Engineering another class of anti-tubercular lead: Hit to lead optimization of an intriguing class of gyrase ATPase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2016.06.042

A structure based medium throughput virtual screening campaign of BITS-Pilani in house chemical library to identify novel binders of Mycobacterium tuberculosis gyrase ATPase domain led to the discovery of a quinoline scaffold. Further medicinal chemistry explorations on the right hand core of the early hit, engendered a potent lead demonstrating superior efficacy both in the enzyme and whole cell screening assay. The binding affinity shown at the enzyme level was further corroborated by biophysical characterization techniques. Early pharmacokinetic evaluation of the optimized analogue was encouraging and provides interesting potential for further optimization.

Full text of DOI:10.1016/j.ejmech.2016.06.042

Accepted Manuscript
Engineering another class of anti-tubercular lead: Hit to lead optimization of an
intriguing class of gyrase ATPase inhibitors
Variam Ullas Jeankumar, Rudraraju Srilakshmi Reshma, Rahul Vats, Renuka
Janupally, Shalini Saxena, Perumal Yogeeswari, Dharmarajan Sriram
PII:
S0223-5234(16)30532-3
10.1016/j.ejmech.2016.06.042
EJMECH 8703
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 January 2016
Revised Date: 21 June 2016
Accepted Date: 22 June 2016
Please cite this article as: V.U. Jeankumar, R.S. Reshma, R. Vats, R. Janupally, S. Saxena, P.
Yogeeswari, D. Sriram, Engineering another class of anti-tubercular lead: Hit to lead optimization of
an intriguing class of gyrase ATPase inhibitors, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.06.042.
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Engineering another class of anti-tubercular lead: Hit to lead
optimization of an intriguing class of Gyrase ATPase
inhibitors
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Variam Ullas Jeankumar , Rudraraju Srilakshmi Reshma , Renuka Janupally , Shalini Saxena , Rahul
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Vats , Perumal Yogeeswari , Dharmarajan Sriram *
A structure based medium throughput virtual screening of in house chemical library to identify novel
binders of Mycobacterium tuberculosis gyrase ATPase domain led to the discovery of a quinoline
scaffold. Initial hit is further optimized to study SAR and biological evaluation.

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Engineering another class of anti-tubercular lead: Hit to lead optimization of
an intriguing class of Gyrase ATPase inhibitors
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Variam Ullas Jeankumar , Rudraraju Srilakshmi Reshma , Rahul Vats , Renuka Janupally ,
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Shalini Saxena , Perumal Yogeeswari , Dharmarajan Sriram *
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Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Shameerpet, R.R. District, Hyderabad-500078, Andhra Pradesh, India.
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# Equal contribution
Corresponding Author*
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D. Sriram
Chair Professor,
Department of Pharmacy,
Birla Institute of Technology & Science-Pilani, Hyderabad Campus,
Jawahar Nagar, R.R. Dist., Hyderabad- 500 078.
INDIA
Telephone: +91-40663030506
Fax: +91-4066303998
Email: dsriram@hyderabad.bits-pilani.ac.in.
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Electronic supplementary information (ESI) available.
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Abstract:
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A structure based medium throughput virtual screening campaign of BITS-Pilani in house
chemical library to identify novel binders of Mycobacterium tuberculosis gyrase ATPase domain
led to the discovery of a quinoline scaffold. Further medicinal chemistry explorations on the
right hand core of the early hit, engendered a potent lead demonstrating superior efficacy both in
the enzyme and whole cell screening assay. The binding affinity shown at the enzyme level was
further corroborated by biophysical characterization techniques. Early pharmacokinetic
evaluation of the optimized analogue was encouraging and provides interesting potential for
further optimization.
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Keywords: Medium throughput virtual screening, Mycobacterium tuberculosis, DNA Gyrase,
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differential scanning fluorimetry.
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1. Introduction:
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Poor patient compliance and irrational prescribing practices have ruined the effectiveness of the
currently available drug regime for tuberculosis. The emergence of the drug resistant strains and
TB co-infection in HIV/AIDS patient are other factors that have made the treatment for TB more
challenging [1-3]. To combat these issues and to meet future therapeutic needs, structurally
unique chemical entities that target new targets/pathways are needed.
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The druggability of DNA Gyrase has been well established clinically and its inhibition has
shown catastrophic effect on bacterial cell growth and survival. DNA gyrase is an essential
enzyme that introduces negative supercoils into DNA and regulates the superhelical state of the
bacterial chromosomes. The functional DNA gyrase enzyme exists as a heterotetramer with two
A subunits and two B subunits (A B ). The A subunit (90 to 100 kDa, depending on the bacterial
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species) carries the breakage-reunion active site, whereas the B subunit (70 to 90 kDa) promotes
ATP hydrolysis, providing sufficient amount of energy for the DNA super coiling.
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Unlike other bacterial genome DNA-gyrase is the sole Type II topoisomerase in Mycobacterium
tuberculosis, making it more attractive from a drug discovery perspective as it makes the enzyme
more vulnerable to inhibition and hence a novel DNA gyrase lead can be effectively nurtured
into an anti-tubercular drug [4-7].
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Flouroquinolones (FQs), that acts through the inhibition of gyrA domain of DNA gyrase are the
most researched anti-tubercular DNA gyrase inhibitors, with two candidates (Moxifloxacin
[MXF] and Gatifloxacin [GAT] being trailed at Phase III of current anti-TB clinical portfolio [8-
10]. MXF has demonstrated promising activity against both drug sensitive and drug resistant
strains of MTB and had shown indication of its usefulness in reducing the length of TB treatment
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regimens in their early in vitro and murine studies [11-12]. However a recent meta-analysis for
clinical trials of MXF or GAT containing regimen to evaluate their treatment efficacy and safety
as part of first line therapy of drug sensitive tuberculosis have indicated that MXF of GAT might
not have the ability to shorten treatment duration in the initial therapy for tuberculosis [13].
These coupled with prevalence of pre-existing resistance to FQs suggest that, although
flouroquinolones could probably replace isoniazid in the first line therapy of tuberculosis
because of their superior bactericidal activity and may also help in treating drug-resistant
tuberculosis but they are not going to revolutionize the treatment of tuberculosis. [14].
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The ATPase domain; that makes the other half of functional gyrase heterotetramer (A B )
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complex promotes ATP hydrolysis, providing sufficient amount of energy for the DNA super
coiling activity. In the absence of the ATP, DNA gyrase catalyzes only the relaxation of
supercoiled DNA but not the introduction of negative supercoils. Depriving the enzyme the
source of energy via inhibition of GyrB domain should still exert the same phenotypic effect on
the bacterial viability to the one exhibited by fluoroquinolones, that inhibits the gyrA domain.
Moreover mutations in gyrase that confer resistance to fluoroquinolones are outside the 43 kDa
N-terminal domain that is required for ATPase activity. However, there has not been any
effective therapeutics developed against this target for TB. Thus inhibitors that target ATPase
domain, the focus of the present study may prove beneficial to replace the fluoroquinolones
when resistance for that class of drug becomes regnant [4-7, 15-20].
2. Results and Discussion:
Combination of ligand based modeling and virtual screening is an emerging concept in drug
design for identification of newer chemotypes as attractive starting points for medicinal
chemistry SAR exploration. In the present work we report the development of a novel class of
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anti-tubercular agent acting through the inhibition of the mycobacterial Gyrase ATPase domain
identified through structure based screening of diverse compound collection of BITS Pilani in
house database. Recent efforts from Astra Zeneca reports the identification of novel class of
pyrrolamides as potential Gyrase ATPase inhibitors with promising antimicrobial potency
against Mycobacterium tuberculosis H37Rv strain as well as 99 drug resistant, clinical isolates of
Mycobacterium tuberculosis [16]. Reported crystal structure of the Mycobacterium smegmatis
GyrB protein co-crystallized with most potent ligand from this study [Gyrase inhibitory IC50 < 5
nM, PDB entry 4BAE] [16] retrieved from the Protein Data Bank (www.rcsb.org) was utilized as
a structural framework in our docking explorations to identify newer chemotypes as putative
binders of Gyrase ATPase. Glide XP (extra precision) module of Schrödinger 9.2 (Glide,
version 5.7, Schrödinger, LLC, New York, NY, 81 2011) [21] was utilized for docking. An
initial validation of the active site pocket was performed by redocking the crystal ligand 2-[4-(3-
bromo-4-chloro-5-methyl-1H-pyrrole-2-amido)-3-methoxypiperidin-1-yl]-4-(1-methyl-1H-1,2,4-
triazol-5-yl)-1,3-thiazole-5-carboxylic acid with the active site residues of the Mycobacterium
smegmatis GyrB protein. Redocking results showed that the compound exhibited similar
interactions [Figure S1 in supplementary information] as that of the original crystal structure
which was further confirmed with RMSD of 1.45 Å. Later, BITS Pilani chemical library
consisting of little more than 3000 diverse compounds were first subjected to a Glide based
Medium Throughput Virtual Screening (MTVS), a method specifically proven to discard
noticeable nonbinders with minimal computational time. The molecules that came out of this
exercise were then subjected to a second round of filtering for medchem tractability based on
physicochemical parameters as predicted using QikProp module of Schrödinger, LLC, and
undesirable chemical features. The basic goal of this strategy was to decrease the enormous
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virtual chemical space of small organic molecules to a manageable number of compounds that
could inhibit the protein with the highest chance to lead to a drug candidate. Four hundred eighty
ligands that satisfied the above criteria and thus were drug like were further flexibly re-docked
together with the crystal ligand using the more computationally expensive Glide standard
precision (SP) scoring; this led to the selection of 120 compounds. To evaluate precisely the
binding interactions that these ligands maintained in the active site cavity, the hits obtained from
Glide SP docking were further evaluated with Glide XP. The Glide XP combines accurate,
physics-based scoring terms and thorough sampling, and the results gave scores ranging from
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−5.01 to −7.10 kcal mol . Final short listing of possible hit compounds was based on visual
inspection of the important amino acid residues in the active site cleft involved in binding that
included hydrogen bonds to Asp79, Arg141 and Arg82 and hydrophobic interactions with Ala53,
Ile171, Val49, Val77, Val50, Val125, Met100, Val99, Ile84 and Val123, analogues to the one
observed with the reported pyrrolamide ligand used as template in this study [PDB entry 4BAE,
Figure S1 in supplementary information].
The selected hits retrieved from the BITS Pilani database were then experimentally evaluated for
their in vitro Mycobacterium smegmatis ATPase inhibitory potency at a single concentration of
50 µM in triplicate by using malachite green based assay adapted to a 96-well plate format and
finally for a dose–response estimation in more detail as described previously[19,22-24]. The
ATPase assay was performed on Mycobacterium smegmatis DNA gyraseB protein due to the
low specific activity of Mycobacterium tuberculosis ATPase. The use of the Mycobacterium
smegmatis GyrB protein as a surrogate for the GyrB protein from Mycobacterium tuberculosis
has been well demonstrated in the literature [22] Novobiocin, which has been previously
demonstrated to be a potent inhibitor of DNA GyrB,[18] was used as a positive control in this
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study. Negative controls (0% inhibition) did not contain any inhibitory compounds. Compounds
were also tested in the presence of Brij-35, a nonanionic detergent, to ascertain whether these
inhibitions were an artifact due to sequestration of the enzyme by drug aggregates. Other
artifacts, like auto absorbance of the drug, were also ruled out by nullifying its absorbance during
the reaction. The working outline utilized for identifying the inhibitors has been depicted in
Figure 1 and the lig. plot representation of the best six ligands (hits) together with their docking
scores, fitness and hydrogen bonding has been depicted in Figure 2.
Among the small number of hits identified Compound 1, [2-(4-(7-chloroquinolin-4-
yl)piperazin-1-yl)-N-(pyridin-2-ylmethyl)acetamide with gyr B inhibitory IC50 = 12.2 ± 0.09
µM emerged as the most promising candidate. To have an structural insight into the orientation
and the possible binding interactions that the hit molecule maintained in the active site of the
protein that could possibly be exploited in the subsequent hit expansion step to deliver more
potent and selective inhibitor, the ligand was analyzed in more detail. In the docking
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explorations, Compound 1 exhibited a docking score of -6.26 kcal/mol and was found to be in
the vicinity of the amino acid Ala53, Asp79, Val125, Val49, Ile171, Val77, Val98,Val99, Pro85,
Asn52, Glu56 and Gly83 amino acid residues (which is also characterized to be the active site
pocket). A closer look at the interaction profile diagram of the molecule [Figure 2, ligand 2 and
Figure S2 in supplementary information] showed the quinoline nitrogen (N-4) to be involved in
a prominent hydrogen bonding interaction with Asp 79, analogues to the one observed in the
crystal ligand [PDB entry 4BAE, Figure S1 in supplementary information]. This interaction is
believed to be critical in retaining the activity. An additional hydrogen bonding interaction was
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observed between the pyridyl nitrogen on the right hand core and NH of His121.Furthermore
the compound was also found to be stabilized by the hydrophobic interaction with Val125,
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Val49, Ile171, Val77, Ala53, Ile84, Val98, Val99, and Val123 amino acid residues. However the
molecule failed to retain hydrogen bonding interaction with Arg141 and the pi-stacking
interaction with Arg82 as observed in the reference crystal structure; probably critical
interactions that might have reduced the potency of the molecules in the present study compared
to the reference pyrrolamide ligand that exhibited in vitro GyrB IC in the nanomolar range.
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Based on the findings from the protein-ligand interaction of compound 1 in the active site of
protein, the following modification (and combinations thereof) were explored in the first hit
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expansion step (i) replacing the 7-chloro substituted quinoline nucleus with a 7–CF 7-OCH
substituted and a un- substituted quinoline core. (ii) extending/reducing the chain length of the
amino-pyridyl nucleus on the right hand side (iii) introducing various aryl/heteroaryl nucleus of
varied chain length on the right hand core as a possible replacement for the 2-aminomethyl
pyridine nucleus.
Thus a series of 13 molecules were synthesized using the synthetic protocol described in Scheme
1 in the first stage of hit expansion as steps towards the derivation of structure-activity
relationships and hit optimization.
The synthesized derivatives (cmpd. 1, 2 – 14) were then evaluated for the GyrB inhibitory
potency using the previously described malachite green assay. Among the 13 derivatives
evaluated for their GyrB inhibitory potency, eight compounds showed an inhibitory IC of less
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than 25 µM; out of which two compounds exhibited strong inhibition of GyrB activity with IC50
less than 10 µM [Table 1]. Compound 6 and 13 emerged as the most promising leads with an
inhibitory IC of 9.1 ± 0.5 µM and 7.6 ± 0.7 µM respectively.
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C-7 position of the quinolone nucleus (cmpd. 2–4) was detrimental to bioactivity as none of the
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modified molecules showed any significant effect on the GyrB activity. Docking experiments
revealed that unlike the chloro substituted analogue that oriented nicely into the hydrophobic
pocket, the various substitutions attempted at the C-7 position changed the orientation of these
molecules (cmpd. 3–4) completely, taking it out of the active site pocket thereby loosing the
important hydrophobic interactions, a fatal determinant of inhibitory potency. A similar trend
was also observed for un-substituted analogue (cmpd. 1) as well. These findings emphasized the
importance of chloro group at this position; primarily due to the hydrophobic interaction that this
group maintained in the active site pocket.
With respect to the modifications attempted to understand the effect of chain length on the
aminopyridyl ring in activity determination (cmpd. 5 – 6). It was found that an increase in chain
length by one carbon atom as in the case of compound 5 was seen to significantly hamper the
activity, this as understood from the docking studies was due to the loss of two important
hydrogen bonding that the hit molecule (cmpd. 1) maintained with Asp79 and His 121 [Figure
S3 in supplementary information]. However chopping the chain length of the aminopyridyl ring
by one carbon atom (as in compound 6) did not have notable reduction in potency. The molecule
could still hold on to the important interaction with Asp 79 observed in the active site pocket and
the additional hydrogen bond with His 141 outside the active site cavity, analogues to the one
observed in the hit molecule compound 1; accounting for its good GyrB inhibitory IC of 9.1 ±
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0.5 µM.
Among the various aryl/heteroaryl substituted amides attempted in synthesis (cmpd. 7 - 14);
compound 13 emerged as the most promising lead with a GyrB inhibitory IC of 7.6 ± 0.7 µM.
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molecule oriented nicely into the active site cavity in a pattern similar to the crystal ligand and
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was found to be involved in three hydrogen bonding interactions with Asp79, Arg141 and Arg82
amino acid residues, in addition to the hydrophobic interactions with the active site residues.
Keeping these findings in mind, a second subset was synthesized by introducing various
substituents on the right hand core of the 7-chloro-4-(piperazin-1-yl)quinoline nucleus that
mimicked the pyridyl nucleus (cmpd. 15 – 27) in the hit molecule compound 1. A further in vitro
GyrB evaluation of the synthesized molecules showed that 9 out of 13 molecules tested under
these conditions showed IC50 less than 15 µM [Table 1] with thiocarbamide (cmpd. 24) and
carbamide (cmpd. 25) derivatives emerging as most potent leads with an IC50 of 5.5 ± 0.1 µM
and 6.1 ± 1.3 µM respectively. A closer look at the interaction profile diagram of these
molecules [Figure S5 in supplementary information] showed that molecule oriented deeply into
the hydrophobic cavity in the active site pocket; exhibiting good fitness score and retaining the
critical hydrogen bonding interaction with Asp79 as well as the non-polar interaction in the
hydrophobic pocket.
Based on these observations, another 18 derivatives exploring various aliphatic and aromatic
carbamide and thiocarbamide derivatives as the right hand core of the 7-chloro-4-(piperazin-1-
yl)quinoline nucleus (cmpd. 28 – 45) were synthesized and evaluated for their GyrB inhibitory
potency. In vitro characterization of these analogues revealed the molecules to exhibit good to
very promising GyrB inhibitory potency in the lower micromolar range. Compound 29 and 36,
the 4-fluorophenyl substituted thiocarbamide and the carbamide derivatives emerged as the most
promising optimized derivatives with a GyrB inhibitory IC of 2.5 ± 0.1 µM and 3.1 ± 0.2 µM
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respectively. The docking studies [Figure S6 & S7 in supplementary information] showed the
molecules to orient in a manner analogues to that virtual screening hit and also to that of the
second generation thiocarbamide/carbamide analogues (cmpd. 24 and 25); retaining the critical
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interaction with Asp 79. The molecules nicely oriented into the hydrophobic cavity and were
found to be stabilized there by hydrophobic interactions with Val49, Val, Ala53, Ile171, Val125,
and Val99 amino acid residues. A closer look at the interaction pattern of the less active
analogue (cmpd. 34 ) [Figure S8 in supplementary information] in this class showed that the
introduction of the bulkier acetyl group at the para position of the right hand phenyl core
changed the orientation completely, thus the molecule though retained the hydrophobic
interaction but failed to maintain the hydrogen bonding interaction with Asp79 thus accounting
for the loss in activity.
The aliphatic thiocarbamide/carbamide derivatives (cmpd. 42 - 45) attempted in synthesis also
turned out to be completely inactive. An in-silico investigation into the same [Figure S9 in
supplementary information] revealed that the molecules oriented in a completely opposite
manner to the active analogues, throwing the quinoline nucleus out of the pocket. Although, the
molecules still retained the interaction with Asp 79 but this was rather observed with the right
hand carbamide and thiocarbamide NH and not with the quinoiline nitrogen (N-4) as in the case
of hit compound 1. Also these molecules failed in maintaining hydrophobic interaction with
amino acid residues present in the active site of protein highlighting the presence of hydrophobic
ring on the right hand core as an important determinant of inhibitory potency.
Thus the hydrogen bonding interaction with Asp 79 in the active site pocket and the non polar
interactions retained in the hydrophobic cavity of the active site could be considered as the
critical factors that drove the bioactivity.
Furthermore, the binding affinity of the most potent analogue was evaluated by measuring the
thermal stability of the protein–ligand complex using biophysical differential scanning
fluorimetry experiments (Figure 3) using a previously demonstrated protocol [23-25].
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Compound 29 displayed a T shift of 4.1°C (T = 48.1°C) compared with the native protein (T
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= 44°C), a repercussion of strong binding of the ligand to the protein and highly correlating with
its GyrB IC50 of 2.5 ± 0.1 µM.
All the synthesized molecules were also subjected to a number of secondary screenings that
included the Mycobacterium tuberculosis DNA supercoiling assay; followed by in vitro
evaluation of their antimycobacterial potency and safety profile.
The DNA supercoiling assay would be an indirect measurement of their GyrB inhibitory potency
as any inhibition of ATPase activity conferred by the DNA GyrB subunit should also inhibit the
supercoiling activity performed by gyrA domain. Thus all the compounds were further evaluated
for their supercoiling inhibition studies using Mycobacterium tuberculosis DNA gyrase
supercoiling kit from Inspiralis (Inspiralis, Norwich) [26]. In general, a good correlation was
observed between the in vitro GyrB potency and in vitro supercoiling activity [Table 1]. The hit
molecule compound 1 exhibited a supercoiling inhibitory IC50 of 6.25±0.8 µM. Out of 44
molecule studied, 35 molecules exhibited an inhibitory IC of < 25 µM, out of which 23
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molecules exhibited an inhibitory IC of < 10 µM and 12 molecules exhibited an inhibitory IC
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of < 5 µM [Table 1]. The optimized analogues Compound 29 and 36 showed an IC50 of 2.7 ±
0.14 µM and 3.125 ± 0.9 µM, respectively well correlating with and GyrB inhibitory IC50 of 2.5
± 0.1 µM and 3.1 ± 0.2 µM, respectively.
The antimycobacterial potency of these molecules were evaluated by in vitro MABA assay [27].
Out of the 44 molecules tested 33 molecules showed MIC less than 50 µM; out of which 16
molecules showed MIC less than 25 µM and 2 molecules exhibited MIC less than 10 µM [Table
1]. The optimized analogues, compounds 29 and 36 showed an in vitro Mycobacterium
tuberculosis MIC of 7.8 µM and 15.3 µM respectively synchronizing well with their GyrB
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inhibitory IC50 of 2.5 ± 0.1and 3.1 ± 0.2 µM and supercoiling IC50 of 2.7 ± 0.14 and 3.125 ± 0.9
µM.
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Finally the toxicity profile of all the compounds were also tested against the mouse macrophage
RAW 264.7 cell lines at 100 µM concentration using (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay [28]. All the compounds displayed relatively good
safety profile except for the few nitro substituted analogues that exhibited slight inhibition at this
tested concentration, with Compound 38 exhibiting maximum inhibition of 42%, but can be
considered relatively insignificant at this stage of the study as their gyrase inhibitory IC50 were
less than 20 µM.
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that showed slight toxicity at the concentration tested [Table 1].
Pharmacokinetic profile of potent analogue compound 29 was evaluated in male Wistar rats
following intravenous (1 mg/kg) and oral (20 mg/kg) administration using a protocol as detailed
in the experimental section of supplementary information. Post to intravenous administration,
compound 29 exhibited favorable pharmacokinetic properties with moderate distribution outside
of vascular system (0.99 L/kg) and low systemic clearance (Figure 5 and Table 2). Following
oral administration (Figure 5), compound 29 was rapidly absorbed (Tmax~ 2h) and showed high
plasma exposure resulting fairly good oral bioavailability (68-75 %). From the oral profile, it is
evident that concentration levels of compound 29 were well above the IC (~1.0 µg/ml) for
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more than 6h. Such a high exposure could ensure therapeutic effectiveness of the compound
against TB infections for extended duration. It is well known that the free drug concentration in
the blood influences pharmacokinetic and pharmacodynamic properties of a molecule [29].
Therefore, we investigated protein binding ability of compound 29 in human and rat plasma
matrices. Compound 29 showed extensive protein binding (> 90 %) across the species and the
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binding rate of compound 29 with plasma protein was concentration-dependent (Table 2). In
order to predict metabolic clearance in humans, we investigated in vitro metabolic stability
profile of compound 29 in human liver microsomes and extrapolated for human clearance
(CLblood). Microsomal stability study predicts slow hepatic clearance (10.01 mL/min/kg) of
compound 29 in humans. In conclusion, our studies demonstrate favorable pharmacokinetic
properties of compound 29, encouraging further in vivo studies and consideration of compound
29 as a suitable candidate to be worked out from a pharmaceutical point of view as potential anti-
tubercular lead.
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3. Conclusion:
The present study describes the identification of novel structural motif from a medium
throughput virtual screening campaign of an in-house 3000-member compound library for
Mycobacterium tuberculosis DNA gyraseB inhibitors. Synthesis and structure activity
relationship (SAR) studies around initial hit led to several analogues, the most potent of which
displayed an in vitro gyrB inhibitory IC50 value of 2.5 ± 0.1 µM. A correlation between in vitro
gyrB inhibitory activity and in vitro antimycobacterial activity against the lab sensitive H37Rv
strain of Mycobacterium tuberculosis has been demonstrated. A preliminary evaluation of
ADME parameters; demonstrated favorable pharmacokinetic properties of compound 29 and
provides an important advance within the context of new antituberculosis chemotypes.
4. Experimental section:
4.1. Chemistry:
4.1.1. General:
3
3
18
19
All commercially available chemicals and solvents were used without further purification. TLC
experiments were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt,
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34
Germany). Homogeneity of the compounds was monitored by thin layer chromatography (TLC)
on silica gel 40 F254 coated on aluminum plates, visualized by UV light and KMnO treatment.
4
Flash chromatography was performed on a Biotage Isolera with prepackaged disposable normal
1
13
phase silica columns. All H and C NMR spectra were recorded on a Bruker AM-300 (300.12
MHz, 75.12 MHz) NMR spectrometer, Bruker BioSpin Corp, Germany. Chemical shifts were
reported in ppm (δ) with reference to the internal standard TMS. The signals were designated as
follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet. Molecular weights
of the synthesized compounds were checked by LCMS 6100B series Agilent Technology.
Elemental analyses were carried out on an automatic Flash EA 1112 Series, CHN Analyzer
(Thermo). The purity of the final compounds was examined by HPLC (Shimadzu, Japan, (on
Phenomenex C8 (150 * 4.6 mm, 5µm, 100 Å) double end-capped RP-HPLC column)) and was
greater than 95%. 4,7-dichloroquinoline, the precursor for preparing compounds 1, 5-45 was
procured from Sigma-Aldrich (Cas no: 86-98-6). The precursor 4-chloroquinoline, 4-chloro-7-
(trifluoromethyl)quinoline, 4-chloro-7-methoxyquinoline utilized for generating compounds 2, 3
and 4 were synthesized utilizing the literature protocol [30-32] respectively.
3
3
3
3
3
3
3
3
35
36
37
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39
40
41
42
4.1.2. 7-Chloro-4-(piperazin-1-yl)quinoline: To a suspension of 4,7-dichloroquinoline (2.5g,
12.6 mmol) and potassium carbonate (2.1, 15.1 mmol) in acetonitrile (20 mL) was added
o
o
piperazine (1.1g, 12.6 mmol) at 30 C. The reaction mixture was then heated to 80 C for 1-2 h
o
(monitored by TLC and LCMS for completion), cooled to 30 C. The mixture was then filtered
through celite bed, and acetonitrile was evaporated in vacuo. The resultant residue was diluted
with water (10 mL) and dichloromethane (20 mL) and the layers separated. The aqueous layer
was re-extracted with dichloromethane (2 x 25 mL). The combined organic extract was washed
with brine, dried over sodium sulphate, and evaporated in vacuo. The resultant residue was the
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57
58
59
60
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64
purified by column chromatography on neutral alumina using hexane:ethylacetate as eluent to
1
give 7-chloro-4-(piperazin-1-yl)quinoline (1.9g, 61.3%) as an off-white solid. H NMR
(DMSO-d ): δ 2.92 - 3.03 (m, 4H), 3.05 - 3.13, (m, 4H), 6.95 (d, J = 5.1Hz, 1H), 7.52 (dd, J =
6
H.
1
3
8.7 Hz, J = 1.8 Hz, 1H), 7.92 - 8.04 (m, 2H), 8.67 (d, J = 4.8 Hz, 1H). C NMR (DMSO-d ): δc.
6
156.7, 152.1, 149.6, 133.4, 128, 125.9, 125.5, 121.3, 109.2, 52.8, 45.3. ESI-MS m/z 248.1
+
(M+1) . Anal Calcd for C H ClN ; C, 63.03; H, 5.70; N, 16.96; Found: 62.99; H, 5.67; N,
1
3
14
3
16.93
4.1.3. Methyl 2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetate: To a suspension of 7-chloro-
4-(piperazin-1-yl)quinoline (2.5g, 10 mmol) and potassium carbonate (1.66 g, 12 mmol) in
o
acetonitrile (20 mL) was added methylbromoacetate (1.53 g, 10 mmol) at 30 C. The reaction
o
mixture was then heated to 60 C for 1h (monitored by TLC and LCMS for completion), cooled
o
to 30 C. The mixture was then filtered through celite bed, and acetonitrile was evaporated in
vacuo. The resultant residue was diluted with water and dichloromethane, and the layers
separated. The aqueous layer was re-extracted with dichloromethane (2 x 40 mL). The combined
organic extract was washed with brine, dried over sodium sulphate, and evaporated in vacuo The
resultant residue was the purified by column chromatography on neutral alumina using hexane:
ethylacetate as eluent to give methyl 2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetate
1
(2.23g, 67.6%) as an off-white solid. H NMR (DMSO-d ): δ 2.73– 2.82 (m, 4H), 3.01 – 3.12
6
H.
1
3
(m, 4H), 3.36 (s, 2H), 3.68 (s, 3H), 6.98 (d, J = 5.1Hz, 1H), 6.93 – 8.61 (m, 4H). C NMR
(DMSO-d ): δc. 170.3, 156.5, 151.9, 149.7, 133.3, 128.1, 125.7, 125.3, 121.5, 108.9, 57.1, 55.3,
6
+
53.9, 51.2. ESI-MS m/z 320.1 (M+H) . Anal Calcd for C H ClN O ; C, 60.09; H, 5.67; N,
1
6
18
3
2
13.14; Found: C, 60.15; H, 5.64; N, 13.11
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65
66
67
68
69
70
71
72
73
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75
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77
78
79
80
81
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85
86
87
4.1.4. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)acetic acid: To a solution of methyl 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetate (1.5g, 4.7 mmol) in THF:H O:CH OH system (1:1:1)
2
3
o
was added lithium hydroxide (0.3 g, 7.1 mmol) at 0 C. The reaction mixture was slowly warmed
o
o
to 30 C then stirred at 30 C for 3-4h (monitored by TLC and LCMS for completion). The
o
reaction mixture was then cooled to 0 C and acidified to a pH of 3-4 with 1N HCl. and extracted
with dichloromethane (3 x 50mL). The combined organic extract was successively washed with
water and brine, dried over sodium sulphate, and evaporated in vacuo to give 2-(4-(7-
1
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.7g, 49%) as white solid. H NMR (DMSO-
6 H
d ): δ 2.63 – 2.69 (m, 4H), 2.98 – 3.09 (m, 4H), 3.24 (s, 2H), 6.97 – 8.63 (m, 5H) 12.1 (s, 1H).
1
3
C NMR (DMSO-d ): δc. 175.8, 156.9, 151.8, 149.3, 133.6, 127.8, 126, 125.6, 121.1, 109, 60.6,
6
+
55.6, 54.1. ESI-MS m/z 304.1 (M-H) . Anal Calcd for C H ClN O ; C, 58.92; H, 5.27; N,
1
5
16
3
2
13.74; Found: C, 58.87; H, 5.24; N, 13.78.
4.1.5. General procedure for the synthesis of amide derivatives (1 –14): To a solution of 2-(4-
(7-sub:quinolin-4-yl)piperazin-1-yl)acetic acid (1 mmol) in dry dichloromethane (3 mL) was
o
added triethyl amine (1.5 mmol) and corresponding amine (1 mmol) at 0 C. Propylphosphonic
anhydride (2 mmol) was then added drop wise to the reaction mixture and the reaction mixture
o
was stirred at 30 C for 6h, (monitored by TLC & LCMS for completion). The reaction mixture
was washed with water (2 mL), brine (2 mL), dried over anhydrous sodium sulphate and
evaporated in vacuo to give the desired product as mentioned below
4.1.5.1. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(pyridin-2-ylmethyl)acetamide (1):
The compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), pyridin-2-ylmethanamine
(0.089g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride
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7

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3
3
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3
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88
89
90
91
92
93
(0.51g, 1.6mmol) to afford 1 (0.25g, 75%) as off white solid. M.p: 155-157 C. H NMR
(DMSO-d ): δ 2.56 - 2.69 (m, 4H), 3.07 – 3.16 (m, 4H), 3.36 (s, 2H), 4.51 (s, 2H), 6.93 (d, J =
6
H.
1
3
5.2Hz, 1H), 7.24 – 8.64 (m, 9H). C NMR (DMSO-d ): δc. 171.4, 156.9, 155.8, 152.7, 150.2,
6
149.3, 140, 136.9, 132.6, 128.6, 125.8, 124.3, 123.1, 121.2, 109.6, 60.3, 52.3, 47.6, 45.9 ESI-MS
+
m/z 396.1 (M+H) . Anal Calcd for C H ClN O; C, 63.71; H, 5.60; N, 17.69; Found: C, 63.66;
2
1
22
5
H, 5.63; N, 17.74.
3
3
3
3
3
3
4
4
4
94
95
96
97
98
99
00
01
02
4.1.5.2. 2-(4-(Quinolin-4-yl)piperazin-1-yl)-N-(pyridin-2-ylmethyl)acetamide (2): The
compound was synthesized according to the above general procedure using 2-(4-(quinolin-4-
yl)piperazin-1-yl)acetic acid (0.25g, 0.92mmol), pyridin-2-ylmethanamine (0.099g mmol,
0.92mmol), triethylamine (0.139g, 1.38 mmol), propylphosphonic anhydride (0.41g, 1.8mmol) to
1
afford 2 (0.23g, 69.7%) as off white solid. H NMR (DMSO-d ): δ 2.49 - 2.57 (m, 4H), 3.01 –
6
H.
1
3
3.11 (m, 4H), 3.32 (s, 2H), 4.47 (s, 2H), 6.98 (d, J = 5.1 Hz, 1H), 7.24 – 8.64 (m, 10H). C
NMR (DMSO-d
6
): δc. 171.1, 157.3, 156, 151.6, 148.9, 140.4, 139.9, 130.6, 130, 128.7, 128.2,
+
127, 124.3, 121.1, 114.9, 60.1, 52.6, 47.8, 46.1. ESI-MS m/z 362.1 (M+H) . Anal Calcd for
21 23 5
C H N O; C, 69.78; H, 6.41; N, 19.38; Found: 69.84; H, 6.36; N, 19.34
4
4
4
4
4
4
4
4
03
04
05
06
07
08
09
10
4.1.5.3.
2-(4-(7-Trifluoromethyl
quinolin-4-yl)piperazin-1-yl)-N-(pyridin-2-
ylmethyl)acetamide (3): The compound was synthesized according to the above general
procedure using 2-(4-(7-trifluoromethyl quinolin-4-yl)piperazin-1-yl)acetic acid (0.25g,
0.74mmol), pyridin-2-ylmethanamine (0.08g mmol, 0.74mmol), triethylamine (0.11g, 1.11
mmol), propylphosphonic anhydride (0.32g, 1.4mmol) to afford 3 (0.18g, 58%) as off white
1
solid. H NMR (DMSO-d
6
): δH. 2.52 - 2.63 (m, 4H), 3.04 – 3.17 (m, 4H), 3.29 (s, 2H), 4.51 (s,
1
3
2H), 7.08 (d, J = 5.2 Hz, 1H), 7.32 – 8.78 (m, 9H). C NMR (DMSO-d ): δc. 171.3, 157.7,
6
156.2, 152.9, 148.8, 147.9, 139.8, 132.7, 129.1, 128.8, 126.5, 124.3, 124.1, 121.8, 121, 120.4,
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ACCEPTED MANUSCRIPT
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4
4
11
12
59.9, 52.4, 47.9, 46. I-MS m/z 430.2 (M+H) . Anal Calcd for C22
H
22
F
3
N
5
O; C, 61.53; H, 5.16; N,
16.31; Found C, 61.48; H, 5.17; N, 16.27.
4
4
4
4
4
4
4
4
4
4
13
14
15
16
17
18
19
20
21
22
4.1.5.4. 2-(4-(7-Methoxy quinolin-4-yl)piperazin-1-yl)-N-(pyridin-2-ylmethyl)acetamide (4):
The compound was synthesized according to the above general procedure using 2-(4-(7-methoxy
quinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.83mmol), pyridin-2-ylmethanamine (0.09g
mmol, 0.83mmol), triethylamine (0.13g, 1.3 mmol), propylphosphonic anhydride (0.37g,
1
1.6mmol) to afford 4 (0.24g, 75%) as off white solid. H NMR (DMSO-d
6
): δH. 2.46 - 2.55 (m,
4H), 2.98 – 3.09 (m, 4H), 3.34 (s, 2H), 3.86 (s, 3H), 4.49 (s, 2H), 6.73 (d, J = 5.1 Hz, 1H), 7.27 –
1
3
8.78 (m, 9H). C NMR (DMSO-d ): δc. 170.9, 156.9, 156, 152.1, 150.9, 148.9, 147.6, 139.9,
6
126.4, 123.8, 123.3, 121.3, 117.6, 116.2, 107.9, 60, 56.2, 52.3, 47.6, 46.1. ESI-MS m/z 392.1
+
(M+H) . Anal Calcd for C H N O ; C, 67.5; H, 6.44; N, 17.89; Found C, 67.45; H, 6.46; N,
22
25
5
2
17.93.
4
4
4
4
4
4
4
4
4
4
23
24
25
26
27
28
29
30
31
32
4.1.5.5. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(2-(pyridin-2-yl)ethyl)acetamide (5):
The compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), 2-(pyridin-2-yl)ethanamine
(0.1g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride (0.51g,
1
1.6mmol) to afford 5 (0.25g, 73.5%) as off white solid. H NMR (DMSO-d ): δ 2.54 – 2.62 (m,
6
H.
4H), 3.06 – 3.19 (m, 6H), 3.27 (s, 2H), 3.63 (t, J = 7.1 Hz, 2H), 6.93 (d, J = 8.1Hz, 1H), 7.24 –
1
3
6
8.67 (m, 9H). C NMR (DMSO-d ): δc. 170.6, 158.1, 157.6, 152.3, 149.8, 147.9, 136.6, 133.7,
129.6, 129, 125.9, 123, 122.6, 121.2, 109.3, 60.2, 52.6, 47.9, 40.9, 35.8. ESI-MS m/z 410.1
+
(M+H) . Anal Calcd for C22
5
H24ClN O; C, 64.46; H, 5.90; N, 17.09; Found: C, 64.41; H, 5.94; N,
17.13.
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4.1.5.6. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(pyridin-2-yl)acetamide (6): The
compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), pyridin-2-amine (0.77g,
0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride (0.51g, 1.6mmol) to
1
afford 6 (0.2g, 63%) as solid. H NMR (DMSO-d ): δ 2.59 – 2.71 (m, 4H), 3.08 – 3.16 (m, 4H),
6
H.
1
3
3.31 (s, 2H), 7.01 (d, J = 5.2 Hz, 1H), 7.28 – 8.71 (m, 9H). C NMR (DMSO-d ): δc. 168.7,
6
157.8, 152.6, 151.5, 149.9, 146.4, 139.2, 133.9, 129.8, 129, 125.9, 124.7, 122.7, 115.4, 109.4,
+
62.9, 52.8, 47.6. SI-MS m/z 382.1 (M+H) . Anal Calcd for C20
H20ClN
5
O; C, 62.91; H, 5.28; N,
18.34; Found: C, 62.86; H, 5.32; N, 18.28.
4
4
4
4
4
4
4
4
4
42
43
44
45
46
47
48
49
50
4.1.5.7. N-Benzyl-2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetamide (7): The compound
was synthesized according to the above general procedure using 2-(4-(7-chloroquinolin-4-
yl)piperazin-1-yl)aceticacid (0.25g, 0.82mmol), phenylmethanamine(0.088g mmol, 0.82mmol),
triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride (0.51g, 1.6mmol) to afford 7
1
(0.27g, 84%) as solid. H NMR (DMSO-d
6
): δH. 2.49 – 2.56 (m, 4H), 2.96 – 3.08 (m, 4H), 3.23
1
3
6
(s, 2H), 4.21 (s, 2H), 6.93 (d, J = 5.1 Hz, 1H), 7.16 – 8.59 (m, 10H). C NMR (DMSO-d ): δc.
171.3, 157.2, 152.4, 149.8, 138.2, 133.1, 129.8, 128.6, 128.8, 127.1, 126.8, 125.8, 122.3, 109.2,
+
59.2, 52.4, 47.4, 43.2. ESI-MS m/z 395.1 (M+H) . Anal Calcd for C H ClN O; C, 66.91; H,
2
2
23
4
5.87; N, 14.19; Found: C, 66.95; H, 5.91; N, 14.23.
4
4
4
4
4
51
52
53
54
55
4.1.5.8. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-phenylacetamide (8) The compound
was synthesized according to the above general procedure using 2-(4-(7-chloroquinolin-4-
yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), aniline (0.076g mmol, 0.82mmol), triethylamine
(0.125g, 1.23 mmol), propylphosphonic anhydride (0.51g, 1.6mmol) to afford 8 (0.14g, 45%) as
1
solid. H NMR (DMSO-d ): δ 2.53 – 2.61 (m, 4H), 3.07 – 3.16 (m, 4H), 3.37 (s, 2H), 6.89 (d, J
6
H.
2
0

ACCEPTED MANUSCRIPT
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4
4
4
4
56
57
58
59
= 5.1 Hz, 1H), 7.13 – 8.62 (m, 10H). C NMR (DMSO-d
6
): δc. 168.3, 157.6, 152.7, 149.6, 138.3,
133.3, 129.8, 129.2, 128.8, 127.9, 126.2, 122.3, 121.6, 109.6, 63.4, 52.6, 47.8. ESI-MS m/z 381.1
+
(M+H) . Anal Calcd for C H ClN O; C, 66.22; H, 5.56; N, 14.71; Found: C, 66.27; H, 5.59; N,
21
21
4
14.75.
4
4
4
4
4
4
4
4
4
60
61
62
63
64
65
66
67
68
4.1.5.9. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(thiophen-2-ylmethyl)acetamide (9):
The compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), thiophen-2-ylmethanamine
(0.093g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride
1
(0.51g, 1.6mmol) to afford 9 (0.21g, 64%) as pale brown solid. H NMR (DMSO-d ): δ 2.49 –
6
H.
1
3
2.56 (m, 4H), 2.91 – 3.03 (m, 4H), 3.23 (s, 2H), 5.02 (s, 2H), 6.83 – 8.49 (m, 9H). C NMR
(DMSO-d ): δc. 171.1, 157.4, 152.8, 149.7, 141.1, 133.2, 129.8, 129, 127.2, 126.6, 125.8, 125.3,
6
+
122.9, 109.8, 59.8, 52.7, 47.6, 42.8. ESI-MS m/z 401.3 (M+H) . Anal Calcd for C H ClN OS;
2
0
21
4
C, 59.91; H, 5.28; N, 13.97; Found: C, 59.86; H, 5.25; N, 13.91.
4
4
4
4
4
4
4
4
4
69
70
71
72
73
74
75
76
77
4.1.5.10. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(furan-2-ylmethyl)acetamide (10)
The compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)aceticacid(0.25g, 0.82mmol), furan-2-ylmethanamine (0.079g
mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride (0.51g,
1
1.6mmol) to afford 10 (0.19g, 59%) as solid. H NMR (DMSO-d ): δ 2.57 – 2.64 (m, 4H),
6
H.
2.97 – 3.07 (m, 4H), 3.27 (s, 2H), 5.23 (s, 2H), 6.33 – 6.46 (m, 2H), 6.89 (d, J = 5.2 Hz, 1H),
1
3
7.39 – 8.59 (m, 6H). C NMR (DMSO-d
6
): δc. 170.8, 157.2, 152.7, 149.9, 146.3, 141.8, 133.4,
+
129.8, 129, 125.8, 122.6, 110.3, 110.1, 109.7, 59, 52.5, 47.2, 36.8. ESI-MS m/z 385.2 (M+H) .
Anal Calcd for C H ClN O ; C, 62.42; H, 5.50; N, 14.56; Found: C, 62.37; H, 5.55; N, 14.52.
2
0
21
4
2
2
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4.1.5.11. 2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(5-nitrothiazol-2-yl)acetamide (11)
The compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), 2-amino-5-nitrothiazole
(0.093g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride
1
(0.51g, 1.6mmol) to afford 11 (0.24g, 68.6%) as solid. H NMR (DMSO-d ): δ 2.61 – 2.73 (m,
6
H.
1
3
4H), 3.06 – 3.16 (m, 4H), 3.31 (s, 2H), 6.89 (d, J = 5.1Hz, 1H), 7.32 – 8.61 (m, 6H). C NMR
(DMSO-d
6
): δc. 168.7, 162.8, 157.6, 152.8, 150.1, 147.6, 136.2, 133.3, 130.2, 129.3, 126.1,
+
122.8, 109.6, 64.1, 52.9, 47.8. ESI-MS m/z 433.2 (M+H) . Anal Calcd for C18
H
17ClN
6
O
3
S; C,
49.94; H, 3.96; N, 19.41; Found: C, 49.88; H, 3.91; N, 19.37.
4
4
4
4
4
4
4
4
4
4
87
88
89
90
91
92
93
94
95
96
4.1.5.12.
2-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-N-(6-nitrobenzo[d]thiazol-2-
yl)acetamide (12) The compound was synthesized according to the above general procedure
using 2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol),6-nitro
benzo[d]thiazol-2-amine (0.16g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol),
1
propylphosphonic anhydride (0.51g, 1.6mmol) to afford 12 (0.32g, 81%) as solid. H NMR
6
(DMSO-d ): δH. 2.56 – 2.64 (m, 4H), 3.01 – 3.09 (m, 4H), 3.27 (s, 2H), 6.96 (d, J = 5.1 Hz, 1H),
1
3
7.26 – 8.82 (m, 8H). C NMR (DMSO-d ): δc. 175.4, 168.8, 158.3, 157.8, 152.8, 150, 143.8,
6
133.2, 131.6, 130.1, 129.1, 125.8, 122.3, 121.1, 118.6, 117.6, 109.8, 63.8, 52.7, 47.6. ESI-MS
+
m/z 483.2 (M+H) . Anal Calcd for C H ClN O S; C, 54.71; H, 3.97; N, 17.40; Found: C,
2
2
19
6
3
59.77; H,4.02; N, 17.45.
4
4
4
5
97
98
99
00
4.1.5.13. N-(4-Acetylphenyl)-2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetamide (13) The
compound was synthesized according to the above general procedure using 2-(4-(7-
chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), 1-(4-aminophenyl)ethanone
(0.11g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol), propylphosphonic anhydride
2
2

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5
5
5
5
5
01
02
03
04
05
(0.51g, 1.6mmol) to afford 13 (0.28 g, 80%) as solid. H NMR (DMSO-d
6
): δH. 2.46 – 2.54 (m,
4H), 2.58 (s, 3H), 2.96 – 3.05 (m, 4H), 3.37 (s, 2H), 6.93 (d, J = 5.1 Hz, 1H), 7.29 – 8.63 (m,
1
3
8H). C NMR (DMSO-d ): δc. 189.3, 168.7, 157.6, 152.7, 149.8, 141.8, 136.9, 133.3, 129.8,
6
+
128.9, 128.7, 125.8, 122.8, 121.3, 109.7, 63.9, 52.8, 47.3, 26.1. SI-MS m/z 423.3 (M+H) . Anal
Calcd for C H ClN O ; C, 65.32; H, 5.48; N, 13.25; Found: C, 65.39; H, 5.51; N, 13.28
2
3
23
4
2
5
5
5
5
5
5
5
5
5
5
06
07
08
09
10
11
12
13
14
15
4.1.5.14. N-(2-Chloro-5-(trifluoromethyl)phenyl)-2-(4-(7-chloroquinolin-4-yl)piperazin-1-
yl)acetamide (14) The compound was synthesized according to the above general procedure
using 2-(4-(7-chloroquinolin-4-yl)piperazin-1-yl)acetic acid (0.25g, 0.82mmol), 2-chloro-5-
(trifluoromethyl) benzenamine (0.16g mmol, 0.82mmol), triethylamine (0.125g, 1.23 mmol),
1
propylphosphonic anhydride (0.51g, 1.6mmol) to afford 14 (0.25g, 64%) as solid. H NMR
(DMSO-d ): δ 2.51 – 2.59 (m, 4H), 2.99 – 3.07 (m, 4H), 3.37 (s, 2H), 6.99 (d, J = 5.2 Hz, 1H),
6
H.
1
3
7.27 – 8.61 (m, 8H). C NMR (DMSO-d ): δc. 169.1, 157.9, 152.6, 150, 138.1, 133.5, 129.8,
6
129.6, 129.4, 129.1, 126.1, 125.9, 124.3, 122.8, 121.9, 117.9, 109.7, 64.2, 52.9, 47.8. ESI-MS
+
m/z 484.2 (M+H) . Anal Calcd for C22
H19Cl
2
F
3
N
4
O; C, 54.67; H, 3.96; N, 11.59; Found: C,
54.71; H, 4.01; N, 11.63.
5
5
5
5
5
5
5
16
17
18
19
20
21
22
4.1.6. General procedure for the synthesis of amide derivatives (15 – 20): To a solution of 7-
chloro-4-(piperazin-1-yl)quinoline (1 mmol) in dry dichloromethane (3 mL) was added triethyl
o
amine (1.5 mmol) and corresponding acid (1 mmol) at 0 C. propylphosphonic anhydride (2
mmol) was then added drop wise to the reaction mixture and the reaction mixture was stirred at
o
30 for 6h (monitored by TLC & LCMS for completion). The reaction mixture was washed with
water (2 mL), brine (2 mL), dried over anhydrous sodium sulphate and evaporated in vacuo to
give the desired product as mentioned below.
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24
25
26
27
28
29
30
31
4.1.6.1. (4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(1H-indol-2-yl)methanone (15): The
compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), indole-2-carboxylic acid (0.16g 1 mmol),
triethylamine (0.15 g, 1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford 15
1
(0.33g, 84.6%) as off white solid. H NMR (DMSO-d ): δ 3.29 – 3.33 (m, 4H), 3.96 – 4.09 (m,
6
H.
1
3
4H), 6.91 – 8.72 (m, 10H), 10.91 (bs, 1H). C NMR (DMSO-d ): δc. 162.3, 155.8, 152.6,
6
149.8, 139.6, 133.7, 133.3, 131.6, 129.8, 128.9, 125.8, 122.7, 121.5, 120.9, 119.4, 114.2, 110.8,
+
109.9, 51.6, 43.2. ESI-MS m/z 391.2 (M+H) . Anal Calcd for C22
4
H19ClN O; C, 67.60; H, 4.90;
N, 14.33; Found C, 67.55; H, 4.95; N, 14.37.
5
5
5
5
5
5
5
5
5
5
32
33
34
35
36
37
38
39
40
41
4.1.6.2. (4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(5-fluoro-1H-indol-2-yl)methanone (16):
The compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), 5-fluoro-1H-indole-2-carboxylic acid (0.18g 1
mmol), triethylamine (0.15 g, 1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford
o
1
16 (0.26g, 78.8%) as off white solid. M.p: 229-231 C. H NMR (DMSO-d
6
): δH. 3.23 – 3.29 (m,
4H), 3.98 – 4.12 (m, 4H), 6.86 (s, 1H), 7.03 – 7.12 (m, 2H), 7.34 – 7.48 (m, 2H), 7.55 – 7.62 (m,
1
3
2H), 7.98 – 8.03 (m, 1H), 8.12 – 8.18 (m, 1H), 8.70 – 8.76 (m, 1H). C NMR (DMSO-d ): δc.
6
161.9, 156, 152.1, 149.6, 133.7, 132.7, 131.5, 128, 126.9, 126.8, 126, 121.3, 113.3, 112.1, 111.8,
+
109.6, 105.6, 104.2, 51.8, 42.9. ESI-MS m/z 409.2 (M+H) . Anal Calcd for C H ClFN O; C,
2
2
18
4
64.63; H, 4.44; N, 13.70, Found, C, 64.69; H, 4.41; N, 13.75.
5
5
5
5
42
43
44
45
4.1.6.3. (4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(5-chloro-1H-indol-2-yl)methanone (17):
The compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol),5-chloro-1H-indole-2-carboxylic acid (0.2g 1 mmol),
triethylamine (0.15 g, 1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford 17
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4

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5
5
5
5
5
46
47
48
49
50
(0.32g, 74.4%) as off white solid. H NMR (DMSO-d
6
): δH. 3.27 – 3.32 (m, 4H), 3.93 – 4.03 (m,
1
3
4H), 6.89 – 8.68 (m, 9H), 10.71 (bs, 1H). C NMR (DMSO-d ): δc. 162.3, 156.4, 152.3, 149.8,
6
136.4, 133.6, 133.3, 132.5, 129.6, 128.7, 125.8, 124.9, 122.7, 122.4, 121.9, 114.3, 113.8, 109.6,
+
51.9, 43.1. ESI-MS m/z 426.1 (M+H) . Anal Calcd for C H Cl N O; C, 62.13; H, 4.27; N,
2
2
18
2
4
13.17; Found C, 62.17; H, 4.31; N, 13.22.
5
5
5
5
5
5
5
5
5
51
52
53
54
55
56
57
58
59
4.1.6.4.
(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(5-methoxy-1H-indol-2-yl)methanone
(18): The compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol),5-methoxy-1H-indole-2-carboxylic acid (0.19g 1
mmol), triethylamine (0.15 g, 1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford
1
18 (0.29g, 69%) as off white solid. H NMR (DMSO-d ): δ 3.26 – 3.31 (m, 4H), 3.86 (s, 3H),
6
H.
1
3
3.99 – 4.09 (m, 4H), 6.83 – 8.56 (m, 9H), 10.89 (bs, 1H). C NMR (DMSO-d ): δc. 162.1,
6
156.4, 152.4, 149.8, 136.4, 133.6, 133.1, 132.6, 129.6, 128.7, 125.8, 124.9, 122.7, 122.4, 121.9,
+
114.3, 113.8, 109.8, 51.9, 43.1. ESI-MS m/z 421.11 (M+H) . Anal Calcd for C23
H21ClN
4
O
2
; C,
65.63; H, 5.03; N, 13.31; Found C, 65.56; H, 5.07; N, 13.26.
5
5
5
5
5
5
5
5
60
61
62
63
64
65
66
67
4.1.6.5. (4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(1H-pyrrol-2-yl)methanone (19): The
compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), 1H-pyrrol-2-carboxylic acid (0.11g 1 mmol),
triethylamine (0.15 g, 1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford 19
1
(0.26g, 76.5%) as off white solid. H NMR (DMSO-d
6
): δH. 3.31 – 3.37 (m, 4H), 3.96 – 4.03 (m,
1
3
4H), 6.79 – 8.71 (m, 9H). C NMR (DMSO-d
6
): δc. 161.8, 156.6, 152.3, 149.7, 133.5, 129.8,
+
128.6, 126.5, 125.7, 122.3, 120.7, 110.3, 109.8, 109.3, 52, 42.8. ESI-MS m/z 341.1 (M+H) .
Anal Calcd for C H ClN O; C, 63.44; H, 5.03; N, 16.44; Found C, 63.49; H, 4.98; N, 16.47.
1
8
17
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75
76
4.1.6.6. (4-(7-Chloroquinolin-4-yl)piperazin-1-yl)(pyridin-2-yl)methanone (20):
The
compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), picolinic acid (0.12g 1 mmol), triethylamine (0.15 g,
1.5 mmol), propylphosphonic anhydride (0.64g, 2 mmol) to afford 20 (0.27g, 71.4%) as off
1
white solid. H NMR (DMSO-d ): δ 3.26 – 3.32 (m, 4H), 3.99 – 4.10 (m, 4H), 7.06 (d, J =
6
H.
1
3
5.2Hz, 1H), 7.39 – 8.89 (m, 8H). C NMR (DMSO-d ): δc. 161.9, 156.8, 156.1, 152.1, 149.8,
6
147.6, 137.1, 133.4, 129.8, 128.7, 126.4, 125.7, 122.3, 121.8, 109.3, 51.7, 42.9. ESI-MS m/z
+
353.1 (M+H) . Anal Calcd for C19
4
H17ClN O; C, 64.68; H, 4.86; N, 15.88; Found C, 64.77; H,
4.89; N, 15.82.
5
5
5
5
5
5
5
5
5
5
5
5
5
77
78
79
80
81
82
83
84
85
86
87
88
89
4.1.7. General procedure for the synthesis of N-alkyl derivatives (22 & 23):
To a solution of 7-chloro-4-(piperazin-1-yl)quinoline (1 mmol) in dry dichloroethane (6 mL)
was added the corresponding aldehyde (1.1 mmol), freshly activated 3 Å molecular sieves (0.25
o
g) and catalytic amount of acetic acid. The reaction mixture was stirred at 30 C for 6 h and
filtered through celite bed (under N
2
atmosphere). The solvent was removed under reduced
o
pressure and residue further diluted with dry methanol (6 mL) and cooled to 0 C. Sodium
o
triacetoxy borohydride (1.5 mmol) was added (portion-wise) and the reaction was stirred at 30 C
for 6 h (monitored by TLC & LCMS for completion for completion). The solvent was then
removed under reduced pressure and the residue diluted with water (5 mL) and extracted with
dichloromethane (10 mL). The aqueous phase was back-extracted with dichloromethane (2 x 10
mL) and dried over sodium sulphate. The combined organic phases were concentrated under
reduced and residue purified by column chromatography using hexane: ethylacetate as eluent to
give the desired product in good yield.
2
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4.1.7.1. 4-(4-((1H-Indol-3-yl)methyl)piperazin-1-yl)-7-chloroquinoline (22): The compound
was synthesized according to the above general procedure using 7-chloro-4-(piperazin-1-
yl)quinoline (0.25g, 1 mmol), indole-2-carboxaldehyde (0.16g, 1.1mmol), to afford 22 (0.23g,
1
60.5%) as buff coloured solid. H NMR (DMSO-d ): δ 2.59 – 2.64 (m, 2H), 3.12 – 3.19 (m,
6
H.
1
3
4H), 3.56 (s, 2H), 7.06 – 8.63 (m, 10H), 9.78 (bs, 1H). C NMR (DMSO-d ): δc. 155.9, 152.5,
6
149.6, 136.9, 133.4, 129.9, 128.6, 127.5, 125.7, 122.8, 122.3, 121.4, 119.3, 118.2, 111.9, 110.7,
+
109.8, 55.6, 51.8, 43.2. ESI-MS m/z 377.2 (M+H) . Anal Calcd for C22
H21ClN
4
; C, 70.11; H,
5.62; N, 14.87; Found C, 70.19; H, 5.67; N, 14.91.
5
5
6
6
6
6
6
6
98
99
00
01
02
03
04
05
4.1.7.2. 4-(4-((1H-Pyrrol-2-yl)methyl)piperazin-1-yl)-7-chloroquinoline (23): The compound
was synthesized according to the above general procedure using 7-chloro-4-(piperazin-1-
yl)quinoline (0.25g, 1 mmol), 1H-pyrrole-2-carboxaldhyde(0.1g, 1.1mmol), to afford 23 (0.25g,
1
75.7%) as buff coloured solid. H NMR (DMSO-d ): δ 2.61 – 2.67 (m, 4H), 3.13 – 3.19 (m,
6
H.
1
3
6
4H), 3.72 (s, 2H), 5.76 – 5.86 (m, 2H), 6.79 – 8.63 (m, 7H). C NMR (DMSO-d ): δc. 156.4,
152.5, 150, 133.6, 130.2, 129.8, 128.6, 126.1, 122.7, 117.3, 109.6, 108.3, 107.2, 56.9, 51.9, 42.7.
+
ESI-MS m/z 327.1 (M+H) . Anal Calcd for C18
H
19ClN
4
; C, 66.15; H, 5.86; N, 17.14; Found C,
66.19; H, 5.83; N, 17.21.
6
6
6
6
6
6
06
07
08
09
10
11
4.1.8. General procedure for the synthesis of N-alkyl derivatives (21, 26 & 27): To a
suspension of 7-chloro-4-(piperazin-1-yl)quinoline (1mmol) and potassium carbonate (1.5mmol)
o
in acetonitrile was added the corresponding alkyl halide (1mmol) at 30 C. The reaction mixture
o
was then heated to 80 C for 1h (monitored by TLC and LCMS for completion) and cooled to
o
30 C. The mixture was then filtered through celite bed, and acetonitrile was evaporated in vacuo.
The resultant residue was diluted with water and dichloromethane, and the layers separated. The
2
7

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6
6
6
6
6
6
6
6
12
13
14
15
16
17
18
19
20
21
22
aqueous layer was re-extracted with dichloromethane (2 x 5 mL). The combined organic extract
was washed with brine, dried over sodium sulphate and evaporated in vacuo.
4.1.8.1.
4-(4-((1H-Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-7-chloroquinoline (21):
The compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), potassium carbonate (0.21g, 1.5mmol) and 2-
(chloromethyl)-1H-benzo[d]imidazole (0.17g, 1 mmol) to afford 21 (0.21g, 55.2%) as solid.
o
1
M.p: 230-231 C. H NMR (CDCl
3
): δH. 2.72 – 2.81 (m, 4H), 3.07 – 3.18 (m, 4H), 4.47 (s, 2H),
1
3
6.97 (d, J = 5.2 Hz, 1H), 7.23 – 8.47 (m, 8H). C NMR (CDCl
3
): δc.157.6, 152.6, 149.8, 141.2,
139.3, 133.4, 129.8, 129.1, 125.7, 123.1, 122.4, 115.4, 109.9, 63.3, 54.2, 49.3. ESI-MS m/z
+
378.1 (M+H) . Anal Calcd for C H ClN C, 66.75; H, 5.33; N, 18.53; Found C, 66.83; H,
2
1
20
5
5.39; N, 18.49.
6
6
6
6
6
6
6
6
23
24
25
26
27
28
29
30
4.1.8.2. 7-Chloro-4-(-4-(5-nitrothiazol-2-yl)piperazin-1-yl)quinoline (26): The compound
was synthesized according to the above general procedure using 7-chloro-4-(piperazin-1-
yl)quinoline (0.25g, 1 mmol), potassium carbonate (0.21g, 1.5mmol) and 2-chloro-5-
1
nitrothiazole(0.17g, 1 mmol) to afford 26 (0.29g, 76.3%) as orange solid. H NMR (DMSO-d
6
):
δH. 3.21 – 3.28 (m, 8H), 6.97 (d, J = 5.1 Hz, 1H), 7.53 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.86 (s,
1
3
1H), 8.11 – 8.67 (m, 3H). C NMR (DMSO-d ): δc. 157.6, 154.3, 152.7, 150.1, 147.3, 136.4,
6
+
133.6, 130, 129, 126, 122.3, 109.6, 47.3, 43.6. ESI-MS m/z 376.1(M+H) . Anal Calcd for
C H ClN O S C, 51.13; H, 3.75; N, 18.63; Found C, 51.06; H, 3.69; N, 18.68.
16
14
5
2
6
6
6
6
31
32
33
34
4.1.8.3. 4-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-7-nitrobenzo[c][1,2,5]oxadiazole (27):
The compound was synthesized according to the above general procedure using 7-chloro-4-
(piperazin-1-yl)quinoline (0.25g, 1 mmol), potassium carbonate (0.21g, 1.5mmol) and 4-chloro-
7-nitrobenzo[c][1,2,5]oxadiazole (0.2g mmol, 1 mmol) to afford 27 (0.27g, 65.8%) as dark red
2
8