Synthesis, antimicrobial activity and molecular docking studies of pyrano[2,3-d]pyrimidine formimidate derivatives

Article abstract of DOI:10.1007/s11164-015-2243-7

A new series of ten poly-functionalized pyrano[2,3-d]pyrimidine formimidate derivatives were successfully synthesized. All the synthesized compounds were characterized by ¹H NMR, ¹³C NMR, HRMS, and FT-IR spectral analysis. All the sy

Full text of DOI:10.1007/s11164-015-2243-7

Res Chem Intermed
DOI 10.1007/s11164-015-2243-7
Synthesis, antimicrobial activity and molecular docking
studies of pyrano[2,3-d]pyrimidine formimidate
derivatives
1
1
1
•
Kotaiah Yalagala
Surya Narayana Maddila
•
Suresh Maddila
1
•
Surjyakanta Rana
2
2
•
•
Sukesh Kalva
1
•
Adam Arnold Skelton
Sreekantha Babu Jonnalagadda
Received: 9 July 2015 / Accepted: 25 August 2015
Ó Springer Science+Business Media Dordrecht 2015
Abstract A new series of ten poly-functionalized pyrano[2,3-d]pyrimidine formimi-
date derivatives were successfully synthesized. All the synthesized compounds were
1
13
characterized by H NMR, C NMR, HRMS, and FT-IR spectral analysis. All the
synthesized compounds were evaluated for their antimicrobial activity by using the well
plate method and MIC by the broth micro dilution method against the strains of bacteria
as well as fungus. Four compounds exhibited good to excellent antimicrobial activity.
The theoretical binding mode of the target molecules was evaluated by docking studies,
which revealed a new molecular scaffold for enhancing the antimicrobial activity of
compounds.
Graphical Abstract Studies on synthesis, characterization, antimicrobial activity, and
molecular docking of novel pyrano[2,3-d]pyrimidine formimidate derivatives (10) are
reported. Four of the compounds exhibited excellent activity.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-015-2243-7)
contains supplementary material, which is available to authorized users.
&
Sreekantha Babu Jonnalagadda
jonnalagaddas@ukzn.ac.za
1
2
School of Chemistry and Physics, University of KwaZulu-Natal, Westville Campus, Chiltern
Hills, Durban 4000, South Africa
Discipline of Pharmaceutical Sciences, University of KwaZulu-Natal, Westville Campus,
Chiltern Hills, Durban 4000, South Africa
123

K. Yalagala et al.
Keywords Pyrano[2,3-d]pyrimidine formimidates Á Synthesis Á Antimicrobial
activity Á Molecular docking studies
Introduction
Despite significant improvement in the treatment of infectious diseases caused by
bacteria and fungi, it still remains a major worldwide health problem due to the
rapid development of resistance against the existing antimicrobial drugs. Emerging
novel antimicrobial agents with various action modes than current drugs are some of
the important approaches to handle the antimicrobial resistance. In view of these
facts, it is essential to develop more effective antimicrobial agents [1]. The current
approach in antibacterial drug discovery is largely focused on the modification of
existing classes of antibiotics. Such compounds may be prone to inactivation by
existing bacterial resistance mechanisms. Therefore, in order to combat the rise and
spread of antibiotic resistant strains, the discovery and development of novel classes
of antibiotics, and the elucidation of their molecular targets have to be urgently
pursued. Thus, the synthesis and innovation of better antimicrobial agents has been
intensively sought after during the last decade.
Pyrano[2,3-d]pyrimidines are fused heterocyclic scaffolds that represent a
privileged structural motif distributed in many naturally occurring compounds [2].
Due to their widespread assortment of pharmacological activities, these compounds
received significant attention in the past few decades [3–6]. Moreover, the six
membered nitrogen-containing heterocyclic compounds as fused pyranes and
pyrimidines are also of biological importance due to their anti-cancer [7],
antimicrobial [8], and antioxidant [9] properties. In addition, polyfunctionalized
pyrano[2,3-d]pyrimidine derivatives are common structural subunits in a variety of
important natural products [10], and they can be used as cognitive enhancers, for the
treatment of neurodegenerative disorders [11]. On the other hand, the Formimidate
Intermediates (FI’s) play a key role in the synthesis, and these have also facilitated
the production of various targeted biologically potent molecules [12]. However, the
synthesis and bioactivity of the formimidate derivatives is less explored and is still
attractive.
1
23

Synthesis, antimicrobial activity and molecular docking…
In the recent past, we have reported the synthesis and activity evaluation of
various heterocyclic compounds, such as thiadiazole for anti-inflammatory activity
[
13], triazoles [14, 15] and 1,3,4-thiadiazoles for antimicrobial activity [16, 17].
Recently, we have also reported the computational investigation of the synthesis of
pyrrole-3-carbonitriles [18]. To the best of our knowledge, there are no reports on
the synthesis of pyrano[2,3-d]pyrimidine formimidates. In this communication, we
report the synthesis of a new series of pyrano[2,3-d]pyrimidine formimidates and
their structural evaluation. All the synthesized compounds were screened for their
in vitro antimicrobial activity and molecular docking studies were implemented.
Results and discussion
Chemistry
Compounds (4a–j), amino-nitriles were prepared from aromatic aldehydes (1a–j),
malononitrile (2), and N,N-dimethylbarbituric acid (3) with urea as a catalyst, in 1:1
mixture (v/v) of ethanol and water solvent as described in the literature [19]. Those
amino-nitriles (4a–j) reacted with orthoformate to afford the target compounds
(
5a–j). The structures of all the products were identified and confirmed by H NMR,
3
C NMR, HR-MS, and FT-IR spectral analysis. The spectra data of (5a–j) showed
1
-
characteristic absorption bands in the ranges between 2209 and 2218 cm due to
1
1
–
C:N stretchings. The H NMR spectral data showed the absence of the –NH
2
peaks and the presence of triplets and quartets in the range between d 1.31–1.40 and
d 4.36–4.46 for –CH and –OCH respectively, which were related to the ethoxy
group. This strongly supports the formation of the formidates. In addition to this,
3
2,
there are several prominent peaks, which were related to other functional and alkyl
1
3
groups that were observed. The C NMR spectral data also reinforced the
formation of the titled derivatives. Furthermore, the structures of the compounds
were also confirmed on the basis of their HR-MS spectral data analysis. This
evidence strongly endorses the formation of the titled compounds (Scheme 1).
CN
R
R
CN
O
N
O
N
O
N
H
O
2
CN
NC
N
a
N
b
N
O
O
O
O
O
NH
2
N
O
O
R
1
a-j
3
4a-j
5a-j
Scheme 1 Reagents and conditions: a 10 % Urea, EtOH:H
R = 2-Cl (5a); 2-Br (5b); 2-F (5c); 4-Cl (5d); 4-Br (5e); 4-F (5f); 2-OMe (5g); 4-OMe (5h); 2, 5-Di-OMe
5i); 2, 3-Di-OMe (5j)
2 3
O (v/v: 1:1), rt, 4 h. b CH(OEt) , reflux, 8 h.
(
123

K. Yalagala et al.
Antimicrobial studies
In the quest for new antibacterial and antifungal agents, all the newly synthesized
compounds 5a–j were screened for their in vitro antibacterial activity against
Staphylococcus aureus, E. coli, K. pneumonia and P. aeruginosa bacterial strains
and in vitro antifungal activity against A. flavus and C. albicans fungal strains. The
antibacterial and antifungal activities were measured by minimum inhibitory
concentration (MIC) using the broth dilution method [20, 21].
During the antibacterial screening, compounds 5d, 5f, 5i, and 5j displayed
significant activity against various bacterial strains. The corresponding MIC values
for compounds 5d, 5f, 5i, and 5j against all the tested bacterial strains were 6.25,
-
1,
6
.25, 6.25, and 6.25 lg ml
respectively, and values were comparable to the
standard drug, ciprofloxacin. 5d, 5f, 5i, and 5j with 4–Cl, 4–F, 2,5–OMe, and 2,3–
OMe groups, respectively, on the phenyl ring have exhibited good activity.
Compounds 5a, 5c, 5g, and 5h have also exhibited moderate activity against all the
tested bacterial strains, relative to ciprofloxacin. The antibacterial evaluation clearly
indicates the derivatives. The results are tabulated in Table 1.
The results of antifungal activity summarized in Table 1 clearly show that 5a, 5c,
g, and 5h have significant activity against A. flavus and C. albicans with a MIC
value of 3.12 lg ml , which is as good as to the standard drug, Fluconazole. The
other derivatives showed moderate antifungal activity against all the tested
microbial strains. It may be generalized from the antifungal activity that electron
5
-
1
Table 1 Antimicrobial activity of the compounds (5a–j): Minimum inhibitory concentrations
-1
(MIC)/ lg ml
Compound
Antibacterial activity
Antifungal activity
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
A. flavus
C. albicans
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
12.5
[100
12.5
6.25
N.A
6.25
6.25
12.5
6.25
6.25
3.12
N.t
12.5
[100
12.5
6.25
N.A
6.25
12.5
12.5
6.25
6.25
3.12
N.t
12.5
[100
12.5
6.25
N.A
6.25
12.5
25
50
12.5
100
6.25
3.12
N.A
3.12
25
6.25
100
6.25
3.12
N.A
3.12
25
[100
12.5
6.25
N.A
6.25
12.5
6.25
6.25
12.5
3.12
N.t
g
h
i
12.5
3.12
3.12
N.t
12.5
3.12
6.25
N.t
6.25
6.25
3.12
N.t
j
Ciprofloxacin
Fluconazole
Blank
1.56
–
1.56
–
–
–
–
–
Lower MIC values indicate higher antimicrobial activity
Bold values indicate the reference values using standard drugs
N.t Not tested, N.A Not Active
1
23

Synthesis, antimicrobial activity and molecular docking…
withdrawing and electron donating groups such as Cl, F and OMe on the phenyl ring
exerted significant activity, when compared to other groups.
Molecular docking
The molecular docking studies were carried out to understand the binding mode and
mechanism of active inhibitors with the crystal structure of the C(30) carotenoid
dehydrosqualene synthase (PDB ID: 3ACX). Docking energy data for all the
synthesized compounds along with standard ciprofloxacin is included in Table 2.
Docking results show that ciprofloxacin had less docking energy compared with the
new target compounds. The docking energy for all the compounds ranged from
–
6.43 to –10.16 kcal/mol. All the compounds could dock in the active site of
dehydrosqualene synthase effectively. The binding mode of the most potent
compounds 5d, 5f, 5i and 5j, is shown in Fig. 1. All the four compounds were
involved in H-bonding with the active site residues Asp48, Tyr41, and His18 with
the high docking energies indicating that those are directly involved in inhibiting the
dehydrosqualene synthase. The binding modes of these compounds are discussed
below.
Binding mode of compound 5d
Figure 1a shows the H-bond interactions with the active site residues of
dehydrosqualene synthase, where NH1 and OH group of compound 5d donates
˚
the hydrogen bond interactions with the Asp 48 and Val 133 (2.7 A). The O4 group
of compound, 5d accepts the hydrogen bond with the NH2 group of Arg171,
whereas the N4 group of compound 5d donates the H-bond interaction with Asn168.
Binding mode of compound 5f
The NH1 and N4 group of compound 5f establishes trivalent H-bond interactions
with the side chain amino acid residue Asp48 and OH group of compound 5f forms
Table 2 Docking score of the
compounds (5a–j) and
ciprofloxacin
Compounds
Docking energy (Kcal/mol)
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
-7.27
-6.42
-7.57
-8.91
-6.91
-8.50
-9.28
-7.01
-9.48
-10.16
-8.712
g
h
i
j
Ciprofloxacin
123

K. Yalagala et al.
Fig. 1 Docking study of active molecules
bivalent interactions with the His 18 and Arg171. The OH and NH group of Tyr248
and Asn168 accepts the H-bond with the OH group of compound 5f (Fig. 1b).
Binding mode of compound 5i
The NH1 group of compound 5i also forms similar H-bond interactions with Asp 48
like compound 5d, whereas the main chain amino acid residue Val133 accepts the
H-bond with the N1 group of compound 5i. The OH group of the compound shows
the bivalent H-bond interaction with the side chain amino acid residue of Arg171,
whereas Tyr129 forms the H-bond interaction with the O4 group of compound 5i.
The O3 and N3 groups of compound 5i share the H-bond interactions with the side
chain amino acid residues of Tyr248 and Asn168 (Fig. 1c).
Binding mode of compound 5j
The higher binding energy of compound 5j indicates that this can bind more tightly
with the active site residues of dehydrosqualene synthase. The O1 group of
compound 5j forms the H-bond interaction with the active site residue of Tyr41
whereas the N1 group donates the H-bond with the side chain amino acid residue of
Val133. The O2 and O3 groups of the compound 5j shares H-bond with the Arg171
and Asn168 (Fig. 1d).
1
23

Synthesis, antimicrobial activity and molecular docking…
Docking results infer that two oxygen atoms of 1,3-dimethylpyrimidine-
,4(1H,3H)-dione, and oxygen and one nitrile group of 4H-pyran-3-carbonitrile
2
among all compounds form interactions with the active site residues of
dehydrosqualene synthase. This indicates that introduction of acceptor and donor
groups at that position in the moiety may enhance antimicrobial activity.
Conclusion
We have synthesized a new series of pyrano[2,3-d]pyrimidine formimidate
derivatives and evaluated them for their in vitro antimicrobial activity. Compounds
5
d, 5f, 5i, and 5j have shown significant antibacterial activity against S. aureus,
E. coli, K. pneumonia, and P. aeruginosa (MIC 6.25 lg/ml) relative to
ciprofloxacin. Antifungal activity data revealed that compounds 5d, 5f, 5i, and 5j
exhibited excellent activity against two fungal strains, A. flavus, and C. albicans
with (MIC 3.25 lg/ml). Thus, the above compounds can be considered as lead
compounds for enhancing their activity and development of more potent antimi-
crobial agents. Furthermore, molecular docking studies revealed the essential
groups, which bind with the active site of the dehydrosqualene synthase.
Experimental
Chemistry
All chemicals and reagents required for the reactions were of analytical grade,
obtained from Sigma-Aldrich and were used without any further purification.
1
A Bruker AMX 400 MHz NMR spectrometer was used to record the H NMR and
1
3
C NMR spectral values. High-resolution mass data were obtained using a Bruker
micro TOF-Q II ESI instrument operating at ambient temperature. The CDCl3
solution was utilized for this while TMS served as the internal standard. TMS was
further used as an internal standard for reporting the all chemical shifts in d (ppm).
The FT-IR spectrum for the samples was established using a Perkin Elmer Perkin
-
1
Elmer Precisely 100 FT-IR spectrometer at the 400–4000 cm area. Purity of all
the reaction products was confirmed by TLC using aluminum plates coated with
silica gel (Merck Kieselgel 60 F254).
General procedure for the synthesis of compounds 4a–j A mixture of chosen
aromatic aldehyde (1a–j) (1 mmol), malononitrile 2 (1.1 mmol) and urea
(
10 mol%) in a specific volume of equal ratio of EtOH:H O (1:1 v/v) was stirred
2
at room temperature (RT) for 30 min. A solution of the N,N-dimethylbarbituric acid
(1 mmol) in ethanol (10 vol) was added to the above mixture. The resulting
3
mixture was further stirred for 5 h at RT. The progress of the reaction was
monitored by TLC. After completion of the reaction, the total mixture was poured
into ice cold water, stirred for 15 min and then filtered, and solid was washed with
123

K. Yalagala et al.
water (100 mL). Further, it was recrystallized by a mixture of ethanol and water
(
80:20) [19].
General procedure for the synthesis of target compounds 5a–j A mixture of
compound (4a–j) (1 mmol) and triethyl orthoformate (10 mmol) was refluxed for
8
h under inert conditions. After completing the reaction (monitored by TLC), the
crude product was cooled to room temperature, and the unreacted triethyl
orthoformate was evaporated under reduced pressure. The solid product obtained
was recrystallized from ethanol. Structures of all the products (5a–j) were
1
13
established and confirmed on the basis of their spectral data, H NMR, C NMR,
HRMS and FTIR. The details of the product characterization are presented in the
ESI.
(
E)-ethyl-N-5-(2-chlorophenyl)-6-cyano-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-
m.p.:
1
H-pyrano[2,3-d]pyrimidin-7-ylformimidate:
-
(5a) Yield:
76 %,
1
1
2
08–210 °C; FTIR (Neat) t (cm ): 2960, 2212, 1716, 1686, 1652, 1612; H
NMR (400 MHz, CDCl ) d: 1.31 (t, 3H, J = 8.0 Hz –OCH CH ), 3.17 (s, 3H,
3
2
3
CH ), 3.42 (s, 3H, CH ), 4.36 (q, 2H, J = 8.0 Hz, –OCH CH ), 5.03 (s, 1H, –CH),
3
3
2
3
7
J = 4.0 Hz, Ar–H), 7.30 (dd, 1H, J = 8.0 Hz, Ar–H), 8.18 (s, 1H, –N=CH);
.14 (dd, 1H, J = 4.0 Hz, Ar–H), 7.18 (dd, 1H, J = 4.0 Hz, Ar–H), 7.23 (d, 1H,
1
3
C
NMR (100 MHz, CDCl ) d: 13.8, 28.2, 29.6, 37.0, 65.0 (–C–H), 82.5, 88.2, 115.7
3
(
–C:N), 127.3, 129.3, 130.5, 133.6, 137.4, 150.3, 151.4, 155.3, 159.4 (–C=O),
? ?
60.7 (–C=O); TOF–MS–ES : m/z = 423 [M ? Na] ; HR–MS calcd. For
1
4
23.0836 found: 423.0843. C H ClN O .
1
9
17
4 4
(
E)-ethyl-N-5-(2-bromophenyl)-6-cyano-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-
H-pyrano[2,3-d]pyrimidin-7-ylformimidate:
1
(5b) Yield:
74 %,
m.p.:
1
-
1
1
98–200 °C; FTIR (Neat) t (cm ): 2953, 2209, 1712, 1682, 1656, 1610; H
NMR (400 MHz, CDCl ) d: 1.39 (t, 3H, J = 8.0 Hz –OCH CH ), 3.23 (s, 3H,
3
2
3
CH ), 3.25 (s, 3H, CH ), 4.42 (q, 2H, J = 8.0 Hz, –OCH CH ), 5.17 (s, 1H, –CH),
3
3
2
3
7
J = 4.0 Hz, Ar–H), 7.58 (dd, 1H, J = 8.0 Hz, Ar–H), 8.26 (s, 1H, –N=CH);
.15 (dd, 1H, J = 4.0 Hz, Ar–H), 7.21 (dd, 1H, J = 4.0 Hz, Ar–H), 7.56 (d, 1H,
1
3
C
NMR (100 MHz, CDCl ) d: 14.2, 28.4, 29.2, 44.4, 62.7 (–C–H), 82.6, 85.9, 112.6
3
(
–C:N), 125.4, 128.2, 129.8, 133.3, 134.7, 150.5, 151.6, 157.1, 160.3 (–C=O),
?
69.1 (–C=O); TOF–MS–ES : m/z = 467 [M ? Na] , 469 [M ? 2 ? Na] ;
?
?
1
HR–MS calcd. For 467.0826 found: 467.0834. C H BrN O .
1
9
17
4 4
(
E)-ethyl-N-6-cyano-5-(2-fluorophenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-
H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5c) Yield: 68 %, m.p.: 194–196 °C;
1
-
1
1
FTIR (Neat) t (cm ): 2962, 2211, 1714, 1680, 1653, 1613; H NMR (400 MHz,
CDCl ) d: 1.42 (t, 3H, J = 8.0 Hz –OCH CH ), 3.19 (s, 3H, CH ), 3.29 (s, 3H, CH ),
3
2
3
3
3
4
.46 (q, 2H, J = 8.0 Hz, –OCH CH ), 5.09 (s, 1H, –CH), 7.10 (m, 1H, Ar–H), 7.24
2 3
(
m, 1H, Ar–H), 7.63 (d, 1H, J = 4.0 Hz, Ar–H), 7.71 (dd, 1H, J = 8.0 Hz, Ar–H),
3
1
8
.31 (s, 1H, –N=CH); C NMR (100 MHz, CDCl ) d: 13.4, 28.2, 29.5, 44.1, 62.3
3
(
–C–H), 83.1, 86.7, 111.9 (–C:N), 124.3, 127.1, 128.9, 132.8, 133.6, 148.4, 152.8,
?
54.6, 162.5 (–C=O), 164.0 (–C=O); TOF–MS–ES : m/z = 407 [M ? Na] ;
?
1
HR–MS calcd. For 407.1208 found: 407.1215. C H FN O .
1
9
17
4 4
1
23

Synthesis, antimicrobial activity and molecular docking…
(
E)-ethyl-N-5-(4-chlorophenyl)-6-cyano-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-
H-pyrano[2,3-d]pyrimidin-7-ylformimidate:
1
(5d) Yield:
81 %,
m.p.:
1
-
1
2
11–213 °C; FTIR (Neat) t (cm ): 2971, 2218, 1713, 1681, 1644, 1614; H
NMR (400 MHz, CDCl ) d: 1.32 (t, 3H, J = 8.0 Hz –OCH CH ), 3.19 (s, 3H,
3
2
3
CH ), 3.25 (s, 3H, CH ), 4.37 (q, 2H, J = 8.0 Hz, –OCH CH ), 4.58 (s, 1H, –CH),
3
3
2
3
7
–
8
.19 (d, 2H, J = 4.0 Hz, Ar–H), 7.25 (d, 2H, J = 4.0 Hz, Ar–H), 8.18 (s, 1H,
1
3
N=CH); C NMR (100 MHz, CDCl ) d: 13.8, 28.3, 29.1, 33.0, 65.1 (–C–H), 84.1,
3
9.1, 115.9 (–C:N), 129.2, 133.8, 139.5, 150.2, 150.9, 154.9, 159.3 (–C=O), 160.3
?
–C=O); TOF–MS–ES : m/z = 423 [M ? Na] ; HR–MS calcd. For 423.0836
?
(
found: 423.0842. C H ClN O .
1
9
17
4 4
(
E)-ethyl-N-5-(4-bromophenyl)-6-cyano-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-
74 %,
1
H-pyrano[2,3-d]pyrimidin-7-ylformimidate:
-
(5e) Yield:
m.p.:
1
1
2
08–210 °C; FTIR (Neat) t (cm ): 2955, 2217, 1712, 1681, 1644, 1613; H
NMR (400 MHz, CDCl ) d: 1.32 (t, 3H, J = 8.0 Hz –OCH CH ), 3.19 (s, 3H,
3
2
3
CH ), 3.41 (s, 3H, CH ), 4.37 (q, 2H, J = 8.0 Hz, –OCH CH ), 4.56 (s, 1H, –CH),
3
3
2
3
7
–
8
.14 (d, 2H, J = 8.0 Hz, Ar–H), 7.40 (d, 2H, J = 8.0 Hz, Ar–H), 8.18 (s, 1H,
1
3
N=CH); C NMR (100 MHz, CDCl ) d: 13.8, 28.3, 29.1, 38.1, 65.1 (–C–H), 84.0,
3
9.0, 115.9 (–C:N), 122.0, 129.6, 132.0, 140.0, 150.9, 154.9, 159.3 (–C=O), 160.7
?
–C=O) TOF–MS–ES : m/z = 467 [M ? Na] , 469 [M ? 2 ? Na] ; HR–MS
?
?
(
calcd. For 467.0331 found: 467.0338. C H BrN O .
1
9
17
4 4
(E)-ethyl-N-6-cyano-5-(4-fluorophenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5f) Yield: 70 %, m.p.:
-
1
1
2
01–203 °C; FTIR (Neat) t(cm ): 2963, 2215, 1714, 1684, 1642, 1616;
H
NMR (400 MHz, CDCl ) d: 1.34 (t, 3H, J = 8.0 Hz –OCH CH ), 3.20 (s, 3H,
3
2
3
CH ), 3.40 (s, 3H, CH ), 4.41 (q, 2H, J = 8.0 Hz, –OCH CH ), 4.62 (s, 1H, –CH),
7
3
3
2
3
1
.18 (m, 2H, Ar–H), 7.32 (m, 2H, Ar–H), 8.20 (s, 1H, –N=CH); C NMR
3
(
(
100 MHz, CDCl ) d: 13.9, 28.6, 29.4, 38.6, 65.4 (–C–H), 84.6, 88.9, 115.7
3
–C:N), 122.1, 130.2, 132.1, 139.4, 151.2, 154.5, 159.6 (–C=O), 161.3 (–C=O);
?
?
TOF–MS–ES : m/z = 407 [M ? Na] ; HR–MS calcd. For 407.1208 found:
07.1214. C H FN O .
4
1
9
17
4 4
(
E)-ethyl-N-6-cyano-5-(2-methoxyphenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5g) Yield: 67 %, m.p.:
-
1
1
1
96–198 °C; FTIR (Neat) t (cm ): 2940, 2213, 1708, 1687, 1657, 1601; H
NMR (400 MHz, CDCl ) d: 1.38(t, 3H, J = 8.0 Hz –OCH CH ), 3.25 (s, 3H, CH ),
3
2
3
3
3
.49 (s, 3H, CH ), 3.82 (s, 3H, –OCH ) 4.42 (q, 2H, J = 8.0 Hz, –OCH CH ), 4.95
3 3 2 3
(
s, 1H, –CH), 6.93 (m, 2H, J = 4.0 Hz, Ar–H), 7.22 (dd, 1H, J = 4.0 Hz, Ar–H),
1
3
7
2
1
.27 (m, 1H, Ar–H), 8.23 (s, 1H, –N=CH); C NMR (100 MHz, CDCl ) d: 13.8,
3
8.2, 29.1, 34.1, 55.7, 64.8 (–C–H), 83.4, 88.7, 111.5, 116.3 (–C:N), 120.9, 128.4,
29.2, 150.4, 151.4, 155.3, 157.4, 158.8 (–C=O), 161.1 (–C=O); TOF–MS–ES :
?
?
m/z = 419 [M ? Na] ; HR–MS calcd. For 419.1331 found: 419.1339. C H N O .
20 20 4 5
(E)-ethyl-N-6-cyano-5-(4-methoxyphenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahy-
dro-1H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5h) Yield: 73 %, m.p.:
-
1
1
2
21–223 °C; FTIR (Neat) t (cm ): 2946, 2209, 1711, 1682, 1651, 1604; H
123

K. Yalagala et al.
NMR (400 MHz, CDCl ) d: 1.40(t, 3H, J = 8.0 Hz –OCH CH ), 3.27 (s, 3H, CH ),
3
2
3
3
3
.51 (s, 3H, CH ), 3.86 (s, 3H, –OCH ) 4.46 (q, 2H, J = 8.0 Hz, –OCH CH ), 5.02
3 3 2 3
(
s, 1H, –CH), 7.18 (d, 2H, J = 8.0 Hz, Ar–H), 7.28 (d, 2H, J = 8.0 Hz, Ar–H), 8.20
13
(
s, 1H, –N=CH); C NMR (100 MHz, CDCl ) d: 13.8, 28.3, 29.2, 34.3, 65.2
3
(
–C–H), 84.1, 89.6, 115.9 (–C:N), 121.2, 126.7, 128.4, 132.9, 141.8, 150.5, 154.8,
?
59.1 (–C=O), 160.8 (–C=O); TOF–MS–ES : m/z = 419 [M ? Na] ; HR–MS
?
1
calcd. For 419.1331 found: 419.1337. C H N O .
2
0 20 4 5
(E)-ethyl-N-6-cyano-5-(2,5-dimethoxyphenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-te-
trahydro-1H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5i) Yield: 70 %, m.p.:
-
1
1
2
19–221 °C; FTIR (Neat) t (cm ): 2955, 2211, 1697, 1667, 1634,1595; H NMR
(
400 MHz, CDCl ) d: 1.38 (t, 3H, J = 8.0 Hz –OCH CH ), 3.26 (s, 3H, CH ), 3.48
3
2
3
3
(
s, 3H, CH ), 3.75 (s, 3H, –OCH ), 3.79 (s, 3H, –OCH ), 4.42 (q, 2H, J = 8.0 Hz,
3 3 3
–
8
5
1
OCH CH ), 4.94 (s, 1H, –CH), 6.73–6.77 (m, 2H, Ar–H), 6.82 (m, 1H, Ar–H),
2 3
1
3
.21 (s, 1H, –N=CH); C NMR (100 MHz, CDCl ) d: 13.8, 28.2, 29.0, 33.7, 55.6,
3
6.5, 64.8 (–C–H), 83.5, 88.7, 113.0, 116.2 (–C:N), 130.0, 150.4, 151.4, 151.8,
53.7, 158.8 (–C=O), 160.8 (–C=O); TOF–MS–ES : m/z = 449 [M ? Na] ;
?
?
HR–MS calcd. For 449.1437 found: 449.1432 C H N O .
21 22 4 6
(E)-ethyl-N-6-cyano-5-(2,3-dimethoxyphenyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-te-
trahydro-1H-pyrano[2,3-d]pyrimidin-7-ylformimidate: (5j) Yield: 73 %, m.p.:
-
1
1
2
20–222 °C; FTIR (Neat) t (cm ): 2952, 2213, 1698, 1669, 1661,1593; H NMR
(
400 MHz, CDCl ) d: 1.36 (t, 3H, J = 8.0 Hz –OCH CH ), 3.25 (s, 3H, CH ), 3.50
3
2
3
3
(
s, 3H, CH ), 3.78 (s, 3H, –OCH ), 3.81 (s, 3H, –OCH ), 4.41 (q, 2H, J = 8.0 Hz,
3 3 3
–
J = 4.0 Hz, Ar–H), 7.18 (d, 1H, J = 4.0 Hz, Ar–H), 8.24 (s, 1H, –N=CH);
OCH CH ), 4.98 (s, 1H, –CH), 6.69 (d, 1H, J = 4.0 Hz, Ar–H), 7.12 (d, 1H,
2 3
1
3
C
NMR (100 MHz, CDCl ) d: 13.6, 28.1, 29.2, 34.1, 56.0, 56.8, 64.9 (–C–H), 83.4,
3
8
7.2, 113.6, 117.1 (–C:N), 131.4, 150.2, 151.6, 152.0, 154.5, 159.2 (–C=O), 161.4
? ?
–C=O); TOF–MS–ES : m/z = 449 [M ? Na] ; HR–MS calcd. For 449.1437
(
found: 449.1434 C H N O .
21 22 4 6
Antimicrobial activity
A microdilution broth susceptibility assay was used for the antibacterial evaluation
of the compounds [20], while antifungal susceptibility of the fungus yeasts was
examined according to the NCCLS reference method for broth dilution antifungal
susceptibility testing of yeasts [21]. Ciprofloxacin was used as the antibacterial
standard, whereas Fluconazole was used as the antifungal reference.
Molecular docking
Ligand preparation
Ligand and protein preparation were performed using the structure editing option of
Chimera software [22, 23]. This module adds hydrogens and assigns Gasteriger
charges for the ligands. Later each ligand was minimized for 100 minimization
1
23

Synthesis, antimicrobial activity and molecular docking…
steps. After ensuring that hydrogens and charges have correctly added, the ligands
are saved as mol2 format.
Protein preparation
Dehydrosqualene synthase of S. aureus was considered as a major enzyme for the
bacterium to survive inside the host cell [24]. In this study the 3-D crystal structure of
dehydrosqualene synthase (PDB ID: 3ACX) complexed with inhibitor, BPH-673, was
retrieved from the protein data bank to study the binding mode of inhibitors. Prior to
protein preparation, the inhibitor, BPH-673, was deleted from the protein using
chimera. The solvent molecules in the protein were removed, hydrogen atoms, and
Kollmann charges were added using the AutoDock program. Furthermore, the protein
structure was fixed and energy minimized using the steepest descent algorithm for 100
minimization steps using UCSF Chimera. The purpose of this step was to relieve bad
contact involving solvent molecules in the initially solvated system [22, 23].
Receptor grid generation
A grid was generated using the Auto Dock 4.0 by selecting the active site residues in
the protein dehydrosqualene synthase (PDB ID: 3ACX) [25, 26]. Active site
residues of PDB ID: 3ACX to perform docking studies were chosen from the
PDBsum website. Docking studies were carried out using grid sizes 48, 72, and 52
along with the x, y, and z axes with 0.375 angstroms spacing.
Docking using AutoDock
Rigid docking was performed to predict the interacting mode of a series of inhibitors
with receptors using AutoDock v 4.0 [25, 26]. The ligands were docked to the
receptor using the default settings of the Lamarckian genetic algorithm. After
docking, the individual binding conformations of each ligand were observed, and
the interactions with the protein were studied. The best and the most energetically
favorable conformation of each ligand were selected based on the fitness function
that was optimized by the genetic algorithm. The results of the docking studies were
quantified in terms of the binding energy (kcal/mol). The predicted binding free
energy was used as criteria for ranking the compounds.
Acknowledgments The authors are thankful to the School of Chemistry and Physics, University of
KwaZulu-Natal, Durban, South Africa for the facilities and financial support.
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