Novel lead generation of an anti-tuberculosis agent active against non-replicating mycobacteria: Exploring hybridization of pyrazinamide with multiple fragments

Article abstract of DOI:10.1007/s00044-015-1352-6

The key to shortening tuberculosis (TB) drug regimen lies in eliminating the reservoir of non-replicating persistent (NRP) Mycobacterium tuberculosis (Mtb). Pyrazinamide (PZA) is the only known drug used as part of a combination therapy that is believed to kill NRP Mtb and achieve sterilization. PZA is active only under low pH screening conditions. Screening and identification of NRP-active anti-TB compounds are severely limited because compounds are usually inactive under regular assay conditions. In an effort to design novel NRP-active anti-TB compounds, we used pyrazinamide as a core and hybridized it with the fragments derived from marketed drugs. One of these designs, compound 8, was a hybrid with fluoroquinolone. This compound exhibited >10 fold improvement in NRP activity under low pH condition as compared to pyrazinamide and a modest activity (0.8 log₁₀ kill) under nutritionally starved NRP condition. Furthermore, compound 8 was active against fluoroquinolone-resistant strains and did not show any activity in a DNA supercoiling assay (gyrase inhibition), suggesting that its mechanism of action is not that of the parent fluoroquinolone. These results provide a novel avenue in the exploration of new chemotypes that are active against non-replicating Mtb.

Full text of DOI:10.1007/s00044-015-1352-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1352-6
ORIGINAL RESEARCH
Novel lead generation of an anti-tuberculosis agent active against
non-replicating mycobacteria: exploring hybridization
of pyrazinamide with multiple fragments
•
Shankar D. Markad Parvinder Kaur B. K. Kishore Reddy
•
•
•
Murugan Chinnapattu Anandkumar Raichurkar
•
•
•
Radha Nandishaiah Manoranjan Panda Pravin S. Iyer
Received: 6 June 2014 / Accepted: 27 February 2015
Ó Springer Science+Business Media New York 2015
Abstract The key to shortening tuberculosis (TB) drug
regimen lies in eliminating the reservoir of non-replicating
persistent (NRP) Mycobacterium tuberculosis (Mtb). Pyr-
azinamide (PZA) is the only known drug used as part of a
combination therapy that is believed to kill NRP Mtb and
achieve sterilization. PZA is active only under low pH
screening conditions. Screening and identification of NRP-
active anti-TB compounds are severely limited because
compounds are usually inactive under regular assay con-
ditions. In an effort to design novel NRP-active anti-TB
compounds, we used pyrazinamide as a core and hy-
bridized it with the fragments derived from marketed
drugs. One of these designs, compound 8, was a hybrid
with fluoroquinolone. This compound exhibited [10 fold
improvement in NRP activity under low pH condition as
compared to pyrazinamide and a modest activity (0.8 log10
kill) under nutritionally starved NRP condition. Further-
more, compound 8 was active against fluoroquinolone-re-
sistant strains and did not show any activity in a DNA
supercoiling assay (gyrase inhibition), suggesting that its
mechanism of action is not that of the parent fluoro-
quinolone. These results provide a novel avenue in the
exploration of new chemotypes that are active against non-
replicating Mtb.
Keywords Tuberculosis (TB) ꢀ Non-replicating (NRP) ꢀ
Pyrazinamide (PZA) ꢀ Fluoroquinolone (FQ) ꢀ
Hybridization
Tuberculosis (TB) causes approximately 2 million deaths
per year. An estimated one-third of the world’s population
is thought to be infected with dormant or latent forms of
Mycobacterium tuberculosis (Dye and Williams, 2010;
Russel et al., 2010). Infections with multidrug-resistant
(MDR) and extensively drug-resistant (XDR) mycobacte-
rial strains as well as co-infection with HIV create a global
health challenge (Gandhi et al., 2010; Mandavilli et al.,
2007). Currently, treatment of TB involves administration
of a combination of isoniazid, rifampin, pyrazinamide and
ethambutol antibiotics for the first 2 months, followed by
an additional 4-month treatment with isoniazid and ri-
fampin (Mitchison, 2005). In the case of MDR tuberculo-
sis, the antibacterial chemotherapy is extended to
12–24 months. This long treatment duration coupled with
poor patient compliance contributes directly to the emer-
gence of MDR and XDR strains of M. tuberculosis limiting
the efficacy of standard therapy (Check, 2007; Dye, 2009).
Screening of new antimycobacterial agents against actively
replicating bacteria is more popular and high throughput
than non-replicating bacteria. However, it is generally be-
lieved that a non-replicating persistence (NRP) is respon-
sible for antimicrobial tolerance. In TB, the key to
shortening the 6-month regimen lies in targeting the NRP
subpopulation (Cho et al., 2007). Herein, we present a
novel approach to target NRP Mtb for the development of
new anti-TB drugs. Pyrazinamide (PZA), one of the
frontline anti-TB drugs, is known to be highly effective
against experimental murine TB in combination with iso-
niazid (McCune et al., 1956). PZA is effective against NRP
in vivo, but under in vitro conditions it shows anti-TB
Shankar D. Markad and Parvinder Kaur have contributed equally to
this work.
S. D. Markad (&) ꢀ P. Kaur ꢀ B. K. Kishore Reddy ꢀ
M. Chinnapattu ꢀ A. Raichurkar ꢀ R. Nandishaiah ꢀ M. Panda ꢀ
P. S. Iyer (&)
AstraZeneca India Pvt. Ltd., Avishkar, Kirloskar Business Park,
Bellary Road, Hebbal, Bangalore 560 024, Karnataka, India
e-mail: shankar.markad@yahoo.com
P. S. Iyer
e-mail: praviniyer@yahoo.com
123

Med Chem Res
2. (R)-1-Boc-3-(hydroxymethyl)piperazine II was treated
with chloroacetyl chloride to give an amide III which
was cyclized to intermediate IV under basic conditions.
Tert-butyloxycarbonyl (Boc) deprotection of interme-
activity only under acidic pH conditions (Heifets and
Lindholm-Levy, 1992; Heifets and Sanchez, 2000). Several
attempts have been made to make pharmacologically su-
perior analogs of PZA without success (Zimhony et al.,
Scheme 2a
2a/5a
Synthesis of fragment
O
H
H
H
H
Cl
1. TFA/DCM
2. LAH/THF
t-BuOK, THF
HN
O
Cl
BocN
BocN
O
OH
BocN
OH
(R)
N
(R)
N
(R)
N
(R)
NH
NEt3/DCM
Cl
O
O
II
III
IV
2a/5a
diate IV was carried out by using trifluoroacetic acid
(TFA) followed by reduction with lithium aluminum
hydride (LAH) to give the fragment 2a/5a (Safina et al.,
2012) of GSK-13222322 (Scheme 2a).
2007). Recently, we have shown Fragmentation Intelligent
Re-combination Enumeration (F.I.R.E.) to synthesize vir-
tual libraries and in silico screening to generate hits (Iyer
and Panda, 2014). In this approach, marketed drugs and
clinical candidates were fragmented into cores and
daughters, and recombined in various permutation and
combinations to regenerate the full molecules. These
molecules could potentially contribute to the biological
activity. In the present study, we have used PZA as a core
and designed a set of compounds by hybridizing it with
specific fragments. Each of these compounds was screened
on Mtb under NRP conditions (acidic stressed and nutrient
starvation) to find a potential lead candidate.
3. The nucleophilic aromatic substitution (SNAr) of 3,4-
difluoronitrobenzene V with thiomorpholine followed
by palladium catalyzed hydrogenation afforded the
required fragment 3a (Ryukou et al., 2007) of Sute-
zolid (Scheme 3a).
Scheme 3a
3a
Synthesis of fragment
S
S
N
F
F
1. MeCN, HN
2. H2, Pd/C, MeOH
H2N
N
F
O2
Syntheses of fragments
V
3a
The required fragments have been synthesized by follow-
ing literature procedures.
4. The nucleophilic substitution reaction of 1,3-bis(tert-
butoxycarbonyl)-2-methyl-2-thiopseudourea VI with
1,4-diaminobutane in tetrahydrofuran (THF)/water
provided the required fragment 4a (Carmignani
et al., 2001) of PMX-30063 (Scheme 4a).
1. The selective reductive amination of 2-adamantanone
I with ethylenediamine using sodium borohydride (NaBH4)
in 1,2-dichloroethane (DCE) provided the required frag-
ment 1a (Meng et al., 2007) of SQ-109 (Scheme 1a).
Scheme 4a
4a
Synthesis of fragment
THF/H2O
NHBoc
NHBoc
N
MeS
NHBoc
NH2
H2N
+
H2N
NBoc
VI
VII
4a
5. The commercially available 2,4-dinitroimidazole VIII
was treated with (S)-glycidol to afford the intermediate
IX. The resulting secondary hydroxyl group of the
intermediate IX was protected with the tetrahydropy-
ranyl (THP) ether by using dihydropyran (DHP)
and pyridinium p-toluenesulfonate (PPTS) to give
the intermediate X. The selective deprotection of
Scheme 1a Synthesis of fragment 1a
H
N
O
NH2
H2N
H2N
DCE, NaBH4, RT
I
1a
123

Med Chem Res
tert-butyldimethyl (TBS) ether of intermediate X with
7. The commercially available b-ketoester XVI was
treated with N,N-dimethylformamide dimethyl acetal
(DMF.DMA) followed by cyclopropylamine to give
the intermediate XVII. The base-mediated cyclization
of intermediate XVII followed by ester hydrolysis
under acidic conditions afforded the required fragment
8a (Matsumoto et al., 1986) of Moxifloxacin.
tetra-n-butyl ammonium fluoride (TBAF) followed by
cyclization gave the intermediate XI. Finally, the THP
deprotection was carried out by using aqueous acetic
acid to give the required fragment 6a (Orita et al.,
2007) of PA-824 (Scheme 6a).
Scheme 8a
8a.
Synthesis of fragment
O
O
O
O
O
N
O
F
F
1. DMF.DMA/Ac2O, CH2Cl2
F
1. K3PO4, CH3CN
2. cHCl/AcOH
Et
O
Et
O
H
O
2. cyclopropylamine, EtOH
l
C
N
l
C
l
C
N
l
C
NH
l
C
N
XVI
XVII
8a
Scheme 6a
6a
Synthesis of Fragments
N
N
N
O2
N
O2
N
O2
OTBS
EtOH
N
NO2
O2
N
DHP, PPTS,
DCM
TBAF/THF
O
NO2
N
OTBS
N
OTBS
N
H
VIII
H
O
OTHP
IX
X
O
O
N
N
AcOH/THF/H2O
N
O2
N
O2
(S)
(S)
N
N
OTHP
OH
XI
6a
6. The free hydroxyl group of piperidin-3-ol XII was
activated by mesylation after protecting the free amine
with tert-butyloxycarbonyl (Boc) under standard con-
ditions to give the intermediate XIII. The mesyloxy
(OMs) group of intermediate XIII was displaced with
ethyl thioacetate to give the intermediate XIV. Tert-
butyloxycarbonyl (Boc) deprotection using tri-
fluoroacetic acid (TFA) followed by coupling with
Boc-L-valine gave amide XV. Finally, the ethyl ester
was hydrolyzed to give the corresponding acid 7a
(Berner and Kerber, 2004) of BC-3205 (Scheme 7a).
Synthesis of pyrazinamide derivatives
The chlorination followed by esterification of 5-oxo-4,5-
dihydropyrazine-2-carboxylic acid 10 by using literature
procedure gave the methyl 5-chloropyrazine-2-carboxylate
11 (Coe et al., 2013) in 55 % yield. The chloro substitution
of compound 11 was displaced with various fragments 1a,
2a, 3a and 4a followed by ammonolysis to give compounds
1, 2, 3 and 4, respectively (Scheme 1).
In order to prepare 6-substituted pyrazinamide deriva-
tives, methyl pyrazine-2-carboxylate 12 was treated with
Scheme 7a
7a.
Synthesis of fragment
O
H
O
S
OMs
1. (Boc)2O/NEt3, CH2Cl2
2. MsCl/NEt3, CH2Cl2
H
H
Et
SC 2CO2
,
Et
O
s
C
N
H
2CO3 DMF
,
N
N
Boc
Boc
XII
XIII
XIV
O
O
1. TFA/CH2Cl2
LiOH,
S
S
Et
O
H
O
2. HATU, DIPEA,
THF/H2O
N
N
H
l
C
2C 2
O
NHBoc
NHBoc
(R)
O
O
H
O
R
(
R
(
)
)
NHBoc
XV
7a
123

Med Chem Res
O
O
O
N
N
N
N
(a)
(b)
(c)
H
O
O
NH2
S
N
H
10
O
l
C
N
R1
N
F
11
H
N
=
1
,
R
=
3
R
,
1
1
N
N
H
H
NHBoc
NHBoc
NH2
NH2
N
=
4b
R
R
,
1
N
H
H
=
2
,
R
(d)
1
N
O
(R)
N
=
4
,
N
1
N
H
Scheme 1 Reagents and conditions. (a) (i) SOCl2, PhCH3, DMF (cat.), 110 °C, 2 h; (ii) MeOH, RT, 16 h; (b) RR0NH, K2CO3, DMF, 120 °C,
MW, 1 h; (c) NH3 in MeOH (8 M), 120 °C, 16 h; (d) HCl in dioxane (4 M), RT, 1 h
Scheme 2 Reagents and
O
O
O
conditions: (a) m-CPBA,
ClCH2CH2Cl, 60 °C, 16 h;
(b) SOCl2, 75 °C, 8 h;
(c) K2CO3, DMF, 120 °C, 1 h
(for 5); Cs2CO3, DMF, MW,
100 °C, 1 h (for 6); (d) NH3 in
MeOH (8 M), 80 °C, 1 h
R2
N
N
l
C
N
N
(c)
(d)
NH2
(a)
(b)
O
O
N
N
H
12
13
=
=
5
R
R
,
N
O
(R)
N
O
N
NO2
(S)
6
,
N
O
m-CPBA followed by thionyl chloride to give methyl
6-chloropyrazine-2-carboxylate 13 (Scanio et al., 2011) by
using literature procedures. The chloro substitution of
compound 13 was displaced with fragments 5a and 6a
followed by ammonolysis to give compounds 5 and 6, re-
spectively (Scheme 2).
The required 5-(aminomethyl)pyrazine-2-carboxamide 16
was obtained from methyl 5-methylpyrazine-2-carboxylate
14. The bromination of compound 14 by using NBS/AIBN
(N-bromosuccinimide/azoisobutyronitrile) gave compound
15 (Aditya and Kodadek, 2012). Finally, ammonolysis of
the compound 15 gave the compound 16. The amine
O
O
O
N
N
N
NH2
O
O
(a)
(b)
Br
H2N
N
N
N
14
15
16
O
O
N
O
F
H
O
N
S
N
H
(c)
(d)
N
N
(e)
N
N
H
NH2
16
N
N
NH2
O
NH2
O
(R)
7
8
O
Scheme 3 Reagents and conditions: (a) NBS, AIBN, CCl4, 80 °C, 16 h; (b) NH3 in MeOH (8 M), RT, 2 h; (c) 7a, HATU, DIPEA, DCM, RT,
16 h; (d) HCl in dioxane (4 M); (e) 8a, CH3CN, NEt3, 80 °C, 2 h
123

Med Chem Res
functionality of the compound 16 was coupled with the
carboxylic acid of fragment 7a to give the required amide
which was treated with HCl to afford the compound 7.
The chloro functionality of the 7-chloro-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydro-1,8-naphthyridine-3-carboxylic
acid 8a was displaced with amine 16 to give compound 8
(Scheme 3).
that work specifically under acidic pH, thereby killing
bacilli that have gone into a non-replicating state following
acidic stress. Recently, novel assays to screen for com-
pounds that disrupt intrabacterial pH homeostasis have
been described (Vandal et al., 2009; Darby et al., 2013).
Therefore, in the present study, the hybridized compounds
of PZA with multiple fragments along with PZA reference
compound 9 as a control were screened on Mtb (M. tu-
berculosis H37Rv ATCC 27294) for their minimum in-
hibitory concentrations (MIC) and minimum bactericidal
concentrations (MBC) using an in vitro low pH (6.3 0.1)
assay. The compound 8 showed MIC = 100 lM which
was tenfold better than that of PZA compound 9,
MIC = 1039 lM. The other compounds turned out to be
inactive at the maximum concentration tested (MIC [
100 lM) (Table 1).
To investigate whether the improved activity of com-
pound 8 could be attributed to the fluoroquinolone core,
activity against fluoroquinolone-resistant isolates was
measured. Since activity was retained against fluoro-
quinolone-resistant isolates, the improved activity is un-
likely to be mediated through the biological target of
fluoroquinolones (Table 2). Fluoroquinolones are known to
inhibit DNA gyrase, and therefore, it was important to
understand the activity of compound 8 against DNA gy-
rase. In fact compound 8 was found inactive in a DNA
supercoiling assay indicating novel mechanism of action
(Table 3).
Biological screening
Depending on macrophage activation status, intracellular
Mtb is exposed to numerous stresses including reactive
oxygen/nitrogen species, low pH (in the range of pH
3.0–6.5) and reduced access to micronutrients following
activation with gamma interferon. This critical immuno-
logical event, results in the fusion of lysosomes with
phagosomes thereby exposing intra-phagosomal Mtb to an
acidic environment (ranging from pH of 3.0 to 6.5) and to
numerous hydrolases that can potentially degrade micro-
bial nucleic acids and other cellular components. During
many years of evolution with the human host, Mtb has
developed several mechanisms to combat the stresses
present within the infected macrophage. One of the ap-
proaches to target the Mtb would be to identify compounds
Table 1 MIC and MBC under low pH conditions
Comp. No. Mtb-MIC (lM) Mtb-MBC (lM) 2 Log10 reduction
Compound 8 showed better MBC (*2 log10 reduction)
results than PZA (100 vs. 2078 lM under low pH condi-
tions on day 14 (Fig. 1), which indicated that the
1
[100
[100
[100
[100
[100
[100
[100
100
[100
[100
[100
[100
[100
[100
[100
100
2
3
4
Table 3 Mtu gyrase supercoiling assay
5
Compound
IC50 (lM)
6
8
[100
7.3
7
Moxifloxacin
Novobiocin
8
0.028
PZA, 9
1039
2078
Table 2 MIC (lM) under low pH conditions and fluoroquinolone-resistant strains (Ofloxres Mtb strainsa,b from AstraZeneca strain bank)
Neutral pH 7.2
7H9 broth
H37Rv
Low pH 6.3
7H9-MonoKPO4
H37Rv
OfloxÒb
MoxÒa
OfloxÒb
MoxÒa
8
50
50
50
100
200
100
Moxi
Oflox
PZA
Inh
a
1.14
1.38
[2078
0.23
1.14
[18.2
1.38
2.28
2.76
1039
2.28
[18.2
5.52
[11.04
[2078
0.23
[11.04
1039
[2078
0.23
1039
Mutant
b
Clinical isolate
123

Med Chem Res
hybridized compound of FQ and PZA looks promising for
further optimization.
Table 5 Broad spectrum panel MIC data of compound 8
Broad spectrum MIC data of compound 8
Compound 8 was also evaluated for its activity in the
nutrient starvation model (Table 4) along with pyr-
azinamide as a positive control and isoniazid as a negative
control. Compound 8 shows net bactericidal activity as 0.8
log10 cfu kill vs. pyrazinamide which shows 1.6 log10 cfu
kill at 1009 MIC concentrations tested (Huang et al.,
2007). Compound 8 exhibited a good dose response from
109, 309 and 1009 MIC concentrations tested. Isoniazid,
which is known to act only on replicating bacilli, did not
show any kill even at 1009 MIC as expected.
Strain
MIC (lM)
E. coli WT
50
E. coli efflux mutant
H. influenzae WT
H. influenzae efflux mutant
S. pneumoniae
0.78
3.12
1.56
100
S. aureus MRQR
P. aeruginosa WT
P. aeruginosa efflux mutant
K. pneumoniae
[200
[200
12
In addition, the data from a panel of broad spectrum
bacteria (from AstraZeneca strain bank) suggest that
compound 8 is active in both gram-positive and gram-
negative pathogens (Table 5). Reference antibiotic controls
were used (Ofloxacin O-8757, Moxifloxacin S-1465, Iso-
niazid I-3377, Novobiocin N-1628, from SIGMA Chemical
Co.) as appropriate in the various assays.
140
Table 6 Physicochemical properties of compound 8
Physicochemical properties of compound 8
AZ LogD
-0.405
68.3
The in vitro DMPK data for 8 suggest that it has de-
sirable physicochemical properties, modest aqueous
Solubility dried DMSO (pH = 7.4) (lM)
PPB (% free)
54.03
Rat Clint (lL/min/mg)
Hu Caco2 ABBA (1E - 6.cm/s)
6.44
1.01; 5.96
solubility and human Caco2 (ATCC Catalog # HTB-37)
permeability, low rat in vitro microsomal clearance and
excellent PPB (Table 6).
In conclusion, we have demonstrated a novel strategy of
generating hits active against NRP mycobacteria using a
hybridization approach of pyrazinamide with multiple
fragments. The identified hit showed good physicochem-
ical properties and bactericidal effect resulting in *2 log10
kill at low pH and *1 log10 kill under nutrient starvation
condition. Furthermore, this hit was also active against a
broad spectrum panel of bacteria.
Fig. 1 MBC data of compound 8 in the low pH assay condition
Acknowledgments Authors are thankful to Dr. Vasan Samban-
damurthy for providing MoxR Mtb strain and Dr. Prashanthi Mad-
havapeddi for interpretation of DNA supercoiling assay data for this
study. We are grateful to Dr. Boudewijn deJonge and Dr. Vasan
Sambandamurthy for proofreading this manuscript. The analytical
support provided by Suresh Rudrapatna and Menasinakai Sreeniva-
saiah is deeply acknowledged.
Table 4 Activity of compound 8 under nutrient starvation
Activity of compound 8 in nutrient starvation model
Control
8
CCntrl
log10 Kill D14
log10 Kill D28
109
309
1009
109
309
1009
109
309
1009
0.1
0.3
0.4
1.0
1.1
1.2
0.1
0.1
0.1
0.2
0.4
Conflict of interest None.
0.8
PZA
1.4
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