Synthesis and antitumor activity of new sulfonamide derivatives of thiadiazolo[3,2-a]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2011.05.037

New series of sulfonamide derivatives of [1,3,4]thiadiazolo[3,2-a] pyrimidine were synthesized and investigated as antitumor agents. Some of the newly prepared compounds were tested for their in vitro and in vivo antitumor activities. Preliminary biological studies revealed that compounds 4c, 4f, and 4j exhibited the highest affinity to DNA, while compounds 4h,i, 6a-c, 8 and 12-14 exhibited moderate activity. Also, compounds 4j, 4f and 4c showed the highest percentage increase in lifespan of mice inoculated with Ehrlich ascites cells over 5-flurouracil (positive control). The detailed synthesis, spectroscopic and biological data are reported.

Full text of DOI:10.1016/j.ejmech.2011.05.037

European Journal of Medicinal Chemistry 46 (2011) 3714e3720
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antitumor activity of new sulfonamide derivatives of thiadiazolo
[3,2-a]pyrimidines
*
Nadia S. El-Sayed, Eman R. El-Bendary , Saadia M. El-Ashry, Mohammed M. El-Kerdawy
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
New series of sulfonamide derivatives of [1,3,4]thiadiazolo[3,2-a]pyrimidine were synthesized and
investigated as antitumor agents. Some of the newly prepared compounds were tested for their in vitro
and in vivo antitumor activities. Preliminary biological studies revealed that compounds 4c, 4f, and 4j
exhibited the highest affinity to DNA, while compounds 4h,i, 6aec, 8 and 12e14 exhibited moderate
activity. Also, compounds 4j, 4f and 4c showed the highest percentage increase in lifespan of mice
inoculated with Ehrlich ascites cells over 5-flurouracil (positive control). The detailed synthesis, spec-
troscopic and biological data are reported.
Received 11 December 2010
Received in revised form
10 April 2011
Accepted 14 May 2011
Available online 30 May 2011
Keywords:
Ó 2011 Elsevier Masson SAS. All rights reserved.
Sulfonamide derivatives
[1,3,4]Thiadiazolo[3,2-a]pyrimidines
DNA-binding affinity
Antitumor activity
1. Introduction
annelated pyrimidines have attracted a great deal of interest owing
to their medicinal activities [26e28]. These medicinal activities
Sulfonamides constitute an important class of drugs. They
possess various types of pharmacological activities such as anti-
bacterial [1e3], anti-carbonic anhydrase [4,5], high-ceiling diuretic
[6], hypoglycemic [7,8], antithyroid [7], anti-inflammatory [9], and
antiglaucoma [6,7]. It is also known that aryl/heteroaryl sulfon-
amides may act as antitumor agents through perturbation of cell
cycle in the G1 phase, distribution of microtubule assembly or
angiogenesis inhibition [10e13]. Moreover, numerous sulfon-
amides were found to act as antitumor agents through carbonic
anhydrase (CA) inhibition [13e16]. It has been shown that two
carbonic anhydrase isozymes (CA IX and CA XII) are prominently
associated with, and over expressed, in many tumors [17,18]. They
are involved in crucial processes connected with cancer progres-
sion and response to therapy [19e21]. Aromatic or heteroaromatic
sulfonamides have been shown to reverse the effect of tumor
acidification, consequently they inhibit the growth of cancer cells
and suppress tumor invasion mediated by the carbonic anhydrases
[22e25]. These compounds contain the classical recognition frag-
ment (AreSO2NH2) that coordinates the carbonic anhydrase active
site (Zn2þ) and so inhibits the catalytic ability of this enzyme
[14,16]. On the other hand, pyrimidine derivatives and heterocyclic
include anticancer [28], antiviral [29], antitumor [30], anti-
inflammatory [31] and others. All these findings encouraged us to
explore the synthesis of sulfonamides containing thiadiazolo[3,2-a]
pyrimidine moieties and examine their activities as antitumor
agents. Herein we report their synthesis, preliminary DNA-binding
assay and antineoplastic evaluation against Ehrlich ascites carci-
noma (EAC) in mice.
2. Results and discussion
2.1. Chemistry
A general approach to synthesize the designed compounds is
outlined in Schemes 1e5. 2-Amino-1,3,4-thiadiazole-5-sulfo-
namide (2), the key intermediate necessary for this study, was
synthesized from acetazolamide 1 via hydrochloric acid hydrolysis
followed by aqueous sodium carbonate neutralization [32].
Compound 2 was reacted with the appropriate 1,3-di (substituted
phenyl)-2-propen-1-one 3ael (chalcone analogues) [33e38] in
refluxing propylene glycol to afford the corresponding 5,7-di
(substituted
phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamides (4ael) (Scheme 1).
The reaction of the intermediate 2 with the appropriate
(4-substituted phenyldiazenyl)butyrate 5aef [39e41] in refluxing
glacial acetic acid afforded the 6-(4-substituted phenyldiazenyl-
* Corresponding author. Tel.: þ20 10 5174242; fax: þ20 50 2247496.
E-mail address: emanelbendary@yahoo.com (E.R. El-Bendary).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.037

N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
3715
R1
O
N
N
R1
N
N
1)
2)
HCl
Na CO
R
H2NO2S
NHCOCH3
H2NO2S
NH2
2
3
S
3a-l
Propylene glycol
S
N
S
N
2
1
H2NO2S
R
N
.
4a-l
R = H ; 4- NH2 ; 3- CH3
1
R = H ; 4- Br ; 4- Cl ; 4- NO2 ; 4- OCH3; 3,4- Di- OCH3 ; 2,6- Di-Cl ;
2-Cl- 5- NO
2
Scheme 1.
[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamides
(6aef))
2.2.1. DNA-binding assay
(Scheme 2).
In this method a fixed amount of the ligand is spotted on the RP-
18 TLC plates followed by the addition of DNA on the same spot at
the origin. The plates were then developed and the position of
unbound DNA was determined by spraying the plates with ani-
saldehyde reagent [43]. The free DNA was detected as a blue spot
(Rf, MeOHeH2O, 8:2) on RP-18 TLC. It was demonstrated that, when
DNA was mixed with compound known to interact with it, e.g.
ethidium bromide, the complex was retained at the origin.
Compounds with high binding affinity to DNA remained on the
base line or migrated for a very short distance, while compounds
with poor binding affinity did not cause DNA to be retained at the
origin.
New derivatives of 5-oxo-7-substituted-5H-[1,3,4]thiadiazolo
[3,2-a]pyrimidine-2-sulfonamide were prepared through the
reaction of compound 2 with different dicarbonyl compounds as in
Schemes 3 and 4. 6,7-Dihydro-5,7-dioxo-5H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-2-sulfonamide (7) was obtained through the reaction
of compound 2 with diethyl malonate in refluxing glacial acetic
acid. Compound 7 upon heating with phosphorus oxychloride in
the presence of N,N-dimethylaniline furnished the corresponding
7-chloro derivative 8. Similarly, reaction of compound 2 with ethyl
acetoacetate in refluxing glacial acetic acid furnished the 5-oxo
derivative 9 in 60% yield (Scheme 3).
The key intermediate 2 was treated with dimethyl acetylene
dicarboxylate in methanol to provide compound 10. The structure
of this compound was established on the basis of its elemental
analysis and spectral data. For example, the 1H-NMR spectrum
showed a signal of the methyl ester at 3.95 ppm. Compound 10 was
subsequently reacted with phenylhydrazine in refluxing absolute
ethanol to give the corresponding 5-oxo-2-sulfamoyl-N0-phenyl-
5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-7-carbohydrazide (11). On
the other hand, hydrolysis of the ester 10 using 12% sodium
hydroxide followed by acidification with hydrochloric acid
produced compound 12 in 45% yield (Scheme 4).
In addition, reaction of compound 2 with acetylacetone using
formic acid-phosphorous pentoxide as a condensation reagent,
followed by addition of perchloric acid to provide the cyclic product
13 as perchlorate salt. Finally, treatment of compound 2 with ethyl
cyanoacetate in methanolic solution of sodium methoxide
produced 6,7-dihydro-5-imino-7-oxo-5H-[1,3,4]thiadiazolo[3,2-a]
pyrimidine-2-sulfonamide mono sodium salt (14) which on acidi-
fication provided the corresponding derivative 15 (Scheme 5).
2.2.1.1. Results from DNA-binding assay. Compounds 4c, 4f and 4j
showed the highest binding affinity to DNA which was demon-
strated by retaining the complex at the origin or by migrating for
a very short distances. Compounds 4h, 4i, 6aec, 8, 9, 12, and 14
showed moderate activities. While compounds 4d, 4k, 6d, 6e and
6f showed weak activity, compound 6f was the weakest in this
study (Table 1). The results indicated that the diphenyl groups at
positions ꢀ5 and ꢀ7 as in compounds 4c, 4f, and 4j may contribute
in the activity. Meanwhile, the presence of nitro group in the phenyl
ring greatly decreases the binding affinity as in compounds 4d and
4h. In addition, the presence of chlorine atom in the phenyl ring
either at 2- or 4-position increases the activity as in compounds 4c,
4h and 4j. The presence of the dimethoxy groups in the phenyl ring
as in compound 4f produces the same activity as the chlorine atom.
2.2.2. Colorimetric assay for compounds that bind to DNA
Methyl green reversibly binds polymerized DNA forming a stable
complex at neutral pH, whereas free methyl green fades. The
absorption maximum for the DNA/methyl green complex is
642.5e645 nm. This colorimetric assay [44] was used tomeasure the
displacement of methyl green from DNA by compounds having
ability to bind with DNA. The degree of displacement was deter-
mined spectrophotometrically by measuring the change in initial
absorbance of DNA/methyl green solution in the presence of refer-
ence compound(ethidium bromide). Theactivityof compounds that
showed high affinity for DNA have been determined and expressed
2.2. Biology
DNA as an affinity probe useful in evaluating the biologically
active compounds: A variety of methods have been utilized for
determining the interaction of small molecular weight compounds
with DNA [42] such as DNA-binding assay and DNA/methyl green
displacement assay.
O
N
N
COOC2H5
COCH3
N
N
R
N
S
glacial acetic acid
R
N
H2NO2S
N
N HC
NH2
+
S
H2NO2S
2
N
CH3
6a- f
5a- f
R = H; Br; Cl; CH3; OCH3; NO2
Scheme 2.

3716
N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
COOC2H5
COOC2H5
NH
N
O
N
CNCH2COOC2H5
CH3OH / CH3ONa
N
N
H2C
N
S
N
N
N
N
S
H2NO2S
NH2
NaHNO2S
S
N
H2NO2S
H2NO2S
NH2
O
2
glacial acetic acid
S
14
O
2
7
1) CH3COCH2COCH3/HCOOH, P2O5
HCl
CH3COCH2COOC2H5
glacial acetic acid
2) HClO4
/
POCl3
DMA
NH
CH3
ClO4
N
O
O
N
N
+
N
H2NO2S
N
N
S
N
N
H2NO2S
S
H2NO2S
O
H2NO2S
N
S
CH3
N
S
N
Cl
N
CH3
15
13
8
9
Scheme 5.
Scheme 3.
significant reduction of viable tumor cells but mice received
compound 4j showed the best results as shown in (Table 5).
as IC50 (Concentrations required for a 50% decrease in the initial
absorbance of the DNA/methyl green solution) as shown in (Table 2).
These results indicate that compound 4c possessed the highest
binding affinity to DNA.
2.2.3.3. Discussion. The results of the present study showed an
antitumor activity of compounds 4j, 4f, and 4c against EAC in Swiss
albino mice. In the 5th day after inoculation of EAC in mice, increase
in body weight and ascites was observed clearly, also the mice
became slow and inactive. Mice received 4j and 5-fluorouracil were
more protected against ascites and showed slight increase in body
weight unlike the control group. Mice received 4c showed slight
toxic symptoms such as dizziness, erection of tail, and became slow.
2.2.3. Antineoplastic activity against Ehrlich ascites carcinoma
(EAC) in mice
The prolongation of lifespan of EAC bearing hosts, the reduction
in viable tumor cell count and the recovery of normal hematolog-
ical and biochemical profiles are three important measures that
have been used in this in vivo testing for the evaluation of the
antineoplastic activity for the compounds that showed the highest
binding affinity to DNA. The results were comparable to that of the
result obtained from the animals treated with the standard drug
5-flurouracil (20 mg/kg bw) [45,46].
3. Experimental
3.1. Chemistry
All melting points (ꢂC) were determined on fisher john melting
point apparatus and are uncorrected. Infrared spectra were recor-
ded in KBr disc using a Unicam SP 1000 infrared spectrophotometer
and expressed in wave number (cmꢀ1). 1H-NMR spectra were
obtained on FT-NMR spectrometer at 200 MHz; the chemical shifts
2.2.3.1. Results. Effect on survival time [47]: The mean survival
time (MST) of each group, consisting of 10 mice was noted. The
antitumorefficacyof the test compounds 4c, 4f and 4j was compared
with 5-fluorouracil (as a positive control) (5-fluorouracil, 20 mg/kg/
day, i.p. for 9 days) as shown in (Table 3). The MST of the treated
groups was compared with that of the control group using the
following calculations: % Increase in lifespan over control ¼ (MST
of treated group/MST of control group ꢀ 1) ꢁ 100, where
MST ¼ survival time (days of each mouse in a group)/Total no. of
mice. Compound4j showed the highest% increasein lifespan ofmice
inoculated with EAC.
are expressed in
d (ppm) units using tetramethylsilan (TMS) as
internal reference and DMSO-d6 or CDCl3 as solvent. Mass spectra
were recorded on JEOL JMS-600H spectrometer using electron
impact technique at 70 eV. Microanalyses (C, H, N) were in agree-
ment with the proposed structures within ꢃ0.4% of the theoretical
values.
Effect on the hematological and biochemical parameters:
Compound 4j showed more or less normal hemoglobin (HB), RBCs,
WBCs and ALT levels were improved as shown in (Table 4).
3.1.1. Chemical synthesis
3.1.1.1. 2-Amino-1,3,4-thiadiazole-5-sulfonamide (2). 2-Acetamido-
1,3,4-thiadiazole-5-sulfonamide (1) (8.5 g, 0.0383 mol) was heated
under reflux with concentrated hydrochloric acid (50 ml) for 7 h.
The reaction mixture was concentrated and neutralized with
2.2.3.2. Determination of viable cell count of Ehrlich ascites cells after
five days of treatment. Both compounds 4j and 4f which showed
N
N
H2NO2S
NH2
S
2
H3COOC C C COOCH3
MeOH
O
O
O
1) 12% NaOH/ 12 h
N
N
S
N
S
PhNHNH2
EtOH
N
N
N
H2NO2S
H2O/ MeOH
H2NO2S
H2NO2S
S
2) HCl
N
N
CONHNHPh
N
10
COOCH3
12
11
Scheme 4.

N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
3717
Table 1
Table 3
DNA-binding affinity of the selected compounds.
Effect of test compounds 4c, 4f, and 4j and 5-fluorouracil on the survival time of
mice inoculated with EAC.
Comp. No
DNA-binding affinity
Group
Normal Control
(Ehrlich only)
4j
4f
4c
5-fluorouracil
4c
4d
4f
4h
4j
High
Weak
High
Moderate
High
% Increase in
lifespan over
control
71.43
0
71.43 57.14 42.86 42.86
4k
4l
Weak
Moderate
Moderate
Moderate
Moderate
Weak
6a
6b
6c
6d
6e
6f
8
11H, 9-AreH, NH2, D2O exchange). Anal. for C17H13ClN4O2S2 (404.89)
C, H, N.
3.1.1.2.4. 5-(4-Nitrophenyl)-7-phenyl-5H-[1,3,4]thiadiazolo [3,2-
Weak
Weak
a]pyrimidine-2-sulfonamide (4d). Yield 0.29
g
(70%); mp.
100e102 ꢂC; 1H-NMR (DMSO-d6):
d
3.80 (s, 1H, C5-H), 5.98 (s, 1H,
Moderate
Moderate
Weak
Moderate
Moderate
C6-H), 7.57e8.58 (m, 11H, 9-AreH, NH2, D2O-exchange). Anal. for
C17H13N5O4S2 (415.45) C, H, N.
9
11
12
14
3.1.1.2.5. 5-(4-Methoxyphenyl)-7-phenyl-5H-[1,3,4]thiadiazolo
[3,2-a]pyrimidine-2-sulfonamide (4e). Yield 0.29
g (73%); mp.
75e77 ꢂC; 1H-NMR (CDCl3):
d
3.70 (s, 1H, C5-H), 3.95 (s, 3H, OCH3),
6.47 (s, 1H, C6-H), 7.26e8.17 (m, 9H, 9-AreH), 8.61 (s, 2H, NH2, D2O-
exchange). Anal. for C18H16N4O3S2 (400.47) C, H, N.
aqueous sodium carbonate solution. The separated solid was
collected by filtration, dried and recrystallized from ethanol to yield
5.38 g (78%) of compound 2, m.p. 218e220 ꢂC (reported m.p. for the
hydrochloride salt ¼ 199e200 ꢂC [32]. IR; 3305e3038 (2-NH2). MS
m/z (%); 181 (0.10, Mþ þ1); 180 (0.70, Mþ); 117 (100.00).
3.1.1.2.6. 5-(3,4-Dimethoxyphenyl)-7-phenyl-5H-[1,3,4]thiadia-
zolo [3,2-a]pyrimidine-2-sulfonamide (4f). Yield 0.30 g (70%); mp.
105e107 ꢂC; 1H-NMR (CDCl3):
d 3.82 (s, 1H, C5-H), 3.98 (s, 6H,
2eOCH3), 6.60e7.83 (m, 11H, C6-H, 8-AreH, NH2, D2O-exchange).
Anal. for C19H18N4O4S2 (430.50) C, H, N.
3.1.1.2. 5,7-Di (substituted phenyl)-5H-[1,3,4]thiadiazolo[3,2-a]pyrim-
idine-2-sulfonamides (4ael). A mixture of 2-amino-1,3,4-thiadia-
zole-5-sulfonamide (2) (0.45 g, 0.0025 mol) and the appropriate
chalcone analogue 3ael [33e38] (0.0025 mol) in propylene glycol
(10 ml) was heated for 3e5 h at 200e220 ꢂC. The mixture was cooled
and diluted with water (50 ml) with vigorous stirring. The separated
solid was collected by filtration, washed with water, dried and
recrystallized from ethanol to afford compounds 4a,h or recrystal-
lized from aqueous ethanol to produce compounds 4beg, iel.
3.1.1.2.1. 5,7-Diphenyl-5H-[1,3,4]thiadiazolo [3,2-a]pyrimidine-2-
sulfonamide (4a). Yield 0.20 g (55%); mp. 60e62 ꢂC; MS m/z (%):
369 (0.94, Mþ ꢀ1), 368 (1.82, Mþ e2), 181 (100). Anal. for
C17H14N4O2S2 (370.45) C, H, N.
3.1.1.2.7. 5-(2,6-Dichlorophenyl)-7-phenyl-5H-[1,3,4]thiadiazolo
[3,2-a]pyrimidine-2-sulfonamide (4g). Yield 0.33
g
(75%); mp.
4.03 (s, 1H,
80e82 ꢂC; IR: 3341 (NH2) cmꢀ1. 1H-NMR (DMSO-d6):
d
C5eH), 5.94 (s, 1H, C6-H), 7.09e8.07 (m, 10H, 8-AreH, NH2, D2O-
exchange). Anal. for C17H12Cl2N4O2S2 (439.34) C, H, N.
3.1.1.2.8. 5-(2-Chloro-5-nitrophenyl)-7-phenyl-5H-[1,3,4]thiadia-
zolo [3,2-a]pyrimidine-2-sulfonamide (4h). Yield 0.36 g (80%); mp.
65e67 ꢂC; 1H-NMR (CDCl3):
d 3.70 (s, 1H, C7-H), 6.16 (s, 1H, C6-H),
7.25e8.20 (m, 8H, 8-AreH), 8.62 (s, 2H, NH2, D2O-exchange). Anal.
for C17H12ClN5O4S2 (449.89) C, H, N.
3.1.1.2.9. 4-(5-Phenyl-2-sulfamoyl-5H-[1,3,4]thiadiazolo [3,2-a]pyr-
imidin-7-yl)benzenamine (4i). Yield 0.25 g (65%); mp. 65e67 ꢂC;
1H-NMR (DMSO-d6):
d 3.62 (s, 1H, C5-H), 4.64 (s, 2H, AreNH2, D2O-
3.1.1.2.2. 5-(4-Bromophenyl)-7-phenyl-5H-[1,3,4]thiadiazolo [3,2-
exchange), 6.50 (s, 1H, C6-H), 7.18e7.90 (m, 11H, 9-AreH, SO2NH2,
D2O-exchange). Anal. for C17H15N5O2S2 (385.46) C, H, N.
a]pyrimidine-2-sulfonamide (4b). Yield 0.27
g
(60%); mp.
113e115 ꢂC; MS m/z (%): 451 (17.30, Mþ þ2), 450 (9.10, Mþ þ1), 449
(34.20, Mþ), 105 (100). Anal. for C17H13BrN4O2S2 (449.34) C, H, N.
3.1.1.2.3. 5-(4-Chlorophenyl)-7-phenyl-5H-[1,3,4]thiadiazolo [3,2-
a]pyrimidine-2-sulfonamide (4c). Yield 0.27 g (67%); mp. 88e89 ꢂC;
3.1.1.2.10. 4-(5-(4-Chlorophenyl)-2-sulfamoyl-5H-[1,3,4]thiadia-
zolo [3,2-a]pyrimidin-7-yl)benzenamineþ(4j). Yield 0.29 g þ(70%); mp.
117e120 ꢂC; MS m/z (%): 420 (5.70, M ), 419 (10.70, M ꢀ1), 418
(14.00, Mþ ꢀ2), 101 (100). Anal. for C17H14ClN5O2S2 (419.91) C, H, N.
3.1.1.2.11. 4-(5-(4-Methoxyphenyl)-2-sulfamoyl-5H-[1,3,4]thia-
diazolo [3,2-a]pyrimidin-7-yl)benzenamine (4k). Yield 0.26 g (62%);
1H-NMR (CDCl3):
d 3.89 (s,1H, C7-H), 6.60 (s,1H, C6-H); 7.21e8.07 (m,
mp. 115e117 ꢂC; 1H-NMR (CDCl3):
d 3.78 (s, 1H, C5-H), 3.96 (s, 3H,
OCH3), 4.20 (s, 2H, AreNH2, D2O-exchange), 5.91(s, 1H, C6-H),
6.70e7.94 (m, 10H, 8-AreH, SO2NH2, D2O-exchange). Anal. for
C18H17N5O3S2 (415.49) C, H, N.
Table 2
Activity of compounds in the DNA/methyl green displacement assay.a
DNA-active compounds
DNA/methyl green IC50, mg/ml
3.1.1.2.12. 5-Phenyl-7-m-tolyl-5H-[1,3,4]thiadiazolo [3,2-a]pyrimi-
dine-2-sulfonamide (4l). Yield 0.23 g (60%); mp. 86e88 ꢂC; 1H-NMR
4c
4f
4h
4j
4l
6a
6b,6c
8
40 ꢃ 1
62 ꢃ 4
80 ꢃ 3
64 ꢃ 2
72 ꢃ 2
77 ꢃ 1
74 ꢃ 2
79 ꢃ 3
81 ꢃ 4
70 ꢃ 1
76 ꢃ 2
1.4 ꢃ 2
(CDCl3):
d 2.42 (s, 3H, CH3), 3.95 (s, 1H, C5-H), 5.70 (s, 1H, C6-H),
Table 4
Effect of test compound 4j and 5-fluorouracil on the hematological and biochemical
parameters of mice inoculated with EAC.
11, 4d, 4k, 6def
12
9,14
Parameter
Normal Control
4j (100
mg/ 5-fluorouracil
(Ehrlich only) mouse)
(20 mg/kg)
Hb/g%
13.4
5.1
4.35
3.7
5.7
3.15
47.4
14.4
11.1
5.96
9.45
6.7
15.6
5.87
14.7
6.9
Ethidium bromide
RBCs/106 mmꢀ3
Total WBCs/103 mm3
a
Values represent the concentration (mean ꢃ SD, n ¼ 3e5 separate determina-
tions) required for a 50 % decrease in the initial absorbance of the DNA/methyl green
solution.
ALT ꢁ 0.1/
m
Lꢀ1

3718
N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
Table 5
thiadiazolo[3,2-a]pyrimidine-2-sulfonamide (7) (1.24 g, 0.005 mol),
phosphorus oxychloride (5 ml) and N,N-dimethylaniline (0.5 ml)
was heated under reflux for 5 h. After cooling, the mixture was
poured gradually on crushed ice and extracted with chloroform
(3 ꢁ10 ml). Thecombined organic extractwas evaporated todryness
and residue obtained was recrystallized from ethanol/water. Yield
Viable cell count of EAC after 5 days of treatment with compounds 4c, 4f, 4j, and
5-fluorouracil.
Group number
Group name
Count (cells)/100 m.L
1
2
3
4
5
4j
4f
4c
17.6 ꢁ 106
18.6 ꢁ 106
99.2 ꢁ 106
83.6 ꢁ 106
192.8 ꢁ 106
0.6 g (45%); mp. 253e255 ꢂC; IR: 3303 (NH2), 1680 (C]O) cmꢀ1
.
5-fluorouracil
Ehrlich only
1H-NMR (DMSO-d6):
d 6.73 (s, 1H, C6-H), 7.79 (s, 2H, NH2, D2O-
exchange). MS m/z (%): 268 (0.10, Mþ þ2), 267 (0.70, Mþ þ1), 135
(100). Anal. for C5H3ClN4O3S2 (266.69) C, H, N.
7.23e7.94 (m, 11H, 9-AreH, NH2, D2O-exchange). Anal. for
C18H16N4O2S2 (384.48) C, H, N.
3.1.1.6. 7-Methyl-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide (9). Ethyl acetoacetate (0.65 g, 0.005 mol) was added
3.1.1.3. 7-Methyl-5-oxo-6-(4-substituted phenyldiazenyl)-5H-[1,3,4]
thiadiazolo[3,2-a]pyrimidine-2-sulfonamides (6aef). A mixture of
2-amino-1,3,4-thiadiazole-5-sulfonamide (2) (0.45 g, 0.0025 mol)
and the appropriate ethyl 3-oxo-2-(4-substituted phenyldiazenyl)
butyrate 5aef [39e41] (0.0025 mol) in glacial acetic acid (10 ml)
was heated under reflux for 12 h and refrigerated overnight. The
separated solid was filtered, dried and recrystallized from aqueous
ethanol to afford compounds 6a, c or recrystallized from acetic
acid/water to yield compounds 6b, def.
dropwise to a stirred solution of 2-amino-1,3,4-thiadiazole-5-
sulfonamide (2) (0.9 g, 0.005 mol) in glacial acetic acid (10 ml). The
reaction mixture was heated under reflux for 12 h and refrigerated
overnight. The separated solid was collected by filtration, dried and
recrystallized from ethanol/water to give the desired compound 9.
Yield 0.74 g (60%); mp. 253e255 ꢂC; IR: 3300 (NH2), 1679 (C]O).
1H-NMR (DMSO-d6):
d 2.26 (s, 3H, CH3), 7.43 (s,1H, C6-H), 8.36 (s, 2H,
NH2, D2O-exchange). Anal. for C6H6N4O3S2 (246.27) C, H, N.
3.1.1.3.1. 7-Methyl-5-oxo-6-(phenyldiazenyl)-5H-[1,3,4]thiadia-
zolo [3,2-a]pyrimidine-2-sulfonamide (6a). Yielꢀd1 0.16 g (45%); mp.
.
1H-NMR (DMSO-
3.1.1.7. Methyl 5-Oxo-2-sulfamoyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimi-
dine-7-carboxylate (10). Dimethyl acetylene dicarboxylate (DMAD)
(0.71 g, 0.005 mol) was added dropwise to a stirred solution of 2-
amino-1,3,4-thiadiazole-5-sulfonamide (2) (0.9 g, 0.005 mol) in
methanol (15 ml). The reaction mixture was heated under reflux for
12 h. After cooling, the separated solid was collected by filtration,
dried and recrystallized from ethanol/water to afford the desired
compound 10. Yield 1.09 g (75%); mp. 188e190 ꢂC; IR: 3265 (NH2):
255e257 ꢂC; IR: 3207 (NH2), 1656 (C]O) cm
d6):
d 2.18 (s, 3H, CH3), 7.35e7.78 (m, 7H, 5-AreH, NH2, D2O-
exchange). Anal. for C12H10N6O3S2 (350.38) C, H, N.
3.1.1.3.2. 7-Methyl-5-oxo-6-(4-bromophenyldiazenyl)-5H-[1,3,4]
thiadiazolo [3,2-a]pyrimidine-2-sulfonamide (6b). Yield 0.26
g
(60%); mp. 247e249 ꢂC; 1H-NMR (DMSO-d6):
d 2.22 (s, 3H, CH3);
7.36e7.79 (m, 6H, 4-AreH, NH2, D2O-exchang.). Anal. for
C12H9BrN6O3S2 (429.27) C, H, N.
1750 (COOCH3): 1628 (C]O). 1H-NMR (DMSO-d6):
d 3.95 (s, 3H,
COOCH3), 6.71 (s, 1H, C6-H), 8.58 (s, 2H, NH2, D2O-exchange). Anal.
for C7H6N4O5S2 (290.28) C, H, N.
3.1.1.3.3. 7-Methyl-5-oxo-6-(4-chlorophenyldiazenyl)-5H-[1,3,4]
thiadiazolo [3,2-a]pyrimidine-2-sulfonamide (6c). Yield 0.25
g
(65%); mp. 258e260 ꢂC; 1H-NMR (DMSO-d6):
d
2.22 (s, 3H, CH3),
3.1.1.8. 5-Oxo-2-sulfamoyl-N0-phenyl-5H-[1,3,4]thiadiazolo[3,2-a]
pyrimidine-7-carbohydrazide (11). Phenylhydrazine (0.39 g, 0.0036
mmol) was added to a stirred solution of methyl 5-oxo-2-
sulfamoyl-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-7-carboxylate
(10) (0.87 g, 0.003 mol) in absolute ethanol (10 ml). The reaction
mixture was heated under reflux for 8 h. After cooling, the sepa-
rated solid was collected by filtration, dried and recrystallized from
ethanol to afford the desired compound 11. Yield 0.64 g (58%); mp.
262e264 ꢂC; IR: 3409 (2eNH), 3303 (NH2), 1698 (C]O), 1641 (C]
7.40e8.25 (m, 6H, 4-AreH, NH2, D2O-exchange). Anal. for
C12H9ClN6O3S2 (384.82) C, H, N.
3.1.1.3.4. 7-Methyl-5-oxo-6-(4-methylphenyldiazenyl)-5H-[1,3,4]
thiadiazolo [3,2-a]pyrimidine-2-sulfonamide (6d). Yield 0.24
g
(67%); mp. 250e252 ꢂC; 1H-NMR (DMSO-d6):
d
2.08 (s, 3H, CH3),
2.21(s, 3H, CH3), 7.30e7.45 (m, 4H, AreH), 7.63 (s, 2H, NH2, D2O-
exchange). Anal. for C13H12N6O3S2 (364.40) C, H, N.
3.1.1.3.5. 7-Methyl-5-oxo-6-(4-methoxyphenyldiazenyl)-5H-
[1,3,4]thiadiazolo [3,2-a]pyrimidine-2-sulfonamide (6e). Yield 0.27 g
O).1H-NMR (DMSO-d6):
d 6.82e7.48 (m, 6H, C6-H, 5-AreH), 8.56 (s,
(72%); mp. 250e252 ꢂC; 1H-NMR (DMSO-d6):
d
2.21 (s, 3H, CH3),
2H, NH2, D2O-exchange), 11.56 (s, 1H, CONHNH, D2O-exchange),
11.95 (s, 1H, CONHNH, D2O-exchange). Anal. for C12H10N6O4S2
(366.38) C, H, N.
3.92 (s, 3H, OCH3), 7.06e7.21 (m, 4H, AreH), 7.59 (s, 2H, NH2, D2O-
exchange). Anal. for C13H12N6O4S2 (380.40) C, H, N.
3.1.1.3.6. 7-Methyl-5-oxo-6-(4-nitrophenyldiazenyl)-5H-[1,3,4]
thiadiazolo [3,2-a]pyrimidine-2-sulfonamide (6f). Yield 0.28 g (70%);
mp. 262e265 ꢂC; MS m/z (%): 393 (2.66, Mþ ꢀ2), 368 (22.50), 180
(100). Anal. for C12H9N7O5S2 (395.37) C, H, N.
3.1.1.9. 5-Oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide
(12). 5 ml (12%) Sodium hydroxide solution was gradually added to
a solution of methyl 5-oxo-2-sulfamoyl-5H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-7-carboxylate (10) (0.87 g, 0.003 mol) in methanol
(15 ml). The reaction mixture was heated under reflux for 12 h. The
solvent was evaporated; the obtained residue was triturated with
water (5 ml) and acidified with hydrochloric acid. The separated
solid was collected by filtration, washed with water, dried and
recrystallized from methanol to yield 0.31 g (45%) of compound 12;
mp. >300 ꢂC. IR: 3344 (NH2), 1631 (C]O). MS m/z (%): 234 (4.30,
Mþ þ2), 233 (3.40, Mþ þ1), 232 (20.10, Mþ), 60 (100). Anal. for
C5H4N4O3S2 (232.24) C, H, N.
3.1.1.4. 6,7-Dihydro-5,7-dioxo-5H-[1,3,4]thiadiazolo [3,2-a]pyrimi-
dine-2-sulfonamide (7). Diethyl malonate (0.8 g, 0.005 mol) was
added to
a stirred solution of 2-amino-1,3,4-thiadiazole-5-
sulfonamide (2) (0.9 g, 0.005 mol) in glacial acetic acid (10 ml). The
mixture was heated under reflux for 12 h and refrigerated overnight.
The separated solid was filtered, dried and recrystallized from acetic
acid/water. Yield 0.81 g (65%); mp. 253e255 ꢂC; IR: 3422 (NH2), 1693
(C]O), 1654 (C]O) cmꢀ1. 1H-NMR (DMSO-d6):
d 3.06 (s, 2H, CH2),
8.22(s, 2H, NH2, D2O-exchange). MSm/z(%):248(1.60,Mþ), 247(3.30,
M
þ ꢀ1), 117(100). Anal. for C5H4N4O4S2 (248.24) C, H, N.
3.1.1.10. 5,7-Dimethyl-[1,3,4]thiadiazolo[3,2-a]pyrimidin-4-ium-2-
sulfonamide perchlorate (13). Acetylacetone (0.33 g, 0.0033 mmol),
2-amino-1,3,4-thiadiazole-5-sulfonamide 2 (0.54 g, 0.003 mol) and
were added to a solution of phosphorus pentoxide (0.43 g,
3.1.1.5. 7-Chloro-5-oxo-5H-[1,3,4]thiadiazolo [3,2-a]pyrimidine-2-
sulfonamide (8). A mixture of 6,7-dihydro-5,7-dioxo-5H-[1,3,4]

N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
3719
0.003 mol) in formic acid (10 ml). The reaction mixture was heated
under reflux for 10 h and the solvent was evaporated. The obtained
residue was triturated with water (5 ml), mixed with 70% perchloric
acid in excess. The precipitated product was collected by filtration,
dried and recrystallized from methanol to afford the desired
compound 13. Yield 0.55 g (53%); mp. >300 ꢂC; IR: 3293 (NH2).
normalized as a % of the untreated DNA/methyl green absorbance
value. Concentrations required for a 50% decrease in the initial
absorbance of the DNA/methyl green solution (IC50’s) were deter-
mined for each compound as shown in (Table 2).
3.2.3. Antineoplastic activity against Ehrlich ascites carcinoma
(EAC) in mice
1H-NMR (DMSO-d6):
d 2.75 (s, 3H, CH3), 2.91(s, 3H, CH3), 7.97 (s, 1H,
C6-H), 8.32 (s, 2H, NH2, D2O-exchange). MS m/z (%); 344 (47.10, M
þ), 342 (100, Mþ ꢀ2), 330 (66.20), 303 (36.80), 193 (45.60). Anal. for
C7H9ClN4O6S2 (344.75) C, H, N.
3.2.3.1. Materials
3.2.3.1.1. Experimental animals. Adult Swiss male albino mice
(20e25 g) were procured from pharmacology department, Man-
soura University, Egypt and used throughout the study. They were
housed in microlon boxes in a controlled environment (tempera-
ture 25 þ 2 ꢂC and 12 h dark/light cycle) with standard laboratory
diet and sterilized water.
3.2.3.1.2. Tumor cells. Ehrlich ascites carcinoma (EAC) was
obtained from National Cancer Institute, Cairo, Egypt. After har-
vesting and preparation of the cells, their total number and viability
were determined by counting using trypan blue [47]. The desired
concentration of tumor cells (2 ꢁ 106 cells per 0.2 ml) was obtained
by dilution with saline (0.9% sodium chloride solution). Viability of
tumor cells obtained and used in this experiment was always
higher than 90.0%. Below this percentage the cells were discarded
and the entire procedure was repeated.
3.1.1.11. 6,7-Dihydro-5-imino-7-oxo-5H-[1,3,4]thiadiazolo[3,2-a]
pyrimidine-2-sulfonamide mono sodium salt (14). To a solution of
sodium metal (0.23 g, 0.01 mol) in dry methanol (15 ml), 2-amino-
1,3,4-thiadiazole-5-sulfonamide (2) (0.9 g, 0.005 mol) and ethyl
cyanoacetate (0.56 g, 0.005 mol) were added and the resulting
mixture was refluxed for 8 h. After cooling, the separated solid
was collected by filtration, dried and recrystallized from methanol
to afford the desired compound 14. Yield 0.9 g (67%); mp. >300 ꢂC;
IR: 451 (2eNH), 1630 (C]O). MS m/z (%): 268 (7.40, Mþ ꢀ1), 267
(6.40, Mþ ꢀ2), 253 (6.40), 57 (100). Anal. for C5H4N5NaO3S2
(269.24) C, H, N.
3.1.1.12. 6,7-Dihydro-5-imino-7-oxo-5H-[1,3,4]thiadiazolo[3,2-a]
pyrimidine-2-sulfonamide (15). To a solution of sodium metal
(0.23 g, 0.01 mol) in dry methanol (15 ml), 2-amino-[1,3,4-
thiadiazole-5-sulfonamide (2) (0.9 g, 0.005 mol) and ethyl cya-
noacetate (0.56 g, 0.005 mmol) were added and the resulting
mixture was heated under reflux for 8 h. The solution was evap-
orated and the obtained residue was triturated with water (5 ml)
and acidified with hydrochloric acid. The separated solid was
collected by filtration, washed with water, dried and recrystallized
from ethanol/water to give the desired compound 15. Yield 0.74 g
(60%); mp. 178e180 ꢂC; IR: 3401 (NH2), 3313 (NH), 1679 (C]O).
3.2.3.2. Procedure. Male Swiss albino mice were divided into 6
groups (n ¼ 10).
Group I e Normal control.
Group II e Disease control, EAC cell line only.
Group III e EAC cell line treated with 4c.
Group IV e EAC cell line treated with 4f.
Group V e EAC cell line treated with 4j.
Group VI e EAC cell line treated with standard [5-fluorouracil
(20 mg/kg)]
1H-NMR (CDCl3):
d 3.25 (s, 2H, CH2), 7.52 (s, 2H, NH2, D2O-
exchange), 7.76 (s, 1H, NH, D2O-exchange). Anal. for C5H5N5O3S2
(247.25) C, H, N.
All the groups were inoculated with 2 ꢁ 106 cells/mouse intra-
peritoneally except normal group; this was taken as day zero. The
treatment started 24 h after inoculation (100
mg/mouse). The
3.2. Biology
control group was treated with the same volume of 0.9% sodium
chloride solution. 5-Fluorouracil (20 mg/kg) was used as a standard
drug. All the treatments were given for nine days.
3.2.1. DNA-binding assay
Analysis of the DNA-binding affinity of the tested compounds
was performed using RP-18 TLC plates. TLC plates (RP-18 F254
;
3.2.3.3. Determination of viable cell count of Ehrlich ascites cells after
0.25 mm; Merck) were predeveloped with MeOHeH2O (8:2). Test
compounds (5 mg/ml in MeOH) were then applied at the origin,
followed by the addition of DNA (1 mg/ml in H2O and MeOH
mixture) at the same positions at the origin. The plates were then
developed with the same solvent system and the position of the
DNA was determined by spraying with anisaldehyde reagent [43].
The reagent yields a blue color reaction with DNA, and the intensity
of the color was proportional to the quantity of DNA added to the
plate. Ethidium bromide was used as a positive control.
five days of treatment. From each group take 100 mL sample of
Ehrlich ascites cells (from three mice) and make 20 fold dilution in
saline. The cells were stained by giemsa stain and measure the
number of viable cells under microscope.
Acknowledgements
The authors would like to express their sincere thanks to Prof.
Dr. Farid A. Badria, Pharmacognosy Department, Faculty of Phar-
macy, Mansoura University, Egypt, for carrying out the DNA-
binding assay and antitumor screening.
3.2.2. Colorimetric assay for compounds that bind to DNA (DNA/
Methyl green displacement assay)
DNA/methyl green (20 mg, Sigma, St. Louis, MO, USA) was sus-
pended in 100 ml of 0.05 M Tris-HCl buffer, pH 7.5, containing
7.5 mM MgSO4 and stirred at 37 ꢂC with a magnetic stirrer for 24 h.
Unless otherwise indicated, samples to be tested were dissolved in
EtOH in eppendorf tubes. Solvent was removed under vacuum and
References
[1] A.K. Gadad, C.S. Mahajanshetti, S. Nimbalkar, A. Raichurkar, Eur. J. Med. Chem.
35 (9) (2000) 853e857.
[2] V.S. Misra, V.K. Saxena, R.J. Srivastava, Ind. Chem. Soc. 59 (1982) 781.
[3] F. Zani, P. Vicini, Arch. Pharm. 331 (1998) 219e223.
200 ml of DNA/methyl green solution was added to each tube. The
ꢀ
[4] Z. Brzozowski, J. S1awinski, F. Sa˛czewski, A. Innocenti, C.T. Supuran, Eur. J. Med.
Chem. 45 (2010) 2396e2404.
[5] G. Renzi, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 10 (2000)
673e676.
[6] T.H. Maren, Physiol. Rev. 47 (1967) 595e781.
[7] T.H. Maren, Ann. Rev. Pharmacol. Toxicol. 16 (1976) 309e327.
absorption maxima for the DNA/methyl green complex is
642.5e645 nm. Samples were incubated in the dark at ambient
temperature. After 24 h, the final absorbance of samples was
determined. Readings were corrected for initial absorbance and

3720
N.S. El-Sayed et al. / European Journal of Medicinal Chemistry 46 (2011) 3714e3720
[8] J. Drew, Science 287 (2000) 1960e1964.
[25] A. Cecchi, A. Hulikova, J. Pastorek, S. Pastorekova, A. Scozzafava, J.Y. Winum,
J.L. Montero, C.T. Supuran, J. Med. Chem. 48 (2005) 4834e4841.
[26] D.J. Brown, Pyrimidines and their benzo derivatives. in: A.R. Katritzky,
C.W. Rees (Eds.), Comprehensive Heterocyclic Chemistry, vol. 3. Pergamon
Press Oxford, 1984, p. 443.
[27] X. Qin, X. Chen, K. Wang, L. Polin, E.R. Kern, J.C. Drach, E. Gullen, Y.C. Cheng,
J. Zemlicka, Bioorg. Med. Chem. 14 (2006) 1247e1254.
[28] H.G. Diaz, D. Vina, L. Santana, E. Clercq, E. Uriarate, Bioorg. Med. Chem. 14
(2006) 1095e1107.
[29] M.N. Nasr, M.M. Gineinah, Arch. Pharm. 335 (2002) 289e295.
[30] R. Lin, S.G. Johnson, P.J. Connolly, S.K. Wetter, E. Binnun, T.V. Hughes,
W.V. Murray, N.B. Pandey, S.J. Moreno-Mazza, M. Adams, A.R. Fuentes-Pes-
quera, S.A. Middleton, Bioorg. Med. Chem. Lett. 19 (2009) 2333e2337.
[31] S.M. Sondhi, M. Johar, S. Rajvanshi, S.G. Dastidar, R. Shukla, R. Raghubir,
J.W. Lown, Aust. J. Chem. 54 (2001) 69e74.
[32] V. Petrow, O. Stephenson, A.J. Thomas, A.M. Wild, J. Chem. Soc (1958) 1508e1513.
[33] W. Dilthey, L. Neuhaus, E. Reis, W. Schommer, J. Prakt. Chem. 124 (1930) 81 C.
A., (1930) 24, 1368.
[9] J.J. Li, D. Anderson, E.G. Burton, J.N. Cogburn, J.T. Collins, D.J. Garland,
S.A. Gregory, H.C. Huang, P.C. Isakson, C.M. Koboldt, E.W. Logusch,
M.B. Norton, W.E. Perkins, E.J. Reinhard, K. Seibert, A.W. Veenhuizem, Y. Zang,
D.B.J. Reitz, Med. Chem. 38 (1995) 4570e4578.
ꢀ
[10] F. Sa˛czewski, A. Innocenti, J. S1awinski, A. Kornicka, Z. Brzozowski, El
.
Pomarnacka, A. Scozzafava, C. Temperini, C.T. Supuran, Bioorg. Med. Chem. 16
(2008) 3933e3940.
[11] V. Garaj, L. Puccetti, G. Fasolis, J.Y. Winum, J.L. Montero, A. Scozzafava,
D. Vullo, A. Innocenti, C.T. Supuran, Bioorg. Med. Chem. Lett. 15 (2005)
3102e3108.
ꢀ
[12] F. Sa˛czewski, J. S1awinski, A. Kornicka, Z. Brzozowski, E. Pomarnacka,
A. Innocenti, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. Lett. 16 (2006)
4846e4851.
[13] C.T. Supuran, A. Scozzafava, A. Casini, Med. Res. Rev. 23 (2003) 146e189.
[14] C.T. Supuran, Carbonic anhydrases: catalytic and inhibition mechanisms,
distribution and physiological roles, in: Carbonic Anhydrase. Its Inhibitors and
Activators. CRC Press, Boca Raton, 2004, pp. 1e23.
[15] A.J. Kivela, J. Kivela, J. Saarnio, S. Parkkila, J. World Gastroenterol. 11 (2005)
155e163.
[34] N.K. Kochetkov, V.F. Belyaev, Zhur. Obshchei Khim 30 (1960) 1495 C.A., 48
(1954) 10665i; 55 (1961) 1519c.
[16] C.T. Supuran, A. Scozzafava, Bioorg. Med. Chem. 15 (2007) 4336e4350.
[17] J.Y. Winum, A. Scozzafava, J.L. Montero, C.T. Supuran, Anticancer Agents Med.
Chem. 9 (2009) 693e702.
[18] SJ.Y. Winum, M. Rami, A. Scozzafava, J.L. Montero, C.T. Supuran, Med. Res. Rev.
28 (2008) 445e463.
[19] S. Pastoreková, J. Pastorek, Cancer-related carbonic anhydrase isozymes and
their inhibition. in: C.T. Supuran, et al. (Eds.), Carbonic Anhydrase. Its Inhib-
itors and Activators. CRC Press, 2004, pp. 255e281.
[20] Ö Tureci, U. Sahin, E. Vollmar, S. Siemer, E. Göttert, G. Seitz, A.-K. Parkkila,
G.N. Shah, J.H. Grubb, M. Pfreundschuh, W.S. Sly, Proc. Natl. Acad. Sci. U.S.A 95
(1998) 7608e7613.
[21] S. Pastorekova, Z. Závadová, M. Kost’ál, O. Babusiková, J. Závada, Virology 187
(1992) 620e626.
[22] C.C. Wykoff, J.N. Beasley, H.P. Watson, K.J. Turner, J. Pastorek, A. Sibtain,
D.G. Wilson, H. Turley, K.L. Talks, H.P. Maxwell, W.C. Pugh, J.P. Ratcliffe,
L.A. Harris, Cancer Res. 60 (2000) 7075e7083.
[35] M. Dzurilla, P. Kristian, Collect. Czech. Chem. Commun. 35 (1969) 417 C.A.,
72(1970) 66551y.
[36] N.L. Silver, D.W. Boykin, J. Org. Chem. 35 (1970) 759e764.
[37] B.S. Furniss, A.J. Hannaford, P.W.G. Smith, A.R. Tatchell, Vogel’s Text Book of
Practical Organic Chemistry, fifth ed. Spottiswoode, Ballantyne, and Co, Lon-
don, 1991, p. 1032.
[38] D.G. Batt, R. Goodman, D.G. Jones, J.S. Kerr, L.R. Mantegna, C. Mc Allister,
R.C. Newton, Sh. Nurnberg, P.K. Welch, M.B. Covington, J. Med. Chem. 36
(1993) 1434e1442.
[39] R. Wizinger, H. Herzog, Helv. Chem. Acta 34 (1951) 1202e1205.
[40] C. Bullow, F. Schaub, Chem. Ber 41 (1908) 2355e2365.
[41] Kjellin, Chem. Ber. 30 (1897) 1965e1969.
[42] J.M. Pezzuto, S.K. Antosiak, W.M. Messmer, M.B. Slaytor, G.R. Honig, Chem.
Biol. Interact 43 (1983) 323e339.
[43] J.M. Pezzuto, C.T. Che, D.D. McPherson, J.P. Zhu, G. Topcu, C.A. Erdelmeier,
G.A. Cordell, J. Nat. Prod. 54 (1991) 1522e1530.
[23] S. Parkkila, H. Rajaniemi, A.K. Parkkila, J. Kivelä, A. Waheed, S. Pastorekova,
J. Pastorek, W.S. Sly, Proc. Natl. Acad. Sci. U.S.A 97 (2000) 2220e2224.
[24] E. Svastova, A. Hulikova, M. Rafajova, M. Zatovicova, A. Gibadulinova, A. Casini,
A. Cecchi, A. Scozzafava, C.T. Supuran, J. Pastorek, S. Pastorekova, FEBS Lett.
577 (2004) 439e445.
[44] N.S. Burres, A. Frigo, R.R. Rasmussen, J.B. McAlpine, J. Nat. Prod. 55 (1992)
1582e1587.
[45] B.D. Clarkson, J.H. Burchenal, Prog. Clin. Cancer 1 (1965) 625e629.
[46] C. Oberling, M. Guerin, Adv. Cancer Res. 2 (1954) 353e423.
[47] K.R. Sheeja, G. Kuttan, R. Kuttan, Amala Res. Bull. 17 (1997) 73e76.