Facile synthesis and quantitative structure-activity relationship study of antitumor active 2-(4-Oxo-thiazolidin-2-ylidene)-3-oxo-propionitriles

Article abstract of DOI:10.1248/cpb.c12-00498

2-(5-Arylidene-4-oxo-3-phenyl-thiazolidin-2-ylidene)-3-oxo-propionitriles 4a-j were prepared via condensation of aromatic aldehydes with 4-thiazolidinones 3a, b. The latter was obtained via electrophilic attack of phenylisothiocyanate on 3-oxo-propionitri

Full text of DOI:10.1248/cpb.c12-00498

September 2012
Regular Article
Chem. Pharm. Bull. 60(9) 1195–1206 (2012)
1195
Facile Synthesis and Quantitative Structure–Activity Relationship Study
of Antitumor Active 2-(4-Oxo-thiazolidin-2-ylidene)-3-oxo-propionitriles
Mona Maurice Hanna and Riham François George*
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University; El-Kasr El-Aini Street, Cairo 11562,
Egypt. Received June 3, 2012; accepted June 25, 2012
2-(5-Arylidene-4-oxo-3-phenyl-thiazolidin-2-ylidene)-3-oxo-propionitriles 4a–j were prepared via con-
densation of aromatic aldehydes with 4-thiazolidinones 3a,b. The latter was obtained via electrophilic attack
of phenylisothiocyanate on 3-oxo-propionitriles 1a,b followed by reaction with chloroacetyl chloride under
basic condition. Additionally, 2-(5-heteroalicyclic methylene) analogues 5a–h were prepared via Mannich re-
action of the appropriate secondary amines and formaldehyde with 4-thiazolidinones 3a,b. Many of the syn-
thesized compounds exhibited promising antitumor properties against colon HCT116 and breast T47D cell
lines. 3D-Pharmacophore modeling and quantitative structure–activity relationship (QSAR) analysis were
combined to explain the observed antitumor properties.
Key words thiazolidinone; quantitative structure–activity relationship; antitumor activity; 3-oxo-propio-
nitrile; HCT116; T47D
Cancer ranks second in diseases leading to mortality, fol- (breast cancer, GI50=0.00089mM), HL-60 (TB) (leukemia,
lowing only cardiovascular diseases. One-quarter of all deaths GI50=0.00085mM), SNB-75 (CNS cancer, GI50=0.00185mM),
in the United States are caused by cancer.1) Out of the many OVCAR-5 (ovarian cancer, GI50=0.00055mM), EKVX (non-
cancer diseases, breast cancer is the most prevalent cancer in small lung cell cancer, GI50=0.00202mM) and PC-3 (prostate
women and represents the second highest leading cause of cancer, GI50=0.00087mM).21)
cancer death in this population after lung cancer.2) Colorec-
Quantitative structure–activity relationship (QSAR) will be
tal cancer is the third most common cancer in both men and also taken into consideration during the present work in order
women, 91% of cases are diagnosed in individuals 50 years to study the pharmacophoric features of the antitumor active
of age and older.1) Chemotherapy is widely used to treat and compounds and to determine the parameters controlling the
control cell growth and limit the spread of cancer cells to pharmacological properties.
other sites. Although, there is a success with certain forms of
cancer, drug therapy has only limited impact against the three
major killers: carcinoma of lung, breast and colorectal system.
Therefore, there is a need to develop novel antitumor agents to
treat and combat this disease.
Several promising antitumor agents containing thiazoli-
dine and thiazolidinone scaffolds have been identified to
have a broad range of anticancer activities.3–14) Previously,
we reported promising antitumor properties of a variety of
5-arylidene thiazolidinone derivatives Ia–c (Fig. 1) exhibit-
ing considerable cytotoxic activity against colon HCT116
cancer cell lines compared with Doxorubicin (reference
standard, IC50=0.00686mM).15) In continuation of these
previous findings and in order to optimize novel antitumor
active agents possessing the same thiazolidinone core, it
is intended in the present work to perform some modifica-
tions in the adopted structures via inserting heteroalicyclic
amines (morpholinyl or piperidinyl functions, 4a–j) instead
of the aromatic amines Ia–c due to their hydrophilic prop-
erties. Moreover, a series of heteroalicyclic methylene con-
taining compounds 5a–h will be also prepared replacing
the arylidene moiety of Ia–c (Fig. 1). Additionally, many
morpholine and piperidine containing compounds were
known as anticancer active agents that exerted their actions
via inhibition of different targets such as phosphatidylinosi-
tol 3-kinases (PI3Ks) and histone deacetylase (HDAC).16–20)
Moreover, the piperidine derivatives IIa–e (Fig. 2) were re-
ported through antitumor activity data obtained by US-NCI
to be active against different cell lines exemplified by T47D
Fig. 1. Reported (Ia–c) and Proposed (4a–j, 5a–h) Thiazolidinone
The authors declare no conflict of interest.
Derivatives
*To whom correspondence should be addressed. e-mail: rihamfgeorge@yahoo.com
© 2012 The Pharmaceutical Society of Japan

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Vol. 60, No. 9
Fig. 2. Structures of Some Antitumor Active Piperidine Derivatives
Results and Discussion
Chemistry The target 2-(5-arylidene-4-oxo-3-phenyl-thi-
azolidin-2-ylidene)-3-oxo-propionitriles 4a–j were prepared as
depicted in Charts 1 and 2. The starting 3-(morpholin-4-yl)-
1a and 3-(piperidin-1-yl)-3-oxo-propionitriles 1b were syn-
thesized according to the previously reported procedures.22)
Electrophilic attack of phenylisothiocyanate on the active
methylene of compounds 1a,b in dry tetrahydrofuran (THF)
in the presence of potassium hydroxide afforded 3-mercapto-
2-carbonyl-3-phenylamino-acrylonitriles 2a,b. The structures
Chart 2. Synthetic Pathway for Preparation of Compounds 4a–j and
5a–h
1
of the isolated compounds 2a,b were confirmed by H-NMR
spectral data, that lacked any signal assigneable for the active
methylene function and exhibited signals due to the aromatic
ring protons at δ=7.11–7.78, and two exchangeable signals (pyrrolidine, piperidine, morpholine and N-methylpiperazine)
corresponding to NH and SH protons at δ=5.34–5.37 and through Mannich reaction yielded 5a–h. The structures of the
1
10.54–11.08 respectively. Reaction of 2a,b with chloroacetyl obtained products 5a–h were confirmed by H-NMR that re-
chloride in the presence of triethylamine (TEA) in dry THF vealed the expected heteroalicyclic protons signals in addition
gave the 4-thiazolidinone derivatives 3a,b. IR spectra of 3a,b to the other skeleton protons (cf. Experimental).
revealed two C=O bands at ν=1736–1724 and 1636cm−1 re-
Furthermore, reaction of the 3-mercapto-3-phenylamino-
gions. H-NMR spectra of 3a,b exhibited the methylene func- acrylonitriles 2a,b with 3-chloropropionyl chloride in THF in
1
tion as a sharp singlet signal at δ=3.90.
the presence of TEA afforded 3-oxo-2-(4-oxo-3-phenyl-[1,3]-
The target arylidenes 4a–j were obtained by condensation thiazinan-2-ylidene)propionitriles 6a,b (Chart 3). Single X-ray
of the appropriate aromatic aldehydes with 3a,b in dimethyl- crystallography of 6a (Fig. 3) allowed good confirmation for
formamide (DMF) in the presence of TEA. The structures of the assigned structure confirming that the isolated product is
4a–j were established through different spectroscopic tech- E-isomer.
1
niques (IR, H-NMR, MS) and elemental analyses data. The
Many trials had been performed toward preparation of
disappearance of the singlet signal due the methylene protons 2-(5-arylidene-4-oxo-3-phenyl-[1,3]thiazinan-2-ylidene)-3-oxo-
and the presence of the sharp singlet signal due to the ylidene propionitriles 7 via condensation reaction of different aromatic
1
proton in H-NMR spectra added a good confirmation for the aldehydes (benzaldehyde, p-anisaldehyde, p-chlorobenzalde-
assigned structures 4a–j. The appearance of the ylidene pro- hyde, salicylaldehyde or vanillin) with 6a,b under different
ton of compounds 4a–c, 4f–h at δ=7.78–7.86 confirmed the reaction conditions. When this reaction was conducted in
formation of Z-isomers.9,15,23–28) On the other hand, the ylidene DMF/TEA, absolute ethanol/piperidine or absolute ethanol/
proton of compounds 4d and 4i were revealed at δ=8.00–8.16, potassium hydroxide at room temperature, or either warming
slightly downfield shifted than the other synthesized ana- the reaction in DMF/TEA at 60°C, no reaction was occurred.
logues, which could be attributed to the anisotropic effect of However, upon conducting the reaction at reflux temperature
the hydroxyl group oriented at the o-position of the arylidene in absolute ethanol/piperidine or absolute ethanol/potassium
function. Furthermore, reaction of 4-thiazolidinones 3a,b hydroxide, the hydrolysed products 2a,b were isolated instead
with formaldehyde and the appropriate heteroalicyclic amines of the expected arylidene derivatives 7 (where the structures
Chart 1. Synthetic Pathway for Preparation of 3a,b

September 2012
1197
Fig. 3. ORTEP Projection of Single Crystal X-Ray Diffraction of 6a
Chart 3. Synthetic Pathway for Preparation of Compounds 6a,b
were established by comparative IR and melting point data).
These results could be attributed to the lower activity of the antitumor activity having IC50 values ranging from 0.00586
methylene group neighbouring to the cyclic ketonic function to 0.04848mM against HCT116 cancer cell lines and from
of the six membered ring system than the corresponding five 0.00646 to 0.05059mM against T47D cell lines. In most cases,
membered thiazolidinone system.
the 5-arylidene derivatives 4a–j exerted promising antitumor
Antitumor Activity In-vitro antitumor activity of the activity against both cell lines than the 5-heteroalicyclic
tested compounds was screened utilizing Sulfo-Rodamine B methyl analogues 5a–h.
(SRB) standard method15,29–35) in the National Cancer Insti-
Considering the observed antitumor screening data of the
tute, Cairo University, Egypt. All the prepared target com- synthesized compounds against HCT116 “colon” cancer cell
pounds (4a–j, 5a–h) were tested for their antitumor proper- line, the arylidene piperidine derivatives 4f–i exerted higher
ties against HCT116 “colon” and T47D “breast” cancer cell potency than the morpholine analogues 4a–d. Compound 4g
lines. From the observed antitumor activity data (Table 1), was the most effective one with IC50=0.00586mM compared
(see also Figs. 1, 2 of the Supplementary data), it has been with Doxorubicin (IC50=0.00686mM). This derivative was
noticed that the tested compounds showed moderate to potent selected by US-NCI for more antitumor activity screening
Table 1. IC50 of the Tested Compounds against Human Tumor Cell Lines
Tested human tumor cell lines, IC50 µg/mL (mM)
Compound
X
R
Colon (HCT 116)
Breast (T47D)
Doxorubicin
—
O
—
H
3.73 (0.00686)
3.64 (0.00872)
3.64 (0.00813)
3.48 (0.0077)
7.65 (0.01407)
14.09 (0.03375)
16.25 (0.03631)
2.92 (0.00646)
3.81 (0.00879)
17.92 (0.03866)
2.86 (0.00688)
3.18 (0.00714)
3.33 (0.0074)
4a
4b
4c
4d
4e
4f
O
4-OCH3
4-Cl
O
O
2-OH
10.91 (0.02517)
12.17 (0.02626)
2.92 (0.00703)
2.61 (0.00586)
2.92 (0.00649)
3.64 (0.00844)
15.00 (0.0325)
O
3-OCH3, 4-OH
H
CH2
CH2
CH2
CH2
CH2
4g
4h
4i
4-OCH3
4-Cl
2-OH
8.10 (0.01877)
14.50 (0.03142)
4j
3-OCH3, 4-OH
5a
5b
5c
5d
5e
5f
O
O
20.00 (0.04848)
13.64 (0.03198)
6.67 (0.01557)
6.96 (0.01576)
11.67 (0.02843)
15.91 (0.03747)
14.09 (0.03303)
13.18 (0.02998)
15.45 (0.03754)
15.91 (0.0373)
8.10 (0.0189)
O
O
5.83 (0.0132)
CH2
CH2
CH2
CH2
12.11 (0.0295)
20.95 (0.04934)
21.58 (0.05059)
14.21 (0.03233)
5g
5h

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Vol. 60, No. 9
utilizing 56 cell lines belonging to nine types of cancers (breast cancer), GI50=0.00133, 0.00113mM, respectively.
(leukemia, non-small cell lung cancer, colon cancer, CNS can- On the other hand, the antitumor activity of the tested
cer, melanoma, ovarian cancer, renal cancer, prostate cancer compounds against T47D breast cell lines exhibiting that com-
and breast cancer).30–35) The observed data expressed as GI50 pound 4c was the most active with IC50 value=0.00646mM
(the concentration resulting in a 50% growth inhibition of compared with Doxorubicin (IC50=0.01407mM), the 5-aryl-
the tumor compared with the control experiments), TGI (the idene morpholine derivatives 4c and 4d (IC50=0.00646,
concentration resulting in a 100% growth inhibition of the 0.00879mM, respectively) were more potent than their pi-
tumor compared with the control experiments) and LC50 were peridine analogues 4h and 4i (IC50=0.0074, 0.01877mM,
presented in Table 2. It showed promising activity against 38 respectively) (Table 1). However, the piperidine containing
cell lines especially, HOP-92, NCI-H226 (non-small cell lung compounds 4f and 4g exhibited higher antitumor activity
cancer), GI50=0.000865, 0.00127mM, respectively, SF-539, (IC50=0.00688, 0.00714mM, respectively) than the morpholine
SNB-75 (CNS cancer), GI50=0.00144, 0.000811mM, respec- derivatives 4a and 4b (IC50=0.03375, 0.03631mM, respective-
tively, MALME-3M, SK-MEL-5 (melanoma), GI50=0.0016, ly). In addition, compound 5d showed good activity with IC50
0.0015mM, respectively, OVCAR-4, SK-OV-3 (ovarian cancer), values=0.0132mM.
GI50=0.00102, 0.00115mM, respectively, 786-0, A498, RXF
QSAR Study. 3D-QSAR Pharmacophore Model-
393, UO-31 (renal cancer), GI50=0.00112, 0.00111, 0.00118, ing This study was performed using Discovery Studio 2.5
0.00148mM, respectively and MDA-MB-231/ATCC, HS 578T software (Accelrys Inc., San Diego, CA, U.S.A.). A given
Table 2. Antitumor Screening Data of 4g Expressed in GI50, TGI, LC50 Values (mM) Utilizing Human Tumor Cell Lines
Panel/cell lines
GI50
TGI
LC50
Panel/cell lines
GI50
TGI
LC50
Leukemia
HL-60(TB)
K-562
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
>0.05
>0.05
>0.05
NDa)
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
0.00277
>0.05
>0.05
NDa)
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
0.00195
0.00102
MOLT-4
RPMI-8226
SR
>0.05
>0.05
>0.05
>0.05
0.0113
0.00167
0.00233
0.00115
Non-small cell lung cancer
A549/ATCC
EKVX
0.00263
0.0041
>0.05
>0.05
0.00938
0.00286
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
0.00257
HOP-62
0.00214
0.000865
0.00127
0.00771
0.00308
0.00112
0.00111
0.00247
0.0025
0.00496
0.0135
>0.05
>0.05
>0.05
>0.05
HOP-92
A498
NCI-H226
ACHN
NCI-H23
CAKI-1
>0.05
>0.05
0.00357
>0.05
>0.05
>0.05
NCI-H460
RXF 393
SN12C
0.00118
0.00225
0.00371
0.00148
0.0325
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-15
>0.05
>0.05
>0.05
>0.05
TK-10
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
UO-31
>0.05
>0.05
>0.05
>0.05
>0.05
Prostate cancer
PC-3
NDa)
>0.05
>0.05
>0.05
>0.05
HT29
DU-145
>0.05
KM12
Breast cancer
MCF7
SW-620
>0.05
0.00133
>0.05
0.00434
>0.05
>0.05
CNS cancer
MDA-MB-231/
ATCC
SF-268
SF-295
0.00374
>0.05
>0.05
NDa)
>0.05
>0.05
>0.05
>0.05
HS 578T
0.00113
0.0223
0.00379
>0.05
>0.05
>0.05
>0.05
>0.05
0.00776
0.00144
0.00409
0.000811
0.00297
BT-549
SF-539
MDA-MB-468
0.00343
SNB-19
>0.05
0.00214
SNB-75
0.0105
U251
>0.05
>0.05
Melanoma
LOX IMVI
MALME-3M
M14
0.00257
0.0016
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
>0.05
0.00417
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
>0.05
0.00405
0.00241
0.0015
0.0412
>0.05
>0.05
>0.05
>0.05
>0.05
0.00211
a) ND=not determined.

September 2012
1199
Fig. 4. (A) Constraint Distances (Å) and (B) Constraint Angels (°) between Features of the Generated Pharmacophore, (C) Mapping of 4g in the
Generated Pharmacophore, (D) Mapping of 5c in the Generated Pharmacophore
Table 3. Constraint Distances (Å) and Angles (°) between Features of the Generated Pharmacophore for Colon HCT116 Cancer Cell Lines
Constraint distances (Å)
Constraint angles (°)
(HBA)–(H1)=3.905; (HBA)–(H2)=6.656; (H2)–(H1)=7.045Å
(H2)–(H1)–(HBA)=68.04°; (H2)–(HBA)–(HBA vector)=55.16°;
(HBA vector)–(H2)–(H1)=70.48°
hypothesis could be combined with a known activity data to affinity and 3D search queries, their predictive value as 3D-
create a 3D-QSAR model that identifies overall aspects of mo- QSAR models is generally limited by steric shielding and
lecular structure governing activity. 3D-QSAR based on phar- bioactivity-modulating auxiliary groups (electron donating or
macophore was constructed using collections of molecules withdrawing functionalities).15,35–38) Thus, a classical QSAR
with activities ranging over a number of orders of magnitude. analysis was employed to search for the best combination of
Pharmacophores explain the variability of bioactivity with orthogonal pharmacophores using a fit value and other struc-
respect to the geometric localization of the chemical features tural descriptors (connectivity, topological, etc.) capable of
present in the molecules.15,35)
explaining bioactivity variation across a collected list of the
Twenty nine compounds (4a–f, 4h–j, 5a,b, 5d–h and the descriptors, allowing different pharmacophoric models com-
previously prepared Ia–j and IIIa–c15)) were taken as a train- peting within the 3D-QSAR framework.
ing set and the remaining two compounds, 4g (with potent
A set of 29 compounds (4a–f, 4h–j, 5a,b, 5d–h and the pre-
antitumor activity) and 5c (with mild activity) were used as a viously reported Ia–j and IIIa–c15)) was used as a training set
test set. The observed HYPOGEN identifies a 3D array of a for a QSAR modeling. The remaining 2 compounds (4g and
maximum of three chemical features common to the training 5c) were adopted as an external test subset for validating the
set that provide relative alignment for each input molecule, QSAR model. Many molecular descriptors were calculated for
consistent with its binding mode to a proposed common re- each compound employing a calculated molecular properties
ceptor site. The chemical features considered were: two hydro- module. The calculated descriptors including various simple
phobic sites (H1, H2) and one hydrogen bond acceptors (HBA) and valence connectivity indices, electro-topological state
(Figs. 4A,B, Table 3 exhibit constraint distances and angles indices, single point quantum-mechanical descriptors (via
between features of the generated pharmacophore). Through the AM1 model) and other molecular descriptors, were con-
the pharmacophore mapping study, it was found that the major sidered. Furthermore, the training set compounds were fitted
structural factors affecting the potency of these compounds against the corresponding pharmacophore hypotheses gener-
were related to their basic skeleton. The controlling features ated by the HYPOGEN automatic runs and their fit values
were two hydrophobic sites represented by the arylidene and (produced by the best-fit command) were added as additional
morpholine or piperidine moieties together with a hydrogen molecular descriptors. Genetic function approximation (GFA)
bond acceptor of the amide carbonyl group for 4a–j. While was employed to search for the best possible QSAR regression
compounds 5a–h had not been fitted well to the generated equation capable of correlating the variations in biological
pharmacophore as they lacked the arylidene moiety, therefore, activities of the training set compounds with variations in the
their fit values were smaller than those of 4a–j that explained generated descriptors, i.e. multiple linear regression model-
their mild antitumor activity (Table 4).
ing (MLR).15,35,39) Equation 1 shows our best-performing
QSAR Modeling Despite of the significance of phar- QSAR model (Fig. 5 exhibits the corresponding scatter plots
macophoric hypotheses for understanding ligand molecule of experimental versus estimated bioactivity values for the

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Vol. 60, No. 9
Table 4. Best Fit Values and Estimated Activities for 29 Compounds of the Training Set Mapped with the Generated 3D-Pharmacophore Model
Compound
X
R
Observed activity
Estimated activity
Fit value
4a
4b
4c
4d
4e
4f
O
O
H
4-OCH3
4-Cl
8.72×10−3
8.13×10−3
7.7×10−3
18.04×10−3
10.91×10−3
10.80×10−3
11.65×10−3
11.64×10−3
10.74×10−3
10.22×10−3
11.41×10−3
11.41×10−3
5.86
5.92
6.05
5.88
5.93
6.08
6.08
6.04
5.48
O
O
2-OH
25.17×10−3
26.26×10−3
7.03×10−3
6.49×10−3
8.44×10−3
32.5×10−3
O
3-OCH3, 4-OH
H
CH2
CH2
CH2
CH2
4h
4i
4-Cl
2-OH
4j
3-OCH3, 4-OH
5a
5b
5d
5e
5f
O
48.48×10−3
31.98×10−3
15.76×10−3
28.43×10−3
37.47×10−3
33.03×10−3
29.98×10−3
35.16×10−3
38.58×10−3
32.54×10−3
39.23×10−3
38.17×10−3
23.29×10−3
30.46×10−3
5.74
5.75
5.69
5.51
5.78
5.74
5.89
O
O
CH2
CH2
CH2
CH2
5g
5h
Ia
Ib
H
CH3
Cl
4-OCH3
2-OH
2-OH
2-OH
4-Cl
4-OCH3
H
6.61×10−3
6.92×10−3
6.92×10−3
7.80×10−3
9.15×10−3
9.95×10−3
10.97×10−3
28.39×10−3
33.29×10−3
38.87×10−3
14.86×10−3
46.35×10−3
113.77×10−3
10.57×10−3
8.88×10−3
7.93×10−3
12.84×10−3
16.33×10−3
14.15×10−3
12.99×10−3
16.01×10−3
16.59×10−3
8.06×10−3
27.45×10−3
44.07×10−3
72.71×10−3
6.09
6.16
6.21
6.00
5.90
5.96
6.00
5.91
5.89
6.21
5.67
5.47
5.25
Ic
Id
H
Ie
Cl
If
CH3
CH3
CH3
H
Ig
Ih
4-Cl
H
Ii
Ij
Cl
H
IIIa
IIIb
IIIc
CH3
H
Cl
OCH3
H
H
training set compounds against HCT116 tumor cell lines). The
goodness of the model was validated by squared correlation
coefficient (R2) and residuals between the predicted and ex-
perimental activity of the training set (Table 5).
Potency (IC50) against HCT116 (colon cancer) cell line (N
“number of molecules in the training set”=29, R2 “squared
correlation coefficient value”=0.726)
IC50=604.41  0.298×[Molecular_SurfaceArea]
 398.59×[Jurs_RNCG]  67.65×[FitValue] (1)
Fig. 5. Estimated Activity versus Observed Activity (IC50) of the Tested
Compounds against HCT116 (Colon) Human Tumor Cell Line
Where the Jurs_RNCG is a relative negative charge descriptor
that described the charge of most negative atom divided by
total negative charge.
Searching for set descriptors (D), containing D descrip- standard deviation (S), by means of multivariable linear re-
≪
tors of optimal subset (d), where d D ones with minimum gression (MLR) technique.

September 2012
1201
Table 5. Estimated Activity Data of the Training Set against HCT116 (Colon Cancer) Cell Line and Calculated Descriptors Governing Activity Accord-
ing to Eq. 1
Compound
Mol. surface area
Jurs_RNCG
Fit value
Observed activity
Estimated activity
Residual
4a
4b
4c
4d
4e
4f
391.60
424.15
414.67
404.88
437.43
392.95
416.03
406.23
438.78
392.02
407.99
429.29
393.37
409.34
407.99
430.64
425.47
425.61
429.27
406.20
439.06
444.88
412.34
435.41
392.92
415.99
446.85
436.91
404.37
0.163
0.149
0.165
0.148
0.134
0.144
0.145
0.156
0.139
0.186
0.181
0.165
0.167
0.162
0.176
0.145
0.145
0.147
0.149
0.148
0.130
0.144
0.127
0.128
0.128
0.129
0.115
0.131
0.115
5.86
5.92
6.05
5.88
5.93
6.08
6.08
6.04
5.48
5.74
5.75
5.69
5.51
5.78
5.74
5.89
6.09
6.16
6.21
6.00
5.90
5.96
6.00
5.91
5.89
6.21
5.67
5.47
5.25
8.72×10−3
8.13×10−3
7.7×10−3
26.27×10−3
17.88×10−3
5.93×10−3
26.88×10−3
19.14×10−3
18.14×10−3
11.10×10−3
9.28×10−3
9.25×10−3
42.31×10−3
21.76×10−3
21.48×10−3
35.41×10−3
44.57×10−3
21.20×10−3
29.57×10−3
7.76×10−3
1.85×10−3
−3.51×10−3
17.96×10−3
22.29×10−3
11.05×10−3
24.94×10−3
23.77×10−3
37.42×10−3
8.89×10−3
41.48×10−3
51.81×10−3
82.72×10−3
−17.55×10−3
−9.75×10−3
1.77×10−3
25.17×10−3
26.26×10−3
7.03×10−3
6.49×10−3
8.44×10−3
32.5×10−3
48.48×10−3
31.98×10−3
15.76×10−3
28.43×10−3
37.47×10−3
33.03×10−3
29.98×10−3
6.61×10−3
6.92×10−3
6.92×10−3
7.80×10−3
9.15×10−3
9.95×10−3
10.97×10−3
28.39×10−3
33.29×10−3
38.87×10−3
14.86×10−3
46.35×10−3
113.77×10−3
−1.71×10−3
7.12×10−3
−11.11×10−3
−4.61×10−3
−0.84×10−3
23.25×10−3
6.17×10−3
4h
4i
4j
5a
5b
5d
5e
5f
10.22×10−3
−5.72×10−3
−6.98×10−3
−7.10×10−3
11.83×10−3
0.41×10−3
5g
5h
Ia
−1.15×10−3
5.07×10−3
Ib
Ic
10.43×10−3
−10.16×10−3
−13.14×10−3
−1.10×10−3
−13.97×10−3
4.62×10−3
Id
Ie
If
Ig
Ih
Ii
−4.13×10−3
29.98×10−3
−26.62×10−3
−5.46×10−3
31.05×10−3
Ij
IIIa
IIIb
IIIc
Table 6. External Validation for the Established QSAR Models Utilizing Promising 4g and 5c Mild Antitumor Active Agents
3D-QSAR pharmacophore
Classical QSAR
Cell lines
HCT116
Compound
Experimental activity Predicted activity
Experimental activity Predicted activity
Fit value
(IC50)
(IC50)
(IC50)
(IC50)
4g
5c
5.86×10−3
7.65×10−3
5.31
5.04
5.86×10−3
6.76×10−3
15.57×10−3
22.14×10−3
15.57×10−3
13.13×10−3
N
R(d)2(N  d 1)
( N +d2)(1  R2)
1
FIT =
S =
resi
(N  d 1) 
i=1
Where; N, is the number of molecules of the training set; resi, Where N=29, R=0.852, S=0.544, FIT=2.91.
is the residual for molecule; i, is the difference between the Validation of QSAR External validation of the deter-
experimental property (p) and predicted property (ppred). mined QSAR equations was performed utilizing two of our
More precisely, the Kubinyi function (FIT)15,35) is a statisti- synthesized analogues exhibiting promising (4g) and mild
cal parameter which is closely related to the Fisher ratio (F), (5c). The observed activities and those provided by QSAR
but avoids the main disadvantage of the latter of being too study are presented in Table 6.
sensitive to changes in small d values, and poorly sensitive
to changes in large d values. The FIT (d) criterion has a low
sensitivity to changes in small d values and a substantially
Conclusion
3-Oxo-2-(4-oxo-3-phenyl-thiazolidin-2-ylidene)propionitriles
increasing sensitivity for large d values. The greater the FIT 3a,b were synthesized via reaction of 3-mercapto-3-oxo-
value the better the linear equation.15,35,40) It is given by the 3-phenylamino-acrylonitriles 2a,b with chloroacetyl chlo-
following equation, “where R(d) is the correlation coefficient ride in the presence of TEA. Reaction of 3a,b with either
for a model with (d) descriptors.” The observed FIT value is aromatic aldehydes or heteroalicyclic secondary amines
2.91 corresponding to model due to HCT116 cancer cell lines. and formaldehyde afforded 2-(5-arylidene-4-oxo-3-phenyl-
thiazolidin-2-ylidene)-2-cyano-3-oxo-propionitriles 4a–j and
2-(5-heteroalicyclic methyl) analogues 5a–h, respectively.

1202
Vol. 60, No. 9
Furthermore, (2E) 3-oxo-2-(4-oxo-3-phenyl-[1,3]thiazinan-2- 1H-NMR (CDCl3, 300MHz) δ: 3.45–3.82 (8H, m, morpholine
ylidene)-propionitriles 6a,b were prepared stereoselectively protons), 5.37 (1H, s, NH exch. D2O), 7.11–7.77 (5H, m, aro-
via reaction of 2a,b with 3-chloropropionyl chloride in the matic H), 11.08 (1H, s, SH exch. D2O). IR (KBr) cm−1: 3271
presence of TEA. Many trials to obtain the arylidene deriva- (NH), 3050–3010 (aromatic CH), 2967–2859 (aliphatic CH),
tives 7 via condensation of different aromatic aldehydes with 2172 (C≡N), 1650 (C=O), 1632 (bending NH), 1593–1535 (C=
6a,b were unsuccessful.
C). Anal. Calcd for C14H15N3O2S (289.36): C, 58.11; H, 5.23; N,
The synthesized 4a–j and 5a–h were tested for their anti- 14.52. Found: C, 57.90; H, 5.30; N, 14.51.
cancer activity against colon (HCT116) and breast (T47D)
3-Mercapto-3-phenylamino-2-(piperidine-1-carbonyl)-acry-
cancer cell lines. It was concluded that the 5-arylidene moiety lonitrile (2b)41): Yellow crystals, 75% yield, mp 160–162°C.
is essential for the antitumor activity, and in most cases, the 1H-NMR (CDCl3, 300MHz) δ: 1.61–1.74 (6H, m, piperidine
piperidine derivatives exhibited better activity than morpho- protons), 3.57–3.76 (4H, m, (CH2)2N piperidine protons), 5.34
line analogues. Compound 4g was the most active compound (1H, s, NH exch. D2O), 7.24–7.78 (m, 5H, aromatic H), 10.54
against colon HCT116 with IC50=0.00586mM compared with (1H, s, SH exch. D2O). IR (KBr) cm−1: 3237 (NH), 3063–3017
Doxorubicin (IC50=0.00686mM) and 4c had the highest activ- (aromatic CH), 2990–2862 (aliphatic CH), 2172 (C≡N), 1650
ity against breast T47D with IC50=0.00646mM compared with (C=O), 1632 (bending NH), 1593–1535 (C=C). Anal. Calcd
Doxorubicin (IC50=0.01407mM). QSAR study confirmed the for C15H17N3OS (287.39): C, 62.69; H, 5.96; N, 14.62. Found:
obtained antitumor results, where the three pharmacophoric C, 62.83; H, 6.11; N, 15.08.
features for HCT116 activity were two hydrophobic sites and
General Procedure for the Preparation of (3a,b) A so-
a hydrogen bond acceptor. Classical QSAR studies of HCT116 lution of 2a,b (3mmol) and TEA (0.61g, 0.84mL, 6mmol) in
(colon) cancer cell lines delivered Eq. 1 of three descriptors dry THF (15mL) was cooled to −5°C in an ice/salt bath and
with R2=0.726. The most important descriptor in the equa- a solution of chloroacetyl chloride (0.34g, 0.24mL, 3mmol) in
tion was the fit value derived from mapping of the synthe- dry THF (5mL) was added dropwise. The mixture was stirred
sized analogues into the generated pharmacophore. The other overnight at room temperature, and the formed precipitate was
controlling descriptors were the molecular surface area and filtered and dried. The residue was suspended in water, stirred
Jurs_RNCG. for 5min, and filtered. The crude product was crystallized
External validation of the established QSAR models utiliz- from ethanol.
ing two of our synthesized analogues exhibiting promising
3-Morpholin-4-yl-3-oxo-2-(4-oxo-3-phenyl-thiazolidin-2-
(4g) and mild (5c) antitumor properties, revealed good agree- ylidene)propionitrile (3a): White crystals, 80% yield, mp
ment between the experimental and the calculated data. It can 188–190°C. 1H-NMR (CDCl3, 300MHz) δ: 3.56 (4H, t,
be concluded that combination of 3D-pharmacophore model- J=4.2Hz, (CH2)2N morpholine protons), 3.67 (4H, t, J=4.2Hz,
ing and QSAR provides an effective technique for understand- (CH2)2O morpholine protons), 3.90 (2H, s, CH2), 7.28–7.59
ing the observed pharmacological properties and thus could be (5H, m, aromatic H). IR (KBr) cm−1: 3059 (aromatic CH),
adopted for developing effective lead structures.
2974–2855 (aliphatic CH), 2199 (C≡N), 1736 (C=O thia-
zolidinone), 1636 (C=O), 1551–1512 (C=C). Anal. Calcd for
C16H15N3O3S (329.38): C, 58.35; H, 4.59; N, 12.76. Found: C,
Experimental
Chemistry Melting points were measured with an Elec- 58.13; H, 4.69; N, 12.93.
trothermal Stuart SMP3 digital melting point apparatus. IR
3-Oxo-2-(4-oxo-3-phenyl-thiazolidin-2-ylidene)-3-piperidin-
spectra (KBr disc) were recorded on Shimadzu FT-IR 8400S 1-yl-propionitrile (3b): White crystals, 86% yield, mp
infrared spectrophotometer. NMR spectra were recorded on a 216–218°C. 1H-NMR (CDCl3, 300MHz) δ: 1.55–1.62 (6H,
Varian Mercury VX 300 spectrometer (1H: 300, 13C: 75MHz) m, piperidine protons), 3.49–3.51 (4H, m, (CH2)2N piperidine
using TMS as an internal standard and on JOEL (Eclipse) protons), 3.90 (2H, s, CH2), 7.26–7.61 (5H, m, aromatic H).
400 (1H: 400, 13C: 100MHz). Mass spectra were measured on IR (KBr) cm−1: 3050 (aromatic CH), 2993–2855 (aliphatic
a Shimadzu GCMS-QP2010 Plus spectrometer (EI, 70eV). CH), 2191 (C≡N), 1724 (C=O thiazolidinone), 1636 (C=O),
Elemental analyses were carried out at the Microanalytical 1558-1497 (C=C). Anal. Calcd for C17H17N3O2S (327.41): C,
center, Faculty of Science, Cairo University, Egypt, and at the 62.37; H, 5.23; N, 12.83. Found: C, 62.49; H, 5.19; N, 13.14.
Regional center for mycology and biotechnology, Al-Azhar
General Procedure for the Preparation of (4a–j) A
University, Egypt. Reagents and solvents used in synthesis solution of 3a,b (1mmol), the appropriate aldehyde (1mmol)
were purchased from Sigma-Aldrich. Compounds 1a,b were and TEA (0.10g, 0.14mL, 1mmol) in DMF (5mL) was stirred
prepared according to the reported procedures.22)
at 70°C for 6h. The mixture was cooled and the obtained
General Procedure for Preparation of (2a,b) To an ice precipitate was filtered, washed and crystallized from ethanol.
cold mixture of 1a,b (5mmol) and fine powdered potassium 2-(5-Benzylidene-4-oxo-3-phenyl-thiazolidin-2-ylidene)-
hydroxide (0.28g, 5mmol) in dry THF (25mL) was added 3-morpholin-4-yl-3-oxo-propionitrile (4a): Yellow crystals,
1
dropwise a solution of phenylisothiocyanate (0.68g, 0.61mL, 30% yield, mp 310–312°C (dec.). H-NMR (CDCl3, 300MHz)
5mmol) in dry THF (10mL). The mixture was stirred at room δ: 3.61 (4H, t, J= 4.2Hz, (CH2)2N morpholine protons), 3.71
temperature for 48h, and it was poured on water (200mL) (4H, t, J=4.2Hz, (CH2)2O morpholine protons), 7.27–7.66
with stirring. The obtained solution was neutralized with (10H, m, aromatic H), 7.86 (1H, s, =CH). 13C-NMR (DMSO-
dilute HCl and the obtained precipitate was filtered, washed d6, 75MHz) δ: 43.8 [(CH2)2N of morpholine], 66.1 [(CH2)2O of
with water and crystallized from ethanol to obtain 2a,b in a morpholine], 77.8 (C=C–CN), 112.0 (CN), 118.0, 122.1, 122.8,
pure form.
124.6, 125.8, 127.6 (aromatic carbons), 129.4 (C of thiazolidi-
3-Mercapto-2-(morpholine-4-carbonyl)-3-phenylamino-ac- none attached to olefinic CH), 130.5 (aromatic C attached to N
rylonitrile (2a): Yellow crystals, 61% yield, mp 122–124°C. of the 4-thiazolidinone ring), 132.1 (C=CH), 133.9 (aromatic C

September 2012
1203
attached to olefinic CH), 161.7 (C=O of thiazolidinone), 162.2 morpholine protons), 4.00 (3H, s, OCH3), 7.03–7.62 (8H, m, ar-
(C=O), 166.3 (C=C–CN). IR (KBr) cm−1: 3050–3028 (aro- omatic H), 7.78 (1H, s, OH exch. D2O), 8.03 (1H, s, =CH). IR
matic CH), 2967–2859 (aliphatic CH), 2191 (C≡N), 1717 (C= (KBr) cm−1: 3291 (OH), 3067–3021 (aromatic CH), 2963–2855
O thiazolidinone), 1647 (C=O), 1547–1493 (C=C). Anal. Calcd (aliphatic CH), 2195 (C≡N), 1709 (C=O thiazolidinone),
for C23H19N3O3S (417.49): C, 66.17; H, 4.59; N, 10.06. Found: 1663 (C=O), 1620–1516 (C=C). Anal. Calcd for C24H21N3O5S
C, 66.33; H, 4.62; N, 10.32.
(463.52): C, 62.19; H, 4.57; N, 9.07. Found: C, 62.42; H, 4.69;
2-[5-(4-Methoxybenzylidene)-4-oxo-3-phenyl-thiazolidin- N, 9.31.
2-ylidene]-3-morpholin-4-yl-3-oxo-propionitrile (4b): Yellow
crystals, 35% yield, mp 295–297°C (dec.). H-NMR (CDCl3, 3-oxo-3-piperidin-1-yl-propionitrile (4f): Yellow crystals, 40%
2-(5-Benzylidene-4-oxo-3-phenyl-thiazolidin-2-ylidene)-
1
300MHz) δ: 3.59–3.61 (4H, m, (CH2)2N morpholine protons), yield, mp 272–274°C (dec.). 1H-NMR (CDCl3, 300MHz) δ:
3.69–3.71 (4H, m, (CH2)2O morpholine protons), 3.90 (3H, s, 1.64 (6H, brs, piperidine protons), 3.55 (4H, brs, (CH2)2N pi-
OCH3), 7.02 (2H, d, J=8.7Hz, protons o-OCH3), 7.35–7.63 peridine protons), 7.36–7.65 (10H, m, aromatic H), 7.83 (1H, s,
(7H, m, aromatic H), 7.81 (1H, s, =CH). 13C-NMR (DMSO-d6, =CH). IR (KBr) cm−1: 3040–3028 (aromatic CH), 2932–2847
100MHz) δ: 45.3 [(CH2)2N of morpholine], 55.5 (OCH3), 66.0 (aliphatic CH), 2195 (C≡N), 1717 (C=O thiazolidinone),
[(CH2)2O of morpholine], 69.7 (C=C–CN), 115.1 (CN), 119.7, 1655 (C=O), 1597–1493 (C=C). Anal. Calcd for C24H21N3O2S
120.0, 121.9, 122.5, 123.6, 124.7 (aromatic carbons), 126.9 (aro- (415.52): C, 69.38; H, 5.09; N, 10.11. Found: C, 69.48; H, 5.18;
matic C attached to olefinic CH), 129.4 (C of thiazolidinone N, 10.43.
attached to olefinic CH), 132.4 (aromatic C attached to N of
2-[5-(4-Methoxybenzylidene)-4-oxo-3-phenyl-thiazolidin-
the 4-thiazolidinone ring), 134.9 (C=CH), 153.1 (aromatic 2-ylidene]-3-oxo-3-piperidin-1-yl-propionitrile (4g): Yellow
1
C attached to OCH3 group), 162.2 (C=O of thiazolidinone), crystals, 45% yield, mp 268–270°C (dec.). H-NMR (CDCl3,
163.0 (C=O), 166.1 (C=C–CN). IR (KBr) cm−1: 3065–3020 300MHz) δ: 1.64 (6H, brs, piperidine protons), 3.55 (4H, brs,
(aromatic CH), 2965–2865 (aliphatic CH), 2191 (C≡N), 1713 (CH2)2N piperidine protons), 3.89 (3H, s, OCH3), 7.01 (2H, d,
(C=O thiazolidinone), 1655 (C=O), 1589–1508 (C=C). MS J=8.7Hz, o-OCH3), 7.35–7.62 (7H, m, aromatic H), 7.78 (1H,
m/z: 448.35 (M+), 449.30 (M++1), 450.30 (M++2), 361, 334.20, s, =CH). 13C-NMR (DMSO-d6, 75MHz) δ: 23.8 (C3,C5 of
164.10, 77.10. Anal. Calcd for C24H21N3O4S (447.52): C, 64.41; piperidine), 25.3 (C4 of piperidine), 44 [C2,C6 of piperidine],
H, 4.73; N, 9.39. Found: C, 64.59; H, 4.80; N, 9.56.
2-[5-(4-Chlorobenzylidene)-4-oxo-3-phenyl-thiazolidin- 125.0, 126.1, 129.3 (aromatic carbons), 127.0 (aromatic C at-
55.5 (OCH3), 76.0 (C=C–CN), 115.1 (CN), 118.7, 120.0, 121.9,
2-ylidene]-3-morpholin-4-yl-3-oxo-propionitrile (4c): Yellow tached to olefinic CH), 129.4 (C of thiazolidinone attached to
1
crystals, 32% yield, mp 334–336°C (dec.). H-NMR (CDCl3, olefinic CH), 132.3 (aromatic C attached to N of the 4-thia-
300MHz) δ: 3.61 (4H, t, J=4.2Hz, (CH2)2N morpholine pro- zolidinone ring), 133.9 (C=CH), 159.1 (aromatic C attached
tons), 3.71 (4H, t, J=4.2Hz, (CH2)2O morpholine protons), to OCH3 group), 160.2 (C=O of thiazolidinone), 164.0 (C=
7.35–7.63 (9H, m, aromatic H), 7.79 (1H, s, =CH). IR (KBr) O), 166.0 (C=C–CN). IR (KBr) cm−1: 3060–3020 (aromatic
cm−1: 3051 (aromatic CH), 2982–2855 (aliphatic CH), 2191 CH), 2928–2839 (aliphatic CH), 2195 (C≡N), 1713 (C=O thia-
(C≡N), 1701 (C=O thiazolidinone), 1647 (C=O), 1593–1543 zolidinone), 1636 (C=O), 1593–1508 (C=C). Anal. Calcd for
(C=C). MS m/z: 451.30 (M+), 452.30 (M++1), 453.30 (M++2), C25H23N3O3S (445.54): C, 67.40; H, 5.20; N, 9.43. Found: C,
365.20 (42.93), 367.20 (13.38), 168.05 (41.75), 170.05 (18.77), 67.30; H, 5.47; N, 9.81.
77.10 (100). Anal. Calcd for C23H18ClN3O3S (451.94): C, 61.13;
2-[5-(4-Chlorobenzylidene)-4-oxo-3-phenyl-thiazolidin-
H, 4.01; N, 9.30. Found: C, 61.41; H, 4.13; N, 9.68.
2-[5-(2-Hydroxybenzylidene)-4-oxo-3-phenyl-thiazolidin- crystals, 35% yield, mp 319–321°C (dec.). H-NMR (DMSO-
2-ylidene]-3-oxo-3-piperidin-1-yl-propionitrile (4h): Yellow
1
2-ylidene]-3-morpholin-4-yl-3-oxo-propionitrile (4d): Orange d6, 300MHz) δ: 1.49–1.57 (6H, m, piperidine protons), 3.43
1
crystals, 30% yield, mp 248–250°C (dec.). H-NMR (DMSO- (4H, brs, (CH2)2N piperidine protons), 7.56–7.72 (9H, m, aro-
d6, 300MHz) δ: 3.48–3.57 (8H, m, morpholine protons), matic H), 7.79 (1H, s, =CH). 13C-NMR (DMSO-d6, 100MHz)
6.99–7.55 (9H, m, aromatic H), 8.00 (1H, s, =CH), 10.62 (1H, δ: 24.0 (C3,C5 of piperidine), 25.4 (C4 of piperidine), 44.1
s, OH exch. D2O). 13C-NMR (DMSO-d6, 75MHz) δ: 46.8 [C2,C6 of piperidine], 69.7 (C=C–CN), 113.1 (CN), 118.7,
[(CH2)2N of morpholine], 66.0 [(CH2)2O of morpholine], 78.0 120.0, 121.9, 123.5, 125.1, 126.0, 127.2 (aromatic carbons),
(C=C–CN), 112.8 (CN), 116.2 (aromatic C attached to olefinic 129.5 (C of thiazolidinone attached to olefinic CH), 131.7
CH), 118.7, 119.8, 120.2, 120.9, 127.8, 128.9, 129.0 (aromatic (aromatic C attached to N of the 4-thiazolidinone ring), 133.7
carbons), 130.4 (C of thiazolidinone attached to olefinic CH), (C=CH), 134.4 (aromatic C attached to olefinic CH), 134.7
132.4 (aromatic C attached to N of the 4-thiazolidinone ring), (aromatic C attached to Cl), 153.6 (C=O of thiazolidinone),
134.8 (C=CH), 157.2 (aromatic C attached to OH), 159.8 155.0 (C=O), 166.4 (C=C–CN). IR (KBr) cm−1: 3059–3028
(C=O of thiazolidinone), 162.2 (C=O), 166.1 (C=C–CN). (aromatic CH), 2928–2855 (aliphatic CH), 2195 (C≡N), 1713
IR (KBr) cm−1: 3445 (OH), 3028 (aromatic CH), 2965–2865 (C=O thiazolidinone), 1636 (C=O), 1585–1489 (C=C). MS
(aliphatic CH), 2195 (C≡N), 1713 (C=O thiazolidinone), m/z: 449.30 (M+), 450.30 (M++1), 451.30 (M++2), 338.20,
1636 (C=O), 1585–1520 (C=C). Anal. Calcd for C23H19N3O4S 168.05, 77.10. Anal. Calcd for C24H21ClN3O2S (449.96): C,
(433.49): C, 63.73; H, 4.42; N, 9.69. Found: C, 63.89; H, 4.36; 64.06; H, 4.48; N, 9.34. Found: C, 64.19; H, 4.71; N, 9.62.
N, 9.97.
2-[5-(2-Hydroxybenzylidene)-4-oxo-3-phenyl-thiazolidin-
2-[5-(4-Hydroxy-3-methoxy-benzylidene)-4-oxo-3-phenyl- 2-ylidene]-3-oxo-3-piperidin-1-yl-propionitrile (4i): Yellow
1
thiazolidin-2-ylidene]-3-morpholin-4-yl-3-oxo-propionitrile crystals, 30% yield, mp 235–237°C (dec.). H-NMR (DMSO-
1
(4e): Yellow crystals, 30% yield, mp 248–250°C (dec.). H- d6, 300MHz): δ 1.48–1.67 (6H, m, piperidine protons),
NMR (CDCl3, 300MHz) δ: 3.60 (4H, t, J=4.5Hz, (CH2)2N 3.43–3.59 (4H, m, (CH2)2N piperidine protons), 6.99–8.02
morpholine protons), 3.71 (4H, t, J=4.5Hz, (CH2)2O (9H, m, aromatic H), 8.16 (1H, s, =CH), 10.90 (1H, s, OH

1204
Vol. 60, No. 9
exch. D2O). IR (KBr) cm−1: 3350 (OH), 3051 (aromatic CH),
3-Morpholin-4-yl-2-(5-morpholin-4-ylmethyl-4-oxo-3-phe-
2932–2851 (aliphatic CH), 2191 (C≡N), 1701 (C=O thia- nyl-thiazolidin-2-ylidene)-3-oxo-propionitrile (5c): Orange
1
zolidinone), 1636 (C=O), 1597–1493 (C=C). Anal. Calcd for crystals, 69% yield, mp 230–232°C (dec.). H-NMR (CDCl3,
C24H21N3O3S (431.52): C, 66.80; H, 4.91; N, 9.74. Found: C, 400MHz) δ: 2.59 (4H, brs, (CH2)2N of morpholine), 2.76 (4H,
66.90; H, 5.03; N, 9.93.
brs, (CH2)2N–CO of morpholine), 2.94–3.04 (2H, m, CH2),
2-[5-(4-Hydroxy-3-methoxybenzylidene)-4-oxo-3-phenyl- 3.49–3.66 (9H, m, 8H (CH2)2O of 2 morpholine rings+CH of
thiazolidin-2-ylidene]-3-oxo-3-piperidin-1-yl-propionitrile (4j): thiazolidinone), 7.48–7.92 (5H, m, aromatic protons). 13C-NMR
Yellow crystals, 33% yield, mp 272–274°C (dec.). 1H-NMR (CDCl3, 100MHz) δ: 46.0 [(CH2)2N morpholine], 52.1 (C of
(DMSO-d6, 300MHz) δ: 1.49–1.57 (6H, m, piperidine pro- thiazolidine attached to CH2), 55.8 [(CH2)2N morpholine],
tons), 3.30 (1H, brs, OH exch. D2O), 3.44 (4H, brs, (CH2)2N 58.3 (CH2), 66.5 [(CH2)2O morpholine], 67.4 [(CH2)2O mor-
piperidine protons), 3.84 (3H, s, OCH3), 6.98–7.71 (8H, m, aro- pholine], 69.2 (C=C–CN), 113.3 (CN), 120.8, 123.3, 124.2,
matic H), 7.79 (1H, s, =CH). 13C-NMR (DMSO-d6, 100MHz) 125.8, 130.4 (aromatic carbons), 134.2 (aromatic C attached to
δ: 23.9 (C3,C5 of piperidine), 25.3 (C4 of piperidine), 44.3 N of thiazolidinone), 159.8 (C=O), 164.5 (C=O of thiazolidi-
[C2,C6 of piperidine], 55.9 (OCH3), 78.3 (C=C–CN), 113.0 none), 178.3 (C=C–CN). IR (KBr) cm−1: 3060 (aromatic CH),
(CN), 116.5, 117.4, 123.8, 124.5, 126.9 (aromatic carbons), 2963–2855 (aliphatic CH), 2199 (C≡N), 1732 (C=O thiazolidi-
128.8 (C of thiazolidinone attached to olefinic CH), 129.3 none), 1628 (C=O), 1566–1493 (C=C). MS m/z: 428 (M+), 341,
(aromatic C attached to olefinic CH), 133.3 (aromatic C at- 242, 176. Anal. Calcd for C21H24N4O4S (428.51): C, 58.86; H,
tached to N of the 4-thiazolidinone ring), 135.1 (C=CH), 148.1 5.65; N, 13.07. Found: C, 58.95; H, 5.71; N, 13.32.
(aromatic C attached to OH), 150.2 (aromatic C attached to
2-[5-(4-Methylpiperazin-1-ylmethyl)-4-oxo-3-phenyl-thia-
OCH3), 158.8 (C=O of thiazolidinone), 162.8 (C=O), 166.2 zolidin-2-ylidene]-3-morpholin-4-yl-3-oxo-propionitrile (5d):
1
(C=C–CN). IR (KBr) cm−1: 3526 (OH), 3062–3032 (aromatic Yellow crystals, 43% yield, mp 222–224°C (dec.). H-NMR
CH), 2940–2851 (aliphatic CH), 2195 (C≡N), 1717 (C=O thia- (CDCl3, 400MHz) δ: 1.86 (3H, s, CH3), 2.25 (4H, brs,
zolidinone), 1663 (C=O), 1616–1516 (C=C). Anal. Calcd for (CH2)2N piperazine), 2.70 (4H, brs, (CH2)2N piperazine), 3.00
C25H23N3O4S (461.54): C, 65.06; H, 5.02; N, 9.10. Found: C, (2H, brs, CH2), 3.50–3.61 (9H, m, 4H (CH2)2N morpholine
65.18; H, 5.18; N, 9.44.
+ 4H (CH2)2O morpholine+CH of thiazolidinone), 7.38–7.72
General Procedure for Preparation of Compounds (5a– (5H, m, aromatic protons). IR (KBr) cm−1: 3060 (aromatic
h) To a mixture of 3a,b (1mmol), formaldehyde (0.10mL, CH), 2963–2801 (aliphatic CH), 2199 (C≡N), 1732 (C=O thia-
37% in water, 1.2mmol) in absolute ethanol (5mL), the ap- zolidinone), 1628 (C=O), 1558–1493 (C=C). Anal. Calcd for
propriate secondary amines (1.2mmol) was added dropwise C22H27N5O3S (441.56): C, 59.84; H, 6.16; N, 15.86. Found: C,
and the mixture was refluxed for 5h. The precipitate obtained 59.93; H, 6.30; N, 16.12.
on cooling was filtered, washed and crystallized from ethanol/
water to obtain 5a–h in a pure form.
3-Oxo-2-(4-oxo-3-phenyl-5-pyrrolidin-1-ylmethyl-thiazoli-
din-2-ylidene)-3-piperidin-1-yl-propionitrile (5e): Yellow crys-
3-Morpholin-4-yl-3-oxo-2-(4-oxo-3-phenyl-5-pyrrolidin-1- tals, 46% yield, mp 184–186°C (dec.). 1H-NMR (CDCl3,
ylmethyl-thiazolidin-2-ylidene)propionitrile (5a): Yellow crys- 400MHz) δ: 1.63–1.85 (10H, m, 4H pyrrolidine protons+6H
tals, 66% yield, mp 226–227°C (dec.). 1H-NMR (CDCl3, piperidine protons), 2.68–2.87 (4H, m, (CH2)2N pyrrolidine
400MHz) δ: 1.76–1.87 (4H, m, pyrrolidine protons), 2.68–2.80 protons), 3.14–3.16 (2H, m, CH2), 3.42–3.55 (4H, m, (CH2)2N
(4H, m, (CH2)2N pyrrolidine protons), 3.14–3.16 (2H, m, CH2), of piperidine), 3.68–3.80 (1H, m, CH of thiazolidinone),
3.46–3.48 (4H, m, (CH2)2N of morpholine), 3.58–3.60 (5H, m, 7.16–7.55 (5H, m, aromatic protons). IR (KBr) cm−1: 3060
(CH2)2O of morpholine+CH of thiazolidinone), 7.14–7.60 (5H, (aromatic CH), 2936–2808 (aliphatic CH), 2199 (C≡N), 1732
m, aromatic protons). 13C-NMR (CDCl3, 100MHz) δ: 24.6 (C=O thiazolidinone), 1628 (C=O), 1558–1493 (C=C). Anal.
[(CH2)2 pyrrolidine], 45.4 [(CH2)2N morpholine], 47.5 (C of Calcd for C22H26N4O2S (410.54): C, 64.36; H, 6.38; N, 13.65.
thiazolidine attached to CH2), 56.5 (CH2), 56.6 [(CH2)2N pyr- Found: C, 64.47; H, 6.41; N, 13.78.
rolidine], 66.5 [(CH2)2O morpholine], 68.8 (C=C–CN), 113.2
3-Oxo-2-(4-oxo-3-phenyl-5-piperidin-1-ylmethyl-thiazolidin-
(CN), 125.0, 129.7, 130.0, 131.2, 131.3 (aromatic carbons), 2-ylidene)-3-piperidin-1-yl-propionitrile (5f): Yellow crystals,
1
134.6 (aromatic C attached to N of thiazolidinone), 161.7 (C= 46% yield, mp 183–185°C (dec.). H-NMR (CDCl3, 400MHz)
O), 163.7 (C=O of thiazolidinone), 177.0 (C=C–CN). IR (KBr) δ: 1.25–1.57 (12H, m, two piperidine rings protons), 2.49–2.67
cm−1: 3067 (aromatic CH), 2967–2859 (aliphatic CH), 2203 (4H, m, (CH2)2N of piperidine), 2.90–3.00 (2H, m, CH2),
(C≡N), 1732 (C=O thiazolidinone), 1628 (C=O), 1558–1493 3.45–3.56 (4H, m, (CH2)2N of piperidine), 4.20–4.22 (1H, m,
(C=C). Anal. Calcd for C21H24N4O3S (412.51): C, 61.15; H, CH of thiazolidinone), 7.16–7.53 (5H, m, aromatic protons).
5.86; N, 13.58. Found: C, 61.19; H, 5.92; N, 13.74.
13C-NMR (CDCl3, 100MHz) δ: 24.6 (C3,C5 of piperidine),
3-Morpholin-4-yl-3-oxo-2-(4-oxo-3-phenyl-5-piperidin-1- 25.3 (C4 of piperidine), 25.8 (C3,C4,C5 of piperidine), 40.5
ylmethyl-thiazolidin-2-ylidene)propionitrile (5b): Yellow crys- [C2,C6 piperidine], 51.2 (C of thiazolidinone attached to CH2),
tals, 45% yield, mp 214–216°C (dec.). 1H-NMR (CDCl3, 51.5 [C2,C6 piperidine], 57.0 (CH2), 70.7 (C=C–CN), 114.2
400MHz) δ: 1.50–1.88 (6H, m, piperidine protons), 2.5–2.7 (CN), 120.0, 123.3, 124.4, 128.9, 129.4 (aromatic carbons),
(4H, m, (CH2)2N of piperidine), 2.90–3.00 (2H, m, CH2), 133.3 (aromatic C attached to N of thiazolidinone), 158.0
3.59–3.71 (8H, m, morpholine), 4.21–4.22 (1H, m, CH of (C=O), 164.0 (C=O of thiazolidinone), 178.8 (C=C–CN);
thiazolidinone), 7.15–7.56 (5H, m, aromatic protons). IR (KBr) IR (KBr) cm−1: 3050 (aromatic CH), 2936–2855 (aliphatic
cm−1: 3060 (aromatic CH), 2924–2855 (aliphatic CH), 2199 CH), 2199 (C≡N), 1728 (C=O thiazolidinone), 1628 (C=O),
(C≡N), 1732 (C=O thiazolidinone), 1628 (C=O), 1558–1493 1570–1493 (C=C). Anal. Calcd for C23H28N4O2S (424.57): C,
(C=C). Anal. Calcd for C22H26N4O3S (426.54): C, 61.95; H, 65.07; H, 6.65; N, 13.20. Found: C, 65.14; H, 6.73; N, 13.51.
6.14; N, 13.14. Found: C, 62.08; H, 6.22; N, 13.43.
2-(5-Morpholin-4-ylmethyl-4-oxo-3-phenyl-thiazolidin-

September 2012
1205
2-ylidene)-3-oxo-3-piperidin-1-yl-propionitrile (5g): Orange For X-ray crystallographic studies, compound 6a was recrys-
1
crystals, 50% yield, mp 224–226°C (dec.). H-NMR (CDCl3, tallized as prismatic colourless crystals from ethanol. The
400MHz) δ: 1.28–1.49 (6H, m, piperidine protons), 2.57–2.74 crystallographic data were collected at T=298K on a Kappa
(4H, m, (CH2)2N of morpholine), 2.85–2.95 (2H, m, CH2), CCD Enraf Nonius FR 590 diffractometer using a graphite
3.38–3.40 (4H, m, (CH2)2N of piperidine), 3.65–3.68 (5H, m, monochromator with Mo-Kα radiation (λ=0.71073Å). The
(CH2)2O of morpholine+CH of thiazolidinone), 7.35–7.75 (5H, crystal structures were determined by SIR9243) and refined
m, aromatic protons). IR (KBr) cm−1: 3050 (aromatic CH), by maXus44) (Bruker Nonius, Delft and MacScience, Japan).
2940–2855 (aliphatic CH), 2199 (C≡N), 1732 (C=O thia- Chemical formula C17H17N3O3S, Mr=343.405, monoclinic,
zolidinone), 1624 (C=O), 1570–1493 (C=C). Anal. Calcd for crystallizes in space group P21/c, cell lengths “a=8.2306 (2),
C22H26N4O3S (426.54): C, 61.95; H, 6.14; N, 13.14. Found: C, b=17.5581 (4), c=12.8758 (4)Å,” cell angles “α=90.00°, β=12
62.04; H, 6.12; N, 13.39.
(18)×101 °, γ=90.00°,” V=1617.86 (13)Å3, Z=4, Dc=1.392mg/
2-[5-(4-Methylpiperazin-1-ylmethyl)-4-oxo-3-phenyl-thiazo- m3, θ values 2.910–27.485°, absorption coefficient μ (Mo-Kα)=
lidin-2-ylidene]-3-oxo-3-piperidin-1-yl-propionitrile (5h): Off 0.22mm−1, F(000)=720. The unique reflections measured 4148
white crystals, 46% yield, mp 198–200°C (dec.). 1H-NMR of which 2406 reflections with threshold expression I>3σ(I)
(CDCl3, 400MHz) δ: 1.45–1.65 (6H, m, piperidine protons), were used in the structural analysis. Convergence for 217
1.95 (3H, s, CH3), 2.25–2.30 (4H, m, (CH2)2N of piperazine), variable parameters by least-squares refinement on F2 with
2.60–2.65 (4H, m, (CH2)2N of piperazine), 2.90–3.10 (2H, m, w=1/[σ2(Fo2)+0.10000Fo2]. The final agreement factors were
CH2), 3.40–3.47 (4H, m, (CH2)2N of piperidine), 3.75–3.77 R=0.044 and wR=0.175 with a goodness-of-fit of 1.119.
(1H, m, CH of thiazolidinone), 7.33–7.76 (5H, m, aromatic
Antitumor Activity The potential cytotoxicity of the
protons). 13C-NMR (CDCl3, 100MHz) δ: 24.5 (2C of pi- tested compounds was evaluated using the method of Skehan
peridine), 25.7 (C of piperidine), 43.3 (CH3), 45.8 [(CH2)2N et al.15,29–35) Cells were plated in 96-multiwell plate (104 cells/
piperidine], 48.5 (C of thiazolidinone attached to CH2), 52.1 well) for 24h before treatment with the prepared compounds
[(CH2)2N piprazine], 56.3 [(CH2)2N piprazine], 59.2 (CH2), to allow the attachment of cells to the wall of the plate.
69.2 (C=C–CN), 113.8 (CN), 120.0, 125.0, 128.9, 129.0, 129.9 The tested compounds were dissolved in dimethylsulfoxide
(aromatic carbons), 135.0 (aromatic C attached to N of thia- (DMSO) and diluted 1000-fold in the assay. Concentrations 0,
zolidinone), 157.1 (C=O), 163.3 (C=O of thiazolidinone), 177.9 5, 12.5, 25, and 50µg/mL of the tested compounds were added
(C=C–CN). IR (KBr) cm−1: 3050 (aromatic CH), 2940–2801 to the cell monolayer. The monolayer cells were incubated
(aliphatic CH), 2199 (C≡N), 1732 (C=O thiazolidinone), 1628 with the compounds for 48h at 37°C, in atmosphere of 5%
(C=O), 1570–1493 (C=C). MS m/z: 439.30 (M+), 327, 211, 177. CO2. After 48h, the cells were fixed, washed and stained with
Anal. Calcd for C23H29N5O2S (439.58): C, 62.84; H, 6.65; N, Sulfo-Rhodamine-B stain (SRB). Excess stain was washed
15.93. Found: C, 62.78; H, 6.70; N, 16.18.
General Procedure for the Preparation of (6a,b)
with acetic acid. The attached stain was recovered with Tris
EDTA buffer. Cell survival and drug activity were determined
A
solution of 2a,b (6mmol) and TEA (1.22g, 1.65mL, 12mmol) by measuring color intensity using an enzyme-linked immu-
in dry THF (15mL) was cooled to −5°C in an ice/salt bath nosorbent assay (ELISA) reader. Data are representative of the
and a solution of 3-chloroapropionyl chloride (0.76g, 0.60mL, individual experiment, performed in three replicates for each
6mmol) in dry THF (10mL) was added dropwise. The mix- individual dose and measured by SRB assay. Control values
ture was stirred overnight at room temperature, and the did not exhibit significant changes compared to the DMSO ve-
formed precipitate was filtered and dried. The residue was hicle. The IC50 was determined by using a program Graph-Pad
suspended in water, stirred for 5min, and filtered. The crude PRISM version 5. Mean and standard error were determined
product was crystallized from ethanol.
by SPSS 11 software.
(2E)
3-Morpholin-4-yl-3-oxo-2-(4-oxo-3-phenyl-[1,3]-
thiazinan-2-ylidene)-propionitrile (6a): White crystals, 71%
Acknowledgement The authors are thankful to Dr. Nasser
1
yield, mp 196–198°C. H-NMR (CDCl3) δ: 2.95–3.06 (m, 2H, S. M. Ismail, Pharmaceutical Chemistry Department, Faculty
COCH2CH2S), 3.14–3.16 (m, 2H, COCH2CH2S), 3.27–3.29 (m, of Pharmacy, Ain Shams University, for his assistance in per-
4H, morpholinyl (CH2)2 N), 3.35–3.40 (m, 4H, morpholinyl forming the QSAR study.
(CH2)2 O), 7.21–7.50 (m, 5H, aromatic protons). IR (KBr)
cm−1: 3040 (aromatic CH), 2982–2862 (aliphatic CH), 2199
(C≡N), 1713 (C=O thiazinanone), 1643 (C=O), 1531–1490
(C=C). Anal. Calcd for C17H17N3O3S (343.41): C, 59.46; H,
4.99; N, 12.24. Found: C, 59.06; H, 5.29; N, 12.39.
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