolidine with reversed stereoselectivity which reveals the merits of our
moieties. The investigation of cytotoxic effect mechanism by Cetin- synthetic protocol to generate both stereoisomers in a single
2
3
Atalay et. al. using fluorescence-activated cell sorting (FACS) attempt. More interestingly, the compound 2e was itself obtained
analysis using propidium iodide stain revealed that the thiazolidine with ‘2R,4R’ stereochemistry and also these absolute configurations
derivative led apoptotic property which triggers caspase-9 activity of chiral centres identified to be retained on BOC derivatization in
and arrested Huh7 hepatocellular carcinoma (HCC) cell lines at 3e. The all functionalized derivatives were completely characterized
1
13
SubG1/G1phase. All these efforts recommend that the thiazolide by IR, H NMR, C NMR, MS spectroscopic techniques and the
based moieties are highly effective anticancer agents with induced spectroscopic data found to be in agreement with their structures.
1
13
apoptosis mechanism.
The representative H and C NMR spectra of compound 3e are
In cognizance of tremendous anti influenza potential, the shown in Fig. 1 and Fig. 2 respectively.
thiazolidine class has been also subjected to computational studies
2
4-25
to establish their structural activity relation.
Besides of
Cl
versatile biological activities, the compounds of thiazolidine
class are imperative as spacers to built metal-organic
frameworks pertinent in the fields of luminescence, magnetism,
R
H N
SH
3
(R)
HO
H
O
2
6
CO
2
storage and heterogeneous catalysis.
The x-ray
O
crystallography is perhaps an unambiguous, state of art tool for
confirmation of the stereochemistry of chiral centers within a
bioactive chiral molecule. By taking in to account the enormous
applications, it is widely employed by crystallographers to ascertain
the chirality in prominent thiazolidine class of organosulfur
NaHCO3, aq. DMSO
R.T., 4 ꢀ16 Hrs.
2
7-30
31-33
1: H (90%)
compounds
and their transition metal complexes.
R
2
2
2
a: 'R,S' = phenyl (82%)
b: 'R,S' = 2ꢀhydroxy phenyl (78%)
c: 'R,S' = 2ꢀmethoxy phenyl (83%)
In present article, we report the synthesis of diastereomeric ATCAs
and their chirally pure N-BOC derivatives towards their anti breast
cancer activity using MCF7 cell lines by MTT assay method. Also the
morphological analysis of cells is reported using fluorescence
microscopy. The activity results of the compounds have been
correlated with their absolute structures.
2
HN
(R/S)
3
S
1
2d: 'R,S' = 2ꢀnitro phenyl (91%)
4
2e: 'R' = 2,6ꢀdichlorophenyl (85%)
(R) 5
HO
O
(
1-2 )
i) NaHCO , aq. Dioxane,
3
O
0
ꢀ 5 C, 1.5 h.
2
. Results and discussion
ii) BOC O, R.T., 12 h.
2
2.1 Chemistry
R
The syntheses of TCA, ATCAs (1, 2b-2f) were accomplished by using
our previously reported protocol using commercially available
3
4
O
(
R/S)
aldehydes and L-Cysteine.HCl with NaHCO as an inexpensive and
3
S
O
N
readily available base in aqueous 20% DMSO solvent medium with
excellent yields as shown in Scheme 1.
(
R)
HO
Since the parent diastereomeric amino acids (2b-2e) were only
soluble in DMSO, their resolution was not feasible by conventional
column chromatographic separation technique. The tert-
butoxycarbonyl (BOC) is known to be a wonderful protecting group;
besides this, it can also build up a variety of weak intermolecular
forces of attraction essential for packing of molecules during the
O
Column
Chromatographic
separation/purification
R
R
3
5
crystal growth.
In order to achieve the resolution of
O
O
(
R)
(
S)
S
diastereomers by making them soluble in silica column compatible
volatile organic solvents and considering the x-ray crystallographic
stereo structure correlated anticancer study approach, we aimed
BOC derivatization of the diastereomeric amino acids (2b-2e). The
compounds 2b-2e were subjected for BOC derivatization in 50%
S
O
N
O
N
(R)
(
R)
HO
HO
O
O
*34
3b: 'R' = 2ꢀhydroxy phenyl (15%)*
34
3
a: 'S' = 2ꢀhydroxy phenyl (85%)
aqueous 1, 4-Dioxane where we used NaHCO
3
as a mild base
3c: 'S' = 2ꢀmethoxy phenyl (60%)*
3d: 'R' = 2ꢀmethoxy phenyl (40%)*
3e: 'R' = 2,6ꢀdichlorophenyl (66%)
3f : 'R' = 2ꢀnitro phenyl (72%)
instead of any strong base like NaOH. The crude oily products
obtained after BOC protection were either separated or purified by
using silica column chromatography to give the fine solids of 3a-3f
(
*presents isolated yields)
3
6
with significant yields. Zhu et. al. have very recently reported the
synthesis of similar N-BOC analogues as exclusively single ‘2R,4R’
diastereomers on the basis of kinetic dynamic resolution having
significant antibacterial activities. In our attempt, the ‘2R/S,4R’
precursor 2d gave exclusively ‘2R,4R’ diastereomer 3f on N-BOC
protection and it is in agreement with Zhu et. al. On contrary to the
Scheme 1. Synthesis of 3-t-butyloxy-2-aryl thiazolidine- 4-
carboxylic acids
2
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1
13
Fig. 1 H NMR spectra of compound 3e
Fig. 2 C NMR spectra of compound 3e
2.2 X-ray crystallographic structures of compound (2e, 3b, 3c, 3e
and 3f)
In order to confirm the absolute structures of synthesized moieties
by X-ray single crystal studies, all the ATCA and N-BOC-ATCAs were
crystallized by vapor deposition technique. Providentially
compounds 2e and 3a-3f were crystallized with appropriate quality
to collect the diffraction data. Out of the total seven diffracted
molecules, we have very recently elaborated the crystal structure of
34
compound 3a (CCDC-977400) in support to stereoselectivity and
mechanistic investigations in the formation of ATCAs. The x-ray
crystal structure of compound 3d (CCDC-703635) has been lately
3
6
studied by Zhu et. al. and thus not incorporated here. However, to
the best of our knowledge, we are first time reporting the x-ray
crystal structures for rest five moieties (2e, 3b, 3c, 3e and 3f).
The crystal data of the compounds 2e, 3b, 3c, 3e and 3f are
(3b)
elaborated in Table 1 and their thermal ellipsoids at 50
%
probability level are shown in Fig.3. The compound 2e, 3b, 3c, 3e
and 3f are crystallized in Orthorhombic, Tetragonal, Monoclinic,
Orthorhombic and Monoclinic crystal systems respectively. The
observed space groups are as P2(1)2(1)2(1), I4, C2, P2(1)2(1)2(1)
and P2(1) respectively. A perspective view of the compounds 2e,
3b, 3c, 3e and 3f through ORTEP diagram at 50 % probability are
shown in Figure 3 and the detailed crystallographic characteristics
are given in supplementary data. Each asymmetric unit of 2e, 3b,
3c, 3e and 3f contains a chiral molecule with substituted phenyl ring
and functionalized thiazolidine ring oriented orthogonal to each
other. All the compounds show the retained absolute configuration
(3c)
(3e)
‘
R’ at COOH substituted chiral carbon (C4 as per scheme 1) of
thiazolidine ring as that of precursor L-Cysteine. Whereas, the
suggested absolute configuration at aromatic ring substituted chiral
carbon (C
2
as per scheme 1) of thiazolidine ring is ‘S’ for compound
3c and ‘R’ for compounds 2e, 3b, 3d, 3e,3f respectively.
(
2e)
(3f)
Fig. 3 Crystal structures (thermal ellipsoids at the 50% probability
level) of compounds 2e, 3b, 3c, 3e and 3f.
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Table 1: Crystal structure data of compounds 2e, 3b, 3c, 3e and 3f
New Journal of Chemistry
Identification code
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
2e
3b
3c
3e
3f
C10 H9 Cl2 N O2 S
278.14
296(2) K
0.71073 Å
Orthorhombic
P2(1)2(1)2(1)
C15 H19 N O5 S
325.37
100(2) K
0.71073 Å
Tetragonal
I4
C16 H21 N O5 S
339.40
296(2) K
0.71073 Å
Monoclinic
C2
C15 H17 Cl2 N O4 S
378.26
296(2) K
0.71073 Å
Orthorhombic
P2(1)2(1)2(1)
C15 H18 N2 O6 S
354.37
100(2) K
0.71073 Å
Monoclinic
P2(1)
Unit cell dimensions
a = 6.9790(7) Å
α= 90°.
a = 22.5029(5) Å
α = 90°.
a = 31.2006(17) Å
α = 90°.
a = 9.4352(11) Å
α = 90°.
a = 10.0776(4) Å
α = 90°.
b = 8.4645(9) Å
β= 90°.
b = 22.5029(5) Å
β = 90°.
b = 10.1101(5) Å
β = 93.505(4)°
c = 5.9607(4) Å
γ = 90°.
b = 10.4526(12) Å
β = 90°.
b = 6.3731(3) Å
β = 90.4180°.
c = 13.2240(6) Å
γ = 90°.
c = 19.895(2) Å
c = 6.4423(2) Å
γ = 90°.
c = 18.336(2) Å
γ = 90°.
γ = 90°.
3
Volume
3
3
3
3
1
4
175.2(2) Å
3262.26(18) Å
1876.73(19) Å
1808.3(4) Å
849.30(6) Å
Z
8
4
4
2
Density (calculated)
3
3
3
3
3
1
.572 Mg/m
1.325 Mg/m
1.201 Mg/m
1.389 Mg/m
1.386 Mg/m
Absorption coefficient
F(000)
-1
-1
-1
-1
-1
0
568
.712 mm
0.220 mm
1376
0.194 mm
720
0.491 mm
784
0.224 mm
372
Crystal size
0.42 x 0.32 x
0.40 x 0.30 x
0.46 x 0.36 x
0.42 x 0.36 x
0.36 x 0.30 x
3
3
3
3
3
0
.28 mm
0.25 m
0.27 mm
0.30 mm
0.27 mm
Theta range for data
collection
2.047 to 24.998°
3.622 to 34.372°
2.118 to 24.991°
2.221 to 24.86°
6.073 to 40.150.
Index ranges
-8/8, -9/10,
-32/29, -27/35,
-10/10
-36/36, -12/12,
-7/7
-11/11, -12/12,
-21/21
-16/17, 11/11,
-20/23
-22/23
Reflections collected
7473
27810
13496
18873
34554
Independent reflections
2058
5796
3287
3186
9531
[
R(int) = 0.0243]
[R(int) = 0.0391]
multi-scan
[R(int) = 0.0414]
multi-scan
[R(int) = 0.0270]
multi-scan
[R(int) = 0.0197]
multi-scan
Absorption correction
Refinement method
multi-scan
Full-matrix least-
squares on F
Full-matrix least-
squares on F
Full-matrix least
squares on F
Full-matrix least-
squares on F
Full-matrix least-
squares on F
2
2
2
2
2
Data / restraints /
parameters
2058 / 0 / 151
5796 / 1 / 204
3287 / 1 / 213
3186 / 0 / 212
9531 / 1 / 221
2
1.083
1.111
1.097
1.094
1.010
Goodness-of-fit on F
Final R indices
R1 = 0.0239,
wR2 = 0.0642
R1 = 0.0245,
wR2 = 0.0648
0.02(2)
R1 = 0.0596,
wR2 = 0.1567
R1 = 0.0681,
wR2 = 0.1608
0.03(2)
R1 = 0.0492,
wR2 = 0.1182
R1 = 0.0537,
wR2 = 0.1210
-0.01(4)
R1 = 0.0372,
wR2 = 0.0933
R1 = 0.0387,
wR2 = 0.0945
0.032(16)
R1 = 0.0237,
wR2 = 0.0646
R1 = 0.0239,
wR2 = 0.0648
0.024(6)
[I>2sigma(I)]
R indices (all data)
Absolute structure
parameter
Largest diff. peak and
hole
0.240 and -0.236
1.456 and -0.451
0.663 and -0.194
0.274 and -0.231
0.313 and -0.328
e.Å-3
-3
-3
-3
-3
e.Å
e.Å
e.Å
e.Å
4
1
2
.3 In vitro anticancer activity
respectively. The established trend reveals that the +R effect
offered by ortho position group in aromatic substituents plays a
vital role to enhance the anticancer potential of the tested
compounds. Although, 2b contains ortho-OH which can give strong
The anticancer activities of ATCA and N-BOC ATCAs moieties viz.
compounds 1 to 3f were tested against breast cancer cell lines:
MCF7. Doxorubicin (DOX) was selected as a standard drug for
+R effect showed surprising higher IC50 values (even higher than 2d
comparison. The IC50 data for 48, 72 and 96 hrs along with their with ortho-NO ) perhaps due to ring opening tautomerism in DMSO
2
3
4
absolute stereochemistry are summarized in Table 2. The under the influence of –OH group, generating E-Z imines during
compounds 1-3f exhibited good antiproliferative activity in micro the activity studies.
The activity results were more interesting for N-BOC derivatized
compounds. The diastereomeric pair 3a with (2S,4R) stereocentres
and 3b with (2R,4R) stereocentres having 2-OH phenyl group, the
IC50 values at 96 hrs were found as 51.3 and 53.8 respectively.
Whereas, for another pair 3c with (2S,4R) stereocentres and 3d
molar concentration. Especially the activity of parent ATCAs was
observed to be enhanced on N-BOC derivatization. This outcome
may be due to locking of C-2 chiral centre in a fix stereochemistry
that can avoid ring opening tautomerism which leads to the
3
4
formation of conformational isomers
The observed higher IC50 values for compound 1, 2a as 76.4, 66.2 at
6 hrs is attributed to absence of the aromatic ring and the
.
with (2R,4R) stereocentres having 2-OCH phenyl group as 36.5 and
3
3
8.3 respectively. These observations are very promising proof
towards the necessity of ‘S’ stereochemistry at C-2 chiral centre in
a, 3c than ‘R’ stereochemistry in 3b, 3d to have better anticancer
9
substitution on aromatic ring respectively. Among the parent
diastereomeric ATCAs 2b to 2e, the observed trend of IC50 values at
3
properties. The trans geometry of the C-2 and C-4 substituents
having lower IC50 values is may be corroborated to possible strong
3 2
96 hrs was 2e (2,6-Cl) < 2c (2-OCH ) < 2d (2-NO ) < 2b (2-OH)
4
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ARTICLE
directional interactions. Among the 3e (IC50 16.7, 96 hrs) and 3f (IC50
8.6, 96 hrs); the later showed the higher activity which may be
1
ascribed to the strong H bonding capacity of two –Cl groups. Thus
six of total twelve studied moieties (2b-2e, 3e, 3f) exhibited
significant anticancer activity; even marginally higher than DOX
drug molecule (IC50 33.4, 96 hrs) against MCF cell line.
Table 2. Anticancer activity of all synthesized compounds (1-3f)
4
8 hrs
IC50
µM)
72 hrs
IC50
(µM)
81.3
96 hrs
IC50
(µM)
76.4
C-2,C-4
Absolute
Configuration
4R
Compound
No.
(
1
90.7
83.9
46.7
42.6
49.3
37.2
67.0
72.2
55.4
62.7
39.7
37.2
58.6
2
a
b
77.4
40.0
32.1
37.7
29.0
61.4
66.6
49.4
46.7
26.9
27.2
46.1
66.2
26.9
24.9
24.6
21.3
51.3
53.8
36.5
38.3
16.7
18.6
33.4
2R/S,4R
2R/S,4R
2R/S,4R
2R/S,4R
2R,4R
2S,4R
2
2
c
2d
2e
3a
3b
2R,4R
2S,4R
3
c
3
d
e
2R,4R
2S,4R
Fig. 4 Fluorescence microscopic images of MCF7 cancer cells at 48
hrs.
3
3
f
2R,4R
--
3
. Experimental Section
DOX
3.1 Materials and methods
(
control)
The TLC monitoring of the reactions was done using Merck DC
precoated TLC plates coated by 0.25 mm Kieselgel 60 F254 and
visualization was done using 254 nm UV lamp. The spectroscopic
*
Cells were incubated with DMSO solution of the selected compounds,
washed and then cell proliferation was evaluated by MTT assay; IC50 was
determined from dose-response curves by sigmoid decay fitting using Origin
1
13
characterization of compounds was done by H and C NMR
spectroscopy in ppm scale (400 and 100 MHz NMR
spectrometers, VARIAN Mercury and Bruker instruments) and IR
spectroscopy in cm (FTIR-8400 spectrometer, SHIMADZU). The
LCMS analysis was done using Ultra Fast Mass Spectrometer (LCMS-
8
.0 software.
δ
-
1
2
.4 Cell morphology studies by Fluorescence Microscopy
In view of the high toxicity exhibited by compounds 1-3e against
MCF7 cell lines, the morphological analysis of treated cells were
done by fluorescence microscopy as shown in Figure 4.
8
030, SHIMADZU) with Chiracel OD column and isobutane as an
eluting solvent. Bruker SMART APEX II single-crystal X-ray CCD
diffractometer was used for X-ray crystal structure studies.
The
cells
were
stained
blue
with
1-Chloro-9,10-
bis(phenylethynyl)anthracene. The studies were performed with 3.2 Chemistry
3
4
IC50 values of these compounds at 48 h to determine the viable cell
3
5
.2.1 Synthesis of compounds 1, 2a-2e
3
.0 mM L-Cysteine hydrochloride (0.875 g), 5.0 mM NaHCO (0.42 g)
mass. All the compounds exhibited a decrease in viable cell
population. This was in corroboration with the data obtained by the
MTT assay. When compared to the control cells, there was no
significant change in the morphology of the stained cells indicating
induced apoptosis mode of cell death.
and 4.75 mM of respective aldehyde were stirred at R.T. in 150 ml
of 20 % aq. DMSO. The reactions were monitored by TLC in terms of
consumption of aldehyde and finally the reaction mixture was
quenched in crushed ice. The precipitated solid products were
filtered under suction and dried to get the compound 1 and
diastereomeric (2R/2S,4R)-2-aryl thiazolidine-4-carboxylic acids
(
1,2a-2e) with fair purity and significant yields. As per the x-ray
crystal structure investigation, the compound 2e is obtained as a
single diastereomer with 2R,4R stereochemistry.
3
.2.2 Synthesis and separation/purification of compounds 3a-3f
The 2.0 mM 2b-2e and 5.0 mM NaHCO (0.224 g) were taken in a
00 ml two neck flask and stirred in 50 ml 50 % aqueous 1,4-
3
1
O
dioxane for 1.5 hrs at 0 C. To the resultant yellowish suspensions
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ARTICLE
New Journal of Chemistry
2
.8 mM (0.610 g) of di-t-butyloxy carbonyl was added drop wise 55.39(C CH ), 37.50(C-5), 37.81(C-5). MS (APCI) m/z 239.90 (M+1),
3
with constant stirring and it was continued to stir by natural heating 239.90 (M+1).
to R.T. for 12 h. During workup, the reaction mixtures were acidified 3.3.5(2R/2S,4R)-2-(2-nitrophenyl) thiazolidine-4-carboxylic acid
3
4
by addition of ice cold 2N HCl up to pH 4.0 and extracted in ethyl
(2d).
-
1
acetate. The ethyl acetate extract were washed by cold saturated C10H10N O S, Yellow solid, yield 1.151 g (91%). IR (KBr,υ,cm )
2
4
3
NaHCO up to alkaline pH 9. The resultant aqueous layers were 2797(broad, COOH), 1633(C=O), 1433(N-O), 1348(C-N), 1195(C-O)
−1 1
further acidified up to pH 4.0 by cold 2N HCl. The acidic medium cm . H NMR (400 MHz, DMSO-d +TMS) δ 8.10–8.03 (m, 2H, H Ar),
6
solutions were extracted finally in DCM, which on evaporation gave 7.97–7.91 (m, 2H, H Ar), 7.85–7.81 (m, 2H, H Ar), 7.76–7.66 (m, 2H,
the oily solids of N-BOC derivatized compounds.
The mixture 3a+3b was separated on silica column using 40 % ethyl 1H, H-4), 3.03 (m, 2H, H-5), 2.72(m, 2H, H-5). C NMR (100MHz,
acetate in hexane gave white solids 3a (2S,4R diastereomer) and 3b DMSO-d6) δ 172.97(C=O), 172.80(C=O), 148.80, 147.90, 139.22,
H Ar), 6.76 (s, 1H, H-2), 6.33 (s, 1H, H-2), 4.02 (t, 1H, H-4), 3.19 (q,
1
3
(2R,4R diastereomer) with isolated yields 85
%
and 15
%
135.86, 134.22, 133.79, 129.59, 128.92, 127.85, 125.77, 124.65(C
respectively. Whereas, the mixture of 3c+3d was separated on silica Ar), 66.77(C-2), 65.96(C-2), 65.67(C-4), 65.32(C-4), 37.31(C-5),
column using 20 % ethyl acetate in hexane which afforded the 3c 36.99(C-5). MS (APCI) m/z 254.00 (M+1), 254.00 (M+1).
(
2S,4R diastereomer) and 3d (2R,4R diastereomer) with isolated 3.3.6(2R,4R)-2-(2,6-dichlorophenyl)thiazolidine-4-carboxylic acid
3
4
yields 60 % and 40 % respectively. The crude products 3f (2R,4R
diastereomer) and 3e (2S,4R diastereomer) were purified on silica
(2e).
2
Cl NO S, White solid, yield 1.18 g (84%). IR (KBr,υ,cm )
-1
C
10
H
9
2
column using 10 % ethyl acetate in hexane with 66 % and 72 % 3454(COOH), 3298(N-H), 1726(C=O), 1330(C-S), 1271(C-O), 912,
-
1 1
yields respectively.
781(aromatic C-H) cm . H NMR (400 MHz, DMSO-d
6
+TMS) δ 7.50
(
dd, 1H, H Ar ), 7.39 (t, 2H, H Ar), 6.42(s, 1H, H-2 ), 3.88 (dd, 1H, H-
13
3
.3 Characterization data
4), 3.46 (dd, 1H, H-5), 2.96 (t, 1H, H-5). C NMR (75 MHz, DMSO-
+TMS) δ 173.00 (C=O), 134.00, 131.00, 130.00, 129.00, 128.60,
2
S, White solid, yield 0.598 g (90%), IR (KBr,υ,cm ) 128.00 (C Ar), 70.00 (C-2), 63.41 (C-4), 38.58 (C-5). MS (APCI) m/z
3
4
3.3.1(4R)-thiazolidine-4-carboxylic acid (1).
d
6
-
1
C
4
H
7
NO
-
1 1
3377(COOH), 3049(N-H), 1627(C=O), 1383(C-S), 1340(C-O) cm .
H
278 (M + 1).
6
+TMS) δ 4.22 (d, 1H, H-2), 4.02 (d, 1H, H- 3.3.7(2S,4R)-3-(tert-butoxycarbonyl)-2-(2-hydroxyphenyl)
NMR (400 MHz, DMSO-d
1
3
34
2
(
), 3.84 (t, 1H, H-4), 3.09 (dd, 1H, H-5), 2.82 (dd, 1H, H-5). C NMR
75 MHz, DMSO-d +TMS) δ 172.45 (C=O), 65.51 (C-2), 54.27 (C-4),
thiazolidine-4-carboxylic acid (3a).
S, white solid, isolated yield 1.27 g (85%), IR (KBr,υ,cm )
3089 (COOH), 1718(C=O), 1672 (C=O), 1396 (C-S), 1280, 1282 (C-O),
-1
6
15 5
C H19NO
3
3
6.34 (C-5). MS (APCI) m/z 134 (M + 1).
.3.2(2R/2S,4R)-2-phenyl thiazolidine-4-carboxylic acid (2a).
3
4
-1 1
3
1003, 754 (aromatic C-H) cm . H NMR (400 MHz, CDCl + TMS) δ
-1
C
3
10
H
11NO
2
S, White solid, yield 0.861 g (82%). IR (KBr,υ,cm ) 10.31 (s, 1H, H COOH), 7.83 (d, 1H, H Ar ), 7.17 (t, 1H, H Ar), 6.83 (m,
431(COOH), 1577(C=O), 1435(aromatic C-C), 1381(C-S), 1305(C-N), 2H, H Ar), 5.93 (s, 1H, H-2), 4.86 (dd, 1H, H-4), 3.43 (dd, 1H, H-5),
−1 1
1236(C-O), 1024, 895, 808 (aromatic C-H) cm . H NMR (400 MHz, 3.43 (3, 3H,trapped MeOH), 2.73 (dd, 1H, H-5), 1.38 + 1.25 (2s, 9H,
1
3
DMSO-d
6
+TMS) δ 7.35-7.50 (m, 5H, H Ar), 7.20-7.33 (m, 5H, H Ar),
3 3 6
H C(CH ) ). C NMR (100 MHz, DMSO-d + TMS) δ 172.69,
5
.64 (s, 1H, H-2), 5.47 (s, 1H, H-2), 4.22 (t, 1H, H-4), 3.90 (t, 1H, H-4), 172.24(C=O), 154.60, 130.29, 127.50, 120.47, 119.90, 115.13 (C Ar),
1
3
3
.39–3.31 (m, 2H, H-5), 3.25–3.01 (m, 2H, H-5). C NMR (75 MHz, 83.35(C(CH
+TMS) 172.92(C=O), 172.24(C=O), 141.25, 138.88, 28.10 (C(CH
28.42, 128.23, 128.15, 127.49, 127.20,126.85 (C Ar), 71.72(C-2), 3.3.8(2R,4R)-3-(tert-butoxycarbonyl)-2-(2-hydroxyphenyl)
3
)
3
), 60.89 (C-2), 49.43 (C-4) ,(C-5,merged to solvent),
DMSO-d
6
δ
3 3
) ). MS (APCI) m/z 326 (M + 1).
1
7
3
4
1.02(C-2), 65.57(C-4), 64.91(C-4), 38.98(C-5), 38.50(C-5). MS (APCI)
thiazolidine-4-carboxylic acid (3b).
S, White solid, isolated yield 0.22 g (15%). IR (KBr,υ,cm )
.3.3(2R/2S,4R)-2-(2-hydroxyphenyl) thiazolidine-4-carboxylic acid 3309 (COOH), 1722 (C=O),1683(C=O), 1390 (C-S), 1390, 1242 (C-O),
-1
m/z 210.0 (M + 1), 210.0 (M + 1).
3
15 5
C H19NO
3
4
-1 1
(2b).
756 (aromatic C-H) cm. H NMR (400 MHz, DMSO d
6
+TMS) δ 9.66
-1
C
10 3
H11NO S, White solid, yield 0.877 g (78%). IR (KBr,υ,cm ) (s, 1H,H COOH), 7.81 (d, 1H, H Ar), 7.03 (m, 1H, H Ar), 6.73 (m, 2H, H
3
097(COOH), 3010(N-H), 1627(C=O), 1383(C-S), 1332(C-N), 1280(C- Ar), 6.11 (s, 1H, H-2), 4.45-4.41 (m,1H, H-4), 3.40-3.35 (m, 1H, H-5),
−1 1
O), 1238(C-O), 1097, 1093, 761 (aromatic C-H) cm
MHz, DMSO-d
. H NMR (400 3.30 (s, 3H,trapped MeOH), 3.03 – 2.91 (m, 1H, H-5), 1.32 +1.10 (2s,
13
6 3 3 6
+TMS) δ 7.10–7.33 (m, 4H, H Ar), 6.73–6.82 (m, 4H, 9H, H C(CH ) ). C NMR (100 MHz, DMSO d +TMS) δ 172.20,
H Ar), 5.83 (s, 1H, H-2), 5.64 (s, 1H, H-2), 4.21 (t, 1H, H-4), 3.83 (t, 171.82 (C=O), 153.58, 152.52, 127.97, 118.43, 114.51(C Ar),
1
3
1
H, H-4), 3.4 (q, 2H, H-5), 3.33 (q, 1H, H-5). C NMR (75 MHz, 80.42(C(CH
3
)
)
3
), 64.60(C-2), 61.06(C-4), (C-5,merged to solvent),
). MS (APCI) m/z 324 (M - 1).
DMSO-d +TMS) 172.92(C=O), 172.24(C=O), 155.13, 154.54, 27.65(C(CH
6
δ
3
3
1
1
3
3
28.99, 128.60, 127.84, 127.49, 126.04, 124.14, 118.99, 118.70, 3.3.9(2S,4R)-3-(tert-butoxycarbonyl)-2-(2-methoxyphenyl)
15.62, 115.00 (C Ar), 67.62(C-2), 65.54(C-2), 65.13(C-4), 64.72(C-4),
8.00(C-5), 36.00(C-5). MS (APCI) m/z 226.0 (M + 1), 226.0 (M + 1).
thiazolidine-4-carboxylic acid (3c).
16 5
C H21NO S, White solid, isolated yield 1.02 g (60 %). IR (KBr,υ,cm )
-1
.3.4(2R/2S,4R)-2-(2-methoxyphenyl)
thiazolidine-4-carboxylic 3365 (COOH), 1718 (C=O),1691(C=O), 1359 (C-S), 1240 (C-O), 758
3
4
-1 1
acid (2c).
6
(aromatic C-H) cm . H NMR (400 MHz, DMSO-d ) δ 7.24 – 7.18 (m,
-1
C
11 3
H13NO S , White solid, yield 0.934 g (83%). IR (KBr,υ,cm ) 1H, H Ar), 7.05-714 (m, 1H, H Ar), 6.98-6.94 (m, 1H, H Ar), 6.90-6.84
3
500(broad) 1642(C=O), 1490(aromatic C-C), 1334(C-N), 1244(C-O) (m, 1H, H Ar), 6.09+6.05 (s, 1H, H-2), 4.89 (dd, 1H, H-4), 3.79+3.77
−1 1
cm . H NMR (400 MHz, DMSO-d
6
+TMS) δ 7.51–7.20 (m, 4H, H Ar), (s, 3H, H CH
.05–6.85 (m, 4H, H Ar), 5.82 (s, 1H, H-2), 5.62 (s, 1H, H-2), 4.22 (t, 3.18 – 2.08 (m, 1H, H-5), 1.30 +1.03 (2s, 9H,H C(CH
H, H-4), 3.86 (t, 1H, H-4), 3.82 (s, 3H, H CH ), 3.72 (s, 3H, H CH ), (100 MHz, DMSO-d +TMS) δ 172.29, 171.90(C=O), 155.79, 155.68,
.18–3.05 (m, 2H, H-5), 3.04–2.85(m, 2H, H-5). C NMR (75 MHz, 153.00, 152.77, 132.65, 131.60, 128.75, 124.98, 124.21, 120.62,
172.75(C=O), 172.24(C=O), 156.5, 156.1, 120.57, 111.23(C Ar), 80.50, 80.07, 79.58 (C(CH ) ),63.68, 59.79(C-
3
), 3.42-3.32 (m, 1H, H-5), 3.29 (s, 3H,trapped MeOH),
1
3
7
1
3
3 3
) ). C NMR
3
3
6
1
3
DMSO-d +TMS)
δ
6
3 3
1
3
30.1,129.2, 128.1, 127.2, 126.4, 125.2, 120.4, 120.1, 111.2, 110.70 2), 58.38, 56.07(C-4), 56.00 (C-OCH ), 32.49,31.47(C-5),
1
3
(C Ar), 66.20 (C-2), 65.30 (C-2), 64.98(C-4), 64.91(C-4), 55.43(C CH3), 28.22+27.95(C(CH ) ). [The replications of C signals for 3c may be
3
3
6
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ARTICLE
attributed to formation of cis/trans imines by thiazolidine ring scan width of 0.5° at different settings of φ and ω with a frame time
6
opening and their coexistence with unopened 3d in DMSO-d ].
of 40 secs keeping the sample-to-detector distance fixed at 5.00 cm.
MS (APCI) m/z 338 (M - 1).
37
The X-ray data acquisition was monitored by APEX2 program suit
3.3.10(2R,4R)-3-(tert-butoxycarbonyl)-2-(2-methoxyphenyl)
All the data were corrected for Lorentz-polarization and absorption
effects using SAINT and SADABS programs integrated in APEX2
thiazolidine-4-carboxylic acid (3d).
C H21NO S, White solid, isolated yield 0.69 g (40 %). IR (KBr,υ,cm )
16 5
-1
3
7
2
(
974 (COOH), 1732 (C=O),1637(C=O), 1406 (C-S), 1246 (C-O), 758 package The structures were solved by direct method and refined
-1 1
2
38
aromatic C-H) cm . H NMR (400 MHz, CDCl
3
+ TMS) δ 7.94 (m, 1H, by full matrix least squares, based on F , using ShelXL-2014/7.
H Ar), 7.21 (m, 1H, H Ar), 6.91 (m, 2H, H Ar), 6.12 (s, 1H, H-2), Molecular diagrams were generated using Mercury programs.
3
9
4
.52(m, 1H, H-4), 3.79 (s, 3H, H CH
3
), 3.41–3.39 (m, 1H,H-5 ), 3.29
Geometrical calculations were performed using SHELXTL39 and
(s, 3H,trapped MeOH), 3.10-2.89 (s, 1H,H-5), 1.32 + 1.09 (2s, 9H,H
40
PLATON. Crystal data for the structures have been deposited in
13
3 3 6
C(CH ) ). C NMR (100 MHz, DMSO-d +TMS) δ 174.17(C=O),
the Cambridge Crystallographic Data Center (CCDC) with numbers
1
6
2
3
56.05,128.79, 126.69, 120.59, 110.31(C Ar), 82.23(C(CH ) ),
4.40(C-2), 61.14(C-4),
7.97(C(CH ) ). MS (APCI) m/z 338 (M -1).
.3.11(2R,4R)-3-(tert-butoxycarbonyl)-2-(2,6-dichlorophenyl)
thiazolidine-4-carboxylic acid (3e).
3 3
(
compound numbers) 1565464 (2e), 1565470 (3b), 1565479 (3c),
565478 (3e) and 1565476 (3f).
3.5 In vitro anticancer activity studies
.5.1 Cell viability assay (MTT assay)
55.46(C -OCH
3
),
31.83(C-5), 29.72+
1
3
3
4
1
3
-1
C15H17Cl NO S, White solid, yield: 1.36 g (72 %). IR (neat, υ,cm ) The MCF-7 cell lines were obtained from National Centre for Cell
2
4
2
981 (COOH), 1760 (C=O),1610(C=O), 1406 (C-S), 1132 (C-O), 783 Sciences Repository, Savitribai Phule Pune University, Pune 411007.
-1 1
(aromatic C-H) cm . H NMR (400 MHz, CDCl +TMS) δ 7.39-7.29 (m, The cells were maintained in RPMI 1640 w/ 10% Fetal bovine serum
3
o
2H, H Ar), 7.20 (t, 1H, H Ar), 6.61 (s, 1H, H-2), 4.84 (d, 1 H, H-4), 3.84 (FBS), 2% P/S, 1.25% L-glut, and 1% sodium pyruvate at 37 C (5%
13
(d, 1 H, H-5), 3.33 (dd, 1 H, H-5), 1.19 (s, 9 H,H C(CH ) ). C NMR CO ) in the steri-cycle CO2 incubator with HEPA Class 100 filters,
3 3
2
3
(100 MHz, CDCl +TMS) δ 169.42(C=O), 156.76, 131.37, 129.69 (C Thermo Electron Corporation.
Ar), 84.42 (C(CH ) ), 63.64(C-2), 62.83(C-4), 30.53(C-5), 27.69 The number of viable cells remaining after appropriate treatment
3
3
(
3
C(CH
.3.12(2R,4R)-3-(tert-butoxycarbonyl)-2-(2-nitrophenyl)
thiazolidine-4-carboxylic acid (3f).
3
)
3
). MS (APCI) m/z 376 (M-1).
was
determined
by
3-(4,5-dimethylthiazol-2-yl)-2,5-
i
diphenyltetrazolium bromide (MTT; Sigma Chemical Co.) assay.
Briefly, cells were plated (4000 cells/well per 0.2 mL medium) in 96-
-1
C15H18N O S, Yellowish solid, yield 1.17 g (66 %). IR (KBr, υ,cm ) well microtiter plates and incubated overnight. The compounds 1-3f
2
6
2
977 (COOH), 1734 (C=O),1645(C=O), 1401 (C-S), 1249 (C-O), 733 was then added at indicated concentrations to quadruplicate wells.
-1 1
(aromatic C-H) cm . H NMR (400 MHz, CDCl +TMS) δ 8.38 – 8.21 After 24 hrs MTT was added to each well at a final volume of 0.5
3
o
(m, 1H, H Ar), 8.18-8.03 (m, 1H, H Ar), 7.70 (m, 1H, H Ar), 7.49 (m, mg/mL and the microplates were incubated at 37 C for 3hrs. After
1
H, H Ar), 6.89+6.60 (s, 1H, H-2), 4.94-4.75 (m, 1H, H-4), 3.41-3.11 the supernatant was removed, the formazan salt resulting from the
13
(m, 2H, H-5), 1.27 +1.25 (2s, 9H,H C(CH
3
)
3
). C NMR (CDCl
3
+TMS) δ reduction of MTT was solubilized in dimethyl sulfoxide (DMSO,
1
6
73.52(C=O), 133.91, 128.60, 128.02, 125.05 (C Ar), 82.94(C(CH ) ), Sigma Chemical Co.) and the absorbance was read at 570 nm using
3
3
4.61(C-2), 62.62(C-4), 31.82(C-5), 29.72+27.97 (C(CH
3
)
3
). MS (APCI) an automatic plate reader (Thermo Corporation). The cell viability
was extrapolated from optical density (OD) values and expressed as
percent survival. The percent cell death was calculated considering
the untreated cells as 100 percent viable. The results allowed
establishing dose-response curves to calculate IC50 values, the
m/z 354 (M + 1).
3.4 Crystallographic studies (2e, 3b, 3c, 3e and 3f)
The single crystals of chiraly pure compounds 2e and 3b, 3c, 3e, 3f
were crystallized in DMF-MeOH and DCM-MeOH co-solvents
respectively using Hexane vapour deposition technique.
concentration required to inhibit cell proliferation by 50%.
.5.2 Fluorescence microscopy
MCF7 cells were seeded on to glass cover slips at a density of 5×10
3
4
The single-crystal structure of compounds 2e, 3c and 3e were cells to achieve ~ 80 confluences. The cells were treated with
o
determined by measuring X-ray intensity data on a Bruker SMART compounds 1-3f for 48 hrs at 37 C. After 48 hrs of incubation the
cells
were
stained
with
1-Chloro-9,10-
APEX II single-crystal X-ray CCD diffractometer equipped with
graphite-monochromatized (Mo-K" = 0.71073 A° ) radiation at 296
K. The X-ray generator was operated at 50 kV and 30 mA. A
preliminary set of cell constants and an orientation matrix were
calculated from total 36 frames. The optimized strategy used for
data collection consisted different sets of ɸ and ω scans with 0.5°
steps in ɸ/ω. Data were collected with a time frame of 10 s keeping
bis(phenylethynyl)anthracene from Sigma Aldrich staining dye
reagents, at a dilution of 1:20 for 30 min at 37 °C. The excess
staining solution was removed and fixed in 4% para-formaldehyde
(
(
PFA). The microscopic images were observed under Carl Zeiss
Magnification 40x) with a filter set excitation/emission at 536 nm.
the sample-to-detector distance fixed at 5.00 cm. X-ray intensity Conclusions
data measurements of 3b and 3f were carried out on a Bruker D8
In summary, ATCA 2e, N-BOC ATCA’s 3b, 3c, 3e and 3f were
VENTURE Kappa Duo PHOTON II CPAD diffractometer equipped
with Incoatech multilayer mirrors optics. The intensity
measurements were carried out with Mo micro-focus sealed tube
diffraction source (Mo-Kα = 0.71073 Å) at 100(2) K temperature.
The X-ray generator was operated at 50 kV and 1.4 mA. A
preliminary set of cell constants and an orientation matrix were
calculated from three sets of 12 frames. Data were collected with ω
obtained in chirally pure forms in excellent yields and
diffracted by X-ray single crystal technique. The broad
spectrum of completely characterized compounds (1-3f) was
studied for their in vitro anti breast cancer activity by MTTS
assay using MCF7 cell lines. Among the resolved diastereomers
(
3a-3d), the ‘2S’ stereochemistry is essential for marginally
higher anticancer activity over ‘2R’ stereocenters on
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DOI: 10.1039/C7NJ02961F
ARTICLE
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Graphical Abstract & Highlighting Statement
X-ray crystal structures and anti breast cancer property of 3-tert-butoxycarbonyl-2-
arylthiazolidine-4-carboxylic acids
a
b
b
a
Rohidas M. Jagtap, Shridhar H. Thorat, Rajesh G. Gonnade, Ayesha A. Khan and Satish K.
a
Pardeshi*
a
Department of Chemistry, Savitribai Phule Pune University (formerly University of Pune),
Ganeshkhind, Pune-411007, India. Email: skpar@chem.unipune.ac.in;
Center for Materials Characterization (CMC), National Chemical Laboratory, Dr. Homi Bhabha
Road, Pune-411008, India.
b
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