Synthesis of some novel substituted purine derivatives as potential anticancer, anti-HIV-1 and antimicrobial agents

Article abstract of DOI:10.1002/ardp.200600118

In search of novel purine antimetabolites, a series of 8-substituted methylxanthine derivatives was prepared in order to explore their in vitro anticancer, anti-HIV-1 and antimicrobial activities. The target compounds include: 8-[(3-substituted-4-oxo-thia

Full text of DOI:10.1002/ardp.200600118

Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
S. M. Rida et al.
185
Full Paper
Synthesis of Some Novel Substituted Purine Derivatives As
Potential Anticancer, Anti-HIV-1 and Antimicrobial Agents*
Samia M. Rida, Fawzia A. Ashour, Soad A.M. El-Hawash, Mona M. El-Semary, and Mona H. Badr
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
In search of novel purine antimetabolites, a series of 8-substituted methylxanthine derivatives
was prepared in order to explore their in vitro anticancer, anti-HIV-1 and antimicrobial activities.
The target compounds include: 8-[(3-substituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-1,3-dime-
thyl (or 1,3,7-trimethyl)-3,7-dihydropurine-2,6-diones 5a–e, 8-[(3,4-disubstituted 2,3-dihydro-
thiazol-2-ylidene)hydrazino]-1,3,7-trimethyl-3,7-dihydropurine-2,6-diones 6a–d and 8-(5-amino-3-
arylpyrazol-1-yl)-1,3-dimethyl- (or 1,3,7-trimethyl)-3,7-dihydropurine-2,6-diones 7a–g. The in vitro
anticancer results revealed that compound 5d exhibited a super sensitivity profile towards leu-
kemia K-562 with a GI50 value of a0.01 lM. Compound 7c showed significant activity against
colon cancer HCT-15 and renal cancer CAKI-1 (GI50 values of 0.47 and 0.78 lM, respectively). Com-
pound 7a displayed high activity against colon cancer HCT-15 (GI50 = 0.8 lM). The anti-HIV-1
results indicated that compound 6b displayed a good reduction of viral cytopathic effect
(56.69%). The antimicrobial results showed that compound 5a was four times more active than
ampicillin against P. aerugenosa (MIC = a25 lg/mL), compound 5b had twice the activity of ampi-
cillin, while compounds 5d, 7c and 7f were equipotent to ampicillin. On the other hand, com-
pound 7a was equipotent to ampicillin against P. vulgaris (MIC = 50 lg/mL).
Keywords: Anticancer / Anti-HIV / Antimicrobial activity / Methylxanthines / Purines /
Received: July 29 2006; accepted: December 19, 2006
DOI 10.1002/ardp.200600118
Moreover, methylxanthines (caffeine, pentoxifylline
and theophylline) are well known to enhance the cytoci-
dal and growth inhibitory effects of DNA-damaging
agents such as radiation, UV light, and anticancer agents
on tumor cells [19, 20]. On the other hand, it has been
reported that methylxanthines may protect cells against
the cytostatic or cytotoxic effects of several aromatic
compounds and significantly decrease the mutagenicity
of the anticancer aromatic drugs such as daunomycin,
doxorubicin, and mitoxantrone [20].
In view of the above mentioned findings and in the
search for novel antimetabolite purine derivatives, our
work has been focused on design and synthesis of a novel
series of 8-substituted methylxanthine derivatives in
order to evaluate their potential as anticancer, anti-HIV,
and/or antimicrobial agents. The target compounds were
Introduction
The purine ring system is considered to be one of the
most important heterocyclic ring system in nature as it
has the distinction of being the parent ring in countless
derivatives of biological relevance. Therefore, it is not sur-
prising that modified purines possess diverse biological
activities, and lead to a better understanding of some bio-
logical effects of DNA-damaging agents as well as
enzymes-substrate interactions. This lead has been used
in the development of many potent medicinal agents.
For example, the antineoplastic [1–4], antileukemic [5–
9], anti-HIV-1 [10–12], antiviral [13–17], antibacterial and
antifungal [3, 18] purine derivatives.
Correspondence: Soad A. M. EL-Hawash, Department of Pharmaceuti-
cal Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria
AR Egypt
E-mail: soadhawash@yahoo.com
Fax: +20 3 487 3273
* Parts of this work have been presented at Assiut University 4th Pharma-
ceutical Sciences Conference, Assiut, Egypt, March 6-7, 2004; Poster P
97-98.
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Scheme 1. Synthetic route for compounds 3–7.
designed to incorporate a five-membered heterocyclic 3a was prepared in a good yield, as previously reported
ring of biological interest either directly linked to the [21], by refluxing the corresponding 8-chloro derivative 1
purine nucleus at position 8 or through a two-nitrogen with hydrazine hydrate in ethanol. On the other hand,
atom spacer. The prepared compounds were biologically reaction of 8-chloro-1,3-dimethyl-3,7-dihydropurine-2,6-
evaluated to test the effect of such molecular modifica- dione with hydrazine hydrate did not afford the respec-
tion of purines on the anticipated pharmacological tive 8-hydrazino derivative 3b, instead, a hydrazine salt
effects.
of 8-chloro1,3-dimethyl-3,7-dihydropurine-2,6-dione was
produced because of the acidic nature of hydrogen at
position 7 as previously described [21].
Here, we report a new synthetic procedure to prepare
3b in a good yield by reduction of the respective 8-diazo
derivative 2 with stannous chloride in hydrochloric acid.
Results and discussion
Chemistry
The target compounds were prepared following the syn- The 8-diazo-1,3-dimethyl-3,7-dihydropurine-2,6-dione 2
thetic pathway depicted in Scheme 1. The intermediate was prepared following the previously reported proce-
8-hydrazino-1,3,7-trimethyl-3,7-dihydropurine-2,6-dione
dure [22] by diazotization of 8-amino-1,3-dimethyl-3,7-
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Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
Novel Substituted Purine Derivatives
187
dihydropurine-2,6-dione hydrochloride with sodium (a0.01 lM). Compound 7c exhibited moderate to strong
nitrite in 5% hydrochloric acid at 0–58C. Refluxing etha- activity against all of the tested cell lines with GI50 of 10– 6
nolic solution of 3a or 3b with the selected isothiocya- to 10– 5 lM. It showed significant activity against colon
nate afforded the respective 8-(N-substituted-thiocarba- cancer HCT-15 and renal cancer CAKI-1 (GI50 values, 0.47
moylhydrazino)-1,3-dimethyl (or 1,3,7-trimethyl)-3,7- and 0.78 lM, respectively). Compound 7a showed activity
dihydropurine-2,6-diones 4a–e. Reaction of 4a–e with against most of the tested cell lines. It exhibited signifi-
ethyl bromoacetate in a refluxing mixture of absolute cant activity against colon cancer HCT-15 (GI50 = 0.8 lM).
ethanol and dry dioxan yielded the corresponding 8-[(3- Compound 7e displayed moderate activity against some
substituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-3,7-
of the tested cell lines, especially CNS cancer SF-268, mel-
dihydropurine-2,6-diones 5a–e as previously reported for anoma MALME-3M, melanoma SK-MEL-5 and renal cancer
the preperation of related compounds [23]. In addition, 786-0 (GI50 values 9.91, 7.13, 6.36, and 6.90 lM, respec-
refluxing a mixture of 4c or 4d with the appropriate phe- tively). In addition, compound 5a showed considerable
nacylbromide gave the respective 8-[(3,4-disubstituted- activity towards some of the tested cell lines. For exam-
2.3-dihydrothiazol-2-ylidene)hydrazino]-3,7-dihydropur-
ple, lung cancer HOP-62, NCI H322M, colon cancer KM12,
ine-2,6-diones 6a-d as previously discussed for the pre- ovarian cancer SK-OV-3, and CNS cancer SF-268, SF-295
peration of analogous compounds [24, 25]. On the other (GI50 values, 4.63, 5.09, 6.36, 6.47, 7.47, and 7.35 lM,
hand, 8-(5-amino-3-arylpyrazol-1-yl)-3,7-dihydropurine- respectively). On the other hand, compounds 5b and 7g
2,6-dione derivatives 7a–g were obtained in good yield showed weak activity with GI50 of 10-5 to 10-4 lM. Tables 2
by refluxing 3a or 3b and the appropriate x-cyanoaceto- and 3 indicate the GI50, TGI, LC50 subpanel and full panel
phenone in ethanol containing acetic acid as previously mean-graph midpoint (MG-MID) values for the active
reported for the preperation of related compounds [26]. compounds respectively.
The structures of the synthesized compounds were con-
firmed by microanalyses, IR, H-NMR, and mass spectral panel MG-MID (lM) by its individual subpanel MG-MID
The ratio obtained by dividing the compound full
1
data (Experimental section).
(lM) is considered as a measure for selectivity [29]. Ratios
greater than 6 indicate selectivity toward the correspond-
ing subpanel cell line, while ratios between 3 and 6 refer
to moderate selectivity. Accordingly, the tested com-
Biology
Anticancer activity
Antitumor activity was performed at the National Cancer pounds proved to be non-selective except compound 5d
Institute (NCI), Bethesda, Maryland, USA. Nine of the which had moderate selectivity for leukemia with ratio
synthesized compounds 5a–d and 7a, c, d, e, g were of 2.64 (Table 4).
selected by the NCI and subjected to the NCI in vitro dis-
From the above mentioned results it could be con-
ease-oriented human cells screening panel assay [27, 28]. cluded that the antitumor activity was associated with 8-
About 60 cell lines of nine tumor subpanels were incu- [(3-substituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-1,3-
bated with five concentrations (0.01–100 lM) for each dimethyl (or 1,3,7-trimethyl )-3,7-dihydropurine -2,6-
compound and were used to create log concentration ver- diones 5a, b, d and 8-(5-amino-3-arylpyrazol-1-yl)-1,3-
sus% growth inhibition curves. Three response param- dimethyl (and 1,3,7-trimethyl)-3,7-dihydropurine-2,6-
eters (GI50, TG1, and LC50) were calculated for each cell diones 7a–g. Regarding the 8-(5-amino-3-arylpyrazol-1-yl)
line. The GI50 value corresponds to the compound's con- series 7a–g, compound 7a showed significant activity
centration causing 50% decreases in net cell growth. The with a broad spectrum of activities. Substitution at posi-
TG1 value is the compound's concentration resulting in tion 4 of the phenyl moity with methyl group resulted in
total growth inhibition and the LC50 is the compound's an increase in activity 7c, while substitution with chlor-
concentration causing a net 50% loss of initial cells at the ine atom decreased the activity 7e. On the other hand,
end of the incubation period (48 h). Subpanel and full substitution at N-7 of purine nucleus with methyl group
panel mean-graph midpoint values (MG-MID) for certain greatly decreased or destroyed the activity 7e and 7g. By
agents are the average of individual real and default GI50, comparing the 8-(3-substituted-4-oxo-thiazolidinyl) deriv-
TGI or LC50 values of all cell lines in subpanel and fullpa- atives 5a, b, d, maximum activity was observed when the
nel, respectively.
substituent at position 3 of the thiazolidine ring was a
In this study, the preliminary screening data of NCI butyl group 5a. Also, substitution at N-7 of the purine
indicated that seven compounds 5a, b, d and 7a, c, e, g nucleus with a methyl group greatly decreased the activ-
showed antitumor activity (Tables 1–4). Compound 5d ity 5d.
exhibited super sensitivity profile towards leukemia K-
From these findings, we could concluded that main-
562 with GI50 value resides in the nanomolar range taining a hydrogen at position 7 of purine nucleus might
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Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
Table 1. Growth inhibitory action (GI50) of some selected in vitro tumor cell lines (lM).
No
5a
NCS
Panel
Subpanel cell lines (cytotoxicity GI50 in lM)a)
720616
Leukemia
CCRF-CEM (22.20), MOLT-4 (21.80, SR (11.90)
Lung Cancer
Colon Cancer
CNS Cancer
Melanoma
HOP-62 (6.63), NCI-H322M (5.09), NCI-H460 (13.70)
HCT-116 (15.20), HT29 (23.90), KM12 (6.36)
SF-268 (7.74), SF-295 (7.35), U 251 (15.00)
SK-MEL-28 (20.00), M14 (28.5)
Ovarian Cancer
Renal Cancer
Prostate Cancer
Breast Cancer
Leukemia
OVCAR-4 (19.50), OVCAR-5 (14.50), SK-OV-3 (6.47)
786-0 (17.30)
DU-145 (19.20
MCF7 (13.70), MDA-MB-435 (14.70), MDA-N (14.6)
5b
720617
CCRF-CEM (22.10), SR (24.90)
Lung Cancer
CNS Cancer
Renal Cancer
Breast Cancer
Leukemia
Lung Cancer
CNS Cancer
Ovarian Cancer
Leukemia
HOP-92 (15.70)
SF-295 (21.20), SF-539 (29.10)
786-0 (27.10)
MCF7(19.40), HS 578T(17.10), MDA-MB-435 (19.10), MDA-N (16.40)
K-562 (a 0.01), RPMI-8226 (17.50)
HOP-92 (5.91)
SF-539 (25.60)
OVCAR-3 (21.00), OVCAR-4 (26.30)
CCRF-CEM (2.64), K-562 (6.45), MOLT-4 (7.15), SR (6.31)
5d
7a
720619
720611
Lung Cancer
A549/ATCC (5.78), EKVX (5.42), HOP-92 (6.46), NCI-H226 (5.54), NCI-322M (4.93),
NCI-H460 (3.30)
Colon Cancer
HCC-2998 (8.67), HCT-116 (6.49), COLO 205 (7.05), HCT-15 (0.80), HT29 (4.15), KM12
(6.58)
CNS Cancer
Melanoma
SF-295 (4.92), SF-539 (5.08), U251(6.77)
SK-MEL-5 (5.39), UACC-62 (6.37)
Ovarian Cancer
Renal Cancer
Prostate Caner
Breast Cancer
Leukemia
IGROV1(1.34), OVCAR-3 (5.35), OVCAR-4 (2.91), OVCAR-8 (4.86), SK-OV-3 (4.03)
786-0 (6.18), ACHN (3.66), CAKI-1(2.12), RXF 393 (2.68), SN12C (4.60), UO-31(6.89)
DU-145 (7.92)
NCI/ADR-RES (2.40), MDA-MB-231/ATCC (10.70), MDA-N (11.7), T-47D (11.30)
CCRF-CEM (2.65), HL-60(TB) (5.04), K-562 (4.03), MOLT-4 (2.06), RPMI-B226 (6.80), SR
7c
720612
(2.43)
Lung Cancer
Colon Cancer
A549/ATCC (4.68), EKVX (3.09), HOP-62 (3.28), NCI-H226 (6.71), NCI-322M (4.04),
NCI-H460 (1.71)
HCC-2998 (7.68), HCT-116 (2.68), COLO 205 (3.62), HCT-15 (0.47), HT29 (4.53), KM12
(4.88)
CNS Cancer
Melanoma
SF-268 (3.59), SF-295 (2.69), SF-539 (4.23), SNB-19 (8.56), U251(5.36)
MALME-3M (3.86), M14 (4.32), SK-MEl-2 (6.64), SK-MEl-28 (6.13),SK-MEL-5 (3.35),
UACC-62(5.37)
Ovarian Cancer
Renal Cancer
IGROV1(2.20), OVCAR-3 (4.55), OVCAR-4 (4.01), OVCAR-5 (8.05), OVCAR-8 (3.37), SK-
OV-3 (3.31)
786-0 (2.08), A498 (4.87), ACHN (1.92), CAKI-1(0.78), SN12C (5.32), TK-10 (9.84), UO-
31(2.78)
Prostate Cancer
Breast Cancer
DU-145 (6.35)
MCF7(6.78), NCI/ADR-RES (1.55), MDA-MB-231/ATCC (8.97), MDA-N (5.99), T-47D
(4.77), BT-549 (6.95), MDA-MB-435 (7.72)
SK-MEl-28 (23.10),SK-MEL-5 (32.40)
MOLT-4 (11.60), S (13.00)
7g
7e
720615
720614
Melanoma
Leukemia
Lung Cancer
Colon Cancer
CNS Cancer
Melanoma
Ovarian Cancer
Renal Cancer
Breast Cancer
HOP-62 (10.50)
HCT-116 (19.10)
SF-268 (9.910), SF-295 (25.50), SF-539 (17.30)
MALME-3M (7.13), SK-MEL-5 (6.36)
OVCAR-4(5.60), OVCAR-8 (18.10), SK-OV-3 (18.10)
786-0 (6.90)
HS 578T(22.40)
a)
Data obtained from NCI in vitro disease-oriented human cell screen.
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Novel Substituted Purine Derivatives
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Table 2. Median growth inhibitory concentrations (GI50, lM) of in vitro subpanel tumor cell lines and GI50 (lM) full panel mean-graph
mid-points (MG-MID).
No
Subpanel tumor cell linesa)
Full panel
GI50
MG-MIDb)
I
II
III
IV
V
VI
VII
VIII
59.6
82.8
89.1
13.8
11.3
IX
5a
5b
5d
7a
7c
7e
7g
38.1
36.4
21.5
58.7
74.7
77.7
6.28
8.25
74.4
80.4
100
21.1
47.8
88.2
7.02
4.89
50.5
69.3
48.8
67.8
79.6
10.3
6.42
68.4
71.6
40.9
60.9
54.2
6.63
4.25
53.6
80.6
81.4
90.9
100
9.22
5.65
88.4
92.8
43.9
45.6
79.8
11.8
8.20
67.9
94.9
35.5
54.9
57.5
6.76
4.68
53.7
81.3
73.8
79.8
5.62
3.98
86.5
57.6
7.53
3.84
60.3
100
100
100
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.
GI50 full panel mean-graph midpoint (lM).
b)
Table 3. Median total growth inhibitory concentrations (TGI, lM) of in vitro subpanel tumor cell lines, TGI (lM) full panel mean-graph
mid-points (MG-MID) and LC50 (lM) full panel mean-graph mid-points (MG-MID).
No
Subpanel tumor cell linesa)
Full panel TGI
MG-MIDb)
I
II
III
IV
90.6
100
100
81.8
55.1
93.9
V
VI
VII
VIII
IX
5a
5b
5d
7a
7c
7e
7g
76.3
90.9
81.1
92.2
92.1
98.6
97.6
73.3
40.2
98.0
96.0
100
100
100
98.1
49.2
100
100
100
100
100
48.4
41.1
97.6
88.5
100
98.4
74.8
44.8
98.5
92.7
98.6
100
72.5
33.3
100
100
100
72.5
60.2
100
100
84.7
79.1
100
82.9
61.5
85.1 (97.7)c)
93.3 (100)
97.7 (100)
69.2 (97.7)
38.0 (85.1)
97.7 (100)
100 (100)
100
100
100
100
100
100
100
100
100
100
a)
For subpanel tumor cell lines see footnote ({) of Table 2.
TG1 (lM) full panel mean-graph mid-point (MG-MID) = the average sensitivity of all cell lines towards the test agent.
LC50 (lM) full panel mean-graph mid-point (MG-MID).
b)
c)
Table 4. Selectivity ratios of the active compounds toward the nine tumor cell lines.
No
Subpanel tumor cell linesa)
I
II
III
IV
V
VI
VII
VIII
IX
5a
5b
5d
7a
7c
7e
7g
0.93
1.51
2.64
0.90
1.22
0.89
0.81
0.60
0.73
0.74
1.08
0.57
0.72
1.01
0.36
0.74
0.72
1.20
1.18
0.62
1.41
1.68
1.15
0.65
0.96
0.96
1.06
1.17
0.73
0.81
0.72
0.66
0.73
0.79
1.14
0.87
0.90
1.06
1.02
1.10
1.00
1.01
0.44
0.60
0.58
0.73
0.83
0.61
0.88
0.60
0.66
0.65
0.49
0.41
0.54
0.81
0.81
1.20
0.72
0.57
0.57
0.79
0.86
a)
For subpanel tumor cell lines see footnote a) of Table 2.
be essential for the formation of hydrogen bonds impor- genicity in a human T4-lymphocyte cell line (CEM) [30].
tant for the interaction with polymerases and the key Activity is expressed as% of protection which represents
enzymes of nucleic acids metabolism.
the percentage of surviving HIV-infected cells treated
with the test compound (at the indicated concentration)
relative to the same uninfected untreated controls. The
Anti-HIV-1 activity
Four compounds 6a–d have been selected by NCI and effective concentration 50% (EC50), represents the concen-
evaluated for their effects on HIV-1 induced cytopatho- tration of the test agent resulting in 50% reduction of
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Table 5. Maximum% protection, the corresponding dose (molar)
and IC50 (molar) of the selected compounds.
Their inhibition zones using the agar cup diffusion tech-
nique [31] were measured. Ampicillin was used as refer-
ence antibacterial, while clotrimazole was used as anti-
fungal reference. Compounds which showed growth
inhibition F20 mm were further evaluated for their
minimal inhibitory concentration (MIC) using the two-
fold serial dilution technique [32].
The results recorded in Table 7 revealed that the tested
compounds exhibited promising activity towards the
Gram-negative P. aerugenosa and P. vulgaris. Compound
5a was the most active, it was four times as active as ampi-
cillin against P. aerugenosa (MIC a25 lg/mL). Compound
5b had twice the activity of ampicillin. In addition, com-
pounds 5d, 7c, and 7f were equipotent to ampicillin.
Compounds 5c, 5e, 6b, 6c, 7a, 7d, 7e, and 7g showed half
the activity, while compounds 6a, 6d, and 7a were devoid
of activity against P. aerugenosa.
No
NCS No.
Maximum% Dose
IC50 (molar)
protection (molar)
6a
6b
6c
6d
722237
722238
722227
722236
16
57
47
45
2.01610– 6 1.09610– 5
6.34610– 6 1.05610– 5
6.34610– 6 1.20610– 5
6.34610– 6 1.08610– 5
Table 6. In vitro anti-HIV-1 activity parameters of compound 6b
(NCS 722238).
Compound
IC50 (Molar)
EC50 (Molar)
TI50 (IC/EC)
6b
AZT
1.05610– 5
5.51610– 6
2.57610– 9
1.91
A1.00610– 6
3.89610– 2
On the other hand, compound 7c was the most active
viral cytopathic effect. The 50% inhibitory concentration against P. vulgaris, it was equipotent to ampicillin (MIC =
(IC50), represent the toxic concentration of drug resulting 50 lg/mL). Compound 7b displayed half the potency of
in 50% growth inhibition of normal uninfected cells. The that of ampicillin. Compounds 5a, 5d, 7c, 7d, 7f, and 7g
therapeutic index (TI50) was determined by dividing (IC50) displayed weak activity (one fourth the activity), while
by (EC50).
the other tested compounds were inactive. Moreover,
Among the tested compounds, compound 6b was con- compounds 6a–d exhibited weak activity towards the
firmed to have moderate in vitro anti-HIV activity. It Gram-positive S. aureus, compound 6c was the active
showed a good reduction of viral cytopathic effect (57%). member.
Compounds 6c and 6d showed mild reduction of viral
Considering the antifungal activity, the tested com-
cytopathic effect by 47 and 45% respectively, while com- pounds were devoid of activity except 7a, which showed
weak activity against C. albicans.
pound 6a exhibited weak activity. It reduced the viral
cytopathic effect by 16% (Table 5). The recorded IC50 and
From the previously mentioned results it could be
EC50 for 6b were 1.05 6 10-5 and 5.51610– 6, respectively, deduced that significant antibacterial activity was asso-
however the resulting therapeutic index (TI) is 1.91 ciated with the 8-(3-substituted-4-oxothiazolidin-2-yli-
which is not sufficient for further in vivo testing as com- dene) series 5a–e. Maximum activity was achieved when
pared with AZT (TI = A3.89610– 2, Table 6).
the substitutent at position 3 was butyl group 5a. Substi-
The aforementioned results indicated that maximum tution at N-7 of purine nucleus greatly decreased the
activity was observed when the the position 3 of the thia- activity (compounds 5c, 5d, and 5e). Replacement of 3-
zole ring was substituted with a butyl group and position substituted-4-oxothiazolidine moity by 3,4-disubstituted-
4 of the phenyl moiety with a chlorine atom.
dihydrothiazol (compounds 6a–d) decreased the activity
towards the Gram-negative P. aerugenosa and P. vulgaris
and slightly increased the activity towards the the Gram-
Antimicrobial activity
Compounds 5a–e, 6a-d and 7a–g were preliminary eval- positive S. aureus. The 5-amino-3-substituted-pyrazolyl
uated for their in vitro antibacterial activity against Sta- series 7a–d showed promising activity against the spore
phylococcus aureus (NCTC 4163) and Bacillus subtilis as forming Gram-negative bacteria P. vulgaris. Also maxi-
mum activity was obtained when the substituent at posi-
Gram-positive bacteria and Pseudomonas aerugenosa
(ATTC 9027), Escherichia coli (5933), and Salmonella typhi tion 3 was unsubstituted phenyl group and at N-7 of pur-
(ATCC 13311) as Gram-negative bacteria and Proteus vul- ine was hydrogen. Substitution at position 4 of phenyl
garis (ATTC 49132) as spore forming Gram-negative bac- group with chlorine atom slightly decreased the activity,
teria. They were also evaluated for their in vitro antifun- while substitution with CH3 group greatly decreased the
gal activity against four types of fungi, Candida albicans activity. In addition, as previously mentioned in the anti-
(NCTC 2708) and Saccharamyces cercvisiae (ATTC 9763) as tumor and anti-HIV activity, substitution at N-7 of purine
examples of yeast, Aspergillus niger (ATTC 16404) and with methyl group greatly decreased or abolished the
Aspergillus terreus (local isolate) as examples of true fungi. activity. Accordingly, it could be concluded that the pre-
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Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
Novel Substituted Purine Derivatives
191
Table 7. The inhibition zones (IZ) in mm diameter and minimal inhibitory concentration (MIC) in lg/mL of the tested compounds.
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
5a
5b
5c
5d
5e
6a
6b
6c
6d
7a
7b
7c
5d
7e
7f
7g
17
–
–
16
16
20
20
24
20
–
–
–
–
–
16
14
13
–
15
14
13
14
15
19
16
15
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5
–
–
–
–
16
–
–
12
–
21
16
16
12
–
–
–
–
–
–
–
–
–
–
–
–
33
32
21
22
21
19
20
20
18
20
19
21
20
21
22
21
–
a25
a50
a200
a100
a200
–
a200
a200
–
14
14
16
14
12
12
12
12
12
20
16
16
12
–
–
–
–
–
–
–
–
–
–
20
19
19
22
18
18
18
19
18
24
22
21
20
18
21
21
–
a200
–
–
a200
–
–
–
–
–
a50
a100
a200
a200
–
a200
a200
50
–
a200
a200
a100
a200
–
–
–
–
–
–
–
–
5
a200
–
–
–
–
–
–
100
a200
–
a200
–
–
–
–
–
–
10
–
17
16
17
18
17
–
a100
a200
a200
a100
a200
100
–
12
–
11
14
–
Ampicillin
Clotrimazole
–
5
Table 8. Physical and analytical data of the synthesized compounds (4–7).
No
R
R1
Yield [%]
M. p. [8C] Cryst. solvent
Mol. formulaa) (Mol. Wt.)
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
H
H
CH3
CH3
CH3
H
(CH2)3CH3
CH2C6H5
(CH2)3CH3
CH2C6H5
C6H11 (cyclo)
(CH2) 3CH3
CH2C6H5
(CH2)3CH3
CH2C6H5
C6H11 (cyclo)
(CH2)3CH3
(CH2)3CH3
CH2C6H5
CH2C6H5
H
65
62
68
73
84
48
55
79
77
56
77
79
51
54
64
62
57
87
79
93
84
>350 Dioxan
>350 Dioxan
240-242 Dioxan
>350 Dioxan
>350 Dioxan
C12H19N7O2S (325.39)
C15H17N7O2S (359.41)
C13H21N7O2S (339.42)
C16H19N7O2S (373.43)
C15H23N7O2S (365.46)
C14H19N7O3S (365.41)
C17H17N7O3S (399.43)
C15H21N7O3S (379.44)
C18H19N7O3S (413.46)
C17H23N7O3S (405.48)
C21H25N7O2S.HBr (520.45)
256–258 EtOH
253–255 EtOH
215–217 EtOH
230–232 EtOH
252–254 EtOH
200–202 EtOH
196–198 EtOH/diethyl ether C21H24ClN7O2S N HBr (554.89)
183–185 EtOH C24H23N7O2S N HBr (554.47)
180–182 EtOH/diethyl ether C24H22ClN7O2S N HBr (588.91)
340–342 EtOH
348–350 EtOH
322–324 EtOH
243–245 EtOH
265–267 EtOH
270–272 EtOH
287–289 EtOH
H
CH3
CH3
CH3
H
Cl
H
Cl
H
H
H
C16H15N7O2 (337.34)
C16H14ClN7O2 (371.78)
C17H17N7O2 (351.36)
C17H17N7O2 (351.36)
C17H16ClN7O2 (385.81)
C17H16BrN7O2 (430.26)
C18H19N7O2 (365.39)
Cl
CH3
H
Cl
Br
CH3
CH3
CH3
CH3
CH3
7g
a)
Analyzed for C, H, N and the results are within l0.4% of the theoretical values.
sence of a H atom at N-7 of purine is essential for maxi- acknowledge the staff of the Department of Health and Human
mum biological activity. Services, National Cancer Institute, Bethesda, Maryland, USA
The physical and analytical data of the synthesized for the anticancer and anti-HIV reports.
compounds 4–7 are given in Table 8.
Experimental
The authors wish to thank Dr. Hesham A. El-Enshasy, Genetic
Engineering and Biotechnology Research Institute(GEBRI),
Mubarak City for Scientific Research and Technology Applica-
tion, Borg El-Arab, Alexandria, Egypt for performing the anti-
bacterial and antifungal screening. The authors also wish to
Chemistry
All melting points were determined in open-glass capillaries on
a Gallenkamp melting point apparatus (Sanyo) and are uncor-
rected. The IR spectra were recorded using KBr discs on a Perkin-
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192
S. M. Rida et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
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 TMS as internal
standard (chemical shift in d ppm). MS were run on a Finnigan
mass spectrometer model SSQ/7000 (70 eV, Thermo Electron
Corporation). The microanalyses were performed at the microa-
nalytical laboratory, National Research Center, Cairo, and the
data were within l0.4% 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 k 254 nm
for few seconds.
IR of compounds 5a–e (KBr, cm– 1): 3193–3100 (NH); 1727–
1713 (C=O thiazolidinone); 1694–1685 (C=O purine); 1665–
1638, 1620–1606, 1535–1531, 1508–1494 (C=N, NH bending,
C=C); 1227–1217, 1053–1038 (C-S-C).
1H-NMR of compound 5a (DMSO-d6, d ppm, JNM-LA 400 FT):
0.89 (t, J = 7.3 Hz, 3H, (CH2)3 –CH3); 1.28 (m, 2H, CH2 –CH2 –CH2 –
CH3); 1.55 (m, 2H, CH2 –CH2 –CH2 –CH3); 3.20 (s, 3H, purine-N3 –
CH3); 3.37 (s, 3H, purine-N1 –CH3); 3.80 (t, J = 7.2 Hz, 2H, CH2 –
(CH2)2 –CH3); 4.01 (s, 2H, thiazolidinone-C5 –H2); 10.08, 11.92 (two
s, each 1H, NH-N, NH (purine), D2O exchangeable).
1H-NMR of compound 5c (DMSO-d6, d ppm, JNM-LA 400 FT):
0.88 (t, J = 7.3 Hz, 3H, (CH2)3 –CH3); 1.28 (m, 2H, CH2 –CH2 –CH2 –
CH3); 1.59 (m, 2H, CH2 –CH2 –CH2 –CH3); 3.17 (s, 3H, purine-N3 –
CH3); 3.34 (s, 3H, purine-N1 –CH3); 3.47 (s, 3H, purine-N7 –CH3);
3.67 (t, J = 7.2 Hz, 2H, CH2 –(CH2)2 –CH3); 4.03 (s, 2H, thiazolidi-
none-C5 –H2); 9.61 (s, 1H, NH–N, D2O exchangeable). MS of 5c m/z
(relative abundance%): 379 [M+] (43.7), 208 (90.2), 151 (15.0), 94
(17.1), 82 (100).
8-Hydrazino-1,3-dimethyl-3,7-dihydropurine-2,6-dione 3b
To a well stirred ice-cooled solution of 8-diazo-1,3-dimethyl-3,7-
dihydropurine-2,6-dione 2 (20.6 g, 100 mmol) in a mixture of
concentrated hydrochloric acid/water (8:1) (80 mL), a solution
of stannous chloride (48 g) in concentrated hydrochloric acid
(60 mL) was added dropwise with stirring over a period of 1 h.
The formed precipitate was filtered, dissolved in water, and neu-
tralized with saturated solution of sodium acetate. The precipi-
tated product was filtered, washed with water, dried, and crys-
tallized from dimethylformamide/water, m.p. 322–3328C
(reported 3208C) [21]; yield 11.2 g (53%).
8-[(3,4-Disubstituted-2,3-dihydrothiazol-2-
ylidene)hydrazino]-1,3,7-trimethyl-3,7-dihydropurine-2,6-
diones hydrobromides 6a-d
A mixture of 4c or 4d (2 mmol) and the appropriate phenacyl
bromide (2 mmol) in absolute ethanol (20 mL) was heated under
reflux for 30 minutes. The reaction mixture was then concen-
trated and left to cool to room temperature. The separated crys-
talline product was filtered, dried, and recrystallized from the
proper solvent. (Table 8).
8-(N-Substituted-thiocarbamoylhydrazino)-1,3-dimethyl
(and 1,3,7-trimethyl)- 3,7-dihydropurine-2,6-diones 4a–e
To a well stirred suspension of 3a or 3b (5 mmol) in absolute
ethanol (100 mL), the appropriate isothiocyanate (5 mmol) was
added. The reaction mixture was heated under reflux for 6–
12 h. The separated product was filtered while hot, washed with
diethyl ether, dried, and crystallized from dioxane. (Table 8).
IR of compounds 4a–e (KBr, cm– 1): 3293–3265, 3257–3208
(NH); 1690–1685 (C=O); 1650–1649, 1627–1624, 1534–1533,
1501–1498 (C=N, NH bending, C=C); 1551–1535, 1285–1278,
1075–1038, 989–947 (N-C=S amide I, II, III, IV bands).
IR of compounds 6a–d (KBr, cm– 1): 3288-3095 (NH); 1703–
1692 (C=O); 1664–1646, 1618–1608, 1599–1534 (C=N, NH bend-
ing, C=C); 1222–1212, 1092–1035 (C-S-C).
1H-NMR of compound 6b (DMSO-d6, d ppm, Varian Gemini
200 MHz): 0.71 (dist. t, 3H, (CH2)3 –CH3); 1.15 (m, 2H, CH2 –CH2 –
CH2 –CH3); 1.59 (m, 2H, CH2 –CH2 –CH2 –CH3); 3.21 (s, 3H, purine-
N3 –CH3); 3.35 (s, 3H, purine-N1 –CH3); 3.81 (s, 3H, purine-N7 –
CH3); 4.00 (dist. t, 2H, CH2 –(CH2)2 –CH3); 7.16 (s, 1H, thiazoline-
C5 –H); 7.40–7.66 (m, 4H, Ar-H); 10.53 (s, 1H, NH, D2O exchange-
able).
1H-NMR of compound 4a (DMSO-d6, d ppm, Varian Gemini
200 MHz): 0.89 (t, J = 7.2 Hz, 3H, (CH2)3-CH3); 1.26 (m, 2H, CH2 –
CH2 –CH2 –CH3); 1.48 (m, 2H, CH2 –CH2 –CH2-CH3); 3.19 (s, 3H,
purine-N3 –CH3); 3.37 (s, 3H, purine-N1 –CH3); 3.49 (m, 2H, CH2 –
(CH2)2 –CH3); 8.15 (t, 1H, NH–CH2, D2O exchangeable); 9.12, 9.27
(two s, each 1H, NH-NH, D2O exchangeable); 12.27 (s, 1H, NH, D2O
exchangeable). MS of 4b m/z (relative abundance%): 361[M + 2]
(5.5), 262 (6.2), 236 (5.2), 195 (30.2), 149 (8.8), 91 (76.6), 56 (100).
1H-NMR of compound 4d (DMSO-d6, d ppm, Varian Gemini
200 MHz): 3.14 (s, 3H, purine-N3 –CH3); 3.34 (s, 3H, purine-N1 –
CH3); 3.56 (s, 3H, purine-N7 –CH3); 4.77 (d, 2H, NH-CH2 –C6H5);
7.21–7.32 (m, 5H, Ar-H); 8.79 (t, 1H, NH–CH2, D2O exchange-
able); 9.33, 9.61 (two s, each 1H, NH-NH, D2O exchangeable).
1H-NMR of compound 6c (DMSO-d6, d ppm, JNM-LA 400 FT):
3.19 (s, 3H, purine-N3 –CH3); 3.39 (s, 3H, purine-N1 –CH3); 3.49 (s,
3H, purine-N7 –CH3); 5.19 (s, 2H, CH2 –C6H5); 6.89 (d, 2H, Ar-C2,6
–
H of benzyl group); 7.09 (s, 1H, thiazoline-C5 –H); 7.28–7.50 (m,
8H, Ar-H); 9.74 (s, 1H, NH, D2O exchangeable).
MS of 6d m/z (relative abundance%): 507 [M+] (1.2), 434 (1.0),
381 (1.39), 255 (2.47), 201 (7.28), 153 (23.1), 109 (100), 58 (89.7).
8-(5-Amino-3-arylpyrazol-1-yl)-1,3-dimethyl (and 1,3,7-
trimethyl)-3,7-dihydropurine-2,6-diones 7a-g
To a solution of 3a or 3b (2 mmol) in ethanol/acetic acid (4:1)
(10 mL), the appropiate x-cyanoacetophenone (2 mmol) was
added. The reaction mixture was heated under reflux for 6 h
and left to cool to room temperature. The separated crystalline
product was filtered, dried, and recrystallized from ethanol.
IR of compounds 7a–g (KBr, cm– 1): 3423–3343, 3327–3265,
3187–3179 (NH2, NH); 1710–1696 (C=O); 1665–1647, 1548–
1540, 1513–11507 (C=N, NH bending, C=C).
8-[(3-Substituted-4-oxo-thiazolidin-2-ylidene)hydrazino]-
1,3-dimethyl-(and 1,3,7-trimethyl)-3,7-dihydropurine-2,6-
diones 5a–e
To a suspension of the selected 4 (2 mmol) in absolute ethanol/
dry dioxane (1:1) (20 mL), ethyl bromoacetate (0.33 g, 2 mmol)
was added. The reaction mixture was heated under reflux for 4–
8 hours then concentrated under reduced pressure. The precipi-
tate formed after addition of a few drops of water was filtered,
dried, and crystallized from ethanol. (Table 8).
1H-NMR of compound 7b (DMSO-d6, d ppm, Varian Gemini
200 MHz): 3.27 (s, 3H, purine-N3 –CH3); 3.51 (s, 3H, purine-N1 –
CH3); 5.90 (s, 1H, pyrazole-C4 –H); 6.75 (s, 2H, NH2, D2O exchange-
able); 7.50 (d, J = 8 Hz, 2H, Ar-C2,6 –H); 7.94 (d, J = 8 Hz, 2H, Ar-
C3,5 –H); 13.60 (s, 1H, NH, D2O exchangeable).
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Arch. Pharm. Chem. Life Sci. 2007, 340, 185–194
Novel Substituted Purine Derivatives
193
1H-NMR of compound 7c (DMSO-d6, d ppm, JNM-LA 400 FT):
2.32 (s, 3H, 4-CH3 –C6H4); 3.27 (s, 3H, purine-N3 –CH3); 3.48 (s, 3H,
purine-N1 –CH3); 5.82 (s, 1H, pyrazole-C4 –H); 6.70 (s, 2H, NH2, D2O
exchangeable); 7.22 (d, J = 8 Hz, 2H, Ar-C3,5 –H); 7.77 (d, J = 8 Hz,
2H, Ar-C2,6 –H); 12.06 (s, 1H, NH, D2O exchangeable). MS of 7c m/z
(relative abundance%): 351 [M+] (100), 265 (12.3), 240 (10.4), 184
(19.1), 142 (25.6), 115 (40.6), 82 (44.5).
Antimicrobial activity
Inhibition zones measurement
The compounds were dissolved in DMSO in a concentration of
1 mg/mL by the agar cup diffusion technique [31] using a 1 mg/
mL solution in DMSO. Each 100 mL of sterile molten agar (at
458C) received 1 mL of 6 h broth, 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 1 mg/mL solu-
tion of the test compounds. The plates were then incubated at
378C for 24 h or 48 h for fungi. A control using DMSO without
the test compound was included for each organism. Ampicillin
was used as standard antibacterial, while clotrimazole was used
as antifungal reference. The resulting inhibition zones are
recorded (Table 7).
1H-NMR of compound 7d (DMSO-d6, d ppm, JNM-LA 400 FT):
3.18 (s, 3H, purine-N3 –CH3); 3.39 (s, 3H, purine-N1 –CH3); 4.10 (s,
3H, purine-N7-CH3); 5.85 (s, 1H, pyrazole-C4 –H); 6.47 (s, 2H, NH2,
D2O exchangeable); 7.31–7.40 (m, 3H, Ar-C3,4,5 –H); 7.76 (d, 2H,
Ar-C2,6 –H).
1H-NMR of compound 7e (DMSO-d6, d ppm, JNM-LA 400 FT):
3.17 (s, 3H, purine-N3 –CH3); 3.33 (s, 3H, purine-N1 –CH3); 4.08 (s,
3H, purine-N7 –CH3); 5.86 (s, 1H, pyrazole-C4 –H); 6.50 (s, 2H, NH2,
D2O exchangeable); 7.40 (d, J = 8 Hz, 2H, Ar-C2,6 –H); 7.73 (d, J = 8
Hz, 2H, Ar-C3,5 –H). MS of 7e m/z (relative abundance %): 387 [M +
2] (37.5), 385 [M+] (54.5), 343 (2.5), 272 (3.0), 247 (5.9), 178 (10.9),
136 (48.6), 111 (15.3), 67 (100).
Minimal inhibitory concentration (MIC) measurement
The minimal inhibitory concentrations (MIC) of the most active
compounds were measured using the two fold serial broth dilu-
tion method [32]. The test organisms were grown in their suita-
ble broth for 24 hours for bacteria and 48 hours for fungi at
378C. Two-fold serial dilutions of the test compounds solution
were prepared using the suitable broth to obtain concentrations
200, 100, 50, 25, and 12.5 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 378C for 48 hours. Then, the
tubes were observed for the presence or absence of microbial
growth. The MIC values of the tested compounds are listed in
Table 7.
1H-NMR of compound 7f (DMSO-d6, d ppm, JNM-LA 400 FT):
3.20 (s, 3H, purine-N3 –CH3); 3.41 (s, 3H, purine-N1 –CH3); 4.09 (s,
3H, purine-N7 –CH3); 5.89 (s, 1H, pyrazole-C4 –H); 6.49 (s, 2H, NH2,
D2O exchangeable); 7.56 (d, J = 8 Hz, 2H, Ar-C2,6 –H); 7.71 (d, J = 8
Hz, 2H, Ar-C3,5 –H).
Biological evaluation
Anticancer activity
The prepared compounds were tested for their in vitro antican-
cer activity against 60 human tumor cell lines, derived from
nine clinically isolated cancer types (leukemia, non-small cell
lung cancer, colon cancer, CNS cancer, melanoma, ovarian can-
cer, renal cancer, prostate cancer, breast cancer) following the
NCI preclinical antitumor drug discovery screen. Each com-
pound was tested at five, ten-fold dilution, 48 h continuous drug
exposure protocol was used and a sulforodamine B (SRB) protein
assay was used to estimate cell viability or growth [27].
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