Novel pyrrolidinone and pyrazolo[1,5-a][1,3,5]triazine derivatives bearing a biologically active sulfamoyl moiety as a new class of antitumor agents

Article abstract of DOI:10.1007/s00706-011-0517-3

Sulfonamides possess many types of biological activities and have recently been reported to show substantial antitumor activity in vitro and/or in vivo. There are a variety of mechanisms for the anticancer activity. The present work reports the synthesis

Full text of DOI:10.1007/s00706-011-0517-3

Monatsh Chem (2011) 142:935–941
DOI 10.1007/s00706-011-0517-3
ORIGINAL PAPER
Novel pyrrolidinone and pyrazolo[1,5-a][1,3,5]triazine derivatives
bearing a biologically active sulfamoyl moiety as a new class
of antitumor agents
Hala M. Aly
Received: 31 May 2010 / Accepted: 2 May 2011 / Published online: 8 June 2011
Ó Springer-Verlag 2011
Abstract Sulfonamides possess many types of biological
activities and have recently been reported to show sub-
stantial antitumor activity in vitro and/or in vivo. There are
a variety of mechanisms for the anticancer activity. The
present work reports the synthesis of some novel pyrrole,
oxopyrrole, and related pyrroloacetamide derivatives,
hydrazones, aminopyrazolinone, thiocarbamoyl, and pyr-
azolo[1,5-a][1,3,5]triazine derivatives bearing a substituted
sulfonamide moiety. All newly synthesized compounds
were evaluated for their in vitro anticancer activity against
the breast cancer cell line MCF7. Most of the screened
compounds showed interesting cytotoxic activities com-
pared with doxorubicin as a reference drug.
representatives of this class of pharmacological agents are
widely used in the clinic as antibacterial [1], hypoglycemic
[2], diuretic, anticarbonic anhydrase [3–5], and antithyroid
[6] drugs. Recently, a host of structurally novel sulfon-
amide derivatives were reported to show substantial
antitumor activity in vitro and/or in vivo [7–11]. The
pyrrole nucleus and pyrrolo[2,3-d]pyrimidine play a vital
role in many biological activities [12], and aminopyrazoles
possess a wide variety of biological activities [13–16].
Also, 3-aminopyrazole was used as a starting material for
the synthesis of some novel purine analogues such as
pyrazolo[1,5-a]pyrimidine. Substituted pyrazolo[1,5-a]-
pyrimidines were prepared as anticancer, antipyretic,
hypotensive, and anxiolytic agents [17]. In addition, pyr-
azolotriazines are applied as herbicides [18]. Having the
above facts in mind and in continuation of my effort to
synthesize heterocyclic compounds containing a sulfon-
amide moiety [7, 8, 19], I was interested in the synthesis of
novel 3-aminopyrazole and pyrazolo[1,5-a][1,3,5]triazine
derivatives containing a sulfonamide moiety to investigate
their anticancer activity.
Keywords Pyrrolidinone Á Aminopyrazolinone Á
Pyrazolo[1,5-a][1,3,5]triazine Á Antitumor activity
Introduction
Cancer is becoming the biggest health hazard for the world.
The development of resistance against existing anticancer
drugs highlights the importance of finding new anticancer
molecules. However, it is rather difficult to search for a
molecule that can selectively inhibit the proliferation of
abnormal cells with only minimal or no effect on normal
cells. Therefore, the search for anticancer agents continues
in various laboratories worldwide.
Results and discussion
Chemistry
A series of novel pyrrole, oxopyrrole, and related
pyrroloacetamide derivatives, hydrazones, aminopyrazoli-
none, thiocarbamoyl, and pyrazolo[1,5-a][1,3,5]triazine
derivatives bearing a substituted sulfonamide moiety were
synthesized (Schemes 1, 2, 3, 4) and biologically evaluated
for their in vitro antitumor activity. The starting material
N4-chloroacetylsulfaphenazole (2) was synthesized from
4-amino-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide
Different authors have reported several sulfonamides
possessing many types of biological activities and
H. M. Aly (&)
Department of Chemistry, Faculty of Science (Girl’s),
Al-Azhar University, Nasr City, Cairo, Egypt
e-mail: hala_mali@yahoo.com
123

936
H. M. Aly
Scheme 1
Cl
Cl
O
O
S
O
1
O
S
O
Cl
N
NH
NH2
N
NH
N
H
DMF
N
N
O
Ph
Ph
2
OCH3
CN
DMF/
K2CO3
H2N
CN
OCH3
O
S
O
C
O
S
H
N
H
N
O
N
NH
CH2 NH
N
NH
N
N
O
O
N
C
Ph
Ph
3
4
CN
O
O
N
N
NH
S
N
O
Ph
H2N
CN
5
(sulfaphenazole, 1) by acetylation with chloroacetyl chlo-
ride [19]. The reactivity of N4-chloroacetylsulfaphenazole
(2) towards some nitrogen nucleophiles and active meth-
ylene compounds was investigated. Thus, the nucleophilic
substitution reaction of 2 with m-anisidine in the presence
of anhydrous potassium carbonate in absolute ethanol
under reflux afforded the acetamide derivative 3. The
structure of this compound was deduced from elemental
analysis and spectral data. The 1H NMR spectrum of
compound 3 revealed singlet signals at d = 3.6, 5.9, and
6.3 ppm corresponding to methoxy and pyrazole protons
and three singlet signals at d = 8.0, 8.20, and 10.9 ppm
due to three NH protons. These three NH groups have three
N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide (7,
Scheme 2).
Attempts to cyclize the hydrazide derivative 7 by
refluxing in ethanol containing piperidine and/or pyridine
to afford pyrrolo[2,3-d]pyrimidine derivative 8 were
unsuccessful on the basis of analytical and spectral data.
The 1H NMR spectrum of compound 7 (DMSO-d6)
exhibited the following signals: 4.3 (s, 2H, CH2), 5.8, 6.6
(2 s, 2H, pyrazole-H), 7.0–8.3 (m, 10H, Ar–H ? NH), 9.4
(s, H, CH=N), 10.8 (s, 1H, SO2NH), 11.3 (s, 2H, NH2). The
mass spectrum of compound 7 had a molecular ion peak at
m/z = 464 [M ? 2] and a base peak at m/z = 77. Its IR
spectrum was characterized by the presence of NH2
absorption bands. Compound 5 reacted with acetic anhy-
dride to afford the product of direct acetylation (9) rather
bands in the IR spectrum at 3,350, 3,223, and 3,193 cm-1
;
an intense band at 1,735 cm-1 was due to the carbonyl
group. The mass spectrum of 3 revealed a molecular ion
peak at m/z = 477 [M ? 2] and a base peak at m/z = 281.
4-(5-Amino-4-cyano-2-oxo-2,3-dihydropyrrol-1-yl)-N-
(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide (5) was
prepared according to the previously reported procedure [19]
(Scheme 1). The o-aminonitrile function of compound 5
was exploited to synthesize some new oxopyrrole
and related pyrroloacetamide derivatives containing a
sulfonamide moiety. Thus, the alkylation reaction of pyr-
rolidinone derivative 5 with excess triethyl orthoformate led
to the formation of 4-[4-cyano-5-(ethoxymethyleneamino)-
2,3-dihydro-2-oxo-1H-pyrrol-1-yl]-N-(1-phenyl-1H-pyra-
zol-5-yl)benzenesulfonamide (6), whose hydrazinolysis
with hydrazine hydrate gave 4-[4-cyano-5-(hydrazi-
nylmethyleneamino)-2,3-dihydro-2-oxo-1H-pyrrol-1-yl]-
than the expected compound 10, which was ruled out due
-1
ꢀ
to the presence of a C:N band at m = 2,200 cm in the
IR spectrum of the product in addition to the H NMR
1
spectrum showing a signal due to the acetyl group which
appeared at d = 2.45 ppm (Scheme 2). Diazotization
of sulfaphenazole (1) with sodium nitrite and hydrochlo-
ric acid at 5–10 °C followed by coupling with the
corresponding nucleophiles such as active methylene
compounds (namely, malononitrile and ethyl cyanoacetate)
[19] furnished the novel hydrazones 11a and 11b, respec-
tively (Scheme 3). The reactions of hydrazones 11a, 11b
with hydrazines (namely, hydrazine hydrate and thiosem-
icarbazide) were studied with the aim of forming pyrazole
derivatives with potential biological activities [7, 19].
Thus, hydrazinolysis of hyrazo derivative 11b with
123

Novel pyrrolidinone and pyrazolo[1,5-a][1,3,5]triazine derivatives
937
Scheme 2
O
S
CH(OC2H5)3
O
N
N
NH
N
O
Ph
C2H5OCH
N
CN
6
NH2NH2.H2O
O
S
O
5
O
O
N
N
N
NH
N
NH S
O
N
N
O
N
Ph
Ph
H2N-HNCH
N
CN
N
NH
7
8
NH2
O
O
N
NH
S
N
N
O
H3C
Ph
C
O
HN
(CH3CO)2O
CN
9
O
O
N
NH
S
N
N
O
N
Ph
H3C
N
H
O
10
Scheme 3
O
S
O
1
O
S
NaNO2/HCl
N
NH
NH2
N
NH
N2Cl
N
N
O
Ph
Ph
X
EtOH
CH3COONa H2C
Y
O
O
X
Y
X
Y
H
C
H
N
NH
S
N
N
N
NH
S
N C
N
N
N
O
O
Ph
Ph
11a,
X=Y= CN
11b, X=CN, Y=COOC2H5
N2H4.H2O
11b
EtOH
O
NH2
N
NH
S
N
N
N
O
NH
O
N
H
Ph
12
123

938
H. M. Aly
Scheme 4
S
O
S
H2NHN C NH2
NH2
NH2
N
11a
N
NH
N
N
N
O
HN
N
Ph
H
13
S
(CH3CO)2O
CH(OC2H5)3
O
O
N
CH3
NH
N
S
N
NH
S
N
N
N
NH
S
N
N
N
N
O
N
O
N
NH
H2N
N
Ph
H2N
N
Ph
14
S
15
hydrazine hydrate in ethanol under reflux followed by
cyclocondensation afforded 3-amino-5-pyrazolinone
its mass spectrum showed a molecular ion peak at
m/z = 492 [M?] and a base peak at m/z = 159.
derivative 12 (Scheme 3). The structure of the latter
product was confirmed on the basis of analytical and
spectral data. Its IR spectrum showed the following
absorption bands: 3,310, 3,305 (NH2/NH), 1,720 (C=O),
and 1,360, 1,187 cm-1 (SO2).
In vitro antitumor screening test
Doxorubicin, the reference drug used in this study, is one
of the most effective antitumor agents used to produce
regression in acute leukemias, Hodgkin’s disease, and other
lymphomas. The cytotoxic effects of the newly synthesized
compounds were evaluated in the MCF7 breast cancer cell
line and doxorubicin (AdriablastinaÒ) was used as a ref-
erence drug in three different concentrations. The primary
anticancer assay was performed in accordance with the
protocol of the Drug Evaluation Branch, the National
Institute of Cancer, Cairo, Egypt [20]. The relationship
On the other hand, the iminopyrazole derivative 13 was
successfully obtained by fusing the hyrazone derivative
11a with thiosemicarbazide in dioxane containing a cata-
1
lytic amount of triethylamine (Scheme 4). The H NMR
spectrum of compound 13 (DMSO-d6) exhibited the fol-
lowing signals: 5.5, 6.0 (2 s, 2H, pyrazole-H), 6.54, 6.67
(2 s, 4H, 2NH2), 7.0–7.7 (m, 10H, Ar–H ? NH), 10.5
(s, 1H, SO2NH) ppm. Its 13C NMR spectrum revealed
15 carbon types with the most important signals at
d = 177.00, characteristic for C=S, and 60.32, due to C-4
of the newly formed pyrazole ring. The IR spectrum of 13
showed the following absorption bands: 3,450, 3,354,
3,209 (NH2), 3,031 (CH-arom.), 1,330, 1,167 (SO2), 1,088
(C=S) cm-1. Its mass spectrum displayed a molecular ion
peak at m/z = 482 [M?] corresponding to the molecular
formula C19H18N10O2S2 with a base peak at m/z = 164.
The iminopyrazole derivative 13 proved to be a versatile
starting material for the synthesis of some novel pyrazolo
[1,5-a][1,3,5]triazines. Thus, treatment of thiocarbamoyl
derivative 13 with acetic anhydride afforded the pyra-
zolotriazine 14. The structure of compound 14 was con-
firmed on the basis of analytical and spectral data. Its mass
spectrum showed a molecular ion peak at m/z = 506 [M?]
and a base peak at m/z = 138. Finally, cyclocondensation
of compound 13 with triethyl orthoformate as carbon donor
moiety under reflux yielded another novel pyrazolo[1,5-a]-
[1,3,5]triazine derivative 15. The structure of compound 15
was proved by the presence of a signal at d = 8.21 ppm
characteristic for the triazine proton in the 1H NMR
spectrum. The IR spectrum showed the disappearance of
the characteristic bands for one amino functional group and
Table 1 In vitro cytotoxic activity of some selected synthesized
compounds
Compd.
Non-viable cells (%)
IC50
(lg cm-3
)
Concentration (lg cm-3
)
100
50
25
10
2
0
0
0
0
0
0
0
0
[100a
[100a
2.14
[100a
2.48
[100a
3.02
[100a
1.88
3
5
24
0
20
0
52
0
78
0
6
7
21
0
22
0
51
0
81
0
9
11a
11b
12
13
14
15
27
0
31
0
59
0
81
0
21
29
0
19
38
0
43
55
0
67
72
0
2.95
[100a
[100a
51.0
0
0
0
0
Doxorubicin (reference) 40
51
54
58
a
IC50 [ 100 lg cm-3 is considered to be inactive
123

Novel pyrrolidinone and pyrazolo[1,5-a][1,3,5]triazine derivatives
939
7.4–8.2 (m, Ar–H ? 2NH), 10.9 (s, NHSO2) ppm; 13C
NMR (300 MHz, DMSO-d6): d = 50.12 (CH2, aliphatic),
55.05 (CH3, aliphatic), 91.57 (pyrazole-4-CH), 105.62,
110.85, 121.78, 125.05, 127.55, 127.85, 129.11, 130.59,
135.70, 138.09, 139.23, 140.26, 140.69, 141.19, 142.54
(15C, aromatic carbons), 146.78 (pyrazole-3-CH), 169.32
(C=O, amide carbon) ppm; IR (KBr): ꢀm = 3,350, 3,223,
3,193 (3NH), 3,050 (CH-arom.), 2,968 (CH-aliph.), 1,735
(C=O), 1652 (C=N), 1,388, 1,150 (SO2) cm-1; MS (70 eV):
m/z = 477 ([M ? 2]?, 31), 281 (100). Calcd. C 60.36, H
4.85, N 14.67, S 6.71. Found: C 60.23, H 4.55, N 14.87, S
6.31.
between surviving fraction and drug concentration was
plotted to obtain the survival curve of the MCF7 breast
cancer cell line. The response parameter calculated was the
IC50 value which corresponds to the compound concen-
tration causing 50% mortality in net cells as depicted in
Table 1. The results showed that among the compounds
tested in this study, 5, 7, 11a, 12, and 13, all of them
containing a sulfonamide moiety, exhibit the highest in
vitro antitumor activity against MCF7 and are more
effective than the positive control (doxorubicin) with IC50
values of 2.14, 2.48, 3.02, 1.88, and 2.95 lg cm-3
,
whereas the other compounds are inactive.
4-[4-Cyano-5-(ethoxymethyleneamino)-2,3-dihydro-2-oxo-
1H-pyrrol-1-yl]-N-(1-phenyl-1H-pyrazol-5-yl)benzenesul-
fonamide (6, C23H20N6O4S)
Conclusions
Compound 5 (840 mg, 2 mmol) in excess triethyl ortho-
formate (60 cm3) was heated under reflux for 5 h. The
reaction mixture was cooled and triturated with cold
ethanol, and the product was separated, collected by
filtration, washed with petroleum ether (40–60 °C), and
recrystallized from ethanol to give 6 (70%) as a brown
This study reports the synthesis of pyrrolidinone 5, cya-
nohydrazinylmethyleneaminone 7, hydrazone 11a, 3-
amino-5-pyrazolinone 12, and thiocarbamoyl 13, which all
contain a biologically active sulfonamide moiety. These
compounds exhibit the highest in vitro antitumor activity
against MCF7 and are more effective than the positive
control doxorubicin. On the other hand compounds 2, 3, 6,
9, 11b, 14, and 15 showed no activity.
1
powder. M.p.: 242 °C; H NMR (300 MHz, DMSO-d6):
d = 1.02 (t, 3H, CH2CH3, J = 6.6 Hz), 3.4 (q, 2H,
CH2CH3, J = 6.6 Hz), 4.1 (s, 2H, CH2), 5.8, 6.4 (2 s,
2H, pyrazole-H), 7.4-8.1 (m, 9H, Ar–H), 9.6 (s, H, CH=N),
10.2 (s, 1H, SO2NH) ppm; 13C NMR (300 MHz, DMSO-
d6): d = 10.9 (CH3, aliphatic), 19.88 (CH2, aliphatic),
29.66 (pyrrole-3-CH2), 75.79 (pyrrole-4-C–CN), 90.98
(pyrazole-4-CH), 117.09 (C:N), 120.30, 121.90, 127.50,
127.50, 129.40, 136.49, 138.20, 139.79, 140.53 (9C,
aromatic carbons), 146.52 (pyrazole-3-CH), 162.98
(N=CHCH2CH3), 163.78 (pyrrole-5-C–N=CHCH2CH3),
Experimental
Melting points were determined on a Stuart melting point
apparatus. IR spectra were recorded on a Shimadzu-440 IR
spectrophotometer using the KBr technique (Shimadzu,
Japan). NMR spectra were measured on a Bruker Avance
300 (300 MHz) in DMSO-d6 as a solvent, using tetra-
methylsilane (TMS) as an internal standard. The mass
spectra were recorded on Shimadzu GCMS-QP-1000EX
mass spectrometers at 70 eV. Elemental analyses were
carried out by the Microanalytical Research Centre, Cairo
University. Analytical results for C, H, N, and S were
within 0.4% of the calculated values. All reagents were
of commercial quality and used without purification.
Compounds 2, 5, 11a, and 11b were prepared according to
the procedure reported in the literature [19].
ꢀ
168.99 (C=O) ppm; IR (KBr): m = 3,243 (NH), 3,065
(CH-arom.), 2,996 (CH-aliph.), 2,218 (C:N), 1,683
(C=O), 1,636 (C=N), 1,376, 1,161 (SO2) cm-1. Calcd. C
57.97, H 4.23, N 17.64, S 6.73. Found: C 58.17, H 3.93, N
17.74, S 7.03.
4-[4-Cyano-5-(hydrazinylmethyleneamino)-2,3-dihydro-2-
oxo-1H-pyrrol-1-yl]-N-(1-phenyl-1H-pyrazol-5-yl)ben-
zenesulfonamide (7, C21H18N8O3S)
To a solution of 238 mg 6 (0.5 mmol) in 50 cm3 dioxane
was added 5 cm3 hydrazine hydrate (100 mmol) and the
reaction mixture was stirred for 30 min. The solid obtained
was recrystallized from ethanol to give 7 (75%) as yellowish
2-(3-Methoxyphenylamino)-N-[4-[N-[(1-phenyl-1H-pyrazol-
5-yl)amino]sulfonyl]phenyl]acetamide
(3, C24H23N5O4S)
1
brown solid. M.p.: 232 °C; H NMR (300 MHz, DMSO-
A suspension of 391 mg 2 (1 mmol) and 113 mg m-anisi-
dine (1 mmol) in 60 cm3 dioxane containing three drops of
triethylamine was heated under reflux for 2 h. The reaction
mixture was poured into crushed ice/water and acidified
with 2 N HCl. The solid product was collected and
recrystallized from dioxane to give pale yellow crystals of
3 (81%). M.p.:[350 °C; 1H NMR (300 MHz, DMSO-d6):
d = 3.6 (s, OCH3), 4.3 (s, CH2), 5.9, 6.3 (2 s, pyrazole-H),
d6): d = 4.3 (s, CH2), 5.8, 6.6 (2 s, pyrazole-H), 7.0–8.3
(m, Ar–H ? NH), 9.4 (s, CH=N), 10.8 (s, SO2NH), 11.3
(s, NH2) ppm; 13C NMR (300 MHz, DMSO-d6): d = 29.76
(pyrrole-3-CH2), 75.83 (pyrrole-4-C–CN), 91.58 (pyrazole-
4-CH), 117.12 (C:N), 121.78, 125.49, 127.20, 127.29,
129.18, 135.89, 138.20, 140.49, 141.13 (9C, aromatic
carbons), 146.22 (pyrazole-3-CH), 162.78 (N=CHNHNH2),
123

940
H. M. Aly
1
163.33 (pyrrole-5-C–N=CHNHNH2), 169.35 (C=O) ppm;
ꢀ
13 (82%). M.p.: 241 °C; H NMR (300 MHz, DMSO-d6):
d = 5.5, 6.0 (2 s, pyrazole-H), 6.54, 6.67 (2 s, 2NH2), 7.0–
7.7 (m, Ar–H ? NH), 10.5 (s, SO2NH) ppm; 13C NMR
(300 MHz, DMSO-d6): d = 60.32 (pyrazole-4-CH), 91.30,
121.33, 125.48, 127.35, 127.92, 129.50, 135.50, 138.25,
140.85, 141.32, 146.89, 164.04 (12C, aromatic car-
bons), 165.08 (pyrazole-5-C=NH), 177.00 (C=S) ppm;
IR (KBr): m = 3,336, 3,310, 3,109 (NH ? NH2), 3,068
(CH-arom.), 2,919 (CH-aliph.), 2,203 (C:N), 1,623 (C=O),
1,659 (C=N), 1,383, 1,156 (SO2) cm-1; MS (70 eV):
m/z = 464 ([M ? 2]?, 2.65), 77 (100). Calcd. C 54.54, H
3.92, N 24.23, S 6.93. Found: C 54.34, H 3.80, N 23.83, S
6.83.
ꢀ
IR (KBr): m = 3,450, 3,354, 3,209 (NH2), 3,031 (CH-arom.),
1,330, 1,167 (SO2), 1,088 (C=S) cm-1; MS (70 eV):
m/z = 482 (M?, 22), 164 (100). Calcd. C 47.29, H 3.76, N
29.03, S 13.29. Found: C 47.49, H 3.86, N 29.43, S 13.59.
N-[3-Cyano-4,5-dihydro-5-oxo-1-[4-[N-[(1-phenyl-1H-
pyrazol-5-yl)amino]sulfonyl]phenyl]-1H-pyrrol-2-yl]-
acetamide (9, C22H18N6O4S)
Compound 5 (588 mg, 1.4 mmol) was refluxed in 50 cm3
acetic anhydride for 3 h. The product was precipitated,
collected, and recrystallized from benzene to give yellow
4-[2-(7-Amino-3,4-dihydro-2-methyl-4-thioxopyrazolo-
[1,5-a][1,3,5]triazin-8-yl)diazenyl]-N-(1-phenyl-1H-pyra-
zol-5-yl)benzenesulfonamide (14, C21H18N10O2S2)
Pyrazole 13 (482 mg, 1 mmol) was refluxed in 80 cm3
acetic anhydride for 3 h. The solid product was
precipitated, collected, and recrystallized from N,N-
dimethylformamide (DMF) to give 14 (62%) as a white
1
needles of 9 (80%). M.p.: 202 °C; H NMR (300 MHz,
DMSO-d6): d = 2.45 (s, COCH3), 5.9, 6.8 (2 s, pyrazole-
H), 7.1–7.9 (m, Ar–H ? NH), 9.9 (s, SO2NH) ppm; 13C
NMR (300 MHz, DMSO-d6): d = 23.42 (CH3, aliphatic),
30.25 (pyrrole-3-CH2), 58.33 (pyrrole-4-C–CN), 91.65
(pyrazole-4-CH), 117.50 (C:N), 121.72, 125.23, 127.50,
127.25, 129.32, 135.89, 138.45, 140.39, 141.02 (9C,
aromatic carbons), 146.19 (pyrazole-3-CH), 162.78 (pyr-
role-5-C–NHCOCH3), 166.15, 169.95 (2 C=O) ppm; IR
1
powder. M.p.: 282 °C; H NMR (300 MHz, DMSO-d6):
d = 0.9 (s, CH3), 5.8, 6.4 (2 s, pyrazole-H), 6.5 (s, NH2),
7.4–7.7 (m, Ar–H ? NH) ppm; 13C NMR (300 MHz,
DMSO-d6): d = 23.99 (CH3, aliphatic), 88.6 (pyrazolotri-
azine-8-C), 91.32, 120.05, 121.20, 125.42, 127.27, 127.71,
129.61, 130.90, 135.55, 138.33, 140.79, 145.15, 152.89,
168.29 (14C, aromatic carbons), 188.12 (C=S) ppm; IR
ꢀ
(KBr): m = 2,200 (C:N), 1,710 (C=O), 1,320, 1,146
(SO2) cm-1. Calcd. C 57.13, H 3.92, N 18.17, S 6.93.
Found: C 57.43, H 4.12, N 18.47, S 7.23.
ꢀ
(KBr): m = 3,234, 3,159 (NH2), 3,010 (CH-arom.), 1,342,
1,149 (SO2), 1,085 (C=S) cm-1; MS (70 eV): m/z = 506
(M?, 34), 138 (100). Calcd. C 49.79, H 3.58, N 27.65, S
12.66. Found: C 49.99, H 3.88, N 28.05, S 12.86.
4-[2-(3-Amino-2,5-dihydro-5-oxo-1H-pyrazol-4-yl)diaze-
nyl]-N-(1-phenyl-1H-pyrazol-5-yl)benzenesulfonamide
(12, C18H16N8O3S)
A mixture of 438 mg 11b (1 mmol) and 500 mg hydrazine
hydrate (10 mmol) in 50 cm3 ethanol was heated under
reflux for 2 h. The solid product which formed on heating
was collected and recrystallized from ethanol to give 12
(64%) as a white powder. M.p.: 238 °C; 1H NMR
(300 MHz, DMSO-d6): d = 5.9, 6.5 (2 s, pyrazole-H),
4-[2-(7-Amino-3,4-dihydro-4-thioxopyrazolo[1,5-a][1,3,5]-
triazin-8-yl)diazenyl]-N-(1-phenyl-1H-pyrazol-5-yl)-
benzenesulfonamide (15, C20H16N10O2S2)
Pyrazole 13 (482 mg, 1 mmol) was refluxed with 70 cm3
triethyl orthoformate for 10 h. The excess of reagent was
removed and ether was added to the cold mixture. The
obtained solid was filtered and recrystallized from ethanol to
give 15 (86%) as a green powder. M.p.:[350 °C; 1H NMR
(300 MHz, DMSO-d6): d = 5.9, 6.2 (2 s, pyrazole-H), 6.9
(s, NH2), 7.4–7.9 (m, Ar–H), 8.20 (s, CH triazine), 11.3 (s,
SO2NH) ppm; 13C NMR (300 MHz, DMSO-d6): d = 88.9
(pyrazolotriazine-8-C), 91.20, 120.36, 121.28, 125.52,
127.47, 127.77, 129.65, 130.85, 135.55, 138.35, 140.81,
145.35, 145.89, 152.39 (14C, aromatic carbons), 188.02
6.4 (s, NH2), 7.5–7.7 (m, Ar–H), 10.9 (s, SO2NH) ppm; 13
C
NMR (300 MHz, DMSO-d6): d = 77 (NH-N–C, 3-amino-
pyrazole), 92.55 (pyrazole-4-CH), 120.58, 125.99, 127.69,
127.89, 129.78, 131.80, 139.05, 140.00, 140.89 (9C,
aromatic carbons), 143.99 (pyrazole-3-CH), 166.5 (ketonic
ꢀ
carbon), 173.10 (C-NH2) ppm; IR (KBr): m = 3,310, 3,305
(NH2/NH), 1,720 (C=O), 1,360, 1,187 (SO2) cm-1. Calcd.
C 50.94, H 3.80, N 26.40, S 7.55. Found: C 50.74, H 3.70,
N 26.00, S 7.65.
ꢀ
(C=S) ppm; IR (KBr): m = 3,200, 3161 (NH2), 3,020 (CH-
arom.), 1,332, 1,146 (SO2), 1,080 cm-1 (C=S); MS (70 eV):
m/z = 492 (M?, 16), 159 (100). Calcd. C 48.77, H 3.27, N
28.44, S 13.02. Found: C 49.17, H 3.37, N 28.54, S 13.32.
3-Amino-5-imino-4-[2-[4-[N-[(1-phenyl-1H-pyrazol-5-yl)-
amino]sulfonyl]phenyl]diazenyl]-1H-pyrazole-2(5H)-
carbothioamide (13, C19H18N10O2S2)
A mixture of 391 mg hydrazone 11a (1 mmol), 91 mg
thiosemicarbazide (1 mmol), and 0.5 cm3 triethylamine in
50 cm3 dioxane was heated under reflux for 12 h. The solid
product which formed on heating was collected and
recrystallized from ethanol to give off-white crystals of
Antitumor activity (in vitro study)
The potential in vitro cytotoxic activity of the newly syn-
thesized compounds was measured using the Sulfo-
123

Novel pyrrolidinone and pyrazolo[1,5-a][1,3,5]triazine derivatives
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Rhodamine-B stain (SRB) assay by the method of Skehan
et al. [20]. The in vitro anticancer screening was done by
the pharmacology unit at the National Cancer Institute,
Cairo University. Cells were plated in 96-multiwell
microtiter plates (104 cells/well) for 24 h before treatment
with the compounds to allow attachment of cell to the wall
of the plate. Test compounds were dissolved in DMSO and
diluted with saline to the appropriate volume. Different
concentrations of the compound under test (0, 1, 2.5, 5, and
10 mg cm-3) were added to the cell monolayer. Triplicate
wells were prepared for each individual dose. Monolayer
cells were incubated with the compounds for 48 h at 37 °C
and in an atmosphere of 5% CO2. After 48 h, cells were
fixed, washed, and stained for 30 min with 0.4% (wt/vol)
with SRB dissolved in 1% acetic acid. Excess unbound dye
was removed by four washes with 1% acetic acid and
attached stain was recovered with Tris–EDTA buffer.
Color intensity was measured in an ELISA reader. The
relation between surviving fraction and drug concentration
is plotted to get the survival curve for the breast tumor cell
line after the specified time. The molar concentration
required for 50% inhibition of cell viability (IC50) was
calculated and the results are given in Table 1.
References
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Eur J Med Chem 45:118
123