Exploring MDR-TB Inhibitory Potential of 4-Aminoquinazolines as Mycobacterium tuberculosis N-Acetylglucosamine-1-Phosphate Uridyltransferase (GlmUMTB) Inhibitors

Article abstract of DOI:10.1002/cbdv.202000237

Drug resistance tuberculosis is one of the challenging tasks that dictates the desperate need for the development of new antitubercular agents which operate via novel modes of action. Here, we are reporting on 4-aminoquinazolines as M. tuberculosis N-acetylglucosamine-1-phosphate uridyltransferase (GlmU^MTB) inhibitors to overcome the problem of the MDR-TB. Amongst the synthesized compounds, two of them were observed to be the effective compounds of the series (IC₅₀=6.4 μM (H37Rv), MIC=25 μM (MDR-TB) and IC₅₀=2.9 μM (H37Rv), MIC=6.25 μM (MDR-TB), respectively).

Full text of DOI:10.1002/cbdv.202000237

Title: Exploring MDR-TB inhibitory potential of 4-amino quinazolines as
Mycobacterium tuberculosis
N -acetylglucosamine-1-phosphate uridyltransferase (GlmU MTB)
inhibitors
Authors: Harun Patel, Rajshekhar Karpoormath, and Mahesh Palkar
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000237
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10.1002/cbdv.202000237
Chemistry & Biodiversity
Exploring MDR-TB inhibitory potential of 4-amino
quinazolines as Mycobacterium tuberculosis
N-acetylglucosamine-1-phosphate uridyltransferase
(GlmU MTB) inhibitors
*
Harun M. Patel a,b , Mahesh Palkar a, Rajshekhar Karpoormath a
a Department of Pharmaceutical Chemistry, University of KwaZulu-Natal (Westville Campus), Private Bag
X54001, Durban- 4000, South Africa.
b Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and Research,
Shirpur, Maharashtra-425405, India
Corresponding Author*
Tel: +91-8806621544, Fax: + 91-2563251808; Email: hpatel_38@yahoo.com
Tel: + 27-312607179; Fax: + 27-312607792; Email: karpoormath@ukzn.ac.za
2
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10.1002/cbdv.202000237
Chemistry & Biodiversity
Abstract
Drug resistance tuberculosis is one of the challenging tasks that dictates the desperate need for
the development of new anti-tubercular agents which operate via novel modes of action. Here,
we are reporting the 4-amino quinazolines as M. tuberculosis N-acetylglucosamine-1-
phosphate uridyltransferase (GlmU MTB) inhibitors to overcome the problem of the MDR-
TB. Amongst the synthesized compounds HMP-05 and HMP-15 was observed to be the
effective compound of the series [IC50 = 6.4 µM (H37Rv), MIC = 25 µM (MDR-TB) and IC50
= 2.9 µM (H37Rv), MIC = 6.25 µM (MDR-TB) respectively].
Keywords: 4-amino quinazoline; N-acetylglucosamine-1-phosphate uridyltransferase (GlmU
MTB); MDR-TB; Cytotoxicity
3
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10.1002/cbdv.202000237
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1. Introduction
Tuberculosis (TB) is an infectious bacterial disease caused by Mycobacterium tuberculosis
(MTB) which commonly affects the lungs and respiratory system. [1-5] The TB morbidity was
reported decreasing, but it remains alarming due to an increase in incidence and prevalence of
[6-10]
Multi-Drug Resistance-TB (MDR-TB).
Worldwide, TB is one of the top 10 causes of
death and the leading cause from a single infectious agent (above HIV/AIDS) as per the WHO
2018 reports. [1,11] Approximately 10.0 million people were infected with TB in 2018, which
includes 57% male, 32% female, 11% children, and 8.6% HIV positive. [1] MDR phenotype is
defined as resistance at least to isoniazid and rifampicin which are the most effective drugs
recommended by the World Health Organization (WHO) and being used as the first-line
treatment in TB patients. [12-13] Therefore, resistance towards these two drugs has become major
problems in the treatment of TB patients. [14]
The development of compounds that target enzymes responsible for the biosynthesis and asse
mbly of the mycobacterial cell wall is desperately needed for the development of effective
anti-tubercular drugs. Examples include currently employed first and second-line TB drugs
[15,16]
such as isoniazid, ethambutol and ethionamide.
These agents act by disrupting the
biosynthesis or the incorporation of mycolic acid, a key lipid which constitutes a major
component of the mycobacterial cell wall (Figure 1).
Another important component of the mycobacterial cell wall is the peptidoglycan layer which
gives the bacterium structural strength and rigidity to withstand internal osmotic pressure.[16]
Peptidoglycan biosynthesis interference has been an extremely successful way for the
development of antibacterial agents against gram-positive and gram-negative bacteria. The
enzymes responsible for the development of the peptidoglycan biosynthesis were not
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10.1002/cbdv.202000237
Chemistry & Biodiversity
[16]
thoroughly investigated as drug targets.
This is due to the broad-spectrum β-lactamase
activity of MTB, which inactivates β-lactam antibiotics. Conversely, other enzymes that are
responsible for the peptidoglycan biosynthesis remained vital targets for anti-tubercular drug
development. One such novel target is N-acetylglucosamine-1-phosphate uridyltransferase
(GlmU). [16-18] This bi-functional enzyme, which is found exclusively in bacteria, catalyzes the
development of Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), an essential
intermediate in peptidoglycan biosynthesis in MTB. [16-18] The crystal structure of the GlmU
MTB has been recently resolved which provides further insight into the two active sites of the
enzyme (N-terminal and C-terminal). N-terminal posses the uridyltransferase and C-terminal
have acetyltransferase activities. The acetyltransferase domain, found in the C-terminus of the
protein is responsible for the first step of the reaction, in which an acetyl group is transferred
from Acetyl Co-A (AcCoA) to Glucosamine-1-phosphate (GlcN-1-P) forming N-acetyl
[16]
glucosamine-1-phosphate (GlcNAc-1-P).
GlcNAc-1-P then serves as a substrate for the
uridyltransferase active site in the N-terminus of the enzyme which forms Uridine diphosphate-
N-acetylglucosamine (UDP-GlcNAc) (Figure 1). Computational kinetic modeling studies on
GlmU MTB have shown uridyltransferase reaction to be the rate-limiting step and offer more
flexibility to target for inhibition of GlmU MTB. [19]
Based on the above reviews in the current study, we are reporting the 4-aminoquinazolines as
N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) MTB inhibitors to overcome the
problem of MDR-TB.
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10.1002/cbdv.202000237
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Figure 1. Role of N-acetyl-glucosamine uridyltransferase (GlmU) in the synthesis of peptidoglycan of M. tuberculosis (Modified from Organic and Bio-molecular Chemistry with permission from Royal Society of
Chemistry)
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Figure 2. Reported and synthesized N-acetyl-glucosamine uridyltransferase (GlmU) MTB inhibitors
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10.1002/cbdv.202000237
Chemistry & Biodiversity
2. Result and Discussion
2.1 Designing of the Compounds (Rationale)
Recently, Doig and co-workers from AstraZeneca reported the promising 4-aminoquinazoline
GlmU MTB inhibitors against M. tuberculosis GlmU uridyltransferase (Fig.2). [20]
Figure 3. Proposed structural modification of Compound ii
Specifically, compound ii exhibited 44% inhibition of the uridyltransferase activity at 50 μM
while compound i was slightly less potent with 36% inhibition at the same concentration.
Further structural modification of the compound i and ii were made by the Tran et al that leads
to the compound iii with 38% inhibition of the uridyltransferase. [16] Encouraged by the activity
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10.1002/cbdv.202000237
Chemistry & Biodiversity
of the compound i, ii, iii and to explore further structural activity relationship (Figure 2 and 3),
we replaced the hydrophobic group containing hydrogen bond acceptor and donor of
compound ii at 4th position with different heterocyclic/homocyclic hydrophobic group with
hydrogen bond acceptor and donor functionality. The substituted heterocyclic/homocyclic
[21]
hydrophobic group at the 4th position of quinazoline ring includes; sulphonamides
,
[25]
piperidine [22], piperazine [23], thiazole [24], thiadiazole
and various aliphatic and aromatic
[26,27]
amines.
These groups were introduced to evaluate the effect of incorporating hydrogen
bond donors and acceptors as well as conformational flexibility at the 4th position of quinazoline
on GlmU uridyltransferase inhibitory activity. We also substituted compounds with a nitro
group at the 6th position of quinazolines since it has significant anti-mycobacterial potential as
shown in Figure 3.[28]
2.2. Chemistry
The synthesis of final compounds HMP (3-23) was carried out according to Scheme 1, where
N'-(2-cyano-4-nitrophenyl)-N,N-dimethylformamide (2) was synthesized by reacting 2-amino-
5-nitrobenzonitrile (1) with dimethylformamide dimethyl acetal to give 2 (Scheme 1).
Cyclization of the intermediate 2 was performed by different primary amino groups containing
aromatic, aliphatic and heterocyclic moieties to afford the desired final compounds HMP (3-
23). All the newly synthesized compounds showed acceptable analysis of their anticipated
structures, which are outlined in the experimental section.
In general, the IR spectrum of final synthesized compounds revealed typical absorption bands
-1
around 1350 cm-1 and 1550 cm for NO2 group, and NH stretching around 3200 cm-1. This
was further substantiated from the 1H-NMR spectrum, which showed the presence of a singlet
signal around 10 to 11 ppm for the N–H proton, confirming the cyclization of final compounds.
Further 13C NMR spectra and Mass data are in full agreement with their delineated structures.
LogP value which denotes the lipophilicity of a drug, plays a vital role in the infiltration of a
compound into bacterial cells. LogP values (the log of the octanol/water partition coefficient)
were calculated for all the synthesized compounds using ChemBioDraw Ultra 14, which
decides the hydrophobicity of the substituents as given in Table 1.
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10.1002/cbdv.202000237
Chemistry & Biodiversity
Scheme 1
Where R =
Compound
Code
Compound
Code
Compound
Code
Compound
R
R
R
R
Code
HMP-03
HMP-04
HMP-05
HMP-06
HMP-07
HMP-11
HMP-08
HMP-12
HMP-09
HMP-13
HMP-10
HMP-14
HMP-15
HMP-19
HMP-16
HMP-20
HMP-17
HMP-21
HMP-18
HMP-22
HMP-23
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2.3. Bio-evaluation and in-silico study
In vitro assessment of the anti-mycobacterial activity of the newly synthesized compounds, HMP (3-
23) was completed at the National Institute of Allergy and Infectious Diseases (NIAID) (Bethesda,
MD, USA). The MIC was established against H37Rv MTB strain grown under aerobic conditions,
utilizing a dual readout (OD 590 and fluorescence) assay. [29,30] The MDR-TB inhibitory activity was
evaluated against the phenotypic MDR-TB strain at the Micro Care Laboratory and Tuberculosis
Research Center Surat, India.
Figure 4. Structure-activity correlationship of quinazolines
All the targeted synthesized compounds demonstrated an interesting anti-mycobacterial activity
profile, with IC50 values from 2.9 to 200 µM against the mycobacterial H37Rv strain and MIC 6.25
µM to 200 µM against the MDR-TB strain. We additionally considered the impacts of various
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10.1002/cbdv.202000237
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aromatic/ heteroaromatic substituents at the 4th position of the quinazoline ring. The structure anti-
mycobacterial relationship at 5th position of 1,3,4- thiadiazole substituted at the 4th position of
quinazoline uncovers that direct attachment of electron-withdrawing group to the 5th position on 1,3,4-
thiadiazole is more active (HMP-10) (H37Rv IC50 = 21 µM and MDR-TB MIC = 50 µM) than any
other substitution (HMP-06, HMP-08, HMP-09, HMP-11, HMP-12). Among the sulphonamides;
sulphaguanidine substituted quinazoline derivatives HMP-05 (H37Rv IC50 = 6.4 µM and MDR-TB
MIC = 25 µM) was found to be the active, followed by sulphacetamide HMP-04 (H37Rv IC50 = 28
µM and MDR-TB MIC = 50 µM) (Figure 4). N-substituted piperidine containing quinazoline
derivative HMP-15 was standout amongst the most effective compound of the series (H37Rv IC50 =
2.9 µM and MDR-TB MIC = 6.25 µM). Likewise, piperazine substitution at the 4th position of the
quinazoline (HMP-16) indicated moderate anti-mycobacterial activity (H37Rv IC50 = 194 µM).
Similar to HMP-15; HMP-21 (H37Rv IC50 = 10.4 µM and MDR-TB MIC = 25 µM) showed
significant anti-mycobacterial activity. Further we have correlated the LogP value with the IC50 value
(H37Rv Mycobacterium strain) and we observed that the logP value higher than 3.3 seemed to be not
favorable for antimycobacterial activity as shown in Figure 5. The synthesized potent compounds of
the series were further screened for cytotoxicity (IC50) in a mammalian Vero cell line (Table 1). All
the tested derivatives showed lower toxicity with IC50 values >238 µM and none of the synthesized
compounds displayed significant activity against the mammalian Vero cell line at concentrations <100
µM.
The crystal structure of GlmU MTB (PDB: 3ST8) was processed for the addition of hydrogens, bond
order correction etc. [31] Water molecules interacting with UDP-GlcNAc and those present around 5
Å of these substrates were retained to maintain the thermodynamic stability. In the case of UDP-
GlcNAc, the diphosphate tail was found to be stabilized by polar contacts with Asn 239, Arg 19 and
one coordinate covalent bonds with Mg 496. Uridyl moiety of UDP-GlcNAc is showing hydrogen
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10.1002/cbdv.202000237
Chemistry & Biodiversity
bonding interaction with Gly 15, Ala 14, Gln 83, and Gly 88. Glucosamine of UDP-GlcNAc is
showing polar interaction with Thr 89, Glu166, Asn181, Gly151 and with water molecules as shown
in (Figure 6a). Potent compound 5 of the series with sulphaguanidine moiety at 4th position of
quinazoline forms a polar hydrogen bond with Glu 166 and Gln205 via guanidine moiety. Lys 26
forms pi-pi stacking with phenyl ring of sulphaguanidine and hydrogen bonding with a nitro functional
group at 6th position of quinazoline ring (Figure 6b). Similarly, nitro functional group of compound
15 shows hydrogen bond interaction with Gln 83 and Gly88 (Figure 6c).
These outcomes are vital, as compounds with increased cytoliability are appealing in the development
of new chemical entities for the treatment of tuberculosis. This is primarily because the management
of tuberculosis requires an extensive course of drug treatment, leading to a number of side-effects
with a high margin of safety.
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Table 1. Physical and Anti-mycobacterial data of the synthesized compounds against the H37Rv and MDR-TB strains
Anti-mycobacterial Activity against
H37Rv (µM)
Under Aerobic Condition
MDR-TB
Compound Code
Melting Point
Molecular Formula
Molecular Weight
Cytotoxicity
IC50 (µM) d
Log P
MIC (µM) e
a
b
IC50
26
IC90
50
MICc
200
45
HMP-03
HMP-04
HMP-05
HMP-06
HMP-07
HMP-08
HMP-09
HMP-10
HMP-11
HMP-12
HMP-13
HMP-14
HMP-15
HMP-16
HMP-17
HMP-18
HMP-19
HMP-20
HMP-21
HMP-22
HMP-23
Isoniazid
Rifampicin
291
311
297
>300
271
>300
>300
200
>300
>300
180
190
232
256
195
212
170
338
194
>300
250
--
C18H13N7O4S
423.4053
423.4053
423.4053
384.7997
273.2706
288.2852
350.3546
342.2566
364.3812
366.3540
294.3080
278.2640
363.4130
288.3051
230.2227
281.2694
272.3024
310.2643
281.2694
348.3123
356.3327
--
2.15
1.61
1.12
4.19
2.23
1.08
4.01
2.60
4.47
3.65
3.79
0.37
3.30
0.84
1.86
1.53
3.55
2.98
1.91
3.42
2.81
---
---
238
246
---
242
---
---
278
---
---
50
25
---
50
---
---
50
---
---
200
25
6.25
---
---
---
---
---
25
C18H13N7O4S
C18H13N7O4S
C16H9ClN6O2S
C11H7N5O2S
C11H8N6O2S
C16H10N6O2S
C11H5F3N6O2S
C17H12N6O2S
C16H10N6O3S
C16H14N4O2
C12H14N4O4
C20H21N5O2
C13H16N6O2
C11H10N4O2
C14H11N5O2
C14H16N4O2
C15H10N4O4
C14H11N5O2
C18H12N4O
C17H16N4O5
--
28
43
6.4
8.3
12
>200
16
>200
18
>200
25
>200
>200
21
>200
>200
38
>200
>200
40
>200
>200
52
8.7
2.9
>200
>200
110
14.2
4.4
>200
>200
119
20
---
241
289
287
---
---
---
---
---
287
---
---
5.4
194
121
23
141
42
10.4
>200
21
>200
198
141
>200
108
18
>200
98
---
>200
200
181
>200
178
23
>200
200
---
---
---
> 200
---
---
---
---
--
--
--
0.0063
0.0034
0.0059
---
LogP value is calculated using the ChemBioDraw Ultra 14 [32]
a IC50= concentration at which growth is inhibited by 50%; b IC90 value = concentration at which growth is inhibited by 90%; c MIC = minimum inhibitory concentration at which M.
tuberculosis H37Rv growth was completely inhibited; d Cytotoxicity activity was determined on mammalian Vero cell line; --- = not determined; e Resistant phenotypic MDR-TB strain
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Figure 5. Correlation of LogP value and IC50 value of the potent compounds against the H37Rv Mycobacterium strain
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Chemistry & Biodiversity
Figure 6a. Binding of N-acetyl-glucosamine UDP in N-acetyl-glucosamine uridyltransferase
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10.1002/cbdv.202000237
Chemistry & Biodiversity
Figure 6b. Interaction of Compound 5 in N-acetyl-glucosamine uridyltransferase
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Chemistry & Biodiversity
Figure 6c. Interaction of Compound 15 in N-acetyl-glucosamine uridyltransferase
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3. Conclusion
To overcome the problem of drug resistance tuberculosis there is a desperate need of new anti-
tubercular agents that operate via novel modes of action. The rigidity to the MTB cell wall is
attributed by the peptidoglycan layer. UDP-GlcNAc serves as the starting material in the
biosynthesis of this peptidoglycan layers. This UDP-GlcNAc is synthesized by N-
acetylglucosamine-1-phosphate uridyltransferase (GlmU MTB). Conclusively in the current
study, we have reported the synthesis and in vitro anti-mycobacterial activity of twenty two, 4
-substituted quinazoline analogues (HMP 3-23) as M.tuberculosis N-acetylglucosamine-1-
phosphate uridyltransferase (GlmU MTB) inhibitors. Among the tested compounds, N-
substituted piperidine containing quinazoline derivative, HMP-15 was observed to be the most
effective compound of the series against the H37Rv and MDR-TB strain [IC50 = 2.9 µM against
H37Rv and MIC = 6.25 µM against the MDR-TB]. Similar to the compounds HMP-15,
compound HMP-05 was observed to be the other effective compound of the series [IC50 = 6.4
µM (H37Rv), MIC = 25 µM (MDR-TB)]. The structure activity-relationship study uncovered
that the N-substituted piperidine and sulphaguanidine ring at the 4th position of quinazoline ring
is decisive for the activity. The synthesized compounds, which demonstrated significant anti-
mycobacterial action, additionally evaluated for their cytotoxicity (IC50) against the
mammalian Vero cell line, utilizing the MTT assay. The outcomes demonstrated that these
compounds showcase anti-mycobacterial activity at non-cytotoxic level. The docking study
has been carried out to get the further insight of GlmU MTB inhibition. Potent compound 5 of
the series with sulphaguanidine moiety at 4th position of quinazoline forms polar hydrogen
bond with Glu 166 and Gln 205 via guanidine moiety. Lys 26 forms pi-pi stacking with phenyl
ring of sulphaguanidine and hydrogen bonding with nitro functional group at 6th position of
quinazoline ring.
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4. Experimental
All the solvents and chemicals have been provided by, Spectrochem and Sigma-Aldrich. The
reactions have been supervised with the aid of pre-coated silica gel TLC aluminium sheets.
Melting points were established using an Analab Scientific Melting point apparatus. FTIR
spectrum was documented utilizing FTIR-8400S Shimadzu spectrometer. 1HNMR
(DMSO/CDCl3) spectra of the compounds have been determined by Bruker Avance-II
spectrometer at 400 MHz (Punjab University-Chandigarh). Chemical shift have been assessed
relative to the internal standard TMS and are reported in δ ppm. Mass spectra of the compounds
were determined at Oxygen Heath Care Pvt.Ltd.at Ahmadabad, Gujarat.
4.1 N'-(2-cyano-4-nitrophenyl)-N,N-dimethylformimidamide (2)
It is prepared as per the procedure reported by Samar Mowafy et al. [33]
4.2 6-nitro-N-substituted quinazolin-4-amine HMP (3-23)
A mixture of N’-(2-cyano-4-nitrophenyl)-N,N-dimethylformamide (2) (13.7 mmol) and
appropriate aniline (15.1 mmol) in glacial acetic acid (30 mL) was stirred and refluxed for 3 h,
reaction was monitored by TLC. After completing the reaction, mixture was filtered to get the
final compound, which was further recrystallized from ethanol.
4.2.1 4-(6-nitroquinazolin-4-ylamino)-N-(pyrimidin-2-yl)benzenesulfonamide (HMP-03 )
Solid, 81% yield; mp 291-293 °C; IR (KBr, ѵmax, cm-1): 3212 (NH), 2945 (CH), 1345 and 1543
(NO2); 1H NMR (DMSO-d6) δ ppm: 11.29 (s, 1H, SO2NH), 8.82 (s, 1H, NH), 6.38-8.71 (m,
13
11H, Ar-H); C NMR (DMSO-d6) δ ppm: 171.12, 168.34, 159.78, 156.34, 151.45, 145.78,
143.34, 130.78, 129.23, 127.12, 126.34, 120.23, 115.46, 114.89, 114.23, 112.68; EI-MS: m/z
423.1 [M+] and 425.1 [M+2]
4.2.2. N-(4-(6-nitroquinazolin-4-ylamino)phenylsulfonyl)acetamide (HMP- 04)
Solid, 62% yield; mp > 300 °C; IR (KBr, ѵmax, cm-1): 3211 (NH), 3012 (CH), 1664.43 (C=O),
1347.90 and 1548.54 (NO2); 1H NMR (DMSO-d6) δ ppm: 11.13 (s, 1H, SO2NH), 8.56 (s, 1H,
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10.1002/cbdv.202000237
Chemistry & Biodiversity
NH), 7.73-8.78 (m, 8H, Ar-H) 2.90 (s, 3H,CH3); 13C NMR (DMSO-d6) δ ppm: 171.78, 170.23,
159.45, 151.56, 145.67, 143.67, 130.67, 129.78, 128.34, 126.45, 115.34, 114.56, 112.67, 21.34;
EI-MS: m/z 387.1 [M+] and 389.1 [M+2]
4.2.3 N-carbamimidoyl-4-(6-nitroquinazolin-4-ylamino)benzenesulfonamide (HMP-05 )
Solid, 58% yield; mp 296-298 °C; IR (KBr, ѵmax, cm-1): 3249 and 3235 (NH2), 3072 (CH), 1338
and 1556 (NO2); 1H NMR (DMSO-d6) δ ppm: 10.79 (s, 1H, C=NH), 10.75 (s, 1H, NH), 7.79-
8.77 (m, 8H, Ar-H ), 6.73 (s, 2H, NH) 3.31 (s, 1H, NH) ; 13C NMR (DMSO-d6) δ ppm: 172.12,
158.61, 158.08, 157.39, 152.97, 144.60, 141, 139.79, 129.54, 126.73, 126.19, 122.02, 120.83;
EI-MS: m/z 387.0 [M+] and 389.0 [M+2]
4.2.4 5-(2-chlorophenyl)-N-(6-nitroquinazolin-4-yl)-1,3,4-thiadiazol-2-amine (HMP- 06)
Solid, 78% yield; mp > 300 °C; IR (KBr, ѵmax, cm-1): 3220 (NH), 2923 (CH), 1345 and 1551
(NO2), 728.67 (C-Cl); 1H NMR (DMSO-d6) δ ppm: 8.53 (s, 1H, NH), 7.60-8.79 (m, 8H, Ar-
13
H); C NMR (DMSO-d6) δ ppm: 175.23, 173.45, 162.46, 154.67, 152.78, 148.23, 138.89,
133.34, 131.23, 130.56, 129.34, 128.45, 127.56, 126.78, 117.57, 114.34; EI-MS: m/z 384.0
[M+] and 386.0 [M+2]
4.2.5 N-(6-nitroquinazolin-4-yl)thiazol-2-amine (HMP-07 )
Solid, 82% yield; mp 272-274 °C; IR (KBr, ѵmax, cm-1): 3224 (NH), 2913 (CH), 1350 and 1552
1
13
(NO2); H NMR (DMSO-d6) δ ppm: 8.56 (s, 1H, NH), 6.70-8.79 (m, 6H, Ar-H); C NMR
(DMSO-d6) δ ppm: 173.56, 162.34, 158.23, 153.23, 147.56, 138.12, 132.56, 128.88, 117.56,
116.23, 113.24; EI-MS: m/z 273.2 [M+] and 275.2 [M+2]
4.2.6 5-methyl-N-(6-nitroquinazolin-4-yl)-1,3,4-thiadiazol-2-amine (HMP-08 )
Solid, 84% yield; mp > 300 °C; IR (KBr, ѵmax, cm-1): 3242 (NH), 2920 (CH), 1341 and 1552
(NO2); 1H NMR (DMSO-d6) δ ppm: 8.57 (s, 1H, NH), 7.62-8.80 (m, 4H, Ar-H), 2.89 (s, 3H,
CH3); 13C NMR (DMSO-d6) δ ppm: 173.34, 162.46, 153.12, 151.78, 147.68, 143.69, 131.34,
128.23, 117.68, 114.34, 20.23; EI-MS: m/z 288.2 [M+]
4.2.7 N-(6-nitroquinazolin-4-yl)-5-phenyl-1,3,4-thiadiazol-2-amine (HMP-09)
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Chemistry & Biodiversity
Solid, 79% yield; mp > 300 °C; IR (KBr, ѵmax, cm-1): 3226 (NH), 2921 (CH), 1358 and 1544
1
13
(NO2); H NMR (DMSO-d6) δ ppm: 8.40 (s, 1H, NH), 6.78-8.87 (m, 9H, Ar-H); C NMR
(DMSO-d6) δ ppm: 175.45, 173.46, 159.23, 153.89, 151.23, 147.67, 134.89, 131.67, 130.23,
129.12, 127.89, 126.34, 117.23, 114.34; EI-MS: m/z 350.0 [M+] and 352.0 [M+2]
4.2.8 N-(6-nitroquinazolin-4-yl)-5-(trifluoromethyl)-1,3,4-thiadiazol-2-amine (HMP- 10)
Solid, 63% yield; mp 200-202 °C; IR (KBr, ѵmax, cm-1): 3260 (NH), 2927 (CH), 1356 and 1534
(NO2), 1098 (C-F); 1H NMR (DMSO-d6) δ ppm: 8.48 (s, 1H, NH), 6.82-7.87 (m, 4H, Ar-H);
13C NMR (DMSO-d6) δ ppm: 174.45, 162.45, 158.76, 153.89, 151.89, 147.98, 131.34, 126.56,
118.78, 117.56, 114.34; EI-MS: m/z 342.4 [M+] and 344.2 [M+2]
4.2.9 N-(6-nitroquinazolin-4-yl)-5-p-tolyl-1,3,4-thiadiazol-2-amine (HMP-11)
Solid, 54% yield; mp >300 °C; IR (KBr, ѵmax, cm-1): 3262 (NH), 2918 (CH), 1345 and 1573
(NO2); 1H NMR (DMSO-d6) δ ppm: 8.97 (s, 1H, NH), 6.65-8.89 (m, 8H, Ar-H), 2.89 (s, 3H,
CH3); 13C NMR (DMSO-d6) δ ppm: 175.45, 173.34, 162.78, 154.78, 153.78, 147.34, 132.56,
130.67, 128.34, 127.68, 126.23, 120.67, 117.68, 1154.56, 22.23; EI-MS: m/z 364.0 [M+] and
366.0 [M+2]
4.2.10 4-(5-(6-nitroquinazolin-4-ylamino)-1,3,4-thiadiazol-2-yl)phenol (HMP-12 )
Solid, 58% yield; mp >300 °C; IR (KBr, ѵmax, cm-1): 3380 (OH), 3253 (NH), 2920 (CH), 1374
and 1557 (NO2); 1H NMR (DMSO-d6) δ ppm: 9.75 (s, 1H, OH), 8.70 (s, 1H, NH), 6.67-8.82
(m, 8H, Ar-H); 13C NMR (DMSO-d6) δ ppm: 175.45, 171.78, 162.34, 159.89, 154.34, 152.34,
147.68, 131.34, 129.23, 128.56, 126.24, 117.78, 116.34, 114.45; EI-MS: m/z 366.0 [M+] and
368.0 [M+2]
4.2.11 N-(2,4-dimethylphenyl)-6-nitroquinazolin-4-amine (HMP-13)
Solid, 65% yield; mp 180-182 °C; IR (KBr, ѵmax, cm-1): 3271 (NH), 2918 (CH), 1376 and 1574
(NO2), 1H NMR (DMSO-d6) δ ppm: 8.28 (s, 1H, NH), 6.72-8.80 (m, 7H, Ar-H), 2.30 (s, 3H,
CH3), 2.16 (s, 3H, CH3); 13C NMR (DMSO-d6) δ ppm: 173.45, 162.89, 153.34, 147.89,
22
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Chemistry & Biodiversity
140.34, 138.45, 132.34, 130.36, 128.48, 127.34, 125.57, 118.34, 116.78, 114.89, 22.34, 19.14;
EI-MS: m/z 294.2 [M+]
4.2.12 2-(2-(6-nitroquinazolin-4-ylamino)ethoxy)ethanol (HMP-14)
Solid, 58% yield; mp 190-192 °C; IR (KBr, ѵmax, cm-1): 3263 (NH), 2920 (CH), 1342 and 1549
(NO2), 1176.43 (C=O); 1H NMR (DMSO-d6) δ ppm: 9.97 (s, 1H, NH), 6.75-8.88 (m, 4H, Ar-
H), 5.45 (s, 1H, OH), 3.20-3.50 (m, 8H, CH2CH2-O-CH2CH2); 13C NMR (DMSO-d6) δ ppm:
163.46, 161.68, 153.34, 147.22, 132.56, 128.58, 117.34, 114.12, 71.46, 69.58, 63.12, 45.10;
EI-MS: m/z 278.2 [M+]
4.2.13 N-(1-benzylpiperidin-4-yl)-6-nitroquinazolin-4-amine (HMP-15)
Solid, 55% yield; mp 232-234 °C; IR (KBr, ѵmax, cm-1): 3235 (NH), 2920 (CH), 1372 and 1584
(NO2); 1H NMR (DMSO-d6) δ ppm: 9.12 (s, 1H, NH), 6.68-8.90 (m, 9H, Ar-H), 1.50-2.60 (m,
9H, CH2 Piperidine), 3.12 (s, 2H, CH2); 13C NMR (DMSO-d6) δ ppm: 162.46, 159.12, 154.78,
147.34, 137.24, 132.58, 130.34, 129.56, 127.24, 126.12, 117.56, 114.88, 65.46, 57.89, 52.34,
32.23; EI-MS: m/z 363.2 [M+]
4.2.14 N-(3-methylpiperazin-1-yl)-6-nitroquinazolin-4-amine (HMP-16)
Solid, 58% yield; mp 256-258 °C; IR (KBr, ѵmax, cm-1): 3272 (NH), 2936 (CH), 1336 and 1544
(NO2); 1H NMR (DMSO-d6) δ ppm: 9.85 (s, 1H, NH), 6.72-8.88 (m, 4H, Ar-H), 1.56-2.85 (m,
6H, CH2 piperazine), 1.89 (s, 1H, NH piperazine ), 1.10 (s, 3H, CH3); 13C NMR (DMSO-d6) δ
ppm: 173.35, 161.12, 153.78, 147.34, 132.24, 128.46, 117.24, 114.46, 69.24, 63.12, 52.34,
40.58, 15.12; EI-MS: m/z 288.3 [M+]
4.2.15 N-cyclopropyl-6-nitroquinazolin-4-amine (HMP-17)
Solid, 59% yield; mp 195-197 °C; IR (KBr, ѵmax, cm-1): 3263 (NH), 2974 (CH), 1371 and 1541
(NO2); 1H NMR (DMSO-d6) δ ppm: 9.74 (s, 1H, NH), 8.20-8.80 (m, 4H, Ar-H), 0.50-2.87 (m,
13
5H, CH2); C NMR (DMSO-d6) δ ppm: 162.24, 159.12, 153.11, 147.34, 131.45, 128.12,
117.56, 116.56, 27.22, 08.78; EI-MS: m/z 230.2 [M+]
23
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Chemistry & Biodiversity
4.2.16 6-nitro-N-(pyridin-2-ylmethyl)quinazolin-4-amine (HMP- 18)
Solid, 52% yield; mp 212-214 °C; IR (KBr, ѵmax, cm-1): 3290.13 (NH), 2964.42 (CH),1350 and
1504 (NO2); 1H NMR (DMSO-d6) δ ppm: 9.96 (s, 1H, NH), 6.78-8.79 (m, 8H, Ar-H), 4.68 (s,
13
2H, CH2); C NMR (DMSO-d6) δ ppm: 162.24, 159.24, 157.89, 153.34, 148.78, 145.34,
140.24, 132.68, 129.24, 122.57, 119.34, 117.34, 114.24, 49.89; EI-MS: m/z 281.1 [M+]
4.2.17 N-cyclohexyl-6-nitroquinazolin-4-amine (HMP-19)
Solid, 56% yield; mp 170-172 °C; IR (KBr, ѵmax, cm-1): 3278 (NH), 3026 (CH), 1317 and 1571
(NO2); 1H NMR (DMSO-d6) δ ppm: 9.09 (s, 1H, NH), 8.35-8.80 (m, 4H, Ar-H), 1.10-1.75 (m,
10H, CH2 Cyclohexyl); 13C NMR (DMSO-d6) δ ppm: 162.34, 159.12, 153.46, 147.89, 131.34,
126.89, 117.24, 116.80, 56.23, 33.34, 26.68, 25.24; EI-MS: m/z 272.3 [M+]
4.2.18 4-(6-nitroquinazolin-4-ylamino)benzoic acid (HMP-20)
Solid, 64% yield; mp > 300 °C; IR (KBr, ѵmax, cm-1): 3294 (NH), 3486 (OH) 2961 (CH), 1708
1
(C=O), 1345 and 1530 (NO2); H NMR (DMSO-d6) δ ppm: 12.10 (s, 1H, OH), 8.89 (s, 1H,
NH), 7.45-7.83 (m, 8H, Ar-H); 13C NMR (DMSO-d6) δ ppm: 173.34, 168.24, 159.46, 153.58,
147.24, 132.12, 130.38, 129.24, 126.28, 121.12, 117.38, 114.12, 112.58; EI-MS: m/z 310.1
[M+]
4.2.19 N1-(6-nitroquinazolin-4-yl)benzene-1,2-diamine (HMP-21)
Solid, 73% yield; mp 194- 96 °C; IR (KBr, ѵmax, cm-1): 3476 (NH), 2958 (CH), 1371 and 1544
(NO2); 1H NMR (DMSO-d6) δ ppm: 9.54 (s, 2H, NH), 3.91 (s, 1H, NH2), 6.78-7.80 (m, 8H,
Ar-H); 13C NMR (DMSO-d6) δ ppm: 173.46, 159.24, 153.12, 147.34, 143.58, 137.24, 133.80,
131.34, 130.12, 128.34, 127.12, 120.34, 117.54, 114.34; EI-MS: m/z 281.2 [M+]
4.2.20 4-methyl-7-(6-nitroquinazolin-4-ylamino)-2H-chromen-2-one (HMP-22)
Solid, 61% yield; mp >300 °C; IR (KBr, ѵmax, cm-1): 3045 (NH), 2933 (CH), 1374 and 1508
(NO2), 1711 (C=O); 1H NMR (DMSO-d6) δ ppm: 8.92 (s, 1H, NH), 6.20-7.78 (m, 8H, Ar-H),
13
2.45 (s, 3H, CH3); C NMR (DMSO-d6) δ ppm: 173.56, 161.34, 159.90, 154.45, 153.56,
24
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Chemistry & Biodiversity
151.34, 147.58, 143.89, 131.34, 128.34, 125.89, 117.68, 116.68, 113.48, 110.48, 109.89,
107.46, 20.34; EI-MS: m/z 348.2 [M+]
4.2.21 6-nitro-N-(3,4,5-trimethoxyphenyl)quinazolin-4-amine (HMP- 23)
Solid, 65% yield; mp 250 °C; IR (KBr, ѵmax, cm-1): 3273 (NH), 2986 (CH) 1087 (C=O), 1340
and 1574 (NO2); 1H NMR (DMSO-d6) δ ppm: 8.45 (s, 1H, NH), 6.48-8.79 (m, 6H, Ar-H), 3.83
13
(s, 3H, OCH3), 3.89 (s, 3H, OCH3); C NMR (DMSO-d6) δ ppm: 173.56, 162.78, 155.68,
153.48, 147.46, 137.87, 131.34, 128.47, 126.38, 117.38, 114.58, 97.93, 62.85, 54.83; EI-MS:
m/z 356.2 [M+]
4.3. Antimycobacterial activity
In vitro assessment of the anti-mycobacterial activity of the newly synthesized compounds
HMP (3-23) was completed at the National Institute of Allergy and Infectious Diseases
(NIAID) (Bethesda, MD, USA). The MIC was established against M. tuberculosis strain
H37Rv grown under aerobic conditions, utilizing a dual readout (OD590 and fluorescence)
assay. [34]
4.4. MDR-Inhibitory Activity
MDR-TB inhibitory activity has been performed at the Micro Care Laboratory and
Tuberculosis Research Center Surat, India screening program utilizing by Löwenstein-Jensen
method against the clinically isolated phenotype MDR strain as per the procedure reported by
Soni et al . [35]
4.5. Cytotoxicity
The selected set of compounds which showed potent activity against MTB (H37Rv) strains
were also evaluated for their cytotoxicity on VERO cell Lines by MTT assay as per the
procedure reported by Falzari et al. [36]
4.6 Molecular Docking
The crystal structure of GlmU MTB (PDB: 3ST8) containing substrates, N-acetyl glucosamine-
1-phosphate (NAcGlc-1-P) and uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) was
25
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10.1002/cbdv.202000237
Chemistry & Biodiversity
processed using Protein Preparation Wizard module of Schrödinger 9.3. [31] This involved some
parameters such as assigning bond orders as the protein is large and contain two substrates,
addition of hydrogens as pdb file does not contain hydrogens, adjusting bond orders and formal
charges for any cofactors if needed, correcting mislabeled elements, adjusting the ionization
and tautomerization states of both protein and substrates etc. The crystallographic water
molecules at distance above 5 Å were removed. However, the water molecules interacting with
the active sites were retained. In the next approach of protein preparation, minimization of the
protein was performed using OPLS_2005 force field up to maximum deviation of 0.8 Å before
[31]
it was processed for generating grid.
To soften the potential for non-polar parts of the
receptor, all atoms were scaled for van der Waal radii of 1.0 Å with partial atomic charges less
than the cut-off value of 0.25 electron unit. The binding region (grid) was defined
corresponding to X, Y, and Z coordinates, over the co-crystallized ligands uridine diphosphate-
N-acetylglucosamine (UDP-GlcNAc) present in the active site respectively. [31]
Acknowledgements
We thank the Department of Pharmaceutical Chemistry, Discipline of Pharmaceutical
Sciences, College of Health Sciences, University of KwaZulu-Natal, South Africa for their
constant support, encouragement and financial assistance. The authors are grateful to the
National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA for
antimycobacterial screening and to Mr. Dilip Jagjivan (UKZN, South Africa) for his assistance
with the NMR experiments.
Authors Contribution
Harun M. Patel and Rajshekhar Karpoormath have performed the experiments, analyzed the
data and wrote the paper. Mahesh Palkar analyzed the data.
26
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