Antibacterial activity of synthetic pyrido[2,3-d]pyrimidines armed with nitrile groups: POM analysis and identification of pharmacophore sites of nitriles as important pro-drugs

Article abstract of DOI:10.1039/C8NJ02081G

A clean and simple one-pot multi-component methodology was developed for the preparation of 7-amino-2,4-dioxo-5-aryl-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carbonitriles via the reaction of 6-amino uracil, aromatic aldehydes and malononitrile using triethylamine (TEA) base as a catalyst in aqueous ethanol medium at NTP. The reaction protocol has assorted advantages viz.; mild reaction conditions, short reaction time, environmentally friendly procedure, low cost chemicals, and easy isolation of derivatives with excellent yields of bioactive products (85-95%). All the synthesised pyrido[2,3-d]pyrimidines bearing nitrile groups (4a-4h) showed comparatively good in vitro antibacterial activities against Staphylococcus and Bacillus cereus (Gram-positive bacteria) and P. merabitis and S. maresens (Gram-negative bacteria). Nevertheless, compound 4h exhibited the highest antibacterial activity and had an excellent % inhibition as compared to standard streptomycin. Overall, compound 4h had enhanced antibacterial activity compared to its positional isomers compound 4f and compound 4g. This increase in bioactivity going from 4f (R = 4-nitro) to 4h (R = 2-nitro) is attributed to the hydrolysis of the CN to an amide which restores the vital intramolecular interaction between the ortho-nitro-phenyl and amide linkages (ONO^δ?...H^δ+N) and offers a crucial template for antibacterial NH, O-pharmacophore sites. The synthesized compound 4h was tested to verify that it had fewer side effects than the standard/streptomycin. But, its possible inclusion in selective fungal/viral media viz. HIV or Hepatitis B/C is a subject of further research. This multi-component synthetic protocol uses cheap ingredients like Et₃N catalyst and ethanol as the green solvent.

Full text of DOI:10.1039/C8NJ02081G

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Antibacterial activity of synthetic pyrido[2,3-d]pyrimidines armed with
nitrile group: POM analyses and identification of pharmacophore sites of
nitriles as important pro-drugs
Rajendra Sukhdeorao Dongre,*,a Jyostna S. Meshram,a Rupali Sudhakarrao Selokar,a
Faisal A. Almalki,b Taibi Ben Hadda*, c
The clean and simple oneꢀpot multiꢀcomponent methodology was developed for the
preparation
of
7ꢀaminoꢀ2,4ꢀdioxoꢀ5ꢀarylꢀ1,2,3,4ꢀtetrahydropyrido[2,3ꢀd]pyrimidineꢀ6ꢀ
carbonitriles via reaction of 6ꢀamino uracil, aromatic aldehydes and malononitrile using
triethylamine (TEA) base as a catalyst in aqueous ethanol medium at NTP. The reaction
protocol own assorted advantageous viz; mild reaction conditions, short reaction time,
environmentally friendly procedure, low cost chemicals, and easy isolation of derivatives with
excellent yields of bioactive products (85ꢀ95%). All the synthesised pyrido[2,3ꢀd]pyrimidines
bearing nitrile compounds (4a-4h) showed comparatively good in-vitro antibacterial activities
against Staphylococcus, Bacillus cereus (Gramꢀpositive bacteria) and P. merabitis S.maresens
(Gramꢀnegative bacteria). Nevertheless, compound 4h exhibited highest antibacterial activity
and owing excellent % inhibition as compared to standard Streptomycin. Overall, this
compound 4h enhanced antibacterial activity as compared to its positional isomers compound
4f and compound 4g. This increase bioactivity going from 4f (R = 4ꢀNitro) to 4h (R = 2ꢀ
Nitro) is attributed to hydrolysis of C≡N to amide which restores vital intramolecular
interaction between orthoꢀNitroꢀphenyl and amide linkages (ONOδ−ꢀꢀHδ+N) and offers crucial
template for antibacterial NH, Oꢀphramacophore sites. The synthesized compound 4h was
tested to verify it fewer side effects than that of standard/Streptomycin. But, it’s possible
inclusion in selective fungal/viral media viz HIV, Hepatitis B/C phenomenon is a subject of
further research. This multiꢀcomponent synthetic protocol is using cheap ingredients like Et3N
catalyst and ethanol as the green solvent.
Keywords: Pyrido[2,3ꢀd]pyrimidines, uracil, one pot, multiꢀcomponent, triethylamine,
antibacterial, PetraꢀOsirisꢀMolinspiration (POM) analyses, nitrile, prodrug, pharmacophore
site identification.
Introduction
1

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Multi component reaction (MCR) is a powerful tool in organic reactions to be used for
making more than one carbon–carbon or carbon–heteroatom bond in oneꢀpot single step
synthetic procedures. Worldwide organic chemists are ever enthusiastic to design new
environmentally clean and economically useful synthetic methods which avoids large solid
wastes after each step of multiꢀstep reactions.1
Uracil and its derivatives own diverse therapeutic properties which makes them biomimetic
reactive pharmacophore sites and imparts the vital role in natural and synthetic organic
chemistry.2,3 Such, heterocyclic compounds have received considerable attention in recent
years due to their assorted biological activities viz; HIVꢀ1 reverse transcriptase,4 especially as
inhibitors of PDE5 extracted from human platelets,5 human EPK2,6 and cyclinꢀdependent
kinase.7 In fact the basic skeleton of the genetic materials is made up of adenine, guanine,
cytosine, thymine and uracil heterocycles illustrates their significance. Furthermore, these
nitrogen containing heterocyclic rings plays pivotal role in medicinal chemistry.
Pyrido[2,3ꢀd]pyrimidine is a bicyclic nitrogen containing heterocyclic compounds based on
uracil skeleton are investigated thoroughly for their inherent antibacterial and antifungal
activities,8 antihypertensive,9 antimicrobial,10 cardiotonic,11 tyrosine kinase,12 antiꢀ
inflammatory,13 and analgesic,14 calcium channel antagonists,15 tuberculostatic,16
antilieshmanial activity,17. These pyrido[2,3ꢀd]pyrimidine derivatives were synthesized using
various catalytic systems like diammonium hydrogen phosphate (DHAP),18 palladiumꢀ
catalysed oxidative coupling,19
Lꢀproline,20
KFꢀAl2O3,21 nucleophilicꢀinduced ring
transformation,22 and sometimes without catalyst,23, 24
On other hand, most of the frequently used drugs such as amphotericin B, bacitracin,
cephaloridine, cisplatin, colistin, cyclosporine, ethacrynic acid, furosemide, paromomycin,
polymyxin B, vancomycin, viomycin and streptomycin causes nerve, kidney, hearing issues,
besides severe muscle and severe breathing problems. Especially, streptomycin own greatest
sides effects and three violations of Lipinski five rule’s; N violations = 3, as shown in below
Figure 1. For these major reasons, we have prepared some pyrido[2,3ꢀd]pyrimidine
derivatives and investigated their antibacterial activities, besides compared them with virtual
Petra/Osiris/Molinspiration (POM) analysis. To the best of our knowledge, this is the first
research study exhibiting quantitative structure activity relationship of pharmacophore sites of
pyrido[2,3ꢀd]pyrimidine asan important proꢀdrugs.
2

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δ
+
H
-
N
δ
NH2
NH
O
δ
-
O
H3C
δ
+
HO
O
H3C
H
+
δ
HO
O
O
H
N
H
δ
+
HO
O
HO
NH2
H
δ
-
N
δ
-
H
+
O
δ
NH
HO
Streptomecin
δ
+
: Antibacterial Pharmacophore site
δ
-
Molecular properties
Drug score
TPSA
MW
333
581
19
N OHNH
N violations
volume
16
GPCR ligand
0.17
Nuclear receptor ligand ꢀ0.32
3
Ion channel modulator
Kinase inhibitor
ꢀ0.09 Protease inhibitor
0.78
0.46
nON
497.40
ꢀ0.24
Enzyme inhibitor
Fig. 1 Structure of Streptomycin (SD1) bearing multi combined antibacterial (Xδ+ꢀꢀꢀꢀYδ−)
pharmacophore sites.
Materials and methods
All chemicals and solvents were obtained from Aldrich Chemical Co. and S. D. Fine chem
Co., and are used in research as such without further purification. The melting points were
1
determined by open capillary method and were uncorrected. All the HꢀNMR spectra of
products were obtained on a BRUKER instrument (300 MHz). FTIR spectra were recorded
on a Perkin–Elmer 298 spectrophotometer using KBr disc and all 13CꢀNMR (100 MHz)
spectra were recorded in DMSOꢀd6 as solvent with TMS as internal standard. Chemical shifts
are reported in ppm and the mass spectra were measured using high resolution GC–MS (DFS)
thermo spectrometers with EI (70 EV). All the reactions were monitored frequently by thin
layer chromatography (TLC) on 0.2ꢀmm preꢀcoated plates of silica gel G60 F254 (Merck).
Spectral data for synthesised pyrido[2,3-d]pyrimidine products
7-Amino-5-(4-methyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4tetrahydropyrido[2,3-d]pyrimidine-6-
carbonitrile (4a)
Yellow powder. Yield 85 %. IR (KBr, cmꢀ1): 3395 (ꢀNH2), 2220 (ꢀCN), 1799 (ꢀC=O), 1715 (ꢀ
C=O);1H NMR (300 MHz, DMSOꢀd6, ppm): 2.36 (s, 3H, CH3), 3.25 (s, 3H, CH3), 3.45 (s,
3H, CH3), 7.12 (d, 2H, HAr), 7.20 (d, 2H, HAr), 7.68 (br s, 2H, NH2), ppm; 13C NMR (100
MHz, DMSOꢀd6, ppm): 160.8, 159.9, 155.5, 150.1, 137.4, 129.7, 128.2, 125.4, 114.1, 98.3,
88.7, 57.3, 31.2, 29.5, 20.9ppm; EIꢀMS (m/z) = 321 (M+)
3

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7-Amino-5-(phenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-
carbonitrile (4b)
White powder. Yield 90%. IR (KBr, cmꢀ1): 3400 (ꢀNH2), 2222 (ꢀCN),1710 (ꢀC=O), 1625 (ꢀ
C=O);1H NMR (300 MHz, DMSOꢀd6, ppm): 3.35 (s, 3H, CH3), 3.65 (s, 3H, CH3),7.24
(d,2H,HAr), 7.45 (t, 3H, HAr), 7.60 (br s, 2H, NH2) ppm; 13C NMR (100 MHz, DMSOꢀd6,
ppm): 160.9 160.1, 159.0, 158.3, 150.7, 134.2, 127.9, 127.7, 116.7, 115.1, 88.8, 40.1, 35.0,
24.1, ppm; EIꢀMS(m/z) = 307 (M+1).
7-Amino-5-(4-methoxyphenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-6-carbonitrile (4c)
Yellow powder. Yield 93%. IR (KBr, cmꢀ1): 3420(ꢀNH2), 2215(ꢀCN), 1760 (ꢀC=O), 1635 (ꢀ
1
C=O); H NMR (300 MHz, DMSOꢀd6, ppm): 3.15 (s, 3H, CH3), 3.47 (s, 3H, CH3), 4.25 (s,
3H, OCH3), 7.05 (d, 2H, HAr), 7.25 (br s, 1H, NH2), 7.9 (d, 2H, HAr); 13C NMR (100 MHz,
DMSOꢀd6, ppm): 160.8, 160.0, 159.9, 158.8, 155.5, 150.1, 129.5, 128.6, 115.7, 112.9, 98.3,
88.8, 62.5, 23.4, 28.3 ppm; EIꢀMS (m/z) = 337 (M+).
7-Amino-5-(4-hydroxyphenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-
tetrahydropyrido[2,3d]pyrimidine-6-carbonitrile (4d)
Yellow powder. Yield 91%. IR (KBr, cmꢀ1): 3391 (ꢀNH2), 2237 (ꢀCN), 1786 (ꢀC=O), 1624 (ꢀ
C=O); 1H NMR (300 MHz, DMSOꢀd6, ppm): 3.1 (s, 3H, CH3), 3.3 (s, 3H, CH3), 6.59 (s, 1H,
HAr), 6.62 (d, 1H, HAr), 6.78 (d, 1H, HAr), 7.18 (t, 1H, HAr), 7.58 (br s, 2H, NH2), 9.46 (s, 1H,
13
OH); C NMR (100 MHz, DMSOꢀd6, ppm): 160.8, 159.8, 155.4, 150.2, 130.9, 121.8, 118.1,
115.4, 115.2, 98.3, 88.5, 28.3, 26.4 ppm.; EIꢀMS (m/z) = 323 (M+).
7-Amino-5-(4-chlorophenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-6-carbonitrile (4e)
White powder. Yield 92%. IR (KBr, cmꢀ1): 3377 (ꢀNH2), 2206 (ꢀCN), 1759 (ꢀC=O), 1618 (ꢀ
C=O); 1H NMR (300 MHz, DMSOꢀd6, ppm): 3.2 (s, 3H, CH3), 3.55 (s, 3H, CH3), 7.34 (d, 1H,
HAr), 7.50 (d, 1H, HAr), 7.72 (br s, 1H, HAr),7.81 (br s, 2H, NH2); 13C NMR (100 MHz,
DMSOꢀd6, ppm): 170, 165, 155.4, 154.6, 150.1, 135.0, 133.7, 131.7, 70, 55, 28, 25; EIꢀMS
(m/z) = 341 (M+).
7-Amino-5-(4-nitrophenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-6-carbonitrile (4f)
White powder. Yield 95%. IR (KBr, cmꢀ1): 3297 (ꢀNH2), 2222 (ꢀCN),, 1758 (ꢀC=O), 1624 (ꢀ
C=O); 1H NMR (300 MHz, DMSOꢀd6, ppm): 3.3 (s, 3H, CH3), 3.7 (s, 3H, CH3), 5.78 ( s, 2H,
NH2), 7.45 (d, 2H, HAr), 8.38 (d, 2H, HAr) ppm; 13C NMR (100 MHz, DMSOꢀd6, ppm):
166.1, 164.9, 162.5, 157.8, 156.7, 148.8, 143.7, 127.3, 123.1, 113.2, 98.4, 84.2, 36.8 and 28.7;
EIꢀMS (m/z) = 353 (M+).
4

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7-Amino-5-(3-nitrophenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-6- carbonitrile (4g)
View Article Online
DOI: 10.1039/C8NJ02081G
White powder. Yield 86%. IR (KBr, cmꢀ1): 3384 (ꢀNH2), 2216 (ꢀCN), 1768 (ꢀC=O), 1622 (ꢀ
C=O); 1H NMR (300 MHz, DMSOꢀd6, ppm): 3.3 (s, 3H, CH3), 3.7 (s, 3H, CH3), 7.75 (m, 2H,
HAr), 7.77 (br s, 2H, NH2), 8.19 (s, 1H, HAr), 8.29 (qd, 1H, HAr), ppm; 13C NMR (100 MHz,
DMSOꢀd6, ppm): 160.8, 160.2, 156.1, 159.0, 155.5, 150.1, 147.1, 138.4, 129.4, 134.8, 98.3,
88.3, 25.4, 29.3; EIꢀMS (m/z) = 353 (M+).
7-Amino-5-(2-nitrophenyl)-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-
d]pyrimidine-6-carbonitrile (4h)
Yellow powder. Yield 89%. IR (KBr,cmꢀ1): 3403 (ꢀNH2), 2225 (ꢀCN) , 1707 (ꢀC=O), 1613 (ꢀ
1
C=O); H NMR (300 MHz, DMSOꢀd6, ppm): 3.3 (s, 3H, CH3), 3.7(s, 3H, CH3), 7.75 (s, 2H,
NH2), 7.51 (d,1H,HAr), 7.43 (dt, 1H, HAr), 7.38 (t, 1H, HAr), 7.28 (dd, 1H, HAr); 13C NMR
(100 MHz, DMSOꢀd6, ppm), 167.50, 173.50, 158.15, 156.02, 130.63, 129.53, 128.70, 128.37,
126.65, 114.50, 75, 53.3 ,29.49, 27.56; EIꢀMS (m/z) = 353 (M+).
Minimum Inhibitory concentration: (MIC)
Pyrido[2,3ꢀd]pyrimidines were dissolved in DMF to prepare stock solutions of 20 mg/mL for
antimicrobial activity. Agar plates were uniformly inoculated with fresh culture of both
°
strains. These impregnated disks were incubated at 30 C for 60 mints to permit good
diffusion and transfer in to incubator for 24 hrs at 37±2 °C. The inhibition zone of compounds
around the disk was measured in mm scale as shown in Table 3 and these observations are
compared with standard Streptomycine.
Results and discussion
Substituted aromatic aldehydes 1a-1h (1 mmol), malononitrile 2 (1 mmol), 6ꢀamino 1, 3ꢀ
dimethyl uracil 3 (1 mmol) and triethyl amine (10 mol%) as organo catalyst were taken in an
R.B. flask with solvent aqueous ethanol (1:1 ratio, 10 mL) and stirred for 30–50 min at room
temperature. The reaction was monitored by thin layer chromatography. The solid product
was filtered, washed with cold water several times, and reꢀcrystallized from ethanol to obtain
pure pyrido[2,3ꢀd]pyrimidine derivatives with excellent yield in below Scheme 1.
5

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Scheme 1 General procedure for the preparation of pyrido[2,3ꢀd]pyrimidines 4a-4h.
Annulated pyrido[2,3ꢀd]pyrimidines (4a-4h) synthesised via KnoevenagelꢀMichael
condensation reaction pathways. (Table 1) In initial studies, 4ꢀnitrobenzaldehyde 1 (1.0
mmol), malononitrile 2 (1.0 mmol), 6ꢀaminoꢀ1,3ꢀdimethyl uracil 3 (1.0 mmol) with aqueous
ethanol (5:5) were taken in Round bottom (R.B) flask mounted over magnetic stirrer. The
catalyst triethylamine (TEA) with 10 mol % concentrations showed the best result among
these different used catalyst in terms of reaction time and product yield (95%).
Table 1 Synthesis of pyrido[2,3ꢀd]pyrimidine derivatives 4a-4h
Product
R
Time (mins) Yield (%)a
Mp (°C)
>300
>300
>300
>300
>300
>300
>300
>300
Mp (Lit.)
>300
>300
>300
___
4ꢀMethyl
H
50
45
55
58
55
50
65
75
85
90
93
91
92
95
86
89
4a
4b
4ꢀMethoxy
4ꢀHydroxy
4ꢀChloro
4ꢀNitro
3ꢀNitro
2ꢀNitro
4c
4d
>300
>300
>300
___
4e
4f
4g
4h
a isolated yields
In the absence of catalyst, the reaction was completed in 185 minutes with product yield mere
35%. Therefore, 10% triethylamine (TEA) catalyst concentration found superior to other mole
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% of the catalysts. The focused is given on systematic usage of different solvents
combinations like ethanol:water, DMF, CH2Cl2 and 1,4ꢀDioxane for the model reaction 4f
using 4ꢀnitrobenzaldehyde 1f (1.0 mmol), malononitrile 2 (1.0 mmol) and 6ꢀamino 1,3ꢀ
dimethyl uracil 3 (1.0 mmol) in presence of 10 mol % triethylamine (TEA) catalyst as shown
in Table 2.
Table 2 Optimization of different solvents for the model product 4f
Entry
Solvents
Time (mins)
Yield (%)a
1
EtOHꢀH2O
DMF
50
95
61
55
65
57
2
70
3
CH2Cl2
105
85
4
1,4ꢀDioxane
Solvent less
5
195
a isolated yields
The optimization results of different solvents for the model product indicated major role of
the solvent on the yield of product and overall reaction span. It was found that the best
conversion was observed when the reaction performed in Ethanol: water/aqueous ethanol.
Aqueous ethanol solvent imparts key advantages such as, environment safety, clean/green
solvent, cheap operation cost, simple workꢀup and harmful effects. Mild base TEA catalyst
facilitates proton removal from active methylene compound/malononitrile 2 thereby enhances
reaction rate and ultimately facilitating Knoevenagel condensation between aryl aldehydes
and active methylene compound, proceeds via intermediate, undergoes dehydration and
finally heterocyclization and thus, its successfully synthesis of annulated pyrido[2,3ꢀ
d]pyrimidine in quantitative yields.
Biological evaluation
Antibacterial activity: All the synthesised compounds (4a-4h) were screened in vitro for their
antibacterial activity against Staphylococcus, Bacillus cereus (Gramꢀpositive bacteria) and P.
merabitis, S. maresens (Gramꢀnegative bacteria). The minimum inhibitory concentration
(MIC) of µg/mL values is calculated out by the discꢀdiffusion technique in order to check the
activity of all synthesised pyrido[2,3ꢀd]pyrimidines. The synthesized pyrido[2,3ꢀ
d]pyrimidines were dissolved in dimethyl sulfoxide (DMSO) for dilution purpose to prepare
stock solution of 1 mg/mL and then were impregnated onto Whatman filter papers (No.1).
The impregnated Whatman filter papers were placed on the surface of solidified nutrient agar
dishes seeded by the test bacteria. Medium containing plates were allowed to stand at room
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temperature for 10 minutes and were set to solidify in a refrigerator for 30 mints. The MICs of
studied pyrido[2,3ꢀd]pyrimidines were measured in millimetres by the end of the incubation
period 48 h carried at 37 °C. Streptomycin (25µg/mL) is used as standard for bacterial studies.
The zones of inhibition were measured on mm scale and shown in following Table 3.
Table 3 Antibacterial activity values of pyrido[2,3ꢀd]pyrimidines [Minimum inhibitory
concentration (MIC) in µg/mL]
Gram positive bacteria
Gram negative bacteria
Compd.
R
cLog P
B. cereus
Staphylococcus
P. merabitis
S.maresens
4ꢀCH3
H
1.54
1.10
1.15
0.62
1.77
1.05
1.03
1.01
ꢀꢀꢀ
12
11
12
18
13
11
9
11
12
11
15
10
9
14
12
13
16
10
8
13
14
17
17
11
10
9
4a
4b
4ꢀOCH3
4ꢀOH
4ꢀCl
4c
4d
4e
4ꢀNO2
3ꢀ NO2
2ꢀ NO2
ꢀꢀꢀ
4f
7
11
10
22
4g
7
6
7
4h
SD1[a]
21
23
22
[a] SD1: Standard drug (Streptomycin).
The pyrido[2,3ꢀd]pyrimidines 4a-4d showed the maximum antibacterial activity against
Staphylococcus, B.cereus and P.merabitis, S.maresens. These maximum activities exerted due
to extensive effect on the membrane potential associated with bactericidal activity attributed
pharmacologically active electron donating functionalities like benzaldehyde, ꢀOH, ꢀCH3 and ꢀ
OCH3 attached to phenyl ring on the fused pyridine skeleton of annulated pyrido[2,3ꢀ
d]pyrimidines. There are no antibacterial pharmacophore sites in structure of compounds 4a-
4h bearing nitrile group as the simple and evident. Rather compounds 4e and 4f showed
moderate activity and 4g and 4h shown relatively low bioactivity due to electron withdrawing
groups. Such contrasting bioactivity pattern of pyrido[2,3ꢀd]pyrimidines led us to investigate
and identify real pharmacophore site by using bioinformatics PetraꢀOsirisꢀMolinspiration
(POM) analysis. The data evaluated in Table 3, noted that 8/8 structures are supposed to be
more efficient than clinical drugs which make this research more curious to study their side
effects.
POM Analyses of Compounds 4a-4h and their metabolites 4aa-4hh
The analysis of antibacterial activity for the series 4a-4h using the PetraꢀOsirisꢀMolinspiration
(POM) programs showed that compound 4h and certainly the rest of series 4a-4h of
pyrido[2,3ꢀd]pyrimidines are more active than standard clinical drug/Streptomycin (SD1) as
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shown in Figure 1. Pyrido[2,3ꢀd]pyrimidines 4a-4h represent no violation (NV = 0) of five
Lipinski rules, so compounds 4a-4h can be used as antibiotics with some limited restriction as
depicted in Table 4.
Table 4 Molinspiration calculations of compounds bearing nitrile group (4a-4h)
Molinspiration calculations [a]
Drugꢀlikeness [b]
Compd.
TPSA NONH
NV
0
VOL
282
GPCRL
ꢀ0.07
ICM
KI
NRL
ꢀ0.65
PI
EI
107
107
116
127
107
153
153
2
2
2
3
2
2
2
ꢀ0.70
0.00
ꢀ0.56
4a
4b
4c
4d
4e
4f
0.07
0
0
0
0
0
0
265
300
273
279
289
289
ꢀ0.04
ꢀ0.07
0.01
ꢀ0.65
ꢀ0.67
ꢀ0.58
ꢀ0.63
ꢀ0.63
ꢀ0.64
0.05
ꢀ0.01
0.09
ꢀ0.65
ꢀ0.60
ꢀ0.47
ꢀ0.64
ꢀ0.67
ꢀ0.67
ꢀ0.54
ꢀ0.53
ꢀ0.49
ꢀ0,55
ꢀ0.60
ꢀ0.60
0.13
0.07
0.18
0.09
0.00
0.02
ꢀ0.03
ꢀ0.18
ꢀ0.18
0.03
ꢀ0.10
ꢀ0.07
4g
153
334
2
0
3
289
497
ꢀ0.23
ꢀ0.64
ꢀ0.09
ꢀ0,11
ꢀ0.24
ꢀ0.78
ꢀ0.32
ꢀ0.65
ꢀ0.04
4h
SD1 [c]
16
0.17
0.78
0.46
[a] TPSA: Total molecular polar surface area; NONH: number of OHꢀꢀꢀN or OꢀꢀꢀNH interaction, NV: number of
violation of five Lipinsky rules; VOL: volume. [b] GPCRL: GPCR ligand; ICM: Ion channel modulator; KI:
[c]
Kinase inhibitor; NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme inhibitor.
Streptomycin/standard drug (Antibacterial).
SD1:
The pyrido[2,3ꢀd]pyrimidines 4aꢀ4h series needs to be tested for their possible mutagenicity,
irritating and reproductive effects as should have lower risks comparable with used standard
drugs. The hydrophilicity character of each compound of4aꢀ4h series has been expressed in
terms of the cLogP value. Rather, absorption/permeation of drug gets greatly affected by the
hydrophilicity i.e., value of cLogP. Accordingly, when cLogP is higher than 5, the absorption
or permeation of drug found to be decreased. On this basis, compound 4a–4h series and their
corresponding metabolites 4aa-4hh have cLogP values within the acceptable criteria, yet few
crucial parameters should be considered. Since, the geometrical conformation of
pharmacophore sites has not been concerned because it’s fixed for all compounds 4a–4h
series. The distribution characteristics, absorption pattern and bioactivity proven depend on
the geometrical parameter and the aqueous solubility of each compound of 4a–4h series.
Further, drugꢀlikeness of compounds 4a–4h series (Table 4) can’t lies in the comparable zone
with standard/Streptomycin drugs. The overall drugꢀscore (DS) have been calculated for
compounds 4a–4h series and compared with that of Streptomycin (SD1). Furthermore, DS
combines drugꢀlikeness, cLogP, logS, molecular weight, and toxicity risks, in one handy
9

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values, all are used to judge the synthesized compound’s overall potential to qualify it for a
marketed drug. The reported compounds 4a–4h series showed low to moderate DS as
compared to standard/Streptomycin drug (Table 4).
All these research findings and POM analyses suggest that compounds 4a-4h series are
worthy for its screening against other pathogenic bacteria and for future clinical trials. Any
pathogenic bacterium is a common mould pathogen of human causing persistent diseases e.g.,
4% patients dying in European hospitals hold persistent aspergillosis which is the leading
transferable cause of death in leukaemia conditions. The compounds 4a-4h series own low
drugꢀscore (DS) and weak drugꢀlikness (DL) features as compared to the standard
Streptomycin and such divergent values makes 4a-4h quite at par with experimental
antibacterial screening data. Indeed, supplementary POM calculations of metabolites of
compounds 4a-4h needs to clarify contrasting results. Thus, this research study suggested the
real bioactive metabolites coexistence.
Stability of 4a–4h series nitrile derivatives withmicroorganisms
The bioꢀactivity evaluation in the literature states that structures bearing nitrile on
enzymatic/chemical hydrolysis undergoes transformation to yield corresponding amide and
carboxylic acids as shown in Scheme 2.25ꢀ30 Since 2001, Wegman et al.25 identified
Rhodococcus sp. as highly active bacterial strain, able to hydrolyse nitrile by using the nitrile
hydratase enzyme.
Scheme 2 Hydration and subsequent hydrolysis of D,Lꢀphenylglycine nitrile.25 (i) Nitrile
hydralase, (ii) Amidase.
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More recently, Kinfe et al.26 confirmed the nitriles gets transformed to the corresponding
carboxyamides and carboxyacids at NTP under bacterial ( Rhodococcus rhodochrous ATCC
BAA-870) hydrolysis. The bacteria expressed nitrile hydratase and amidase activities as
responsible for this bioꢀconversion of nitriles shown in Scheme 3.
DOI: 10.1039/C8NJ02081G
OH
R
O
R
O
OH
R
O
OH
(i)
+
O
O
H2N
HO
N
Scheme 3 (i) R. Rhodochrous sp., MeOH/potassium phosphate buffer (1:10), 30 °C, 3 h.26
Osprian et al. 27 indicated a second bacteria; the Rhodococcus erythropolis NCIMB11540 as a
source of highly active nitrile hydratase/amidase enzymatic hydrolysis of nitriles to amides
and carboxylic acids as shown in Scheme 4.
Scheme 4 Cyanohydrin hydrolysis catalyzed by R. erythropolis NCIMB 11540.27
Cells of Rhodococci strains converted naproxen nitrile via naproxen amide to naproxen.
Rhodococcus sp. MP50 converted racemic naproxen nitrile predominantly to Rꢀnaproxen
amide and racemic naproxen amide to Sꢀnaproxen as shown in Scheme 5.
Scheme 5 Naproxen nitrile hydrolysis catalyzed by Rhodococcus sp. MP50.28
The bacterial strains viz; Rhodococcus sp. C3II and Rhodococcus erythropolis MP50 family
are especially selected for enantioselective hydrolysis of pharmaceutically interesting 2ꢀ
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arylpropionitriles like naproxen nitrile by Effenberger, Graef for hydrolysis of various
aliphatic and aromatic nitriles and carboxylic acids and amides as shown in Scheme 6.29
Scheme 6 Dinitriles hydrolusis catalyzed by hydratase and amidase of Rhodococcus sp. C3II
and Rhodococcus erythropolis MP50 bacterial strains.29 (i) Nitrile hydratase (ii) Amidase.
The amidase from Rhodococcus rhodochrous J1, which hydrolyzes amide to an acid and
ammonium, was surprisingly found to catalyze the hydrolytic cleavage of the CꢀN triple bond
in a nitrile,30 to form an acid and ammonium stoichiometrically. An amidase plays a crucial
role in the hydrolysis of nitriles as well as amides. The βꢀaminoꢀnitrile compounds undergoes
enzymatic hydrolysis to the corresponding amides,31 using the nitrile biocatalytic activity of
Rhodococccus rhodochrous ATCC BAA-870. Results showed that the nitrile hydratase
enzyme was enantioselective for these compounds as shown in Scheme 7.
Scheme 7 Biocatalytic conversion of _ꢀaminonitriles to _ꢀamino amides and acids,31 (i)
Nitrile hydratase, (ii) Amidase, (iii) Nitrilase.
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The contradiction between experimental screening data (Table 3) and POM virtual screening
data of series 4a-4h (Table 4) is of a great importance for both Organic Chemists and
Pharmacologists as explained on via enzymatic and bioinformatics approaches. The analysis
of theoretical drug scores for compounds 4a-4h series using the PetraꢀOsirisꢀMolinspiration
(POM) program showed that the antibacterial pharmacophore site Xδ+ꢀꢀꢀꢀY δ− (where X, Y =
O, N, S, P) is completely missing and this series represent awful antibacterial pharmacophore
site. Its due to large distance between terminal nitrile and amine groups at antibacterial
pharmacophore site (dNꢀꢀꢀN > 2.8 Å) as affixed by POM theory.32ꢀ38
While the experimental antibacterial screening data as shown in Table 1 indicated more bioꢀ
activity of compounds 4a-4h series than clinical drug standard/Streptomycin wherein
antibacterial Xδ+ꢀꢀꢀꢀY δ− pharmacophore sites gets combind as shown in Figure 1. The
hydrophilicity of metabolites 4aa-4hh is expressed in terms of the cLogP value. The
absorption or permeation of 4a-4h series are greatly affected by the hydrophilicity (value of
cLogP). Accordingly, when cLogP is higher than 5, the absorption or permeation decreases.
On this basis, most of tested 4a-4h compound series have cLogP values within the acceptable
criteria and are active at different concentration because another crucial parameter is
considered. This does not concern the geometrical conformation of pharmacophore because it
is resolved and fixed for compound 4aa-4hh via C=Oδ−ꢀꢀꢀ δ+HNH intramolecular interaction
as shown in Scheme 8. This intraꢀtemplate effect in favour of constitution of vital O1ꢀꢀꢀHN1
antibacterial pharmacophore as hypothesized by POM theory.32ꢀ38 This calculated overall
drugꢀscore (DS) for metabolites 4aa-4hh (Scheme 8) now compared with that of standard
drug as shown in Table 5. The DS combines drugꢀlikeness, cLogP, logS, molecular weight,
and toxicity risks, in one handy value that may be used to judge the compound’s overall
potential to qualify for a drug. The reported compounds 4aa-4hh showed excellent DS as
compared with the standard drug used in commerce.32ꢀ38
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Scheme 8 Mechanism of transformation of proꢀdrugs 4a-4h to 4aa-4hh via bio catalyzed
hydrolysis of nitrile group and identification of antibacterial pharmacophore site.
Table 5 Molinspiration calculations of metabolites (4aa-4hh)
Molinspiration calculations [a]
Drugꢀlikeness [b]
Compd.
TPSA NONH
NV
0
VOL
295
GPCRL
ꢀ0.09
ICM
KI
NRL
ꢀ0.02
PI
EI
126
126
135
146
126
172
172
4
4
4
5
4
4
4
ꢀ0.65
ꢀ0.02
ꢀ0.68
4aa
4bb
4cc
4dd
4ee
4ff
0.10
0
0
0
0
1
1
278
304
286
292
302
302
ꢀ0.04
ꢀ0.09
ꢀ0.00
ꢀ0.05
ꢀ0.19
ꢀ0.18
ꢀ0.60
ꢀ0.62
ꢀ0.53
ꢀ0.53
ꢀ0.59
ꢀ0.59
ꢀ0.03
ꢀ0.01
ꢀ0.07
ꢀ0.01
ꢀ0.11
ꢀ0.09
ꢀ0.68
ꢀ0.64
ꢀ0.51
ꢀ0.67
ꢀ0.70
ꢀ0.70
ꢀ0.37
ꢀ0.39
ꢀ0.34
ꢀ0.39
ꢀ0.47
ꢀ0.47
0.16
0.10
0.20
0.12
0.03
0.04
4gg
172
334
4
1
3
302
497
ꢀ0.23
ꢀ0.60
ꢀ0.09
ꢀ0.12
ꢀ0.24
ꢀ0.81
ꢀ0.32
ꢀ0.51
4hh
0.01
0.46
SD1 [c]
16
0.17
0.78
[a] TPSA: Total molecular polar surface area; NONH: number of OHꢀꢀꢀN or OꢀꢀꢀNH interaction, NV: number of
violation of five Lipinsky rules; VOL: volume. [b] GPCRL: GPCR ligand; ICM: Ion channel modulator; KI:
[c]
Kinase inhibitor; NRL: Nuclear receptor ligand; PI: Protease inhibitor; EI: Enzyme inhibitor.
Streptomycin (Antibacterial).
SD1:
Conclusion
In this research study, a clean, simple facile and conveniently practical methodology was
developed for synthesis of annulated pyrido[2,3ꢀd]pyrimidines/uracils using mild base TEA as
a novel organoꢀcatalyst in green solvent aqueous ethanol. This synthesized methodology own
few advantageous like, performed unedr mild conditions, excellent yields, operational
simplicity, simple separation, clean, energyꢀefficiency, high atomꢀeconomy, no special work
up required, products are filtered and purified only by simple crystallization.
The Quantitative structure–activity relationship (QSAR) of synthesized compounds 4a-4d
series own excellent antibacterial profile and pyrido[2,3ꢀd]pyrimidines 4a-4d series can serve
as the prospective prodrugs. They are substances described in an inactive form that is then
metabolized via enzymatic action into the active compounds 4aa-4dd. The rationale behind
administering prodrugs is to optimize absorption, distribution, metabolism, and excretion of
these drugs. In addition, these prodrugs undergo enzymatic biotransformation in the presence
of fungus which makes them good therapeutic agents against fungus.39
Pyrido[2,3ꢀd]pyrimidines 4a-4d series have exhibited varying degree of inhibitory effect on
the growth of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50 bacterial strains and
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found superior than that of clinical drug standard/Streptomycin. Calculated overall drugꢀscore
for metabolites are superior to standard/streptomycin drug. Drug score combines drugꢀ
likeness, cLogP, logS, molecular weight, and toxicity risks to judge overall potential as a
prodrug. These synthesized compounds showed excellent drug score than that of standard
drug streptomycin as supported by POM analyses.32ꢀ38 This research work provided with
structure–activity information, indeed proved a simple control of nature of few numbers of
substituents leads to product/metabolites owing high bioꢀactivity.
Acknowledgments
R.S.D. and T.B.H. wish to extend their sincere appreciation to Head of Department of
Chemistry, RTM Nagpur, University Nagpurꢀ33, M.S., India for providing the essential
laboratory facilities needed to carry out this research work. Professor T. Ben Hadda would
like to thank ACTELION; the Biopharmaceutical Company of Swiss, for the molecular
properties calculations.
Conflicts of interest
There are no conflicts to declare.
Notes and References
a
Department of Chemistry, RTM Nagpur university, 440033 Nagpur, India. Tel: +91-
8087723120; E-mail: rsdongre@hotmail.com
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Umm Al-Qura University,
Makkah, Saudi Arabia.
c
Materials Chemistry Laboratory, Faculty of Sciences, University Mohammed Premier,
60000 Oujda, Morocco. E-mail: taibi.ben.hadda@gmail.com
Fax: (+212)0536500603; Tel: (+212)0666134178; E-mail: taibi.ben.hadda@gmail.com
1.
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Graphical Abstract
The clean and simple oneꢀpot multiꢀcomponent methodology was developed for the
preparation of 7ꢀaminoꢀ2,4ꢀdioxoꢀ5ꢀarylꢀ1,2,3,4ꢀtetrahydropyrido[2,3ꢀd]pyrimidineꢀ6ꢀ
carbonitriles with excellent yields (85ꢀ95%) of compounds (4a-4h) is described. All the
synthesised pyrido[2,3ꢀd]pyrimidines bearing nitrile compounds (4a-4h) showed
comparatively good in-vitro antibacterial activities against Staphylococcus, Bacillus cereus
(Gramꢀpositive bacteria) and P. merabitis S.maresens (Gramꢀnegative bacteria). Pyrido[2,3ꢀ
d]pyrimidines 4a-4d series have exhibited varying degree of inhibitory effect on the growth of
Rhodococcus sp. C3II and Rhodococcus erythropolis MP50 bacterial strains and found
superior than that of clinical drug standard/Streptomycin. Calculated overall drugꢀscore for
metabolites are superior to standard/streptomycin drug. Drug score combines drugꢀlikeness,
cLogP, logS, molecular weight, and toxicity risks to judge overall potential as a prodrug.
These synthesized compounds showed excellent drug score than that of standard drug
streptomycin as supported by bioꢀinformatic POM analyses.
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List of Figures
Fig. 1 Structure of Streptomycin (SD1) bearing multi combined antibacterial (Xδ+ꢀꢀꢀꢀYδ−)
pharmacophore sites.
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List of Schemes
Scheme 1 General procedure for the preparation of pyrido[2,3ꢀd]pyrimidines 4a-4h.
Scheme 2 Hydration and subsequent hydrolysis of D,Lꢀphenylglycine nitrile.25 (i) Nitrile
hydralase, (ii) Amidase.
Scheme 3 (i) R. Rhodochrous sp., MeOH/potassium phosphate buffer (1:10), 30 °C, 3 h.26
Scheme 4 Cyanohydrin hydrolysis catalyzed by R. erythropolis NCIMB 11540.27
Scheme 5 Naproxen nitrile hydrolysis catalyzed by Rhodococcus sp. MP50.28
Scheme 6 Dinitriles hydrolusis catalyzed by hydratase and amidase of Rhodococcus sp. C3II
and Rhodococcus erythropolis MP50 bacterial strains.29 (i) Nitrile hydratase (ii) Amidase.
Scheme 7 Biocatalytic conversion of _
ꢀaminonitriles to _ꢀamino amides and acids,31 (i)
Nitrile hydratase, (ii) Amidase, (iii) Nitrilase.
Scheme 8 Mechanism of transformation of proꢀdrugs 4a-4h to 4aa-4hh via bio catalyzed
hydrolysis of nitrile group and identification of antibacterial pharmacophore site.
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List of Tables
Table 1 Synthesis of pyrido[2,3ꢀd]pyrimidine derivatives 4a-4h
Table 2 Optimization of different solvents for the model product 4f
Table 3 Antibacterial activity values of pyrido[2,3ꢀd]pyrimidines [Minimum inhibitory
concentration (MIC) in µg/mL]
Table 4 Molinspiration calculations of compounds bearing nitrile group (4a-4h)
Table 5 Molinspiration calculations of metabolites (4aa-4hh)
21