Novel 1,3,4-heterodiazole analogues: Synthesis and in-vitro antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2011.11.013

The synthesis of some new heterodiazole and their annulated imidazo[2,1-b]1,3,4-oxa/thiadiazolone 6a-d, 7a-d; 1,3,4-oxa or thiadiazole[3,2-a]pyrimidine diamine 8a-d and 1,3,4-oxa or thiadiazole-3- piperidino-1-propamide 11a,b derivatives have been describ

Full text of DOI:10.1016/j.ejmech.2011.11.013

European Journal of Medicinal Chemistry 47 (2012) 445e451
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 1,3,4-heterodiazole analogues: Synthesis and in-vitro antitumor activity
,
c
Azza T. Taher a, Hanan H. Georgey b , Hussein I. El-Subbagh
*
a Departments of Organic Chemistry, Faculty of Pharmacy, Cairo University, P.O.Box 11562 Cairo, Egypt
b Department of pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, El-Kasr El-Eini Street, P.O.Box 11562Cairo, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences & Pharmaceutical Industries, Future University, 12311 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of some new heterodiazole and their annulated imidazo[2,1-b]1,3,4-oxa/thiadiazolone 6a
ed, 7aed; 1,3,4-oxa or thiadiazole[3,2-a]pyrimidine diamine 8aed and 1,3,4-oxa or thiadiazole-3-
piperidino-1-propamide 11a,b derivatives have been described. The obtained compounds were evalu-
Received 29 September 2011
Received in revised form
3 November 2011
Accepted 5 November 2011
Available online 15 November 2011
ated for their in-vitro antitumor activity. A single dose (10 mM) of the test compounds were used in the
full National Cancer Institute (NCI) 60 cell lines panel assay. Compounds 6c and 6d displayed appreciable
anticancer activity against leukemia, non-small cell lung, CNS and showed moderate activity against
colon, melanoma, and breast cancer cells lines. Compound 6c possessed remarkable broad-spectrum
antitumor activity which almost 4 fold more active than the known drug 5-FU with GI50, TGI, and
Keywords:
Heterodiazole
1,3,4-Oxadiazole
Thiadiazole,[3,2-a]pyimidine
Antitumor activity
LC50 values of 6.0, 17.4, and 55.1 mM, respectively.
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
[2,1-b][1,3,4]thiadiazole analogues (4) have been reported as
potential antitumor agents [22,23], Fig. 1. In view of above
mentioned facts and as an attempt to obtain new potent antitumor
agents with good bioavailability and low toxicity, we would like to
describe herein the synthesis and anticancer activity of a new series
of N-substituted 2-amino-5-aryl-1,3,4-oxa or thiadiazole and 7,7a-
dihydro-imidazo[2,1-b][1,3,4]oxa or thiadiazole analogues.
Cancer is continuing to be a major health problem in developing
as well as undeveloped countries [1e5]. The great cancer incidence
worldwide increases the search for new, safer and efficient anti-
cancer agents, aiming the prevention or the cure of this illness. In
spite of all the efforts to combat cancer, the success of the treatment
of certain types of tumors has shown little progress due to their
aggressiveness and the mechanisms of malignant cell metastasis.
Moreover, there has been wide interest in compounds containing
1,3,4-oxa/thiadiazole scaffold because of their unique chemical
structure and the broad-spectrum of their biological properties
[6e16]. They displayed interesting in-vitro and in-vivo antitumor
activity [17e19]. Although many classes of drugs are being used for
the treatment of cancer, the need for more potent selective anti-
tumor agents is still not precluded.
Literature survey revealed that, N-substituted 2-amino-5-aryl-
1,3,4-thiadiazole analogues (1) specially its phenyl substituent,
exhibit significant in-vitro antiproliferative activity [20]. Moreover,
Levamisole (2) appears to be the most effective in patient’s drug
against small tumor burdens as it acts by stimulating the respon-
siveness of lymphocytes toward tumor antigens [21]. The imidazo
[2,1-b]thiazole derivatives of Levamisole (3) and the other imidazo
2. Results and discussion
2.1. Chemistry
The target compounds 6e8, 10, and 11 were synthesized as
outlined in Schemes 1 and 2. 5-Aryl-1,3,4-oxa or thiadiazol-2-
amine exemplified in this manuscript (5aed) were prepared
according to reported methods [24,25]. Acylation of the amino
group of 5aed with chloroacetyl chloride [26] or oxalyl chloride in
dry benzene in presence of potassium carbonate followed by
cyclization on N3 gave the targets (6aed) and (7aed), respectively.
Moreover, reaction of 5aed with malononitrile in absolute ethanol
and calculated amount of triethylamine [27] afforded 2-phenyl-
8aH-[1,3,4]oxa or thiadiazolo[3,2-a]pyrimidine-5,7-diamine 8aed,
Scheme 1. On the other hand, reaction of 5a,b with 3-
chloropropionyl chloride using different reaction conditions yiel-
ded 3-chloro-N-(5-(4-chlorophenyl)-1,3,4-oxa or thiadiazol-2-yl)
propanamide (10a,b) rather than the expected cyclized product
tetrahydro[1,3,4]oxa or thiadiazolo[3,2-a]pyrimidine-7-one (9).
* Corresponding author. Tel.: þ201275600699; fax: þ2023628426.
E-mail address: hanan-hanna@hotmail.com (H.H. Georgey).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.11.013

446
A.T. Taher et al. / European Journal of Medicinal Chemistry 47 (2012) 445e451
Regarding the activity toward individual cell lines; compound
6c and 6d showed selective potency against leukemia cell line
CCRF-CEM with GI values of 88.2 and 62.5%, respectively. Mean-
while, leukemia cell line RPMI-8226; non-small cell lung EKVX;
CNS cancer SF-295, SNB-75, and U251 proved to be selectively
sensitive to 6c with GI values of 72.2, 94.5, 90.6, 88.3 and 58.3%,
respectively. In addition, compounds 6c and 6d proved lethal to the
melanoma cell line LOX IMVI, ovarian cancer IGORV1, renal cancer
cell lines CAKI-1 and UO-31. The same was observed in case of 6c
toward the melanoma cell line MALME-3M. With regard to broad-
spectrum antitumor activity; close examination of the data pre-
sented in Table 1, revealed that compounds 6c and 6d are the most
active members of this study, showing effectiveness toward
numerous cell lines belong to different tumor subpanels. The same
analogy indicated that 8a, 8b and 11d possess moderate antitumor
activity. Compounds 7b, 7d and 11a posses’ selective potency
toward leukemia cell lines, while 6a posses’ selectivity toward renal
cancer cell lines. Compound 6c passed the primary anticancer assay
Fig. 1. Structures of some literature active antitumor agents.
Meanwhile, reaction of 10a,b with piperidine in dry acetonitrile
[28] afforded 11a,b (Scheme 2). The structures of the obtained
compounds was established through spectroscopic (IR, 1H NMR,
Mass) as well as elemental analyses data.
at an arbitrary concentration of 100
mM. Consequently, this active
compound was carried over and tested against a panel of 60
different tumor cell lines at a 5-log dose range [29e32]. Three
response parameters, GI50, TGI, and LC50 were calculated for each
cell line, using the known drug 5-Fluorouracil (5-FU) as a positive
control. Compound 6c is almost four fold more active than the
positive control 5-FU, with GI50, TGI, and LC50 values of 6.0,17.4, and
2.2. Preliminary in-vitro antitumor screening
55.1 mM, respectively (Fig. 2, Table 2).
The synthesized compounds (6aed, 7aed, 8a,b and 11aed)
were subjected to the NCI’s disease-oriented human cell lines
screening assay to be evaluated for their in-vitro antitumor activity.
2.3. Structure-activity correlation
A single dose (10 mM) of the test compounds were used in the full
NCI 60 cell lines panel assay which includes nine tumor subpanels
namely; leukemia, non-small cell lung, colon, CNS, melanoma,
ovarian, renal, prostate, and breast cancer cells [29e32]. The data
reported as mean-graph of the percent growth of the treated cells,
and presented as percentage growth inhibition (GI %). The obtained
results of the tested oxadiazole and thiadiazole analogues 6aed,
7aed, 8a,b, and 11aed, Table 1; showed distinctive potential
pattern of selectivity, as well as broad-spectrum antitumor activity.
Structure-activity correlation, based on the number of cell lines
proved sensitive toward each of the synthesized individual
compounds, revealed that, 7,7a-dihydro-imidazo[2,1-b][1,3,4]thia-
diazol-5(6H)-ones (6c,d) are more active antitumors than their
7,7a-dihydro-imidazo[2,1-b][1,3,4]oxadiazol-5(6H)-one
(6a,b)
counterparts. The introduction of a carbonyl group at position 6- of
compound 6 produced 7,7a-dihydro-imidazo[2,1-b][1,3,4]oxadia-
zole-5,6-diones (7) with a dramatic decrease in the antitumor
Scheme 1. Synthesis of the target compounds 6e8: (i) ClCOCH2Cl, K2CO3, toluene; (ii) ClCOCOCl, toluene; (iii) CH2(CN)2, NEt3, EtOH.

A.T. Taher et al. / European Journal of Medicinal Chemistry 47 (2012) 445e451
447
Scheme 2. Synthesis of the target compounds 10 and 11: (i) ClCO(CH2)2Cl, DMF; (ii) piperidine, K2CO3, acetonitrile.
activity and enhanced selectivity toward leukemia cell lines as
4.1. Chemistry
shown in compounds 7b and 7d. Replacement of the imidazo[2,1-b]
[1,3,4]thiadiazol-5(6H)-one nucleus by [1,3,4]oxadiazolo[3,2-a]
pyrimidine-5,7-diamine produced compound 11 with nearly abol-
ished activity except the 4-chlorophenyl analogue 11d which
retained a moderate broad-spectrum antitumor activity.
4.1.1. General procedure for the preparation of 2-phenylimidazo
[2,1-b][1,3,4]oxa/thiadiazol-6(5H)-one (6aed)
A mixture of 5aed (10 mmol) and chloroacetyl chloride
(15 mmol, 1.69 g, 1.20 mL) was refluxed in dry toluene (30 mL) in
the presence of anhydrous potassium carbonate (1.37 g, 10 mmol)
for 8 h. The reaction mixture was then filtered while hot and the
filtrate was evaporated to dryness under vacuum. The residual mass
was washed with water, dried and crystallized from aqueous
ethanol.
3. Conclusion
Compound
2-phenyl-7,7a-dihydro-imidazo[2,1-b][1,3,4]thia-
diazol-5(6H)-one (6c), Fig. 2; is a broad-spectrum antitumor agent
showing effectiveness toward numerous cell lines belong to
different tumor subpanels. Compound 6c is the most active
member of this study with GI50, TGI, and LC50 values of 6.0,17.4, and
4.1.1.1. 2-Phenylimidazo [2,1-b][1,3,4]oxadiazol-6(5H)-one (6a). Yield:
73%; mp 125e127 ꢂC; IR (KBr, cmꢀ1): 3062 (CH aromatic), 2924, 2854
1
(CH aliphatic), 1693 (C]O); H NMR (DMSO-d6):
d 3.02, 3.15 (s, 2H,
55.1 mM, respectively. The synthesized 7,7a-dihydro-imidazo[2,1-b]
CH2), 7.37e8.57 (m, 5H, AreH); 13C NMR (DMSO): 166.0.155.3, 130.0,
129.3, 129.0, (2) 128.6, (2) 125.6, 65.3; Anal. Calcd. For C10H7N3O2
(201.18): C, 59.70; H, 3.51; N, 20.89. Found: C, 59.40; H, 3.12; N, 20.44.
[1,3,4]thiadiazol-5(6H)-one analogues could be considered as
a useful template for future development to obtain more potent
antitumor agent(s).
4.1.1.2. 2-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]oxadiazol-6(5H)-one
(6b). Yield: 78%; mp 320e322 ꢂC; IR (KBr, cmꢀ1): 3078 (CH
aromatic), 2951, 2858 (CH aliphatic), 1683 (C]O), 833 (CeCl); 1H
4. Experimental
Unless specified all chemicals were of commercial grade, used
without further purification and were obtained from Aldrich
Chemical Co. (Milwaukee, WI). Melting points were carried out by
the open capillary tube method using a Gallenkamp digital melting
point Griffin apparatus 1901 and they are uncorrected. Elemental
Microanalyses were recorded using Heraew and Vario El III (ele-
mntar), CHNS analyzer (Germany) at the Micro Analytical Center,
Faculty of Science, Cairo University. Infrared Spectra were recorded
on Jasco FT.IR plus 460 Japan, and expressed in wave number
(cmꢀ1), using potassium bromide discs. 1H NMR Spectra were
carried out using a Varian Gemini 300 MHz spectrophotometer. The
NMR (DMSO-d6): d 3.44 (s, 2H, CH2), 7.28e7.93 (m, 4H, AreH);
EIMS, m/z: 237 (Mþ þ 2, 8.00%), 236 (Mþ þ 1, 15.21%), 235 (Mþ,
2.68%); Anal. Calcd. For C10H6ClN3O2 (235.63): C, 50.97; H, 2.57; N,
17.83. Found: C, 51.18; H, 2.76; N, 18.20.
4.1.1.3. 2-Phenylimidazo [2,1-b][1,3,4]thiadiazol-6(5H)-one(6c). Yield:
85%; mp 212e214 ꢂC; IR (KBr, cmꢀ1): 3032 (CH aromatic), 2947, 2835
(CH aliphatic), 1705 (C]O); 1H NMR (DMSO-d6):
d 4.48 (s, 2H, CH2),
7.47e7.85 (m, 3H, AreH), 7.94 (dd, 2H, AreH); 13C NMR (DMSO):
165.3, 162.2, 158.0, 130.6, 129.8, (2)129.2, (2)126.8, 42.3; EIMS, m/z:
219 (Mþ þ 2, 0.26%), 218 (Mþ þ 1, 1.50%), 217 (Mþ, 0.24%); Anal. Calcd.
For C10H7N3OS (217.25): C, 55.29; H, 3.25; N, 19.34. Found: C, 55.62;
H, 3.14; N, 19.71.
chemical shifts were expressed in d ppm units using trimethylsilane
as the internal standard. The exchangeable protons were
exchanged by D2O. Mass Spectra was recorded on Shimadzu QP-
2010 plus. All reactions were monitored by thin layer chromatog-
raphy. Silica gel/TLC-cards DC- Alufolien-Kieselgel with fluorescent
indicator 254 nm; layer thickness 0.2 mm; 20 ꢁ 20 cm aluminum
cards were used. Petroleum ether: ethyl acetate (1:1) or (1:2) was
the adopted solvent system. Compounds 5aed [24,25] were
prepared according to reported procedures.
4.1.1.4. 2-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-6(5H)-one
(6d). Yield: 83%; mp 246e248 ꢂC; IR (KBr, cmꢀ1): 3024 (CH
aromatic), 2947, 2831 (CH aliphatic), 1705 (C]O), 759 (CeCl); 1H
NMR (DMSO-d6):
d 4.48 (s, 2H, CH2), 7.62 (dd, 2H, AreH), 8.00 (dd,
2H, AreH); 13C NMR (DMSO): 165.3, 161.0, 158.3, 135.2, 129.3, (2)
128.7, (2)128.5, 42.2; EIMS, m/z: 254 (Mþ þ3, 5.20%), 252 (Mþ þ 1,

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A.T. Taher et al. / European Journal of Medicinal Chemistry 47 (2012) 445e451
Table 1
Percentage growth inhibition (GI %) of in-vitro subpanel tumor cell lines at 10 mM concentration of compounds 6e11. Bold values represents to point out the active compounds
and lethal effect.
Subpanel tumor cell lines
% Growth Inhibition (GI %)a
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
11b
11d
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
e
e
e
e
e
e
e
88.2
21.0
34.8
37.5
72.2
36.3
62.5
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
15.0
13.6
e
e
15.2
12.7
10.6
e
15.6
e
34.1
26.9
20.6
nt
e
e
17.2
e
18.8
24.0
43.5
32.5
e
e
26.0
19.8
e
e
10.4
e
e
e
nt
12.1
12.6
e
e
e
11.1
10.4
19.9
11.9
13.4
Non-small cell lung cancer
A549/ATCC
EKVX
NCI-H226
NCI-H23
e
e
e
e
e
e
e
39.2
94.5
27.7
21.6
22.1
46.5
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
20.4
e
e
e
11.4
14.9
31.0
32.4
e
e
e
e
13.1
e
e
e
e
e
e
e
e
e
NCI-322M
NCI-H522
e
12.5
e
10.8
12.4
e
e
e
e
12.9
20.9
18.9
19.7
14.0
19.9
19.5
Colon cancer
HCT-116
HCT-15
HT29
e
e
e
e
e
e
e
e
42.6
44.8
36.2
21.8
39.9
51.2
31.1
38.3
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
11.8
e
KM12
CNS cancer
SF-268
SF-295
SNB-19
SNB-75
U251
e
e
e
e
e
e
20.0
90.6
23.2
88.3
58.3
16.5
38.6
18.2
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
14.7
e
15.6
e
e
11.0
e
e
e
e
e
e
11.6
e
e
e
e
19.9
e
12.0
11.9
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
L
L
L
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
22.0
31.4
11.7
e
e
e
e
e
e
10.7
e
30.2
30.4
14.1
10.4
35.2
48.4
27.8
e
e
e
e
e
e
e
e
e
e
e
e
e
10.3
e
e
27.5
17.4
e
e
e
e
e
e
e
21.2
e
e
e
e
e
24.7
e
15.2
e
15.5
e
e
16.4
e
e
20.7
e
e
e
Ovarian cancer
IGORV1
OVCAR-8
e
e
e
e
e
e
L
35.3
17.9
L
39.8
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
15.8
e
22.3
e
e
e
e
e
e
e
NCI/ADR-RES
e
e
Renal cancer
786e0
A498
e
e
25.1
nt
29.5
L
32.9
25.4
L
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
10.9
e
e
e
e
e
e
e
e
e
e
11.6
e
12.7
e
ACHN
e
17.3
L
20.1
e
e
e
e
e
e
e
e
CAKI-1
SN12C
TK-10
21.2
e
e
e
e
e
e
20.5
e
10.7
e
e
18.8
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
UO-31
20.4
26.8
L
20.5
11.7
25.7
27.0
23.8
36.6
21.9
28.6
16.5
Prostate cancer
PC-3
e
e
30.1
25.7
e
e
10.7
e
e
16.3
e
14.3
e
14.7
Breast cancer
MCF7
BT-549
T-47D
MDA-MB-468
e
e
e
e
e
e
e
e
46.4
53.4
33.2
61.5
25.7
39.5
13.0
e
e
e
e
e
e
e
e
e
e
e
e
e
12.1
12.2
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
17.3
e
e
e
e
a
-, GI <10%; nt, not tested; L, compound proved lethal to the cancer cell line.
17.20%); Anal. Calcd. For C10H6ClN3OS (251.69): C, 47.72; H, 2.40; N,
16.70. Found: C, 48.08; H, 2.52; N, 16.98.
washed twice with 15% potassium carbonate solution (20 mL),
dried, and crystallized from methanol.
4.1.2. General procedure for the preparation of 2-phenylimidazo
[2,1-b][1,3,4]oxa/thiadiazol-5,6-dione 7a-d
To a suspension of the appropriate oxa/thiadiazole 5aed
(10 mmol) in toluene (40 mL) at 60e65 ꢂC, oxalyl chloride
(15 mmol, 1.90 g, 1.27 mL) added dropwise with stirring, then
refluxed for 6 h. The solid product separated on cooling was filtered,
4.1.2.1. 2-Phenylimidazo [2,1-b][1,3,4]oxadiazol-5,6-dione (7a). Yield:
69%; mp > 350 ꢂC; IR (KBr, cmꢀ1): 3062 (CH aromatic), 1774, 1708
(C]Os); 1H NMR (DMSO-d6):
d 7.46e7.62 (m, 3H, AreH), 7.75 (dd,
2H, AreH); EIMS, m/z: 217 (Mþ þ 2, 0.14%), 215 (Mþ, 0.11%); Anal.
Calcd. For C10H5N3O3 (215.16): C, 55.82; H, 2.34; N, 19.53. Found: C,
55.72; H, 2.54; N, 19.22.

A.T. Taher et al. / European Journal of Medicinal Chemistry 47 (2012) 445e451
449
4.1.3.2. 2-(4-Chlorophenyl)-8 aH-[1,3,4]oxadiazolo [3,2-a]pyrimidine-
5,7-diamine (8b). Yield: 61%; mp 248e250 ꢂC; IR (KBr, cmꢀ1): 3330,
3251, 3112 (NH2S), 3030 (CH aromatic), 829 (CeCl); 1H NMR
(DMSO-d6):
d 7.2 (br. s, 4H, NH2s exchangeable by D2O), 7.52e7.79
(m, 6H, AreH); EIMS, m/z: 265 (Mþ þ 2, 20.0%), 264 (Mþ þ 1, 20.0%),
263 (Mþ, 12.0%); Anal. Calcd. For C11H10ClN5O (263.68): C, 50.10; H,
3.82; N, 26.56. Found: C, 50.35; H, 3.55; N, 26.20.
4.1.3.3. 2-Phenyl-8aH-[1,3,4]thiadiazolo [3,2-a]pyrimidine-5,7-diamine
(8c). Yield: 59%; mp 146e149 ꢂC; IR (KBr, cmꢀ1): 3421, 3348,
Fig. 2. Structure of the active antitumor agent 6c.
3213(NH2S), 3086 (CH aromatic); 1H NMR (DMSO-d6):
d 7.22e7.85
(m, 7H, AreH), EIMS, m/z: 245 (Mþ); Anal. Calcd. For C11H11N5S
4.1.2.2. 2-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]oxadiazol-5,6-dione
(7b). Yield: 73%; mp > 350 ꢂC; IR (KBr, cmꢀ1): 3062 (CH aromatic),
(245.30): C, 53.86; H, 4.52; N, 28.55. Found: C, 53.37; H, 4.11; N, 28.89.
1750, 1693 (C]Os), 756 (CeCl); 1H NMR (DMSO-d6):
d 8.17 (dd, 2H,
AreH), 8.32 (dd, 2H, AreH); 13C NMR (DMSO): 165.6, 156.1, 149.9,
144.03, 136.2, (2)130.5, (2)128.9, 123.6; EIMS, m/z: 250 (Mþ þ 1,
8.48%), 249 (Mþ, 9.32%), 248 (Mþꢀ1, 4.28%); Anal. Calcd. For
C10H4ClN3O3 (249.61): C, 48.12; H, 1.62; N,16.83. Found: C, 48.34; H,
1.85; N, 17.10.
4.1.3.4. 2- (4-Chlorophenyl)-8 aH-[1,3,4]thiadiazolo [3,2-a]pyrimi-
dine-5,7-diamine (8d). Yield: 66%; mp > 350 ꢂC; IR (KBr, cmꢀ1):
3275, 3150(Br., NH2S), 3089 (CH aromatic), 829 (CeCl); 1H NMR
(DMSO-d6):
d 7.38 (br. s, 4H, NH2 exchangeable by D2O), 7.48e7.77
(m, 6H, AreH); 13C NMR (DMSO): 179.2, 168.7, 155.1, 133.8, 129.7, (2)
128.9, (2)127.7, 120.5, 118.7; Anal. Calcd. For C11H10ClN5S (279.74):
C, 47.23; H, 3.60; N, 25.03. Found: C, 47.55; H, 3.67; N, 24.83.
4.1.2.3. 2-Phenylimidazo [2,1-b][1,3,4]thiadiazole-5,6-dione (7c). Yield:
81%; mp 327e329 ꢂC; IR (KBr, cmꢀ1): 3016 (CH aromatic), 1746, 1693
(C]Os); 1H NMR (DMSO-d6):
d 7.10e7.53 (m, 3H, AreH), 7.71 (dd, 2H,
4.1.4. General procedure for the preparation of 3-chloro-N-(5-(4-
chlorophenyl)-1,3,4-oxa/thiadiazol-2-yl)propanamide 10a,b
A solution of the appropriate oxa or thiadiazole 5b and/or 5d
(10 mmol) in dry dimethyl formamide (DMF, 10 mL) containing 3-
chloropropionyl chloride (15 mmol, 1.90 g, 1.43 mL) was stirred at
room temperature (25e30 ꢂC) for 24 h. The solution was poured
onto crushed ice (20 mL) and the resulting solid was filtered,
washed and crystallized from benzene/methanol (7:3).
AreH); 13C NMR (DMSO): 160.5,156.1, 149.3, 135.9, 132.4, (2)129.9, (2)
129.0, 126.3; EIMS, m/z: 232 (Mþ þ 1, 0.17%), 231 (Mþ, 0.57%); Anal.
Calcd. For C10H5N3O2S (231.23): C, 51.94; H, 2.18; N, 18.17. Found: C,
52.23; H, 2.24; N, 18.58.
4.1.2.4. 2-(4-Chlorophenyl)imidazo[2,1-b][1,3,4]thiadiazol-5,6-dione
(7d). Yield: 88%; mp 204e205 ꢂC; IR (KBr, cmꢀ1): 3012 (CH
aromatic), 1748, 1697 (C]Os); 1H NMR (DMSO-d6):
d 7.37, 7.40 (dd,
2H, AreH), 7.88,7.91 (dd, 2H, AreH),; 13C NMR (DMSO): 168.8, 161.1,
159.8, 156.8, 135.3, (2) 133.9, (2) 129.3, 127.8; EIMS, m/z: 267
(Mþ þ 2, 16.00%); Anal. Calcd. For C10H4ClN3O2S (265.68): C, 45.21;
H, 1.52; N, 15.82. Found: C, 44.92; H, 1.41; N, 15.99.
4.1.4.1. 3-Chloro-N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)prop-
anamide (10a). Yield: 77%; mp 164e166 ꢂC; IR (KBr, cmꢀ1): 3278
(NH), 3062 (CH aromatic), 2900, 2853 (CH aliphatic), 1693 (C]O);
1H NMR (DMSO-d6): 2.26 (t, 2H, COCH2), 3.12 (t, 2H, CH2Cl), 7.57
(dd, 2H, AreH), 7.98 (dd, 2H, AreH), 10.21 (s, 1H, NH exchangeable
by D2O); EIMS, m/z: 286 (Mþ1, 37.49%), 285 (Mþ, 11.57%); Anal.
Calcd. For C11H9Cl2N3O2 (286.11): C, 46.18; H, 3.17; N, 14.69. Found:
C, 46.25; H, 3.31; N, 14.88.
4.1.3. General procedure for the preparation of 2-phenyl-8aH-[1,3,4]
oxa/thiadiazolo[3,2-a]pyrimidine-5,7-diamine 8a-d
A mixture of 5a,b (5 mmol), malononitrile (5 mmol, 0.33 g) and
triethylamine (2 mL) in absolute ethanol (30 mL) was heated under
reflux for 12 h. The precipitate formed was filtered, dried and
crystallized from acetic acid.
4.1.4.2. 3-Chloro-N-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl)prop-
anamide (10b). Yield: 86%; mp >350 ꢂC; IR (KBr, cmꢀ1): 3163 (NH),
3051 (CH aromatic), 2945, 2854 (CH aliphatic), 1685 (C]O), 833
(CeCl); 1H NMR (DMSO-d6):
d 2.28(t, 2H, COCH2), 3.38 (t, 2H,
4.1.3.1. 2- Phenyl-8aH-[1,3,4]oxadiazolo [3,2-a]pyrimidine-5,7-diamine
(8a). Yield: 58%; mp 295e297 ꢂC; IR (KBr, cmꢀ1): 3332, 3244, 3178,
CH2Cl), 7.41 (dd, 2H, AreH), 7.91 (dd, 2H, AreH), 10.33 (s, 1H, NH
exchangeable by D2O); 13C NMR (DMSO): 162.9, 160.9, 158.6, 135.0,
130.5, (2)129.3, (2)128.5, 41.1, 33.5; EIMS, m/z: 304 (Mþ þ 3, 1.57%),
(NHs), 3030 (CH aromatic); 1H NMR (DMSO-d6):
d 7.21e7.92 (m, 7H,
AreH), 8.72 (s, 4H, 2NH2 exchangeable by D2O); EIMS, m/z: 231
(Mþ þ 2, 0.44%), 230 (Mþ þ 1, 0.76%), 229 (Mþ, 0.32%); Anal. Calcd.
For C11H11N5O (229.23): C, 57.63; H, 4.84; N, 30.55. C, 57.69; H, 4.97;
N, 31.02.
303 (Mþ
þ
2, 10.62%), 301 (Mþ, 15.30%); Anal. Calcd. For
C11H9Cl2N3OS (302.17): C, 43.72; H, 3.00; N, 13.91. Found: C, 43.93;
H, 3.21; N, 14.24.
Table 2
Compound 6c median growth inhibitory (GI50
,
m
M), total growth inhibitory (TGI,
m
M) and median lethal (LC50
,
m
M) concentrations of in-vitro subpanel tumor cell lines.
Compound
Activity
Subpanel tumor cell linesa
MG-MIDb
I
II
III
IV
V
VI
VII
VIII
IX
6c
GI50
TGI
LC50
GI50
TGI
4.1
9.7
20.7
9.4
24.9
67.1
8.7
18.8
51.3
8.0
20.1
51.4
7.7
33.5
83.9
8.9
16.6
42.3
7.6
18.3
50.8
6.9
23.7
71.3
6.0
17.4
55.1
25.9
c
55.7
c
5-FU
15.1
8.4
72.1
70.6
61.4
45.6
22.7
76.4
22.60
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
LC50
a
I, leukemia; II, non-small cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer; VIII, prostate cancer; IX, breast cancer.
b
c
Full panel mean-graph midpoint (
m
M).
M.
Compounds showed values > 100
m

450
A.T. Taher et al. / European Journal of Medicinal Chemistry 47 (2012) 445e451
dihydrofolate reductase inhibition, antitumor testing, and molecular
modeling study of some new 4(3H)-quinazolinone analogs, Bioorg. Med.-
Chem. 14 (2006) 8608e8621.
4.1.5. Generalprocedure forthepreparation ofN-(5-(4-chlorophenyl)-
1,3,4-oxa/thiadiazol-2-yl)-3-(piperidin-1-yl)propamide 11a,b
To a solution of compounds 10a and/or 10b (10 mmol) in dry
acetonitrile (30 mL) in the presence of anhydrous potassium
carbonate (1.37g, 10 mmol), piperidine (12 mmol, 1.02 g,1.18 mL)
was added dropwise with stirring. Then, the mixture was refluxed
for 24 h. The reaction mixture was filtered while hot, the filtrate
was evaporated to dryness .The obtained solid was crystallized
from acetone.
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4.1.5.1. N-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-3-(piperidin-1-
yl)propamide (11a). Yield: 62%; mp 295e297 ꢂC; IR (KBr, cmꢀ1):
3155 (NH), 3032 (CH aromatic), 2924, 2854 (CH aliphatic), 1761(C]
O); 1H NMR (DMSO-d6):
d 1.14e1.16 (m, 2H, CH2 (C4)), 1.20e1.22 (m,
4H, CH2 (C3,5)), 2.86e3.06 (m, 6H, CH2 (C2,6), þ COCH2), 3.11 (t, 2H,
CH2N, J ¼ 8.4), 7.46 (dd, 2H, AreH), 7.93 (dd, 2H, AreH), 11.64 (s, 1H,
NH exchangeable by D2O); EIMS, m/z: 334 (Mþ, 9.58%), 333 (Mþꢀ1,
2.54%); Anal. Calcd. For C16H19ClN4O2 (334.80): C, 57.40; H, 5.72; N,
16.73. Found: C, 57.18; H, 5.89; N, 17.06.
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yl)propamide (11b). Yield: 68%; mp 143e145 ꢂC; IR (KBr, cmꢀ1):
3178 (NH), 3020 (CH aromatic), 2935, 2854 (CH aliphatic),1693 (C]
O), 829 (CeCl); 1H NMR (DMSO-d6):
d 1.51e1.69 (m, 2H, CH2 (C4)),
1.71e1.77 (m, 4H, CH2 (C3,5)), 2.92e3.03 (m, 6H, CH2 (C2,6) þ COCH2),
3.35 (t, 2H, CH2N, J ¼ 8.4), 7.57 (dd, 2H, AreH), 7.94 (dd, 2H, AreH),
10.2 (s, 1H, NH exchangeable by D2O); EIMS, m/z: 354 (Mþ þ 4,
2.00%), 353 (Mþ þ 3, 4.12%), 352 (Mþ þ 2, 3.62%), 351 (Mþ þ 1, 5.00%),
350 (Mþ, 14.37%); Anal. Calcd. For C16H19ClN4OS (350.86): C, 54.77;
H, 5.46; N, 15.97. Found: C, 54.43; H, 5.21; N, 16.02.
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Under a sterile condition, cell lines were grown in RPMI 1640
media (Gibco, NY, USA) supplemented with 10% fetal bovine serum
(Biocell, CA, USA), 5 ꢁ 105 cell/ml was used to test the growth
inhibition activity of the synthesized compounds. The concentra-
tions of the compounds ranging from 0.01 to 100 mM were prepared
in phosphate buffer saline. Each compound was initially solubilized
in dimethyl sulfoxide (DMSO), however, each final dilution con-
tained less than 1% DMSO. Solutions of different concentrations
(0.2 ml) were pipetted into separate well of a microtiter tray in
duplicate. Cell culture (1.8 ml) containing a cell population of
6 ꢁ 104 cells/ml was pippeted into each well. Controls, containing
only phosphate buffer saline and DMSO at identical dilutions, were
also prepared in the same manner. These cultures were incubated
in a humidified incubator at 37 ꢂC. The incubator was supplied with
5% CO2 atmosphere. After 48 h, cells in each well were diluted 10
times with saline and counted by using a coulter counter. The
counts were corrected for the dilution [28e31].
Acknowledgments
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Thanks are due to the NCI, Bethesda, MD, USA for performing
the antitumor testing of the synthesized compounds.
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