Synthesis and in vitro anti-tumor activity of new oxadiazole thioglycosides

Article abstract of DOI:10.1016/j.ejmech.2010.11.008

A facile, convenient and high yielding synthesis of novel thioglycosides incorporating 1,3,4-oxadiazole, triazole and or triazine moieties from readily available starting materials has been described. The key step of this protocol is the formation of 3-isobutyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3) via condensation between methyl iso-butyl ketone and phenylhydrazine followed by application of Vilsmeier-Haack reaction. 3 was converted either to 1,3,4-oxadiazole derivative or condensed with O-aminothiols to give the bases 8, 19 and 20 in good yields, respectively. The aglycons 8, 19, and 20 were coupled with different activated halosugars in the presence of basic medium. Pharmacological evaluation of compounds 8, 14, 16 and 22 in vitro against 2-cell lines MCF-7 (breast) and HEPG2 (liver) revealed them to possess high anti-tumor activities with IC₅₀ values ranging from 2.67-20.25 (μg/mL) for breast cell line (MCF-7) and 4.62-43.6 (μg/mL) for liver cell line (HEPG2). None of the tested compounds exhibited any toxicity in doses up to 500 mg kg^-1 of the animal body weight.

Full text of DOI:10.1016/j.ejmech.2010.11.008

European Journal of Medicinal Chemistry 46 (2011) 229e235
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and in vitro anti-tumor activity of new oxadiazole thioglycosides
,
M.A. Abu-Zaied a, E.M. El-Telbani a, G.H. Elgemeie b, G.A.M. Nawwar a
*
a National Research Centre, Green Chemistry Department, 12622 Dokki, Cairo, Egypt
b Chemistry Department, Faculty of Science, Helwan University, Ain-Helwan, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile, convenient and high yielding synthesis of novel thioglycosides incorporating 1,3,4-oxadiazole,
triazole and or triazine moieties from readily available starting materials has been described. The key
step of this protocol is the formation of 3-isobutyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3) via
condensation between methyl iso-butyl ketone and phenylhydrazine followed by application of Vils-
meiereHaack reaction. 3 was converted either to 1,3,4-oxadiazole derivative or condensed with O-
aminothiols to give the bases 8, 19 and 20 in good yields, respectively. The aglycons 8, 19, and 20 were
coupled with different activated halosugars in the presence of basic medium. Pharmacological evaluation
of compounds 8, 14, 16 and 22 in vitro against 2-cell lines MCF-7 (breast) and HEPG2 (liver) revealed
Received 12 April 2010
Received in revised form
3 November 2010
Accepted 5 November 2010
Available online 12 November 2010
Keywords:
1,3,4-Oxadiazole
Thiogycosides
Tamoxifen
5-Flurouracil
Anti-tumor activities
Toxicity
them to possess high anti-tumor activities with IC50 values ranging from 2.67e20.25 (
cell line (MCF-7) and 4.62e43.6 ( g/mL) for liver cell line (HEPG2). None of the tested compounds
exhibited any toxicity in doses up to 500 mg kgꢀ1 of the animal body weight.
Ó 2010 Elsevier Masson SAS. All rights reserved.
mg/mL) for breast
m
1. Introduction
In view of the above mentioned findings and our previous
reports [22e26], the purpose of the present work was to design,
synthesize and investigate the anti-tumor activity of some novel
1H-pyrazolo-1,3,4-oxadiazole derivatives carrying carbohydrate
residues through S-glycosidic bond formation.
The incidence and mortality of cancer patients have become one
of the important issues discussed worldwide. Unfortunately, devel-
opment of resistance to chemotherapeutic agents is a common
obstacle in the treatment of different types of cancers [1,2]. Several
important drugs including tamoxifen (TAM), 5-flurouracil (5FU),
adriamycin (ADR) and vincristin (VCR) with different structures and
mechanisms of anti-tumor activities fail to end these problems
completely. Due totheseveral side effects, drug resistance andfailure
of anti-tumor drugs to exert their effects in certain cases of cancers
[3e5], looking for new chemotherapeutic agents with synthetic or
natural origins is one of the hot topics in cancer research laboratories.
1,3,4-Oxadiazole derivatives have attracted significant attention
in the field of drug discovery because of their wide array of phar-
macological activities, including antibacterialeantifungal, anal-
gesic, anti-inflammatory, antihypertension, muscle relaxing and
anticancer activities [6e16]. Additionally, pyrazole and N-arylpyr-
azole derivatives are very important class of heterocyclic
compounds that have remarkable pharmacological activities as
antibacterialeantifungal, hypoglycemic, hyperlipidemia, kinase
inhibitor for treatment of type 2 diabetes, anti-inflammatory and
tumor necrosis inhibitor [17e21].
2. Chemistry
The synthesis of our desired pyrazole derivatives began with
commercially available aliphatic ketones 1a,b (namely, 2-butanone
and methyl iso-butyl ketone) and phenylhydrazine as starting
materials. Condensation between the ketone and hydrazine
provided a hydrazones 2a,b in high yields. The VilsmeiereHaack
reaction using 2.5 eq of reagent performed a double addition of
reagent to afford, ultimately after hydrolysis, the desired cyclized
aldehyde 3 in 80% yield unfortunately, the application of the Vils-
meiereHaack reaction at the hydrazone 2a did not led to desired
product, instead 3,4-dimethyl-1-phenyl pyrazole (4) was obtained
as a sole product (Scheme 1).
The reaction sequence of preparing the desired aglycon [5-
(3-Isobutyl-1-phenyl-1H-pyrazole-4-yl)-1,3,4-
oxadiazle-2-thiol
(8)] is some what long and linear with few common intermediates.
To this aim, aldehyde 3 is oxidized using a mixture of sulphuric acid
(30%) and K2Cr2O7 to yield the acid 5 which esterified using abso-
lute ethanol in presence of few drops of conc. sulphuric acid,
affording 6 in fairly good yield. The 1H NMR of acid 5 showed the
* Corresponding author. Tel.: þ202 333711211/1428; fax: þ202 33370931.
E-mail address: gnawwar@yahoo.com (G.A.M. Nawwar).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.11.008

230
M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
CHO
R= iso-butyl-
O
CH3
NH
R
N
N
3
4
N
POCl3/DMF
PhNHNH2
AcOH/H2O
R
CH3
Ph
CH3
H3C
1
a,
R= ethyl-
R= ethyl-
2
a, b
b, R= iso-butyl-
N
N
Ph
Scheme 1. Synthesis of 1-phenylpyrazoles 3 and 4.
absence of aldehydic proton which detected at the parent 3, while,
the ester 6 showed the ethyl protons at their expected locations.
Treatment of 6 with hydrazine hydrate in boiling ethanol
afforded the hydrazide derivative 7 in good yield. Finally the oxa-
diazole derivative 8 was achieved following our previously repor-
ted method [27] by adopting a simple one pot procedure that
involves reacting of 7 with carbon disulphide under strong basic
conditions followed by acidification with diluted hydrochloric acid
(Scheme 2).
anomeric proton as a doublet at
d
0
¼ 5.68 and 6.24 ppm with
0
a spinespin coupling constant (J1 ,2 ¼ 9.95 and 10.39 Hz) corre-
sponding to a trans orientation of H-10 and H-20 protons indi-
cating the b-configuration. On the other hand, the formation of
S-glycosides 14 and 15 and not the corresponding N-glycosides 13
were proved using 13C NMR spectroscopy which revealed the
absence of a signal at
appearance of a signal at
sponding to the C-2 carbon of the 1,3,4-oxadiazole ring, whose
chemical shift is the same as that of the corresponding S-methyl
derivative [28e30], the same results were obtained with
compounds 16 and 17 (Scheme 3).
d
178 ppm for the thione carbon and the
¼ 162.21 and 161.55 ppm corre-
d
The coupling between the aglycon and the activated sugars was
achieved in the presence of basic medium. Thus, aglycon 8 was
reacted with NaH in dry DMF followed by addition of the activated
cyclic sugars (20,30,40,60-tetra-O-acetyl-
a
-D
-gluco (or galacto)) pyr-
Additionally, when compound 3 was condensed with selected
examples of bidentate ligands (18a, b), the condensing adducts 19,
20 were obtained in high yields. The 1H NMR of 19, 20 showed the
methine CH proton at 7.97 and 8.22 ppm supported the condensing
adduct structures. Finally, the S-glycosides 21, 22 were obtained in
fairly good yields, on treatment of compounds 19 and 20 with 9 in
the presence of NaH/DMF following the above mentioned condi-
anosyl bromide 9 and 10, or the activated acyclic sugar analogues,
chloromethyl methylthioether (11), or chloromethyl ethyl ether
(12), to give the corresponding thioglycoside derivatives 14e17 in
a good yields, respectively.
The structures of thioglycosides 14 and 15 were established
and confirmed for the reaction products on the basis of their
elemental analysis and spectral data (1H NMR, 13C NMR and MS,
cf. experimental). Thus, their 1H NMR spectrum showed the
0
0
tions whereas the
9.20 Hz) (Scheme 4).
b
-isomers were obtained (J1 ,2 ¼ 9.75 and
Scheme 2. Synthetic pathway for the synthesis of 5-(3-isobutyl-1-phenyl-1H-pyrazol-4-yl)-1,3,4-oxadiazole-2-thiol 8.

M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
231
Scheme 3. Synthesis of thioglycosides 14e17.
3. Pharmacology
2.5, 5 and 10 mg/mL) were added to the cell monolayer triplicate
wells were prepared for each individual dose. Monolayer cells
were incubated with the compound(s) for 48 h at 37 ꢁC and in
atmosphere of 5% CO2. After 48 h, cells were fixed, washed and
stained with sulfo-rhodamine B strain. Excess stain was washed
with acetic acid and attached stain was recovered with tris EDTA
buffer. Coor intensity was measured in an ELISA reader. Finally, the
relation between surviving fraction and drug conc. is plotted to get
the survival curve of each tumor cell line after the specified
compound.
3.1. Materials and methods
Potential cytotoxicity effect of the newly synthesized
compounds in four concentrations, were evaluated in the National
Institute of Cancer, Cairo Egypt by SRB assay [31]. Cells were plated
in 96-multiwell plate (104 cells/well) for 24 h before treatment
with the compounds to allow attachment of cell to the wall of
plate. Different concentrations of each compound under test (0, 1,
N
SH
EtOH/ pip.
N
NaH, DMF
18a,b
3
+
N
X
N
9
N
N
Ph
N
N
19
, X = CH
20
, X = C(Me)-C(O)
AcO
O
a =
18
SH
N
N
N
N
OAc
OAc
NH2
S
N
N
AcO
Me
N
X
N
N
N
Ph
21
b =
18
O
SH
NH2
, X= CH
, X = C(Me)-C(O)
22
Scheme 4. Synthesis of pyrazolo (triazole and/or triazine) thioglycosides 21 and 22.

232
M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
3.2. Anticancer screening studies
1.2
TAM
8
14
16
22
1
0.8
0.6
0.4
0.2
0
Four of the newly synthesized compounds were screened for
their anticancer activities, the two cell lines one dose assay has
been done for compounds 8, 14, 16 and 22. The 2-cell lines used in
the present investigation are MCF-7 (breast) and HEPG2 (liver). IC50
was calculated with regard to saline control group and potency was
calculated with regard to percentage of change of tamoxifen or
5-flurouracil and tested compounds, as depicted in Table 1.
Our SAR study shows that all the tested compounds have high or
moderate anti-tumor activity towards both the two cell lines with
IC50 values ranging from 2.67e20.25 (
mg/mL) for breast cell line
(MCF-7) and 4.62e43.6 g/mL) for Liver cell line (HEPG2)
(m
0
10
20
30
Conc :µg/ml
40
50
60
compared with both standard drugs. Oxadiazoles carrying two
substituents (14 and 16) especially when contain thioglycosidic
linkage at C2 are more active than those carrying one substituent
(compound 8). Compound 16 was highly specific and potent for
both the two cell lines (MCF-7 and HEPG2). The substituents at C2
and C5 of 1, 3, 4-oxadiazole ring shown to be vital for potency.
Suggesting specific interaction of these groups with biological
targets. Compound 22 has very excellent and specific anti-tumor
activity for HEPG2 this may be attributed to the presence of
pyrimidine skeleton similar to 5-flurouracil Fig. 1.
Fig. 1. Representative Graph showing survival of MCF-7 cell grown compared to
Tamoxifen (TAM) in the presence of increasing concentrations of compounds 8, 14, 16
and 22.
4.2. Acute oral toxicity
Our results in the present study showed that no mortality in rats
traded with compound 8 while compounds 14, 16 and 22 caused
25%, 25% and 37.50% mortality at 500 mg/kg b.wt, respectively.
These results indicated that the lethal doses (LD50) of tested
compounds are >500 mg/kg b.wt and all compounds are consid-
ered a moderate toxicity based on oral LD50 values (no label, >500
<2000 mg/kg), which recommended by the Organization for
Economic Co-operation and Development [30] Fig. 2.
4. Toxicity studies
4.1. Animals and treatment
All the compounds tested for their anticancer activity 8, 14, 16
and 22 showed very good or moderate anticancer activity in terms
of growth inhibitory effect on cancer cell lines. Hence, it could be
a potential drug candidate for cancer treatment. Hence these
particular compounds were analyzed for its acute toxicity (lethal
dose) which is one of the basic requirements in fixing the thera-
peutic dose in drug development. Male albino rats of the Wistar
strain (Rattus norvegicus) weighing 110e115 g were obtained from
Animal Breeding House of the National Research Centre (NRC),
Dokki, Cairo, Egypt. Animals were housed in clean plastic cages in
the laboratory animal room (23 ꢂ 2 ꢁC) on the standard pellet diet
and tap water ad-libitum, a minimum relative humidity of 40% and
a 12 h dark/light cycle. Rats were allowed to acclimate to laboratory
conditions for 7 days prior to dosing. All compounds were dissolved
in DMSO and administered by gavages at a fixed volume of 0.5 mL/
rats. Animals were randomly divided into five groups, eight rats of
each. Four groups were used for single dose treatments of
compounds 8, 14, 16, and 22 at 500 mg/kg body weight, and one
served as control (0.5 mL DMSO/rat). Then mortality of treated-rats
was recorded after 24 h. The experimental protocols and proce-
dures were approved by the Local Ethics Committee at the National
Research Centre (NRC), Dokki, Cairo, Egypt, and confirmed with the
Guide for the Care and Use of Laboratory Animals (NRC, 1999).
5. Conclusion
We have achieved the synthesis of 1H-pyrazolo-1,3,4-oxadia-
zole derivatives having cyclic and acyclic carbohydrate residues
through S-glycosidic bond formation in an efficient manner. Phar-
macological evaluation of compounds 8, 14, 16 and 22 against 2-cell
lines MCF-7 (breast) and HEPG2 (liver) revealed them to possess
high or moderate anti-tumor activities. Compounds 14, 16 and 22
showed very good anticancer activity in terms of growth inhibitory
effect on both cancer cell lines. Hence, it could be a potential drug
candidate for cancer treatment. None of the tested compounds
presented any toxicity in doses ranging from 50 to 500 mg kgꢀ1 of
1.2
5FU
8
14
16
22
1
0.8
0.6
0.4
0.2
0
Table 1
Cytotoxcity of the synthesized candidates on breast (MCF-7) and liver (HEPG2)
cancer cell lines.
Compd.
No.
IC50
(
m
g/mL) Breast
IC50 (mg/mL) Liver
cell line (HEPG2)
cell line (MCF-7)
8
14
16
22
17.9
7.1
2.67
20.25
8.31
e
43.6
13.9
4.62
9.1
0
10
20
30
Conc:µg/ml
40
50
60
(TAM)a
(5FU)b
e
25
Fig. 2. Representative Graph showing survival of HEPG2 cell grown compared to 5-
Flurouracil (5FU) in the presence of increasing concentrations of compounds 8, 14, 16
and 22.
a
TAM (tamoxifen), standard drug for breast cancer.
5FU (5-flurouracil), standard drug for liver cancer.
b

M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
233
the animal body weight. The lethal doses (LD50
compounds are >500 mg/kg b.wt.
)
of tested
6.2.2. 3, 4-Dimethyl-1-phenyl-1H-pyrazole (4)
Colorless solid (ethanol); yield 76%; m.p. 86e88 ꢁC 1H NMR
(DMSO-d6): 1.94 (s, 3H, CH3), 2.11 (s, 3H, CH3), 7.11e7.42 (m, 5H,
d
PheH), 8.09 (s, 1H, pyrazole H-5). Anal. Calcd. For. C11H12N2
(172.23): C, 76.71; H, 7.02; N, 16.27. Found: C, 76.90; H, 7.20; N,
16.35.
6. Experimental
All melting points were determined on an Electrothermal 9100
digital melting point apparatus. NMR and mass spectra were
recorded on: 1H, 13C NMR spectra were determined using Jeol
JMS-AX 500 MHz and TMS as internal standard. Chemical shifts are
6.3. 3-Isobutyl-1-phenyl-1H-pyrazole-4-carboxylic acid (5)
Compound 3 (10 mmol) was dissolved in 20% sulphuric acid
(10 mL) then, aqueous solution of 30% K2Cr2O7 (20 mL) was added
slowly within 30 min. The reaction mixture was refluxed for
another 1 h, allowed to cool to room temperature and poured onto
ice water. A solid product precipitate was filtered off, washed with
water (20 mL) dried and crystallized from ethanol to give ꢀ51 as
expressed as
d (ppm). Mass spectra were recorded on Varian MAT
311 A at 70 ev. Elemental analyses were carried out at the micro-
analytical unit, Faculty of Science, Cairo University. Precoated silica
gel 60 F254plats with a layer thickness 0.25 nm from Merck were
used for thin layer chromatography. Yields are not optimized.
colorless crystals. Yield 82%, m.p. 158e159 ꢁC; IR (KBr, cm
) y
3046e3399 (OH), 2954, 2869(CH), 1696(CO), 1600(C]N); 1H NMR
6.1. General procedure for synthesizing of 2a and 2b
(DMSO-d6):
d
0.88, 0.90 (2s, 6H, 2ꢃ CH3), 2.01 (m, 1H, CH), 2.70 (d,
2H, J ¼ 6.9 Hz, CH2), 7.30e7.85 (m, 5H, PheH), 8.84 (s, 1H, pyrazole
H-5), 12.82 (brs., 1H, OH). MS, m/z (%): 244 [Mþ] (50), 201.8 (100),
158 (25), 142.2 (9), 77 (4). Anal. Calcd. For. C14H16N2O2 (244.29): C,
68.83; H, 6.60; N, 11.47. Found: C, 68.70; H, 6.81; N, 11.35.
To 2-butanone or methyl iso-butyl ketone (10 mmol) in acetic
acid (100 mL) and water (10 mL) was added phenylhydrazine
(11 mmol). The reaction mixture was stirred at room temperature
until completion (TLC, 2 h). The reaction mixture was diluted with
an additional 50 mL of water and subsequently extracted with ethyl
acetate (3 ꢃ 50 mL). The organic layer was separated, washed with
water, dried and evaporated under reduced pressure to give:
6.4. Ethyl-3-isobutyl-1-phenyl-1H-pyrazole-4-carboxylate (6)
Compound 5 (10 mmol) in absolute ethanol (20 mL) contains
sulphuric acid (5 drops) was refluxed for 2 h. the reaction mixture
was then cooled to room temperature poured onto ice water,
neutralized to pH 6e7 by adding NaHCO3 in small potions and
extracted using ethyl acetate (50 mL). The two phases were sepa-
rated and aqueous phase was extracted with ethyl acetate
(3 ꢃ 10 mL), the organic phase was dried over anhydrous sodium
sulphate then evaporated under reduced pressure and the residue
was crystallized from ethanol to give 6 as a white solid. Yield 72%;
6.1.1. 1-(Butan-2-ylidene)-2-phenylhydrazine (2a)
As viscous yellow oil, yield 78%; 1H NMR (CDCl3):
d 0.96 (t, 3H,
CH3), 1.75 (q, 2H, CH2), 2.35 (s, 3H, CH3), 6.90e7.35 (m, 6H, PheH,
NH). MS, m/z (%): 162 [Mþ] (90), 147.6 (5), 133 (4), 85.2 (12), 77 (86).
Anal. Calcd. For. C10H14N2 (162.28): C, 74.03; H, 8.70; N, 17.27.
Found: C, 74.22; H, 8.51; N, 17.40.
6.1.2. 1-(4-Methylpenten-2-ylidene)-2-phenylhydrazine (2b)
As yellow crystals (ethanol), yield 75%; m.p. 131e132 ꢁC; 1H
m.p. 49e50 ꢁC; IR (KBr, cmꢀ1
1597 (C]C); 1H NMR (500 MHz, CDCl3):
)
y
2959, 2868 (CH), 1698 (COOEt),
0.98, 0.99 (2s, 6H, 2ꢃ
d
NMR (CDCl3):
d
0.86, 0.88 (2s, 6H, 2ꢃ CH3), 1.96 (m, 1H, CH), 2.04 (s,
CH3), 1.36 (t, 3H, CH3-ester), 2.11 (m, 1H, CH), 2.82 (d, 2H,
J ¼ 6.85 Hz, CH2), 4.31 (q, 2H, CH2 -ester), 7.45e7.69 (m, 5H, PheH),
8.34 (s, 1H, pyrazole H-5). Anal. Calcd. For. C16H20N2O2 (272.34): C,
70.56; H, 7.40; N, 10.29. Found: C, 70.71; H, 7.22; N, 10.40.
3H, CH3), 2.97 (d, 2H, J ¼ 6.90 Hz, CH2), 6.98e7.65 (m, 5H, PheH),
7.91(s, 1H, NH). MS, m/z (%): 190 [Mþ] (100), 175.9 (8), 148.8 (5),
134.9 (4), 97(4), 76.7 (86). Anal. Calcd. For. C12H18N2 (190.28): C,
75.74; H, 9.53; N, 14.72. Found: C, 75.90; H, 9.41; N, 14.55.
6.5. 3-Isobutyl-1-phenyl-1H-pyrazole-4-carbohydrazide (7)
6.2. General procedure for synthesizing of 3 and 4
Compound 6 (10 mmol) and hydrazine hydrate (10 mmol) in
250 mL round-bottomed flask was refluxed in ethanol (20 mL) in
the presence of catalytic amount of triethyl amine for 8 h. The
reaction mixture was allowed to cool to room temperature and
poured onto ice water. The resulting brown solid was filtered off
and crystallized from ethanol to affꢀo1rd 7as yellow crystals. Yield
Phosphorous oxychloride (25 mmol) was added to DMF
(100 mL) at 0 ꢁC and stirred for 30 min 2b or 2a (10 mmol) were
added slowly to this mixture and stirred for 5 h. The crude reaction
was then quenched into water (1 L) and stirred for an additional 1 h
and extracted with ethyl acetate (3 ꢃ 50 mL). The organic layer was
separated, washed with water, dried and evaporated under reduced
pressure. The crude was purified using column chromatography
(pet.ether/ethyl acetate 8:2, Rf ¼ 0.35) to afford:
80%; m.p. 270e272 ꢁC; IR (KBr, cm
) y 3409, 3320 (NH2), 3135
(NH), 2969, 2878 (CH), 1668 (CO), 1597 (C]C); 1H NMR (500 MHz,
DMSO-d6):
d
0.86, 0.88 (2s, 6H, 2ꢃ CH3), 2.01 (m, 1H, CH), 2.73 (d,
2H, J ¼ 6.8 Hz, CH2), 4.20 (s, 2H, NH2), 7.47e7.70 (m, 5H, PheH), 8.73
(s, 1H, pyrazole H-5), 9.18 (s, 1H, NH). Anal. Calcd. For. C14H18N4O
(258.32): C, 65.09; H, 7.02; N, 21.69. Found: C, 65.20; H, 7.11; N,
21.80.
6.2.1. 3-Isobutyl-1-phenyl-1H-pyrazole-4-carbaldehyde (3)
Colorless crystals (ethanol); yield 80%; m.p. 168e170 ꢁC. IR (KBr,
cmꢀ1
d6):
J ¼ 6.90 Hz, CH2), 7.43e7.83 (m, 5H, PheH), 9.07 (s, 1H, pyrazole H-
5), 9.89 (s, 1H, CHO). 13C NMR:
22.54 (2C, CH3), 28.82(CH), 36.12
) y
2950, 2877 (CH), 1682 (CO), 1594 (C]C); 1H NMR (DMSO-
d
0.86, 0.88(2s, 6H, 2ꢃ CH3), 1.96 (m, 1H, CH), 2.71 (d, 2H,
6.6. 5-(3-Isobutyl-1-phenyl-1H-pyrazole-4-yl)-1,3,4-oxadiazle-
2-thiol (8)
d
(CH2),119.71 (2C, AreC),123.15 (pyrazole C4),127.68 (PheC),129.69
(2C, AreC), 131.71(pyrazole C5), 139.21 (AreC), 155.48 (pyrazole
C2), 184.48 (CHO). MS, m/z (%): 228 [Mþ] (50), 213.2 (30), 199 (3),
183 (63), 157.3 (100), 142.7 (20), 93(73), 77(6). Anal. Calcd. For.
C14H16N2O (228.29): C, 73.66; H, 7.06; N, 12.27. Found: C, 73.50; H,
7.22; N, 12.40.
To a solution of compound 7 (10 mmol) in ethanolic potassium
hydroxide (20 mL), carbon disulphide (2 mL) was dropped in over
period of half hour at room temperature (25 ꢁC). After an additional
half hour of stirring, a precipitate was formed and collected by
filtration. The salt formed was dissolved in water, then acidify with

234
M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
HCl the precipitate thus formed was filtered off and crystallized
from methanol to give 8 as pale yellow solid. Yield 72%, m.p.
PheH), 8.89 (s, 1H, pyrazole H-5); 13C NMR:
d 14.47 (CH3), 22.58 (2C,
2CH3), 28.56 (CH), 36.46 (CH2), 60.12 (CH2), 114.23 (pyrazole C4),
119.52 (2C, AreC), 127.15 (AreC), 129.57 (2C, AreC), 131.34 (pyrazole
C5), 139.52 (AreC), 151.22 (pyrazole C3), 155.66 (oxadiazole C5),
163.58 (oxadiazole C2). Anal. Calcd. For. C17H20N4OS2 (360.50): C,
56.64; H, 5.59; N, 15.54. Found: C, 56.80; H, 5.40; N, 15.60.
160e161 ꢁC; 1H NMR (500 MHz, DMSO-d6):
d
0.91, 0.92 (2s, 6H, 2ꢃ
CH3), 2.01 (m, 1H, CH), 2.73 (d, 2H, J ¼ 6.90 Hz, CH2) 7.33e7.90 (m,
5H, PheH), 9.15 (s, 1H, pyrazole H-5), 14.50 (brs, 1H, SH). Anal.
Calcd. For. C15H16N4OS (300.38): C, 59.98; H, 5.37; N, 18.65. Found:
C, 59.80; H, 5.50; N, 18.70.
6.7.4. 5-(Ethoxymethylthio)-2-(3-isobutyl-1-phenyl-1H-pyrazol-
6.7. General procedure for synthesizing of 14e17
4-yl)-1,3,4-oxadiazole (17)
White solid; yield 77%, m.p. 112e114 ꢁC; IR (KBr, cmꢀ1
) y 3060,
To a solution of 8 (10 mmol) in dry DMF (20 mL), NaH (15 mmol)
was added portionwise through 15 min and the solution stirred at
room temperature for another 30 min. Then, a solution of 2,3,4,6-
2920, (CH), 1600 (C]C); 1H NMR (500 MHz, DMSO-d6) 0.88, 0.89
(2s, 6H, 2ꢃ CH3), 2.01 (m, 1H, CH), 2.60 (s, 3H, CH3), 2.66e2.73 (m,
4H, 2ꢃ CH2), 4.20 (s, 2H, CH2), 7.25e7.88 (m, 5H, PheH), 8.90 (s, 1H,
H-5 pyrazole). Anal. Calcd. For. C18H22N4O2S (358.46): C, 60.31; H,
6.19; N, 15.63. Found: C, 60.40; H, 6.10; N, 15.50.
tetra-O-acetyl-a-D-gluco (or galacto)pyronosyl bromide 9 or 10, or
chloromethyl methylthioether (11), or chloromethyl ethyl ether
(12) in DMF (10 mL) was dropped within 30 min and the reaction
mixture was stirred at room temperature until completion (TLC,
3e6 h). After completion, the reaction mixture was poured on
water and acidified with diluted acetic acid. The aqueous phase was
extracted with ethyl acetate (3 ꢃ 20 mL), the combined organic
phase was washed with water dried over anhydrous sodium
sulphate. Removal of solvent gave a residue, which was purified by
column chromatography using an appropriate solvent system to
give compounds 14e17.
6.8. General procedure for synthesizing of 19 and 20
Compound 3 (10 mmol) was refluxed with an equimolecular
amount of 18a or 18b in absolute ethanol (30 mL) in the presence of
piperidine (4 drops) for 10 h. A precipitate was formed on hot was
filtered off and recrystallized from acetic acid to give 19 and 20 as
a pale yellow solid, respectively.
6.8.1. 4-[((3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)methylene)
6.7.1. 5-(3-Iso-butyl-1-phenyl-1H-pyrazol-4-yl)-2-(20,30,40,60-tetra-
amino]-4H-1,2,4-triazole-3-thiol (19)
O-aceyl-
White solid; yield 69%, m.p. 95e96 ꢁC; 1H NMR (500 MHz,
DMSO-d6):
b-
D-glucopyranosylthio)-1,3,4-oxadiazole (14)
Yield 77%; m.p. 237e238 ꢁC; IR (KBr, cmꢀ1
)
y
2959, 2868 (CH),
3106(SH), 2950, 2867(CH), 1598(C]N); 1H NMR (500 MHz, DMSO-
d6): 0.94 (s, 3H, CH3), 0.97 (s, 3H, CH3), 2.1 (m,1H, CH), 2.77 (m, 2H,
d
0.91, 0.92 (2s, 6H, 2ꢃ CH3), 1.84e1.93 (4s, 12H, 4ꢃ
d
OAc), 1.96 (m, 1H, CH), 2.81 (d, 2H, J ¼ 6.85 Hz, CH2), 4.01 (dd, 1H,
J ¼ 2.2, 12.2 Hz, 60-H), 4.09 (m, 2H, 50-H, 60-H), 4.97 (t, 1H, J ¼ 9.5 Hz,
40-H), 5.13 (t, 1H, J ¼ 9.3 Hz, 20-H), 5.66 (t, 1H, J ¼ 9.5 Hz, 30-H), 5.68
CH2), 7.44e7.68 (m, 5H, PheH), 7.97 (s, 1H, CH]N), 8.35 (s, 1H,
triazole CH]N), 8.37 (s, 1H, pyrazole H-5), 10.23 (s, 1H, SH). Anal.
Calcd. For. C16H18N6S (326.42): C, 58.87; H, 5.56; N, 25.75. Found: C,
58.70; H, 5.71; N, 25.60.
(d, 1H, J1 ,2 ¼ 9.95 Hz, 10-H), 7.3e7.89 (m, 5H, PheH), 9.11 (s, 1H,
0
0
pyrazole H-5). 13C NMR:
d 20.78e20.84 (4CH3$CO), 22.79 (CH3),
22.82 (CH3), 28.10 (CH), 36.20(CH2), 62.21 (C06), 68.19 (C04), 70.31
(C02), 73.14 (C03), 75.54 (C05), 82.53 (C01), 106.79 (pyrazole C4),
119.28 (2C, AreC), 127.66 (AreC), 129.97(2C, AreC), 130.19 (pyr-
azole C5), 139.28 (AreC), 152.52 (pyrazole C3), 159.43 (oxadiazole
C5), 162.21 (oxadiazole C2), 169.76e170.07 (4COCH3). MS, m/z (%):
630 [Mþ] (50), 285 (22), 258 (100), 228 (82), 77 (3). Anal. Calcd. For.
C29H34N4O10S (630.67): C, 55.23; H, 5.43; N, 8.88. Found: C, 55.40;
H, 5.20; N, 8.70.
6.8.2. 4-[((3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)methylene)
amino]-3-mercapto-6-methyl-1,2,4-triazin-5(4ꢀH1)-one (20)
Yield 82%; m.p. 188e189 ꢁC; IR (KBr, cm
) y 3108(SH), 2958,
2865 (CH), 1698(CO), 1599(C]N); 1H NMR (500 MHz, DMSO-d6):
0.93 (s, 3H, CH3), 0.94 (s, 3H, CH3), 2.01 (s,1H, CH), 2.33 (s, 3H,
d
CH3), 2.79 (d, 2H, J ¼ 6.85 Hz, CH2), 7.43e7.90 (m, 6H, PheH, SH),
8.22 (s, 1H, CH]N), 8.50 (s, 1H, pyrazole H-5). Anal. Calcd. For.
C18H20N6OS (368.46): C, 58.68; H, 5.47; N, 22.81. Found: C, 58.50; H,
5.58; N, 22.70.
6.7.2. 5-(3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)-2-(20,30,40,60-tetra-
O-aceyl-
b
-D-galactopyranosylthio)-1,3,4-oxadiazole (15)
6.9. General procedure for synthesizing of 21 and 22
White solid; yield 65%, m.p. 96e98 ꢁC; 1H NMR (500 MHz,
DMSO-d6):
d
0.90, 0.92 (2s, 6H, 2ꢃ CH3), 1.84e1.93 (4s, 12H, 4ꢃ
To a solution of 19 or 20 (10 mmol) in dry DMF (20 mL), NaH
(15 mmol) was added portionwise through 15 min and the solution
stirred at room temperature for another 30 min. Then, a solution of
OAc), 1.96 (m, 1H, CH), 2.81 (d, 2H, J ¼ 6.85 Hz, CH2), 4.20 (m, 2H, 60,
600-H), 4.37 (m, 1H, 50-H), 5.12 (t, 1H, J ¼ 9.5 Hz, 40-H), 5.25 (t, 1H,
J ¼ 9.3 Hz, 30-H), 5.66 (t, 1H, J ¼ 9.5 Hz, 20-H), 6.24 (d, 1H,
20,30,40,60-tetra-O-acetyl-
a-D-glucopyronosyl bromide (9) in DMF
J1 ,2 ¼ 10.39 Hz, 10-H), 7.31e7.89 (m,5H, PheH), 9.11 (s,1H, pyrazole
(10 mL) was dropped within 30 min and the reaction mixture was
stirred at room temperature until completion (TLC, 6e8 h). After
completion, the reaction mixture was poured on water and acidi-
fied with diluted acetic acid. The aqueous phase was extracted with
ethyl acetate (3 ꢃ 20 mL), the combined organic phase was washed
with water dried over anhydrous sodium sulphate. Removal of
solvent gave a residue, which was purified by column chromatog-
raphy using an appropriate solvent system to give compounds 21
and 22, respectively.
0
0
H-5); 13C NMR:
d 19.98e20.80 (4 CH3 CO), 22.79 (CH3), 22.88 (CH3),
28.25 (CH), 36.15(CH2), 61.90 (C06), 68.20(C04), 68.95 (C02), 72.25
(C03), 75.20 (C05), 81.55(C01), 105.79 (pyrazole C4), 119.28 (2C,
AreC), 127.66 (AreC), 129.97(2C, AreC), 139.28 (AreC),130.55
(pyrazole C5), 152.58 (pyrazole C3), 158.66 (oxadiazole C5), 161.55
(oxadiazole C2), 169.70e170.15 (4COCH3). Anal. Calcd. For.
C29H34N4O10S (630.67): C, 55.23; H, 5.43; N, 8.88. Found: C, 55.30;
H, 5.32; N, 8.90.
6.7.3. 5-(3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)-2-[((methylthio)
methyl)thio]-1,3,4-oxadiazole (16)
6.9.1. N-[(3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)methylene]-3-
(20,30,40,60-tetra-O-aceyl-
b-D-glucopyranosylthio)-4H-1,2,4-triazol-
White solid; yield 66%, m.p. 50e52 ꢁC; 1H NMR (500 MHz,
4-amine (21)
DMSO-d6)
d
0.87, 088 (2s, 6H, 2ꢃ CH3), 1.97 (m, 1H, CH), 2.11 (s, 3H,
Compound 21 was purified by column chromatography
CH3), 2.70 (d, 2H, J ¼ 6.9 Hz, CH2), 3.77 (s, 2H, CH2), 7.29e7.86 (m, 5H,
(pet.ether/ethylacetate 8:2, Rf ¼ 0.45). Yield 75%, m.p. 190e192 ꢁC;

M.A. Abu-Zaied et al. / European Journal of Medicinal Chemistry 46 (2011) 229e235
235
1H NMR (500 MHz, DMSO-d6):
d 0.91 (s, 3H, CH3), 0.93 (s, 3H, CH3),
[6] P. Brown, D.J. Best, N.J.P. Broom, R. Cassels, P.J. O’Hanlon, T.J. Mitchell,
N.F. Osborne, J.M. Wilson, J. Med. Chem. 40 (1997) 2563e2570.
[7] M. Harfenist, D.J. Heuser, C.T. Joyner, J.F. Batchelor, H.L. White, J. Med. Chem.
39 (1996) 1857e1863.
[8] C. Chen, C.H. Senanayake, T.J. Bill, R.D. Larsen, T.R. Verhoeven, P.J. Reider,
J. Org. Chem. 59 (1994) 3738e3741.
[9] M. Amir, K. Shikha, Eur. J. Med. Chem. 39 (2004) 535e545.
[10] S. Vardan, H. Smulyan, S. Mookherjee, R. Eich, Clin. Pharmacol. Ther. 34 (1983)
290e296.
[11] H.L. Yale, K. Losee, J. Med. Chem. 9 (1966) 478e483.
[12] C.R.W. Guimaraes, D.L. Boger, W.L. Jorgensen, J. Am. Chem. Soc. 127 (2005)
1737e1743.
1.90e1.94 (4s, 12H, 4ꢃ OAc),1.95 (m, 1H, CH), 2.76 (d, 2H, J ¼ 6.8 Hz,
CH2), 3.90 (m,1H, 50-H), 4.10 (m, 2H, 60, 60-H), 4.92 (t,1H, J ¼ 9.60 Hz,
40-H), 5.06 (t,1H, J ¼ 9.20 Hz, 20-H), 5.42 (t,1H, J ¼ 9.6 Hz, 30-H), 5.73
(d, 1H, J1 ,2 ¼ 9.75 Hz, 10-H), 7.47e7.86 (m, 5H, PheH), 8.87 (s, 1H,
CH]N), 8.97(s, 1H, triazole CH]N), 9.32 (s, 1H, pyrazole H-5). Anal.
Calcd. For. C30H36N6O9S (656.71): C, 54.87; H, 5.53; N, 12.80. Found:
C, 54.70; H, 5.60; N, 12.70.
0
0
6.9.2. 4-[((3-Isobutyl-1-phenyl-1H-pyrazol-4-yl)methylene)
[13] D. Han, X.B. Meng, L.N. Wang, H. Liu, Y. Yao, Z. Wang, Z.J. Yang, Z.M. Liu, Z.J. Li,
Tetrahedron Asymmetry 20 (2009) 399e410.
[14] L. Mishra, M.K. Said, H. Itokawa, K. Takeya, Bioorg. Med. Chem. 3 (1995)
1241e1245.
[15] D. Kumar, S. Sundaree, E.O. Johnson, K. Shah, Bioorg. Med. Chem. Lett. 19
(2009) 4492e4494.
[16] W.S. Li, S.V. More, C.H. Wang, Y.C. Jen, C.F. Yao, T.F. Wang, C.C. Hung, S.C. Jao,
Bioorg. Med. Chem. Lett. 20 (2010) 1148e1152.
[17] P.G. Baraldi, A. Bovero, F. Fruttarolo, R. Romagnoli, M.A. Tabrizi, D. Preti,
K. Varani, P.A. Borea, A.R. Moorman, Bioorg. Med. Chem. 11 (2003)
4161e4169.
[18] M.C. Cardia, L. Corda, A.M. Fadda, A.M. Maccioni, E. Maccioni, A. Plumitallo,
ILFarmaco 53 (1998) 698e708.
[19] A.M. Farag, A.S. Mayhoub, S.E. Barakat, A.H. Bayomi, Bioorg. Med. Chem. 16
(2008) 881e889.
[20] Y. Xia, C.D. Fan, B.X. Zhao, J. Zhao, D.S. Shin, J.Y. Miao, Eur. J. Med. Chem. 43
(2008) 2347e2353.
amino]-6-methyl-3-(20,30,40,60-tetra-O-aceyl-
b-D-
glucopyranosylthio)-1,2,4-triazin-5(4H)-one (22)
Compound 22 was purified by using column chromatography
(pet.ether/ethylacetate 6:1, Rf ¼ 0.35). Yield 78%, m.p. 140e142 ꢁC;
IR (KBr, cmꢀ1
) y 2958, 2866(CH), 1752(OCOCH3), 1698(CO), 1602
(C]N); 1H NMR (500 MHz, CDCl3):
d 0.94 (s, 3H, CH3), 0.96 (s, 3H,
CH3), 2.00e2.08 (m, 13H, 4ꢃ OAc, CH), 2.33 (s, 3H, CH3), 2.73 (d, 2H,
J ¼ 6.90 Hz, CH2), 3.92 (m, 1H, 50-H), 4.11 (dd, 1H, J ¼ 2.30, 12.30 Hz,
60-H), 4.24 (m, 1H, 600-H), 5.20 (t, 1H, J ¼ 9.60 Hz, 40-H), 5.35 (t, 1H,
J ¼ 9.3 Hz, 20-H), 5.70 (t, 1H, J ¼ 9.50 Hz, 30-H), 6.81 (d, 1H,
J1,2 ¼ 9.20 Hz, 10-H), 7.43e7.68 (m, 5H, PheH), 8.23 (s, 1H, CH]N),
8.44 (s, 1H, pyrazole H-5). 13C NMR:
d 17.17 (CH3), 20.66e20.82
(4CH3CO), 22.62 (2C, 2CH3), 28.80 (CH), 36.21(CH2), 61.72 (C06),
67.93 (C04), 68.98 (C02), 73.96 (C03), 74.55 (C05), 86.78 (C01), 115.32
(pyrazole C4), 119.62 (2C, AreC), 127.48 (AreC), 129.40 (2C, AreC),
129.65 (pyrazole C5), 139.30 (AreC), 146.55, 148.92, 155.35, 164.85,
169.07e170.21 (4 COCH3), 170.66 (CO). MS, m/z (%): 698 [Mþ] (1),
212 (40), 183 (100), 77 (14). Anal. Calcd. For. C32H38N6O10S (698.74):
C, 55.00; H, 5.48; N, 12.03. Found: C, 55.10; H, 5.60; N, 12.20.
[21] F. Bozzo, A. Bassignana, L. Lazzarato, D. Boschi, A. Gasco, C. Bocca, A. Miglietta,
Chem. Biol. Interact. 182 (2009) 183e190.
[22] G.H. Elgemeie, M.M. Hussein, S.A. Al-Khursani, J. Carbohydr. Chem. 23 (2004)
465e481.
[23] G.H. Elgemeie, W.A. Zaghary, K.M. Amin, T.M. Nasr, J. Carbohydr. Chem. 27
(2008) 373e378.
[24] G.H. Elgemeie, W.A. Zaghary, K.M. Amin, T.M. Nasr, J. Carbohydr. Chem. 28
(2009) 161e178.
[25] G.H. Elgemeie, E.H. Eltammy, I.I. Elgawad, N.M. Mahmoud, Synth. Commun. 39
(2009) 443e458.
[26] H.N. Hafez, A.B. El-Gazzar, G.A.M. Nawwar, Eur. J. Med. Chem. 45 (2010)
1485e1493.
References
[27] E.S.M. Yakout, Y.A. Allam, G.A.M. Nawwar, Heteroatom Chem. 10 (1999)
177e182.
[28] A.M. Attia, G.H. Elgemeie, Synth. Commun. 33 (2003) 2243e2255.
[29] I.W. Still, N. Plavac, T.M. Mackinnon, M.S. Chauban, Can. J. Chem. 54 (1976)
280e289.
[1] J. Li, L.Z. Xu, K.L. He, W.J. Juo, Y.H. Zheng, P. Xia, Y. Chen, Breast Cancer Res. 3
(2001) 253e263.
[2] J. Engel, R. Eckel, J. Kerr, M. Schmidt, G. Furstenberger, R. Richter, H. Sauer,
H.J. Senn, Ital. J. Anat. Embryol. 106 (2001) 59e68.
[30] L. Stefaniak, Org. Magn. Reson. 12 (1979) 379e382.
[31] P. Skehan, R. Strong, D. Scadiaro, A. Monks, J. Mc-Mahon, D. Vistica,
J.T. Warren, H. Bokesch, S. Kenney, M.R. Boyed, J. Natl. Cancer Inst. 82 (1990)
1107e1112.
[3] N. Uchiyama-Kokubu, T. Watanabe, Anticancer Drugs 12 (2001) 769e779.
[4] I.A. Cree, L. Knight, F. Di Nicolantonio, S. Sharma, T. Gulliford, Curr. Opin.
Investig. Drugs 3 (2002) 634e640.
[5] I.F. Faneyte, P.M.P. Kristel, M.J. van de Vijver, Intl. J. Cancer 93 (2001) 114e122.