Design and synthesis of novel heterofused pyrimidine analogues as effective antimicrobial agents

Article abstract of DOI:10.1016/j.molstruc.2019.01.105

A total of 66 novel heterofused pyrimidine analogues (pyrazolo[3,4-d]pyrimidine (7-43) and pyrido[2,3-d]pyrimidine (51a-l & 52a-h)) were synthesized by employing suitable methods. The desired structures of all the synthesized compounds were confirmed based on FT-IR, ¹H NMR, ¹³C NMR and HRMS experimental data. Further, ¹⁹F NMR and ¹H-¹⁵N HMBC of the representative compound were presented. All the final compounds were screened for their in vitro antitubercular (Mycobacterium tuberculosis; H₃₇ Rv), antibacterial (S. aureus, B. subtilis, E. coli and P. aeruginosa) and antifungal (C. neoformans, C. albicans and A. niger) activities. Compounds 51d, 51j, 51k, 51l, and 51g displayed good antibacterial and antifungal activity (MIC = 12.5 μg/ml) against bacterial and fungal strains, while moderate inhibition (MIC = 59 μM) was observed for 51l against H₃₇ Rv strain.

Full text of DOI:10.1016/j.molstruc.2019.01.105

Journal of Molecular Structure 1183 (2019) 246e255
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Design and synthesis of novel heterofused pyrimidine analogues as
effective antimicrobial agents
,
,
Balakumar Chandrasekaran a 1, Srinivasulu Cherukupalli a 1, Sivanandhan Karunanidhi a,
Afsana Kajee b, Rajeshwar Reddy Aleti a, Nisar Sayyad a, Babita Kushwaha a,
Srinivas Reddy Merugu a, Koleka P. Mlisana b, Rajshekhar Karpoormath a
,
*
a Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical Sciences, College of Health Sciences, University of KwaZulu-Natal, Westville
Campus, Durban, South Africa
b Department of Microbiology, National Health Laboratory Services (NHLS), Inkosi Albert Luthuli Central Hospital, Durban, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
A total of 66 novel heterofused pyrimidine analogues (pyrazolo[3,4-d]pyrimidine (7-43) and pyrido[2,3-
d]pyrimidine (51a-l & 52a-h)) were synthesized by employing suitable methods. The desired structures
of all the synthesized compounds were confirmed based on FT-IR, 1H NMR, 13C NMR and HRMS exper-
imental data. Further, 19F NMR and 1H-15N HMBC of the representative compound were presented. All
the final compounds were screened for their in vitro antitubercular (Mycobacterium tuberculosis; H37 Rv),
antibacterial (S. aureus, B. subtilis, E. coli and P. aeruginosa) and antifungal (C. neoformans, C. albicans and
A. niger) activities. Compounds 51d, 51j, 51k, 51l, and 51g displayed good antibacterial and antifungal
Received 21 November 2018
Received in revised form
30 January 2019
Accepted 31 January 2019
Available online 1 February 2019
Keywords:
activity (MIC ¼ 12.5
m
g/ml) against bacterial and fungal strains, while moderate inhibition (MIC ¼ 59
mM)
Pyrazolo[3,4-d]pyrimidines
Fluorinated pyrido[2,3-d]pyrimidines
Antitubercular activity
Antibacterial activity
Antifungal activity
was observed for 51l against H37 Rv strain.
© 2019 Published by Elsevier B.V.
1. Introduction
target for the scientific research [3] and in this context several re-
ports dealing with the synthesis of fused pyrimidine compounds as
potential drugs or molecular tools were published [4]. Pyrido[2,3-d]
pyrimidine and pyrazolo[3,4-d]pyrimidine are privileged scaffolds
and well documented for their versatile pharmacological actions
such as antibacterial [5,6], antifungal [7,8], anticancer [9,10], anti-
inflammatory [11,12], antidiabetic [13], antiviral [14,15], herbicidal
and antileukemic [16,17].
The discovery of potential broad spectrum and safe antimicro-
bial leads continues to be a challenge for the researchers worldwide
[1]. Due to numerous factors such as inappropriate and uncon-
trolled use of antibiotics, lack of patient compliance, emergence of
multidrug resistance, HIV co-infection, emergence of new strains of
micro-organisms and long-term multiple chemotherapy regimen
has increased the incidences of life threatening microbial infections
further complicating the treatment [2]. Thus, there has been a
continuous ever-increasing effort by the researchers to identify and
develop novel antimicrobial agents.
Heterofused pyrimidines (pyrido[2,3-d]pyrimidine and pyrazolo
[3,4-d]pyrimidine) are widely distributed in nature and have
attracted the attention of organic/medicinal chemists due to their
biological and chemotherapeutic importance. The structural simi-
larity of fused pyrimidines with purines have made them a prime
a
-Aminonitrile is one of the most captivating functional groups
that attracted the organic medicinal chemists all over the world and
this functionality on different ring systems is frequently utilized for
the synthesis of pyrimidines and their fused derivatives [18e20].
The versatile utility of 2-amino nicotino-nitrile functionality can
well be explored synthetically to yield pyrido[2,3-d]pyrimidines
[21,22]. In most of the cases, pyrido[2,3-d]pyrimidines can be
accomplished via annulations of pyridine over pyrimidine or vice-
versa. Recently, different approaches have been employed to
obtain pyrido[2,3-d]pyrimidines using direct three-component
condensation [23], catalyst-free reactions [24], cascade imination/
Buchwald-Hartwig cross coupling/cycloaddition [25], Knoevena-
gel condensation followed by [4þ2] cycloaddition [26] including
few green chemistry techniques [27e29]. Further, fluoro or
* Corresponding author.
E-mail address: karpoormath@ukzn.ac.za (R. Karpoormath).
B. Chandrasekaran and S. Cherukupalli contributed equally to this work.
1
https://doi.org/10.1016/j.molstruc.2019.01.105
0022-2860/© 2019 Published by Elsevier B.V.

B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
247
trifluoromethyl substituents on heterocyclic molecules impact the
physico-chemical properties of the pharmaceuticals thereby influ-
encing the pharmacological activities, accordingly [30]. These
literature reports embody a convincing evidence to explain the
profound research on the pyrimidine-based compounds. The
structures of pharmacologically active pyrazolo[3,4-d]-pyrimidine
and pyrido[2,3-d]pyrimidine with antimicrobial activity are pre-
sented in Fig. 1.
Rational drug design by bioisosteric modification [31] or mo-
lecular hybridization [32] is widely employed to obtain pharma-
cologically active compounds. In continuation of our efforts to
synthesize novel heterocyclic compounds of medicinal interest
[33,34], we have already synthesized 4,6-disubstituted pyrazolo-
[3,4-d]pyrimidine analogues and screened for their anticancer ac-
tivity [35]. We herein report their antimicrobial (antibacterial and
fungal) activity data. As a bioisosteric modification of pyrazole us-
ing pyridine, we designed and synthesized some novel tri-
fluoromethyl substituted pyrido[2,3-d]pyrimidine analogues from
with triethyl orthoacetate/orthopropionate (45/46) in the presence
of catalytic quantity of acetic acid for 8 h under reflux yielded the
imino ether analogues 47 [38] and 48, respectively. FT-IR spectra of
47 and 48 showed the absence of N-H stretching of primary amino
group and the presence of N¼C stretching at 1652 and 1643 cmꢀ1
,
while retaining the characteristic nitrile bands (2231 and
2227 cmꢀ1). Absence of the singlet signal of NH2 protons and
appearance of multiplet signals at
tively for ethyl protons (-O-CH2-CH3) connected to oxygen atom,
whereas multiplets at 1.14 and 2.42 ppm for ethyl protons (CH3-
d 1.37 and d 4.35 ppm, respec-
d
d
CH2-) in 1H NMR indicated the formation of imino ether function-
ality and established the structure of novel intermediate 48. This
has been further substantiated by the appearance of carbon signals
at
d
10.58 and 24.50 ppm indicated the ethyl carbons (CH3-CH2)
13.77
associated to the quaternary carbon, whereas the signals at
d
and 62.91 ppm attributable to the ethyl carbons connected to ox-
ygen (O-CH2-CH3). The compounds 47 and 48 were independently
reacted with ethanolic solution of ammonia to obtain 4-amino-5-
trifluoro-methyl-2,7-disubstituted pyrido[2,3-d]pyrimidines 49
[38] and 50 in high yields. The major aspect of the reaction is an
amination on the imino carbon subsequent cyclisation with a nitrile
carbon. The disappearance of nitrile stretching and appearance of
N-H stretching of primary amino group at 3355 and 3440 cmꢀ1 in
FT-IR spectra indicated the formation of intermediate compounds
49 and 50. This has been further confirmed by 1H NMR (400 MHz,
CDCl3) in which the characteristic appearance of broad singlet
a
versatile precursor (2-amino-3-cyano-4-trifluoromethyl-6-
phenyl pyridine). Additionally, antitubercular screening of all the
final compounds were performed against Mycobacterium tubercu-
losis H37 Rv strain.
2. Results and discussion
2.1. Chemistry
resonating at
apart from singlet signal at
quartet signals, respectively at
CH2) for 50. Nucleophilic addition/elimination reaction between
free amino group (49 and 50) and different aryl/hetero aryl acid
chlorides in presence of pyridine yielded corresponding final
compounds 51a-l and 52a-h. FT-IR spectra of all the final com-
pounds demonstrated carbonyl (C¼O) stretching in the range of
1582e1620 cmꢀ1 together with the appearance of N-H stretching at
d
6.10e6.34 ppm corresponding to the free NH2 group
2.64 ppm (CH3) for 49 and triplet and
1.4 ppm and 2.9 ppm (CH3 and
The synthesis of a series of 4,6-disubstituted pyrazolo[3,4-d]
pyrimidine hybrids (7-43) was achieved through an efficient and
versatile synthetic route as depicted Scheme 1. The structures and
experimental data of compounds 7e43 were clearly provided in the
previous work [35]. Further, the synthesis of novel pyrido [2,3-d]
pyrimidines were accomplished by simple and conventional
method. The starting material 2-amino-3-cyano-4-trifluoromethyl-
6-phenyl pyridine (44) was prepared as per the reported method in
the literature [36,37]. The starting material 44 reacted individually
d
d
Fig. 1. Structures of the known fused pyrimidines as antimicrobial agents.

248
B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
Scheme 1. Synthetic exploration for the preparation of 4,6-disubstituted pyrazolo[3,4-d]pyrimidine analogues (7-43) [35].
2920e3106 cmꢀ1 indicating the formation of a new amide bond.
This has been further confirmed by 1H NMR spectra (400 MHz,
CDCl3) in which the disappearance of broad NH2 signal and
obtain novel fluorinated pyrido[2,3-d]pyrimidines (51a-l and 52a-
h) is outlined in Scheme 2 and various substitutions are presented
in Table 1.
appearance of new singlet in the range of
d
12.66e15.69 ppm cor-
Reagents and conditions (Scheme 1): (a) hydrazine hydrate,
ethanol, 80 ꢁC, 3 h, 92%; (b) Conc. H2SO4, NH4OH, H2O, 50 ꢁC, 5 h,
90%; (c) potassium ethyl xanthogenate, DMF, 120 ꢁC, 6 h, 82%; (d) 2-
chloroethyl benzene, K2CO3, DMF, 70 ꢁC, microwave, 20 min, 76%;
(e) 1-bromopentane, NaOH, H2O, glacial acetic acid, 50 ꢁC, 5 h, RT,
overnight, 80%; (f) POCl3, DMF, 80 ꢁC, 2 h, 85%; (g) R-NH2, EtOH,
80 ꢁC, 2 h, 75e95%.
responds to the N-H proton are observed which established the
structure of final compounds. This was further supported by 13C
NMR spectra in which the appearance of new and most prominent
carbon signal that resonated at around
d 162.90e180.46 ppm
assigned to the amide carbonyl C¼O that confirmed 51a-l and 52a-
h. The presence of fluorine and nitrogen atoms in final compounds
were confirmed by 19F and 15N NMR (1D and 2D) experiments and
the spectral data of the representative compound 52b in CDCl3 was
presented in the supporting information. No internal standards
(CFCl3 or ammonia) were used for the 19F and 15N NMR experi-
ments. 19F NMR of 52b showed a singlet signal at ꢀ59.19 ppm
Reagents and conditions (Scheme 2): (a) AcOH, reflux, 160 ꢁC,
8 h; (b) NH3, EtOH, rt, 4 h; (c) acid chlorides, pyridine, rt,
30e90 min.
2.2. Antimicrobial evaluation
which evidently confirmed the presence of CF3 group. In 1H-15
N
HMBC NMR experiment, the correlation between amide nitrogen
(NH, ¼ 2.94 ppm) was
¼ 15.19 ppm) and methylene proton (CH2,
observed as a significant correlation and appeared as a cross peak at
155.7 ppm. This result was substantiated by the presence of third
nitrogen atom (N3) in 52b. Similarly, a cross peak observed at
247.3 ppm with methylene proton (CH2, quartet at
¼ 2.94 ppm)
confirmed the presence of N1. Further, the methine proton (CH,
304.7 ppm was attributed to N8 (See
supporting information-Fig. S58). In addition, the final derivatives
were confirmed successively by recording their respective HRMS,
which displayed accurate molecular ion peaks that correlated well
with their expected molecular weights. The synthetic pathway to
In vitro antitubercular evaluation of final compounds 7e43, 51a-
l and 52a-h was carried out at Infectious Disease Research Institute
(IDRI), Bethesda, MD, USA under the National Institute of Allergy
and Infectious Diseases (NIAID) screening program. MIC was
determined against M. tuberculosis strain H37 Rv grown under
aerobic conditions by using a dual read-out (OD590 and fluores-
cence) assay procedure [39e41]. The assay is mainly based on the
measurement of growth in liquid medium of a fluorescent reporter
strain of H37 Rv, where the readout was either optical density (OD)
or fluorescence. The use of two readouts minimizes various draw-
backs caused by the compound precipitation or the generation of
autofluorescence. Screened compounds belonged to two series:
d
d
d
d
d
d
¼ 8.23 ppm) resonating at
d

B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
249
Scheme 2. Synthetic pathway to novel fluorinated pyrido[2,3-d]pyrimidines 51a-l and 52a-h.
Table 1
(Table 2). From the screened, pyrido[2,3-d]pyrimidine series,
compounds 51d, 51j, and 51l demonstrated good antibacterial ac-
Synthesized final compounds with their substituents.
tivity (MIC ¼ 12.5
Gram-positive bacterial strains as compared to the standard
g/mL). None of the compounds in this
series presented any significant inhibition against Gram-negative
bacterial strains.
The in vitro antifungal screening [42] of 7e43, 51a-l and 52a-h
was conducted against C. neoformans (ATCC66031), C. albicans
(ATCC90028) and A. niger (ATCC 16 404) fungal strains using
Amphotericin B as a reference drug. The results (MIC values) of
in vitro antifungal screening of 7e43, 51a-l and 52a-h are sum-
marized in Tables 2 and 3 From the tested pyrazolo[3,4-d]pyrimi-
dine series, all compounds displayed moderate activity
mg/ml) specifically against both of the tested
S. No.
Compd. Code
R1
R2
1
2
3
4
5
6
7
8
51a
51b
51c
51d
51e
51f
51g
51h
51i
51j
51k
51l
52a
52b
52c
52d
52e
52f
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Me
-Et
-Et
-Et
-Et
-Et
-Et
-Et
-Et
-Phenyl
-2-Furyl
-2-Thienyl
-4-Heptyl-phenyl
-Cyclohexyl
-Phenoxyacetyl
ꢀ3,3-Dimethylbutyryl
-4-Nitrophenyl
-Pyridin-4-yl
-Biphenyl
amoxycillin (MIC ¼ 0.39
m
9
10
11
12
13
14
15
16
17
18
19
20
-Benzofuryl
ꢀ3,5-Bistrifluoromethyl
- Phenyl
(MIC ¼ 50
compounds 51k and 51l from pyrido[2,3-d]pyrimidine series
exhibited potent activity with an MIC of 12.5 g/ml against all the
mg/ml) against C. albicans. On the other hand, couple of
-2-Furyl
-2-Thienyl
-4-Heptylphenyl
-Biphenyl
-Benzofuryl
m
fungal strains (Table 3). These biological screening results pro-
pounded that the trifluoromethyl substituted compound 51l
bearing methyl substituent at 2nd position of pyrido[2,3-d]pyrimi-
dine presented good antitubercular, antibacterial and antifungal
activities.
52g
52h
ꢀ3,5-Bistrifluoromethyl
-4-Nitrophenyl
pyrazolo[3,4-d]pyrimidine series (7-43) and pyrido[2,3-d]pyrimi-
dine series (51a-l, 52a-h). From the tested pyrazolo[3,4-d]pyrimi-
dine series, compound 26 displayed the moderate inhibition
3. Experimental
against M. tuberculosis H37 Rv with MIC 120
pound 51l from pyrido[2,3-d]-pyrimidine series exhibited good
inhibition at MIC 59 M. The anti-tubercular activity data of all the
final compounds (7e43, 51a-l and 52a-h) are presented in Tables 2
and 3.
The in vitro antibacterial screening [42] of 7e43, 51a-l and 52a-h
was conducted against 2 Gram-positive [Staphylococcus aureus
(ATCC25923), Bacillus subtilis (ATCC 6051)] and 2 Gram-negative
[Escherichia coli (ATCC35218), Pseudomonas aeruginosa (ATCC
27 853)] bacterial strains using amoxycillin as a reference drug. The
results (MIC values) of in vitro antibacterial screening of 7e43, 51a-l
and 52a-h are summarized in Tables 2 and 3 From the tested pyr-
azolo[3,4-d]pyrimidine series, moderate activity was observed for
mM. Similarly, com-
3.1. Chemistry
m
The analytical grade (AR) chemicals and reagents were obtained
from Merck and Sigma-Aldrich and were used as such without any
further purification. The progress of the reactions and the purity of
the synthesized compounds were monitored by Thin Layer Chro-
matography (TLC) on precoated silica gel 60 F254 (mesh) (E. Merck)
and spots were visualized under UV light (long and short wave-
length). Merck silica gel (60e120 mesh) was used for column
chromatography. Melting points of all the synthesized compounds
were recorded in open capillaries using Thermo Fisher Scientific
(IA9000, UK) digital melting point apparatus and are uncorrected.
Bruker Alpha FT-IR spectrophotometer (Billerica, MA, USA) with
universal ATR sampling accessory was used to record all FT-IR
compounds 31, 35 and 37 (MIC ¼ 25
mg/ml) against E. coli strain

250
B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
Table 2
Antimicrobial evaluation of heterofused pyrimidines (Series-1).
Code
R
M. tuberculosis
37 Rva (MIC in
mM)
Bacterial strains (MIC in
Gram positive
m
g/ml)
Fungal strains (MIC in mg/ml)
H
Gram negative
S. aureus ATCC B. subtilis ATCC E. coli ATCC P. aeruginosa ATCC C. neoformans ATCC C. albicans ATCC A. niger ATCC
25 923
6051
35 218
27 853
66 031
90 028
16 404
7
8
9
Ph
200
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
25
>100
>100
>100
25
>100
25
>100
>100
>100
>100
>100
>100
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
>100
50
50
50
50
>100
50
50
-
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
2-ClPh
3-ClPh
4-ClPh
3-NO2Ph
4-NO2Ph
2-BrPh
3-BrPh
4-BrPh
2,4-(CH3)2Ph >200
SCH3Ph >200
4-Cl-3-CF3Ph >200
4-Br-3-CF3Ph >200
4-Cl-3NO2Ph >200
>200
>200
>200
>200
>200
>200
>200
>200
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
4-F-2-CH3Ph
3-Br-4-CH3Ph >200
2-Cl-4-FPh >200
3,4-(OCH3)Ph >200
4-(ethynyl)Ph >200
>200
Ph
120
2-ClPh
3-ClPh
4-ClPh
3-NO2Ph
4-NO2Ph
2-BrPh
3-BrPh
4-BrPh
2,4-(CH3)2Ph >200
SCH3Ph >200
4-Cl-3-CF3Ph >200
4-Br-3-CF3Ph >200
4-Cl-3-NO2Ph >200
>200
>200
>200
>200
>200
>200
>200
>200
>100
>100
50
50
50
50
50
50
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
e
>100
>100
>100
>100
50
50
4-F-2-CH3Ph
3-Br-4-CH3Ph >200
2-Cl-4-FPh >200
3,4-(OCH3)Ph >200
>200
>100
>100
50
>100
e
Rifampicin
0.0067
Amoxycillin
Amphotericin B
e
<0.39
e
<0.39
e
<0.39
e
<0.39
e
e
e
e
e
<0.39
<0.39
<0.39
a
MIC in mM at which M. tuberculosis H37 Rv growth was completely inhibited and is derived from the inflection point of the fitted curve to the lower asymptote (zero
growth).
spectra. 1H and 13C NMR spectra of all the synthesized compounds
were recorded on Bruker Avance IV NMR spectrometer at 400 and
101 MHz, using CDCl3 and DMSO‑d6, respectively with TMS as an
internal standard and signals were reported in parts per million
(ppm). Further, 19F and 15N NMR experiments of the representative
compound were conducted in CDCl3 without using any internal
standard (CFCl3 or ammonia). All high-resolution mass spectra
(HRMS) were recorded on an Autospec mass spectrometer under
electron spray ionization.
reaction mixture was heated under reflux for 8 h at 160 ꢁC. After
completion of the reaction (as monitored by TLC), the solution was
allowed to cool to room temperature followed by evaporation of
excess of triethyl orthoacetate/propionate in vacuo yielded dark
coloured residue. The crude mixture was purified through column
chromatography using 60e120 mesh silica gel employing 10%
CHCl3 in hexane as suitable eluent to obtain pure 47 and 48.
3.1.2.1. Ethyl
N-[3-cyano-6-phenyl-4-(trifluoromethyl)-2-pyridyl]
ethaneimidate 47. Ethyl N-[3-cyano-6-phenyl-4-(trifluoromethyl)-
2-pyridyl]ethaneimidate 47 was characterized and the data
corroborated well as described in the literature [38]. Yield 81%, m.p.
89e91 ꢁC (93 ꢁC), FT-IR (ATR, Vmax): 2231 (CN), 1652 (N¼C), 1260
3.1.1. Synthesis of 2-amino-3-cyano-4-trifluoromethyl-6-phenyl
pyridine 44
2-Amino-3-cyano-4-trifluoromethyl-6-phenyl pyridine 44 was
synthesized and characterized based on earlier literature reports
[36,37].
(CeO), 1134 (C-F) cmꢀ1
.
3.1.2.2. Ethyl
N-[3-cyano-6-phenyl-4-(trifluoromethyl)-2-pyridyl]
propaneimidate 48. Yield 85%; m.p. 92e94 ꢁC; FT-IR (ATR, Vmax):
3.1.2. Procedure for the synthesis of ethyl N-[3-cyano-6-phenyl-4-
(trifluoromethyl)-2-pyridyl]ethaneimidate (47)/propanimidate (48)
2-Amino-3-cyano-4-trifluoromethyl-6-phenyl pyridine 44 (3g,
11.4 mmol) was taken in excess of triethyl- orthoacetate 45/propi-
onate 46 (8 ml) and added catalytic quantity of acetic acid. The
2227 (CN), 1643 (N¼C), 1252 (CeO), 1142 (C-F) cmꢀ1
(400 MHz, DMSO‑d6):
,
1H NMR
d 1.14 (m, 3H, CH3-CH2), 1.37 (m, 3H, O-CH2
-CH3), 2.42 (m, 2H, CH3-CH2), 4.35 (m, 2H, O-CH2-CH3), 7.56e7.57
(m, 3H, Ar-H), 8.16 (m, 1H, Ar-H), 8.22e8.24 (m, 2H, Ar-H) ppm, 13
C

B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
251
Table 3
Antimicrobial evaluation of heterofused pyrimidines (Series-2).
Code
M. tuberculosis H37 Rva (MIC in
m
M) Bacterial strains (MIC in
Gram positive
m
g/ml)
Fungal strains
Gram negative
S. aureus ATCC B. subtilis ATCC E. coli ATCC P. aeruginosa ATCC C. neoformans ATCC C. albicans ATCC A. niger ATCC
25 923
6051
35 218
27 853
66 031
90 028
16 404
51a
51b
51c
51d
51e
51f
51g
51h
51i
51j
51k
51l
52a
52b
52c
52d
>200
>200
>200
>100
>200
>170
>200
>200
>200
>200
>200
59
200
200
200
200
200
100
100
100
50
50
25
12.5
25
25
50
50
50
12.5
50
12.5
50
50
50
50
50
50
50
50
e
50
50
25
12.5
25
25
50
50
50
12.5
50
12.5
50
50
50
50
50
50
50
50
e
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
e
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
e
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
12.5
12.5
25
25
25
25
25
25
25
25
e
25
25
25
25
25
25
25
25
12.5
25
12.5
12.5
25
25
25
25
25
25
12.5
12.5
12.5
25
25
25
25
25
25
25
52e
52f
52g
52h
25
25
25
e
25
e
Rifampicin
Amoxycillin
Amphotericin B e
0.0067
e
0.39
e
0.39
e
0.39
e
0.39
e
e
e
e
>0.39
>0.39
>0.39
a
MIC in mM at which M. tuberculosis H37 Rv growth was completely inhibited and is derived from the inflection point of the fitted curve to the lower asymptote (zero
growth).
3.1.4. General procedure for the synthesis of final compounds (51a-l
and 52a-h)
NMR (100 MHz, DMSO‑d6):
d 10.58 (CH3-CH2), 13.77 (O-CH2-CH3),
24.50 (CH3-CH2), 62.91 (O-CH2-CH3), 95.31, 111.61, 113.55 (CN),
120.26, 122.99, 127.69, 129.03, 131.34, 135.96, 141.25, 141.58, 160.26,
163.00, 169.38 ppm.
To the key intermediate compounds 49/50 (1 mmol) in pyridine
(2 ml), different acid chlorides (1 mmol) were added dropwise and
stirred at room temperature for about 30e90 min. After completion
of the reaction (as monitored by TLC), the mixture was poured onto
crushed ice and pH of the solution was adjusted to neutral using dil.
HCl. The solid separated was isolated by filtration, dried and puri-
fied by passing through a column packed with silica gel 60e120
using 15% ethyl acetate in n-hexane to yield pure target compounds
51a-l and 52a-h.
3.1.3. Procedure for the synthesis of 2-substituted-7-phenyl-5-
(trifluoromethyl)pyrido[2,3-d]pyrimidin-4-amine (49 and 50)
Compound 47/48 (1.57 mmol) was treated with ethanolic solu-
tion (10 ml) of ammonia (5 ml), and stirred at room temperature for
4 h. During the course of stirring, solid separated out from the so-
lution and excess of ethanol was evaporated under reduced pres-
sure. The crude residue was passed through silica gel column
(60e120 mesh) and eluted with 20% CHCl3 in hexane mixture to
obtain 49 and 50 as pure products.
3.1.4.1. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)benzamide (51a). Pale yellow solid, yield: 250 mg
(62%), mp: 233e235 ꢁC, FT-IR (ATR, Vmax): 2920 (NH), 1582 (C¼O),
1151 (C-F) cmꢀ1 1H NMR (400 MHz, CDCl3):
, d 2.64 (s, 3H, CH3),
3.1.3.1. 2-Methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-amine (49). This intermediate 6 was synthesized and
characterized as per the reported literature [38]. Yield 73%, m.p.
236e237 ꢁC (230 ꢁC), FT-IR (ATR, Vmax): 3355, 3440 (NH2), 1354
7.39e7.43 (m, 2H, Ar-H), 7.47e7.50 (m, 4H, Ar-H), 8.20e8.23 (m, 3H,
Ar-H), 8.31e8.33 (m, 2H, Ar-H), 15.68 (s, 1H, N-H) ppm, 13C NMR
(100 MHz, CDCl3):
d 29.89, 109.22, 117.24, 117.31, 122.90, 127.05,
127.28, 128.11, 129.11, 130.72, 131.80, 135.29, 135.87, 137.09, 137.43,
155.18, 155.85, 159.51, 162.75, 179.86 ppm, HRMS (ESI, m/z)
[MþNa]þ, calculated for C22H15F3N4ONa, 431.1096; found 431.1094.
(CeN) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d
2.63 (s, 3H, CH3), 6.10 (2H,
br s, 2H, NH2), 7.46e7.49 (m, 3H, Ar-H), 8.08 (s, 1H, Ar-H), 8.21e8.24
(m, 2H, Ar-H) ppm, 13C NMR (100 MHz, CDCl3):
26.13, 100.71,
d
115.74, 115.80, 128.01, 129.03, 131.46, 133.92, 134.24, 136.59, 159.92,
161.86, 162.85, 168.68 ppm.
3.1.4.2. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)furan-2-carboxamide (51b). Pale brown solid, yield:
270 mg (69%), mp: 236e238 ꢁC, FT-IR (ATR, Vmax): 3057 (NH), 1586
(C¼O), 1144 (C-F) cmꢀ1
, d 2.70 (s, 3H,
1H NMR (400 MHz, CDCl3):
3.1.3.2. 2-Ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyrimidin-
4-amine (50). Yield 81%, m.p. 228e230 ꢁC, FT-IR (ATR, Vmax): 3647,
CH3), 6.57 (s, 1H, Ar-H), 7.53e7.55 (m, 4H, Ar-H), 7.66 (s, 1H, Ar-H),
8.27e8.28 (m, 2H, Ar-H), 8.29 (s, 1H, Ar-H), 15.09 (s, 1H, N-H) ppm.
3335, (NH2), 1357 (CeN), 1135 (C-F) cmꢀ1 1H NMR (400 MHz,
,
CDCl3):
d
1.36e1.40 (t, J ¼ 7.6 Hz, 3H, CH2CH3), 2.89e2.98 (q,
J ¼ 7.6 Hz, 2H, CH2CH3), 6.34 (2H, br s, 2H, NH2), 7.49e7.52 (m, 3H,
3.1.4.3. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)thiophene-2-carboxamide (51c). Yellow solid, yield:
246 mg (59%), mp: 239e241 ꢁC, FT-IR (ATR, Vmax): 2954 (NH), 1565
Ar-H), 8.11 (s, 1H, Ar-H), 8.24e8.27 (m, 2H, Ar-H) ppm, 13C NMR
(100 MHz, CDCl3):
128.08, 128.60, 129.02, 131.49, 133.96, 134.29, 136.57, 160.06, 161.74,
163.00, 172.46 ppm.
d 12.51 32.61, 100.90, 115.86, 115.93, 124.55,
(C¼O), 1146 (C-F) cmꢀ1
, d 2.61 (s, 3H,
1H NMR (400 MHz, CDCl3):
CH3), 7.05e7.06 (m, 1H, Ar-H), 7.45e7.47 (m, 3H, Ar-H), 7.53e7.54

252
B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
(m, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 8.18e8.20 (m, 3H, Ar-H), 15.28 (s,
1H, N-H), 13C NMR (100 MHz, CDCl3):
29.69, 109.99, 118.17, 118.24,
Ar-H), 8.36 (s, 1H, Ar-H), 8.81e8.82 (m, 2H, Ar-H), 15.62 (s, 1H, -NH)
ppm, 13C NMR (100 MHz, CDCl3):
22.71, 109.76, 118.77, 118.84,
d
d
118.32, 118.38, 128.05, 128.20, 129.11, 131.73, 133.82, 136.28, 138.16,
138.50, 143.05, 156.09, 156.38, 160.44, 163.80, 175.36 ppm, HRMS
(ESI, m/z) [MþNa]þ, calculated for C20H13F3N4OSNa, 437.0660;
found 437.0663.
121.12, 123.36, 123.85, 128.17, 129.02, 129.23, 132.08, 136.08, 138.12,
138.46, 144.49, 149.76, 156.04, 157.86, 160.59, 164.50, 178.80 ppm.
3.1.4.10. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-[1,10-biphenyl]-4-carboxamide (51j).
Greenish-yellow solid, yield: 281 mg (58%), mp: 241e243 ꢁC, FT-IR
3.1.4.4. 4-Heptyl-N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido
[2,3-d]pyrimidin-4-yl)benz-amide (51d). Pale yellow solid, yield:
280 mg (57%), mp: 146e148 ꢁC, FT-IR (ATR, Vmax): 3034 (NH), 1581
(ATR, Vmax): 3051 (NH), 1580 (C¼O), 1127 (C-F) cmꢀ1
,
1H NMR
(400 MHz, CDCl3):
d 2.75 (s, 3H, CH3), 7.38e7.40 (m, 1H, Ar-H),
(C¼O), 1139 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 0.80e0.82 (m,
7.44e7.48 (m, 2H, Ar-H), 7.54e7.56 (m, 3H, Ar-H), 7.65e7.67 (m,
2H, Ar-H), 7.71e7.73 (m, 2H, Ar-H), 8.28e8.30 (m, 2H, Ar-H), 8.32 (s,
1H, Ar-H), 8.44e8.46 (m, 2H, Ar-H), 13C NMR (100 MHz, CDCl3):
3H, CH3), 1.17e1.25 (m, 8H, 4CH2), 1.57 (s, 2H, CH2), 2.57e2.63 (m,
5H, CH3 and CH2), 7.18e7.22 (m, 2H, Ar-H), 7.47e7.50 (m, 3H, Ar-H),
8.21e8.23 (m, 5H, Ar-H), 15.69 (s, 1H, N-H), 13C NMR (100 MHz,
d
22.65, 110.20, 118.32, 118.39, 127.01, 127.29, 128.05, 128.10, 128.90,
CDCl3): d 13.07, 21.64, 28.13, 28.26, 28.67, 30.12, 30.76, 35.09,109.29,
129.15, 130.69, 131.81, 135.64, 136.26, 138.06, 138.40, 140.21, 145.48,
156.50, 156.67, 160.27, 163.81, 180.46 ppm, HRMS (ESI, m/z)
[MþNa]þ, calculated for C28H19F3N4ONa, 507.1409; found 507.1409.
117.12, 117.20, 120.19, 122.91, 127.02, 127.41, 128.08, 129.24, 130.65,
133.43, 135.32, 137.06, 137.40, 147.60, 155.22, 155.60, 159.50, 162.59,
179.87 ppm.
3.1.4.11. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
3.1.4.5. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)benzofuran-2-carboxamide
(51k). Yellowish-green
solid, yield: 258 mg (58%), mp: 261e263 ꢁC, FT-IR (ATR, Vmax): 3014
imidin-4-yl)cyclohexane-carboxamide
(51e). White
solid,
yield:190 mg (48%), mp: 231e232 ꢁC, FT-IR (ATR, Vmax): 3044 (NH),
(NH),1579 (C¼O),1117 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 2.74
,
1620 (C¼O), 1132 (C-F) cmꢀ1 1H NMR (400 MHz, DMSO‑d6):
(s, 3H, CH3), 7.29e7.30 (m, 1H, Ar-H), 7.41e7.43 (m, 1H, Ar-H),
7.54e7.57 (m, 3H, Ar-H), 7.59e7.61 (m, 1H, Ar-H), 7.73e7.75 (m,
1H, Ar-H), 7.86 (s,1H, Ar-H), 8.28e8.30 (m, 2H, Ar-H), 8.32 (s,1H, Ar-
d
1.323e1.327 (m, 5H, cyclohexyl), 1.57e1.68 (m, 5H, cyclohexyl),
2.00 (s, 1H, cyclohexyl CH), 2.05e2.42 (m, 3H, CH3), 7.56e7.58 (m,
3H, Ar-H), 8.24e8.28 (m, 3H, Ar-H), 12.66 (s, 1H, N-H), 13C NMR
H), 15.15 (s, 1H, -NH), 13C NMR (100 MHz, CDCl3):
d 22.68, 112.35,
(100 MHz, DMSO‑d6):
d 21.44, 25.26, 25.47, 29.12, 43.64, 110.12,
115.20, 118.48, 118.54, 123.29, 123.65, 123.94, 127.66, 127.73, 128.11,
129.16, 131.86, 136.27, 152.11, 156.34, 160.50, 164.07, 171.93 ppm,
HRMS (ESI, m/z) [MþNa]þ, calculated for C24H15F3N4O2Na,
471.1045; found 471.1045.
115.71, 115.78, 123.69, 127.69, 129.03, 131.18, 136.45, 137.17, 137.51,
159.32, 159.48, 161.74, 177.34 ppm.
3.1.4.6. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-2-phenoxy-acetamide (51f). Off white solid, yield:
273 mg (63%), mp: 168e170 ꢁC, FT-IR (ATR, Vmax): 3016 (NH), 1640
3.1.4.12. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-3,5-bis-(trifluoromethyl)benzamide (51l). Pale green
solid, yield: 260 mg (49%), mp: 176e178 ꢁC; FT-IR (ATR, Vmax): 3012
(C¼O), 1118 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 2.69e2.94 (s,
3H, CH3), 4.86 (s, 2H, CH2), 6.97e6.99 (m, 3H, Ar-H), 7.32e7.33 (m,
2H, Ar-H), 7.55e7.57 (m, 3H, Ar-H), 8.31e8.32 (m, 3H, Ar-H) ppm,
(NH),1686 (C¼O),1117 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 2.79
(s, 3H, CH3), 7.52e7.54 (m, 3H, Ar-H), 8.08 (s, 1H, Ar-H), 8.20e8.26
(m, 1H, Ar-H), 8.28e8.30 (m, 2H, Ar-H), 8.57e8.58 (m, 2H, Ar-H)
13C NMR (100 MHz, CDCl3):
d 26.58, 67.81, 114.69, 114.81, 121.48,
122.68,128.41,129.69,130.06,132.06,132.26,136.40,158.22,166.99,
168.95, 184.50 ppm.
ppm, 13C NMR (100 MHz, CDCl3):
d 23.28, 100.20, 115.42, 115.49,
120.61, 123.32, 125.26, 127.08, 128.17, 129.05, 130.60, 130.94, 130.97,
131.27, 132.39, 133.45, 137.77, 135.09, 158.97, 159.98, 162.80, 165.47,
167.09 ppm.
3.1.4.7. 3,3-Dimethyl-N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyr-
ido[2,3-d]pyrimidin-4-yl)-butanamide (51g). Pale pink solid, yield:
164 mg (41%), mp: 192e194 ꢁC, FT-IR (ATR, Vmax): 3018 (NH), 1682
3.1.4.13. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)benzamide (52a). Pale yellow solid, yield: 224 mg
(56%), mp: 203e204 ꢁC, FT-IR (ATR, Vmax): 3056 (NH), 1583 (C¼O),
(C¼O), 1156 (C-F) cmꢀ1, 1H NMR (400 MHz, DMSO‑d6):
d 0.91e0.97
(m, 9H, aliphatic), 2.21e2.24 (m, 2H, aliphatic), 2.87 (s, 3H, CH3),
7.61e7.66 (m, 3H, Ar-H), 8.42e8.44 (m, 2H, Ar-H), 8.65 (s, 1H, Ar-H)
1139 (C-F) cmꢀ1 1H NMR (400 MHz, CDCl3):
, d 1.52e1.56 (t,
ppm, 13C NMR (100 MHz, DMSO‑d6):
d 10.82, 10.87, 16.65, 25.56,
J ¼ 7.68 Hz, 3H, CH2CH3), 2.98e3.04 (q, J ¼ 7.63 Hz, 2H, CH2CH3),
7.48e7.52 (m, 2H, Ar-H), 7.55e7.57 (m, 4H, Ar-H), 8.27e8.29 (m, 2H,
110.88, 120.34, 128.36, 129.25, 132.16, 133.13, 135.91, 158.33, 161.57,
163.82, 168.09, 176.60 ppm.
Ar-H), 8.30e8.33 (m, 1H, Ar-H), 8.40e8.42 (m, 2H, Ar-H) ppm, 13
C
NMR (100 MHz, CDCl3): d 11.12, 29.48, 118.43, 128.14, 128.32,129.13,
3.1.4.8. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-4-nitrobenz-amide (51h). Greenish-yellow solid, yield:
260 mg (58%), mp: 250e252 ꢁC; FT-IR (ATR, Vmax): 3043 (NH), 1572
130.14, 131.73, 132.83, 136.41, 136.91, 157.11, 160.62, 163.92 ppm,
HRMS (ESI, m/z) [MþNa]þ, calculated for C23H17F3N4ONa, 445.1252;
found 445.1252.
(C¼O), 1136 (C-F) cmꢀ1
, d 2.78 (s, 3H,
1H NMR (400 MHz, CDCl3):
CH3), 7.54e7.58 (m, 3H, Ar-H), 8.29e8.33 (m, 4H, Ar-H), 8.36 (s, 1H,
Ar-H), 8.51e8.54 (m, 2H, Ar-H), 15.67 (s, 1H, -NH) ppm, 13C NMR
3.1.4.14. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)furan-2-carboxamide (52b). Pale brown solid, yield:
265 mg (69%), mp: 230e232 ꢁC, FT-IR (ATR, Vmax): 3111 (NH), 1581
(100 MHz, CDCl3):
d 22.71, 109.80, 123.41, 128.15, 129.21, 130.94,
132.04, 136.08, 142.25, 150.23, 157.67, 160.57, 164.42, HRMS (ESI, m/
(C¼O), 1136 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 1.46e1.50 (t,
z) [M ꢀ H]- calculated for C22H13F3N5O3, 452.0970; found 452.0953.
J ¼ 7.60 Hz, 3H, CH2CH3), 2.91e2.97 (q, J ¼ 7.73 Hz, 2H, CH2CH3),
6.51e6.53 (m, 1H, Ar-H), 7.46e7.51 (m, 4H, Ar-H), 7.61 (s, 1H, Ar-H),
8.19e8.23 (m, 3H, Ar-H), 15.19 (s, 1H, -NH) ppm, 13C NMR (100 MHz,
3.1.4.9. N-(2-methyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)isonicotinamide (51i). Pale green solid, yield: 220 mg
(54%), mp: 222e224 ꢁC, FT-IR (ATR, Vmax): 3029 (NH), 1574 (C¼O),
CDCl3):
d 10.87, 29.38, 110.18, 112.39, 118.24, 126.66, 128.09, 129.12,
1158 (C-F) cmꢀ1
,
1H NMR (400 MHz, CDCl3):
d
2.77 (s, 3H, CH3),
131.71, 136.37, 137.78, 138.12, 138.46, 138.80, 147.23, 151.55, 156.56,
7.53e7.58 (m, 3H, Ar-H), 8.18e8.20 (m, 2H, Ar-H), 8.21e8.29 (m, 2H,
160.47, 160.55, 163.87, 170.87 ppm, 1H-15N HMBC (400 MHz, CDCl3):

B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
253
d
155.7, 247.3, 304.7 ppm, 19F NMR (376 MHz, CDCl3):
d
ꢀ59.19 ppm,
138.20, 138.95, 158.05, 160.34, 160.75, 164.68, 177.60 ppm.
HRMS (ESI, m/z) [MþNa]þ, calculated for C21H15F3N4O2Na,
435.1045; found 435.1046.
3.1.4.20. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-4-nitrobenz-amide (52h). Yellow solid, yield: 300 mg
(69%), mp: 229e231 ꢁC, FT-IR (ATR, Vmax): 3106 (NH), 1568 (C¼O),
3.1.4.15. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)thiophene-2-carboxamide (52c). Brown solid, yield:
300 mg (75%), mp: 207e208 ꢁC, FT-IR (ATR, Vmax): 3057 (NH), 1587
1136 (C-F) cmꢀ1 1H NMR (400 MHz, CDCl3):
, d 1.54e1.58 (t,
J ¼ 7.64 Hz, 3H, CH2CH3), 3.02e3.08 (q, J ¼ 7.62 Hz, 2H, CH2CH3),
7.56e7.58 (m, 3H, Ar-H), 8.29e8.33 (m, 4H, Ar-H), 8.37 (s, 1H, Ar-H),
8.53e8.56 (m, 2H, Ar-H), 15.82 (s, 1H, -NH) ppm, 13C NMR
(C¼O), 1134 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 1.50e1.54 (t,
J ¼ 7.66 Hz, 3H, CH2CH3), 2.95e3.00 (q, J ¼ 7.63 Hz, 2H, CH2CH3),
7.15e7.17 (m, 1H, Ar-H), 7.18e7.54 (m, 3H, Ar-H), 7.63e7.65 (m, 1H,
Ar-H), 8.08 (s, 1H, Ar-H), 8.27e8.31 (m, 3H, Ar-H), 15.52 (s, 1H, -NH)
(100 MHz, CDCl3):
d 11.06, 29.47, 118.82, 123.43, 128.22, 129.22,
130.96, 132.01, 136.20, 142.35, 150.25, 157.94, 160.41, 164.52,
178.66 ppm, HRMS (ESI, m/z) [M ꢀ H]- calculated for C23H15F3N5O3,
466.1127; found 466.1125.
ppm, 13C NMR (100 MHz, CDCl3):
d 11.07, 29.44, 110.16, 118.36,
118.43, 128.13, 128.22, 129.12, 131.72, 133.73, 133.80, 136.42, 138.25,
143.12, 156.66, 160.47, 160.61, 163.99, 175.50 ppm.
3.2. Pharmacology
3.1.4.16. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-4-heptylbenz-amide (52d). Pale yellow solid, yield:
297 mg (60%), mp: 120e122 ꢁC; FT-IR (ATR, Vmax): 2923 (NH), 1583
3.2.1. In vitro antitubercular evaluation
The in vitro antitubercular activity of synthesized compounds
(7e43, 51a-l and 52a-h) was conducted at Infectious Disease
Research Institute (IDRI) within the National Institute of Allergy
and Infectious Diseases (NIAID) screening program, Bethesda, MD,
USA. The activity was assessed against M. tuberculosis H37 Rv grown
under aerobic conditions by using a dual read-out (OD590 and
fluorescence) assay procedure [39e41]. Test compounds were
prepared as 10-point two-fold serial dilutions in DMSO and diluted
into 7H9-Tw-OADC medium in 96-well plates with a final DMSO
concentration of 2%. The highest concentration of compound was
(C¼O), 1139 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 0.86e0.92 (t,
J ¼ 6.9 Hz, 3H, CH2CH3 of heptyl), 1.25e1.28 (m, 7H, heptyl),
1.51e1.54 (m, 3H, heptyl), 1.62e1.67 (m, 3H, heptyl), 2.66e2.70 (t,
J ¼ 7.72 Hz, 2H, heptyl), 2.97e3.02 (q, J ¼ 7.60 Hz, 2H, CH2CH3),
7.30e7.32 (m, 2H, Ar-H), 7.55e7.57 (m, 3H, Ar-H), 8.28e8.33 (m, 5H,
Ar-H)) ppm, 13C NMR (100 MHz, CDCl3):
d 11.15, 14.88, 22.65, 29.15,
29.28, 29.50, 31.16, 31.78, 36.13, 128.13, 128.46, 129.12, 130.27,
131.67, 136.45, 148.63, 156.88, 160.61, 163.79 ppm.
200
compounds with limited solubility, the highest concentration was
50ꢂ less than the stock concentration e.g. 100 M for 5 mM DMSO
stock, 20 M for 1 mM DMSO stock. For potent compounds, assays
were repeated at lower starting concentrations. Each plate included
assay controls for background (medium/DMSO only, no bacterial
mM and compounds were soluble in DMSO at 10 mM. For
3.1.4.17. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-[1,10-biphenyl]-4-carboxamide (52e). Pale Yellow solid,
yield: 273 mg (58%), mp: 223e224 ꢁC, FT-IR (ATR, Vmax): 3030 (NH),
m
1578 (C¼O), 1126 (C-F) cmꢀ1
,
1H NMR (400 MHz, CDCl3):
m
d
1.54e1.57 (t, J ¼ 7.46 Hz, 3H, CH2CH3), 3.00e3.05 (q, J ¼ 7.55 Hz,
2H, CH2CH3), 7.37e7.38 (m, 1H, Ar-H), 7.39e7.41 (m, 2H, Ar-H),
7.42e7.49 (m, 3H, Ar-H), 7.56e7.57 (m, 2H, Ar-H), 7.67e7.75 (m,
2H, Ar-H), 8.29e8.31 (m, 3H, Ar-H), 8.34e8.49 (m, 2H, Ar-H) ppm,
cells), zero growth (100 mM Rifampicin) and maximum growth
(DMSO only), as well as a rifampicin dose response curve. Plates
were inoculated with M. tuberculosis and incubated for 5 days,
growth was measured by OD590 and fluorescence (Ex 560/Em 590)
using a BioTek™ Synergy 4 plate reader. Growth was calculated
separately for OD590 and RFU. The minimum inhibitory concen-
tration (MIC) was determined on the basis of 10-point dose
response curve which was plotted as % growth. The MIC values
were conveniently calculated from the inflection point of the fitted
curve to the lower asymptote (zero growth).
13C NMR (100 MHz, CDCl3):
d 10.12, 28.45, 109.34, 117.42, 126.00,
126.28, 127.01, 127.14, 127.87, 128.12, 129.69, 130.73, 134.73, 139.25,
144.43, 155.99, 159.51, 159.77, 162.90 ppm, HRMS (ESI, m/z)
[MþNa]þ, calculated for C29H21F3N4ONa, 521.1565; found 521.1569.
3.1.4.18. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)benzofuran-2-carboxamide (52f). Yellow solid, yield:
261 mg (60%), mp: 243e244 ꢁC, FT-IR (ATR, Vmax): 3053 (NH), 1578
(C¼O), 1140 (C-F) cmꢀ1, 1H NMR (400 MHz, CDCl3):
d 1.53e1.57 (t,
3.2.2. In vitro antibacterial evaluation
J ¼ 7.66 Hz, 3H, CH2CH3), 2.99e3.05 (q, J ¼ 7.62 Hz, 2H, CH2CH3),
7.28e7.32 (m, 1H, Ar-H), 7.42e7.44 (m, 1H, Ar-H), 7.55e7.57 (m, 3H,
Ar-H), 7.60e7.62 (m, 1H, Ar-H), 7.74e7.76 (m, 1H, Ar-H), 7.88 (s, 1H,
Ar-H), 8.28e8.32 (m, 2H, Ar-H), 8.34 (s, 1H, Ar-H), 15.35 (s, 1H, -NH)
The synthesized title compounds (7e43, 51a-l and 52a-h) were
screened for their potential antibacterial property against a panel of
pathogenic bacterial strains by following earlier reported MIC assay
method using resazurin dye [42].
ppm, 13C NMR (100 MHz, CDCl3):
d 10.80, 29.38, 110.07, 112.35,
115.13, 118.53, 123.31, 123.65, 127.69, 127.73, 128.13, 129.16, 131.82,
136.35, 152.19, 156.36, 156.75, 160.46, 160.65, 164.18, 172.08 ppm,
HRMS (ESI, m/z) [MþNa]þ, calculated for C25H17F3N4O2Na,
485.1201; found 485.1205.
3.2.2.1. Microorganisms used. Standard cultures of 2 Gram-positive
[S. aureus (ATCC 25 923), B. subtilis (ATCC 6051)] and 2 Gram-
negative [E. coli (ATCC 35 218), P. aeruginosa (ATCC 27 853)] were
used for the antibacterial screening assay. Culturing and sub-
culturing (one day prior to testing) of these microorganisms was
carried out at the Department of Microbiology, Inkosi Albert Luthuli
Hospital, Durban, South Africa.
3.1.4.19. N-(2-ethyl-7-phenyl-5-(trifluoromethyl)pyrido[2,3-d]pyr-
imidin-4-yl)-3,5-bis-(trifluoromethyl)-benzamide (52g). Pale Yellow
solid, yield: 309 mg (59%), mp: 212e213 ꢁC, FT-IR (ATR, Vmax): 3061
(NH), 1575 (C¼O), 1123 (C-F) cmꢀ1
,
1H NMR (400 MHz, CDCl3):
3.2.2.2. Preparation of medium. The nutrient medium was pre-
pared by dissolving 22 g of Muller-Hinton Broth (MHB) containing
acid hydrolysate of casein, beef extract and starch in 1 L of double
distilled water. The pH of this medium was adjusted to 7.4 0.1 and
sterilized by autoclave for 15 min at 121 ꢁC. The solution was
allowed to cool and stored at a temperature of 4 ꢁC. Sterility check
d
1.55e1.59 (t, J ¼ 7.66 Hz, 3H, CH2CH3), 3.03e3.09 (q, J ¼ 7.63 Hz,
2H, CH2CH3), 7.55e7.59 (m, 3H, Ar-H), 8.03 (s, 1H, Ar-H), 8.30e8.32
(m, 2H, Ar-H), 8.32e8.38 (m, 1H, Ar-H), 8.90 (s, 2H, Ar-H), 15.73 (s,
1H, -NH) ppm, 13C NMR (100 MHz, CDCl3):
d
11.01, 29.45, 109.90,
118.92, 121.88, 124.59, 125.72, 128.25, 129.23, 130.16, 131.72, 136.17,

254
B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
was performed by incubating un-inoculated media in an aerobic
an Innovation Post Doctoral Research Fellowship (ID: 99546). RK is
also thankful to NRF-SA for funding this project (Grant No. 103728
and 112079). We are highly grateful to the National Institutes of
Health, Infectious Disease Research Institute (IDRI), National Insti-
tute of Allergy and Infectious Diseases (NIAID) screening program,
Bethesda, MD, USA (antitubercular activity data) and National
Health laboratory services (NHLS), Inkosi Albert Luthuli Central
Hospital (Durban, South Africa) for their expertise in antibacterial
and fungal screening studies. Authors express heartfelt thanks to
Mr. Dilip Jagjivan and Dr. Caryl Janse Van Rensburg (UKZN, South
Africa) for their assistance in the NMR and HRMS experiments.
incubator at 37 ꢁC for 18e24 h.
3.2.2.3. Preparation of test compounds (stock solution and working
standard). An accurately weighed quantity (4.000 mg) of the syn-
thesized compounds and standard amoxycillin were dissolved
individually in 1 mL of DMSO to give stock solution (4000
Further, 100 L of stock solution was diluted with 900 L of double
distilled water to afford working standard solution (400 g/mL).
mg/mL).
m
m
m
3.2.2.4. Preparation of inoculums. One day prior to screening, one
or more identical colonies of microorganisms were suspended in
4.5 mL sterile double distilled water. Inoculates were adjusted to
0.5 McFarland standard (1.5 ꢂ 108 cfu/mL) [43]. A density check
turbidimeter was used to ensure that the inoculum was a 0.5
McFarland standard.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.molstruc.2019.01.105.
3.2.2.5. Broth micro-dilution method. The in vitro antibacterial
evaluation of the newly synthesized title compounds (7e43, 51a-l
and 52a-h) was performed using the broth micro-dilution method.
100 mL of sterile double distilled water was added to all outer-
perimeter wells of a 96-well microliter plates to minimize evapo-
ration of the medium in the test wells during the incubation. To the
References
[1] H.N. Hafez, H.A.R. Hussein, A.B.A. El-Gazzar, Synthesis of substituted thieno
[2,3-d]pyrimidine-2,4-dithiones and their S-glycoside analogues as potential
antiviral and antibacterial agents, Eur. J. Med. Chem. 45 (2010) 4026e4034.
[2] D.M. Morens, G.K. Folkers, A.S. Fauci, The challenge of emerging and re-
emerging infectious diseases, Nature 430 (2004) 242e249.
[3] R. Jorda, K. Paruch, V. Krystof, Cyclin dependent kinase inhibitors inspired by
roscovitine: purine bioisosteres, Curr. Pharm. Des. 18 (2012) 2974e2980.
[4] M. Chauhan, R. Kumar, Medicinal attributes of pyrazolo[3,4-d]pyrimidines: a
review, Bioorg. Med. Chem. 21 (2013) 5657e5668.
[5] M. Shimizu, Y. Takase, S. Nakamura, H. Katae, A. Minami, Pipemidic acid, a
new antibacterial agent active against Pseudomonas aeruginosa: in vitro
properties, Antimicrob. Agents Chemother. 8 (1975) 132e138.
[6] M. Bakavoli, G. Bagherzadeh, M. Vaseghifar, A. Shiri, M. Pordel, M. Mashreghi,
P. Pordeli, M. Araghi, Molecular iodine promoted synthesis of new pyrazolo
[3,4-d]pyrimidine derivatives as potential antibacterial agents, Eur. J. Med.
Chem. 45 (2010) 647e650.
remaining test wells, 100 mL of MHB was added. Two-fold serial
dilutions of the test compounds and standard (amoxycillin) were
made directly on the microplate using MHB. The compounds were
tested at final concentration of 200, 100, 50, 25, 12.5, 6.25, 3.125,
1.56, 0.78, and 0.39 mg/mL. Finally, 10 mL of the freshly prepared
bacterial inoculum was added to the wells. The microplates were
covered and sealed with parafilm and incubated at 37 1 ꢁC for
24 h. After this, 10 mL of freshly prepared resazurin (0.4 mg/mL) was
added to the test wells and incubated further for 5 h. The MIC was
determined as a blue colour in the test well was interpreted as no
bacterial growth and a pink colour was scored as growth. The MIC
was thus defined at the lowest drug concentration that prevented a
colour change from blue to pink. This experiment was conducted in
[7] P. Acosta, B. Insuasty, A. Ortiz, R. Abonia, M. Sortino, S.A. Zacchino, J. Quiroga,
Solvent-free microwave-assisted synthesis of novel pyrazolo[4',3':5,6]pyrido
[2,3-d]pyrimidines with potential antifungal activity, Arab. J. Chem. 9 (2016)
481e492.
[8] B.S. Holla, M. Mahalinga, M.S. Karthikeyan, P.M. Akberali, N.S. Shetty, Syn-
thesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential anti-
microbial agents, Bioorg. Med. Chem. 14 (2006) 2040e2047.
[9] M. Barvian, D.H. Boschelli, J. Cossrow, E. Dobrusin, A. Fattaey, A. Fritsch, D. Fry,
P. Harvey, P. Keller, M. Garrett, F. La, W. Leopold, D. McNamara, M. Quin,
S. Trumpp-Kallmeyer, P. Toogood, Z. Wu, E. Zhang, Pyrido[2,3-d]pyrimidin-7-
one inhibitors of cyclin-dependent kinases, J. Med. Chem. 43 (2000)
4606e4616.
[10] A. Angelucci, S. Schenone, G.L. Gravina, P. Muzi, C. Festuccia, C. Vicentini,
M. Botta, M. Bologna, Pyrazolo[3,4-d]pyrimidines c-Src inhibitors reduce
epidermal growth factor-induced migration in prostate cancer cells, Eur. J.
Cancer 42 (2006) 2838e2845.
[11] H.N. Hafez, H.A.S. Abbas, A.R.B. El-Gazzar, Synthesis and evaluation of anal-
gesic, anti-inflammatory and ulcerogenic activities of some triazolo- and 2-
pyrazolyl-pyrido[2,3-d]pyrimidines, Acta Pharm. 58 (2008) 359e378.
[12] G.N. Tageldin, S.M. Fahmy, H.M. Ashour, M.A. Khalil, R.A. Nassra, I.M. Labouta,
Design, synthesis and evaluation of some pyrazolo[3,4-d]pyrimidine de-
rivatives bearing thiazolidinone moiety as anti-inflammatory agents, Bioorg.
Chem. 80 (2018) 164e173.
triplicate and the average MIC values in mg/mL were determined.
4. Conclusions
In this study, we have generated a small library of pyrazolo[3,4-
d]pyrimidine analogues (7-43) and novel fluorinated pyrido[2,3-d]
pyrimidines (51a-l and 52a-h), quantitatively in a facile manner
employing an attractive starting material 2-amino-6-phenyl-4-
(trifluoromethyl)-nicotinonitrile (44). In vitro antitubercular eval-
uation against Mycobacterium tuberculosis H37Rv showed a mod-
erate inhibition (MIC ¼ 59
antifungal screening data (in vitro) revealed a good inhibition
g/ml) for 51d, 51j, 51k, 51l and 52g exclusively against
mM) for 51l, whereas antibacterial and
(MIC ¼ 12.5
m
[13] A.W.H. Cheung, B. Banner, J. Bose, K. Kim, S. Li, N. Marcopulos, L. Orzechowski,
J.A. Sergi, K.C. Thakkar, B.B. Wang, W. Yun, C. Zwingelstein, S. Berthel,
A.R. Olivier, 7-Phenyl-pyrido[2,3-d]-pyrimidine-2,4-diamines: novel and
highly selective protein tyrosine phosphatase 1B inhibitors, Bioorg. Med.
Chem. Lett 22 (2012) 7518e7522.
Gram-positive bacterial strains and against all fungal strains. Thus,
these research findings can assist future medicinal chemists in
developing novel pyrimidine-based antimicrobial lead compounds.
€
[14] C.M. Lacbay, J. Mancuso, Y.S. Lin, N. Bennett, M. Gotte, Y.S. Tsantrizos, Modular
Conflicts of interest
assembly of purine-like bisphosphonates as inhibitors of HIV-1 reverse
transcriptase, J. Med. Chem. 57 (2014) 7435e7449.
[15] J.H. Chern, K.S. Shia, T.A. Hsu, C.L. Tai, C.C. Lee, Y.C. Lee, C.S. Chang, S.N. Tseng,
S.R. Shih, Design, synthesis, and structure-activity relationships of pyrazolo
[3,4-d]pyrimidines: a novel class of potent enterovirus inhibitors, Bioorg. Med.
Chem. Lett. 14 (2004) 2519e2525.
Authors hereby declare that there are no financial/commercial
conflicts of interest.
[16] H.B. Cottam, C.R. Petrie, P.A. McKernan, R.J. Goebel, N.K. Dalley, R.B. Davidson,
R.K. Robins, G.R. Revankar, Synthesis and biological activity of certain 3,4-
disubstituted pyrazolo[3,4-d]pyrimidine nucleosides, J. Med. Chem. 27
(1984) 1119e1127.
[17] C.R. 3rd Petrie, H.B. Cottam, P.A. McKernan, R.K. Robins, G.R. Revankar, Syn-
thesis and biological activity of 6-azacadeguomycin and certain 3,4,6-
trisubstituted pyrazolo[3,4-d]pyrimidine ribonucleosides, J. Med. Chem. 28
(1985) 1010e1016.
Acknowledgements
Authors are thankful to Discipline of Pharmaceutical Sciences,
College of Health Sciences, University of KwaZulu-Natal (UKZN),
Durban, South Africa for providing the necessary facilities. One of
the authors (CB) gratefully acknowledges National Research Foun-
dation (NRF), South Africa (SA) for research funding in the form of
[18] I. Bergner, C. Wiebe, N. Meyer, T. Opatz, Cyclocondensation of a-aminonitriles

B. Chandrasekaran et al. / Journal of Molecular Structure 1183 (2019) 246e255
and enones: a short access to 3,4-dihydro-2H-pyrrole 2-carbonitriles and Commun. 8 (2017) 1697e1705.
255
2,3,5 tri-substituted pyrroles, J. Org. Chem. 74 (2009) 8243e8253.
[19] K.L. Seley, P. Januszczyk, A. Hagos, L. Zhang, D.T. Dransfield, Synthesis and
antitumor activity of thieno-separated tricyclic purines, J. Med. Chem. 43
(2000) 4877e4883.
[20] J.A. Zoltewicz, T.W. Sharpless, One-step synthesis of fused pyrimidinethiones
from o-aminonitriles and thioamides, J. Org. Chem. 32 (1967) 2681e2685.
[21] A.M.K. El-dean, Synthesis of some pyrimidothienopyrimidine derivatives,
Monatshefte Fur Chemie 129 (1998) 523e533.
[32] C. Viegas-Junior, A. Danuello, V.D.S. Bolzani, E.J. Barreiro, C.A.M. Fraga, Mo-
lecular hybridization: a useful tool in the design of new drug prototypes, Curr.
Med. Chem. 14 (2007) 1829e1852.
[33] G.A. Hampannavar, R. Karpoormath, M.B. Palkar, M.S. Shaikh,
B. Chandrasekaran, Dehydrozingerone inspired styryl hydrazine thiazole hy-
brids as promising class of antimycobacterial agents, ACS Med. Chem. Lett. 7
(2016) 686e691.
[34] S. Cherukupalli, B. Chandrasekaran, R.R. Aleti, N. Sayyad, G.A. Hampannavar,
S.R. Merugu, H.R. Rachamalla, R. Banerjee, R. Karpoormath, Synthesis of 4,6-
disubstituted pyrazolo[3,4-d]pyrimidine analogues: cyclin-dependent kinase
2 (CDK2) inhibition, molecular docking and anticancer evaluation, J. Mol.
Struct. 1176 (2019) 538e551.
[35] S. Cherukupalli, B. Chandrasekaran, V. Krystof, R.R. Aleti, N. Sayyad,
S.R. Merugu, N.D. Kushwaha, R. Karpoormath, Synthesis, anticancer evalua-
tion, and molecular docking studies of some novel 4,6-disubstituted pyrazolo
[3,4-d]pyrimidines as cyclin-dependent kinase 2 (CDK2) inhibitors, Bioorg.
Chem. 79 (2018) 46e59.
[36] B. Narsaiah, A. Sivaprasad, R. V Venkataratnam, An improved synthetic route
to trifluoromethyl-6-substituted-2(1H)-pyridones, Org. Prep. Proced. Int. 25
(1993) 116e117.
[37] B. Narsaiah, A. Sivaprasad, R. V Venkataratnam, A novel synthetic route to 2-
amino-3-cyano-4-trifluoromethyl-6-substituted pyridines, J. Fluorine Chem.
67 (1994) 87e90.
[38] S.R. Kanth, G.V. Reddy, K.H. Kishore, P.S. Rao, B. Narsaiah, U.S.N. Murthy,
Convenient synthesis of novel 4-substitutedamino-5-trifluoromethyl-2,7-
disubstituted pyrido[2,3-d]pyrimidines and their antibacterial activity, Eur. J.
Med. Chem. 41 (2006) 1011e1016.
[22] Y.L. Zhong, J. Lee, R.A. Reamer, D. Askin, New method for the synthesis of
diversely functionalized imidazoles from N-acylated
Lett. 6 (2004) 929e931.
a-aminonitriles, Org.
[23] A.M. Rad, M. Mokhtary, Efficient one-pot synthesis of pyrido[2,3-d]pyrimi-
dines catalyzed by nanocrystalline MgO in water, Int. Nano Lett. 5 (2015)
109e123.
[24] S. Singh, M. Saquib, M. Singh, J. Tiwari, F. Tufail, J. Singh, J. Singh, A catalyst
free, multicomponent-tandem, facile synthesis of pyrido[2,3-d]pyrimidines
using glycerol as a recyclable promoting media, New J. Chem. 40 (2016)
63e67.
[25] Y. Liu, S. Jin, Z. Wang, L. Song, Y. Hu, Microwave assisted tandem Heck-
Sonogashira reactions of N,N-di-Boc-protected 6-amino-5-iodo-2-methyl
pyrimidin-4-ol in an efficient approach to functionalized pyrido[2,3-d]py-
rimidines, Org. Lett. 16 (2014) 3524e3527.
[26] S. Samai, G.C. Nandi, S. Chowdhury, M.S. Singh, L-Proline catalyzed synthesis
of densely functionalized pyrido[2,3-d]pyrimidines via three-component one-
pot domino Knoevenagel aza-Diels-Alder reaction, Tetrahedron 67 (2011)
5935e5941.
[27] M.E. Chizhova, O.Y. Bakulina, A.Y. Ivanov, P.S. Lobanov, D.V. Darin, Facile
synthesis of pyrido[2,3-d]pyrimidines via cyclocondensation of 4,6-dichloro-
[39] J. Ollinger, M.A. Bailey, G.C. Moraski, A. Casey, S. Florio, T. Alling, M.J. Miller,
T. Parish, A dual read-out assay to evaluate the potency of compounds active
against, Mycobacterium tuberculosis, PLoS One. 8 (2013), e60531.
[40] A. Zelmer, P. Carroll, N. Andreu, K. Hagens, J. Mahlo, N. Redinger,
B.D. Robertson, S. Wiles, T.H. Ward, T. Parish, J. Ripoll, G.J. Bancroft,
U.E. Schaible, A new in vivo model to test anti-tuberculosis drugs using
fluorescence imaging, J. Antimicrob. Chemother. 67 (2012) 1948e1960.
[41] R.J. Lambert, J. Pearson, Susceptibility testing: accurate and reproducible
minimum inhibitory concentration (MIC) and non-inhibitory concentration
(NIC) values, J. Appl. Microbiol. 88 (2000) 784e790.
2-methyl-sulfanylpyrimidine-5-carbaldehyde with
b-substituted b-amino-
acrylic esters, Tetrahedron 71 (2015) 6196e6203.
[28] F. Belhadj, Z. Kibou, N. Cheikh, N. Choukchou-Braham, D. Villemin, Convenient
access to new 4-substituted aminopyrido[2,3-d]pyrimidine derivatives, Tet-
rahedron Lett. 56 (2015) 5999e6002.
[29] M.A.H. Ayed, T. Gmiza, J.E. Khiari, H.B. Ben, Efficient one-pot synthesis of
substituted pyrido[2,3-d]pyrimidines from vinamidinium and chlor-
opropeniminium salts, Synth. Commun. 42 (2012) 1824e1831.
ꢀ
ꢀ
~
[30] J. Wang, M. Sanchez-Rosello, J.L. Acena, C. del Pozo, A.E. Sorochinsky,
S. Fustero, V.A. Soloshonok, H. Liu, Fluorine in pharmaceutical industry:
fluorine-containing drugs introduced to the market in the last decade
(2001e2011), Chem. Rev. 114 (2014) 2432e2506.
[42] C.M. Mann, J.L. Markham, A new method for determining the minimum
inhibitory concentration of essential oils, J. Appl. Microbiol. 84 (1998)
538e544.
[31] D. Heimann, C. Lueg, H. de Vries, B. Frehland, D. Schepmann, L.H. Heitman,
B. Wünsch, Bioisosteric replacement of central 1,2,4-oxadiazole ring of high
affinity CB2 ligands by regioisomeric 1,3,4-oxadiazole ring, Med. Chem.
[43] J. Mcfarland, The nephelometer: an instrument for estimating the number of
bacteria in suspensions used for calculating the opsonic index and for vac-
cines, J. Am. Med. Assoc. XLIX 14 (1907) 1176e1178.