Identification of important structural features of thiazolo[4,5-d] pyrimidines required for potent antifungal activity

Article abstract of DOI:10.1007/s00044-010-9378-2

Pyrimidines, either mononuclear or condensed with other heterocycles have established its importance in medicinal chemistry. Variety of biological activities have been reported by thiazolo[4,5-d]pyrimidine derivatives. The present work describes the synthesis and antifungal activity of several 3-(substituted)-5-(substituted)phenylamino- 6-(substituted)phenylthiazolo[4,5-d] pyrimidine-7(6H) -one-2(3H)-thiones. The target compounds were synthesized by cyclocondensation of 4-amino-5-carbethoxy-3-(substituted) thiazolo-2(3H)-thione and S-methyl di(substituted) phenylisothiourea. All synthesized compounds were tested for minimum inhibitory concentration against different fungal strains such as, Aspergillus niger, A. clavatus and Candida albicans and compared with fluconazole and nystatin as reference drug. Some of the compounds have exhibited potent inhibitory activity on all fungal strains, and were found more potent than reference standard. Some of the important structural features required for broad spectrum activity in this series have been derived. Springer Science+Business Media, LLC 2010.

Full text of DOI:10.1007/s00044-010-9378-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1450–1454
DOI 10.1007/s00044-010-9378-2
ORIGINAL RESEARCH
Identification of important structural features of thiazolo[4,5-d]
pyrimidines required for potent antifungal activity
•
Mahesh Chhabria Ishwarsinh Rathod
•
•
Khushbu Vala Paulomi Patel
Received: 9 February 2010 / Accepted: 4 June 2010 / Published online: 27 June 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Pyrimidines, either mononuclear or condensed
with other heterocycles have established its importance in
medicinal chemistry. Variety of biological activities have
been reported by thiazolo[4,5-d]pyrimidine derivatives.
The present work describes the synthesis and antifungal
activity of several 3-(substituted)-5-(substituted)phenyla-
mino-6-(substituted)phenylthiazolo[4,5-d]pyrimidine-7(6H)
-one-2(3H)-thiones. The target compounds were synthesized
by cyclocondensation of 4-amino-5-carbethoxy-3-(substi-
tuted)thiazolo-2(3H)-thione and S-methyl di(substituted)
phenylisothiourea. All synthesized compounds were tested
for minimum inhibitory concentration against different
fungal strains such as, Aspergillus niger, A. clavatus and
Candida albicans and compared with fluconazole and
nystatin as reference drug. Some of the compounds have
exhibited potent inhibitory activity on all fungal strains,
and were found more potent than reference standard. Some
of the important structural features required for broad
spectrum activity in this series have been derived.
Introduction
Invasive fungal infections, particularly in immunosup-
pressed patients, have continued to increase in incidence
during the past 20 years and are now significant cause of
morbidity and mortality (Andre, 2008; Russel, 2002; Beth,
1999). All current agents have some serious liabilities: such
as inadequate spectrum, limited dosage forms, narrow
therapeutic window and rapid emergence of resistance
(Boucher et al., 2004; William and Thomas 2001). The
recent expansion of antifungal drug research has occurred
because there is a critical need for new antifungal agents to
treat these life-threatening invasive infections (Gallagher
et al., 2004; Georgopapadakou and Walsh 1994). Thus,
intense efforts in antifungal drug discovery are still needed
to develop more promising and effective antifungal agents.
It is well known that pyrimidine and condensed pyrim-
idine derivatives are of great biological interest, especially
as antiviral, antitumor and antimicrobial agents. In con-
tinuation of our ongoing work on thiazolopyrimidines, it
was planned to synthesize a series of 3-(substituted)-5-
(substituted)phenylamino-6-(substituted)phenylthiazolo[4,
5-d]pyrimidine-7(6H)-one-2(3H)-thiones as potential anti-
fungal agent and it was also planned to derive important
structural features required for antifungal activity.
Keywords Thiazolo[4,5-d]pyrimidine Á Antifungal Á
MIC
Abbreviations
MIC Minimum inhibitory concentration.
SAR Structural activity relationship
Experimental
Chemistry
M. Chhabria (&) Á I. Rathod Á K. Vala Á P. Patel
Department of Pharmaceutical Chemistry,
L. M. College of Pharmacy, Gujarat University,
Navrangpura, Ahmedabad 380 009, Gujarat, India
e-mail: mahesh.chhabria@rediffmail.com
Earlier, thiazolo[4,5-d]pyrimidine was prepared by reaction of
4-amino-5-carbethoxy-3-(substituted)thiazolo-2(3H)-thione in
the presence of triphenylphosphene, hexachloroethane and
triethylamine to give iminophosphorane which on reaction with
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Med Chem Res (2011) 20:1450–1454
1451
aryl isocyanate afforded carbodiimide. Condensation of car-
bodiimides with alkylamine gave intermediate guanidines,
which get cyclized to thiazolo[4,5-d]pyrimidine (Ying et al.,
2007) in the presence of sodium ethoxide.
7(6H)-one-2(3H)-thiones 3 in moderate to good yield
(Table 1).
Spectral characteristics of synthesized compounds
In present study, we have eliminated the use of trip-
henylphosphene, hexachloroethane and arylisocyanate and
cyclization was affected by weak base. The target com-
pounds were synthesized by cyclocondensation of 4-amino-
5-carbethoxy-3-(substituted)thiazolo-2(3H)-thione (Gewald,
2004) and S-methyldi(substituted)phenylisothiourea (Fur-
niss and Hannaford 2006) in DMF in the presence of
potassium carbonate (Scheme 1). All compounds were
3-(Ethyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7
(6H)-one-2(3H)-thione (3a)
IR (KBr, cm-1): 3357 (N–H), 2860 (C–H), 1685 (C=O),
1247 (C=S). 1H-NMR (d ppm, DMSO-d6): 1.2 (t, 3H,CH3),
2.3 (q, 2H, CH2), 3.7 (s, 3H, OCH3), 3.9 (s, 3H, OCH3), 6.7
(s, 1H, NH), 6.70–7.95 (m, 8H, Ar–H), MS: 441.0 (M?1),
439.1 (M-1).
1
characterized by IR, H-NMR and mass spectroscopy.
General method of synthesis of 3-(substituted)-
5-(substituted)phenylamino-6-(substituted)phenylthiazolo
[4,5-d]pyrimidine-7(6H)-one-2(3H)-thiones
3-(Phenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3b)
A mixture of 4-amino-5-carbethoxy-3-(substituted)thiazolo-
2(3H)-thione 1 (0.3 g, 0.01 mol) and appropriate sym-
metrical S-methyldi(substituted)phenylisothiourea 2 (0.33 g,
0.01 mol) was refluxed in dimethylformamide for 8–10 h in
the presence of anhydrous K2CO3. The reaction mixture was
allowedtocoolandpouredinice-coldHClsolution(10%v/v).
The product obtained was filtered, washed with water and
dried. Recrystallization of the crude product in n-hexane-
dichloromethane afforded 3-(substituted)-5-(substituted)
phenylamino-6-(substituted)phenylthiazolo[4,5-d]pyrimidine-
IR (KBr, cm-1): 3350 (N–H), 2860 (C–H), 1650 (C=O),
1250 (C=S). MS: 489.0 (M?1), 487.1 (M-1).
3-(Phenyl)-5-(2-methylphenyl)amino-6-
(2-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7
(6H)-one-2(3H)-thione (3c)
IR (KBr, cm-1): 3357 (N–H), 2860 (C–H), 1685 (C=O),
1247 (C=S). 1H-NMR (d ppm, DMSO-d6): 3.6 (s, 3H,
CH3), 3.8 (s, 3H, OCH3), 6.5 (s, 1H, NH, D2O exchange-
able), 6.90–7.80 (m, 13H, Ar–H).
3-(Phenyl)-5-(4-methylphenyl)amino-6-
(4-methylphenyl)thiazolo[4,5-d]pyrimidine-7
(6H)-one-2(3H)-thione (3d)
O
O
S
S
NH2
R1
R1
IR (KBr, cm-1): 3374 (N–H), 1687 (C=O), 1250 (C=S).
1H-NMR (d ppm, DMSO-d6): 2.2 (s, 6H, Ar–CH3), 6.6
(s,1H, NH, D2O exchangeable), 6.85–7.95 (m, 13H, ArH).
MS: 456.9 (M?1), 455.0 (M-1).
N
N
N
S
H
R
2
1
DMF, K2CO3, Reflux
3-(Phenyl)-5-(2,6-dimethylphenyl)amino-6-
(2,6-dimethylphenyl)thiazolo[4,5-d]pyrimidine-7
(6H)-one-2(3H)-thione (3e)
O
N
R1
S
N
N
IR (KBr, cm-1): 3374 (N–H), 1685 (C=O), 1250 (C=S).
1H-NMR (d ppm, DMSO-d6): 2.3 (s, 12H, Ar–CH3), 6.7
(s, 1H, NH, D2O exchangeable), 6.9–7.69 (m, 11H, Ar–H).
S
NH
R
3-(Phenyl)-5-phenylamino-6-phenylthiazolo[4,5-d]
pyrimidine-7(6H)-one-2(3H)-thione (3f)
R1
3
IR (KBr, cm-1): 3323 (N–H), 1683 (C=O), 1250 (C=S).
1H-NMR (d ppm, DMSO-d6): 6.8 (s, 1H, NH, D2O
exchangeable), 6.92–7.66 (m, 15H, Ar–H).
Scheme 1 Synthetic scheme for 3-(substituted)-5-(substituted)phe-
nylamino-6-(substituted)phenylthiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thiones
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Table 1 Physical characteristics of 3-(substituted)-5-(substituted)phenylamino-6-(substituted)phenylthiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thiones (3)
O
R1
S
N
S
N
N
NH
R
R1
3
Compound no.
R
R1
Molecular formula
Molecular weight
Melting point (°C)
Yield (%)
3a
3b
3c
3d
3e
3f
C2H4
4-OCH3
4-OCH3
2-OCH3
4-CH3
C21H20N4O3S2
C25H20N4O3S2
C25H20N4O3S2
C25H20N4OS2
494.05
488.09
488.09
456.12
484.10
428.15
518.06
494.05
518.14
616.04
428.15
518.06
488.09
456.12
557.14
655.19
484.10
110–112
236–238
225–227
282–284
278–280
246–248
[300
67.20
76.92
60.00
62.50
44.11
52.60
66.66
67.74
54.54
48.71
54.54
48.48
58.30
31.50
42.55
27.02
54.80
C6H5
C6H5
C6H5
C6H5
2,6-diCH3
C6H5
C27H24N4OS2
C6H5
C23H16N4OS2
3g
3h
3i
4-CH3O-C6H4
4-CH3O-C6H4
4-CH3O-C6H4
4-CH3O-C6H4
2-CH3O-C6H4
2-CH3O-C6H4
3-Cl,4-F-C6H3
3-Cl,4-F-C6H3
3,4-diCl-C6H3
3,4-diCl-C6H3
4-Br-C6H4
4-OCH3
4-F
C26H22N4O4S2
C24H16F2N4O2S2
C26H22N4O4S2
C24H16Br2N4O2S2
C26H22N4O4S2
C28H26N4O2S2
C25H18ClFN4O3S2
C23H12Br2ClFN4S2
C25H18Cl2N4O3S2
238–240
250–252
290–295
250–252
270–272
205–207
202–204
290–295
210–212
245–247
2-OCH3
4-Br
3j
3k
3l
4-OCH3
2,6-diCH3
4-OCH3
4-Br
3m
3n
3o
3p
3q
4-OCH3
4-Br
C26H22Cl2Br2 N4O4S2
C25H19BrN4O3S2
4-OCH3
3-(4-Methoxyphenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3g)
3-(4-Methoxyphenyl)-5-(2-methoxyphenyl)amino-6-
(2-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3i)
IR (KBr, cm-1): 3394 (N–H), 1689 (C=O), 1253 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.6 (s, 3H, OCH3), 3.8 (s,
3H, OCH3), 3.9 (s, 3H, OCH3), 6.7 (s, 1H, NH, D2O
exchangeable), 6.98-7.89 (m, 12H, Ar–H).
IR (KBr, cm-1): 3370 (N–H), 1689 (C=O), 1250 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.6 (s, 3H, OCH3), 3.8 (s,
3H, OCH3), 3.9 (s, 3H, OCH3), 6.7 (s, 1H, NH, D2O
exchangeable), 6.89–7.75 (m, 12H, Ar–H).
3-(4-Methoxyphenyl)-5-(4-fluorophenyl)amino-6-
(4-fluorophenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3h)
3-(4-Methoxyphenyl)-5-(4-bromophenyl)amino-6-
(4-bromophenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3j)
IR (KBr, cm-1): 3323 (N–H), 1683 (C=O), 1250 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.9 (s, 3H, OCH3), 6.5 (s,
1H, NH), 6.96-7.76 (m, 12H, Ar–H). MS: 494.9 (M?1),
493.1 (M-1).
IR (KBr, cm-1): 3305 (N–H), 1677 (C=O), 1251 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.8 (s, 3H, OCH3), 6.7
(s, 1H, NH), 6.92-7.66 (m, 12H, Ar–H).
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3-(2-Methoxyphenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3k)
3-(4-Bromophenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3q)
IR (KBr, cm-1): 3375 (N–H), 1685 (C=O), 1251 (C=S). 1H-
NMR (d ppm, DMSO-d6): 3.6 (s, 3H, OCH3), 3.7 (s, 3H,
OCH3), 3.9 (s, 3H, OCH3), 6.7 (s, 1H, NH, D2O exchange-
able), 6.90–7.85 (m, 12H, Ar–H).
IR (KBr, cm-1): 3289 (N–H), 1681 (C=O), 1251 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.7 (s, 3H, OCH3), 3.9
(s, 3H,OCH3), 6.7 (s, 1H, NH), 6.90–7.65 (m, 12H, Ar–H).
Antifungal screening
3-(2-Methoxyphenyl)-5-(2,6-dimethylphenyl)amino-6-
(2,6-dimethylphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3l)
All synthesized compounds 3a–q were tested in vitro
against three fungal strains Candida albicans, Aspergillus
niger and A. clavatus by broth dilution method using
Sabouraud’s nutrient media (Jennifer 2004) and MIC val-
ues were calculated (Table 2). Fluconazole and nystatin
were taken as the standard drug.
IR (KBr, cm-1): 3371 (N–H), 1693 (C=O), 1253 (C=S).
1H-NMR (d ppm, DMSO-d6):
2.3 (s, 12H, Ar–CH3), 3.8 (s, 3H, OCH3), 6.7 (s, 1H,
NH, D2O exchangeable), 6.80–7.80 (m, 10H, Ar–H).
3-(3-Chloro-4-fluorophenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3m)
Results and discussion
A series of 3-(substituted)-5-(substituted)phenylamino-
6-(substituted)phenylthiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thiones (3) was synthesized (Scheme 1) to carry out
SAR study. Compounds with various substituents at third,
fourth and fifth position of thiazolopyrimidines were syn-
thesized and screened. Phenyl ring was considered essen-
tial at all three positions as it imparts more lipophilicity and
hence more penetration through membrane. All the tested
compounds exhibited significant antifungal activity against
all fungal strains with MIC value ranges from 0.2 to
25.6 lg/mL. Replacement of phenyl ring by alkyl group at
the third position (compound 3a) has also shown significant
antifungal activity, but was found less potent than phenyl
derivatives. Compound with 4-methoxyphenyl group at
third position and 4-fluorophenyl group at fifth and sixth
position exhibited potent activity.
IR (KBr, cm-1): 3350 (N–H), 1698 (C=O), 1248 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.8 (s, 3H, OCH3), 3.9 (s,
3H, OCH3), 6.7 (s, 1H, NH), 6.90–7.80 (m, 11H, Ar–H).
MS: 542.9 (M?2), 539.1 (M-2).
3-(3-Chloro-4-fluorophenyl)-5-(4-bromophenyl)amino-6-
(4-bromophenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3n)
IR (KBr, cm-1): 3310 (N–H), 1683 (C=O), 1261 (C=S).
1H-NMR (d ppm, DMSO-d6): 6.7 (s, 1H, NH), 6.90–7.65
(m, 11H, Ar–H). MS: 638.9 (M?2), 637.1 (M-2).
3-(3,4-Dichlorophenyl)-5-(4-methoxyphenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3o)
Compound 3b with phenyl ring on third position and
4-methoxyphenyl on fourth position was found to be the
most potent compound against all strains of fungi. Com-
pound 3i and q have shown potent activity with MIC value
of 0.4 lg/mL against A. niger and compound 3b, j, m have
shown most potent activity with MIC value of 0.2 lg/mL
against A. clavutus.
IR (KBr, cm-1): 3350 (N–H), 1693 (C=O), 1251 (C=S).
1H-NMR (d ppm, DMSO-d6): 3.7(s, 3H, OCH3), 3.9 (s,
3H, OCH3), 6.6 (s, 1H, NH), 6.92–7.70 (m, 11H, Ar–H).
MS: 559.0 (M?2), 555.1 (M-2).
Study reveals that substitution with electron donating
group on phenyl ring retained potency on all strain. With
electron withdrawing groups on phenyl ring potency on
A. niger and A. clavutus reduced significantly.
3-(3,4-Dichlorophenyl)-5-(4-bromophenyl)amino-6-
(4-methoxyphenyl)thiazolo[4,5-d]pyrimidine-7(6H)-one-
2(3H)-thione (3p)
So for broad spectrum antifungal thiazolopyrimidines, at
third position either unsubstituted phenyl or phenyl with
electron donating group is essential. Similarly at fourth and
fifth position phenyl ring with electron donating group is
required.
IR (KBr, cm-1): 3312 (N–H), 1685 (C=O), 1249 (C=S).
1H-NMR (d ppm, DMSO-d6): 6.6 (s, 1H, NH, D2O
exchangeable), 6.92–7.70 (m, 11H, Ar–H). MS: 657.0
(M?2).
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References
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Minimum inhibitory concentrations (lg/mL)
3a
2
0.5
4
0.8
0.8
12.8
12.8
25.6
25.6
0.5
0.5
0.4
4
16
3b
0.2
12.8
0.8
4
3c
3d
0.8
4
3e
3f
4
25.6
4
3g
4
3h
0.4
12.8
0.8
16
4
0.4
25.6
0.2
0.8
4
3i
3j
3k
4
3l
4
3m
0.4
4
4
0.2
4
3n
4
3o
0.8
0.8
4
4
0.8
16
3p
4
3q
0.4
16
7
12.8
16
Fluconazole
Nystatin
0.5
8
7
123