Studies on synthesis of novel pyrido[2,3-d]pyrimidine derivatives, evaluation of their antimicrobial activity and molecular docking

Article abstract of DOI:10.1016/j.bmcl.2018.03.022

A series of novel pyrido[2,3-d]pyrimidine derivatives 6 were prepared starting from 2-amino-3-cyano-4-trifluoromethyl-6-phenyl pyridine 3 via Grignard's reaction, cyclization followed by coupling with aliphatic and cyclic amines. All the compounds 6 were

Full text of DOI:10.1016/j.bmcl.2018.03.022

Accepted Manuscript
Studies on synthesis of novel pyrido[2,3-d]pyrimidine derivatives, evaluation
of their antimicrobial activity and molecular docking
B. Veeraswamy, D. Madhu, G. Jitender Dev, Y. Poornachandra, G. Shravan
Kumar, C. Ganesh Kumar, B. Narsaiah
PII:
S0960-894X(18)30201-4
https://doi.org/10.1016/j.bmcl.2018.03.022
BMCL 25676
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 January 2018
1 March 2018
9 March 2018
Please cite this article as: Veeraswamy, B., Madhu, D., Jitender Dev, G., Poornachandra, Y., Shravan Kumar, G.,
Kumar, C.G., Narsaiah, B., Studies on synthesis of novel pyrido[2,3-d]pyrimidine derivatives, evaluation of their
antimicrobial activity and molecular docking, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://
doi.org/10.1016/j.bmcl.2018.03.022
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Studies on synthesis of novel pyrido[2,3-d]pyrimidine derivatives, evaluation of their
antimicrobial activity and molecular docking
B. Veeraswamy, D. Madhu, G. Jitender Dev, Y. Poornachandra, G. Shravan Kumar, C. Ganesh Kumar and B.
Narsaiah*
1

Bioorganic & Medicinal Chemistry Letters
Studies on synthesis of novel pyrido[2,3-d]pyrimidine derivatives, evaluation of
their antimicrobial activity and molecular docking
b
B. Veeraswamya, D. Madhua,d, G. Jitender Deva,d, Y. Poornachandrac,d, G. Shravan Kumar , C. Ganesh
Kumarc,d and B. Narsaiaha,d*
aFluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India.
bBioinformatics Wing, Mycology & Plant Pathology lab, Department of Botany, Osmania University, Hyderabad-500007, India.
cMedicinal Chemistry and Biotechnology Division, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India.
dAcademy of Scientific and Innovative Research, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad 500007, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel pyrido[2,3-d]pyrimidine derivatives 6 were prepared starting from 2-amino-3-cyano-
4-trifluoromethyl-6-phenyl pyridine 3 via Grignard’s reaction, cyclization followed by coupling with
aliphatic and cyclic amines. All the compounds 6 were screened for antibacterial, minimum
bactericidal concentration (MBC), biofilm inhibition activity as well as antifungal and minimum
fungicidal concentration (MFC) activities. Among the screened compounds, the compounds 6e, 6f,
and 6m which showed exhibiting promising activity have been identified. The results reveal that the
compound pyrido[2,3-d]pyrimidine derivative 6e altered the sterol profile which may exert its
antifungal activity through inhibition of ergosterol biosynthesis and could be an ideal candidate for
antifungal therapy. The molecular docking results also validated the antifungal results.
Key words:
Grignard’s reaction;
cyclization;
coupling reaction;
antimicrobial activity.
pyrimidine framework. In our earlier studies, we also identified
novel pyrazolo[3,4-b]pyridine and pyrimidine functionalized
1,2,3-triazole derivatives exhibiting promising antimicrobial and
anti-biofilm activities. 6
In most ecological niches, bacterial cells grow on inert or living
surfaces as single or multiple-species communities enclosed by a
self-produced polymeric matrix, which are so-called the biofilms.
The bacterial growth is very poor when floating in water or low
in nutrients and show better growth when adherent to surfaces,
where they can find a protective and nutrient-rich environment.1
Biofilms formed by potentially pathogenic bacteria which are
commonly encountered in chronic and nosocomial infections
provide the bacteria the ability to resist against stress, antibiotics,
In the present context, pyrido[2,3-d]pyrimidine derivatives were
identified to display promising biological activities,7,8 and was
found to be more specific to exhibit dihydrofolate reductase
9-11
inhibition, anti-tumor,
as well as diuretic properties.12 Some
of these compounds also possessed antimicrobial13-16 and
cytotoxic activities.17,18 The strategically positioned fluorine19 or
trifluoromethyl20,21 group in the molecule basically influenced the
change in the reactivity and the properties of molecule in terms of
lipid solubility, oxidative thermal stability and the oral
bioavailability. Considering these above facts, the trend is driving
more towards the synthesis of fluorinated molecules and the
objective of the present study is to identify the promising
molecules exhibiting anti-biofilm and anti-Candida activities.
Keeping in view, the importance of pyrido[2,3-d] pyrimidine
derivatives and in continuation of our efforts,22-24 we have
synthesized a series of novel pyrido[2,3-d] pyrimidine derivatives
2
biocides and host-immunological defenses and in the medical
sector where they colonize through bacterial adhesion and
biofilm formation on several biomedical implants such as stents,
heart valves, vascular grafts and catheters.3 In this context, the
discovery of novel compounds that can specifically target and
inhibit the biofilm formation would be of great interest in
comparison to the rational use of antibiotics and/or biocides.
Such biofilm inhibitors would prove to be of significance for use
in the prevention of biofilm formation in various industrial and
medical environments. Current biofilm preventive strategies are
essentially aimed at the discovery of potential natural anti-
biofilm compounds such as 2-aminoimidazole containing
alkaloids isolated from marine sponges4 and Kocuran-
functionalized silver glyconanoparticles for use as anti-biofilm
coatings on silicone urethral catheters.5 In this regard, we were
interested to explore the anti-biofilm and anti-Candida activities
against some of the novel molecules having the pyrido[2,3-d]
and screened for
antibacterial, minimum bactericidal
concentration (MBC), biofilm inhibition, and anti-Candida
activities. Minimum fungicidal concentration (MFC), and
inhibition of ergosterol biosynthesis aspects were also
investigated. Sterol 14 alpha-demethylase (CYP51) is one of the
enzymes involved in ergosterol biosynthesis pathway, catalyzing
C14-demethylation of lanosterol which is critical for ergosterol
biosynthesis. It transforms lanosterol into 4,4'-dimethyl cholesta-
2

C6H4
8,14,24-triene-3-beta-ol, which is the target of therapeutic
importance for anti-fungal drug development.25 The compounds
which showed promising activity have been identified in each
case.
11
12
13
4-MeO
C6H4
222
176
139
49.40
83.68
42.73
6k
6l
The 2(1H) pyridone 1 was reacted with 2-chloroacetamide in
acetone using K2CO3 as base under reflux condition for 6 h to
obtain exclusively 2-((3-cyano-6-phenyl-4-(trifluoromethyl)
pyridin-2-yl)oxy)acetamide 2, and was treated with potassium
carbonate in N,N-dimethylformamide at 110-120 °C to form 2-
4-MeO
C6H4
amino-3-cyano-4-trifluoromethyl-6-phenyl
pyridine
3.26
Compound 3 was further reacted with freshly prepared different
aryl magnesium bromides in diethyl ether at room temperature
4-MeO
C6H4
6m
and
obtained
the
3-(imino(aryl)methyl)-6-phenyl-4-
was
(trifluoromethyl)pyridin-2-amine 4. The compound
4
independently reacted with ethyl 2-chloro-2-oxoacetate in DCM
using triethylamine as base at room temperature for 1 h which
resulted in the formation of ethyl 4-aryl-7-phenyl-5-
14
15
16
4-MeO
C6H4
132
210
186
74.48
85.98
87.55
6n
6o
6p
(trifluoromethyl)pyrido[2,3-d]pyrimidine-2-carboxylate
5
and
was coupled with different primary aliphatic amines, cyclic
secondary amines to obtain products 6a-p. The reactions are
outlined in Scheme 1 and the products are tabulated in Table 1.
4-MeO
C6H4
C6H5CH2NH
4-MeO
C6H4
a- Isolated yield of the final reaction (i.e., 5 to 6).
Table 1. Physical properties of compounds 6a-p
Compounds 6a-p were screened for antibacterial activity27 in
vitro against different Gram-positive and Gram-negative bacterial
strains. Among all the compounds screened, compound 6e, 6f and
6m showed promising activity against all the bacterial species.
Compounds 6g and 6k exhibited promising activity specifically
towards Staphylococcus aureus MTCC 96, while compound 6l
and 6a showed promising to moderate activity towards Klebsiella
planticola MTCC 530. The structure-activity relationship studies
revealed that these compounds 6e, 6f and 6m have piperazine
moiety with alkyl groups in para-position adjacent to carbonyl
promoting activity due to enhancement of electron density on
carbonyl oxygen which binds to the organism. However, all
other compounds (6c, 6d, 6i, 6j, 6n, 6o and 6p) did not show
antibacterial activity up to the maximum tested concentration of
125 µg/mL. The antibacterial activity results to this regard are
tabulated in table 2.
Reagents and conditions : (a) 2-Chloroacetamide, NaI, acetone, K2CO3,
reflux, 6h; (b) K2CO3, DMF, 110-120OC, 2h; (c) RMgX, Et2O, rt, 1h; (d)
Ethyl-2-chloro-oxo-acetate, Et3N, DCM, rt, 1h; (e) amines, 50-60oC, 2-3h
Scheme 1. Preparation of pyrido[2,3-d]pyrimidine derivatives.
S.
Comp
NR1R2
R
M.P Yielda
No. ound
(oC)
(%)
1
2
NHC2H5
C6H5
C6H5
240
248
47.39
68.49
6a
6b
NHC2H4OH
3
4
NHC3H7
C6H5
C6H5
224
206
72.63
78.12
6c
6d
Table 2. Antibacterial activity of the pyrido[2,3-d]pyrimidine
derivatives
5
6
C6H5
C6H5
117
142
52.41
46.02
6e
6f
Com
pd
Minimum inhibitory concentration (µg/mL)
BS
MTCC
121
SA
SA
ML
KP
EC
PA
MTCC
2453
MTCC
96
MTCC
2940
MTCC
2470
MTCC
530
MTCC
739
a
6a
6b
-
-
-
-
31.2
-
-
-
7
C6H5
C6H5
202
77.35
6g
-
31.2
-
-
-
-
6c
6d
6e
6f
-
-
-
-
-
-
-
8
9
C6H5CH2NH
NHC2H4OH
236
219
79.2
6h
6i
-
-
-
-
-
-
-
4-MeO
C6H4
71.22
15.6
31.2
7.8
7.8
15.6
15.6
15.6
15.6
31.2
15.6
15.6
15.6
31.2
15.6
10
NHC3H7
4-MeO
184
57.22
6j
3

6g
6h
6i
-
7.8
-
-
15.6
-
-
related to a general toxic effect on the bacterial strains. Thus, the
compounds 6b, 6e, 6f, 6g, 6h, 6k, 6l and 6m were screened for
anti-biofilm activity30 against five bacterial species such as
Bacillus subtilis MTCC 121, Staphylococcus aureus MTCC 96,
Staphylococcus aureus MLC 16 MTCC 2940, Pseudomonas
aeruginosa MTCC 2453 and Klebsiella planticola MTCC 530
which are important nosocomial pathogens and have the ability
to form biofilms. The results summarized in Table 4, clearly
confirmed that the promising activity is related to the presence of
piperazine moiety in compounds 6e, 6f and 6m with IC50 values
between 2.7 – 21.6 μg/mL towards all the tested bacterial species.
Among them, compounds (6e, 6f, 6g, 6k and 6m) showed
promising activity (IC50 values ranging between 2.7–5.4 μg/mL)
against Staphylococcus aureus MTCC 96, while compounds (6f,
6g, 6l and 6m) showed promising activity (IC50 values ranging
between 5.4–10.8 μg/mL) specifically against Klebsiella
planticola MTCC 530.
-
31.2
-
-
-
-
-
-
-
-
-
-
-
-
-
6j
-
-
-
-
-
-
-
-
6k
6l
7.8
-
-
-
-
15.6
15.6
-
-
-
-
-
-
-
6m
6n
6o
6p
C
7.8
-
7.8
-
15.6
15.6
15.6
7.8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
0.9
0.9
0.9
0.9
0.9
0.9
0.9
a_ No activity
BS, Bacillus subtilis MTCC 121; SA, Staphylococcus aureus MTCC 96; Staphylococcus aureus
MLS16 MTCC 2940; ML, Micrococcus luteus MTCC 2470; KP, Klebsiella planticola MTCC 530;
In medical environments, the use of contaminated medical
devices and prolonged intensive care units showed increased
prevalence of fungal diseases. Under such episodes, the most
commonly associated fungal strains belong to the genus Candida,
most notably Candida albicans, which causes both superficial
and systematic candidiasis.31 Based on these facts and the
observed antibacterial results, Candida species are important
opportunistic pathogens frequently encountered in infections of
immunocompromised patient groups comprising those on cancer
chemotherapy, broad-spectrum antibiotics and HIV-infected
individuals. Among the many pathogenic Candida species,
Candida albicans is the major fungal pathogen of importance to
humans. Due to its versatility, it can behave as a commensal
organism posing a major problem from a clinical perspective
resulting in infections.32 Different Candida strains have the
ability to produce extracellular polymeric substances (EPS) and
get encased on this matrix to form biofilms which are known to
develop on the surfaces of prosthesis and medical devices, and
exhibit resistance to both antifungal and host immune defenses as
compared to their free-living planktonic counterparts. This is
most likely to be the cause of recalcitrant persistence of Candida
albicans on inert, inserted surfaces or, superficial mucosae.33,34
Considering these above facts, we screened some of the selected
compounds 6b, 6c, 6d, 6e, 6f, 6h and 6m for anti-Candida
activity against different Candida strains. More specifically,
compounds 6d, 6e, 6f and 6m showed promising activity (MIC
values ranging between 3.9–31.2 μg/mL) against C. albicans
MTCC 3017 and was comparable to the standard miconazole
drug. The results on the anti-Candida activity are tabulated in
Table 5.
EC, Escherichia coli MTCC 739; PA, Pseudomonas aeruginosa MTCC 2453;
C Ciprofloxacin
(Standard).
Compounds 6b, 6e, 6f, 6g, 6h, 6k, 6l and 6m were further
evaluated for the minimum bactericidal concentration28 against all
the seven bacterial species in comparison to ciprofloxacin as
standard. Compounds 6e, 6f and 6m consistently showed
promising minimum bactericidal concentration activity. The
activity data is tabulated in Table 3.
Table 3. Minimum bactericidal concentration of compound 5a-r,
6a-h and 7a-f
Test
Minimum bactericidal concentration (µg/ml)
BS
SA
SA
ML
KP
EC
PA
Compd
MTCC
121
MTCC
96
MLS16
MTCC
2940
MTCC
2470
MTCC
530
MTCC
739
MTCC
2453
a
6b
6e
6f
-
62.4
15.6
15.6
15.6
31.2
7.8
-
-
-
-
-
31.2
31.2
15.6
62.4
31.2
31.2
-
31.2
62.4
31.2
31.2
31.2
31.2
31.2
6g
6h
6k
6l
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15.6
31.2
0.58
-
-
6m
C
15.6
0.58
15.6
0.58
15.6
1.17
31.2
1.17
31.2
0.58
15.6
1.17
Compounds 6b, 6c, 6d, 6e, 6f, 6h and 6m were also evaluated for
minimum fungicidal concentrations against different Candida
strains in comparison to the standard miconazole drug. All the
compounds showed minimum fungicidal concentration (MFC)
values ranging between 3.9 - 62.5 μg/mL). However, the standard
miconazole drug exhibited minimum fungicidal concentration
values ranging between 7.8 - 15.6 μg/mL. Among them,
compound 6e proved promising and exhibited a lower MFC
value of 3.9 μg/mL. The activity data is tabulated in Table 6.
aNot active; BS, Bacillus subtilis MTCC 121; SA, Staphylococcus aureus MTCC 96; Staphylococcus
aureus MLS16 MTCC 2940; ML, Micrococcus luteus MTCC 2470; KP, Klebsiella planticola MTCC
530; EC, Escherichia coli MTCC 739; PA, Pseudomonas aeruginosa MTCC 2453; C Ciprofloxacin
(Standard).
Biofilms are structured consortia of bacteria embedded in a self-
produced polymeric matrix causing chronic infections in humans
via hospital and community environments, especially due to the
high antibiotic resistance associated with them as well as the
ability to resist phagocytosis and other components of the body's
defense system. In several biomedical implants such as stents,
heart valves, vascular grafts and catheters, the bacteria colonize
through adhesion mechanism and forms biofilms due to the
contamination of these biomedical implants during post-surgical
infections.29 Considering these facts, the further step undertaken
was to investigate whether these compounds exhibited a specific
anti-biofilm activity or whether this observation was simply
Candida albicans is now recognized as a major cause of hospital-
acquired infections.35 Most of the antifungal drugs currently
available to treat Candida infections target the ergosterol
biosynthetic pathway or its end product ergosterol. Considering
this fact, we further investigated the promising test compound 6e
in comparison to the standard miconazole drug to delineate its
mode of action in the ergosterol biosynthetic pathway for one of
the highly susceptible strain of C. albicans MTCC 183. In this
4

regard, the UV spectral scans of the sterol profiles for one of the
representative strain of C. albicans MTCC 183 36 was determined
(Figure 1), and later the total ergosterol content was quantified
from the data obtained on culturing the C. albicans MTCC 183
strain with different concentrations (0, 2, 4, and 16 µg/mL) of the
test compound 6e and the standard miconazole drug (see Table
7). It was noticed that the ergosterol content decreased
significantly with an increase in the concentration of test
compound 6e. Similarly, a dose-dependent decrease in ergosterol
content was observed when the C. albicans MTCC 183 strain
was cultured in presence of miconazole. Our findings suggest
that the pyrido[2,3-d]pyrimidine derivative 6e altered the sterol
profile which may exert its antifungal activity through inhibition
of ergosterol biosynthesis. The selective cytotoxic behaviour of
this compound hints at its affinity to the specific target site in the
ergosterol biosynthetic pathway. The Candida-cidal activity of
the compound 6e might also be due to the direct damage to the
cell membrane. However, the exact mechanism of action of this
compound needs to be further elucidated.
(X=25.874, Y=18.831 and Z= 15.131). Lamarckian genetic
algorithm (LGA) was selected for freezing, docking and default
parameters used in Autodock 4.2.
The amino acid sequence of lanosterol 14-alpha demethylase was
retrieved from Uniprot.39 A three dimensional model was
generated for lanosterol 14-alpha demethylase. A sequence
similarity search was performed to identify the structural
40
similarity of the query sequence by using Protein BLAST tool
by selecting database against Protein Data Bank (PDB) for
identifying template for homology model building. 4K0F protein
was selected as a template for modeled protein. The template
protein was downloaded from RCSB.41 Comparative sequence
alignment studies were performed with query and template
structure using Clustal X tool and online Clustal W tools.42
MODELLER 9.17 software was used to develop the model. It is
an automated approach for comparative modeling by satisfaction
of spatial restrains.43 After generating the files, the best model
was selected on the basis of Lowest Objective Function. It is
also known as normalized Discrete Optimized Molecule Energy
(DOPE) score. The generated model was validated by
Ramachandran plot and Psi/Phi angles using PROCHECK.44
Active Site Prediction: Active site was predicted for the
developed model by Sybyl Siteid module. The models showed 18
active site pockets. The pocket which is having higher value was
selected as active site. The molecule 6e was sketched in SYBYL
I
I
45
version 6.7 and energetically minimized by adding Gasteiger-
Huckel charges. The molecules were then saved in .mol2 format
for molecular docking purpose.
UV spectrophotometric scans of the sterol profiles of C. albicans MTCC 183.
The culture was grown for 20 h in Sabouraud dextrose broth containing
different concentrations of the (I) test compound, 6e and (II) Miconazole per
mL, such as 0 (curve A), 2 (curve B), 4 (curve C) and 16 (curve D) µg. The
sterols were extracted from the cells, and the spectral profiles were recorded
at the wavelength between 240 and 300 nm
Docking of the 6e synthesized compound is summarized in Table
8. The derivative 6e was selected for molecular docking has some
collective structural features. The comparison of free energies
corresponding to binding of title compounds with target protein
revealed that maximum of the compounds interacted with the
receptor. Compound 6e exhibited binding energy of -10.70
kcal/mol. It showed interaction with Asp233.
Figure 1
Molecular docking study was performed to validate the
antifungal activity results and to explain the binding mode of
proteins and ligand complexes. The compound was docked by
using Autodock 4.2 software.37-38 The modeled three dimensional
structure of lanosterol 14-alpha demethylase protein was
imported to Autodock 4.2 and structurally optimized by adding
hydrogens to protein allocated with Kollman charges. After
adding the hydrogens, the model was saved in PDBQT format,
later ligands were prepared by optimizing the torsion angles and
saved them in PDBQT format. Potential binding site for the
lanosterol 14-alpha demethylase modeled protein was identified.
A grid was generated around active site pocket amino acids
In conclusion,
a series of novel pyrido[2,3-d]pyrimidine
derivatives 6a-p were prepared and screened for antibacterial,
minimum bactericidal concentration (MBC), anti-biofilm, anti-
Candida, minimum Candida-cidal concentration (MFC) and
ergosterol inhibition activities. Compounds 6e, 6f, and 6m which
showed promising activity have been identified. The promising
pyrido[2,3-d]pyrimidine derivative 6e exhibited an important
inhibitory role in the ergosterol biosynthetic pathway and it was
also validated through modeling studies. Further, optimization of
structural modification on the pyridopyrimidine derivatives is in
progress.
Table 4: Biofilm inhibition assay of the selected pyrido[2,3-d]pyrimidine derivatives
Compound
IC50 values (in μg/ml)h
BS
SA
SA MLS-16 MTCC
PA
KP
MTCC 121a
-
MTCC 96b
21.6 ± 0.42
2940C
MTCC 2453d
-
MTCC 530e
-
f
6b
6e
6f
-
10.8 ± 0.15
21.6 ± 0.26
-
5.4 ± 0.12
2.7 ± 0.11
2.7 ± 0.20
10.8 ± 0.32
10.8 ± 0.28
-
21.6 ± 0.33
10.8 ± 0.32
-
21.6 ± 0.41
5.4 ± 0.22
5.4 ± 0.26
6g
5

6h
6k
6l
-
21.6 ± 0.33
2.7 ± 0.19
-
-
-
-
-
-
-
-
-
-
-
10.8 ± 0.32
10.8 ± 0.25
0.29 ± 0.14
6m
Cg
2.7 ± 0.13
0.23 ± 0.14
5.4 ± 0.22
0.21 ± 0.11
10.8 ± 0.16
0.24 ± 0.09
2.7 ± 0.16
0.32 ± 0.12
a
BS, Bacillus subtilis MTCC 121; bSA, Staphylococcus aureus MTCC 96; cSA Staphylococcus aureus MLS16 MTCC 2940; d PA, Pseudomonas aeruginosa MTCC 2453; eKP, Klebsiella planticola MTCC 530; -
fno activity gC Ciprofloxacin (Standard). hBiofilm inhibition assay was performed using crystal violet method. All the biofilms were treated with different concentrations of test compounds
and the respective IC50 values were interpreted from the dose-response curves. Each experiment was performed in triplicate and the results are represented as mean ± S.D (n = 3).
Table 5: Anti-Candida activity of the pyrido[2,3-d]pyrimidine derivatives.
Minimum inhibitory concentration (µg/mL)
C. a
MT
CC
C. a
C. a
C. a
C. a
C. a
C. a
C. a
MTC
C
C. a
MTC
C
C.
C.
C.
C.
Issatchen
MTCC MTCC MTCC MT
227
MTCC MTCC
3017
parapsi aaseri
losis
MTCC
1744
glabrat krusei kia
a
MTCC
3019
compd
854
1637
CC
3018
3958
MTCC
1962
MTC
C
3020
hanoiensi
s
MTCC
4755
183
4748
7315
a
6b
6c
6d
6e
6f
31.2
15.6
7.8
-
-
-
15.6
-
31.2
15.6
7.8
31.2
-
62.5
-
31.2
15.6
15.6
31.2
62.5
62.5
31.2
7.8
-
31.2
15.6
7.8
15.6
7.8
31.2
15.6
31.2
31.2
15.6
15.6
31.2
7.8
31.2
15.6
3.9
7.8
-
31.2
7.8
7.8
7.8
-
-
-
15.6
7.8
7.8
15.6
-
31.2
15.6
-
31.2
7.8
-
62.5
15.6
-
15.6
31.2
31.2
31.2
15.6
7.8
31.2
-
-
3.9
3.9
7.8
-
3.9
3.9
31.2
31.2
15.6
7.8
15.6
15.6
7.8
-
6h
6m
31.2
3.9
-
31.2
3.9
-
62.5
62.5
7.8
15.6
-
31.2
7.8
15.6
7.8
15.6
7.8
31.2
7.8
62.5
7.8
Miconazole
(Standard)
a- No activity
7.8
7.8
7.8
7.8
7.8
Table 6: Minimum Fungicidal Concentration (MFC) of the selected pyrido[2,3-d]pyrimidine derivatives
Candida strains
Minimum fungicidal concentration (µg/mL)
6b
6c
6d
6e
6f
6h
6m
Miconazole
(Standard)
Candida albicans MTCC 183
C. albicans MTCC 227
C. albicans MTCC 854
C. albicans MTCC 1637
C. albicans MTCC 3017
C. albicans MTCC 3018
C. albicans MTCC 3958
C. albicans MTCC 4748
C. albicans MTCC 7315
62.5
31.2
31.2
62.5
-
15.6
31.2
15.6
15.6
15.6
62.5
62.5
62.5
31.2
3.9
7.8
15.6
15.6
31.2
7.8
-
62.5
7.8
7.8
a
-
7.8
-
31.2
31.2
31.2
7.8
7.8
15.6
7.8
-
15.6
15.6
7.8
-
-
-
62.5
31.2
62.5
62.5
62.5
15.6
-
62.5
-
7.8
31.2
15.6
31.2
62.5
62.5
62.5
62.5
62.5
15.6
7.8
-
-
-
-
-
62.5
62.5
7.8
31.2
62.5
15.6
6

C. parapsilosis MTCC 1744
C. aaseri MTCC 1962
-
-
15.6
15.6
31.2
-
62.5
15.6
-
62.5
62.5
31.2
-
62.5
62.5
31.2
31.2
62.5
31.2
31.2
15.6
-
7.8
15.6
7.8
31.2
31.2
62.5
15.6
15.6
15.6
31.2
62.5
C. glabrata MTCC 3019
C. krusei MTCC 3020
-
15.6
15.6
Issatchenkia hanoiensis MTCC 4755
62.5
31.2
31.2
15.6
a- No activity
Table 7: Inhibition of ergosterol biosynthesis in C. albicans MTCC 183 by compound 6e
Test compounds
Mean ergosterol content of cells grown with test compounds at different concentrations (µg/mL)
0
2
4
16
6e
2.27 ± 0.11
2.27 ± 0.11
1.75 ± 0.12
1.81 ± 0.09
1.08 ± 0.08
1.12 ± 0.10
0.41 ± 0.06
0.53 ± 0.04
Miconazole (Standard)
Table 8: Interacting amino acids and their binding energy of compound 6e with modeled lanosterol 14-alpha demethylase protein
No. of
ligand
Interacting amino acids
Grid X-Y-Z coordinates
Binding energy ΔG
Dissociation
constant (kl)
(Kcal/Mol)
6e
Asp233
25.874, 18.831, 15.131
-10.70
14.42 nM
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