Design, synthesis & evaluation of condensed 2H-4-arylaminopyrimidines as novel antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2014.02.066

A small, focussed library of condensed 2H-4-arylaminopyrimidines, with 3-diversity points, based on an initial design by molecular docking study of this scaffold at the active site of the fungal enzyme of cytochrome P ₄₅₀ family, lanosterol 14α

Full text of DOI:10.1016/j.ejmech.2014.02.066

European Journal of Medicinal Chemistry 77 (2014) 166e175
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis & evaluation of condensed 2H-4-
arylaminopyrimidines as novel antifungal agents
,
b
a
c
d
Kishor S. Jain a , Vijay M. Khedkar , Nikhilesh Arya , Prasad V. Rane , Pratip K. Chaskar ,
*
Evans C. Coutinho b
a Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune, 410401 Maharashtra, India
b Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Mumbai, 400098 Maharashtra, India
c Department of Pharmaceutical Chemistry, RJSPM’s College of Pharmacy, Moshi-Alandi Road, Pune, 412105 Maharashtra, India
d Department of Pharmaceutical Chemistry, Vivekanand Education Society’s College of Pharmacy, Chembur, Mumbai, 400074 Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A small, focussed library of condensed 2H-4-arylaminopyrimidines, with 3-diversity points, based on an
initial design by molecular docking study of this scaffold at the active site of the fungal enzyme of cy-
tochrome P450 family, lanosterol 14a-demethylase (CYP51) was synthesized through a one-pot green
chemical synthetic protocol. The screening of the synthesised compounds for antifungal activity against
Candida albicans, Aspergillus fumigatus & Aspergillus niger revealed activity in many of the compounds as
comparable to that of fluconazole. Based on the antifungal activity and physicochemical property data of
these derivatives, a meaningful SAR has been proposed.
Received 15 December 2013
Received in revised form
21 February 2014
Accepted 28 February 2014
Available online 3 March 2014
Keywords:
Ó 2014 Elsevier Masson SAS. All rights reserved.
Antifungal activity
Condensed pyrimidines
Docking
Lanosterol 14-
MWI
a-demethylase
SAR
1. Introduction
through the inhibition of the fungal enzyme, lanosterol 14a-
demethylase (CYP51). This enzyme is a member of the cytochrome
P450 super family and is implicated in the biosynthesis of fungal
In recent years, the incidence and severity of life threatening
fungal infections have increased, particularly in patients with
impaired or compromised immunity [1], signifying the urgent
need for development of new alternative antifungal agents [2]. Of
the various invasive mycotic infections in humans, majority are
caused by Candida and Aspergillus species, of which Candida albi-
cans is one of the main causes of nosocomial fungal infectious
diseases. The other species, Aspergillus fumigatus has become one
of the most prevalent airborne fungal pathogens [3]. Presently, the
most widely employed drugs like amphotericin B and the azole
class of antifungals suffer from drawbacks. While, the former has
poor pharmacokinetics and potential for toxicity, the resistance
development by the fungal strains against the later has become
rampant [4,5]. These reasons highlight the urgent need for the
discovery of newer antifungal drugs, particularly bearing
nonconventional chemical structures and which are effective and
safe [6]. It is well known that the azoles exert their activity
ergosterol, by catalyzing the oxidative removal of the 14
a-methyl
group of lanosterol to give the
D
14,15-unsaturated key in-
termediates [7]. Recent studies have shown that typical azole in-
hibitors fit in the putative active site of CYP51 by a combination of
heme co-ordination, hydrogen bonding,
pep stacking and hy-
drophobic interactions [8]. It is reported that in the active site of
CYP51 the 1,2,4-triazole ring of the antifungal azole is positioned
perpendicularly to the porphyrin plane with a ring nitrogen atom
(N4 of 1,2,4-triazole) coordinated to the heme iron of CYP51. This
co-ordination is of key importance for the antifungal activity. The
halogenated phenyl group sits deep in the same hydrophobic
binding cleft in the active site of the target enzyme CYP51. These
interactions of azoles at the active site of CYP51, provide the basis
for the design of novel N-heterocyclic derivatives as potential
antifungal agents with a broad antifungal spectrum and possibly
with less potential to develop drug resistance.
Condensed pyrimidine ring system exhibits wide range of bio-
logical activities [9]. We have reported some condensed pyrimidine
derivatives, as novel lipid lowering agents (I) [10e13], proton pump
inhibitors (II) [14], and anti-proliferative agents (III) [15].
* Corresponding author.
E-mail address: drkishorsjain@gmail.com (K.S. Jain).
http://dx.doi.org/10.1016/j.ejmech.2014.02.066
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
167
Considerable anti-proliferative and anti-tumour activity poten-
tial observed in condensed 2H-4-(substituted-phenyl)amino-
pyrimidines III [15], prompted us to think of exploring this scaffold
also, for antifungal activity. This thinking had two reasons;
Aspergillus niger (ATCCÒ 9142). These MIC values are represented in
Tables 1 and 2 and compared with fluconazole, as well as, docking
scores of some of these compounds.
2.3. Docking study
1. The urgent need for search of novel structures bearing anti-
fungal activity, due to increase in resistance to the existing azole
drugs by fungal strains &
2. This scaffold though new for antifungal activity, combines the
nitrogen heterocyclic system; pyrimidine & its aromatic amine
side chain, which could afford better binding at the CYP51 active
site (Fig. 1).
A perusal of the geometries of fluconazole and the 2H-4-(4-
fluorophenylamino)-pyrazolo[3,4-d]pyrimidine
(1a),
reveals
spatial differences in their overlap (Fig. 2) and thus, indicating the
possibility of involvement of different sets of amino acid residues at
the active site of the fungal CYP51 during their interactions. This
opens up a possibility of evolving a new scaffold with less pro-
pensity to resistance development by the fungal strains to it.
Indeed a comparison between the docking modes of fluconazole
and these inhibitors (1a & 1b) at the active site of CYP51 (Figs. 3e5),
revealed some promise for the envisaged structures. Like the N-4
atom of the 1,2,4-triazole ring of fluconazole, the N-3 of the
condensed pyrimidin-4-amines, is also bound to the heme iron.
Similarly, like the difluorophenyl moiety of fluconazole, the
substituted-phenyl ring of the arylamino moiety of the condensed
pyrimidin-4-amine, seems to be involved in hydrophobic in-
teractions with a set of amino acids in close vicinity. Few of these
amino acids are common to both fluconazole and the ligands. This
can be explained on the basis of difference in the 3D geometries of
fluconazole and these ligands as discussed above. Further, these
compounds have an amino functionality for additional interactions
in the CYP51 activity. The interaction of the pyrazole ring fused to
the pyrimidine also which cannot be overlooked.
Herein, we report the design of a novel series of condensed 2H-
4-substitutedanilino-pyrimidines based on the molecular docking
studies of a few derivatives, its microwave irradiation (MWI) based
one-pot synthesis and its evaluation for antifungal activity. The
antifungal activity data and physicochemical properties have been
utilized to delineate some optimal structural information for the
activity.
2. Results and discussion
2.1. Chemistry
The synthetic protocol involves the MWI assisted trans-
formation of a variety of o-amino ester substrates, involving their
cyclocondensation and further chlorination with formamide-POCl3
mixture and subsequent nucleophilic displacement reaction with
appropriate substituted aniline, to afford the corresponding
condensed 2H-pyrimidin-4-arylamines (1e6), in around 30e
60 min, in excellent yields and purity (Scheme 1) [16]. This novel
synthetic protocol under MWI is a one pot multicomponent reac-
tion which is high yielding, eco-friendly, rapid, novel as well as,
eliminating intermittent workups. Initially, six compounds, 1ae6a,
were synthesized and evaluated. Thereafter, based on the obser-
vations, the synthesis of remaining derivatives was undertaken.
The receptor (active site of CYP51) interaction maps for
fluconazole
and
representative
condensed
2H-4-
arylaminopyrimidines (1a & 1b) are depicted in Figs. 3e5, while
the interaction data (Glide score, Glide energy and amino acids of
active site involved), as well as, antifungal activity data are given in
Table 1.
The docking scores of these compounds (1a & 1b) were nearly
comparable to that of fluconazole and interestingly their antifungal
activities against three resistant fungal strains were better than
fluconazole (Table 1).
2.2. Antifungal study
Interestingly, when three more derivatives 1c, 1d & 1e were
synthesized and evaluated for their antifungal activity against same
three different pathogenic fungal strains, all of them were found to
exhibit better antifungal activities than the drug. The rank order of
activity being 1c ꢀ 1a ꢀ 1b > 1d > 1e, indicating the influence of
the electron withdrawing groups present on the arylamino ring of
these compounds.
The in vitro antifungal activities of the compounds 1ae6a were
determined by using the serial micro dilution method in 96 multi-
cell microlitre plates in duplicate, in culture media comprising of
potato pulp: 200 g/l, dextrose: 20 g/l and agar: 25 g/l adopting the
reported procedures [17,18]. The minimum inhibitory concentra-
tions (MIC) values (
mg/ml) were obtained from triplicate assay
Analysis of the Glide docking scores showed that these com-
(three of two test tubes with identical results were taken as MIC’s)
pounds docked to the active site of the cytochrome P450 sterol 14
demethylase in a manner comparable to that of fluconazole
a-
against C. albicans (ATCCÒ 90028), A. fumigatus (ATCCÒ 204305) and
Fig. 1. Bioactive condensed pyrimidines.

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K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
Scheme 1. Synthetic protocol for target compounds (1ae6a).
(Table 1). Thus, the antifungal activity of these ligands is reflected
by their interaction with the heme atom as well as amino acids in
the CYP51 active site, where the pyrimidine ring & the arylamino
side chain; comprising of the amino moiety, aryl ring and its sub-
stituent, all play very important roles. Furthermore, the interaction
of these ligands at the active site of CYP51 may also be influenced
by the heterocyclic ring fused to the 2H-4-arylaminopyrimidine
scaffold along with the substituents borne by it. Therefore, to
study the effect of this fused ring along with its substituents, some
more docking experiments were performed with more varied
condensed 2H-4-arylaminopyrimidines, followed by their actual
synthesis and antifungal evaluation. The correlation of the docking
and antifungal activity data could lead to fruitful information. Thus,
compounds 2a, 3a, 4a, 5a & 6a were designed and studied for their
docking interactions and synthesized and evaluated for their anti-
fungal activity (Table 2) (Figs. 6e10).
interactions for the best docking poses. Six out of these seven
compounds (except 5a) exhibit better activity as well as glide scores
as compared to that of fluconazole.
The active site of cytochrome P450 sterol 14a-demethylase is
highly conserved and there are many amino acid residues in it,
participating in close van der Waals and coulombic interactions
with the inhibitor, when bound to it. Detailed analysis of the per-
residue interaction between the synthesized compounds and the
enzyme in comparison with fluconazole revealed some key in-
teractions that appear to play a role in the binding of these mole-
cules to the target.
All the ligands were found coordinated to the iron of the heme
group present in the active site to more or less extent. This is a very
important observation pointing to the fact, that the pyrimidine
derivatives may share the same inhibition mechanism as flucona-
zole which is also coordinated with the metal ion in the enzyme
active site. However, this interaction could not be quantified
because of the coordinating nature of the interaction and the lack of
terms for quantifying metaleligand complexes. Also, it is not al-
ways the pyrimidine ring N3 atom being involved in the interaction
with the heme iron, but also the side chain NH, the ‘S’ of methylthio
All the seven compounds 1a, 1b, 2a, 3a, 4a, 5a & 6a, as well as
fluconazole, were analyzed for their docking modes in the active
site of the enzyme. Although the real interaction profile for the
synthesized compounds discussed herein has not been resolved
experimentally, it is interesting to discuss the predicted
Table 1
Molecular docking data and antifungal data of compounds (1aee).
Compd. no.
R
Glide score Glide energy (kcal/mole) Evdw (kcal/mole) Eelect. (kcal/mole) mMICa
Candida albicans Aspergillus niger Aspergillus fumigatus
1a
1b
4-F
4-Cl
ꢁ8.14
ꢁ6.81
ꢁ48.923
ꢁ34.835
ꢁ36.629
ꢁ36.289
ꢁ12.294
0.0011
0.0024
0.0075
0.0011
0.0028
0.0045
0.0026
0.0012
0.0075
0.0010
0.0022
0.0024
0.0012
0.0013
0.0040
0.0012
0.0022
0.0028
02.041
Fluconazole
ꢁ7.34
ꢁ52.922
ꢁ33.765
ꢁ19.156
b
b
b
b
1c
1d
1e
3-Cl, 4-F
4-Br
H
b
b
b
b
b
b
b
b
a
Molar minimum inhibitory concentration.
Docking experiments not performed for this compounds.
b

K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
169
Table 2
Molecular docking data and antifungal data of compounds (1ae6a).
Sr. No.
Glide score Glide energy Evdw
(rank order) (kcal/mole) (kcal/mole)
Eelect. (kcal/mole) mMIC
Candida albicans Aspergillus niger Aspergillus fumigatus
(Rank order for activity)
Sum of rank orders
(overall rank order
for activity)
1
2
3
4
5
6
7
8
9
10
2a
3a
ꢁ8.28 (4)
ꢁ9.04 (1)
ꢁ47.751
ꢁ46.787
ꢁ37.058
ꢁ31.855
ꢁ10.693
ꢁ14.931
0.0013 (3)
0.0005 (1)
0.0105 (5)
0.0029 (4)
0.0105 (6)
0.0015 (3)
14 (5)
08 (2)
4a
ꢁ8.57 (3)
ꢁ37.397
ꢁ27.381
ꢁ10.016
0.0031 (4)
0.0023 (2)
0.0024 (4)
10 (4)
5a
6a
ꢁ6.33 (6)
ꢁ8.68 (2)
ꢁ20.895
ꢁ46.557
ꢁ19.080
ꢁ32.543
ꢁ01.814
ꢁ14.014
0.0081 (5)
0.0094 (6)
0.0351 (6)
0.0005 (1)
0.0045 (5)
0.0005 (1)
16 (6)
08 (2)
1a
ꢁ8.14 (5)
ꢁ48.923
ꢁ36.629
ꢁ12.294
0.0011 (2)
0.0026 (3)
0.0012 (2)
07 (1)
The rank orders in columns 7e9 are the antifungal activity rank orders for the compounds against corresponding fungal strain indicated under respective columns.
The overall rank orders in column 10 are given after considering the overall antifungal activity of the individual compounds against all 3 fungal strains.
Compound 3a is ranked 2; equal to compound 6a in antifungal activity. Thus, rank order is 1a ‡ 3a ‡6a > 4a > 2a > 5a.
substituents are seen involved in some of the cases. Thus, the target
compounds could be predicted to bind with the heme, but with
different affinities. A perusal of the data in Tables 2 and 3 as well as
the docking poses in Figs. 3e10, reveal that in case of compounds
1a, 2a, 3a, 4a and 6a, either the pyrimidine N3 atom or the side
chain NHe are involved in binding with the heme iron, though the
later is bit at a distance than N3. Also the phenyl ring is also oriented
close to the heme for hydrophobic interactions. In case of com-
pound, 5a, both the N3 atom & the side chain NH are little farther, so
their interaction with heme iron seems to be comparatively,
weaker. In case of 4a, it is the side chain ‘NHe’ and the ‘S’ of
methylthio substituent are seem to be involved in this interaction.
In case of compound 1b, only N3 is oriented close for interaction
with the heme iron. Therefore, the interaction of these ligands with
the amino acids was also studied. A different set of amino acids of
the CYP51 active site are involved in the interactions with the
ligands as compared to those with fluconazole. Only MET-123 &
ALA-110 are common, as far as the sets of amino acids in close vi-
ꢀ
cinity (w5.0 A from the molecular surface of ligands) to the
difluorophenyl group of fluconazole & the 4-fluorophenyl group of
the arylamino moiety of the condensed pyrimidin-4-amines (1ae
6a) are concerned. The amino acid TYR-116 is most prominently
involved in the interaction in case of all ligands.
These interactions together, contribute to the docking energies
and docking scores of these ligands. In case of 5a, the lower glide
score (docking score ꢁ5.95) and possibly its overall lower anti-
fungal activity can be attributed to the steric hindrance of the 4-
chlorophenyl ring attached to its thiophene moiety, causing the
compound to adopt a different conformation in the active site of
CYP51 wherein both, its pyrimidine N3 as well as side chain NH are
a bit drifted away from the heme iron and thus, the interaction at
heme is reduced considerably.

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K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
Fig. 2. Overlap of Fluconazole with condensed 2H-4-(4-fluorophenylamino)pyrazolo-pyrimidine: Difference in spatial geometries.
Fig. 3. Docking mode of Fluconazole in the active site of CACYP51.
Fig. 4. Docking mode of Compound 1a in the active site of CACYP51.
Fig. 5. Docking mode of Compound 1b in the active site of CACYP51.

K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
171
Fig. 6. Docking mode of compound 2a in the active site of CACYP51.
Fig. 7. Docking mode of compound 3a in the active site of CACYP51.
Fig. 8. Docking mode of compound 4a in the active site of CACYP51.
Fig. 9. Docking mode of compound 5a in the active site of CACYP51.

172
K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
Fig. 10. Docking mode of compound 6a in the active site of CACYP51.
On other hand, though the pyrimidine N3 of 4a is also
3. Conclusions
distanced away from the heme iron, this is compensated by the
close proximity of the arylamino side chain NH and the corre-
sponding ‘S’ of the methylthio substituent groups of this ligand to
the heme iron. In case of 6a, the carbonyl eC]O of the ester group
is closely involved in the interaction with the amino acids, MET-
106, TYR-103 and TYR-116. These interactions may be leading to
better docking scores and antifungal activities of these
compounds.
Thus, a new scaffold has been proposed for potential antifungal
activity. The structural difference of these compounds from the
azoles can make them less prone to the development of resistance
by pathogenic fungal strain against them. The good docking scores
as well as antifungal activity of these compounds have been justi-
fied by investigating their interaction at the active site of CYP51. A
meaningful SAR has been developed.
On careful examination of the docking interactions of the three
most active of these compounds; 1a, 3a, and 6a as compared with
those of fluconazole at the enzyme active site, few important things
are evident as described in Table 3.
4. Experimental
4.1. General
Compounds 1a, 3a and 6a showed relatively stronger electro-
static interactions fairly comparable to that of fluconazole, which
could possibly also contribute to their good glide scores and anti-
fungal activities. Compound 6a has poorer Evdw, Eelect as well as
glide scores & predictably lesser antifungal activity as compared to
fluconazole.
Rank order for the compounds with respect to their docking
scores, e9.04 (3a) > ꢁ8.68 (6a) > ꢁ8.57 (4a) > ꢁ8.28 (2a) > ꢁ8.14
(1a) > ꢁ6.33 (5a).
Melting points were determined using a Veego electronic
melting point apparatus model MP-D containing silicon oil bath
with stirrer and are uncorrected. The purity of the synthesized
compounds was tested using precoated TLC plates (silica gel 60 F254
plates, Merck) and visualization was achieved via UV light ab-
sorption. The IR absorption spectra were recorded on Perkin Elmer
Spectrum, BX II FTIR spectrometer, using (KBr) pellets and ab-
sorption frequencies (n) are stated in cmꢁ1. The 1H NMR spectra
(CDCl3) were measured on Varian Mercury 300 MHz spectrometer.
Interestingly, when a systematic analysis of the antifungal ac-
tivity data (mMIC values) of these ligands against the three
different fungal strains was done by assigning them rank orders
(see foot note for Table 2), it is found that the rank order for the
overall antifungal activity is as follows; 1a ꢀ 3a ꢀ 6a > 4a > 2a > 5a.
This is not in agreement with the rank order for the docking
scores for these compounds mainly due to compound 1a, which
though lower in rank docking score, is considerably higher ranked
in the antifungal activity.
The chemical shifts are expressed in parts per million (d ppm).
Tetramethylsilane (TMS) was used as an internal standard. The LC-
ESI/MS analysis was carried out on LCQ-Advantage (Thermo Fin-
nigan, San Jose, CA, USA) ion trap mass spectrometer. Microwave
synthesizer (Questron Technologies Corp., Canada; model-ProM)
having monomode open vessel was used for the synthesis.
Elemental analyses for compounds were obtained using a Flash EA
1112 Thermofinnigan Instrument.
A plausible explanation for this change of rank order in actually
observed overall antifungal activity seems to indicate the role of
some other structural feature or property prominently influencing
the interaction at the CYP51 active site. Interestingly, a consider-
ation on the nature of the ring fusion at the pyrimidine addresses
this anomaly in the correlation of docking scores vs antifungal ac-
tivity, within the series to some extent. The lipophilicity of the ring
fused to the pyrimidine seems to influence the overall antifungal
activity of the compounds, inversely (Table 4). The compounds 1a,
3a, 4a & 5a all have the rings fused to the pyrimidine scaffold with
lower ClogP values in range of 2.68e3.76; while the compounds 2a
& 5a carry more lipophilic rings with ClogP, 4.308 & 5.05,
respectively.
4.2. General procedure for synthesis of condensed 2H-pyrimidin-4-
amines (1ae6a and 1bee)
All the compounds (1ae6a and 1bee) were prepared by taking
respective o-aminoesters and appropriate substituted anilines as
per the procedure reported earlier by us [16] (Yield 80e92%).
4.2.1. N-(4-Fluorophenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (1a)
Yield: 82.35%; mp 174e176 ꢂC; IR (KBr): 3301 (NeH), 2365 (Ce
H), 1584 (N]N), 975 (CeF) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d ppm:
7.2e7.8 (m, 4H, NHeAreH), 8.2e8.4 (s, 5H, NeAreH), 8.6 (s, 1H, Ne
N]CH at 5), 9.2 (s, 1H, N]CHeN at 2), 10.1 (s, 1H, NH at 4); ESI-MS
m/z: 306 (Mþ1), 212,195,112. Anal. Calcd. for C17H12FN5 (305.31): C,
66.88; H, 3.96; N, 22.94. Found: C, 66.74; H, 3.86; N, 22.82.
Close analysis of the results and discussions above lead to the
conclusion for the following key structureeactivity relationship
(SAR) points (Fig. 11):

K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
173
Table 3
Comparison of interactions of compounds (1ae6a) & fluconazole with heme iron in the CYP51 cavity.
Interaction of the ligand at heme iron and amino acidsa (within w5 A from the
Distance of N3 from heme iron (A)
H-bond (A)
ꢀ
ꢀ
ꢀ
Compd. no.
ligands molecular surface) in the CYP51 cavity
1a
Both the pyrimidine N3 and side chain NH are oriented close to the heme iron
atom. The 4-fluorophenyl ring lies close for interaction with heme iron and the
amino acids, that are in close vicinity of the molecule namely; TYR-116,
PHE-110, ALA-115, LEU-127.
The heme iron atom is close to the pyrimidine N3. The side chain NH is oriented
slightly away from the heme iron. The 4-fluorophenyl ring lies close for
interaction with heme iron and the amino acids that are in close vicinity of the
molecule namely; TYR-116, MET-123, PHE-110, ALA-115, ALA-117, LEU-127.
Both the pyrimidine N3 and side chain NH are oriented close to the heme iron
atom. The 4-fluorophenyl ring also lies close for interaction with heme iron. It
and the carbonyl eC]O of the ester group, interact with the amino acids, that
are in close vicinity of the molecule namely; TYR-116, VAL-114, ALA-115,
PHE-110, MET-123, LEU-127, MET-103, MET-106
2.854
2.661
2.824
3.047 (NH with heme iron)
3.125 (NH with heme iron)
4.981 (NH with heme iron)
3a
6a
Fluconazole
The N4 of 1,2,4-triazole coordinates with the heme iron. The difluorophenyl ring
lies close for interaction with heme iron and the amino acids that are in close
vicinity of the molecule namely; TYR-103, MET-106, PHE-110, ALA-115,
LEU-127, PHE-290, ALA-291, ALA-287.
2.392
e
a
ꢀ
All amino acid mentioned are within w5A from the ligands molecular surface.
Table 4
CLogp values of rings fused to the pyrimidine scaffold.
Compound no.
1a
3a
6a
4a
2a
6a
Ring fused
ClogP
2.684
3.364
3.760
3.332
4.308
5.045
4.2.2. N-(4-Chlorophenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (1b)
for C17H11ClFN5 (339.75): C, 60.10; H, 3.26; N, 20.61. Found: C, 60.16;
H, 3.18; N, 20.50.
Yield: 86.95%; mp 156e158 ꢂC; IR (KBr): 3456 (NeH), 2917 (Ce
H), 1691 (N]N), 789 (CeCl) cmꢁ1 1H NMR (300 MHz, CDCl3)
;
4.2.4. N-(4-Bromophenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
4-amine (1d)
d
ppm: 7.18e7.45 (m, 4H, NHeAreH), 7.66e7.68 (s, 5H, NeAreH),
8.25 (s,1H, NeN]CH at 5), 8.70 (s,1H, N]CHeN at 2), 9.7 (s,1H, NH
at 4); ESI-MS m/z: 322 (Mþ1), 212, 195, 128. Anal. Calcd. for
Yield: 81.05%; mp 168e172 ꢂC; IR (KBr): 3428 (NeH), 2924 (Ce
H), 1601 (N]N), 780 (CeBr) cmꢁ1 1H NMR (300 MHz, CDCl3)
;
C
17H12ClN5 (321.76): C, 63.46; H, 3.76; N, 21.77. Found: C, 63.38; H,
d
ppm: 7.40e7.66 (m, 4H, NHeAreH), 7.95e7.98 (s, 5H, NeAreH),
3.52; N, 21.84.
8.56 (s, 1H, NeN]CH at 5), 8.89 (s, 1H, N]CHeN at 2), 10.1 (s, 1H,
NH at 4); ESI-MS m/z: 367 (Mþ1), 212, 195, 173. Anal. Calcd. for
C17H12BrN5 (366.21): C, 55.75; H, 3.30; N, 19.12. Found: C, 55.66; H,
4.2.3. N-(3-Chloro-4-fluorophenyl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4-amine (1c)
3.22; N, 19.19.
Yield: 83.04%; mp 166e170 ꢂC; IR (KBr): 3456 (NeH), 2937 (Ce
H), 1604 (C]N), 1556 (C]C), 731 (CeCl) cmꢁ1; 1H NMR (300 MHz,
4.2.5. N-(Phenyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine
(1e)
CDCl3)
d ppm: 7.06e7.25 (m, 3H, NHeAreH), 7.72e7.75 (s, 5H, Ne
AreH), 8.08 (s, 1H, NeN]CH at 5), 8.55 (s, 1H, N]CHeN at 2), 9.44
(s, 1H, NH at 4); ESI-MS m/z: 340 (Mþ1), 212, 195, 146. Anal. Calcd.
Yield: 80.55%; mp 180e184 ꢂC; IR (KBr): 3452 (NeH), 2956 (Ce
H), 1536 (N]N) cmꢁ1 1H NMR (300 MHz, CDCl3)
; d ppm: 7.15e
Fig. 11. Key structural requirement of condensed pyrimidine for antifungal activity.

174
K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
7.35 (m, 5H, NHeAreH), 7.54e7.56 (s, 5H, NeAreH), 7.99 (s, 1H,
NeN]CH at 5), 8.26 (s, 1H, N]CHeN at 2), 9.0 (s, 1H, NH at 4);
ESI-MS m/z: 288 (Mþ1), 212, 195, 94. Anal. Calcd. for C17H13N5
(287.32): C, 71.06; H, 4.56; N, 24.37. Found: C, 71.22; H, 4.37; N,
24.26.
4.2.6. 2H-4-Fluorophenyl-(5,6,7,8-tetrahydrobenzo(b)thieno[2,3-d]
pyrimidin-4-yl)-amine (2a)
Yield: 86.95%; mp 166e168 ꢂC; IR (KBr): 3432 (NeH), 2924 (Ce
H), 1560 (N]N), 828 (CeF) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d ppm:
1.83 (s, 4H, CH2 at 6 & 7), 2.60 (s, 4H, CH2 at 5&8), 6.89e7.25 (m, 4H,
NHeAreH), 8.1 (s, 1H, CH at 2), 8.8 (s, 1H, NH at 4); ESI-MS m/z: 300
(Mþ1), 206, 189, 112. Anal. Calcd. for C16H14FN3S (299.37): C, 64.19;
H, 4.71; N, 14.04; S, 10.71. Found: C, 64.26; H, 4.63; N, 14.19; S, 10.88.
4.2.7. 2H-(4-Fluorophenyl)-(5,6-dimethylthieno[2,3-d]pyrimidin-
4-yl)-amine (3a)
Yield: 86.31%; mp 166e170 ꢂC; IR (KBr): 3427 (NeH), 2917 (Ce
H),1602 (N]N), 900 (CeF) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d ppm:
1.95 (s, 3H, CH3 at 6), 2.22 (s, 3H, CH3 at 7), 7.05e7.33 (m, 4H, NHe
AreH), 8.15 (s, 1H, CH at 2), 8.77 (s, 1H, NH at 4); ESI-MS m/z: 274
(Mþ1), 180, 163, 112. Anal. Calcd. for C14H12FN3S (273.33): C, 61.52;
H, 4.43; N, 15.37; S, 11.73. Found: C, 61.33; H, 4.50; N, 15.47; S, 11.80.
Fig. 12. Overlay of the best docked conformation of fluconazole over the X-ray
structure in presence of heme.
4.2.8. N-(4-Flurophenyl)-3-(methylthio)-1-phenyl-1H-pyrazolo
[3,4-d]pyrimidin-4-amine (4a)
(0.5) using Mueller-Hinton broth (105 CFU/ml). Antifungal activity
was examined by the disc diffusion method under standard con-
ditions using potato-dextrose agar (according to CLSI guidelines)
[17]. Sterile filter paper discs (5 mm diameter, Whatman no. 3
chromatography paper) were dripped with compound solutions
(DMSO) to load 500 mg of a given compound per disc. Dry discs
were placed on the surface of appropriate agar medium. The results
were read after 30 h of incubation at 36 ꢂC. Compounds which
showed activity in disc diffusion tests were examined by the agar
dilution method to determine their MIC’s minimal inhibitory con-
centration (CLSI) [18].
Yield: 89.10%; mp 152e156 ꢂC; IR (KBr): 3306 (NeH), 2916 (Ce
H), 1590 (N]N), 987 (CeF) cmꢁ1; 1H NMR (300 MHz, CDCl3)
d ppm:
2.45 (s, 3H, SeCH3 at 5), 6.88e7.25 (m, 4H, NHeAreH), 7.63e7.66 (s,
5H, NeAreH), 8.5 (s, 1H, N]CHeN at 2), 9.1 (s, 1H, NH at 4); ESI-MS
m/z: 352 (Mþ1), 258, 241, 112. Anal. Calcd. for C18H14FN5S (351.4): C,
61.52; H, 4.02; N, 19.93; S, 9.12. Found: C, 61.36; H, 4.15; N, 20.05; S,
9.17.
4.2.9. (4-Fluorophenyl)-[5-(4-chlorophenyl)thieno[2,3-d]
pyrimidin-4-yl]-amine (5a)
Yield: 91.85%; mp 185e188 ꢂC; IR (KBr): 3408 (NeH), 1352 (CeF)
The MIC’s of the compounds were studied by disc diffusion
method making serial dilution of the compounds from the range 2e
2048 mg/ml. The optical densities of spores in 0.2% tween 80 solu-
cmꢁ1; 1H NMR (300 MHz, CDCl3)
d ppm: 6.15 (s, 1H, CH at 6), 7.07e
7.42 (m, 4H, m, NHeAreH), 7.78e8.10 (m, 4H, AreH at 5), 8.63 (s,
1H, N]CHeN at 2), 9.0 (s, 1H, NH at 4); ESI-MS m/z: 356 (Mþ1),
262, 245, 112. Anal. Calcd. for C18H11ClFN3S (355.82): C, 60.76; H,
3.12; N, 11.81; S, 9.01. Found: C, 60.68; H, 3.02; N, 11.63; S, 9.19.
tions were adjusted to 50 at 540 nm using a colorimeter.
4.4. Docking studies
All the molecular modelling studies were carried out on Intel
Xeon based system with the Linux Enterprise OS using the Schrö-
dinger molecular modelling package (Schrödinger, Inc., USA).
4.2.10. 4-(4-Fluorophenylamino)-5-methylthieno[2,3-d]pyrimidin-
6-carboxylate (6a)
Yield: 88.51%; mp 182e185 ꢂC; IR (KBr): 3173 (NeH), 2926 (Ce
H), 1689 (C]O), 1372 (CeF) cmꢁ1 1H NMR (300 MHz, CDCl3)
;
4.4.1. Molecular docking
d
ppm: 1.25e1.61 (t, 3H, COOCH2CH3 at 6), 2.96e2.98 (s, 3H, CH3 at
Molecular docking studies were performed using the Glide
module incorporated in the Schrödinger molecular modelling
package. Glide Extra-Precision (XP) scoring function was adopted in
the current study to estimate protein-ligand binding affinities. This
scoring function is equipped with force field-based parameters
accounting for solvation and repulsive interactions, lipophilic,
hydrogen bonding interactions, metal-ligand interactions as well as
contributions from coulombic and van der Waals interaction en-
ergies, all incorporated in the empirical energy functions.
5), 4.37e4.42 (m, 2H, COOCH2CH3 at 5), 7.11e7.38 (m, 4H, m, NHe
AreH), 8.07 (s, 1H, N]CHeN at 2), 8.88 (s, 1H, NH at 4); ESI-MS m/z:
332 (Mþ1), 286, 238, 221, 112. Anal. Calcd. for: C16H14FN3O2S
(331.36): C, 57.99; H, 4.26; N,12.68; S, 9.68. Found: C, 57.84; H, 4.20;
N, 12.47; S, 9.61.
4.3. Determination of antifungal activity
All the target compounds were tested against three pathogenic
fungal strains Candida albicans (ATCCÒ90028), Aspergillus fumigatus
(ATCCÒ204305) & Aspergillus niger (ATCCÒ9142) for their inhibitory
activity, using a Mueller-Hinton broth by serial dilution method in
96 multicell microlitre plates in duplicate. All these organisms were
procured from ATCC, LGC Promochem Pvt., Bangalore, India. The
control strains of same organisms were also developed in suitable
culture media. The inoculums of both, the control strains and
clinical isolates were standardized by adjusting to McFareld scale
4.4.2. Preparation of protein and ligand structures for docking
simulations
The X-ray structure of sterol 14a-demethylase (CYP51) in com-
plex with inhibitor fluconazole (PDB code: 3KHM) [19] was ob-
tained from the RCSB Protein Data Bank (PDB), http://www.rcsb.
org/pdb. The protein-inhibitor complex was prepared for Glide
calculations by running the protein preparation wizard applying

K.S. Jain et al. / European Journal of Medicinal Chemistry 77 (2014) 166e175
175
the OPLS-2005 force field. Any crystallographic water, if present
Appendix A. Supplementary data
was eliminated and hydrogens were added to the structure corre-
sponding to pH 7.0 considering the appropriate ionization states for
both the acidic and basic amino acid residues. After assigning
appropriate charge and protonation state, prepared structure was
subjected to energy minimization until the average root mean
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.02.066.
ꢀ
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ꢀ
ꢀ
ꢀ
ꢀ
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