Novel amide and sulphonamide derivatives of 6-(piperazin-1-yl)phenanthridine as potent Mycobacterium tuberculosis H37Rv inhibitors

Article abstract of DOI:10.1016/j.ejmech.2015.01.013

A series of thirty three novel 6-(piperazin-1-yl)phenanthridine amide and sulphonamide analogues were synthesized, characterized and screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis (MTB) H37Rv strain. These compounds exhibited minimum inhibitory concentration (MIC) between >56 and 50 μg/mL. Out of these derivatives, few compounds 6l, 6r, 7b, 7f, 7g and 7k exhibited moderate activity (MIC = 6.25 μg/mL) and compounds 6b, 6e, 6k, 6n, 7h, 7i and 7n displayed good activity (MIC = 3.13 μg/mL), whereas compounds 6m, 6s and 7d exhibited excellent anti-tubercular activity (MIC = 1.56 μg/mL). In addition, MTT assay was accomplished on the active analogues of the series against mouse macrophage (RAW 264.7) cells to evaluate the toxicity profile of the newly synthesized compounds and selectivity index of the compounds was determined. Additionally, compounds 6b and 7d were docked to the ATPase domain of M. tuberculosis GyrB protein to know the interaction profile and structures of compounds 6b and 7d were further substantiated through single crystal XRD.

Full text of DOI:10.1016/j.ejmech.2015.01.013

Accepted Manuscript
Novel 6-(piperazin-1-yl)phenanthridine amide and sulphonamide derivatives as potent
Mycobacterium tuberculosis H37Rv inhibitors
Kalaga Mahalakshmi Naidu, Hunsur Nagendra Nagesh, Manjeet Singh, Dharmarajan
Sriram, Perumal Yogeeswari, Kondapalli Venkata Gowri Chandra Sekhar
PII:
S0223-5234(15)00033-1
10.1016/j.ejmech.2015.01.013
EJMECH 7629
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2014
Revised Date: 22 November 2014
Accepted Date: 8 January 2015
Please cite this article as: K.M. Naidu, H.N. Nagesh, M. Singh, D. Sriram, P. Yogeeswari, K.V. Gowri
Chandra Sekhar Novel 6-(piperazin-1-yl)phenanthridine amide and sulphonamide derivatives as potent
Mycobacterium tuberculosis H37Rv inhibitors, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.01.013.
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ACCEPTED MANUSCRIPT
Novel 6-(piperazin-1-yl)phenanthridine amide and
sulphonamide derivatives as potent Mycobacterium
tuberculosis H37Rv inhibitors
a
a
b
c
Kalaga Mahalakshmi Naidu, Hunsur Nagendra Nagesh, Manjeet Singh, Dharmarajan Sriram, Perumal
c
a*
Yogeeswari, Kondapalli Venkata Gowri Chandra Sekhar
Department of Chemistry, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar
a
Nagar, Shamirpet Mandal, Hyderabad-500 078, India.
Department of Chemistry, Banaras Hindu University, Varanasi- 221 005, India.
b
c
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad campus, Jawahar
Nagar, Shamirpet Mandal, Hyderabad-500 078, India.
3
3
3 novel compounds are synthesized and evaluated for their anti-TB activity. Amongst these,
compounds exhibited excellent anti-TB activity with MIC 1.56 µg/mL and SI for these
compounds was >46.

ACCEPTED MANUSCRIPT
Novel 6-(piperazin-1-yl)phenanthridine amide and sulphonamide
derivatives as potent Mycobacterium tuberculosis H37Rv inhibitors
a
a
b
Kalaga Mahalakshmi Naidu, Hunsur Nagendra Nagesh, Manjeet Singh, Dharmarajan
c
c
a∗
Sriram, Perumal Yogeeswari, Kondapalli Venkata Gowri Chandra Sekhar
a
c
Department of Chemistry, Birla Institute of Technology & Science-Pilani, Hyderabad
campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, India.
Department of Chemistry, Banaras Hindu University, Varanasi- 221 005, India.
b
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
campus, Jawahar Nagar, Shamirpet Mandal, Hyderabad-500 078, India.
Abstract: A series of thirty three novel 6-(piperazin-1-yl)phenanthridine amide and
sulphonamide analogues were synthesized, characterized and screened for their in vitro
antimycobacterial activity against Mycobacterium tuberculosis (MTB) H37Rv strain. These
compounds exhibited minimum inhibitory concentration (MIC) between 1.56 ˗ ≥50 µg/mL.
Out of these derivatives, few compounds 6l, 6r, 7b, 7f, 7g and 7k exhibited moderate activity
(
MIC = 6.25 µg/mL) and compounds 6b, 6e, 6k, 6n, 7h, 7i and 7n displayed good activity
MIC = 3.13 µg/mL), whereas compounds 6m, 6s and 7d exhibited excellent anti-tubercular
(
activity (MIC = 1.56 µg/mL). In addition, MTT assay was accomplished on the active
analogues of the series against mouse macrophage (RAW 264.7) cells to evaluate the toxicity
profile of the newly synthesized compounds and selectivity index of the compounds was
determined. Additionally, compounds 6b and 7d were docked to the ATPase domain of
Mycobacterium tuberculosis GyrB protein to know the interaction profile and structures of
compounds 6b and 7d were further substantiated through single crystal XRD.
Keywords: Phenanthridine; antimycobacterial activity; piperazine sulphonamide;
Mycobacterium tuberculosis; single crystal XRD.
1
. Introduction
*
Corresponding author; Tel: +91-40-66303527; Fax: +91-40-66303998
E-mail address: kvgc@hyderabad.bits-pilani.ac.in; kvgcs@yahoo.com
1

ACCEPTED MANUSCRIPT
Tuberculosis (TB) is a contagious disease caused by tubercular bacilli. In spite of several
efforts by scientists across the world, this ancient scourge cannot be curtailed. It remains as
one of the major public health concerns after the human immunodeficiency virus. Worldwide
in 2013, 9 million people fell ill with TB, including 1.1 million cases among people living
with HIV. 1.5 million died from TB, including 0.36 million who were HIV-positive. An
estimated 0.48 million people developed multidrug-resistant TB (MDR-TB) and there were
an estimated 0.21 million deaths from MDR-TB. Around 9.0 % of MDR-TB cases have
extensively drug-resistant TB (XDR-TB). Around 100 countries have XDR-TB cases. More
than 50 companies are involved in the development of new TB drugs. Since last four decades
there are no new anti TB drugs except for Bedaquiline and Delamanid which became the first
novel TB drugs approved. These two are used only for the treatment of pulmonary MDR-TB
patients in serious or life-threatening conditions [1,2]. TB drug discovery and development
has not progressed to the extent necessary to annihilate the illness completely. Hence, it is
extremely essential to examine new compounds to cure against drug resistant forms of this
mortal disease along with minimal side effects. Focus should also be on developing
compounds which not only reduce the cost, but also the treatment period.
Phenanthridine derivatives are significant core moieties found in a variety of natural
products, important class of alkaloids and other synthetic vital molecules with a wide range
of biological activities and applications, including antibacterial [3,4], anticancer [5,6],
antimalarial [7], anti-HIV [8], anti-HCV agents [9], antiplasmodial [10] and in particular as
antitubercular agents [11-15]. Recently, Cappoen et al reported 1,2,3,4,8,9,10,11-
Octahydrobenzo[j]phenanthridine-7,12-diones as new leads against Mycobacterium
tuberculosis [11]. Our group also recently reported synthesis and evaluation of anti-tubercular
activity of 6-(4-substitutedpiperazin-1-yl) phenanthridine analogues as anti-TB agents [12].
Lipophilicity of compounds plays an important role in the biological activities which they
display. Attaching an alkyl/aryl amide and sulphonamides enhances the lipophilicity of
compounds [16,17]. Consequently, alkyl/aryl amide and alkyl/aryl sulphonamide derivatives
are known to exhibit wide range of pharmacological activities and physiochemical properties.
Indeed, these alkyl/aryl amide and alkyl/aryl sulphonamide analogues display enhanced
antibacterial and anti-TB activity [17-22].
In the quest for developing novel anti-TB agents we recently reported two compounds with
sulphonamide moiety [13]. Interestingly out of the two compounds, one of them exhibited
excellent anti-TB activity (MIC = 1.56 µg/mL) and this prompted us to explore further
structure activity relationship (SAR) of the compounds and hence prepared this series of
2

ACCEPTED MANUSCRIPT
compounds based on sulphonamides. Since carboxamides are analogous to sulphonamides,
we also decided to synthesise various amide derivatives and study their SAR. Hence, we
designed new (substituted)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone and 6-(4-((4-
substituted)sulfonyl)piperazin-1-yl)phenanthridine derivatives expecting increased biological
activity to combat this fatal disease. Design strategy to achieve title compounds [11-15, 17-
2
2] is depicted in figure 1.
2
. Chemistry
In this report, we chalked out for the synthesis of 6-(4-substitutedpiperazin-1-yl)
phenanthridine derivatives starting from 9-fluorenone as outlined in Scheme 1. We
synthesized compound 5 according to our reported protocol with slight modification [12]. 9-
fluorenone upon treatment with hydroxylamine hydrochloride and sodium acetate afforded N-
hydroxy-9H-fluoren-9-imine (2). Further heating 2 with polyphosphoric acid and phosphorus
pentoxide gave phenanthridin-6(5H)-one (3). 6-chlorophenanthridine (4) was synthesized by
refluxing 3 with phosphorus oxychloride and N,N-dimethylaniline. Cardinal synthon 6-
(piperazin-1-yl)phenanthridine (5) was prepared by irradiating a mixture of 6-
chlorophenanthridine, anhydrous piperazine, triethylamine (TEA) and N,N-
dimethylformamide (DMF) under microwave conditions.
Initially, we tried monitoring the reaction conditions with various bases and solvents as
summarised in Table 1. We set off our investigation for the synthesis of 6b, under various
reaction conditions. As a model reaction, compound (5) was treated with various acids and
amide coupling reagents were employed to get desired compound (entry 1-11). Initially, we
employed TEA as base, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC.HCl) and 1-
hydroxybenzotriazole (HOBt) as amide coupling reagents in the presence of
o
dichloromethane (DCM) as solvent at 0 C to rt. Resultant mixture was stirred for 8h to yield
6
b in about 58% (entry 1). Changing the solvent to DMF under similar reaction conditions
yielded 6b in about 52%. Alternatively, changed amide coupling reagents such as 1-
Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluoro
phosphate (HATU), (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) and (Benzotriazol-1-yloxy)tripyrrolidinophosphonium
[
hexafluorophosphate (PyBOP) and solvent as N,N-diisopropylethylamine (DIPEA) under
similar condition yields are 38-42% (entry 3–5). Next we hope to improve the yields, 1-
Propanephosphonic anhydride (T P) was employed to alleviate the yield (entry 6-11).
3
Initially, the reaction was carried out with various bases and solvents (entry 6-7) to give the
desired compound in moderate yield. The reaction was further optimised with TEA and DCM
3

ACCEPTED MANUSCRIPT
at various intervals of time (entry 8-11), to give 6b in good yield. Found entries 9-10 potently
improved the yield and actuate reaction period 6-8 h and reputable technique. Having the
optimised reaction conditions handy, a series of 19 compounds 6a-s was synthesized in good
yield, thus validating the scope of the present protocol.
Further, compound 5 was reacted with various sulfonyl chlorides, using TEA as base and
DCM as solvent at 0 ºC to rt for 1-2 h to yield 7a-n in good yields. All the title compounds
1
13
were purified by column chromatography. Both analytical and spectral data ( H NMR, C
NMR, FTIR, Elemental analysis and MS) of all the synthesized compounds were confirmed
prior to their evaluation of antimycobacterial activity.
In general, nucleophilic aromatic substitution at 6-chlorophenanthridine was confirmed by
FTIR analysis of key synthon 5 which showed the disappearance of IR absorption peak at
-1
~
754 cm due to aromatic C-Cl stretching frequencies and further disappearance of weak
-1
band at ~3280 cm in the IR spectra of 6-(piperazin-1-yl)phenanthridine (5) confirms the
1
formation of title compounds (6a-s and 7a-n). In general, H NMR spectrum of final
compounds displayed 8 aromatic protons of phenanthridine in the range δ 7.32 - 8.74 and 8
aliphatic piperazine (-CH -) protons were observed in the range δ 3.42 - 4.28. The spectral
2
data of all the title compounds are provided in experimental section.
3
. Results and discussion
3
.1. Antimycobacterial activity
All the synthesized compounds were tested for their ability to inhibit the growth of MTB
H37Rv strain by Microplate Alamar Blue Assay (MABA) with compound concentration
ranging from 50 to 0.78 µg/mL. Isoniazid, Rifampicin and Pyrazinamide were used as the
positive drug standards. The in vitro test results for title compounds tabulated in Table 2
indicate that MIC ranges from 1.56 to ≥ 50 µg/mL. Compounds with MIC 1.56 – 12.5 µg/mL
were further subjected to cytotoxicity studies. In these analogues, compounds 6m, 6s and 7d
are found to be excellent promising candidates by inhibiting 99% growth of MTB H37Rv
(ATCC 27294) strain at 1.56 µg/mL concentration.
Among the synthesized compounds, electronic effects of substituent play an important role in
displaying anti-TB activity. 6-(piperazin-1-yl)phenanthridine (5) was inhibiting 99% growth
of MTB H37Rv strain at 25 µg/mL [12]. Amongst, phenanthridine amides (6a-s) and
phenanthridine sulphonamide (7a-n) derivatives MTB activity summary, SAR studies are
th
described based on activity of compound 5. Introduction of cyanoacetyl group (6a) at the 4
position of compound 5, retained the anti-TB activity with MIC 25 µg/mL. Presence of amine
an electron donating group (EDG) at either ortho (6g) or para (6h) position on phenyl ring
4

ACCEPTED MANUSCRIPT
decreased the activity by ≥2 fold. Introduction of nitro group, an electron withdrawing group
(EWG), at meta (6e) position on phenyl ring greatly enhanced anti-TB activity by 8 fold with
MIC 3.13 µg/mL. On the other hand nitro group at di-meta (6f) and para (6d) position on
phenyl ring, decreased activity by 1-2 folds. Presence of chloro an EWG at para (6c) position
on phenyl ring improved anti-TB activity by 2 fold with MIC 12.5 µg/mL. Benzoyl group
th
(
6b) at 4 position of compound 5 significantly increased anti-TB activity by 8 folds with
th
MIC 3.13 µg/mL; cinnamoyl group (6j) at 4 position of 5 increased activity by 2 folds with
th
MIC 12.5 µg/mL and 2-(naphthalen-1-yl)acetyl group (6i) at 4 position of compound 5
critically decreased the anti-TB activity by >2 folds with MIC >50 µg/mL. To establish
extensive SAR we also varied heterocyclic compounds such as five membered rings (furan
and thiophene), six membered ring (pyridyls) and indole analogues. Furan-2-carbonyl group
th
(
6o) and thiophene-2-carbonyl group (6p) at 4 position of 6-(piperazin-1-yl)phenanthridine
5) retained and decreased anti-TB activity by 1-2 folds with MIC 25 µg/mL and 50 µg/mL
(
respectively. Introduction of six member heterocyclic rings such as nicotinoyl (6l), picolinoyl
th
(
6k), 6-formicacid picolinoyl (6n) and isonicotinoyl groups (6m) at 4 position of 5 greatly
enhanced the anti-TB activity by 4 – 16 folds with MIC 6.25 – 1.56 µg/mL. While, 2-(1H-
indol-3-yl)acetyl (6q), 3-(1H-indol-3-yl)propanoyl (6r) and 4-(1H-indol-3-yl)butanoyl
th
groups (6s) at 4 position of 5 witnessed either retention (6q) or greatly enhanced (6r and
6
s) the anti-TB activity by 1 – 16 folds. In conclusion, 6a-s presence of EDGs on phenyl ring
and five membered heterocyclic rings decreased anti-TB activity while EWGs, six membered
and benzannulated heterocyclic rings increased the activity (exception 6d).
SAR profile of the fourteen phenanthridine sulphonamide (7a-n) analogues is as follows. In
th
the presence of simple methanesulfonyl group (7a) at the 4 position of compound 5 the anti-
th
TB activity is retained with MIC 25 µg/mL. Benzenesulfonyl group (7b) at the 4 position of
compound 5 increased the anti-TB activity by 4 fold with MIC 6.25 µg/mL. Presence of
EDGs in the phenyl ring increased the activity in general exception being (7c). 2,4,6-triethyl
groups (7e) on phenyl ring improved anti-TB activity by 2 fold with MIC 12.5 µg/mL. Tert-
butyl group (7d) at para position on phenyl ring remarkably enriched the anti-TB activity by
1
6 folds with MIC 1.56 µg/mL. Methoxy group at para (7k) position on phenyl ring increased
the anti-TB activity by 4 folds with MIC 6.25 µg/mL. Presence of electron withdrawing
halogens (F (7g), Cl (7h) and Br (7i)) at para position on phenyl ring impressively enhanced
the anti-TB activity by 4–8 folds with MIC 6.25 – 3.13 µg/mL, including compound 7f.
Electron withdrawing nitro (7l) and acetyl (7m) groups at para positions on phenyl ring either
th
retained or decreased activity by 1–2 folds. 5-bromothiophene-2-sulfonyl group at 4
5

ACCEPTED MANUSCRIPT
position of 6-(piperazin-1-yl)phenanthridine (5) prominently augmented the anti-TB activity
by 8 folds with MIC 3.13 µg/mL. In general, EWGs on phenyl ring enhanced or retained the
anti-TB activity exception being 7l. Wrapping up the results, we observe that in the series
synthesized, sulphonamide analogues were more active than the amides.
3
.2. Molecular modelling studies
To scrutinize the interaction profile, the compounds (6b and 7d) were docked to the ATPase
domain of MTB GyrB protein found from protein data bank (PDB ID–3ZKB) [23] with
additional precision mode (XP) of Glide module [24]. The interaction patterns of the
analogues were studied by starting with analysis of co-crystallized ligand docking pattern.
The crystal ligand phosphoaminophosphonic acid-adenylate ester (ANP) was involved in
prominent H-bonding connections, one of the interactions between the amino group of the
ANP moiety and the oxygen atom of Asp79 at bond length was 1.78Å as shown in Figure 2.
The direction of this ligand in the protein active site disclosed the presence of a hydrophobic
pocket where the ANP moiety was stabilized by the interactions with val 99, Ile 84, lys 108
and Tyr 114. The docking pose of compounds 6b and 7d were portrayed in figure 3 and
figure 4 respectively. Compound 6b showed strong hydrogen bond interaction with Lys 108
by1.97Å distance. Both the compounds displayed common potent hydrophobic interactions
with val 98, val 99, Ile 84, Pro 85, ala 113 and Tyr 114, whereas compound 7d showed extra
hydrophobic interaction with Ala 87 and strong π-π stacking interaction (CPK model of
protein in Figure 4) with Arg 141. These hydrophobic interactions lead towards the
specificity of compounds with this protein, assigning to its activity.
3
.3. Cytotoxicity assay
Among the series, most active compounds (MIC ≤12.5 µg/mL) were subjected to Promega
Cell Titer 96 non-radioactive cell proliferation assay to evaluate the in vitro cytotoxicity
against mouse macrophage (RAW264.7) cell lines. The approximate IC50 values and
selectivity index (SI) values [25] are tabulated in Table 3. In these all active compounds
(MIC ≤12.5 µg/mL) displayed SI profile >7. while good anti-TB active compounds 6b, 6e,
6
k, 6n, 7h, 7i and 7n (MIC 3.13 µg/mL) exhibited SI profile >19. The most active anti-TB
compounds 6m, 6s, and 7d exhibited excellent SI profile >46. This outcome indicates that
novel chemophores 6m, 6s and 7d are not toxic and might be considered for further structural
modification of phenanthridine piperazine amide/sulphonamide analogues as leads to
accomplish the treatment of this deadly disease.
3
.4. X-ray crystallographic study
6

ACCEPTED MANUSCRIPT
The X-ray crystallographic analysis of the compounds 6b and 7d was carried out: Crystals
were grown from the slow evaporation of a 1:3:6 ratio of methanol/dichloromethane/ethyl
acetate solvent mixture at rt, to get white flake crystals. A desirable crystal under triclinic
3
system with space groups P
ī
of 6b, crystal size/mm 0.6 × 0.5 × 0.4 and crystal under
3
monoclinic system with space groups P2 /n of 7d, crystal size/mm 0.6 × 0.5 × 0.4
1
respectively. These crystals were kept at 293 K during data collection. Using Computer
programs SHELXL97 (Sheldrick, 2008) the structure was solved with the unknown structure
solution program using unknown and refined with the SHELXS-97 (Sheldrick, 2008) [26].
The crystal data, data collection and structure refinement details are summarized in Table 4
and molecular structure is given as an ORTEP diagram pictured in Figure 5. Crystallographic
data of the compounds 6b and 7d were deposited with the Cambridge Crystallographic Data
Centre (CCDC). These compounds have been assigned the following deposition numbers
CCDC 1012376 and CCDC 1012377 respectively. The X-ray structure parameters and
complete details of compound 6b and 7d are provided in supplementary material.
4
. Conclusion:
In this communication, phenanthridine derivatives were synthesized with moderate to
excellent yields. The preliminary in vitro anti-TB screening results, toxicity studies and SI
profile results of compounds 6b, 6e, 6k, 6n, 7h, 7i and 7n (MIC 3.13 µg/mL) displayed good
anti-TB activity and SI profile >19. Compounds 6m, 6s, and 7d (MIC 1.56 µg/mL) exhibited
excellent anti-TB activity and SI profile >46. Detailed in vivo studies and the exact
mechanistic studies of 6m, 6s, and 7d analogues are scope of future study in our lab. The 6-
(4-substitutedpiperazin-1-yl)phenanthridine skeleton is established to be an important motif
for further development of anti-tubercular agents.
5
. Experimental Section:
5
.1. Materials and methods:
Chemicals and solvents were procured from commercial sources and are analytically pure.
Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates
(Merck 60 F254) with visualization of components by UV light (254 nm). Column
1
chromatography was carried out on silica gel (Merck 230-400 mesh). H NMR spectra and
1
3
C NMR spectra were recorded at 300 MHz using a Bruker AV 300 spectrometer or 400
MHz using a Bruker AV 400 spectrometer (Bruker CO., Switzerland) in CDCl or DMSO-D6
3
solution with tetramethylsilane as the internal standard, and chemical shift values (δ) were
given in ppm. Microwave reactions were performed in closed vessel using Biotage Initiator
7

ACCEPTED MANUSCRIPT
microwave synthesizer (Uppsala, Sweden). IR spectra were recorded on a FT-IR
-1
spectrometer (Schimadzu) and peaks are reported in cm . Melting points were determined on
an electro thermal melting point apparatus (Stuart-SMP30) in open capillary tubes and are
uncorrected. Elemental analyses were analysed by Elementar Analysensysteme GmbH vario
MICRO cube CHNS/O Analyzer. Mass spectra (ESI-MS) were recorded on Schimadzu
MS/ESI mass spectrometer.
5
.2 Synthesis of title compounds (6a-s and 7a-n)
For 6a-s: 6-(piperazin-1-yl)phenanthridine (1.14 mmol) was dissolved in DCM (4 mL) in an
oven dry 25mL two necked RBF. TEA (3.41 mmol) and amide coupling reagent T P (1.36
3
o
mmol) followed by anhydrous substituted alkyl / aryl acids (1.14 mmol) were added at 0 C
and stirred at rt for 6h.
For 7a-n: 6-(piperazin-1-yl)phenanthridine (1.14 mmol) was dissolved in DCM (4 mL) in an
oven dry 25mL two necked RBF. TEA (3.41 mmol) followed by anhydrous substituted aryl
o
sulfonyl chloride (1.36 mmol) were added at 0 C and stirred for 2h at rt.
Completion of the reaction was monitored by TLC using 0 - 6% MeOH in DCM as mobile
phase. After the reaction was complete, reaction mixture was quenched in ice cold water and
extracted with DCM (3 x 10 mL), combined organic layers were washed with saturated brine
solution and dried on anhydrous Na SO then evaporated in vacuo. Obtained residue was
2
4
purified by column chromatography using gradient 0 - 6% MeOH in DCM, to give the title
compounds (6a-s and 7a-n).
5
.2.1. 3-oxo-3-(4-(phenanthridin-6-yl)piperazin-1-yl)propanenitrile (6a)
o
1
Appearance: off white solid; yield = 78%; mp = 139-140 C; H NMR (400 MHz, CDCl ) δ
3
8
.56 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 7.2 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.0
1
3
Hz, 1H), 7.82-7.48 (m, 4H), 3.82 – 3.54 (t, 8H), 3.46 (s, 2H). C NMR (100.61 MHz,
CDCl ) δ 170.14, 161.68, 142.40, 136.22, 134.45, 131.41, 130.37, 128.83, 127.19, 125.14,
3
1
24.17, 122.13, 121.58, 120.26, 119.85, 51.60, 47.12, 31.28. FT-IR: ν 2248 (CN); ESI-MS:
+
(
m/z) calcd. for C H N O, 330.14, found: 331.22 (M + H) . Anal. Calcd for C H N O;
2
0
18
4
20 18
4
(
%) C 72.71, H 5.49, N 16.96; found: C 72.82, H 5.54, N 17.04.
.2.2. (4-(phenanthridin-6-yl)piperazin-1-yl)(phenyl)methanone (6b)
5
o
1
Appearance: white solid; yield = 85%; mp = 108-109 C; H NMR (400 MHz, CDCl ) δ 8.59
3
(d, J = 8.4 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.0 Hz,
13
1
H), 7.82 (t, J = 7.6 Hz, 1H), 7.78 (m, 2H), 7.66-7.45 (m, 6H), 4.12 – 3.51 (t, 8H). C NMR
(
100.61 MHz, CDCl ) δ 170.74, 159.62, 143.50, 135.74, 135.02, 133.41, 130.37, 130.12,
3
8

ACCEPTED MANUSCRIPT
1
4
29.83, 128.83, 128.59, 127.15, 126.95, 126.14, 125.17, 122.83, 121.91, 121.26, 51.63,
+
7.82. ESI-MS: (m/z) calcd. for C H N O, 367.16, found: 368.23 (M + H) . Anal. Calcd for
2
4
21
3
C H N O; (%) C 78.45, H 5.76, N 11.44; found: C 78.61, H 5.81, N 11.53.
24
21
3
5
.2.3. (4-chlorophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6c)
o
1
Appearance: off white solid; yield = 81%; mp = 132-133 C; H NMR (400 MHz, CDCl ) δ
3
8
.59 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.8 Hz, 2H), 8.25 (d, J = 8.2 Hz, 1H), 7.96 (d, J = 10.8
Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.84 (d, J = 8.8 Hz, 2H), 7.81 – 7.48 (m, 4H), 3.67 – 3.44
1
3
(
t, 8H). C NMR (100.61 MHz, CDCl ) δ 170.52, 160.14, 144.22, 136.18, 135.81, 132.87,
3
1
1
31.08, 130.64, 129.54, 128.63, 128.32, 127.11, 126.84, 126.33, 125.46, 122.57, 121.87,
21.18, 52.28, 47.54. ESI-MS: (m/z) calcd. for C H ClN O, 401.12, found: 402.25 (M +
24
20
3
+
H) . Anal. Calcd for C H ClN O; (%) C 71.73, H 5.02, N 10.46; found: C 71.81, H 5.09, N
2
4
20
3
1
0.53.
.2.4. (4-nitrophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6d)
5
o
1
Appearance: yellow solid; yield = 68%; mp = 148-149 C; H NMR (400 MHz, DMSO-d ) δ
6
8
2
8
1
1
.62 (d, J = 8.4 Hz, 1H), 8.48 (d, J = 8.0, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 9.6 Hz,
H), 7.97 (d, J = 7.6 Hz, 1H), 7.92 (d, J = 8.0 Hz, 2H), 7.82 – 7.54 (m, 4H), 3.72 – 3.68 (t,
1
3
H). C NMR (100.61 MHz, DMSO-d ) δ 172.12, 164.51, 146.86, 142.02, 139.53, 137.84,
6
32.07, 130.84, 129.74, 128.44, 128.01, 127.69, 126.80, 126.11, 125.47, 123.78, 122.35,
20.88, 52.23, 48.59. ESI-MS: (m/z) calcd. for C H N O , 412.15, found: 413.22 (M +
24
20
4
3
+
H) . Anal. Calcd for C H N O ; (%) C 69.89, H 4.89, N 13.58; found: C 69.98, H 4.95, N
2
4
20
4
3
1
3.70.
.2.5. (3-nitrophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6e)
5
o
1
Appearance: pale yellow solid; yield = 63%; mp = 230-231 C; H NMR (400 MHz, DMSO-
d6) δ 8.69 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.42 (d, J = 8.0, 1H), 8.26 (d, J = 8.4 Hz, 1H),
8
–
1
1
.21 (d, J = 9.2 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.93 (m, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.82
1
3
7.54 (m, 4H), 3.69 – 3.57 (t, 8H). C NMR (100.61 MHz, DMSO-d ) δ 171.08, 162.31,
6
47.36, 143.47, 141.11, 139.62, 135.24, 132.04, 131.84, 130.02, 129.21, 128.68, 128.32,
27.34, 126.01, 125.82, 125.21, 123.52, 121.76, 120.88, 53.62, 47.84. ESI-MS: (m/z) calcd.
+
for C H N O , 412.15, found: 413.20 (M + H) . Anal. Calcd for C H N O ; (%) C 69.89,
2
4
20
4
3
24 20
4
3
H 4.89, N 13.58; found: C 69.96, H 4.96, N 13.66.
.2.6. (3,5-dinitrophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6f)
5
o
1
Appearance: yellow solid; yield = 64%; mp = 196-197 C; H NMR (400 MHz, DMSO-d ) δ
6
9
.08 – 8.83 (s, 3H), 8.46 (d, J = 8.0, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H),
9

ACCEPTED MANUSCRIPT
1
3
7
.93 (d, J = 8.0 Hz, 1H), 7.85 – 7.48 (m, 4H), 3.71 – 3.62 (t, 8H). C NMR (100.61 MHz,
DMSO-d ) δ 172.32, 159.22, 154.08, 144.75, 142.47, 139.81, 138.02, 133.83, 132.38, 130.39,
6
1
29.21, 128.52, 127.01, 125.44, 124.23, 122.89, 121.22, 119.65, 52.63, 47.82. ESI-MS:
+
(
m/z) calcd. for C H N O , 457.13, found: 458.21 (M + H) . Anal. Calcd for C H N O ;
2
4
19
5
5
24 19
5
5
(
%) C 63.02, H 4.19, N 15.31; found: C 63.11, H 4.24, N 15.39.
.2.7. (2-aminophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6g)
5
o
1
Appearance: white solid; yield = 60%; mp = 164-165 C; H NMR (400 MHz, DMSO-d ) δ
6
8
.38 (d, J = 8.0 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.4
Hz, 1H), 7.80 – 7.18 (m, 6H), 7.12 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 8.4 Hz, 1H), 6.02 (br,
1
3
2
1
1
H), 3.68 – 3.52 (t, 8H). C NMR (100.61 MHz, DMSO-d ) δ 169.78, 158.13, 146.87,
6
42.42, 141.44, 138.23, 137.54, 131.12, 130.66, 130.08, 129.23, 128.84, 126.45, 124.87,
24.12, 123.88, 121.86, 120.22, 119.82, 118.52, 52.42, 46.57. ESI-MS: (m/z) calcd. for
+
C H N O, 382.17, found: 383.26 (M + H) . Anal. Calcd for C H N O; (%) C 75.37, H
24
22
4
24 22
4
5
.80, N 14.65; found: C 75.51, H 5.85, N 14.54.
.2.8. (4-aminophenyl)(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6h)
5
1
Appearance: off white semi solid; yield = 62%; mp = Not determined (ND); H NMR (400
MHz, DMSO-d ) δ 8.57 (d, J = 8.4 Hz, 1H), 8.43 (d, J = 8.0, 1H), 8.22 (d, J = 7.8 Hz, 1H),
6
7
.98 (d, J = 8.4 Hz, 1H), 7.84 (m, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.71 – 7.52 (m, 3H), 7.18 (d,
1
3
J = 8.2 Hz, 2H), 5.84 (br, 2H), 3.78 – 3.54 (t, 8H). C NMR (100.61 MHz, DMSO-d ) δ
6
1
1
70.84, 163.58, 147.18, 146.09, 140.25, 134.28, 131.12, 130.82, 129.98, 128.22, 127.84,
27.02, 126.45, 125.34, 124.72, 123.21, 122.87, 119.24, 52.64, 47.82. ESI-MS: (m/z) calcd.
+
for C H N O, 382.17, found: 383.22 (M + H) . Anal. Calcd for C H N O; (%) C 75.37, H
2
4
22
4
24 22
4
5
.80, N 14.65; found: C 75.54, H 5.86, N 14.52.
5
.2.9. 2-(naphthalen-1-yl)-1-(4-(phenanthridin-6-yl)piperazin-1-yl)ethanone (6i)
o
1
Appearance: pale yellow solid; yield = 75%; mp = 181-182 C; H NMR (400 MHz, DMSO-
d6) δ 8.80 (d, J = 8.4 Hz, 1H), 8.65 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H), 8.14 (d, J =
8
–
.0 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.78
1
3
7.42 (m, 7H), 7.31 (d, J = 8.0 Hz, 1H), 4.28 (s, 2H), 3.93 – 3.40 (t, 8H). C NMR (100.61
MHz, DMSO-d ) δ 172.09, 163.24, 150.86, 148.13, 141.18, 140.45, 138.16, 134.22, 133.72,
6
1
1
32.07, 131.28, 130.88, 129.12, 128.33, 126.57, 125.78, 124.02, 123.31, 123.04, 122.89,
22.23, 121.64, 121.02, 120.58, 50.82, 48.54. ESI-MS: (m/z) calcd. for C H N O, 431.19,
2
9
25
3
+
found: 432.33 (M + H) . Anal. Calcd for C H N O; (%) C 80.72, H 5.84, N 9.74; found: C
2
9
25
3
8
0.79, H 5.87, N 9.81.
1
0

ACCEPTED MANUSCRIPT
5
.2.10. (E)-1-(4-(phenanthridin-6-yl)piperazin-1-yl)-3-phenylprop-2-en-1-one (6j)
o
1
Appearance: off white greenish solid; yield = 72%; mp = 154-156 C; H NMR (400 MHz,
DMSO-d ) δ 8.58 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.88
6
(d, J = 8.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.75 – 7.48 (m, 7H), 7.38 (d, J = 15.2 Hz, 1H),
1
3
7
1
1
.29 (d, J = 15.8 Hz, 1H), 3.68 – 3.54 (t, 8H). C NMR (100.61 MHz, DMSO-d ) δ 172.08,
6
61.22, 154.16, 148.12, 140.82, 138.45, 133.16, 132.84, 131.90, 128.37, 127.78, 125.23,
24.88, 123.65, 122.44, 122.21, 121.84, 121.22, 120.36, 118.41, 51.59, 46.38. ESI-MS: (m/z)
+
calcd. for C H N O, 393.18, found: 394.27 (M + H) . Anal. Calcd for C H N O; (%) C
2
6
23
3
26 23
3
7
9.36, H 5.89, N 10.68; found: C 79.48, H 5.93, N 10.75.
5
.2.11. (4-(phenanthridin-6-yl)piperazin-1-yl)(pyridin-2-yl)methanone (6k)
1
Appearance: off white semi solid; yield = 78%; mp = ND; H NMR (400 MHz, CDCl ) δ
3
8
.59 (d, J = 8.8 Hz, 1H), 8.46 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 8.14 (m, 1H), 7.96
(d, J = 7.8 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.78 – 7.48 (m, 5H),
1
3
3
1
1
.92 – 3.52 (t, 8H).
C NMR (100.61 MHz, CDCl ) δ 171.23, 160.89, 154.04, 151.12,
3
49.34, 142.81, 140.86, 133.17, 132.34, 130.14, 129.13, 128.47, 126.05, 124.60, 123.37,
22.88, 122.14, 121.22, 120.80, 51.74, 46.58. ESI-MS: (m/z) calcd. for C H N O, 368.16,
2
3
20
4
+
found: 369.24 (M + H) . Anal. Calcd for C H N O; (%) C 74.98, H 5.47, N 15.21; found: C
2
3
20
4
7
5.07, H 5.41, N 15.14.
5
.2.12. (4-(phenanthridin-6-yl)piperazin-1-yl)(pyridin-3-yl)methanone (6l)
o
1
Appearance: off white solid; yield = 74%; mp = 153-154 C; H NMR (400 MHz, DMSO-d )
6
δ 8.81 (s, 1H), 8.72 (d, J = 8.4 Hz, 2H), 8.64 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.8 Hz, 1H),
13
7
.94 (m, 2H), 7.83 (d, J = 8.0 Hz, 1H), 7.78 – 7.51 (m, 4H), 3.98 – 3.44 (t, 8H). C NMR
(
100.61 MHz, DMSO-d ) δ 170.84, 162.45, 153.76, 153.23, 151.88, 141.54, 139.72, 132.65,
6
1
5
32.11, 130.81, 129.33, 128.28, 126.32, 124.86, 123.42, 122.46, 122.02, 121.85, 120.43,
+
2.68, 47.54. ESI-MS: (m/z) calcd. for C H N O, 368.16, found: 369.28 (M + H) . Anal.
2
3
20
4
Calcd for C H N O; (%) C 74.98, H 5.47, N 15.21; found: C 75.05, H 5.45, N 15.12.
2
3
20
4
5
.2.13. (4-(phenanthridin-6-yl)piperazin-1-yl)(pyridin-4-yl)methanone (6m)
1
Appearance: off white semi solid; yield = 81%; mp = ND; H NMR (400 MHz, CDCl ) δ
3
8
.74 (d, J = 8.8 Hz, 2H), 8.45 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.8
Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 7.82 (d, J = 7.6 Hz, 1H), 7.75 – 7.44 (m, 4H), 3.88 – 3.54
1
3
(
t, 8H). C NMR (100.61 MHz, CDCl ) δ 172.06, 159.15, 152.74, 150.89, 147.21, 144.39,
3
1
39.82, 133.45, 130.23, 128.06, 127.14, 126.23, 124.18, 123.42, 122.01, 121.79, 120.12,
1
1

ACCEPTED MANUSCRIPT
+
5
2.36, 47.52. ESI-MS: (m/z) calcd. for C H N O, 368.16, found: 369.23 (M + H) . Anal.
23 20 4
Calcd for C H N O; (%) C 74.98, H 5.47, N 15.21; found: C 75.12, H 5.40, N 15.16.
2
3
20
4
5
.2.14. 6-(4-(phenanthridin-6-yl)piperazine-1-carbonyl)picolinic acid (6n)
1
Appearance: off white semi solid; yield = 65%; mp = ND; H NMR (400 MHz, DMSO-d ) δ
6
1
8
–
1
1
1.58 (s, 1H), 8.88 (d, J = 8.4 Hz, 1H), 8.82 (d, J = 9.2 Hz, 1H), 8.61 (m, 1H), 8.57 (d, J =
.4 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 7.8 Hz, 1H), 7.79
1
3
7.46 (m, 4H), 3.82 – 3.52 (t, 8H). C NMR (100.61 MHz, DMSO-d ) δ 172.36, 171.81,
6
60.12, 152.45, 150.86, 148.32, 143.75, 140.48, 135.08, 134.53, 131.24, 129.04, 128.14,
26.87, 124.12, 123.44, 122.81, 122.63, 121.07, 120.24, 52.72, 47.50. ESI-MS: (m/z) calcd.
+
for C H N O , 412.15, found: 413.28 (M + H) . Anal. Calcd for C H N O ; (%) C 69.89,
2
4
20
4
3
24 20
4
3
H 4.89, N 13.58; found: C 69.98, H 4.95, N 13.64.
5
.2.15. furan-2-yl(4-(phenanthridin-6-yl)piperazin-1-yl)methanone (6o)
o
1
Appearance: off white solid; yield = 76%; mp = 118-119 C; H NMR (400 MHz, CDCl ) δ
3
8
.59 (d, J = 8.8 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.8
Hz, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.86 – 7.54 (m, 4H), 7.43 (d, J = 7.8 Hz, 1H), 7.22 (m,
1
3
1
1
1
3
7
5
H), 4.94 – 3.68 (t, 8H). C NMR (100.61 MHz, CDCl ) δ 169.74, 157.68, 148.15, 142.44,
3
35.22, 133.45, 130.87, 130.12, 129.18, 128.84, 128.04, 127.21, 126.90, 126.12, 125.18,
22.18, 121.98, 118.24, 51.68, 48.84. ESI-MS: (m/z) calcd. for C H N O , 357.14, found:
2
2
19
3
2
+
58.25 (M + H) . Anal. Calcd for C H N O ; (%) C 73.93, H 5.36, N 11.76; found: C
22
19
3
2
3.99, H 5.41, N 11.83.
.2.16. (4-(phenanthridin-6-yl)piperazin-1-yl)(thiophen-2-yl)methanone (6p)
o
1
Appearance: white solid; yield = 81%; mp = 143-145 C; H NMR (400 MHz, DMSO-d ) δ
6
8
.72 (d, J = 8.8 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.0
Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.88 – 7.37 (m, 5H), 4.89 – 3.53
1
3
(
t, 8H). C NMR (100.61 MHz, DMSO-d ) δ 170.02, 158.88, 149.22, 141.89, 136.72,
6
1
1
31.93, 130.12, 129.94, 129.34, 128.84, 128.11, 127.26, 126.95, 125.87, 125.03, 122.48,
21.68, 120.87, 52.46, 47.52. ESI-MS: (m/z) calcd. for C H N OS, 373.12, found: 374.27
2
2
19
3
+
(
M + H) . Anal. Calcd for C H N OS; (%) C 70.75, H 5.13, N 11.25; found: C 70.83, H
22 19 3
5
5
.19, N 11.15.
.2.17. 2-(1H-indol-3-yl)-1-(4-(phenanthridin-6-yl)piperazin-1-yl)ethanone (6q)
o
1
Appearance: off white solid; yield = 80%; mp = 162-163 C; H NMR (400 MHz, DMSO-d )
6
δ 8.24 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.88 (d, J = 8.0
Hz, 1H), 7.84 (m, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.58 – 7.48 (m, 3H), 7.42 (d, J = 8.0 Hz,
1
2

ACCEPTED MANUSCRIPT
1
3
1
H), 7.34 (s, 1H), 7.20 (m, 2H), 6.08 (br, 1H), 3.88 (t, 4H), 3.80 (s, 2H), 3.52 (t, 4H).
C
NMR (100.61 MHz, DMSO-d ) δ 168.54, 159.62, 144.83, 143.23, 140.23, 138.64, 135.49,
6
1
35.05, 133.63, 132.84, 129.47, 128.43, 127.19, 126.82, 126.11, 124.46, 123.23, 121.52,
19.88, 119.04, 117.74, 108.04, 52.64, 48.48, 40.68. ESI-MS: (m/z) calcd. for C H N O
1
27
24
4
+
4
20.19, found: 421.15 (M + H) . Anal. Calcd for C H N O: (%) C 77.12, H 5.75, N 13.32;
27 24 4
Found: C 77.24, H 5.88, N 13.41.
5
.2.18. 3-(1H-indol-3-yl)-1-(4-(phenanthridin-6-yl)piperazin-1-yl)propan-1-one (6r)
o
1
Appearance: off white solid; yield = 83%; mp = 120-121 C; H NMR (400 MHz, DMSO-d )
6
δ 8.19 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.2
Hz, 1H), 7.80 (m, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.61 – 7.44 (m, 3H), 7.41 (d, J = 8.0 Hz,
1
3
1
H), 7.38 (s, 1H), 7.22 (m, 2H), 6.84 (br, 1H), 3.92 – 2.70 (t, 12H). C NMR (100.61 MHz,
DMSO-d ) δ 170.22, 162.48, 145.16, 144.06, 141.18, 139.21, 136.42, 135.84, 133.82, 133.08,
6
1
28.92, 128.11, 127.64, 126.74, 126.32, 124.22, 123.68, 120.94, 119.24, 118.48, 117.38,
10.28 52.12, 47.39, 39.94, 32.98. ESI-MS: (m/z) calcd. for C H N O 434.21, found:
1
2
8
26
4
+
4
35.28 (M + H) . Anal. Calcd for C H N O: (%) C 77.39, H 6.03, N 12.89; Found: C 77.46,
28 26 4
H 6.12, N 12.94.
5
.2.19. 4-(1H-indol-3-yl)-1-(4-(phenanthridin-6-yl)piperazin-1-yl)butan-1-one (6s)
o
1
Appearance: off white solid; yield = 75%; mp = 148-149 C; H NMR (400 MHz, DMSO-d )
6
δ 8.22 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 7.6 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.89 (d, J = 7.4
Hz, 1H), 7.84 (m, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.62 – 7.39 (m, 3H), 7.35 (d, J = 8.0 Hz,
1
3
1
H), 7.30 (s, 1H), 7.18 (m, 2H), 6.42 (br, 1H), 3.89 – 2.60 (t, 12H), 1.68 (m, 2H). C NMR
(
100.61 MHz, DMSO-d ) δ 170.84, 163.04, 144.87, 143.21, 141.85, 139.42, 135.08, 134.91,
6
1
1
33.09, 132.44, 129.48, 128.74, 127.04, 126.23, 123.11, 122.41, 122.26, 121.39, 120.12,
19.94, 118.87, 111.46, 52.88, 46.24, 40.37, 30.42, 39.87. ESI-MS: (m/z) calcd. for
+
C H N O 448.22, found: 449.24 (M + H) . Anal. Calcd for C H N O: (%) C 77.65, H
29
28
4
29 28
4
6
.29, N 12.49; Found: C 77.72, H 6.38, N 12.38.
5
.2.20. 6-(4-(methylsulfonyl)piperazin-1-yl)Phenanthridine (7a):
1
Appearance: off white semi solid; yield = 78%; mp = ND; H NMR (400 MHz, CDCl ) δ
3
8
.26 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 7.4
1
3
Hz, 1H), 7.74 – 7.41 (m, 4H), 3.87 – 3.53 (t, 8H), 3.09 (s, 3H). C NMR (100.61 MHz,
CDCl ) δ 164.24, 148.76, 140.08, 133.13, 131.67, 130.43, 129.88, 123.24, 122.41, 121.82,
3
1
20.34, 119.92, 114.34, 53.65, 48.21, 38.98. ESI-MS: (m/z) calcd. for C H N O S 341.12,
18 19 3 2
1
3

ACCEPTED MANUSCRIPT
+
found: 342.14 (M + H) . Anal. Calcd for C H N O S: (%) C 63.32, H 5.61, N 12.31; found:
1
8
19
3
2
C 63.64, H 5.74, N 12.28.
.2.21. 6-(4-(phenylsulfonyl)piperazin-1-yl)phenanthridine (7b)
5
o
1
Appearance: off white solid; yield = 88%; mp = 238-239 C; H NMR (400 MHz, CDCl ) δ
3
8
.57 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 7.8 Hz, 2H), 7.90 (dd, J = 9.0
1
3
Hz, J = 0.8 Hz, 1H), 7.82 (d, J = 7.4 Hz, 1H), 7.77 – 7.39 (m, 7H), 3.68 – 3.44 (t, 8H). C
NMR (100.61 MHz, CDCl ) δ 162.86, 152.08, 141.12, 138.47, 132.24, 131.82, 130.12,
3
1
4
29.64, 124.28, 123.81, 122.89, 121.24, 121.08, 120.42, 119.92, 118.42, 112.47, 50.48,
+
8.07. ESI-MS: (m/z) calcd. for C H N O S 403.13, found: 403.22 (M + H) . Anal. Calcd
2
3
21
3
2
for C H N O S: (%) C 68.46, H 5.25, N 10.41; found: C 68.55, H 5.32, N 10.60.
2
3
21
3
2
5
.2.22. 6-(4-tosylpiperazin-1-yl)phenanthridine (7c)
o
1
Appearance: pale yellow solid; yield = 93%; mp = 188-189 C; H NMR (400 MHz, CDCl )
3
δ 8.48 (d, J = 8.2 Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.91 (dd, J = 8.4 Hz, J = 0.8 Hz, 1H),
7
4
1
1
4
1
5
.88 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 7.8 Hz, 2H), 7.79 (d, J = 8.0 Hz, 2H), 7.74 – 7.46 (m,
1
3
H), 3.64 – 3.38 (t, 8H), 2.94 (s, 3H). C NMR (100.61 MHz, CDCl ) δ 162.42, 153.14,
3
44.46, 139.08, 138.81, 132.11, 131.81, 129.64, 128.43, 124.82, 123.08, 122.28, 121.63,
20.42, 119.92, 118.42, 113.22, 52.18, 47.11, 24.08. ESI-MS: (m/z) calcd. for C H N O S
24
23
3
2
+
17.15, found: 417.24 (M + H) . Anal. Calcd for C H N O S: (%) C 69.04, H 5.55, N
2
4
23
3
2
0.06; found: C 69.11, H 5.64, N 10.12.
.2.23. 6-(4-(4-tert-butylphenylsulfonyl)piperazin-1-yl)phenanthridine (7d)
o
1
Appearance: white solid; yield = 88%; mp = 154-155 C; H NMR (400 MHz, CDCl ) δ 8.58
3
(
d, J = 8.0 Hz, 1H), 8.45 (d, J = 8.2 Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.91 (dd, J = 8.4 Hz, J
0.8 Hz, 1H), 7.78 (dd, J = 6.4 Hz, J = 1.6 Hz, 1H), 7.74 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H),
.76 (m, 1H), 7.74 (d, J = 7.2 Hz, 1H), 7.64 – 7.55 (m, 4H), 7.51 (d, J = 8.0 Hz, 1H), 3.60 –
=
7
3
1
1
1
3
.35 (t, 8H), 1.37 (s, 9H). C NMR (100.61 MHz, CDCl ) δ 159.24, 156.71, 143.45, 134.98,
3
32.83, 130.30, 128.82, 128.62, 127.79, 126.81, 126.17, 125.95, 125.14, 122.85, 122.65,
21.86, 121.03, 50.48, 46.03, 35.23, 31.13. ESI-MS: (m/z) calcd. for C H N O S 459.19,
2
7
29
3
2
+
found: 460.25 (M + H) . Anal. Calcd for C H N O S: (%) C 70.56, H 6.36, N 9.14; found:
2
7
29
3
2
C 70.64, H 6.28, N 9.24.
.2.24. 6-(4-(mesitylsulfonyl)piperazin-1-yl)phenanthridine (7e)
5
o
1
Appearance: white solid; yield = 81%; mp = 118-119 C; H NMR (400 MHz, CDCl ) δ 8.48
3
(d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.98 (dd, J = 8.8 Hz, J = 1.6 Hz, 1H), 7.82 (dd,
J = 7.2 Hz, J = 1.2 Hz, 1H), 7.72 – 7.58 (m, 4H), 7.24 (s, 2H), 3.62 – 3.42 (t, 8H), 2.88 – 2.62
1
4

ACCEPTED MANUSCRIPT
1
3
(
s, 9H). C NMR (100.61 MHz, CDCl ) δ 162.38, 157.24, 148.12, 145.31, 139.45, 138.08,
3
1
5
+
31.78, 131.41, 130.21, 127.09, 125.22, 124.62, 123.26, 122.08, 121.96, 121.14, 120.42,
1.04, 47.21, 31.08, 30.88. ESI-MS: (m/z) calcd. for C H N O S, 445.18, found: 446.23 (M
2
6
27
3
2
+
H) . Anal. Calcd for C H N O S; (%) C 70.08, H 6.11, N 9.43; found: C 70.14, H 6.23, N
2
6
27
3
2
9
.59.
.2.25. 6-(4-(4-chloro-2,5-dimethylphenylsulfonyl)piperazin-1-yl)phenanthridine (7f)
5
o
1
Appearance: white solid; yield = 87%; mp = 163-164 C; H NMR (400 MHz, CDCl ) δ 8.58
3
(d, J = 8.4 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 7.8 Hz,
1
H), 7.85 (s, 1H), 7.78 – 7.53 (m, 4H), 7.34 (s, 1H), 3.56 – 3.46 (t, 8H), 2.62 – 2.42 (s, 6H).
1
3
C NMR (100.61 MHz, CDCl ) δ 164.21, 158.73, 149.08, 142.74, 141.03, 139.22, 137.63,
3
1
1
4
6
5
37.24, 134.09, 131.11, 130.87, 129.32, 128.08, 126.43, 123.28, 122.92, 121.03, 120.25,
19.46, 51.82, 47.04, 36.42, 30.12. ESI-MS: (m/z) calcd. for C H ClN O S, 465.12, found:
2
5
24
3
2
+
66.15 (M + H) . Anal. Calcd for C H ClN O S; (%) C 64.44, H 5.19, N 9.02; found: C
2
5
24
3
2
4.58, H 5.30, N 9.14.
.2.26. 6-(4-(4-fluorophenylsulfonyl)piperazin-1-yl)phenanthridine (7g)
o
1
Appearance: pale yellow solid; yield = 74%; mp = 188-189 C; H NMR (400 MHz, CDCl )
3
δ 8.57 (d, J = 8.2 Hz, 1H), 8.42 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 8.2 Hz, 1H), 7.90 (d, J = 8.2
1
3
Hz, 1H), 7.48 – 7.29 (m, 8H), 3.64 – 3.32 (t, 8H). C NMR (100.61 MHz, CDCl ) δ 161.86,
3
1
1
60.08, 151.11, 149.23, 138.14, 137.67, 135.62, 132.16, 131.23, 128.32, 126.45, 124.82,
23.88, 122.44, 121.21, 120.08, 114.05, 52.24, 46.42. ESI-MS: (m/z) calcd. for
+
C H FN O S, 421.12, found: 422.25 (M + H) . Anal. Calcd for C H FN O S; (%) C
23
20
3
2
23 20
3
2
6
5.54, H 4.78, N 9.97; found: C 65.62, H 4.88, N 10.08.
5
.2.27. 6-(4-(4-chlorophenylsulfonyl)piperazin-1-yl)phenanthridine (7h)
o
1
Appearance: white solid; yield = 82%; mp = 196-197 C; H NMR (400 MHz, CDCl ) δ 8.59
3
(d, J = 10.8 Hz, 1H), 8.46 (dd, J = 10.8 Hz, J = 1.6 Hz 1H), 8.25 (dd, J = 11.2 Hz, J = 1.2 Hz
1
7
1
1
H), 7.96 (dd, J = 10.8 Hz, J = 1.2 Hz 1H), 7.82 – 7.48 (m, 4H), 7.28 (d, J = 6.8 Hz, 1H),
13
.24 (d, J = 7.2 Hz, 1H), 3.67 – 3.44 (t, 8H). C NMR (100.61 MHz, CDCl ) δ 163.14,
3
59.28, 150.88, 142.23, 139.86, 134.07, 132.47, 131.52, 129.71, 128.91, 127.20, 124.88,
24.25, 123.62, 122.89, 121.03, 120.21, 51.72, 46.05. ESI-MS: (m/z) calcd. for
+
C H ClN O S; 437.09, found: 438.15 (M + H) . Anal. Calcd for C H ClN O S; (%) C
23
20
3
2
23 20
3
2
6
3.08, H4.60, N 9.59; found: 63.19, H4.64, N 9.65.
5
.2.28. 6-(4-(4-bromophenylsulfonyl)piperazin-1-yl)phenanthridine (7i)
1
5

ACCEPTED MANUSCRIPT
o
1
Appearance: pale yellow solid; yield = 78%; mp = 224-225 C; H NMR (400 MHz, CDCl )
3
δ 8.64 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 7.6 Hz, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.88 (m, 1H),
7
.84 (d, J = 7.2 Hz, 1H), 7.82 – 7.51 (m, 5H), 7.22 (d, J = 7.2 Hz, 2H), 3.51 – 3.20 (t, 8H).
1
3
C NMR (100.61 MHz, CDCl ) δ 163.80, 159.12, 148.46, 144.02, 139.14, 138.07, 134.61,
3
1
4
33.72, 131.98, 131.34, 127.45, 126.82, 123.74, 122.46, 121.84, 121.03, 120.42, 51.68,
+
7.04. ESI-MS: (m/z) calcd. for C H BrN O S, 481.04, found: 482.13 (M + H) . Anal.
2
3
20
3
2
Calcd for C H BrN O S; (%) C 57.27, H 4.18, N 8.71; found: C 57.34, H 4.23, N 8.85.
2
3
20
3
2
5
.2.29. 6-(4-(4-(trifluoromethyl)phenylsulfonyl)piperazin-1-yl)phenanthridine (7j)
o
1
Appearance: pale yellow solid; yield = 74%; mp = 163-164 C; H NMR (400 MHz, CDCl )
3
δ 8.57 (d, J = 8.2 Hz, 1H), 8.44 (d, J = 7.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.98 – 7.49 (m,
1
3
9
1
1
H), 3.63 – 3.38 (t, 8H). C NMR (100.61 MHz, CDCl ) δ 161.24, 156.68, 149.12, 142.81,
3
38.37, 134.12, 131.28, 130.98, 130.12, 129.35, 128.84, 128.02, 126.87, 124.07, 123.89,
21.25, 120.81, 119.48, 52.38, 48.65. ESI-MS: (m/z) calcd. for C H F N O S; 471.12,
2
4
20
3
3
2
+
found: 472.18 (M + H) . Anal. Calcd for C H F N O S; (%) C 61.14, H 4.28, N 8.91;
2
4
20
3
3
2
found: C 61.18, H 4.31, N 8.86.
5
.2.30. 6-(4-(4-methoxyphenylsulfonyl)piperazin-1-yl)phenanthridine (7k)
o
1
Appearance: pale yellow solid; yield = 78%; mp = 208-209 C; H NMR (400 MHz, CDCl )
3
δ 8.76 (d, J = 8.4 Hz, 1H), 8.63 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.88 (m, 1H),
7
1
3
1
1
.82 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H), 7.78 – 7.74 (m, 2H), 7.69 (dd, J = 8.0 Hz, J = 0.8 Hz,
H), 7.65 (dd, J = 8.0 Hz, J = 0.8 Hz, 1H), 7.55 (m, 1H), 7.22 (d, J = 7.2 Hz, 2H), 3.88 (s,
1
3
H), 3.47 – 3.32 (t, 8H). C NMR (100.61 MHz, CDCl ) δ 164.08, 160.28, 151.42, 140.23,
3
33.86, 132.46, 131.27, 130.52, 128.74, 127.91, 126.24, 124.88, 124.21, 123.63, 122.87,
20.03, 118.77, 51.78, 46.53. ESI-MS: (m/z) calcd. for C H N O S; 433.14, found: 434.23
2
4
23
3
3
+
(
M + H) . Anal. Calcd for C H N O S; (%) C 66.49, H 5.35, N 9.69; found: C 66.54, H
24 23 3 3
5
5
.42, N 9.77.
.2.31. 6-(4-(4-nitrophenylsulfonyl)piperazin-1-yl)phenanthridine (7l)
o
1
Appearance: pale yellow solid; yield = 81%; mp = 198-199 C; H NMR (400 MHz, CDCl )
3
δ 8.57 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 7.8 Hz, 3H), 8.06 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4
1
3
Hz, 2H), 7.91 (dd, J = 8.0 Hz, J = 0.8 Hz, 1H), 7.79 – 7.48 (m, 4H), 3.64 – 3.38 (t, 8H). C
NMR (100.61 MHz, CDCl ) δ 161.25, 154.71, 146.36, 137.11, 132.87, 132.23, 131.08,
3
1
4
28.52, 127.79, 126.82, 126.18, 125.94, 125.14, 123.65, 121.86, 121.02, 120.48, 50.79,
+
7.04. ESI-MS: (m/z) calcd. for C H N O S; 448.12, found: 449.22 (M + H) . Anal. Calcd
23
20
4
4
for C H N O S; (%) C 61.59, H 4.49, N 12.49; found: C 61.64, H 4.61, N 12.54.
2
3
20
4
4
1
6

ACCEPTED MANUSCRIPT
5
.2.32. 1-(4-(4-(phenanthridin-6-yl)piperazin-1-ylsulfonyl)phenyl)ethanone (7m)
o
1
Appearance: off white solid; yield = 76%; mp = 204-205 C; H NMR (400 MHz, CDCl ) δ
3
8
.60 (d, J = 8.8 Hz, 1H), 8.46 (d, J = 8.0 Hz, 2H), 8.40 (d, J = 8.2 Hz, 1H), 8.12 (d, J = 8.4
Hz, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.88 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 7.81 – 7.44 (m, 4H),
1
3
3
.64 – 3.38 (t, 8H), 2.72 (s, 3H). C NMR (100.61 MHz, CDCl ) δ 171.47, 160.68, 152.23,
3
1
45.22, 138.22, 132.46, 132.67, 130.97, 129.42, 128.84, 127.46, 126.17, 124.24, 124.08,
1
23.68, 121.22, 120.88, 120.56, 50.64, 46.03, 29.44. ESI-MS: (m/z) calcd. for C H N O S;
2
5
23
3
3
+
4
45.14, found: 446.25 (M + H) . Anal. Calcd for C H N O S; (%) C 67.40, H 5.20, N 9.43;
25 23 3 3
found: C 67.49, H 5.24, N 9.47.
5
.2.33. 6-(4-(5-bromothiophen-2-ylsulfonyl)piperazin-1-yl)phenanthridine (7n)
o
1
Appearance: pale yellow solid; yield = 80%; mp = 162-163 C; H NMR (400 MHz, CDCl )
3
δ 8.58 (d, J = 8.4 Hz, 1H), 8.45 (d, J = 7.8 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 8.93 (d, J = 8.0
Hz, 1H), 7.81 – 7.50 (m, 4H), 7.38 (d, J = 7.2 Hz, 1H), 7.16 (d, J = 7.4 Hz, 1H), 3.66 – 3.40
1
3
(
t, 8H). C NMR (100.61 MHz, CDCl ) δ 162.24, 151.47, 144.78, 139.41, 134.89, 133.01,
3
1
5
31.22, 128.41, 126.84, 126.12, 125.08, 124.86, 123.25, 123.11, 122.47, 120.78, 119.23,
+
0.48, 46.14. ESI-MS: (m/z) calcd. For C H BrN O S ; 487.00, found: 487.13 (M + H) .
2
1
18
3
2 2
Anal. Calcd for C H BrN O S ; (%) C 51.64, H 3.71, N 8.60; found: C 51.78, H 3.75, N
2
1
18
3
2 2
8
.69.
.3. Microplate Alamar Blue Assay (MABA)
5
The anti-mycobacterial activities of title compounds 6a-s and 7a-n were evaluated against
MTB H37Rv (ATCC 27294) strain using MABA [27,28]. 200µL of sterile deionized water
was added to all outer-perimeter wells of sterile 96-well plates to minimize evaporation of the
medium in the test wells during incubation. The wells in rows B to G in columns 3 to 11
received 100ꢀl of 7H9GC broth. 100µL of 2× drug solutions were added to the wells in rows
B to G in columns 2 and 3. By using a multichannel pipette, 100ꢀl was transferred from
column 3 to column 4, and the contents of the wells were mixed well. Identical serial 1:2
dilutions were continued through column 10, and 100ꢀl of excess medium was discarded
from the wells in column 10.
1
1
00µL of MTB inoculum was added to the wells in rows B to G in columns 2 to 10. Add
00µL of medium to B11 and C11 (media control), 100µL of MTB inoculum to D11 and E11
and 100µL of MTB inoculum with 3-5% DMSO to F11 and G11 (solvent control).
The plates were sealed with parafilm and were incubated at 37°C for 5 days. 50µL of a
freshly prepared 1:1 mixture of 10× Alamar Blue (Accumed International, Westlake, Ohio)
1
7

ACCEPTED MANUSCRIPT
reagent and 10% Tween 80 was added to well D11. The plates were reincubated at 37°C for
2
4 h. If well D11 turned pink, the reagent mixture was added to all wells in the microplate (if
the well remained blue, the reagent mixture would be added to another control well and the
result would be read on the following day). The microplates were resealed with parafilm and
were incubated for an additional 24 h at 37°C, and the colors of all wells were recorded. A
blue color in the well was interpreted as no growth, and a pink color was scored as growth. A
few wells appeared violet after 24 h of incubation, but they invariably changed to pink after
another day of incubation and thus were scored as growth (while the adjacent blue wells
remained blue). The MIC was defined as the lowest drug concentration which prevented a
color change from blue to pink.
5
.4. Molecular modelling studies:
The 2D structures of synthesized compounds (6a-s and 7a-n) were sketched and converted to
D using Ligprep, module of Schrodinger suite package 2013. ATPase domain of
3
Mycobacterium tuberculosis GyrB protein was selected and prepared for docking by protein
preparation wizard which was a part of the Maestro software [29]. Bond orders and formal
charges were added for hetero groups, and hydrogens were added to all atoms in the system.
Water molecules within 5 Å distance were removed. For each structure, a brief relaxation was
performed using an all-atom constrained minimization carried out with the Impact
Refinement module (Impref). (Impact v5.8, Schro¨dinger, LLC, New York, NY) using the
OPLS-2005 force field to alleviate steric clashes that may exist in the original PDB structure.
The minimization was terminated when the energy converged or the RMSD reached a
maximum cut off of 0.30 Å. The binding site was defined by a rectangular box surrounding
the ANP and coordinates of grid box were X = -21.987185, Y=26.604429, Z=-50.838890.
Prepared ligands were docked into the ANP binding pocket of MTB GyrB protein with Glide
5
.0 (Glide v6.0 Schrodinger, LLC, New York, NY) [30]. Glide energy grids were generated
for each of the prepared complexes. Glide XP (Extra Precision) docking with ligand
flexibility was performed. A total of ten ligand conformations were allowed and finally top
score conformation was selected as active conformation. Molecules were analyzed based on
docking score, interacting amino acids and hydrogen bonds.
5
.5. Cytotoxicity assay
Most active anti-TB compounds (MIC ≤ 12.5 µg/mL) were further examined for toxicity in
RAW 264.7 cell lines at the concentration of 50 µg/mL. After 72 h of exposure, viability was
1
8

ACCEPTED MANUSCRIPT
assessed on the basis of cellular conversion of MTT into a formazan product using the
Promega Cell Titer 96 non-radioactive cell proliferation assay [31].
Acknowledgements
The
financial
assistance
provided
by
Department
of
Biotechnology,
(No.BT/PR4801/MED/29/370/2012), New Delhi, India is gratefully acknowledged.
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Halgren, P.C. Sanschagrin, D.T. Mainz, J. Med Chem. 49 (2006) 6177–6196.
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Chem. 64 (2013) 239–251.
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26]. G.M. Sheldrick. SHELX97 Program for Crystallography Refinement; University of
Go¨ttingen: Germany, (1997).
[
[
27]. L.A. Collins, S.G. Franzblau, Antimicrob. Agents Chemother. 41 (1997) 1004-1009.
28]. S.G. Franzblau, R.S. Witzig, J.C. McLaughlin, P. Torres, G. Madico, A. Hernandez,
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Microb. 36 (1998) 362–366.
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29]. Maestro, version 9.5, Schrödinger, LLC, New York, NY, 2013.
2
0

ACCEPTED MANUSCRIPT
[
[
30]. Schrödinger Suite 2013 QM-Polarized Ligand Docking protocol; Glide version 6.0,
Schrödinger, LLC, New York, NY, 2013; Jaguar version 8.1, Schrödinger, LLC, New
York, NY, 2013; QSite version 6.0, Schrödinger, LLC, New York, NY, 2013.
31]. D. Gerlier, N. Thomasset, Immunol. Methods. 94 (1986) 57–63.
2
1

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Figure 1: Design strategy to achieve title compounds
Figure 2: Interaction profile of ANP with ATPase domain of Mycobacterium tuberculosis
GyrB protein. The hydrophobic pocked is highlighted with the residues interacting with the
crystal ligand
Figure 3: Interaction profile of compound 6b with ATPase domain of Mycobacterium
tuberculosis GyrB protein. The various hydrophobic interactions of compound 6b are
noticeable.
Figure 4: Interaction profile of compound 7d with ATPase domain of Mycobacterium
tuberculosis GyrB protein. The different polar contacts, π-π interaction and the hydrophobic
interactions with compound 7d are noticeable
Figure 5: ORTEP plot for the compound 6b and 7d. All the non-hydrogen atoms are
presented by their 50% probability thermal ellipsoids
Scheme 1: General synthetic procedure for title compounds
a
Table 1: Optimisation of reaction conditions of 6b
Table 2: Antimycobacterial activities of compound 6a-s and 7a-n against MTB H37Rv
Table 3: IC (µg/mL) and SI values of active compounds
5
0
Table 4: Crystal data and structure refinement details for 6b and 7d
2
2

ACCEPTED MANUSCRIPT
a
Table 1: Optimisation of reaction conditions of 6b
o
Yield
Entry
Solvent
Base
Reagent
Temperature ( C)
Time
b
(%)
1
2
3
4
5
6
7
8
9
DCM
DMF
DMF
DMF
DMF
DCM
DCM
DCM
DCM
DCM
DCM
TEA
TEA
EDC.HCl, HOBt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
rt
8 h
8 h
8 h
8 h
8 h
4 h
2 h
4 h
6 h
8 h
12 h
58
EDC.HCl, HOBt
52
41
42
38
54
56
68
84
85
84
DIPEA
DIPEA
DIPEA
Pyridine
TEA
HATU
BOP
PyBOP
T3P
T3P
TEA
T3P
TEA
T3P
1
0
TEA
T3P
1
1
TEA
T3P
a
All the reactions were carried out with 5 (1.0 equiv.), base (2.0 equiv.) and reagent (1.0-1.6 equiv.) in solvent,
Isolated yield after column chromatography.
b

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Table 2: Antimycobacterial activities of compounds 6a-s and 7a-n against MTB H37Rv
MIC(µg/mL)
S.No.
Entry
R / R
against MTB
H Rv strain
1
2
37
1
2
6
a
25
6
b
3.13
12.5
6
c
3
4
6d
50
5
6
6
e
3.13
6
f
25
7
6
g
>50
50
6
h
8
9
6
i
>50
6
j
12.5
3.13
6.25
1.56
1
0
6
k
1
1
1
2
6
l
6
m
1
1
3
4
6
n
3.13

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1
5
6o
25
50
6
6
p
q
1
1
6
7
25
1
1
8
9
6
r
s
6.25
1.56
6
2
2
2
2
2
0
1
2
3
4
7a
7b
7c
7d
25
6.25
>50
1.56
7
e
12.5
6.25
2
5
7
f
2
2
2
6
7
8
7g
7h
7i
6.25
3.13
3.13
25
2
9
7j

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3
0
7k
6.25
50
7
l
3
3
1
2
7m
7n
25
3
3
3.13
-
Isoniazid
Rifampicin
Pyrazinamide
-
-
-
0.1
0. 2
6.25
-
-

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Table 3: IC (µg/mL) and SI values of active compounds
5
0
a
MIC (µg/mL) in % cell inhibition
IC50
approximation
112.81
94.33
SI
Entry
MTB H37Rv
3.13
at 50 µg/mL
22.16
26.50
34.64
22.12
42.16
19.16
27.90
26.52
34.64
34.12
30.72
28.92
26.52
34.64
32.76
20.92
28.16
26.52
42.16
values
36.15
7.54
6
b
6
6
c
e
12.5
3.13
72.17
23.13
9.04
6
j
12.5
113.01
59.29
6
k
3.13
19.00
20.87
57.43
30.21
11.54
46.96
13.02
55.41
7.54
6
l
6.25
130.48
89.60
6
m
1.56
6
n
3.13
94.26
6
r
6.25
72.17
6
s
1.56
73.27
7
b
d
6.25
81.38
7
1.56
86.44
7
e
12.5
94.26
7
f
6.25
72.17
11.54
12.21
38.30
28.45
15.08
38.01
7
g
6.25
76.31
7
h
3.13
119.50
88.77
7
i
3.13
7
7
k
n
6.25
94.26
3.13
59.29
a
Selectivity index

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Table 4: Crystal data and structure refinement details for 6b and 7d
Compound
Formula
6b
7d
C H N O
C H N O S
24
21
3
27 29
459.60
293
3
2
Sum formula weight
Temperature/K
Crystal system
Space group
367.44
293
Triclinic
Monoclinic
P2 /n
1
Pī
a (Å)
9.4717 (13)
12.5058(13)
b (Å)
9.6936 (14)
14.6774 (17)
13.3524 (14)
90, 89.451 (9), 90
c (Å)
11.8432 (16)
73.767 (12), 74.207
Angle α, β, γ (°)
(
12), 65.560 (14)
3
Volume (Å )
935.1 (2)
2
2450.8(5)
4
Z
F(000)
Dcalc (g/mm )
µ (mm )
388.0
1.305
0.081
976.0
1.246
0.16
3
-1
Absorption correction:
Radiation wavelength (Å)
Multi-scan
0.71073
Multi-scan
0.71073
MoKα
MoKα
Radiation type
Radiation monochromator
Graphite
Graphite
(h, k, l)max, (h, k, l)min
(12, 13, 14), (−12, −13, (17, 20, 15), (−9, −16,
−14)
−18)
2
Θ range for data collection
6.6 − 58.2°
6.4 − 58.4°
0.922, 1.000
7784, 6663, 3305
Tmin, Tmax
0.246, 1.000
6305, 5019, 2213
No. of measured, independent
and observed [I > 2σ(I)]
reflections
Rint
0.038
0.684
0.6 × 0.5 × 0.4
0.018
0.687
0.6 × 0.5 × 0.4
−
1
(sin θ/λ)max (Å )
3
Crystal size/mm
2
2
2
R[F > 2σ(F )], wR(F ), S
0.0757 ( 2213), 0.2206 0.050 (3305), 0.121
(
4075), 0.95
(5149), 1.02
298
No. of parameters
253
−
3
ꢀρmax, ꢀρmin (e Å )
0.27, −0.28
0.14, −0.25
Structure refinement
SHELXL97 (Sheldrick, 2008)