Synthesis, anticancer, anti-HIV-1, and antimicrobial activity of some tricyclic triazino and triazolo[4,3-e]purine derivatives

Article abstract of DOI:10.1007/s00044-011-9612-6

In an effort to etablish new candidates with improved antineoplastic, anti-HIV-1 and antimicrobial activities, the synthesis of some new triazino and triazolo[4,3-e]purine derivatives is described: 6,8-dimethyl-1, 4-dihydro-1,2,4-triazino[4,3-e]purine-7,9(6H, 8H)-diones 3-6; 5,7,9-trimethyl-1,2,4-triazolo[4,3-e]purine-6,8(5H, 7H, 9H)-diones 11-13, together with the synthesis of the 8-substituted purine derivative: 8-(3,5-diamino-1H-pyrazol-4-yl)diazenyl-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione 7. The prepared compounds were tested for their in vitro anticancer, anti-HIV and antimicrobial activities. The results of the in vitro anticancer screening revealed that compound 3 exhibited considerable activity against melanoma MALME-3 M, non-small lung cancer HOP-92 and breast cancer T-47D (GI₅₀ values of 25.2, 31.8, and 32.9 μM, respectively). The anti-HIV-1 results indicated that compounds 7 and 13c displayed moderate activity (maximum % cell protection 30.52 and 35.54 at 2 × 10^-4 M, respectively). The in vitro antimicrobial data showed that compound 12 was the most active against P. aeruginosa, it was equipotent to ampicillin (MIC < 100 μg/ml). While compound 11d was the most active against P. vulgaris, it was as active as ampicillin (MIC < 50 μg/ml). In addition, compounds 12 and 13c were the most active against S. aureus (MIC <50 and <25 μg/ml, respectively). On the other hand, the tested compounds devoid of antifungal activity except 6b and 11c which showed weak activity against A. niger. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9612-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1107–1119
DOI 10.1007/s00044-011-9612-6
ORIGINAL RESEARCH
Synthesis, anticancer, anti-HIV-1, and antimicrobial activity
of some tricyclic triazino and triazolo[4,3-e]purine derivatives
•
Fawzia A. Ashour Samia M. Rida
•
•
•
Soad A. M. El-Hawash Mona M. ElSemary
Mona H. Badr
Received: 5 May 2010 / Accepted: 21 February 2011 / Published online: 27 March 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In an effort to etablish new candidates with
improved antineoplastic, anti-HIV-1 and antimicrobial
activities, the synthesis of some new triazino and triazol-
o[4,3-e]purine derivatives is described: 6,8-dimethyl-1,
4-dihydro-1,2,4-triazino[4,3-e]purine-7,9(6H, 8H)-diones
3–6; 5,7,9-trimethyl-1,2,4-triazolo[4,3-e]purine-6,8(5H, 7H,
9H)-diones 11–13, together with the synthesis of the
8-substituted purine derivative: 8-(3,5-diamino-1H-pyrazol-
4-yl)diazenyl-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione 7.
The prepared compounds were tested for their in vitro
anticancer, anti-HIV and antimicrobial activities. The
results of the in vitro anticancer screening revealed that
compound 3 exhibited considerable activity against mela-
noma MALME-3 M, non-small lung cancer HOP-92 and
breast cancer T-47D (GI50 values of 25.2, 31.8, and
32.9 lM, respectively). The anti-HIV-1 results indicated
that compounds 7 and 13c displayed moderate activity
(maximum % cell protection 30.52 and 35.54 at 2 9 10-4
M, respectively). The in vitro antimicrobial data showed
that compound 12 was the most active against P. aerugin-
osa, it was equipotent to ampicillin (MIC \ 100 lg/ml).
While compound 11d was the most active against P. vul-
garis, it was as active as ampicillin (MIC \ 50 lg/ml). In
addition, compounds 12 and 13c were the most active
against S. aureus (MIC \50 and \25 lg/ml, respectively).
On the other hand, the tested compounds devoid of
antifungal activity except 6b and 11c which showed weak
activity against A. niger.
Keywords Purines ꢀ Anticancer ꢀ Anti-HIV ꢀ
Antimicrobial activity
Introduction
Purines and condensed purines have received much atten-
tion over the years for their interesting pharmacological
properties as antineoplastic (Peifer et al., 2009; Ito et al.,
2007; Lech-Maranda et al., 2006), antileukemic (Ramas-
amy et al., 1990; Avery et al., 1990; Woo et al., 1992;
Steurer et al., 2006; Jeha and Kantarjian, 2007), anti-HIV-1
(McLaren et al., 1991; Johnson et al., 1991; Valiaeva et al.,
2006), antiviral (Lee et al., 1999; Li et al., 2005; Kmo-
nickova et al., 2006; ElAshry et al., 2006; Chen et al.,
2007) and animicrobial (Zinchenko et al., 1987; Kascatan-
Nebioglu et al., 2006) agents.
This study is a continuation to previous efforts (Rida
et al., 2005, 2007) aiming to locate novel synthetic lead
compounds for future development as anticancer, antiviral
and/or antimicrobial agents. In our earlier study (Rida
et al., 2007), we reported the synthesis and evaluation of in
vitro anticancer, anti-HIV-1 and antimicrobial activities of
a number of new 8-substituted methylxanthines. The
compounds were designed to comprise the purine nucleus
linked at C-8 with various heterocyclic ring systems either
directly or through a two-nitrogen atom spacer. Among
these derivatives, 8-[(3-benzyl-4-oxo-thiazolidin-2-ylidene)
hydrazino]-1,3,7-trimethyl-3,7-dihydropurine-2,6-dione (I,
Fig. 1) exhibited a supersensitivity profile toward leukemia
K-562 with a GI50 value \ 0.01 lM, 8-{[3-butyl-4-(4-
chlorophenyl)-2,3-dihydrothiazol-2-ylidene]hydrazino}-1,3,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-011-9612-6) contains supplementary
material, which is available to authorized users.
F. A. Ashour (&) ꢀ S. M. Rida ꢀ S. A. M. El-Hawash ꢀ
M. M. ElSemary ꢀ M. H. Badr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
University of Alexandria, Alexandria, AR, Egypt
e-mail: Fawziaashour@yahoo.com
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Med Chem Res (2012) 21:1107–1119
Fig. 1 Lead purine structures
O
O
CH3
CH3
N
H3C
O
H3C
O
N
N
N
NH
N
S
N
NH
N
S
N
N
N
N
N
CH3
O
CH3
CH3(CH2)3
Cl
I
II
O
O
H
N
H3C
H3C
O
N
N
N
NH
N
S
N
N
O
N
N
R
N
O
N
CH3
O
CH3
R
III
IV
R = (CH2)3, CH2C6H5
R = (CH2)2N(CH3)2, (CH2)3N(CH3)2
(CH2)2N(CH2CH3), (CH2)3N(CH2CH3)2.
OH
NR1R2
N
N
N
N
S
N
N
N
N
R
V
VI
R = H, CH3; R1R2 = H, CH3 ; -(CH2)5-
7-trimethyl-3,7-dihydro-purine-2,6-dione (II, Fig. 1)
displayed a moderate anti-HIV-1 activity, and 8-[(3-sub-
stituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-1,3-dimethyl-
3,7-dihydropurine-2,6-diones (III, Fig. 1) were 2–4 times
more potent than ampicillin against P. aeruginosa.
In addition, some polycyclic fused purine derivatives
have been reported as potent anticancer or antiviral agents.
For example, 6-dialkylaminoalkyl-8,10-dimethylpurino
[7,8-a]quinazoline-5,9,11(6H, 8H, 10H)triones (IV, Fig. 1)
exhibited significant in vitro cytotoxic activity against
human promyelocytic leukemia and cervix adenocarci-
noma (Settimo et al., 1998). 4-Substituted pyrido[1,2-
e]purine derivatives (V, Fig. 1) showed interesting activity
on multidrug resistant cell lines, MCF7R, which were
shown to have increased resistance to doxorubicin (Pinguet
et al., 1999). 7,8-Dihydrothiazolo[2,3-b]purin-4-ol (VI,
Fig. 1) showed in vitro inhibiting effect on influenza
virus(Hadden et al., 1986). 1,3,8,10-Tetramethylpurino
[7,8-g]-6-azapteridine-2,4,7,9(1H,3H,8H,10H)-tetrone was
found to be active against P 338 Leukemia (Ueda et al.,
1987). 4-Amino-tetrahydroquinazolino [3,2-e] purine deriv-
atives showed antiproliferative effects on the murine leu-
kemia L1210 cell line (Verones et al., 2010). Oligo and
polyribonucleotides containing selected triazolo [2,3-a]
purines were moderately active against HIV but showed
greater potency against human cyclomelagovirus (HCMV)
than ganciclovir (Tutonda et al., 1998). These findings,
together with the fact that the majority of DNA interca-
lating agents comprising a planar tricyclic or tetracyclic
chromophore(Palmer et al., 1988; Filippatos et al., 1994;
Kimura et al., 1992; Abadi et al., 1999), motivated our
interest toward the design and synthesis of some triazino
and triazolo[4,3-e]purine derivatives to explore their
Moreover, methylxanthines, including caffeine, pen-
toxifylline and theophylline are compounds used world-
wide. Many known biological effects of methylxanthines
were reported in the literature. They have been found to
enhance the cytocidal and growth-inhibitory effects of
DNA-damaging agents such as anticancer activity of some
chemotherapeutic agents, UV light and ionizing irradiation
(Saito et al., 2003; Lazarczyk et al., 2004). On the other
hand, methylxanthines have been recently shown to protect
cells against the cytostatic or cytotoxic effects of several
aromatic compounds and significantly decrease the muta-
genicity of the anticancer aromatic drugs such as dauno-
mycin, dixorubicin and mitoxantrone (Piosik et al., 2005).
Furthermore, some reports indicated that methylxan-
thines changed the inhibitory effect of antibacterial agents
(Charles and Rawal, 1973; Banerjee and Chatterjee, 1981;
Hosseinzadeh et al., 2006). Aminophylline and caffeine
potentiated the antimicrobial action of penicillin G, car-
benicillin, ceftizoxime and gentamicin against Staphylo-
cous aureus and Pseudomenous aeruginosa (Charles and
Rawal, 1973; Hosseinzadeh et al., 2006). Also, caffeine
increased the efficacy of furazolidone against Vibrios
(Banerjee and Chatterjee, 1981).
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Med Chem Res (2012) 21:1107–1119
1109
anticancer, antiviral and antimicrobial activities hoping
to go a step forward in the field of antimetabolities. Two
new series of substituted 6,8-dimethyl-1,4-dihydro-1,2,4-
triazino[4,3-e]purine-7,9(6H, 8H)-diones 3; 4; 5 and
6a, b (Scheme 1) and 5,7,9-trimethyl-1,2,4-triazolo[4,3-e]
purine-6,8(5H, 7H, 9H)-diones 11a–d; 12 and 13a–
c (Scheme 2). These compounds are considered as related
structural analogs of the previously reported IV, V and
VI (Fig. 1). In addition, 8-(3,5-diamino-1H-pyrazol-4-
yl)diazenyl-1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione 7
(Scheme 1) was designed as another molecular variant of I,
II, and III (Fig. 1). The prepared compounds were bio-
logically evaluated for their anticancer, anti-HIV-1 and
antimicrobial activities to explore the effect of such
molecular modifications on the anticipated pharmacologi-
cal effects.
The 8-diazo-1,3-dimethyl-3,7-dihydropurine-2,6-dione
hydrochloride 1 was prepared following the previously
reported procedure (Jones and Robins, 1960), by diaz-
otization of 8-amino-1,3-dimethyl-3,7-dihydropurine-2,
6-dione hydrochloride with sodium nitrite in 5% hydro-
chloric acid at 0–5°C. Refluxing ethanolic solution of the
hydrazono derivative 2a afforded the respective 4-imino-
6,8-dimethyl-1,4-dihydro-1,2,4-triazino[4,3-e]purine-7,9-
(6H,8H)-dione-3-carbonitrile 3. However, ethyl (4-imino-
6,8-dimethyl-1,4-dihydro-1,2,4-triazino[4,3-e]purine-7,9
(6H,8H)-dione)-3-carboxylate 4 was obtained by refluxing
2b in dimethyl formamide instead of ethanol. 3-Acetyl-4-
oxo-6,8-dimethyl-1,4-dihydro-1,2,4-triazino[4,3-e]purine-
7,9(6H,8H)-dione 5 was prepared in an excellent yield by
refluxing a solution of the hydrazono derivative 2c in
absolute ethanol in the presence of equivalent amount
anhydrous sodium acetate as a catalyst. However, cycli-
zation failed in absence of sodium acetate, even on using
boiling dimethyl formamide as a solvent. Hydrolysis of
the imino derivative 3 or 4 in 18% hydrochloric acid
afforded the corresponding oxo derivative 6a or b,
respectively. 8-(3,5-diamino-1H-pyrazol-4-yl)diazenyl-1,3-
dimethyl-1H-purine-2,6(3H, 7H)-dione 7 was obtained in a
good yield by treating 2a with hydrazine hydrate in dry
dimethyl formamide at room temperature. Attempts to
prepare compound 8, by cyclocondensation of 4b with
Chemistry
The target compounds were prepared following the syn-
thetic pathways depicted in Schemes 1 and 2. The key
intermediates hydrazono derivatives 2a–c were prepared in
good yields, as previously reported (Jones and Robins,
1960), by coupling an alcoholic suspension of 8-diazothe-
ophylline 1 with the active methylene of malononitrile,
ethyl cyanoacetate or ethyl acetoacetate in dry pyridine.
O
O
O
H3C
N
H3C
N
N
N
H3C
H
v
H
N
-
+
N
N
N
N
N
N
N
O
N
N
O
N
N
N
O
N
CH
CH3
3 HN
CH3
O
CN
R
a; R = CN
1
3
6 a,b
b; R = COOH
i
ii
2a
v
O
O
H
H3C
O
H3C
N
N
N
N
N
H
H
iii
a; R = R1 = CN
b; R = CN, R1= COOC2H5
c; R = COCH3, R1 = COOC2H5
N
R
N
N
N
N
O
N
N
2b
R1
CH3
CH
3 HN
2 a-c
2b
COOC2H5
vi
4
iv
2a
vi
x
2c
O
O
O
H
H3C
N
N
H3C
H2N
H2N
N
H
N
H
N
H3C
O
O
N
N
N
NH
N
N
N
O
N
N
N
N
NH
N
O
N
N
N
N
CH3
CH3
O
COCH3
CH3
7
H2N
5
8
Reagents: i = CH2(RR1); ii = EtOH / ; iii = DMF / ; iv = NaAc / EtOH / ; v = 18 % HCl; vi = NH2NH2.H2O.
Scheme 1 Synthetic route for the synthesis of compounds 2a–c, 3, 4, 5, 6a, b and 7
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Med Chem Res (2012) 21:1107–1119
hydrazine hydrate, following the reaction condition descri-
bed for compound 7, failed and the result was the recovery
of the starting material. However, raising the reaction
temperature from ambient to reflux gave the unexpected
compound 4 (Confirmed by mixed m.p. with compound 4,
12 with different alkyl halides in dimethyl formamide in
the presence of equivalent amount of anhydrous potassium
carbonate gave the respective 3-alkylthio or aralkylthio-
5,7,9-trimethyl-1,2,4-triazolo[4,3-e]purine-6,8(5H,7H,9H)-
diones 13a–c.
1
IR and H-NMR spectra).
The structures of the synthesized compounds were
1
Scheme 2 starts with 8-hydrazinocaffeine 9 which was
prepared in a good yield, as previously reported (Priewe
and Poljak,1955), by refluxing the corresponding 8-chloro
derivative with hydrazine hydrate in ethanol. Condensation
of 9 with the appropriate aromatic aldehydes in boiling
ethanol yielded the corresponding 8-arylidenehydrazino-
3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-diones 10a-d,
as previously described (Klosa, 1956). Oxidative cycliza-
tion of 10a–d using bromine in the presence of equivalent
amount of anhydrous sodium carbonate afforded the cor-
responding 3-aryl-5,7,9-trimethyl-1,2,4-triazolo[4,3-e]pur-
ine-6,8(5H,7H,9H)-diones 11a–d. On the other hand, 5,7,
9-trimethyl-3-thioxo-2,3-dihydro-1,2,4-triazolo[4,3-e]pur-
ine-6,8(5H,7H,9H)-dione 12 was obtained by refluxing
ethanolic solution of 9 with carbon disulfide in the presence
of equivalent amount of sodium hydroxide. Alkylation of
confirmed by microanalyses, IR, H-NMR, 13C-NMR and
1
mass spectral data (experimental section). H-NMR spec-
trum of compound 2c showed two singlets at 7.19 and
12.88 ppm due to two NH protons, indicating the existence
of this compound in the hydrazono form rather than the azo
1
form. H-NMR spectrum of compounds 3 and 4 showed
two deuterium oxide-exchangeable singlets at different
chemical shifts attributed to two NH protons, confirming
that these compounds exist in the imino form rather than
the amino form. IR and 1H-NMR spectra of compounds 6a,
b revealed the existence of three possible tautomeric forms.
The IR spectrum showed OH and NH stretching absorption
bands and the 1H-NMR showed two NH and one OH
signals at different chemical shifts each is integrated for 1/3
proton. IR spectrum of compound 12 revealed a broad band
at 3442 cm-1 due to NH stretching and its 1H-NMR
Scheme 2 Synthetic route for
the synthesis of compounds
10a–d, 11a–d, 12 and 13a–c
O
CH3
CH3
N
N
NHNH2
N
O
N
CH3
iii
i
9
O
O
CH3
CH3
CH3
N
CH3
N
N
N
NH
N
N
N
O
N
N
O
N
NH
CH3
CH3
S
10 a-d
ii
12
R
iv
O
O
CH3
CH3
CH3
N
CH3
N
N
N
N
N
N
N
N
O
N
N
O
N
CH3
CH3
RS
13 a-c
R
R = CH3, C2H5, CH2C6H5
11 a-d
R = H, Cl, Br, OCH3
Reagents: i = R-C6H4CHO, ii = Br2 / Na2CO3, iii = CS2 / NaOH, iv = RX / K2CO3.
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Med Chem Res (2012) 21:1107–1119
1111
showed a deuterium oxide exchangeable singlet at
6.73 ppm attributed to NH proton, confirming the existence
of this compound in the thione rather than the thiol form.
performed at the Microanalytical Laboratory, National
Research Center, Cairo, and the data were within 0.4% of
the theoretical values. Reactions were monitored by thin
layer chromatography on silica gel-protected aluminium
sheets (Type 60 F254, Merck, Darmstadt, Germany) and
the spots were detected by exposure to UV-lamp at c
254 nm for few seconds.
Experimental
All melting points were determined in open-glass capil-
laries on a Gallenkamp melting point apparatus (Sanyo)
and were uncorrected. The IR spectra were recorded using
KBr discs on a Perkin-Elmer 1430 spectrophotometer
(Perkin-Elmer, Norwalk, CT, USA). 1H-NMR spectra were
recorded on a Varian Gemini 200 MHz spectrometer
(Varian Inc., Palo Alto, CA, USA) or JNM-LA 400 FT
NMR system (JEOL, Tokyo, Japan) using tetramethylsil-
ane (TMS) as internal standard and dimethyl sulfoxide
(DMSO-d6) as solvent. Splitting patterns were assigned as
follows: s = singlet, d = doublet, t = triplet, q = quartet,
and m = multiplet (chemical shift d ppm). The 13C-NMR
spectra were performed on Joel spectrometer (500 MHZ)
using tetramethylsilane (TMS) as internal standard and
dimethylsulfoxide (DMSO-d6) as a solvent. MS were run
on a Finnigan mass spectrometer model SSQ/7000 (70 eV,
Thermo Electron Corporation). The microanalyses were
Synthesis of 3,7-dihydro-1,3-dimethyl-2,6-dioxo-1H-
purin-8-ylhydrazono malononitrile 2a, ethyl (3,7-
dihydro-1,3-dimethyl-2,6-dioxo-1H-purin-8-
ylhydrazono)cyanoacetate 2b, and ethyl 2-[(3,7-
dihydro-1,3-dimethyl-2,6-dioxo-1H-purin-8-
yl)hydrazono]-3-oxobutanoate 2c
To an ice-cooled suspension of 8-diazotheophylline 1
(1.03 g, 5 mmole) in absolute ethanol (25 ml), a solution of
malononitrile, ethyl cyanoacetate or ethyl acetoacetate
(7.5 mmole) in dry pyridine (25 ml) was added dropwise
while stirring over a period of half an hour. The reaction
mixture was then stirred at room temperature for 3 h. The
separated product was filtered, washed with water then
ethanol and air dried (Table 1).
Table 1 Physical and analytical data of the synthesized compounds (2–13)
Comp. No.
R
R1
Yield (%)
MP (Crys. Solv.)
Mol. formulaa (mol. wt.)
2a
CN
CN
70
97
41
78
72
94
96
96
67
82
91
89
74
68
58
52
85
45
85
77
63
165–167
209–211b
C10H8N8O2 (272.22)
–
2b
2c
CN
COOC2H5
COCH3
COOC2H5
161–163
C13H16N6O5 (336.31)
3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
[350 (EtOH)
C10H8N8O2 (272.22)
C12H13N7O4 (319.28)
C11H10N6O4 (290.24)
C10H7N7O3 (273.21)
C10H8N6O5.HCl (328.67)
C10H12N10O2 (304.27)
4
–
234–236 (EtOH)
[350 (DMF/EtOH)
272–274 (EtOH)
224–226 (EtOH)
[350 (DMF/EtOH)
273–275c (EtOH)
260–262 (DMF)
265–267 (DMF)
262–264c (EtOH)
240–242 (EtOH)
237–239 (EtOH)
248–250 (EtOH)
225–227 (Dioxane)
281–283 (EtOH/Ether)
188–190 (EtOH)
164–166 (EtOH)
132–134 (EtOH)
5
–
6a
CN
6b
7
COOH
–
10a
10b
10c
10d
11a
11b
11c
11d
12
H
C15H16N6O2 (312.33)
Cl
C
15H15ClN6O2 (346.77)
15H15BrN6O2 (391.22)
Br
C
OCH3
H
C16H18N6O3 (342.35)
C15H14N6O2 (310.31)
C15H13ClN6O2 (344.76)
C15H13BrN6O2 (389.21)
Cl
Br
OCH3
–
C16H16N6O3 (340.34)
C9H10N6O2S (266.28)
C10H12N6O2S (280.31)
C11H14N6O2S (294.33)
C16H16N6O2S (356.40)
13a
13b
13c
a
CH3
CH2CH3
CH2C6H5
Analyzed for C, H, N and the results are within 0.4% of the theoretical values
b
c
Jones and Robins, 1960
Klosa, 1956
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Med Chem Res (2012) 21:1107–1119
IR of compound 2a (m cm-1): 3225 (br.NH); 2206
(C:N); 1711 (C=O purine); 1666, 1552, 1503 (C=N, NH
bending, C=C).
(C4=NH iminotriazinopurine); 135.96(N-C5a=C9a triazin-
opurine) 32.26, 28.98(N3–CH3, N1–CH3 purine, respec-
tively); 151.38, 154.89(C2=O, C4=O purine, respectively);
114.58 (N–C9a=C5a triazinopurine); 145.24(N10–C10a=N1–
H triazinopurine).
IR of compound 2b (m cm-1): 3258, 3164 (NH); 2219
(C:N); 1711 (br.C=O ester and C=O purine); 1656, 1614,
1540 (C=N, NH bending, C=C); 1236, 1140, 1053 (C–O–C).
IR of compound 2c (m cm-1): 3276, 3201(NH); 1726
(C=O ester); 1697 (C=O ketone and C=O purine); 1649,
1563, 1525 (C=N, NH bending, C=C); 1239, 1139, 1067
(C–O–C).
1H-NMR of compound 2c (DMSO-d6, d ppm, Varian
Gemini 200 MHz): 1.29 (t, J = 7 Hz, 3H, CH2–CH3); 2.22
(s, 3H, COCH3); 3.23 (s, 3H, purine-N3–CH3); 3.41 (s, 3H,
purine-N1–CH3); 4.24 (q, J = 7 Hz, 2H, CH2–CH3); 7.19,
12.88 (two s, each 1H, two NH, D2O exchangeable).
Electron impact Mass Spectrum m/z (% abundance):
320 (2) M ?1; 319 (9) M; 275 (6); 247 (30); 195 (100); 178
(5); 163 (9); 152 (11); 138 (16); 120 (9); 110 (10); 109 (8);
108 (7), 106 (7); 94 (9); 93 (12); 83 (5); 82 (40); 81 (12);
80 (13); 78 (9); 69 (5); 68 (28); 67 (49); 66 (6); 58 (13); 57
(5); 56 (10); 55 (8); 54 (14); 53 (29).
Synthesis of 3-acetyl-4-oxo-6,8-dimethyl-1,4-dihydro-
1,2,4-triazino[4,3-e]purine-7,9(6H,8H)-dione 5
To a solution of ethyl 2-[(3,7-dihydro-1,3-dimethyl-2,6-
dioxo-1H-purin-8-yl)azo]-3-oxobutanoate 2c (0.67 g, 2
mmole) in absolute ethanol (10 ml), anhydrous sodium
acetate (0.16 g, 2 mmole) was added. The reaction mixture
was heated under reflux for 1 h and left to cool to room
temperature. The separated crystals were filtered, washed
with water, dried and recrystallized from dimethylforma-
mide/ethanol (Table 1).
IR (m cm-1): 3344, 3257 (NH); 1732 (C=O triazinone);
1695, 1690 (C=O purine and C=O ketone, respectively);
1655, 1590, 1476 (C=N, NH bending, C=C).
1H-NMR (DMSO-d6, d ppm, JNM-LA 400 FT): 2.44 (s,
3H, COCH3); 3.16 (s, 3H, purine-N3-CH3); 3.42 (s, 3H,
purine-N1-CH3); 6.95 (s, 1H, NH, D2O exchangeable). 13C-
NMR (DMSO-d6, dppm):23.14(O=C–CH3 acetyl); 194.57
(C=Oacetyl); 154.17(C3–COCH3 triazinopurine); 193.23
(C4=O oxotriazinopurine); 136.15(N–C5a=C9a triazinopu-
rine) 32.32,29.12 (N3-CH3, N1-CH3 purine, respectively);
151.41, 154.92(C2=O, C4=O purine, respectively); 114.62
Synthesis of 4-imino-6,8-dimethyl-1,4-dihydro-1,2,4-
triazino[4,3-e]purine-7,9-(6H,8H)-dione-
3-carbonitrile 3
A solution of 3,7-dihydro-1,3-dimethyl-2,6-dioxo-1H-purin-
8-ylazomalononitrile 2a (0.54 g, 2 mmole) in absolute eth-
anol (10 ml) was heated under reflux for 15 min then left to
cool at room temperature. The separated crystalline product
was filtered, dried and recrystallized from ethanol (Table 1).
IR (m cm-1): 3310 (br.NH); 2242 (C:N); 1697 (C=O
purine); 1637, 1502, 1471 (C=N, NH bending, C=C).
1H-NMR (DMSO-d6, d ppm, JNM-LA 400 FT): 3.30 (s,
3H, purine-N3-CH3); 3.54 (s, 3H, purine-N1–CH3); 9.99,
10.45 (two s, each 1H, two NH, D2O exchangeable).
Synthesis of ethyl (4-imino-6,8-dimethyl-1,4-dihydro-
1,2,4-triazino[4,3-e]purine-7,9(6H,8H)-dione)-3-
carboxylate 4
(N-C9a=C5a
triazinopurine);
145.12(N10–C10a=N1–H
triazinopurine).
A solution of ethyl (3,7-dihydro-1,3-dimethyl-2,6-dioxo-
1H-purin-8-yl azo)cyanoacetate 2b (0.64 g, 2 mmole) in
dry dimethyl-formamide (10 ml) was heated under reflux
for 2 h then left to cool to room temperature. The precip-
itate formed after addition of few drops of water was fil-
tered, dried and crystallized from ethanol (Table 1).
IR (t cm-1): 3306, 3174 (NH); 1718, 1700 (C=O ester
and C=O purine respectively); 1637, 1592, 1514, 1468
(C=N, NH bending, C=C); 1237, 1171, 1068 (C–O–C).
1H-NMR (DMSO-d6, d ppm, JNM-LA 400 FT): 1.36 (t,
J = 7 Hz, 3H, CH2–CH3); 3.30 (s, 3H, purine-N3–CH3);
3.54 (s, 3H, purine-N1–CH3); 4.41 (q, J = 7 Hz, 2H, CH2–
CH3); 9.25, 10.71 (two s, each 1H, two NH, D2O
Synthesis of 4-oxo-6,8-dimethyl-1,4-dihydro-1,2,4-
triazino[4,3-e]purine-7,9(6H,8H)-dione-3-carbonitrile
6a, and 4-oxo-6,8-dimethyl-1,4-dihydro-1,2,4-
triazino[4,3-e]purine-7,9(6H,8H)-dione-3-carboxylic
acid 6b
A
solution of 4-imino-6,8-dimethyl-1,4-dihydro-1,2,4-
triazino-[4,3-e]purine-7,9(6H,8H)-dione-3-carbonitrile 3
or ethyl (4-imino-6,8-dimethyl-1,4-dihydro-1,2,4-triazino
[4,3-e]purine-7,9(6H,8H)-dione)-3-carboxylate 4 (2 mmole)
in 18% hydrochloric acid (10 ml) was heated under reflux
for 1 h. The reaction mixture was concentrated under
reduced pressure and left to cool to room temperature. The
separated crystalline product was filtered, dried and re-
crystallized from ethanol (Table 1).
exchangeable). 13C-NMR (DMSO-d6,
d ppm):13.77
(O = C–CH2–CH3 ester); 61.13(O–CH2–CH3 ester);162.89
(C=O ester), 153.97(C3-COOEt triazinopurine); 139.47
123

Med Chem Res (2012) 21:1107–1119
1113
IR of compound 6a (m cm-1): 3440, 3166, 3133 (br.OH,
NH); 2243 (C:N); 1727 (C=O triazinone); 1694 (C=O
purine); 1664, 1597, 1500, 1459 (C=N, NH bending, C=C).
1H-NMR of compound 6a (DMSO-d6, d ppm, JNM-LA
400 FT): 3.24 (s, 3H, purine-N3-CH3); 3.46 (s, 3H, purine-
with ethanol, dried and crystallized from the proper solvent
(Table 1).
IR of compounds 10a–d (t cm-1): 3177–3115 (NH);
1697–1691 (C=O); 1645–1624, 1623–1596, 1578–1574,
1548–1538 (C=N, NH bending, C=C).
1H-NMR of compound 10a (DMSO-d6, d ppm, JNM-
LA 400 FT): 3.19 (s, 3H, purine-N3–CH3); 3.37 (s, 3H,
purine-N1–CH3); 3.92 (s, 3H, purine-N7–CH3); 7.37–7.44
(m, 3H, Ar–C3,4,5–H); 7.65 (d, 2H, Ar–C2,6–H), 8.09 (s,
1H, N=CH); 11.45 (s, 1H, NH, D2O exchangeable).
1H-NMR of compound 10b (DMSO-d6, d ppm, JNM-
LA 400 FT): 3.17 (s, 3H, purine-N3–CH3); 3.33 (s, 3H,
purine-N1–CH3); 3.85 (s, 3H, purine-N7–CH3); 7.45 (d,
J = 8.4 Hz, 2H, Ar–C2,6–H); 7.65 (d, J = 8.4 Hz, 2H, Ar–
1
N1-CH3); 6.93, 7.06, 7.19 (three s, each = H, OH, NH,
NH-N, D2O exchangeable).
3
Electron impact mass spectrum of compound 6a m/z
(% abundance): 274 (8) M ? 1; 273 (58) M; 244 (8); 217
(6); 216 (20); 215 (8); 189 (16); 188 (22); 161 (17); 152
(13); 136 (15); 109 (9); 108 (21); 94 (8); 93 (7); 82 (23);
81 (17); 80 (10); 79 (11); 78 (9); 69 (13); 68 (56); 67
(100); 66 (12); 58 (11); 56 (31); 55 (24); 54 (24); 53 (38);
52 (9).
IR of compound 6b (t cm-1): 3522–3441, 3126 (br.OH,
br.NH); 1726 (C=O triazinone); 1699 (br.C=O acid and
C=O purine); 1664, 1646, 1569, 1535 (C=N, NH bending,
C=C).
C3,5–H); 8.07 (s, 1H, N=CH); 11.50 (s, 1H, NH, D2O
exchangeable).
Synthesis of 3-aryl-5,7,9-trimethyl-1,2,4-triazolo[4,3-
1H-NMR of compound 6b (DMSO-d6, d ppm, JNM-LA
e]purine-6,8(5H,7H,9H)-diones 11a–d
400 FT): 3.19 (s, 3H, purine-N3–CH3); 3.38 (s, 3H, purine-
1
N1–CH3); 6.98, 7.11, 7.20 (three s, each = H, OH, NH,
NH–N, D2O exchangeable); 13.19 (s, 1H, COOH, D2O
To a stirred mixture of 8-arylidenehydrazino-3,7-dihydro-
1,3,7-trimethyl-1H-purine-2,6-diones 10a–d (2 mmole)
and anhydrous sodium carbonate (2.3 g, 2 mmole) in
chloroform (20 ml), bromine (0.3 ml) was added. The
reaction mixture was stirred at room temperature for 2 h,
evaporated under reduced pressure. The residue was tritu-
rated with ice-cold water, filtered, washed with water, dried
and crystallized from the proper solvent (Table 1).
IR of compounds 11a–d (t cm-1): 1713–1707 (C=O);
1676–1664, 1645–1637, 1533–1525, 1482–1469 (C=N,
C=C).
1H-NMR of compound 11a (DMSO-d6, d ppm, JNM-
LA 400 FT): 2.88 (s, 3H, purine-N3–CH3); 3.31 (s, 3H,
purine-N1–CH3); 3.83 (s, 3H, purine-N7–CH3); 7.53–7.60
(m, 3H, Ar–C3,4,5–H); 7.68 (d, 2H, Ar–C2,6–H).
1H-NMR of compound 11b (DMSO-d6, d ppm, JNM-
LA 400 FT): 2.95 (s, 3H, purine-N3–CH3); 3.37 (s, 3H,
purine-N1–CH3); 3.83 (s, 3H, purine-N7–CH3); 7.62 (d,
3
exchangeable).
Synthesis of 8-(3,5-diamino-1H-pyrazol-4-yl)diazenyl-
1,3-dimethyl-1H-purine-2,6(3H, 7H)-dione 7
To an ice-cooled suspension of 3,7-dihydro-1,3-dimethyl-
2,6-dioxo-1H-purin-8-yl azomalononitrile 2a (0.54 g, 2
mmole) in dry dimethylformamide (5 ml), hydrazine
hydrate (98%) (0.5 g, 10 mmole) was added dropwise with
stirring. After complete addition, the reaction mixture was
left overnight at room temperature, poured onto ice/water
and neutralized with dilute hydrochloric acid. The sepa-
rated product was filtered, washed with water, dried, and
crystallized from dimethylformamide/water (Table 1).
IR (m cm-1): 3445, 3328, 3239 (NH2, NH); 1677 (C=O
purine); 1645, 1569, 1484 (C=N, NH bending, C=C).
1H-NMR (DMSO-d6, d ppm, Varian Gemini 200 MHz):
3.26 (s, 3H, purine-N3–CH3); 3.47 (s, 3H, purine-N1–CH3);
6.58 (br.s, 4H, two NH2, D2O exchangeable); 11.22, 12.45
(two s, each 1H, NH purine, NH pyrazole, D2O exchange-
able).
J = 8 Hz, 2H, C2,6–Ar–H); 7.72 (d, J = 8 Hz, 2H, C3,5
–
Ar–H).
1H-NMR of compound 11c (DMSO-d6, d ppm, JNM-LA
400 FT): 2.96 (s, 3H, purine-N3–CH3); 3.25 (s, 3H, purine-
N1–CH3); 3.83 (s, 3H, purine-N7–CH3); 7.65 (d, J = 8 Hz,
2H, C2,6–Ar–H); 7.76 (d, J = 8 Hz, 2H, C3,5–Ar–H).
Synthesis of 8-arylidenehydrazino-3,7-dihydro-1,3,7-
trimethyl-1H-purine-2,6-diones 10a–d
Synthesis of 5,7,9-trimethyl-3-thioxo-2,3-dihydro-
1,2,4-triazolo[4,3-e]purine-6,8(5H,7H,9H)-dione 12
To a suspension of 8-hydrazinocaffeine 9 (1.12 g, 5
mmole) in absolute ethanol (20 ml), the appropriate aro-
matic aldehyde (5 mmole) was added. The reaction mix-
ture was heated under reflux for 30 min then cooled to
room temperature. The separated solid was filtered, washed
To a mixture of 8-hydrazinocaffeine 9a (2.24 g, 10 mmole)
and sodium hydroxide (0.4 g, 10 mmole) in absolute eth-
anol (20 ml), carbon disulphide was added (30 ml). The
reaction mixture was heated under reflux for 24 h and then
123

1114
Med Chem Res (2012) 21:1107–1119
the solvent was evaporated under reduced pressure. The
residue was dissolved in water, filtered and the filterate was
neutralized with concentrated hydrochloric acid. The
formed precipitate was filtered, washed with water, dried
and crystallized from ethanol/ether (Table 1).
IR (t cm-1): 3442 (br.NH); 2631 (weak SH); 1715
(C=O); 1684, 1489, 1467 (C=N, C=C); 1549, 1331, 1035,
970 (N–C=S amide I, II, III, IV bands).
1H-NMR (DMSO-d6, d ppm, Varian Gemini 200 MHz):
3.16 (s, 3H, purine-N3–CH3); 3.33 (s, 3H, purine-N1–CH3);
3.60 (s, 3H, purine-N7–CH3); 6.73 (s, 1H, NH, D2O
exchangeable).
growth of the treated cells compared to the untreated
control cells. Compounds which reduced the growth of any
one of the cell lines to 32% or less (negative numbers
indicate cell kill) were passed on for the evaluation in the
full panel in vitro antitumor screen consisting of 60 human
tumor cell lines, derived from nine clinically isolated types
of cancer types (Leukemia, non-small cell lung cancer,
colon cancer, CNS cancer, melanoma, ovarian cancer,
renal cancer, prostate cancer, breast cancer) following the
NCI preclinical antitumor drug discovery screen. Each
compound was tested at five concentrations at ten-fold
dilutions. A 48 h continuous drug exposure protocol was
used and a sulforodamine B (SRB) protein assay was used
to estimate cell viability or growth (Boyed and Paull,
1995).
Synthesis of 3-alkylthio or aralkylthio- 5,7,9-trimethyl-
1,2,4-triazolo[4,3-e]purine-6,8(5H,7H,9H)-diones
13a–c
Results
A. mixture of 5,7,9-trimethyl-3-thioxo-2,3-dihydro-1,2,4-
triazolo[4,3-e]purine-6,8(5H,7H,9H)-dione 12 (0.53 g, 2
mmole), anhydrous potassium carbonate (0.28 g, 2 mmole)
and the appropriate alkyl halide (3 mmole) in dry dimethyl
formamide (5 ml) was stirred at room temperature for 3 h.
The reaction mixture was poured onto ice/water and the
formed precipitate was filtered, washed with water, dried
and crystallized from ethanol (Table 1).
IR of compounds 13a–c (t cm-1): 1711–1698 (C=O);
1664–1652, 1550–1535, 1452–1449 (C=N, C=C);
1220–1215, 1036–1035 (C–S–C).
Four of the synthesized compounds (3; 4; 11a, b) were
selected by the National Cancer Institute (NCI) and eval-
uated for their in vitro antineoplastic activity against three-
cell-line panel consisting of the Breast-MCF-7 cell line, the
lung-NCI-H460 cell line and the CNS-SF-268 cell line.
Only compound 3 showed promising activity. It reduced
the growth of breast cell line to less than 32% (14%)
(Table 2). Compound 3 was then subjected to the NCI in
vitro disease-oriented human cells screening panel assay
(Grever et al., 1992; Boyed and Paull, 1995) to investigate
its antitumor activity. About 60 cell lines of nine tumor
subpanels were incubated with five concentrations
(0.01–100 lM) for each compound and were used to create
log concentration versus % growth inhibition curves. Three
response parameters (GI50, TGI, and LC50) were calculated
for each cell line. The GI50 value corresponds to the
compound’s concentration causing 50% decreases in net
cell growth. The TGI value is the compound’s concentra-
tion resulting in total growth inhibition and the LC50 is the
compound’s concentration causing a net 50% loss of initial
cells at the end of the incubation period (48 h). Subpanel
and full panel mean-graph midpoint values (MG-MID) for
certain agents are the average of individual real and default
GI50, TGI or LC50 values of all cell lines in subpanel and
fullpanel, respectively.
1H-NMR of compound 13a (DMSO-d6, d ppm, JNM-
LA 400 FT): 2.68 (s, 3H, S–CH3); 3.19 (s, 3H, purine-N3–
CH3); 3.40 (s, 3H, purine-N1–CH3); 3.73 (s, 3H, purine-
N7–CH3).
Biological activity
Anticancer screening
Materials and methods
Anticancer screening was performed at the National Can-
cer Institute (NCI), Bethesda, Maryland, USA. The com-
pounds were evaluated in three cell lines in a one-dose
primary anticancer assay subsequent to the NCI preclinical
antitumor drug discovery screen (Grever et al., 1992;
Boyed and Paull, 1995). The three cell lines used were lung
(NCI-H460), breast (MCF-7) and CNS (SF-268). In the
current protocol, each cell is inoculated and preincubated
on microtiter plates. Test agents are then added at a single
concentration (100 lM), and the culture is incubated for
48 h. Endpoint determinations are made with alamar blue.
The results for each agent are presented as the percent of
Compound 3 exhibited considerable activity against
some of the tested cell lines (Table 3). For example, GI50
values of 31.8 lM against non-small cell lung cancer HOP-
92, 25.2 lM against melanoma MALME-3 M and
32.9 lM against breast T-47D.
The GI50, TGI, and LC50 subpanel and full panel mean-
graph midpoint (MG-MID) values, respectively, are shown
(Table 4). The ratio obtained by dividing the compound’s
full panel MG-MID (lM) by its individual subpanel MG-
MID (lM) has been considered as a measure of compound
123

Med Chem Res (2012) 21:1107–1119
1115
Table 2 Growth percentages of the 3-cell line panel in primary anticancer screen of some selected compounds
Comp. no.
NSC no.
Sample concentration
Growth percentages
Lung NCI-H460
Breast MCF7
CNS SF-268
3
S-720606
S-720605
S-720609
S-720610
1.00E-04 Molar
1.00E-04 Molar
1.00E-04 Molar
1.00E-04 Molar
62
99
98
95
14
93
84
82
47
98
90
90
4
11a
11b
Table 3 Growth inhibitory action (GI50) of some selected in vitro tumor cell lines (lM)
Comp. no.
NCS no.
Panel
Subpanel cell lines (cytotoxicity GI50 in lM)a
3
S-720606
Melanoma
MALME-3M (25.2), SK-MEL-5 (45.9)
HOP-92 (31.8), NCI-H226 (54.0)
T-47D (32.9)
Lung cancer
Breast cancer
Renal cancer
Leukemia
ACHN (44.3), A498 (51.4), CAKI-1 (51.7), UO-31 (50.6)
CRF-CEM (67.8)
a
Data obtained from NCI in vitro disease-oriented human cell screen
Table 4 Median growth inhibitory concentrations (GI50
, lM),
was non-selective with ratios ranging between 0.85 and
1.18 (Table 4).
Median total growth inhibitory concentrations (TGI, lM) of in vitro
subpanel tumor cell lines, and selectivity ratios of compound 3 toward
the nine tumor cell lines
In vitro anti-HIV-1 activity
Subpanel tumor
cell linesa
GI50 (lM)
TGI (lM)
Selectivity
ratios
Materials and methods
I
94.6
87.3
100
100
0.90
0.97
0.85
0.87
1.04
0.87
1.18
0.99
0.93
–
II
100
The in vitro anti-HIV drug testing system was performed in
the National Cancer Institute’s Developmental Therapeutics
Program, AIDS antiviral screening program, according to a
reported procedure (Weislow et al., 1989). The assay
involved the killing of T4 lymphocytes by HIV. T4 lym-
phocytes (CEM cell line) were exposed to HIV at a virus-to-
cell ratio of approximately 0.05 and treated with the com-
pounds, dissolved in dimethylformamide, at doses ranging
from 10-8 to 10-4 M. A complete cycle of virus reproduction
is necessary to obtain the required cell killing (incubation at
37°C in a 5% carbon dioxide atmosphere for 6 days).
Uninfected cells with the compound served as a toxicity
control, whereas the infected and uninfected cells without
the compound served as basic controls. After incubation, the
tetrazolium salt XTT was added to all wells, and cultures
were incubated to allow formazan color development by
viable cells. Formazan production was measured spectro-
photometrically and possible protective activity was con-
firmed by microscopic detection of viable cells. The effect of
each compound on cell growth of HIV-infected and unin-
fected cells was compared to that of untreated uninfected
cells. All tests were compared with AZT as positive control
carried out at the same time under identical conditions.
III
100
IV
98.0
81.6
97.9
72.2
86.3
91.6
85.1b
100
V
96.9
100
VI
VII
100
VIII
100
IX
100
100c (100)d
Full panel MG-MID
a
I, Leukemia; II,non-small cell lung cancer; III, colon cancer; IV,
CNS cancer; V, melanoma; VI, ovarian cancer; VII, renal cancer;
VIII, prostate cancer; IX, breast cancer
b
GI50 (lM) full panel mean-graph mid-point (MG-MID) = The
average sensitivity of all cell lines toward the test agent
c
TGI (lM) full panel mean-graph mid-point (MG-MID) = The
average sensitivity of all cell lines toward the test agent
d
LC50 (lM) full panel mean-graph mid-point (MG-MID)
selectivity (Monks et al., 1991). Ratios between 3 and 6
refer to moderate selectivity, ratios greater than 6 indicate
high selectivity toward the corresponding cell line, while
compounds meeting neither of these criteria are rated non-
selective (Monks et al., 1991). Accordingly, compound 3
123

1116
Med Chem Res (2012) 21:1107–1119
Table 5 Maximum % Protection, the corresponding dose (molar) and IC50 (molar) of the selected compounds
Comp. no.
NCS no.
Maximum % protection
Dose (M)
IC50 (M)
5
722234-U/1
722233-T/1
722232-S/1
722235-V/1
722239-Z/1
722240-A/1
1.43
10.90
3.73
2.00 9 10-5
2.00 9 10-4
2.00 9 10-5
2.00 9 10-4
2.00 9 10-4
2.00 9 10-4
[2.00 9 10-4
[2.00 9 10-4
[2.00 9 10-4
[2.00 9 10-4
[2.00 9 10-4
[2.00 9 10-4
6a
6b
7
30.52
4.86
13a
13c
35.54
Results
Candida albicans (NCTC 2708) and Saccharomyces cer-
visiae (ATTC 9763) as examples of yeast; Asperigillus
niger (ATTC 16404) and Asperigillus terreus (local iso-
late) as examples of true fungi. Each 100 ml of sterile
molten agar (at 45°C) received 1 ml of 6 h broth and then
the seeded agar was poured into sterile Petri dishes. Cups
(8 mm in diameter) were cut in the agar. Each cup received
0.1 ml of the 2 mg/ml solution of the tested compounds.
The plates were then incubated at 37°C for 24 h for bac-
teria or 48 h for fungi. A control using DMF without the
test compound was included for each organism. Ampicillin
in DMF was used as standard antibacterial, while clot-
rimazol was used as antifungal reference.
Six compounds (5; 6a, b; 7; 13a, c) have been selected by
NCI and evaluated for their effects on HIV-1 induced cy-
topathogenicity in a human T4 lymphocyte cell line (CEM)
(Weislow et al., 1989). Activity is expressed as % of
protection which represents the percentage of surviving
HIV-infected cells treated with the test compound (at the
indicated concentration) relative to the same uninfected
untreated controls. The effective concentration 50%
(EC50), represents the concentration of the test agent
resulting in 50% reduction of viral cytopathic effect. The
50% inhibitory concentration (IC50), represent the toxic
concentration of drug resulting in 50% growth inhibition of
normal uninfected cells. In this screen, the compounds are
considered to be active if they display complete protection
at a concentration \0.1 lM. Compounds which show
incomplete protection or show protection at a concentration
above 0.1 lM are considered moderately active. As
revealed from (Table 5), compounds 7 and 13c showed
moderate reduction of viral cytopathic effect by 30.52 and
35.54% at 2.00 9 10-4 M, respectively. The other tested
compounds were inactive.
Minimal inhibitory concentration (MIC) measurement
The minimal inhibitory concentration (MIC) of the most
active compounds was measured using the two-fold serial
broth dilution method (Scott, 1989). The test organisms
were grown in their suitable broth for 24 h for bacteria and
48 h for fungi at 37°C. Twofold serial dilutions of the test
compounds solution were prepared using the suitable broth
to obtain concentrations 200, 100, 50, and 25 lg/ml. The
tubes were then inoculated with the test organisms; each
5 ml received 0.1 ml of the above inoculum and were
incubated at 37°C for 48 h. Then the tubes were observed
for the presence or absence of microbial growth.
Antimicrobial evaluation
Materials and methods
Results
Inhibition zones measurement
Compounds (3; 4; 5; 6a, b; 7; 11a–d; 12; 13a–c) were
preliminary evaluated for their in vitro antibacterial activ-
ity. The results recorded in (Table 6) revealed that the
tested compounds exhibited promising activity toward the
Gram-negative P. aeruginosa and P. vulgaris. Compound
12 was the most active against P. aeruginosa, it was
equipotent to ampicillin (MIC \ 100 lg/ml), while com-
pounds 11b, 11c, 11d, and 13a showed half the activity. On
the other hand, compound 11d was the most active against
P. vulgaris, it was as active as ampicillin (MIC \ 50 lg/
ml). Compounds 4, 5, 6a, 11b, and 11c displayed half the
The tested compounds were evaluated by the agar cup
diffusion technique (Conte and Barriere, 1988), using a
2 mg/ml solution in DMF. The test organisms were
Staphylococcus aureus (NCTC 4163) and Bacillus subtilis
(ATTC 6633) as Gram-positive bacteria, Pseudomonas
aeruginosa (ATTC 9027), Escherichia coli (5933), and
Salmonella typhi (ATCC 13311) as Gram-negative bacteria
and Proteus vulgaris (ATTC 49132) as spore forming
Gram-negative bacteria. They were also evaluated for their
in vitro antifungal activity against four types of fungi,
123

Med Chem Res (2012) 21:1107–1119
1117
Table 6 The inhibition zones (IZ) in mm diameter and minimal inhibitory concentration (MIC) in lg/ml of the tested compounds against
different bacterial strains
Comp. no.
S. aureus
B. subtilis
S. typhi
P. aeruginosa
E. coli
P. vulgaris
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
IZ
MIC
3
–
16
–
–
14
14
14
–
–
12
–
–
–
18
20
–
–
14
15
14
12
13
–
–
19
22
23
22
21
14
19
25
22
28
19
19
19
18
–
–
\100
\100
\100
\200
–
4
–
–
–
–
–
5
–
–
–
–
–
6a
14
14
–
–
–
–
–
–
16
16
–
–
–
6b
14
10
15
13
11
20
11
–
–
–
–
–
–
7
–
–
–
–
–
–
11a
11b
11c
11d
12
–
–
–
11
–
–
17
21
20
21
21
20
19
19
–
–
14
12
12
20
12
12
12
12
–
–
–
22
14
14
24
20
20
26
–
\100
–
–
–
\200
\200
\200
\100
\200
–
–
\100
\100
\50
–
–
–
–
–
–
\200
17
–
–
\200
\50
\200
\200
\25
5
–
–
–
–
5
–
–
–
13a
13b
13c
Ampicillin
–
–
–
13
–
–
–
–
–
–
–
–
–
–
–
–
100
100
10
50
Considering the antifungal activity, the tested com-
pounds were devoid of activity except 6b and 11c, which
showed weak activity against A. niger (Table 7).
Table 7 The inhibition zones (IZ) in mm diameter and minimal
inhibitory concentration (MIC) in lg/ml of the tested compounds
against fungi
Comp. no.
C. albicans S. cerevisiae A. niger
A. terreus
IZ
MIC
IZ
MIC
IZ MIC IZ MIC
Discussion
3
12
17
13
15
13
11
12
12
14
18
11
11
11
11
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5
14
15
13
14
14
12
15
14
13
16
12
12
13
14
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5
12
–
–
–
–
–
–
–
–
–
4
From the previously mentioned results, it could be deduced
that compound 3 exhibited considerable activity against
melanoma MALME-3M, non-small lung cancer HOP-92
and breast cancer T-47D. Moreover, significant antibacte-
rial activity was associated with the 3-aryl-1,2,4-triazol-
o[4,3-e]purine series 11a–d. Maximum activity was
5
–
–
–
6a
–
–
–
6b
20 \200
–
–
7
–
16
–
–
–
–
–
–
11a
–
–
11b
–
–
achieved when the substituent at position
3 was
11c
22 \100
–
–
4-methoxyphenyl group 11d. Replacement of 3-aryl moi-
ety in (compounds 11a–d) by 3-alkylthio or aralkylthio
(compounds 13a–c) decreased the activity toward the
Gram-negative P. aeruginosa and P. vulgaris and increased
the activity toward the Gram-positive S. aureus. Maximum
activity was obtained when the substituent at position 3
was benzylthio 13c. Substituted 1,2,4-triazino[4,3-e]pur-
ines 4, 5, and 6a showed promising activity against the
spore forming Gram-negative bacteria P. vulgaris.
It is worthy to mention that compounds 11b, 12, and 13a
exhibited broad spectrum of activity against Gram-positive
and Gram-negative bacteria.
11d
–
–
–
–
–
–
–
–
16
–
–
12
–
13a
–
–
–
13b
–
–
–
13c
–
–
–
Clotrimazole
10
–
10
potency of ampicillin, while compound 6b showed
weak activity (one fourth the activity). Furthermore,
determination of the antibacterial activity against the
Gram-positive S. aureus indicated that compounds 12 and
13c exhibited significant activity but lower than that of
ampicillin (MIC \ 50 and \25 lg/ml, respectively).
Acknowledgments The authors with to thank Dr. Manal A. Sha-
laby, Genetic Engineering and Biotechnology Research Institute
(GEBRI), Mubark City for Scientific Research and Technology
123

1118
Med Chem Res (2012) 21:1107–1119
Application, Borg El-Arab, Alexandria, Egypt for performing the
antibacterial and antifungal screening. The authors are also grateful to
the staff of the Department of Health and Human Services, National
Cancer Institute, Bethesda, Maryland, USA for the anti-cancer and
anti-HIV reports.
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