Fragment Discovery for the Design of Nitrogen Heterocycles as Mycobacterium tuberculosis Dihydrofolate Reductase Inhibitors

Article abstract of DOI:10.1002/ardp.201600066

Fragment-based drug design was used to identify Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitors. Screening of ligands against the Mtb DHFR enzyme resulted in the identification of multiple fragment hits with IC₅₀ values in the range of 38–90 μM versus Mtb DHFR and minimum inhibitory concentration (MIC) values in the range of 31.5–125 μg/mL. These fragment scaffolds would be useful for anti-tubercular drug design.

Full text of DOI:10.1002/ardp.201600066

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Archiv der Pharmazie
Full Paper
Fragment Discovery for the Design of Nitrogen Heterocycles as
Mycobacterium tuberculosis Dihydrofolate Reductase Inhibitors
Rupesh U. Shelke1, Mariam S. Degani1, Archana Raju1, Mukti Kanta Ray2, and Mysore G. R. Rajan2
1
Institute of Chemical Technology, Nathalal Parekh Marg, Matunga, Mumbai, India
Radiation Medicine Centre, Tata Memorial Hospital, Parel, Mumbai, India
2
Fragment-based drug design was used to identify Mycobacterium tuberculosis (Mtb) dihydrofolate
reductase (DHFR) inhibitors. Screening of ligands against the Mtb DHFR enzyme resulted in the
identification of multiple fragment hits with IC50 values in the range of 38–90 mM versus Mtb DHFR
and minimum inhibitory concentration (MIC) values in the range of 31.5–125 mg/mL. These fragment
scaffolds would be useful for anti-tubercular drug design.
Keywords: DHFR / FBDD / Mycobacterium tuberculosis / Nitrogen heterocycles
Received: March 15, 2016; Revised: May 21, 2016; Accepted: May 27, 2016
DOI 10.1002/ardp.201600066
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
which arrests DNA biosynthesis and cause the cell death [6].
This “thymineless death” by DHFR inhibitors is a useful goal in
Introduction
Tuberculosis (TB) remains one of the world’s deadliest
communicable diseases. The worldwide mortality rate of TB
is more than 1.5 million people per year, and now ranks
alongside HIV as a leading cause of death. In addition, multi-
drug resistant TB strains (MDR-TB) have emerged as a
worldwide problem. In 2014, approximately 480000 people
developed MDR-TB worldwide [1]. With the emergence of
MDR-TB and extensively drug-resistant TB (XDR-TB), there is
an urgent need to develop new anti-TB drugs acting on novel
targets having minimum cross-resistance with existing
drugs [2, 3].
Dihydrofolate reductase (DHFR) is an important enzyme in
both mammals and microorganisms including Mycobacterium
tuberculosis (Mtb). It catalyzes folic acid conversion to dihydro
and tetrahydro folic acid (a cofactor involved as a one carbon
donor in purine and pyrimidine de novo synthesis) which is a
crucial step in the folate pathway [4, 5]. Inhibition of the
folate pathway leads to interruption of the thymidine supply,
the therapy of cancer (methotrexate), bacterial (trimetho-
prim), and protozoal infections (pyrimethamine) [7]. A range
of potential anti-tubercular compounds have been designed
based on three-dimensional structure of Mtb DHFR reported
by Li et al. [8]. 2,4-Diamino-substituted pyrimidine scaffold
was found to be the common feature of many reported
Mtb DHFR inhibitors [9–13]. However, several researchers
have attempted to design DHFR inhibitors without this
feature [14–16].
Fragment-based drug design (FBDD) has now emerged as an
alternative to HTS [17, 18]. Zelboraf (vemurafenib) [19] was
approved by FDA in 2012, and is considered the first drug
derived from FBDD, for late stage melanoma. Hajduk et al. [20]
analyzed 45 projects in-house, which underwent both FBDD
and conventional HTS and pointed out that FBDD offers several
attractive aspects compared to HTS. FBDD approach has been
successfully used for development of Mtb thymidine mono-
phosphate kinase inhibitors [21]. Bastien et al. [22] designed
specific inhibitors for bacterial trimethoprim resistant type II
R67 DHFR by FBDD approach, leading to symmetrical bis-
benzimidazolecompoundsandabis-carboxyphenylcompound
which selectively inhibited the target (Ki ¼ 2–4 mM).
Correspondence: Prof. Mariam S. Degani, Institute of Chemical
Technology, Nathalal Parekh Marg, Matunga, Mumbai 400019,
India.
E-mail: ms.degani@ictmumbai.edu.in
Fax: þ91-22-3361-1020
Our research group has been involved in the search of DHFR
inhibitors for several pathogens including Mtb [13, 23–26].
Recently, we have designed 2,4-diamino-s-triazines with
potential activity against Mtb DHFR [27].
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
1

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
In this light, the current study was aimed at FBDD of Mtb DHFR
inhibitors to identify fragment scaffolds as potential hits.
Various nitrogen heterocycles such as triazines [28], 2,4-
diaminopyrimidines [9], purines [29], quinazolinones [30], and
aminoquinazolines [31] are reported to have antimycobacte-
2,4-diaminopyrimidine feature but having structural similar-
ity with the dihdyrofolic acid (Fig. 1).
Results and discussion
rial activity. Hence,
a fragment library was designed
considering various nitrogen heterocycles such as cyanouracil,
The basic principles of FBDD such as “rule of three” [18] were
xanthines, and quinazolinones without the “classical”
followed to virtually screen a library of diverse heterocycles.
OCH3
O
O
N
O
O
N
O
H
N
CN
N
O
HN
N
N
N
NH
N
N
HN
N
O
N
O
N
NH2
aH
O
N
O
4
3
h
2
a
2
O
N
N
N
N
HN
N
1
O
N
H
O
N
O
2b
N
H
2i
O
d
3
OCH3
O
N
HN
N
N
N
N
O
N
c
N
O
2
O
N
H
l
2
2
Cl
b
3
OCH3
O
O
N
N
N
N
N
HN
O
N
O
N
H
N
N
O
O
m
F
c
3
2d
Cl
O
N
N
N
N
HN
N
N
N
O
O
k
2
N
O
O
N
e
2
e
3
Cl
N
N
N
N
N
HN
O
N
2j
O
N
2f
F
O
N
HN
N
O
N
2g
Figure 1. Structures of the fragments.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
2

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Fragment Discovery for the Design of Mtb DHFR Inhibitors
Archiv der Pharmazie
The ADME properties of the designed fragments were
analyzed using Qikprop [32] (Table 1).
obtained may be further optimized to potential anti-
tubercular compounds.
Molecular docking study revealed that all the fragments
fitted well in the active site of the DHFR enzyme. All the
fragments showed hydrogen bonding with Ile94, one of
the key residues present in active site of the enzyme. Few of
the fragments also showed interactions with Asp27. p–p
stacking with Phe31 was observed in case of xanthine and
quinazolin-4-one fragments (Fig. 2).
The reasonably high values of Glide score (ꢀ6.10 to
ꢀ7.85 kcal/mol) and Emodel score (ꢀ37.03 to ꢀ55.83 kcal/
mol) indicate the stability of fragments in the active site.
As a part of our ongoing research, we have developed
several protocols for synthesis of heterocycles like cyanouracil
(1) [33].
Conclusions
The designed and synthesized fragments have shown
promising biological activity with IC50 (38–90 mM) against
Mtb DHFR and MIC (31.5–125 mg/mL) against Mtb. Thus, a
library of fragment scaffolds active against target enzyme is
obtained. These hits can be used as starting points to design
novel compounds as potential leads having significant activity
against M. tuberculosis.
Xanthine derivatives were synthesized using 6-aminouracils
as starting materials [34]. Bromination of 6-aminouracils with
NBS gave 5-bromo-6-aminouracils which upon nucleophilic
substitution by substituted amines gave 5,6-diaminouracils.
This was then cyclized using triethylorthoformate as one
carbon donor to yield xanthine using microwave irradiation
(Scheme 1). 1,3-Dimethylxanthine (2a) was synthesized from
uracils by nitrosation, followed by reduction and
cyclization [35].
Experimental
Design of fragments
Docking studies were carried out using standard Glide
molecular docking protocol implemented within the
€
Maestro molecular modeling suit by Schrodinger, LLC,
New York, NY, 2008 installed on Intel Pentium 4 computer.
The protein structure, PDB code 1DF7 [42] which is the
ternary complex of enzyme, NADPH, and methotrexate,
̊
An efficient and versatile method for the preparation
of a series of 2- and 3-substituted 4-quinazolinones via a
one-pot three component reaction of commercially
available starting materials was developed. The protocol
involved use of microwave as an efficient energy source
(Scheme 2). 2-Substituted quinazolin-4-ones were synthe-
sized using anthranilic acid, substituted aldehyde, ammo-
nium acetate as a source of ammonia, and acetic acid as a
catalyst. The reaction took place within 15–20 min under
microwave irradiation at 80 W. Synthesis of 3-substituted
quinazolin-4-one involves heating anthranilic acid, substi-
tuted amine, ortho ester, and acetic acid as a catalyst. It
was found that among various ortho esters, triethylor-
thoformate gave good yields of the product. This
protocol was better than the reported protocols, which
have several drawbacks such as harsh reaction conditions
(120°C) [36a], use of hazardous catalysts [36b], long
reaction time [36c], and two-step synthesis [36d]. 2-
Aminoquinazolin-4-one (4) was synthesized using
reported methods [37].
Synthesized fragments were screened against Mtb DHFR
and IC50 was determined spectrophotometrically [38] using an
in house protocol [39]. Fragments were also screened against
Mtb (H37Rv) in order to determine the actual minimum
inhibitory concentration (MIC) with Resazurin microtiter assay
(REMA) (Table 2) [40, 41].
Fragments showed moderate to good activity against Mtb
DHFR enzyme (IC50 in the range 38–90 mM). These were also
active against Mtb in whole cell assay (MIC in the range 31.5–
125 mg/mL). Quinaozolin-4-one derivatives were found to be
more active in Mtb whole cell assay compared to cyanouracil
and xanthine fragments. Thus, these active fragments
with a resolution of 1.7 A was retrieved from Brookhaven
Protein Data Bank (www.rcsb.com) and used for the
validation of the docking algorithm. All water molecules
were deleted, bond orders, and charges of co-factor
NADPH and methotrexate were assigned properly in the
protein preparation step. A grid which is the representa-
tion of shape and properties of receptor using several
different sets of fields was generated. The structure of
methotrexate was randomized using Ligprep [43] and
used for the cognate docking studies. The docking study
was carried out using SP mode with “must match” of at
least one hydrogen bond criterion; with Ile94, Ile5, and
Asp27.
Ligand structures were constructed using build option
within Maestro using standard geometries and standard bond
lengths. With the help of Ligprep facility, appropriate
hydrogens were added and
a single, low-energy, 3D
conformation was generated for each of the ligand by energy
minimization using MMFFs with a dielectric constant of 1.0. In
order to further understand the stability of fragments in
active site of enzyme, MM-GBSA post-docking scoring was
employed.
Chemistry
All chemicals and reagents used were of reagent grade.
Thin layer chromatographic (TLC) analyses were per-
formed on E-Merck silica gel 60 F-254 precoated aluminum
TLC plates. Melting points were determined on Therm-
ionic Campbell electronics melting point instrument and
are uncorrected. FTIR spectra were recorded on Perkin-
Elmer infrared spectrometer with KBr discs. 1H NMR
spectra were recorded on 300 MHz Bruker 300
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
3

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
Table 1. Qikprop studies of the fragments.
Rule of
three
Entry
1
Structure
QPlogPo/w
PSA
SASA
FOSA
FISA
PISA
WPSA
0.51
97.41
409.03
0
180.63
228.40
0
0
2a
ꢀ0.02
87.68
369.01
158.09
141.54
69.38
0
0
2b
2c
0.87
1.73
99.46
86.64
429.96
461.26
37.64
167.01
113.97
225.31
232.75
0
0
0
0
114.54
2d
1.65
85.65
459.14
116.59
118.71
223.84
0
0
2e
2f
2.13
2.18
85.65
85.63
491.29
483.08
204.77
116.63
118.71
118.59
167.81
176.22
0
0
0
71.65
2g
1.89
85.63
468.07
116.62
118.60
185.83
47.02
0
2h
2i
1.73
2.21
93.91
71.05
493.32
508.39
209.32
196.15
118.68
88.29
165.33
223.95
0
0
0
0
(Continued)
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
4

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Fragment Discovery for the Design of Mtb DHFR Inhibitors
Archiv der Pharmazie
Table 1. (Continued)
Rule of
three
Entry
2j
Structure
QPlogPo/w
PSA
SASA
FOSA
FISA
PISA
WPSA
2.59
71.05
540.53
284.33
88.29
167.91
0
0
2k
2.46
71.02
517.43
196.21
88.22
185.98
47.02
0
2l
2.25
2.72
79.33
71.01
542.60
532.44
288.88
196.21
88.27
88.21
165.46
176.38
0
0
0
2m
71.65
3a
3b
1.14
2.59
53.44
58.47
377.22
514.35
101.27
92.71
94.26
86.77
181.69
334.87
0
0
0
0
3c
3.06
2.71
50.19
50.19
504.07
480.02
0
0
86.76
86.76
345.66
393.26
71.65
0
0
3d
0
3e
4
2.70
0.09
40.48
74.08
454.95
351.93
0
0
59.77
395.19
194.61
0
0
0
0
157.33
spectrometer using tetramethyl silane (TMS) as internal
reference. The values of chemical shifts (d) are expressed in
ppm. Abbreviations used were, s ¼ singlet, bs ¼ broad
singlet, d ¼ doublet, dd ¼ doublet of doublet, t ¼ triplet,
m ¼ multiplet. Physical and spectral properties were in
accordance with the literature.
The NMR and IR spectra as well as the InChI codes of the
investigated compounds are provided as Supporting Information.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
5

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
Figure 2. Interactions of some fragments with the Mtb DHFR.
Synthesis of 7-benzyl-1,3-dimethyl-1H-purine-2,6(3H,7H)-
dione (2i)
The procedure for the synthesis of 2i is described as an
washed with water. Yield 1.2 g (80%); mp 215–216°C
(214–216°C) [44]; IR (KBr): vmax ¼ 3255.4, 2957.1, 1651.0,
1574.0 cmꢀ1
.
example as follows (2a–m were prepared similarly).
Amination of 6-amino-5-bromo-1,3-dimethylpyrimidine-
2,4(1H,3H)-dione (IIi)
Bromination of 6-amino-1,3-dimethylpyrimidine-
2,4(1H,3H)-dione (Ii)
Triethylamine (2.4 mL, 17.1 mmol) and 6-amino-5-bromo-1,3-
dimethylpyrimidine-2,4(1H,3H)-dione (1 g, 4.2 mmol) was
added to RBF containing N,N-dimethylformamide (2 mL)
and stirred. Benzyl amine (0.514 mL, 4.7 mmol) was added
to the reaction mixture. RM was then heated to 100–120°C
and stirred for 24–28 h. Reaction was monitored by TLC. Upon
completion, RM was cooled to room temperature and
quenched in ice cold water. Solid obtained was filtered and
washed with water. Yield 0.8 g (72%); mp 184–186°C [45]; IR
Benzoyl peroxide (0.312 g, 1.29 mmol) and 6-amino-1,3-
dimethylpyrimidine-2,4(1H,3H)-dione (1 g, 6.45 mmol) were
added to RBF containing N,N-dimethylformamide (4 mL) and
stirred. N-Bromosuccinimide (1.26 g, 7.08 mmol) was added in
portions to the mixture. Reaction mixture (RM) was then
stirred at room temperature (RT) for 15–20 min. Reaction was
monitored by TLC. Upon completion, RM was quenched in
water (20 mL). Solid obtained was collected by filtration and
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
6

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Fragment Discovery for the Design of Mtb DHFR Inhibitors
Archiv der Pharmazie
O
Ar
O
O
O
Ar
N
R'
R'
NH
R'
R'
Br
N
N
N
a
c
N
b
N
N
NH2
O
N
R
NH2
O
N
R
O
N
R
NH2
O
R
I
2b
m
−
II
III
2b-R,R' = H; Ar = Bn
Ib,IIb,IIIb-R,R' = H; IIIb-Ar = Bn
2c-R = Bn; R' = H; Ar = Me
2d-R = Me; R' = H; Ar = Bn
2e-R = Me; R' = H; Ar = pMeBn
2f-R = Me; R' = H; Ar = pClBn
2g-R = Me; R' = H; Ar = pFBn
2h-R = Me; R' = H; Ar = pOMeBn
2i-R,R' = Me; Ar = Bn
2j-R,R' = Me; Ar = pMeBn
2k-R,R' = Me; Ar = pFBn
2l-R,R' = Me; Ar = pOMeBn
2m-R,R' = Me; Ar = pClBn
Ic,IIc,IIIc-R = Bn; R' = H; IIIc-Ar = Me
Id,IId,IIId-R = Me; R' = H; IIId-Ar = Bn
Ie,IIe,IIIe-R = Me; R' = H; IIIe-Ar = pMeBn
If,IIf,IIIf-R = Me; R' = H; IIIf-Ar = pClBn
Ig,IIg,IIIg-R = Me; R' = H; IIIg-Ar = pFBn
Ih,IIh,IIIh-R = Me; R' = H; IIIh-Ar = pOMeBn
Ii,IIi,IIIi-R,R' = Me; IIIi-Ar = Bn
Ij,IIj,IIIj-R,R' = Me; IIIj-Ar = pMeBn
Ik,IIk,IIIk-R,R' = Me; IIIk-Ar = pFBn
Il,IIl,IIIl-R,R' = Me; IIIl-Ar = pOMeBn
Im,IIm,IIIm-R,R' = Me; IIIm-Ar = pClBn
Conditions: a = NBS (1.1eq.) DMF, RT; b = Ar-NH2(1.1 eq.), Triethyl amine (4 eq.) DMF, 100-120oC, 24−28 hrs;
c = Triethyl orthoformate (2 mL), Microwave 100W, 20−30 min.
Scheme 1. Synthesis of xanthines.
(KBr): vmax ¼ 3297.5, 3018.5, 3061, 1681.7, 1652.8, 1624.0,
room temperature; diethyl ether was added to it and stirred.
Solid was filtered and washed with diethyl ether. The pure
product was characterized by melting point determination, IR
and 1H NMR spectroscopy. Yield 0.36 g (70%).
1575.0, 1527.0 cmꢀ1
.
Cyclization of 6-amino-5-(benzylamino)-1,3-
dimethylpyrimidine-2,4(1H,3H)-dione (IIIi)
6-Amino-5-(benzylamino)-1,3-dimethylpyrimidine-
2,4(1H,3H)-dione (0.5 g, 1.9 mmol) was added to triethylortho-
formate (2 mL) and then subjected to microwave irradiation
at 100W for 20–30min. Upon completion, RM was cooled to
7-Benzyl-1,3-dimethyl-1H-purine-2,6(3H,7H)-dione (2i)
Mp 158–160°C [46]; IR (KBr): vmax 3024.5, 1684.7, 1631.8,
1593.3 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.34 (s, 3H),
3.38 (s, 3H), 5.40 (s, 2H), 7.417–8.077 (m, 5H), 8.28 (s, 1H).
O
NH2
Ph
a
N
H
COO
N
3e
O
N
b
NH
3a-R' = Me
3b-R' = pOMePh
3c-R' = pClPh
3d-R' = Ph
R'
3a-d
Conditions: a = Aniline (1.1eq.), Acetic acid (0.2mL), Triethylorthoformate (2mL), Microwave
120 W, 80oC; b = Aldehyde (1.1 eq.), Ammonium acetate (2 eq.), Microwave 120 W, 80oC
Scheme 2. Synthesis of quinazolinones.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
7

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
Table 2. Biological evaluation of the fragments.
Sr. no.
1
Structure
MIC vs. Mtb (mg/mL)
IC50 vs. Mtb DHFR (mM)
125
65.8
2a
>125
NDa)
2b
2c
31.5
38.6
77.2
>125
2d
>125
53.8
2e
2f
>125
>125
85.5
48.5
2g
>125
71.9
2h
2i
>125
>125
63.1
49.5
(Continued)
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
8

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Fragment Discovery for the Design of Mtb DHFR Inhibitors
Archiv der Pharmazie
Table 2. (Continued)
Sr. no.
2j
Structure
MIC vs. Mtb (mg/mL)
IC50 vs. Mtb DHFR (mM)
62.5
53.4
2k
62.5
89.7
2l
>125
>125
41.2
40.0
2m
3a
3b
125
41.4
49.1
31.5
3c
62.5
31.5
65.2
50.8
3d
3e
4
31.5
125
69.5
NDa)
a) Not determined.
1,3-Dimethyl-1H-purine-2,6(3H,7H)-dione (2a)
Mp 290°C [47]; IR (KBr): vmax 3325.1, 3024.5, 1684.7, 1631.8,
1593.3 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.53 (s, 3H),
3.29 (s, 3H), 8.65 (s, 1H), 11.03 (s, 1H).
7-Benzyl-1H-purine-2,6(3H,7H)-dione (2b)
Mp 294°C [48]; IR (KBr): vmax 3123.1, 3026.5, 1688.7, 1632.8,
1591.3 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 5.33 (s, 2H),
7.27–7.53 (m, 5H), 8.48 (s, 1H), 10.0 (s, 1H), 11.0 (s, 1H).
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
9

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
3-Benzyl-7-methyl-1H-purine-2,6(3H,7H)-dione (2c)
1,3-Dimethyl-7-(4-chlorobenzyl)-1H-purine-2,6(3H,7H)-
dione (2m)
Mp 230°C [49]; IR (KBr): vmax 3102.1, 3027.5, 1681.7, 1630.8,
1591.3 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): d ppm 3.96
Mp 220°C [52]; IR (KBr): vmax 3024.5, 1684.7, 1631.8,
(s, 3H), 5.24 (s, 2H), 7.27–7.53 (m, 5H), 8.43 (s, 1H), 11.08
1593.3 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d ppm 3.30
;
(s, 1H).
(s, 3H), 3.41 (s, 3H), 5.40 (s, 2H), 7.25–7.33 (m, 4H), 8.27
(s, 1H).
7-Benzyl-3-methyl-1H-purine-2,6(3H,7H)-dione (2d)
Mp 274°C [50]; IR (KBr): vmax 3122.1, 3022.5, 1680.4, 1631.7,
1593.5 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.40 (s, 3H),
5.33 (s, 2H), 7.27–7.53 (m, 5H), 8.48 (s, 1H), 11.0 (s, 1H).
Synthesis of 2-methylquinazolin-4(1H)-one (3a)
To
a mixture of anthranilic acid (1 g, 7.2 mmol) and
ammonium acetate (1.13 g, 14.5 mmol), acetic acid (0.2 mL)
and triethylorthoacetate (1.48 mL, 8.0 mmol) were added. The
mixture was subjected to microwave irradiation at 120 W at
80°C for 15–20 min. After completion of the reaction, as
monitored by TLC, 20% methanolic solution in water was
added to get precipitation. Solid was filtered and washed
with water to get 2-methyl-4(3H)-quinazolinone. The pure
product was characterized by melting point determination, IR
and 1H NMR spectroscopy. (Corresponding aldehydes were
used for synthesis of other 2-substituted quinazolinones
(3b–d) in place of triethylorthoacetate.) Yield 0.75 g (65%);
2-methylquinazolin-4(1H)-one (3a) mp 236–238°C [54]; IR
3-Methyl-7-(4-methylbenzyl)-1H-purine-2,6(3H,7H)-dione
(2e)
Mp 250°C [51]; IR (KBr): vmax 3222.1, 3022.5, 1684.4, 1632.8,
1589.6 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 2.33 (s, 3H),
3.48 (s, 3H), 5.39 (s, 2H), 7.18–7.26 (m, 4H), 8.05 (s, 1H), 10.13 (s,
1H).
7-(4-Chlorobenzyl)-3-methyl-1H-purine-2,6(3H,7H)-dione
(2f)
Mp 280°C [52]; IR (KBr): vmax 3220.2, 3022.9, 1684.8, 1634.5,
1596.2 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.60 (s, 3H),
5.33 (s, 2H), 6.99–7.52 (m, 4H), 8.11 (s, 1H), 9.96 (s, 1H).
(KBr): vmax 3303, 3185, 3061, 1656, 1612, 1510 cmꢀ1; H NMR
(300 MHz, CDCl3): d ppm 2.34 (s, 3H), 7.23–8.08 (m, 4H), 11.76
1
(s, 1H).
7-(4-Fluorobenzyl)-3-methyl-1H-purine-2,6(3H,7H)-dione
(2g)
2-(4-Methoxyphenyl)quinazolin-4(1H)-one (3b)
Mp 290°C [51]; IR (KBr): vmax 3220.2, 3022.9, 1684.8, 1634.5,
1596.2 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.41 (s, 3H),
5.56 (s, 2H), 7.04–7.29 (m, 4H), 8.24 (s, 1H), 10.20 (s, 1H).
Mp 256°C [55]; IR (KBr): vmax 3303, 3185, 3061, 1656, 1612,
1510, 1203.1 cmꢀ1 1H NMR (300 MHz, CDCl3): d ppm 3.70
;
(s, 3H), 6.62–7.38 (m, 8H), 11.55 (s, 1H).
7-(4-Methoxybenzyl)-3-methyl-1H-purine-2,6(3H,7H)-
dione (2h)
Mp 270°C [53]; IR (KBr): vmax 3220.2, 3022.9, 1684.8, 1634.5,
1596.2, 1202.3 cmꢀ1; 1H NMR (300 MHz, DMSO-d6): d ppm 3.56
(s, 3H), 3.87 (s, 3H), 5.52 (s, 2H), 6.77–7.0 (m, 4H), 8.11 (s, 1H),
10.04 (s, 1H).
2-(4-Chlorophenyl)quinazolin-4(1H)-one (3c)
Mp 302°C [55]; IR (KBr): vmax 3301.3, 3071.4, 1666.2, 1612.2,
1510.2 cmꢀ1 1H NMR (300 MHz, CDCl3): d ppm 7.10–7.61 (m,
;
8H), 12.01 (s, 1H).
2-Phenylquinazolin-4(1H)-one (3d)
Mp 240°C [56]; IR (KBr): vmax 3386.3, 3071.4, 1666.2, 1612.2,
1,3-Dimethyl-7-(4-methylbenzyl)-1H-purine-2,6(3H,7H)-
dione (2j)
1510.2 cmꢀ1 1H NMR (300 MHz, CDCl3): d ppm 7.07–7.55 (m,
;
9H), 12.0 (s, 1H).
Mp 200°C [51]; IR (KBr): vmax 3024.5, 1684.7, 1631.8,
1593.3 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): d ppm 2.33 (s,
Synthesis of 3-phenylquinazolin-4(3H)-one (3e)
3H), 3.48 (s, 3H), 3.60 (s, 3H), 5.39 (s, 2H), 7.18–7.26 (m, 4H),
8.05 (s, 1H).
The mixture of anthranilic acid (1 g, 7.2 mmol) and triethyl-
orthoformate (1.82 mL, 10.9 mmol) and aniline (0.613 mL,
8.01 mmol) and acetic acid (0.2 mL) was subjected to micro-
wave irradiation at 120 W at 80°C for 15–20 min. After
completion of the reaction, as monitored by TLC, 20%
methanolic solution in water was added to get precipitation.
Solid was filtered and washed with water to get 3-phenyl-
quinazolin-4-one. The pure products were characterized by
melting point determination, IR and 1H NMR spectroscopy.
Yield 1.053 g (65%).
1,3-Dimethyl-7-(4-fluorobenzyl)-1H-purine-2,6(3H,7H)-
dione (2k)
Mp 240°C [52]; IR (KBr): vmax 3024.5, 1684.7, 1631.8,
1593.3 cmꢀ1 1H NMR (300 MHz, DMSO-d6): d ppm 3.07 (s,
;
3H), 3.40 (s, 3H), 5.51 (s, 2H), 7.22–7.51 (m, 4H), 8.20 (s, 1H).
1,3-Dimethyl-7-(4-methoxybenzyl)-1H-purine-2,6(3H,7H)-
dione (2l)
Mp 205°C [51]; IR (KBr): vmax 3024.5, 1684.7, 1631.8,
3-Phenylquinazolin-4(3H)-one (3e)
1593.3 cmꢀ1
;
1H NMR (300 MHz, DMSO-d6): d ppm 3.56 (s,
Mp 140°C [57]; IR (KBr): vmax 3180.4, 3050.2, 2936, 2822, 1654,
1
3H), 3.76 (s, 3H), 3.87 (s, 3H), 5.52 (s, 2H), 6.77–7.0 (m, 4H), 8.11
1611, 1509 cmꢀ1; H NMR (300 MHz, CDCl3): d ppm 7.54–8.22
(s, 1H).
(m, 9H), 8.35 (s, 1H).
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
10

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
Fragment Discovery for the Design of Mtb DHFR Inhibitors
Archiv der Pharmazie
2-Aminoquinazolin-4(3H)-one (4)
[9] M. H. R. I. El-Hamamsy, A. W. Smith, A. S. Thompson,
M. D. Threadgill, Bioorg. Med. Chem. 2007, 15,
4552–4576.
Mp 260°C [58]; IR (KBr): vmax 3324,2, 3123.2, 3071.4, 1666.2,
1612.2, 510.2 cmꢀ1 1H NMR (300 MHz, CDCl3): d ppm 7.40–
;
7.73 (m, 4H), 8.23 (s, 2H), 11.93 (s, 1H).
[10] A. L. B. Gerum, J. E. Ulmer, D. P. Jacobus, N.P. Jensen,
D. R. Sherman, C. H. Sibley, Antimicrob. Agents Chemo-
ther. 2002, 46, 3362–3369.
[11] R. C. Reynolds, S. R. Campbell, R. G. Fairchild, R. L. Kisliuk,
P. L. Micca, S. F. Queener, J. Med. Chem. 2007, 50,
3283–3289.
[12] A. Kumar, M. Zhang, L. Zhu, R. P. Liao, C. Mutai, S. Hafsat,
PLoS ONE 2012, 7, e39961.
[13] S. Bag, N. R. Tawari, M. S. Degani, QSAR Comb. Sci. 2009,
28, 296–311.
Biological evaluation
Fragment-based inhibitor screening against purified
enzyme
The purification of Mtb DHFR from recombinant strain of
Saccharomyces cerevisiae, Y182 was carried out using novel
affinity chromatography. The column contained methotrex-
ate immobilized on Sephabeads EB EP 105 through an epoxy
linker and diethyl amine spacer arm. DHFR activity was
determined spectrophotometrically [38], by monitoring the
decreasing absorbance for 2 min at 340 nm and 37°C.
A unit of the enzyme activity is defined as the amount of
the enzyme converting 1.0 mmol of DHF and NADPH to THF
and NADPþ per min and is calculated from the change in
absorbance at 340 nm under specified assay condition.
[14] M. Kumar, R. Vijayakrishnan, G. Subba Rao, Mol. Divers.
2010, 14, 595–604.
[15] G. Mugumbate, K. A. Abrahams, J. A. C. Cox,
ꢀ
G. Papadatos, G. van Westen, J. Lelievre, PLoS ONE
2015, 10, e0121492.
[16] D. S. Gond, R. J. Meshram, S. G. Jadhav, G. Wadhwa,
R. N. Gacche, Drug Invent. Today. 2013, 5, 182–191.
[17] D. Fattori, Drug Discov. Today 2004, 9, 229–238.
[18] M. Congreve, G. Chessari, D. Tisi, A. J. Woodhead, J. Med.
Chem. 2008, 51, 3661–3680.
[19] J. Tsai, J. T. Lee, W. Wang, J. Zhang, H. Cho, S. Mamo,
Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 3041–3046.
[20] P. J. Hajduk, Nat. Rev. Drug Discov. 2007, 6, 211–219.
[21] R. Bueno, N. Toledo, B. Neves, R. Braga, C. Andrade, J.
Mol. Model. 2013, 19, 179–192.
[22] D. Bastien, M. C. C. J. C. Ebert, D. Forge, J. Toulouse,
N. Kadnikova, F. Perron, J. Med. Chem. 2012, 55,
3182–3192.
[23] M. S. Degani, S. Bag, R. Bairwa, N. R. Tawari,
S. F. Queener. J. Heterocyclic Chem. 2010, 47,
558–563.
Anti-tubercular activity
Compounds were tested against M. tuberculosis (H37Rv) in
order to determine the MIC in the broth microdilution assay
using REMA method. Homogenous Mtb (H37Rv) culture
suspension was seeded in microtiter plates at density of 104
cells per well along with serially diluted compounds to a final
volume of 200 mL. The control received equivalent amount of
DMSO. The plates were incubated at 37°C for 7 days in a CO2
incubator. Resazurin dye (0.02%) was added after 7 days and
plates were again incubated for 48 h. MIC was determined
visually by inspection of the dye color (blue to pink). Isoniazid
was used as reference standard.
Financial support from UGC (SAP), Govt. of India, is acknowl-
edged by R.U.S.
[24] S. Bag, N. R. Tawari, S. F. Queener, M. S. Degani, J.
Enzyme Inhib. Med. Chem. 2010, 25, 331–339.
[25] S. Bag, N. R. Tawari, M. S. Degani, S. F. Queener, Bioorg.
Med. Chem. 2010, 18, 3187–3197.
[26] N. R. Tawari, S. Bag, M. S. Degani, Current Pharm. Des.
2011, 17, 712–751.
The authors have declared no conflicts of interest. The authors
are alone responsible for the content and the writing of the
paper.
[27] N. R. Tawari, S. Bag, A. Raju, A. C. Lele, R. Bairwa,
M. K. Ray, Future Med. Chem. 2015, 7, 979–988.
[28] A. B. Patel, K. H. Chikhalia, P. Kumari, Eur. J. Med. Chem.
2014, 79, 57–65.
[29] C. Correia, M. A. Carvalho, M. F. ProencSa, Tetrahedron
2009, 65, 6903–6911.
References
[1] Global Tuberculosis report WHO, 2015.
[2] M. C. Raviglione, I. M. Smith, New Engl. J. Med. 2007,
356, 656–659.
€
[3] G. B. Migliori, C. Lange, R. Centis, G. Sotgiu, R. Mutterlein,
[30] C. G. Bonde, A. Peepliwal, N. J. Gaikwad, Arch. Pharm.
2010, 343, 228–236.
[31] J. Odingo, T. O’Malley, E. A. Kesicki, T. Alling,
M. A. Bailey, J. Early, Bioorg. Med. Chem. 2014, 22,
6965–6979.
H. Hoffmann, Eur. Respir. J. 2008, 31, 1155–1159.
[4] I. M. Kompis, K. Islam, R. L. Then, Chem. Rev. 2005, 105,
593–620.
[5] J. M. Blaney, C. Hansch, C. Silipo, A. Vittoria, Chem. Rev.
1984, 84, 333–407.
€
[32] QikProp. 3.5 ed: Schrodinger, LLC: New York; 2012.
[6] J. J. McGuire, Current Pharm. Des. 2003, 9, 2593–2613.
[7] R. L. Then, J. Chemother. 2004, 16, 3–12.
[8] R. Li, R. Sirawaraporn, P. Chitnumsub, W. Sirawaraporn,
J. Wooden, F. Athappilly, J. Mol. Biol. 2000, 295, 307–323.
[33] Y. Pedgaonkar, M. S. Degani, R. Iyer, Mol. Divers. 2011,
15, 263–267.
[34] S. S. Chavan, M. S. Degani, Green Chem. 2012, 14,
296–299.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
11

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 1–12
R. U. Shelke et al.
Archiv der Pharmazie
[35] R. Dach, W. Goldschmidt (Boehringer Ingelheim KG), US
Patent 5510484, 1996.
[45] J. E. Garst, G. L. Kramer, Y. J. Wu, J. N. Wells, J. Med.
Chem. 1976, 19, 499–503.
[36] a) J.-F. Liu, J. Lee, A. M. Dalton, G. Bi, L. Yu, C. M. Baldino,
E. McElory, M. Brown, Tet. Lett. 2005, 46, 1241–1244; b)
J. F. Liu, M. Kaselj, Y. Isome, P. Ye, K. Sargent, K. Sprague,
D. Cherrak, C. J. Wilson, Y. Si, D. Yohannes, S. C. Ng, J.
Comb. Chem. 2006, 8, 7–10; c) T. M. Potewar, R. N. Nadaf,
T. Daniel, R. J. Lahoti, K. V. Srinivasan, Syn. Comm. 2005,
35, 231–241; d) Y. Kabri, A. Gellis, P. Vanelle, Green
Chem. 2009, 11, 201–208.
[46] C. C. Malakar, D. Schmidt, J. Conrad, U. Beifuss, Org. Lett.
2011, 13, 1378–1381.
[47] Z. Li, M. Day, J. Ding, K. Faid, Macromolecules 2005, 38,
2620–2625.
[48] L. G. Marzilli, L. A. Epps, T. Sorrell, T. J. Kistenmacher, J.
Am. Chem. Soc. 1975, 97, 3351–3358.
[49] W. Hutzenlaub, W. Pfleiderer, Liebigs. Ann. Chem. 1979,
1979, 1847–1854.
[37] X. Zhang, D. Ye, H. Sun, D. Guo, J. Wang, H. Huang,
Green Chem. 2009, 11, 1881–1888.
[38] E. L. White, L.J. Ross, A. Cunningham, V. Escuyer, FEMS
Microbiol. Lett. 2004, 232, 101–105.
[39] A. Raju, M. S. Degani, M. A. Khedkar, S. N. Niphadkar,
Inter. J. Drug Design Discov. 2013, 4, 994–997.
[40] J. C. Palomino, A. Martin, M. Camacho, H. Guerra,
J. Swings, F. Portaels, Antimicrob. Agents Chemother.
2002, 46, 2720–2722.
[50] J. Szolomajer, G. Paragi, G. Batta, C. F. Guerra,
F. M. Bickelhaupt, Z. Kele, NewJ. Chem. 2011, 35, 476–482.
[51] J. Lister, Aust. J. Chem. 1979, 32, 387–97.
[52] H. J. Huang, W. C. Lee, G. P. A. Yap, T. G. Ong, J.
Organomet. Chem. 2014, 761, 64–73.
[53] R. Sakai, K. Konno, Y. Yamamoto, F. Sanae, K. Takagi,
T. Hasegawa, J. Med. Chem. 1992, 35, 4039–4044.
[54] X. Liu, H. Fu, Y. Jiang, Y. Zhao, Angew. Chem. Int. Edit.
2009, 48, 348–351.
[41] R. Bairwa, M. Kakwani, N. R. Tawari, J. Lalchandani,
M. K. Ray, M. G. R. Rajan, Bioorg. Med. Chem. Lett. 2010,
20, 1623–1625.
[42] R. Li, R. Siriwaraporn, P. Chitnumsub, W. Siriwaraporn,
J. Wooden, F. Athappilli, J. Mol. Biol. 2000, 295, 307.
[55] X. S. Wang, K. Yang, M. M. Zhang, C. S. Yao, Synth.
Commun. 2010, 40, 2633–2646.
[56] C. Brouwer, K. Jenko, S. S. Zoghbi, R. B. Innis, V. W. Pike,
J. Med. Chem. 2014, 14, 6240–6251.
[57] S. L. Wang, K. Yang, C. S. Yao, X. S. Wang, Synth.
Commun. 2011, 42, 341–349.
€
[43] LigPrep, version 2.0. New York, NY: Schrodinger, LLC;
2006.
[58] C. Lecoutey, C. Fossey, S. Rault, F. Fabis, Eur. J. Org. Chem.
2011, 15, 2785–2788.
[44] E. F. Schroeder, Google Patents, 1956.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
12