An improved synthesis of pyrido[2,3-: D] pyrimidin-4(1 H)-ones and their antimicrobial activity

Article abstract of DOI:10.1039/c8ob00627j

The screening of a small library of diverse chemical structures resulted in the identification of 2-thioxodihydropyrido[2,3-d]pyrimidine 10a as having broad spectrum antibacterial activity (MIC 0.49-3.9 μg mL^-1), and reasonable antifungal activity (MIC 31.25 μg mL^-1). An expeditious synthesis of 10a was optimized by varying solvents, catalysts and the use of microwave irradiation with the best conditions using DMF as a solvent, I₂ (10 mol%) and a 30 minutes reaction time compared to 15 h for classic conventional heating. The pharmacokinetic properties and calculation of drug likeness of 10a suggested good traditional drug-like properties and led to the synthesis of a small library with seven compounds 10a and 10d-i showing broad antimicrobial activity (MIC = 0.49-7.81 μg mL^-1) and selectivity indices of more than 5.6 against the normal colon cell line (CCD-33Co). The antifungal activity of compounds 10d-i was moderate to strong with MIC values of 1.95-15.63 μg mL^-1.

Full text of DOI:10.1039/c8ob00627j

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DOI: 10.1039/C8OB00627J
Organic and biomolecular chemistry
Communication
An improved synthesis of pyrido[2,3-d]pyrimidin-4(1H)-ones and
their antimicrobial activity
a,b,*
b
c
d
Mohamed Fares
, Soha R. Abd El Hadi , Radwa A. Eladwy , Aly A. Shoun , Marwa M. Abdel-
e
f
g
a,*
Received 00th January 20xx,
Accepted 00th January 20xx
Aziz , Wagdy M. Eldehna , Hatem A. Abdel-Aziz , Paul A. Keller
DOI: 10.1039/x0xx00000x
www.rsc.org/
The screening of a small library of diverse chemical structures resulted in the identification of 2-thioxodihydropyrido[2,3-
-1
d]pyrimidine 10a as having broad spectrum antibacterial activity (MIC 0.49 - 3.9 μg mL ), and reasonable antifungal
-1
activity (MIC 0.49 - 3.9 μg mL ). An expeditious synthesis of 10a was optimized by varying solvents, catalysts and the use
of microwave irradiation with the best conditions using DMF as a solvent, I (10 mol%) and a 30 minute reaction time
2
compared to 15 h for classic conventional heating. The pharmacokinetic properties and calculation of drug likeness of 10a
suggested good traditional drug-like properties and led to the synthesis of a small library with seven compounds 10a and
-
1
1
0d-i showing broad antimicrobial activity (MIC = 0.49 – 7.81 μg mL ) and selectivity indices of more than 5.6 against the
normal colon cell line (CCD-33Co). The antifungal activity of compounds 10d-i was moderate to strong with MIC values of
-
1
1
.95 – 7.81 μg mL .
1
5, 16
a priori followed by determination of the modes of action.
Introduction
Most current antibiotics approved by the FDA for clinical
applications are derivatives of a limited number of chemical
The paucity of novel antimicrobial agents in contemporary
science is highly challenging with only 1.6% of new antibiotic
drugs in clinical trials, a situation arising from the reduction in
relevant drug discovery research, which leads to fewer novel
1
7,
classes or scaffolds, with many discovered in the mid-1980s.
1
Strikingly, most candidates in recent phase 3 clinical trials
8
1
9
were based on known scaffolds, including the tetracycline
derivative omadacycline the macrolide derivative
solithromycin II, trimethoprim analogue iclaprim III and the
quinolones derivatives zabofloxacin IV and delafloxacin
1
-6
I,
registered therapeutics. Since the dawn and overuse of
antibiotics, bacteria have exploited windows to readily develop
3
-12
V
resistance to the established anti-infective agents.
In vitro
1
9
(Figure 1).
target-based approaches have been used widely to discover
potent enzyme inhibitors, but many are devoid of whole cell
antibacterial activity, presumably due either to the inability to
reach the intracellular targets, or as a result of the process of
N
O
OH
O
O
N
N
O
O
H
H
O
O
O
H
1
3, 14
H
F
bacterial active efflux.
More promising is whole-cell
N
NH
2
O
OH
OH
N
OH OH
O
O
O
O
screening, where antimicrobial potencies have been identified
H
2
N
N
NH2
4
N
N
N
N
O
O
Omadacycline (I)
Solithromycin (II)
Iclaprim (III)
NH
2
O
O
O
N
O
F
O
N
O
OH
F
F
OH
R
N
NH
R
N
Cl
F
R
S
N
O
2
H N
F
N
NH2
N
O
N
ACH-702 R =
N
R =
R =
OH
NH
Zabofloxacin (IV)
Delafloxacin (V)
IQT (VI)
Figure 1. Examples of antibacterial agents in phase
isothiazoloquinoline derivatives (VI).
3 clinical trials (I-V),
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Figure 2. Chemical structure of compound PP1 (10a).
Attempts to discover novel antimicrobial scaffolds related to
known classes include the isothiazoloquinolones IQT VI (Figure
2
), which are related to the known quinolones. These
0
1
derivatives showed activity against methicillin sensitive
Staphylococcus aureus (MSSA) (MIC = 0.13 - 0.0020 μg/mL)
against methicillin resistant Staphylococcus aureus (MRSA)
(MIC = 4.0 - 0.060 μg/mL) and against Escherichia coli (E. coli)
(MIC = 0.50 - 0.0040 μg/mL). Examples of ITQ derivatives were
found to have 8- to 16- fold greater activity than the standard
drug against MRSA, and ACH-702 is in pre-clinical
Scheme 1: Reagents and conditions: (i) KOH, methanol, rt, (ii) NaOEt/ethanol, reflux, 4
2
0
h.
development.
The continued need for new anti-infective agents is well Therefore, 5-aryl-7-(thiophen-2-yl/4-chlorophenyl)-2-thioxo-
documented, especially those based on new chemical classes. 2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-ones 10a-j were
As part of an ongoing program searching for new leads, we prepared by heating at reflux equimolar quantities of 6-amino-
report here 2-thioxodihydropyrido[2,3-d]pyrimidine 10a which 2-thiouracil
revealed broad spectrum antibacterial activity against three yields of 44-65% (Scheme 2). In a typical example, analysis of
6 and the chalcones 3a-j in dry DMF for 15-20 h in
1
Gram -ve and two Gram +ve bacteria with MIC values ranging the H NMR spectra of 10b revealed a resonance at 7.22 ppm
from 0.98 to 3.9 μg/mL, (control drug 0.49 - 0.98 μg/mL), and assigned to C5-Ar H3 and H5 with the doublet at 7.49 ppm
interestingly it showed MIC = 31.3 μg/mL against MRSA assigned to C5-Ar H2 and H6. The pyridyl H6 was assigned to
1
control drug 3.9 μg/mL) and two fungi (control drug 0.49 - the singlet at δ 7.67 in the H NMR spectra. To the
corresponding C7 para substituted aryl substituent, Ar H3 and
(
0
.98 μg/mL).
Here we describe the development of this scaffold with the H5 was assigned the resonance at 7.58 ppm, while the
synthesis of a small library of compounds and their screening resonance at 8.25 ppm was assigned to Ar H2 and H6. Two
against Gram +ve, Gram -ve and MRSA bacteria as well as resonances at δ 12.38 and 13.08 were assigned to the two
1
3
antifungal agents. The calculation of the pharmacokinetic pyrimidyl NHs. The C NMR spectrum of the fluorine
properties of all compounds and the assessment of the containing compound 10b showed the splitting pattern of the
1
cytotoxicity of the most active compounds was also fluorine and carbon with coupling constants JF-C = 243.1 Hz at
2
resonance 161.3 ppm, JF-C = 20.9 Hz at resonance 114.2 ppm
performed.
3
and JF-C = 8.6 Hz at resonance 131.0 ppm.
Results and discussions
The reaction is an apparent heterocyclic condensation of the
uracil amino group with the propenone carbonyl 3a-j to afford
the Schiff bases 7a-j (Scheme 2). This mechanism has been
Synthesis of pyrido[2,3-d]pyrimidine derivatives 10a-j:
The required chalcone starting materials 3a-j were
prepared via a typical Claisen-Schmidt condensation of either
p-chloroacetophenone 1a or 2-acetyl thiophene 1b with
aromatic aldehydes 2a-e in alcoholic alkaline solution in yields
2
3
investigated indicating an initial conjugate addition, followed
by fragmentation with subsequent multistep reactions to
produce the ‘1,2-addition’ product. This then undergo
intramolecular cyclization providing the 5,6-dihydropyrido[2,3-
d]pyrimidines 8a-j which tautomerizes to the 5,8-
dihydropyrido[2,3-d]pyrimidines 9a-j which then oxidizes to
2
1
of 62-75% (Scheme 1). The synthesis of 6-amino-2-thiouracil
was accomplished by heating thiourea at reflux with ethyl
cyanoacetate in the presence of sodium ethoxide in 68%
yield (Scheme 1).
6
5
4
2
.
4-26
1
0a-j
It has been reported that condensation of 6-amino-
The classic Skraup synthesis of quinoline uses hot aniline,
nitrobenzene and glycerol in the presence of catalytic sulfuric
2
-thiouracil
6
with chalcone analogues containing Ar or Ar
1
2
2
2, 23 para-substitution that are electron withdrawing groups (eg,
acid and produces low yields of product (Figure 3).
NO
which this position is unsubstituted or is para-methoxy
substituted, the 5,8-dihydropyrido[2,3-d]pyrimidines
2
, Cl), the oxidized form 10 was directly obtained. Cases in
Doebner and von Miller replaced the glycerol with α,β-
unsaturated carbonyl compounds, with heating under acidic
conditions or in the presence of iodine as a catalyst to obtain
9
2
4
2
3
predominated. In our study, all examples were isolated as
quinolines in good yields (Figure 3). A further development
replaced the aniline with 6-amino-2-thiouracil to synthesize
the pyrido[2,3-d]pyrimidine ring instead of quinoline (Figure
the oxidized 10a-j, including the para-methoxy substituent 10i
presumably due to the extended reaction time (20 h) in air.
Microwave-assisted synthesis of the pyrido[2,3-d]pyrimidine
derivative 10a:
,
2
4
3
). We report here the continued evolution of this synthesis,
to synthesize the versatile pyrido[2,3-d]pyrimidine scaffold in
DMF at reflux in the absence of a catalyst.
The Doebner von Miller cyclocondensation reaction of 6-
amino-2-thiouracil with chalcone 3a was also investigated
under microwave conditions. Conventional heating of starting
materials and 3a at reflux in DMF gave 44% yield of 10a
table 1, entry 1). Replacing the solvent with trifluoracetic acid
6
6
(
(
TFA) and maintaining heating for 12 h decreased the yield
2
| J. Name., 2012, 00, 1-3
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(
28%) (table 1, entry 2), with further heating resulting in no formamide, while maintaining catalytic I
significant change. The use of microwave irradiation under provides
Supplementary Information), which supports the proposed
mechanism using either I (entry 8) or the Lewis acid indium
2
at 40 mol% (entry 11)
2-aminopyrido[2,3-d]pyrimidine
12
(see
2
chloride (entry 7) as catalysts.
Table 1. Formation of the pyridopyrimidine 10a under conventional heating or
microwave irradiation in the presence or absence of catalyst
Entr
Solvent
Catalyst
Method
Time
Yield%
y
1
2
3
DMF
TFA
Free
Free
Free
Reflux
Reflux
15 h
12 h
1 h
44
28
18
catalyst free
Figure 3. Skraup and Doebner-Miller synthesis of quinolone and Quiroga et. al synthesis
DMF
MW (160 °C,
of pyrido[2,3-d]pyrimidine.
1
50 W)
O
4
5
6
7
8
9
DMF
DMF
DMF
DMF
DMF
DMF
DMF
Free
Free
MW (240 °C,
150 W)
2 h
4 h
1 h
1 h
1 h
26
Traces
Traces
Traces
15
Ar
2
O
Ar2
O
H
O
H
NH
i
NH
∗
NH
Ar
1
Ar2
+
H
2
N
N
S
Ar
N
N
H
S
H
4
0-65%
1
Ar1 H+N
N
H
S
MW (240 °C,
150 W)
3a-j
6
7
a-j
8a-j
Yb(OTf)
3
(40
MW (160 °C,
150 W)
7
-10 Ar
1
Ar
-C
2
Yield %
44
Tautomerism
H+/+H+
a
b
c
d
e
f
4-ClC
4-ClC
4-ClC
4-ClC
4-ClC
6
6
6
6
6
H
4
6
H
5
mol%)
-
H
4
4-FC
6
H
4
52
InCl
3
(50
MW (160 °C,
150 W)
H
4
4-ClC
6
H
4
6
50
H
4
4-OMeC
4-NO
-C
H
4
46
mol%)
H Ar
2
O
H
4
2
C
6
H
4
61
Ar
2
O
2-thienyl
2-thienyl
2-thienyl
2-thienyl
2-thienyl
6
H
5
65
57
43
40
50
∗
NH
I
I
I
2
2
2
(40 mol%)
MW (160 °C,
150 W)
NH
air oxidation
g
h
i
6 4
4-FC H
Ar
1
N
H
N
S
4-ClC
6
H
4
Ar
1
N
N
S
H
H
4-OMeC
4-NO
6
H
4
10a-j
9a-j
(30 mol%)
(10 mol%)
MW (160 °C,
40
min
30
32
j
2
C
6
H
4
1
50 W)
MW (160 °C,
50 W)
1
0
59
Scheme 2: Reagents and conditions: (i) Dry DMF, reflux 15-20 h.
1
min
1h
conditions for 1 h in DMF at 160 °C gave 10a in low yield (18%)
table 1, entry 3), which improved marginally with increased
11
Forma
mide
I2 (40 mol%)
MW (160 °C,
150 W)
Traces
(
temperature and time (table 1, entry 4). Reaction monitoring Pharmacokinetic properties and drug likeness calculations
by mass spectrometry analysis indicated the reduced form 9a
Drug likeness estimations have been used as tools to minimize
attrition in the process of drug discovery and refers to the
27
was synthesized, presumably as the reaction was performed in
a sealed tube with a limited oxygen supply. Upon workup, the
reaction was left overnight to crystallize resulting in an
efficient oxidation of 9a to 10a. Application of higher
temperature (240 °C) for longer time periods (4 h) in catalyst
free conditions only gave traces of 10a (table 1, entry 5).
similarity of compound properties to existing oral drugs.
Strategies to assess drug likeness include the Lipinsiki
28
guidelines. Interestingly, all compounds fulfil the Lipinsiki
criteria, which indicate the possibility of oral bioavailability
(Table 2). All compounds 10a-j have 2-3 rotatable bonds (Table
The Doebner-Von Miller synthesis of quinoline has been
2
). Reduced molecular flexibility is measured with the number
reported to be catalysed by iodine, Lewis acids (InCl
3
, Yb(OTf)
3
,
,
of rotatable bonds and is greatly linked to the oral
29
bioavailability with the number of rotatable bonds less or
30
equal to ten potentially increases the oral bioavailability.
Sc-(OTf) , ZnCl ) and Brønsted acids (TsOH, HClO
2
3
4
2
3
Amberlite). Therefore, further optimization proceeded using
catalytic ytterbium(III) trifluoromethanesulfonate Yb(OTf) or
indium chloride (InCl ), which resulted in traces of the desired
3
Percentage of the absorbed drugs (%ABS) is inversely
proportional to the molecular volume and topological polar
3
compound, however, the use of iodine (40 mol%) gave a 15%
yield in a 1 h reaction (table 1, entry 8). Reducing the reaction
time enabled the catalytic loading to be reduced to 10 mole%,
providing 10a in 59% isolated yield (table 1, entry 10).
surface area (TPSA) and is calculated using the formula %ABS =
31
1
09 ± 0.345 x TPSA (Table 2). Compounds 10a-j possess %ABS
ranging from 71.96 to 87.77. The number of rotatable bonds
nrot), TPSA, molecular volume and %ABS provide indications
(
2 3
Higher catalytic loadings of I and InCl (entry 8 and entry 7,
to sustain good oral bioavailability of drug candidates.
respectively) resulted in the synthesis of 11 as a by-product in
1
yields of 45% and 38%, respectively. The H NMR data of 11
The drug likeness score (DLS) of all the compounds 10a-g were
32
calculated using MolSoft software and received a positive
(
see Supplementary Information) indicated a resonance for
only one NH of the pyrimidine at 11.20 ppm and a resonance
at 3.14 ppm was assigned to the two CH of the N,N-dimethyl,
drug likeness score from 0.05 to 0.66 (Table 2). Compounds
eliciting positive values should be considered as drug
candidates, while those with zero or negative values might not
3
which suggests a nucleophilic substitution reaction of the thiol
group in 10a yielding compound 11. The proposed mechanism
starts with the generation of dimethylamine, which undergoes
nucleophilic addition to C2 of 10a with the thiol group acting
as the leaving group. The replacement of DMF with
31
be considered as drug-like candidates. The 2-thioxo-2,3-
dihydropyrido[2,3-d]pyrimidinone 10h counterpart emerged
as the most similar to drugs (DLS = 0.66) followed by 2-thioxo-
2,3-dihydropyrido[2,3-d]pyrimidinone 10a and 10d (Table 2).
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Our physicochemical calculation indicated that the compounds likeness model scores, which enables them to be potential oral
0a-j fulfilled the Lipinski guidelines and have positive drug- bioavailable leads.
1
Table 2. Drug likeness calculations and Lipinski parameters of the compounds 10a-10j
n
violations
≤1
Drug-likeness
a
b
c
d
f
g
h
i
Com
Rule
Ar
1
Ar
2
M. Wt
≤500
LogP
HBA
HBD
e
Rule of 5 nrot
TPSA
Volume
%ABS
model score
≤5.0
4.39
≤10
3
≤5
2
1
0a
0b
0c
4-ClC
4-ClC
6
6
H
4
-C
4-FC
4-ClC
4-OMeC
4-NO
6
H
5
365.04
383.03
0
0
Pass
Pass
2
2
61.55
61.55
297.05
301.98
87.77
87.77
0.61
0.36
1
H
4
6
H
4
4.51
3
2
1
4-ClC
4-ClC
4-ClC
6
6
6
H
4
6
H
4
399.00
395.05
411.03
337.03
355.02
371.00
367.04
383.03
4.98
4.46
4.46
3.81
3.94
4.37
3.79
3.96
3
4
5
4
4
4
5
6
2
2
3
2
2
2
5
0
0
0
0
0
0
0
0
Pass
Pass
Pass
Pass
Pass
Pass
Pass
Pass
2
3
3
2
2
2
3
3
61.55
70.78
107.37
61.55
61.55
61.55
70.78
107.37
310.58
322.59
320.38
274.22
279.15
287.76
299.77
87.77
84.58
71.96
87.77
87.77
87.77
84.58
71.96
0.39
0.61
0.22
0.05
0.57
0.66
0.45
1
0d
0e
H
4
6 4
H
1
H
4
2 6 4
C H
1
0f
2-thienyl
2-thienyl
-C
6 5
H
1
0g
6 4
4-FC H
1
0h 2-thienyl
4-ClC
2-thienyl 4-OMeC
2-thienyl 4-NO
6 4
H
10i
6 4
H
1
0j
2
C
6
H
4
c
3
297.56
0.22
a
b
d
e
f
g
Molecular weight; Lipophilicity; Number of hydrogen bond acceptors; Number of hydrogen bond donors, Number of violations, Number of rotatable bonds, Topological
h
i
polar surface area; Molecular volume; Percentage absorption.
unsubstituted 10f reduced activity against B. subtilis and E. coli
−
1
Anti-microbial and Cytotoxicity Evaluation
by a factor of four with an MIC value of 1.95 μg mL . Insertion
of fluorine or chlorine in the Ar2 para-position resulted in 10g
and 10h, with the former maintaining activity against S.
Antibacterial activities and selectivity indices
Compounds 10a
,
10d-10i showed promising inhibition zone
−1
pneumonia (MIC = 0.98 μg mL , SI of 30.9) and B. subtilis (MIC
−1
0.98 μg mL and SI = 30.9), with acceptable activity against
values in comparison to the standard drugs ampicillin (Am), for
Gram +ve bacteria, and gentamycin (Gm), for Gram -ve
bacteria. These compounds were further tested for their
=
S. aureus, P. aeruginosa and E. coli (MIC = 1.95, 3.90 and 1.95
−1
μg mL , respectively). The chloro derivative 10h was
−1
minimum inhibition concentration (MIC) in μg mL and
showed excellent to good activities across the tested Gram +ve
and Gram –ve bacteria (Table 3).
equipotent to the controls against B. subtilis, P. aeruginosa
−1
and E. coli (MIC = 0.49, 0.98 and 0.49 μg mL , respectively)
and possessed half the activity against S. aureus (MIC = 0.98 μg
−1
mL ) with a selectivity index of >13.7. Generally, the chloro
The activity of 2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidinone
1
0a was excellent against S. pneumonia and E.coli (MIC = 0.98
counterpart 10h showed the best activity against all the tested
−1
μg mL ) with a therapeutic index SI = 22.1 (Table 3). It also
strains except for S. pneumonia. Substituting a OMe at the Ar
2
displayed good activity against B. subtilis, S. aureus and P.
C4 of derivative 10f resulted in a halving of activity against S.
−1
pneumonia and B. subtilis (MIC = 1.95 and 0.98 μg mL ,
−1
aeruginosa (MIC = 1.95, 3.9 and 3.9 μg mL , respectively).
Grafting fluorine or chlorine in the Ar2 para-position (10b and
respectively), and a SI >51.3. The observed different activities
1
0c respectively) decreased activity against all strains. The
of 10i towards the rest of the strains were good to fair (MIC =
−1
addition of the para-methoxy group (10d) increased activity
3.9 - 7.81 μg mL , SI > 12.8).
−1
against S. pneumonia (MIC = 0.49 μg mL ), while maintaining
The para-methoxy derivative 10i is the least cytotoxic among
all the tested compounds (IC50 > 100 μM). The presence of the
methoxy group increased the activity of 10i relative to the
unsubstituted 10f towards S. pneumonia and B. subtilis, while
maintaining the activity against S. aureus and p. aeruginosa.
the antibacterial activity of the standard drug against the Gram
+ve bacteria B. subtilis and the Gram -ve bacteria P. aeruginosa
−1
and E. coli (MIC = 0.49, 0.98 and 0.49 μg mL , respectively).
Compound 10d is the least cytotoxic compound with a SI
against all the tested strains >46.8. Compound 10e bearing an
Ar2 4-nitro substituent retained the antibacterial activity of
2
Insertion of a nitro group in C4 of Ar (10j), showed only small
inhibition zones and was not measured for MIC determination
due to reduced antibacterial activity. Generally, the thiophene
containing analogues 10f-j activities showed excellent to
moderate activities, but with different trend to the 7-p-
chlorophenyl analogues 10a-e. The activity decreased in the
order of 4-Cl>4-F>4-OMe>unsubstituted>nitro. Therefore, this
might unravel the particularity of the incorporation of a small
halogen or an electron donating group in the para position of
the phenyl motif of the 5-phenyl-7-(thiophen-2-yl)-2-thioxo-
1
0a and showed excellent potency against S. pneumonia and
−
1
E. coli (MIC = 0.98 μg mL ) and an acceptable selectivity index
35.2) and elicited good activities against the other three
(
−1
bacteria (MIC = 0.98 μg mL ) with SI = 17.7. The effect of
substitution of the 7-para-chloropyrido[2,3-d]pyrimidines 10a-
e, grafting a methoxy or a nitro group onto the Ar2 para-
position is beneficial for the activity, whereas, substitution
with halogens decreased the activity dramatically.
Bioisosteric replacement of the 7-(para-chlorophenyl) motif in
2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one 10f (Table 3).
10a-e with a 2-thienyl gave compounds 10f-j (Table 3). The
4
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Journal Name
ARTICLE
−
1
Table 3. In vitro antibacterial activity screening of the synthesized compounds 10a-j as inhibition Zones (IZ) in millimetres and minimum inhibitory concentrations (MICs) in μg mL
IC50
Gram +ve Bacteria
Gram -ve Bacteria
(
µM)
Sp
Bs
Sa
MRSA
Pa
Ec
Com
(CCD-
I.Z
21.9±0.58
MIC
.98
I.Z
MIC
I.Z
MIC
I.Z
MIC
I.Z
MIC
I.Z
MIC
3
3Co)
0
1.95
(11.1)
NT
3.90
(5.6)
NT
16.3±0.67 31.25
(0.7)
3.90
(5.6)
NT
0.98
(22.1)
NT
1
0a
21.2±0.67
20.1±2.1
20.4±0.46
21.6±1.2
21.65
(
22.1)
1
0b 12.3±0.58
0c 16.3±0.44
NT
14.6±0.67
17.2±1.2
13.5±1.2
NT
NT
NT
NT
14.2±0.58
16.7±0.58
16.3±0.46
18.2 1.2
NT
NT
1
NT
NT
15.4±0.63
NT
NT
NT
0
.49
0.49
>100
1.95
(17.7)
1.95
(46.8)
1.95
(17.7)
19.6±0.58
3.90
(23.4)
7.81
(4.4)
NA
0.98
(93.2)
1.95
(17.7)
0.49
>100
0.98
(35.2)
1
0d 24.2±0.44
23.6±1.2
21.3±0.63
20.6±1.2
20.2±0.25
20.9±0.58
23.5±0.23
20.3±0.67
16.9±1.5
26.2±1.2
22.4±0.58
21.3±0.25
18.3±0.44
19.2±1.2
24.6±1.2
22.4±0.63
21.2±0.44
21.4±1.2
91.30
34.50
NT
>100
0
.98
18.3±0.25
NA
1
0e
0f
0g
22.4±1.2
18.7±0.44
22.3±0.63
21.3±0.25
(35.2)
2
1.3±
.25
1
3.90
1.95
3.9
7.81
1.95
0
0
.98
30.9)
.90
13.7)
0.98
(30.9)
0.49
1.95
(15.5)
0.98
NA
NA
3.90
(7.8)
0.98
1.95
(15.5)
0.49
1
23.4±1.5
24.3±1.2
22.1±1.5
18.1±0.67
30.26
53.50
>100.00
NT
(
3
21.3±1.2
NA
1.95
(27.4)
NA
1
0h 20.4±0.44
23.4±0.63
18.4±0.63
16.3±1.5
24.9±0.58
(
>100
0.98
(54.7)
3.90
(54.7)
7.81
>100
3.90
1
.95
1
0i
21.3±0.44
19.4±0.67
23.8±0.63
20.3±1.2
18.2
>51.3
>100
>25.6
>12.8
>25.6
NT
NT
1
0j
NT
NT
NT
NT
NT
±0.46
3
2.4±
.72
Am
0.98
0.49
0.49
0
Gm
VM
22.3±0.58
0.98
25.4±1.2
0.49
20.3±0.63
3.90
NT: Not tested: Selectivity Indices presented in brackets below the MIC values. The screening organisms, Gram +ve bacteria: Streptococus pneumoniae (RCMB 010010,
Sp), Bacillus subtilis (RCMB 010067, Bs) and Staphylococcus aureus (RCMB) 010028, Sa), MRSA: Methicillin Resistant Staphylococcus aureus. Gram -ve bacteria:
Pseudomonas aeruginosa (RCMB 010044, Pa) and Escherichia coli (RCMB 010053, Ec), Am: Ampicillin, Gm: Gentamicin, Vm: Vancomycin
The encouraging antimicrobial results with compounds 10a
0d-i inspired the further testing of these compounds against
MRSA (Table 3). The 2-thioxo-2,3-dihydropyrido[2,3- 10e demonstrated weak activity (MIC = 15.63 μg mL ). The
d]pyrimidinone 10h provided a two-fold activity increase (MIC selectivity indices of 10d 10h and 10i were within the
1.95 μg mL , SI = 27.4) relative to that of vancomycin (MIC = acceptable range. The compounds showed better activities
,
dihydropyrido[2,3-d]pyrimidinones 10f and 10i showed fair
−
1
1
activity against C. albicans (MIC = 7.81 μg mL ) and compound
−1
,
−
1
=
3
−
1
.90 μg mL ). The 4-OMe derivative 10d maintained the and selectivity indices against A. fumigatus than C. albicans.
−
1
activity of the vancomycin (MIC = 3.90 μg mL , with SI = 23.4)
−1
(Table 3), while, compounds 10a (MIC = 31.25 μg mL and SI =
−1
Aspergillus fumigatus
Candida albicans
0.7) and 10e (MIC = 7.81 μg mL and SI = 4.4) showed modest
Compound
IZ
MIC
31.25 (0.7)
NT
IZ
MIC
31.25 (0.7)
NT
activity (Table 3). The 2-thioxo-2,3-dihydropyrido[2,3-
d]pyrimidinones 10a 10d, 10e and 10h inhibited the growth of
MRSA, however, the remaining compounds were inactive.
Among the tested compounds 10a 10d-i, the 2-thioxo-2,3-
1
0a
0b
19.2 ± 0.6
15.2 ± 1.2
15.2 ± 1.2
21.3 ± 1.5
20.1 ± 0.6
18.6 ± 1.2
21.3 ± 1.5
20.3 ± 1.2
16.4 ± 1.2
14.1 ± 0.3
13.4 ± 1.5
20.8 ± 1.2
17.3 ± 0.6
18.3 ± 0.6
19.2 ± 0.6
19.3 ± 1.5
,
1
1
0c
10d
0e
0f
NT
NT
,
−
1
1.95 (46.8)
3.90 (8.9)
3.90
1.95 (46.8)
15.63 (2.2)
7.81
dihydropyrido[2,3-d]pyrimidinone 10h (MIC = 1.95 μg mL )
emerged as the most active, eliciting double the activity of
vancomycin.
1
1
1
0g
0h
1.95 (15.5)
3.90 (13.7)
3.90 (7.8)
3.90 (13.7)
Anti-fungal activity
1
The antifungal activity of compounds 10a
showed that 2-thioxo-2,3-dihydropyrido[2,3-d]pyrimidinones
0d 10g and 10i as the most active agents against A.
fumigatus with MIC values = 1.95 μg mL followed by 10e
, 10d-i (Table 4)
1
0i
20.6 ± 1.2 1.95 (>51.3) 18.3 ± 0.6 7.81 (>12.8)
1
0j
16.3 ± 0.6
23.7± 1.2
NT
12.6 ± 2.1
25.4± 0.6
NT
1
,
AB
0.98
0.49
−
1
,
10f
−1
and 10h with MIC values = 3.90 μg mL . Selectivity indices of
the tested compounds ranged from 13.7 to >51.3 except for
compound 10a (0.7) and 10e (8.9). The 2-thioxo 10d analogue
−
1
(
MIC = 1.95 μg mL ) was the most active analogue followed by
−1
1
0g and 10h (MIC = 3.90 μg mL ). The 2-thioxo-2,3-
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Journal Name
Table 4. In vitro antifungal activity screening of the synthesized compounds 10a-j as
1
L. L. Ling, T. Schneider, A. J. Peoples, A. L. Spoering, I. Engels,
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−
mL
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zones expressed in millimetres, MIC: Minimum inhibitory concentration
expressed in μg/mL. The screening organisms: Mould: Aspergillus fumigatus
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Conclusions
The synthesis and antimicrobial evaluation of the
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5,7-disubstituted
pyrido[2,3-d]pyrimidine
1
1
derivatives were performed with a microwave assisted
1
synthesis of 10a optimized to produce better yields in shorter
times. Using DMF as the solvent and with 10 mol% of I under
2
microwave radiation for 30 minutes gave the best yield 59% 12 J. M. Blair, M. A. Webber, A. J. Baylay, D. O. Ogbolu and L. J.
Piddock, Nat. Rev. Microbiol. 2015, 13, 42-51.
yield. The compounds were tested in vitro for their
1
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antibacterial activity and moderate to excellent activity was
2
4
observed for compounds 10a and 10d-i with MIC = 0.49–7.81
1
1
−1
μg mL (Figure 4). Compounds 10d-e and 10h displayed
−1
potent inhibition of MRSA (MIC =1.95–3.90 μg mL ). Among
the tested compounds, derivatives 10d 10g and 10i exhibited
Glickman, ACS Chem. Biol. 2014, 9, 2572-2583.
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,
−1
the best anti-aspergillus activity (MIC = 1.95 μg mL ) and
compound 10d represent the best obtained against Candia
1
7 P. Fernandes and E. Martens, Biochem. Pharmacol. 2017,
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excellent in silico pharmacokinetic properties calculation. The 18 A. L. Demain, Chem. Biol. 2011, 18, 939.
−1
albicans (MIC = 1.95 μg mL ). All the compounds showed
1
1
9 WHO report, Antibacterial agents under clinical
development, 2017, WHO/EMP/IAU/2017.11.
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best compounds 10d and 10h will be subjected to a further
cycle of derivatization to explore more about the structure
antimicrobial relationship of this scaffold and to eventually
develop the best derivatives into potentially novel and safe
antimicrobial agents.
2
2
1 Y. Qian, G.-Y. Ma, Y. Yang, K. Cheng, Q.-Z. Zheng, W.-J. Mao,
L. Shi, J. Zhao and H.-L. Zhu, Bioorg. Med. Chem. 2010, 18
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,
4
2
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,
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Ghabbour, A. Abdel-Megeed, W. M. Eldehna, H. A. Abdel-
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Figure 4. SAR of all the compounds against the tested bacterial and fungi.
2
2
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Conflicts of interest
2
9 M. J. Ahsan, J. G. Samy, H. Khalilullah, M. S. Nomani, P.
Saraswat, R. Gaur and A. Singh, Bioorg. Med. Chem. Lett.
There are no conflicts to declare.
2
011, 21, 7246-7250.
3
3
3
0 D. F. Veber, S. R. Johnson, H.-Y. Cheng, B. R. Smith, K. W.
Ward and K. D. Kopple, J. Med. Chem. 2002, 45, 2615-2623.
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Acknowledgements
MF thanks UOW for the University Postgraduate Award and
International Postgraduate Tuition Award scholarships. Patrick
McCosker is highly appreciated for his efforts revising the first
draft of the Manuscript.
2 http://molsoft.com/mprop/
References
6
| J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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DOI: 10.1039/C8OB00627J
An improved synthesis of pyrido[2,3-
d]pyrimidin-4(1H)-ones and their
antimicrobial activity