Reversed isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2017.12.047

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a–c, 7 and 8; H37Rv-MIC₉₉ ≤1.25 μM, R5401-MIC₉₉ ≤2.5 μM, X_61-MIC₉₉ ≤5 μM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a–c and 3b (MIC₉₀ ≤13.42 μM) and inhibition of EB efflux demonstrated by these compounds are encouraging.

Full text of DOI:10.1016/j.bmc.2017.12.047

Accepted Manuscript
Reversed Isoniazids: Design, synthesis and evaluation against Mycobacterium
tuberculosis
Malkeet Kumar, Kawaljit Singh, Andile H. Ngwane, Fahreta Hamzabegovic,
Getahun Abate, Bienyameen Baker, Ian Wiid, Daniel F. Hoft, Peter Ruminski,
Kelly Chibale
PII:
S0968-0896(17)31809-6
https://doi.org/10.1016/j.bmc.2017.12.047
BMC 14152
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 September 2017
20 December 2017
28 December 2017
Please cite this article as: Kumar, M., Singh, K., Ngwane, A.H., Hamzabegovic, F., Abate, G., Baker, B., Wiid, I.,
Hoft, D.F., Ruminski, P., Chibale, K., Reversed Isoniazids: Design, synthesis and evaluation against Mycobacterium
tuberculosis, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.12.047
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Reversed Isoniazids: Design, synthesis and evaluation against
Mycobacterium tuberculosis
Malkeet Kumara, Kawaljit Singha, Andile H. Ngwaneb, Fahreta Hamzabegovicc, Getahun
Abate,c, Bienyameen Bakerb, Ian Wiidb, Daniel F. Hoftc,d, Peter Ruminski,f, Kelly Chibalea,e,g*
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDST-NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for
TB Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and
Health Sciences, Stellenbosch University, P.O. Box 241, Cape Town, 8000, South Africa.
cDepartment of Internal Medicine, Division of Infectious Diseases, Allergy and Immunology,
Saint Louis University, 1100 S. Grand Blvd, 63104, MO, USA
dDepartment of Molecular Biology, Saint Louis University, 1100 S. Grand Blvd, 63104, MO,
USA
eInstitute of Infectious Disease and Molecular Medicine, University of Cape Town,
Rondebosch 7701, South Africa.
fCentre for World Health and Medicine, Saint Louis University, 1100 S. Grand Blvd, 63104,
MO, USA
gSouth African Medical Research Council Drug Discovery and Development Research Unit,
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
Abstract:
Novel reversed isoniazid (RINH) agents have been synthesized by covalently linking
isoniazid with various efflux pump inhibitors (EPIs) cores and their structural motifs. These
RINH agents were evaluated for anti-mycobacterial activity against sensitive, isoniazid
mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of
compounds were also tested ex vivo for intracellular activity as well as in the ethidium
1

bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds
against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤ 1.25 µM, R5401-
MIC99 ≤ 2.5 µM, X_61- MIC99 ≤ 5 µM) demonstrated the viability of reversed anti-TB agent
strategy towards the development of novel anti-mycobacterial agents to address the rapidly
growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and
3b (MIC90 ≤ 13.42 µM) and inhibition of EB efflux demonstrated by these compounds are
encouraging.
KEYWORDS: efflux pumps, chemosensitizers, macrophages, efflux pump inhibitors,
M. tuberculosis, ethidium bromide.
1. Introduction
Tuberculosis (TB) caused by Mycobacterium tuberculosis (M. tuberculosis) is one of the
most fatal infectious diseases. It is more prevalent in poor and under developed countries,
especially in Africa and the South East Asian Region, and is responsible for 1.5 million
deaths annually.1 The rapid and widespread emergence of multi-drug resistant (MDR) and
extremely-drug resistant (XDR) strains have hampered TB control and its eradication.2,3 The
emergence of resistance against first line anti-TB drugs (isoniazid, rifampicin, pyrazinamide
and ethambutol) has complicated treatment both financially as well as socially.4 Therefore,
urgent efforts are required to counter this challenge.
Efflux pumps (EPs) are membrane bound active transporters, which decrease the antibacterial
activity of unrelated drug families by reducing the cytoplasmic concentration of the drugs to
sub-inhibitory levels. The survival of M. tuberculosis in the presence of such sub-inhibitory
drug concentrations is facilitated and results in the development of resistance.5,6 A number of
measures to counter EP-mediated resistance have been adopted in the past including (i)
engineering of the native drug molecule to by-pass EPs,7 and (ii) cocktail use of drugs and
efflux pump inhibitors (EPIs).8 These approaches have shown promising results and
prospects for the development of anti-TB drugs. Recently reported semisynthetic analogues
of spectinomycin demonstrated potent anti-tubercular properties by evading EPs.9 A number
of efflux pump genes (mmpL7, p55, efpA, mmr, Rv1258c and Rv2459) are responsible for low
to high levels of isoniazid (INH) resistance in M. tuberculosis.10 The over expression of these
genes is known to be countered by EPIs leading to increased susceptibility of MDR strains
towards INH.11 However, the combination of anti-TB drugs with EPIs as adjunctive agents
presents additional challenges from a pharmacological perspective, which hinder the further
2

development of this combination strategy in clinical phases. Therefore, covalently linking an
EPI or chemosensitizer moiety to INH to generate hybrid compounds, herein referred to as
reversed INH agents, presents good prospective to overcome this liabilities and may reverse
EP-mediated resistance and revitalize its anti-mycobacterial activity against M(/X)DR-TB.
Reversed hybrid compounds as antibacterial agents have already been reported.12 In this
regard, the dual action antibacterial agent SS14, developed by covalently linking the
antibacterial agent berberine (a substrate of NorA-EP) and an EPI, INF55 (5-nitro-2-
phenylindole) showed enhanced accumulation of berberine in bacteria leading to superior
potency as compared to a cocktail of these two agents (Figure 1). 12,13
On the other hand, the concept of resistance reversal by reversed agents in malaria has been
explored by Peyton and co-workers by developing reversed chloroquine (RCQ) agents
through covalently linking chloroquine (CQ) and its analogues with various chemosensitizer
cores.14,15 When evaluated against CQ resistant Plasmodium falciparum strains, the RCQ
agents showed resistance reversal.16 In an analogous fashion, the concept of reversed anti-TB
agents (RATAs) can also be explored in TB drug discovery (Figure 1) using various anti-TB
drugs and chemosensitizers. This may present a viable strategy to mitigate the emergence of
drug tolerance and/or resistance mediated by EPs.
3

Figure 1: The concept of RCQs, SS14 and design of RINH agents.
In this paper, we report the design, synthesis and biological evaluation of reversed isoniazid
(RINH) agents as novel anti-mycobacterial agents. These RINH agents were designed by
covalently linking INH with various EPIs or chemosensitizer moieties or their structural
motifs derived from phenothiazines (1a,b, 2a,b and 3a,b), iminodibenzyl (1c), thioxanthene
(4a,b), cycloheptadiene (4c), dibenzosuberyl (5), diphenylmethane (6 and 7) and
triphenylmethane (8) via an alkyl chain linker (Figure 2).17,18 A propyl linker was chosen for
initial studies towards establishing a proof-of-concept based on its promise in RCQs. 14,15
.
4

Figure 2: Designed RINH agents by covalently linking INH with various EPIs/chemosensitizers or
their structural motifs.
2. Chemistry:
Schemes 1-3 summarize the synthesis of RINH agents 1-8. The synthesis of compounds 1a-c
and 2a,b commenced with the reaction of phenothiazines 9a,b and iminodibenzyl 9c with 1-
bromo-3-chloropropane using NaH in DMF and NaNH2 in toluene respectively, to obtain the
corresponding intermediates 10a-c, which upon reaction with INH and benzhydrazide
afforded target compounds 1a-e. To accomplish the synthesis of RINH agents 2a,b,
intermediates 10a,b were reacted with 3-amino propanol using K2CO3 in DMF to afford the
hydroxy intermediate 11a,b, which upon reaction with Boc anhydride furnished intermediate
12a,b. The Appel reaction on compound 12a,b furnished the key intermediate 13a,b, which
was reacted with INH in DMF using Et3N at 55 ºC to obtain compound 14a,b in low yield
due to over alkylation at the hydrazine NH of the target compounds. Thereafter, N-Boc
deprotection of compound 14a,b provided the target compounds 2a,b.
5

Scheme 1: Synthesis of RINH agents 1a-e and 2a,b.
Reagents and reaction conditions: (i) 1-bromo-3-chloropropane (1.2 eq.), NaH (1.2 eq.), DMF, 0-25
°C, 11-12 h (10a and 10b), 63-75%; 1-bromo-3-chloropropane (2 eq.), NaNH2 (2.5 eq.), toluene,
reflux (115 °C), 14 h, (10c), 40%; (ii) 3-aminopropanol (1.5 eq.), K2CO3 (1.3 eq.), DMF, 80 °C, 10-12
h; 65-68%; (iii) (Boc)2O (1.3 eq.), Et3N (1.2 eq.), DCM, 0-25 °C, 30-40 min, 93-95%; (iv) Ph3P (1.5
eq.), CBr4 (1.5 eq.), DCM, 25 °C, 1.5-2 h, 82-88%; (v) isoniazid (5 eq.), Et3N (3 eq.), DMF, 25-55 °C,
10-12 h, 30-35%; (vi) TFA/DCM (10% v/v), 25 °C, 1-1.5 h, 66-83% (2a,b); (vii) isoniazid (1.2 eq.),
NaH (1.2 eq.), DMF, 0-25 °C, 5-6 h, (1a,b), 18-24%; isoniazid (4 eq.), Et3N (4 eq.), iso-propanol,
reflux (90 °C), 12-24 h, (1c), 15%; benzhydrazide (4 eq.), Et3N (4 eq.), iso-propanol, reflux (90 °C),
12
h,
(1d
and
1e),
20-25%.
6

Scheme 2: Synthesis of compounds 19a,b, 23a-c, 25, 27, 31 and 33.
Reagents and reaction conditions: (i) (Boc)2O (1.3 eq.), Et3N (1.3 eq.), DCM, 0-25 °C, 1.5 h, 90%;
(ii) methanesulfonyl chloride (1.2 eq.), Et3N (1.2 eq.), DCM, 0-25 °C, 1 h, 97%; (iii) NaH (1.1 eq.),
DMSO, 25-70-100 °C, 18-20 h, 30-32%; (iv) TFA (10% v/v in DCM), 1N-NaOH, 1-1.5 h, 87-95%;
(v) (Boc)2O (1.3 eq.), Et3N (1.5 eq.), DMAP (0.01 eq.), DCM, 0-25 °C, 20 h, 95%; (vi) Zn (4.5 eq.),
TiCl4 (1M in toluene) (2 eq.), 1,4-dioxane, 60 °C-reflux, 10-12 h, 40-55%; (vii) piperazine (1.1 eq.),
DBU (0.25 eq.), MS-4 A°, 0-25 °C, toluene, 10 h, 67%; (viii) piperazine (2 eq.), K2CO3 (1 eq.), NaI
(0.2 eq.), acetonitrile, reflux (90 °C), 8 h, 65%; (ix) NBS (1.1 eq.), H2O Acetone (1:5), 25 °C, 2 h,
7

90%; (x) 1-Boc-piperazine (1.1 eq.), DMSO, Cs2CO3 (2 eq.), 60 °C, 8 h, 40%; (xi) TFA/DCM (10%
v/v), DCM, 25 °C, 1 h, 97%; (xii) piperazine (10 eq.), DCM, 0-25 °C, 1 h, 73.5%.
Scheme 2 outlines the synthesis of various EPI moieties based on phenothiazines (19a,b and
23a,b), dibenzosuberenone 23c, dibenzosuberyl 25 and diphenylmethane 27 , using reported
methods.19 The synthesis of analogue 31 commenced with the treatment of commercially
available diphenylethene 28 with N-bromosuccinimide (NBS) in a mixture of acetone and
water (5:1) to afford intermediate 29 (Scheme 2).20 This intermediate was further reacted with
1-Boc-piperazine in DMSO in the presence of Cs2CO3 to afford 30, which, upon
N-Boc-deprotection, yielded compound 31.The triphenylpiperazine derivative 33 was
obtained by reacting trityl bromide 32 with piperazine in DCM.
The piperazine and piperidine containing EPI moieties (19a,b, 23a-c, 25, 27, 31 and 33) were
further reacted with 1-bromo-3-chloropropane to generate key intermediates 34a-i (Scheme
3), which upon reaction with INH and Et3N in iso-propanol provided target compounds 3-8.
Scheme 3: Synthesis of RINH agents 3-8
Reagents and reaction conditions: (i) Et3N (2.5 eq.), toluene, 65 °C, 8-12 h, 60-90%; (ii)
isoniazid (4eq.), iso-propanol, Et3N (4 eq.), 95 °C, 12-16 h, 30-45%.
3. Results and Discussions:
A total of 14 RINH agents were synthesized along with two benzhydrazide-based compounds
to explore the role of the pyridine nitrogen in RINHs. Primarily, anti-mycobacterial activity
of all the compounds was evaluated against the drug sensitive strain (H37Rv) of M.
tuberculosis (Table 1) using the BACTEC 960 system. The compounds with good potency
against H37Rv (MIC99 ≤ 5 µM) were further evaluated against various low to high level
8

isoniazid mono-resistant strains (R73, R4965 and R5401; experimental section, Table 3) and
four MDR clinical isolates (TT149, X_3, X_60 and X_61; experimental section, Table 3) of
M. tuberculosis to investigate reversal of resistance. The promising compounds with potency
against H37Rv and R5401 strains (MIC99 ≤ 5 µM), were also tested against Chinese Hamster
Ovary (CHO) cell lines to determine their cytotoxicity profile. INH was used as a positive
control for all the in vitro evaluations. A selected number of compounds were further tested
for intracellular bacillus Calmette-Guerin (BCG) bacilli (an attenuated Mycobacterium bovis
vaccine strain) inhibition in macrophages and for EP inhibitory activity in the ethidium
bromide (EB) assay.
3.1. In vitro activity against Mycobacterium tuberculosis
All the RINH agents were found to be active against the H37Rv strain of M. tuberculosis
(MIC99 ≤ 5 µM), compounds 1a-c, 3b, 4a-c, 7 and 8 (MIC99 ≤ 1.25 µM) showed activity
comparable to INH (MIC99 = 0.73 µM) (Table 1). Compounds 1d and 1e, which are
analogues of 1a and 1b, respectively, did not display activity up to the highest tested
concentration (10 µM), demonstrating the importance of the pyridyl nitrogen for
anti-mycobacterial activity. All the compounds with MIC99 ≤ 5 µM were further tested
against clinical isolates of M. tuberculosis with low (R5401; 0.73 µM ≤ INH-MIC99 ≤ 2.93
µM) to high (R73 and R4965; INH-MIC99 ≥ 2.43 µM) levels of INH mono-resistance.
All compounds were found to be inactive up to the highest tested concentration (10 µM)
against high level INH-resistant strains R73 (INH-MIC99 > 10 µM; mutation in inhA promoter
and katG gene; Table 3) and R4965 (INH-MIC99 > 10 µM; mutation in katG gene). However,
compounds 3a,b, 4a-c, and 6-8 demonstrated activity against a low level INH mono-resistant
strain R5401 (MIC99 ≤ 2.5 µM; mutation in inhA promoter) (Table 1). Compounds 3a and 6
displayed 2-fold superior anti-mycobacterial activity against the clinical isolate R5401 as
compared to the H37Rv strain. However, compound 3b displayed analogous activity against
both strains (H37Rv and R5401). On the other hand, compounds 4a-c, 7 and 8 demonstrated
a two-fold reduction in potency against the R5401 strain as compared to H37Rv strain of
M. tuberculosis. These results revealed that chemosensitizers when covalently linked to INH
are able to circumvent low level INH resistance but failed to do so in strains with high levels
of resistance. This is in line with the known fact that EPs contribute toward low level INH
resistance but are dominated by mutations in strains with high levels of resistance.10
9

Table 1: Anti-mycobacterial activity, cytotoxicity and selectivity index of RINH agents.
MIC99 (µM)X
H37Rv R5401 X_61 X_60
IC50 (µM)
SIR5401
Compounds
INH
R
CHO
Cell lines
0.73
2.5
2.5
> 10
aND
>100
aND
>35
aND
aND
1a
1.0
> 10
1b
1c
1d
1e
0.25
1.0
> 10
> 10
bND
bND
aND
aND
bND
bND
aND
aND
bND
bND
aND
aND
bND
bND
aND
aND
bND
bND
> 10
> 10
5.0
5.0
>10
aND
aND
aND
aND
2a
2b
> 10
aND
aND
aND
aND
10

MIC99 (µM)X
H37Rv R5401 X_61 X_60
IC50 (µM)
SIR5401
48.8
37
Compounds
R
CHO
Cell lines
5.0
1.0
2.5
1.0
2.5
2.5
> 10
122
37.4
152
3a
> 10 > 10
3b
4a
1.25
2.5
> 10
60.8
4b
4c
5
0.625
1.25
5.0
1.25
2.5
2.5
5.0
> 10
> 10
aND
296
148
aND
236.8
59.2
aND
> 10
aND
6
7
5.0
2.5
2.5
5
5
> 10
> 10
48.1
174
19.3
139.2
11
1.25

MIC99 (µM)X
H37Rv R5401 X_61 X_60
IC50 (µM)
SIR5401
Compounds
R
CHO
Cell lines
1.25
2.5
2.5
2.5
97.6
78.1
8
MIC99 (µM)X: No significant activity was observed against R73, R4965 (MIC99 > 10 µM) , TT149
and X-3 clinical isolates up to the highest tested concentration (10 µM) of compounds; aND: Not
b
determined as MIC99 > 10 µM against R5401 strain of M. tuberculosis; ND: Not determined as
MIC99 > 10 µM against H37Rv strain of M. tuberculosis; SIR504: Selectivity index of compounds
with respect toMIC99 of M. tuberculosis strain R5401 and IC50 against CHO cell lines.
Compounds 3a-b, 4a-c, 6, 7 and 8 with good potency against R5401 strain (MIC99 ≤ 2.5 µM)
were further evaluated for cytotoxicity against the CHO cell line and were found to have low
cytotoxicity (IC50 > 10 µM) with good selective index values (SI > 10) with respect to the
R5401 strain. These RINH agents were further evaluated against four MDR clinical isolates
TT149 (INH-MIC99 > 10 µM), X_3 (INH-MIC99 > 10 µM), X_61 (INH-MIC99 < 5 µM) and
X_60 (INH-MIC99 > 10 µM). All the compounds were found to be inactive against TT149
and X_3 strains up to the highest tested concentration (10 µM). However, some potency was
displayed by compounds 3a, 4a-c, 6, 7 and 8 against X_61 (MIC99 ≤ 5 µM), and analogue 8
was also potent against X_60 strains (MIC99 = 2.5 µM). The activity of most of these
compounds against the X_61 strain (MIC99 ≤ 5 µM) is comparable to their potency against
the R5401 strain (MIC99 ≤ 2.5 µM). R5401 and X_61 harbours low level INH resistance
(0.73 ≤ MIC99 ≤ 2.93 µM) against INH, therefore, it was unsurprising that compounds 3a, 4a
and 8 showed analogous activity against both strains (MIC99 = 2.5 µM), while two-fold lower
potency was exhibited by compounds 4b-c, 6 and 7 (MIC99 = 5 µM) against X_61 as
compared to the R5401 (MIC99 = 2.5 µM) strain.
Only one RINH agent, 8 showed potency (MIC99 = 2.5 µM) against the X_60 clinical isolate
with high levels of INH resistance (MIC99 > 2.93 µM). This is an encouraging result and may
lay a foundation for the development of RINH agents to counter both low as well as high
levels of INH resistance.
12

In general, phenothiazine- (1a,b, 2a,b and 3a,b), thioxanthene- (4a,b), tricyclic- (1c),
dibenzosuberyl- (5), diphenylmethane (6 and 7) and triphenylmethane- (8) based EPI
compounds showed good to moderate potency against the H37Rv strain of M. tuberculosis
(MIC99 ≤ 5 µM). Chloro-substituted phenothiazine (1b and 3b) and thioxanthene (4b) based
compounds showed superior activity as compared to their unsubstituted analogues (1a, 3a
and 4a). The introduction of piperidine (3a,b and 4a-c) and piperazine (6, 7 and 8) moieties
in EPI structural motifs enhanced activity against drug sensitive (H37Rv), INH mono
resistant (R5401) and MDR clinical isolates (X_61 and X_60) of M. tuberculosis. The
nitrogen atom in the pyridyl moiety of RINH agents was found to be necessary for anti-
mycobacterial activity as a drastic loss of anti-mycobacterial potency was observed with the
replacement of the INH moiety with a benzhydrazide group (1a-b vs 1d-e) (Table 1).
3.2. Ex vivo cytotoxicity and anti-mycobacterial evaluation of RINH agents
It is well known that M. tuberculosis induces a number of EPs during and post macrophage
infection, which assist in the development of EP-mediated tolerance. This tolerance leads to
the emergence of various forms of high to low level resistance both in active as well as in
persistent (non-replicating) forms of M. tuberculosis.21,22 The synthesized RINHs were
hypothesized to reverse macrophage induced tolerance due to the attached EPI or
chemosensitizer moiety or structural motif. Therefore, a subset of compounds (1a-e and 3a)
was evaluated against intracellular BCG in macrophages for proof-of-concept studies.
Amongst these selected compounds, 3b was chosen for the demonstration of retention of in
vitro potency in macrophages while 1a, 1b, 1c, 1d and 1e were selected to explore the
significance of the pyridine nitrogen and sulphur atoms in the anti-mycobacterial activity
both in vitro and in macrophages. All compounds were first evaluated for cytotoxicity against
THP-1 cell lines as THP-1 monocytes were used for macrophage development.
Chlorpromazine (CPZ), a known chemosensitizer and EPI,17,23 and INH were used as positive
controls in these experiments. A low cytotoxicity cut-off IC20 (inhibitory concentration of
20% of THP-1 cell lines) was used to achieve higher selectivity against intracellular BCG
with a minimum effect on the viability of THP-1 macrophages. Therefore, compounds were
evaluated against intracellular BCG at a concentration ≤ IC20.
INH and compounds 1a, 1c, 1d and 1e demonstrated a low cytotoxicity profile with
IC20 > 100 µM, while compounds 1b, 3b and CPZ demonstrated IC20 values of 24.4, 2.2 and
13

4 µM, respectively (Table 2). It appears that piperidine-based compound 3b is more toxic to
THP-1 cells compared to other compounds (Table 2). However, because no other piperidine-
compounds were tested on THP-1 cells, it is unclear whether or not the piperidinyl moiety is
responsible or for the observed THP-1 cytotoxicity of 3b. The results summarized in Table 1
showed that compounds 3b and 6 were more toxic on CHO cell lines than compounds 4a, 4b,
7 and 8 despite all incorporating a piperidinyl moiety, suggesting that this moiety may not be
the reason for cytotoxicity. INH and CPZ displayed 90% and 40% inhibition of intracellular
BCG at a minimum inhibitory concentration (MIC) value of 0.015 and 4 µM (Table 2),
respectively. All the compounds significantly inhibited (> 90%) the growth of intracellular
BCG in macrophages (Table 2) at a concentration ≤ IC20 except 1d and 1e (40% inhibition).
Two RINH agents 1b and 3b displayed MIC 90 inhibition of intracellular BCG at much lower
concentrations of 2.44 and 1 µM, respectively as compared to 1a (13.30 µM) and 1c (13.42
µM), but exhibited lower potency as compared to INH. However, structural modifications,
such as the introduction of a piperidine linker in the EPI moiety (1b vs 3b) improves the
potency by 2-fold. This indicates that the potency of RINH agents can be enhanced with
structural modifications, which may afford compounds with superior activity than INH both
in vitro and in macrophages.
A comparison of activities (1a,b vs 1d,e) demonstrated that the pyridyl nitrogen is required
for the intracellular anti-mycobacterial activity in macrophages as was previously established
during in vitro studies. Additionally, inhibition of intracellular BCG by 1d and 1e is possibly
due to inhibition of EPs as these compounds lack activity against axenic
M. tuberculosis (H37Rv).20 The 2-chloro substituted phenothiazine containing compounds
(1b and 1e) showed superior activity as compared to unsubstituted ones (1a and 1d). All
these compounds were further evaluated in an ethidium bromide assay to assess their efflux
pump inhibition efficacy.
14

Table 2: Cytotoxicity against THP-1 cell lines and intracellular inhibition of BCG by CPZ,
INH, and selected compounds 1a-e and 3b.
IC20 (µM)
% Inhibition
Comp
CPZ
R
X
MIC (µM)
of BCG in
THP-1 cell
line
macrophages
40
4
-
4
-
>100
>100
>90
>90
0.015
13.3
INH
1a
N
N
24.4
>90
2.44
1b
N
C
>100
>100
>90
40
13.42
26.6
1c
1d
C
N
>100
40
12.2
1e
2.2
>90
1
3b
Comp: compound; MIC: Minimum inhibitor concentration corresponding to maximum
15

inhibition of BCG in macrophages.
3.3. Ethidium bromide evaluation of selected RINH agents
The short-listed compounds (Table 2) were also evaluated for their EP inhibition activity in
the Ethidium bromide (EB) assay. The recorded increase in mean fluorescence intensity in
the presence of a compound as compared to the negative control signifies the EP inhibition
efficiency of a compound leading to the higher concentration of EB in the BCG-EB assay.
Therefore, a higher intensity indicates superior inhibition of EPs responsible for EB efflux in
BCG in macrophages.
CPZ, 1b and 3b were tested at concentrations of 25, 25 and 10 µM, respectively, due to their
low IC20 values against the THP-1 cell lines, while other RINH agents (1a, 1c, 1d, 1e and
INH) were evaluated at 50 µM. A 40-90% increase in fluorescent intensity was demonstrated
by most of the RINH agents (1b, 1d, 1e and 3b), which is two-fold higher than the increase in
intensity demonstrated by CPZ while 1a and 1c showed fluorescence intensity slightly less
than CPZ. The negligible effect on EB efflux by INH was demonstrated by very low increase
in fluorescent intensity, and a higher increase in intensity exhibited by RINHs (1b, 1c, 1d, 1e
and 3b). This signifies superior EP inhibition efficiency towards EPs responsible for EB
efflux as compared to the known EP inhibitor CPZ (Figure 3).
These observations highlighted the dual action nature of RINH agents as inhibitors of EPs (as
suggested by the EB assay, Figure 3) and anti-mycobacterial agents per se (demonstrated by
in vitro and macrophage anti-mycobacterial data in table 1 and 2, respectively). The enhanced
EP inhibition activity of 1a and 1b and their analogues 1d and 1e, respectively, as compared
to INH is exclusively due to the phenothiazine core, albeit loss in anti-mycobacterial activity
was observed with 1d and 1e due to the absence of a pyridyl nitrogen. This implies that the
pyridyl nitrogen may not be required for EP inhibition activity but necessary for anti-
mycobacterial potency both in vitro and in macrophages.
16

Figure 3: EP inhibition evaluations of selected RINH agents in the EB assay. (MFI: Mean
fluorescence intensity)
4. Summary and conclusions
A total of 14 RINH agents and two benzhydrazide-based compounds were synthesized and
evaluated against sensitive and resistant strains of M. tuberculosis (Table 1). A selected
number of compounds were also tested ex vivo (Table 2) for intracellular activity, and in the
ethidium bromide assay for efflux pump inhibition efficacy (Figure 3).
The potency of RINH agents against both sensitive and resistant (INH mono-resistant and
MDR) strains of M. tuberculosis demonstrated the viability of the strategy of reversed anti-
TB agents for the development of novel anti-mycobacterial compounds to address the rapidly
growing issue of resistance. In addition to phenothiazine (3a,b) and thioxanthene (4a,b)
based RINH agents, demonstration of good to moderate potency by compounds containing
tricyclic (iminodibenzyl 1c, cyproheptadine 4c and trityl 8) and bicyclic (diphenylmethane, 6
and 7)-based EPI/chemosensitizer moieties further expanded the set of chemosensitizer
motifs, which can also be explored for further development of reversed anti-TB agents.
Macrophage activity and inhibition of EB efflux in the EB-assay by selected compounds
(1a-c and 3b) demonstrated proof of the dual acting nature of RINH agents. Preliminary
structure–activity relationship (SAR) studies (1a,b vs 1d,e) showed the significance of the
pyridyl nitrogen for anti-mycobacterial activity both in vitro and in macrophages. This SAR
also demonstrated proof of retention of EPI characteristics in hybrid compounds by attached
EPI moieties as exemplified by inhibition of intracellular BCG and EB efflux by 1d and 1e.
17

In conclusion, we have demonstrated the potential of dual acting RINH agents as anti-
mycobacterial agents effective against MDR and XDR strains of M. tuberculosis. These
encouraging anti-mycobacterial and resistance reversal activities displayed by RINH agents
lay a foundation for further exploration the concept of reversed anti-TB agents (RATAs) to
counter EP-mediated resistance.
5. Experimental section:
5.1. Chemistry
All commercially available chemicals were purchased from either Sigma-Aldrich or Combi
Blocks. All solvents were dried by appropriate techniques. Unless otherwise stated, all
solvents used were anhydrous. Purification of compounds were carried out by either by
recrystallization or column chromatography on silica gel 60 (Fluka), particle size 0.063−0.2
mm (70−230 mesh), as the stationary phase. All target compounds and intermediates were
1
characterized by H NMR and LC-MS. NMR spectra were recorded on either a Varian
Mercury-300 (1H 300.1 MHz, 13C 75.5 MHz) or Bruker-400 (1H 400.2 MHz, 13C 100.6 MHz)
instrument using CDCl3, CD3OD, and DMSO-d6 as solvents. Liquid chromatograph with
mass spectrometer (LC-MS) analysis was performed using an Agilent® 1260 Infinity binary
pump, Agilent® 1260 Infinity diode array detector (DAD), Agilent® 1290 Infinity column
compartment, Agilent®1260 Infinity standard autosampler, and a Agilent® 6120 quadrupole
(single) mass spectrometer, equipped with APCI and ESI multimode ionization source.
Purities were determined by Agilent® LC-MS using a Kinetex Core C18 2.6 µm column (50
mm × 3 mm). Mobile phase B: 0.4% acetic acid, 10 mM ammonium acetate in a 9:1 ratio of
HPLC grade methanol, and type 1water. Mobile phase A: 0.4% acetic acid in 10 mM
ammonium acetate in HPLC grade (type 1) water, with flow rate = 0.9 mL/min; detector,
diode array (DAD), and all final compounds were confirmed to have ≥95% purity.
5.1.1. General Procedure for the synthesis of compounds 1a and 1b: NaH (1.2 equiv.) was
added to a cooled solution (0 °C) of compound 10a or b (1.0 equiv) and isoniazid (1.2 equiv)
in anhydrous DMF (3 ml). The resulting reaction mixture was stirred at room temperature (25
°C) for 5-6 h. After completion of reaction (TLC), DMF was removed in vacuo and the
residue was taken in EtOAc (25 ml). The organic phase was washed with brine (3×15 ml),
dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash
chromatography on silica gel using MeOH-DCM as eluent afforded product 1a or 1b.
18

5.1.1.1. N'-(3-(10H-phenothiazin-10-yl)propyl)isonicotinohydrazide (1a). Yield (0.24 g,
1
18%) as an oil. H-NMR (400 MHz, CDCl3) δ 8.71 (d, J = 6.0 Hz, 2H), 7.45 (d, J = 6.4 Hz,
2H), 7.19 (m, 4H), 6.98 (dd, J = 7.2 and 1.2 Hz, 2H), 6.94 (dd, J = 7.2 and 1.2 Hz, 2H), 4.08
(t, J = 6.4 Hz, 2H), 3.13 (t, J = 6.4 Hz, 2H), 2.06 (m, H-6,); 13C-NMR (101 MHz, CDCl3) δ
165.2, 150.2 (2C), 145.2 (2C), 140.1, 127.7 (2C), 127.4 (2C), 125.5 (2C), 122.8 (2C), 120.8
(2C), 115.7 (2C), 49.6, 44.7 and 25.3; MS (EI+) m/z calculated for C21H20N4OS: 376.1;
found, 377.3 (M+1). HPLC purity: 96%.
5.1.1.2. N'-(3-(2-chloro-10H-phenothiazin-10-yl)propyl)isonicotinohydrazide (1b). Yield
1
(0.32 g, 24%) as an oil. H-NMR (400 MHz, CDCl3) δ 8.70 (d, J = 6.0 Hz, 2H), 7.44 (d, J =
6.0 Hz, 2H), 7.16 (m, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.92 (td, J = 7.6 and 0.8 Hz, 1H), 6.89
(dd, J = 8.4 and 2.4 Hz, 2H), 6.83 (d, J = 2.0 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 3.09 (t, J = 6.4
Hz, 2H), 2.03 (quin, J = 6.4 Hz, 2H). 13C-NMR (101 MHz, CDCl3) δ 165.1, 150.5 (2C),
144.9 (2C), 142.1, 133.4, 128.5, 127.9 (2C), 125.9, 123.8 (2C), 120.8 (3C), 115.6 (2C), 49.5,
44.6 and 25.1; MS (EI+) m/z calculated for C21H19ClN4OS: 410.10; found, 411.5 (M+1),
412.5 (M+2) and (M+3); HPLC purity: 96%.
5.1.2. General Procedure for the synthesis of compounds 14a and 14b: A solution of
compound 13a or 13b (1 equiv.) in DMF (1 ml) was added to a mixture of isoniazid (5 equiv)
and triethylamine (3 equiv) under an atmosphere of nitrogen at room temperature (25 °C).
The reaction mixture was continuously stirred for an additional hour, after which it was
heated to 55 °C and stirred for another 10-12 hours. After completion of reaction (TLC),
DMF was removed in vacuo, dried over sodium sulfate and concentrated in vacuo. The crude
product was purified by biotage flash chromatography.
5.1.2.1. tert-butyl (3-(10H-phenothiazin-10-yl)propyl)(3-isonicotinoylhydrazinyl)propyl)
1
carbamate (14a)Yield (0.18 g, 35%) as a white solid. m.p. 55-58 °C; H-NMR (400 MHz,
CD3OD) δ 8.67 (d, J = 6.0 Hz, 2H), 7.73 (d, J = 6.4 Hz, 2H), 7.18 (td, J = 7.2 and 1.6 Hz,
2H), 7.12 (dd, J = 7.6 and 1.2 Hz, 2H), 6.96 (dd, J = 7.6 and 0.8 Hz, 2H), 6.95 (td, J = 7.2
and 1.2 Hz, 2H), 3.90 (t, J = 6.4 Hz, 2H), 3.35 (t, J = 7.2 Hz, 2H), 2.81 (t, J = 7.2 Hz, 2H),
2.78 (t, J = 6.8 Hz, 2H), 2.01 (quin, J = 6.4 Hz, 2H), 1.80 (quin, J = 7.2 Hz, 2H), 1.40 (bs,
9H); 13C-NMR (101 MHz, CD3OD) δ 164.5 (2C), 156.1, 149.6 (2C), 145.4, 141.3, 127.1
(2C), 126.9 (2C), 122.6 (2C), 121.7 (2C), 121.4, 115.7 (2C), 79.8, 48.5, 44.6 (2C), 44.0, 27.3
(3C), 26.5 and 26.4; MS (EI+) m/z calculated for C29H35N5O3S: 533.2; found, 534.2 (M+1).
19

5.1.2.2. tert-butyl(3-(2-chloro-10H-phenothiazin-10-yl)propyl)(3-(2-
isonicotinoylhydrazinyl)propyl)carbamate (14b). Yield (0.15 g, 30%) as a white solid; m.p.
62-64 °C; 1H-NMR (400 MHz, CD3OD) δ 7.20 (td, J = 7.6 and 2.0 Hz, 1H), 7.18 (dd, J = 8.0
and 1.6 Hz, 1H), 7.13 (d, J = 8.0 Hz, 1H), 7.01 (m, 3H), 6.99 (dd, J = 8.0 and 1.6 Hz, 1H),
3.93 (t, J = 6.8 Hz, 2H), 3.33 (t, J = 7.2 Hz, 2H), 2.84 (t, J = 7.2 Hz, 2H), 2.80 (t, J = 6.4 Hz,
13
2H), 2.07 (quin, J = 6.4 Hz, 2H), 1.70 (quin, J = 7.2 Hz, 2H), 1.36 (bs, 9H); C-NMR (101
MHz, CD3OD) δ 165.2, 156.5 148.3 (2C), 145.3 142.9, 139.1, 132.1, 126.2, 126.1, 126.0,
124.1, 123.3, 121.8, 121.0, 120.1 (2C), 114.8 and 114.7, 79.5, 48.4, 44.2 (2C), 44.0, 27.1
(3C), 26.5 and 26.4; MS (EI+) m/z calculated for C29H34ClN5O3S: 567.21; found, 568.1
(M+1) and 570.1 (M+3).
5.1.3. General Procedure for the synthesis of compounds 2a and 2b: 10 ml of a 10% mixture
of TFA (10 equiv) in DCM by volume was added to compound 14a and 14b (1 equiv) in a
round bottom flask and stirred at room temperature (25 °C) for 1-1.5 hours. After completion
of reaction (TLC), excess of TFA was removed in vacuo and residue was dissolved in 10%
MeOH in DCM. The amberlyst A-21 was added to scavenger the residual TFA and stirred for
one hour, after which reaction mixture was filtered through celite and washed with a 20%
mixture of methanol in DCM. The combined organic layer was concentrated under reduced
pressure to obtain 2 (a and b).
5.1.3.1. N'-(3-((3-(10H-phenothiazin-10-yl)propyl)amino)propyl)isonicotinohydrazide (2a).
Yield (0.40 g, 66%) as a white solid; m.p. 48-50 °C; 1H-NMR (400 MHz, DMSO-d6) δ 10.30
(s, 1H), 8.70 (d, J = 5.2 Hz, 2H), 7.73 (d, J = 6.0 Hz, 2H), 7.18 (m, 4H), 6.96 (d, J = 8.0 Hz,
2H), 6.95 (t, J = 6.8 Hz, 2H), 3.91 (t, J = 6.8 Hz, 2H), 3.01 (m, 4H), 2.89 (t, J = 6.0 Hz, 2H),
2.04 (quin, J = 7.2 Hz, 2H), 1.70 (quin, J = 6.8 Hz, 2H); 13C-NMR (101 MHz, DMSO-d6) δ
164.3, 150.2 (2C), 144.5 (2C), 140.0, 127.6 (2C), 127.2 (2C), 124.2 (2C), 122.7 (2C), 121.2
(2C), 115.9 (2C), 48.4, 45.8, 44.7, 43.6, 23.7 and 23.3; MS (EI+) m/z calculated for
C24H27N5OS: 433.19; found, 434.2 (M+1); HPLC purity 99%.
5.1.3.2. N'-(3-((3-(2-chloro-10H-phenothiazin-10-
yl)propyl)amino)propyl)isonicotinohydrazide (2b). Yield (0.37 g, 83%) as a white solid; m.p.
61-64 °C; 1H-NMR (400 MHz, CD3OD) δ 8.74 (dd, J = 6.4 and 2.0 Hz, 2H), 7.74 (dd, J = 6.4
and 1.6 Hz, 2H), 7.15 (m, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.90 (m, 2H), 6.95 (td, J = 7.6 and
1.2 Hz, 1H), 6.88 (dd, J = 8.4 and 2.0 Hz, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.22 (m, 4H), 3.09 (t,
20

J = 6.0 Hz, 2H), 2.04 (quin, J = 6.4 Hz, 2H), 1.70 (quin, J = 6.0 Hz, 2H); 13C-NMR (101
MHz, CD3OD) δ 165.1, 148.3 (2C), 145.7, 142.9, 139.2, 132.1, 126.5, 126.1, 125.9, 124.4,
123.3, 121.9, 121.0, 120.1 (2C), 114.8, 114.7, 49.3, 44.1, 42.4 (2C), 22.9 and 21.9; MS (EI+)
m/z calculated for C24H26ClN5OS: 467.15; found, 468.1 (M+1), 469.1 (M+2)+ and 470.1
(M+3); HPLC purity 97.1%.
5.1.4. General Procedure for the synthesis of compounds 1c-e and 3-8. Triethylamine (4
equiv.) was added to a solution of benzhydrazide (4 equiv.; 1d and 1e) or isoniazid (4
equivalent; 1c and 3-8) and corresponding chloro-intermediate (1a-c or 34a-i; 1 equiv.) in
iso-propanol, and the resulting mixture was refluxed for 12-24 hours. After completion of
reaction (TLC), iso-propanol was removed in vacuo and residue was taken in DCM (40 ml),
and washed with water (5 x 15 mL). The aqueous layer was extracted with DCM (5 x 10 mL)
and the combined DCM extracts was washed with brine (1 x 20 ml), dried over sodium
sulphate and concentrated in vacuo. The crude product was purified by column
chromatography to afford the target compound 1c-e and 3-8.
5.1.4.1. N'-(3-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)propyl)isonicotinohydrazide (1c).
Yield (0.11 g, 15%) as an oil. 1H-NMR (400 MHz, CDCl3) δ 7.16 - 7.08 (m, 4H), 7.60 (d, J =
7.2 Hz, 2H), 6.99 (m, 2H), 3.88 (t, J = 6.8 Hz, 2H), 2.99 (t, J = 6.4 Hz, 2H), 3.12 (s, 4H), 2.27
(quin, J = 6.8, Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 165.1, 149.4 (2C), 147.3, 140.6,
134.1, 129.4, 126.1, 122.2, 121.4, 119.6, 49.0 (2C), 31.9 (2C), 26.0; MS (EI+) m/z calculated
for C23H24N4O: 372.2; found, 373.1 (M+1); HPLC purity: 95.1%.
5.1.4.2. N'-(3-(10H-phenothiazin-10-yl)propyl)benzohydrazide (1d): Yield (80 mg, 20%) as
1
an oil; H-NMR (400 MHz, CDCl3) δ 7.72 (d, J = 8.4 Hz, 2H), 7.52 (tt, J = 7.6 and 1.6 Hz,
1H), 7.43 (t, J = 8.0 Hz, 2H), 7.15 (m, 2H), 7.10 (dd, J = 7.6 and 1.2 Hz, 2H), 6.99 (dd, J =
7.6 and 1.2 Hz, 2H), 6.90 (td, J = 7.2 and 1.2 Hz, 2H), 4.05 (t, J = 6.4 Hz, 2H), 3.04 (t, J =
6.4 Hz, 2H), 2.02 (quin, J = 6.8 Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 167.8, 145.4 (2C),
132.9, 131.3 (2C), 128.2 (2C), 127.1 (2C), 126.9 (2C), 126.8 (2C), 125.3, 122.2 (2C), 115.6
(2C), 48.8, 44.4 and 25.0; MS (EI+) m/z calculated for C22H21N3OS: 375.1; found, 376.1
[M+1]+, HPLC purity 96.8%
5.1.4.3. N'-(3-(2-chloro-10H-phenothiazin-10-yl)propyl)benzohydrazide (1e). Yield (75 mg,
1
25%) as an oil; H-NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.2 Hz, 2H), 7.52 (t, J = 7.6 Hz,
1H), 7.43 (t, J = 7.2 Hz, 2H), 7.12 (td, J = 8.0 and 1.6 Hz, 1H), 7.11 (dd, J = 8.0 and 1.6 Hz,
1H), 7.05-7.01 (m, 2H), 7.00 (d, J = 2.0, 2H), 6.95 (td, J = 7.6 and 1.2 Hz, 1H), 6.90 (dd, J =
21

8.4 and 2.4 Hz, 1H), 4.04 (t, J = 6.4 Hz, 2H), 3.05 (t, J = 6.4 Hz, 2H), 2.02 (quin, J = 6.8 Hz,
2H); 13C-NMR (101 MHz, CDCl3) δ 167.8, 146.8, 144.6, 133.1, 132.9, 131.4, 128.1 (2C),
127.6, 127.2, 126.9, 126.8 (2C), 125.0, 124.1, 122.7, 121.9, 116.0, 115.7, 48.7, 44.6 and 24.9;
MS (EI+) m/z calculated for C22H20ClN3OS: 409.1; found: 410.1 [M+1]+, 411.1 [M+2]+,
412.1 [M+3]+; HPLC purity 97%.5.1.4.4.
5.1.4.4. N'-(3-(4-(10H-phenothiazin-10-yl)piperidin-1-yl)propyl)isonicotinohydrazide (3a).
Yield (0.136 g, 30%) as a white solid. m.p. 54-56 °C; 1H-NMR (400 MHz, DMSO-d6) δ 8.74
(d, J = 6.0 Hz, 2H), 7.75 (d, J = 6.0 Hz, 2H), 7.20 (m, 2H), 7.19 (m, 2H), 7.15 (d, J = 7.0 Hz,
2H), 7.03 (m, 2H), 3.75 (t, J = 12.8 Hz, 1H), 3.00 (bd, J = 9.2 Hz, 2H), 2.87 (t, J = 6.8 Hz,
2H), 2.44 (t, J = 7.2 Hz, 2H), 2.18 (m, 4H), 2.1 (bd, J = 10.0 Hz, 2H), 1.65 (quin, J = 6.8 Hz,
2H); 13C-NMR (101 MHz, DMSO-d6) δ 163.8, 150.4 (2C), 145.3 (2C), 140.7, 130.0 (2C),
127.8 (2C), 127.5 (2C), 123.8 (2C), 121.5 (2C), 120.8 (2C), 62.5, 56.2, 53.2 (2C), 50.2, 31.6
(2C) and 25.4; MS (EI+) m/z calculated for C26H29N5OS: 459.2; found, 460.2 (M+1); HPLC
purity 99.8%.
5.1.4.5. N'-(3-(4-(2-chloro-10H-phenothiazin-10-yl)piperidin-1-
yl)propyl)isonicotinohydrazide (3b). Yield (0.17 g, 30%) as a white solid; m.p. 85-87 °C; 1H-
NMR (400 MHz, CD3OD) δ 8.74 (d, J = 6.4 Hz, 2H), 7.75 (d, J = 6.0 Hz, 2H), 7.19 (m, 2H),
7.17 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.02 (m, 1H), 6.99
(dd, J = 8.4 and 2.4 Hz, 1H), 3.79 (m, 1H), 3.10 (bd, J = 10.0 Hz, 2H), 3.00 (t, J = 6.4 Hz,
2H), 2.61 (t, J = 6.8 Hz, 2H), 2.32 (m, 4H), 2.05 (m, 4H), 2.05 (quin, J = 7.2 Hz, 2H); 13C-
NMR (101 MHz, CD3OD) δ 164.5, 149.6 (2C), 146.3, 144.6, 141.3, 132.5, 129.0, 128.4,
127.6, 127.1, 126.9, 123.7, 123.2, 121.4 (2C), 121.3, 121.0, 62.5, 56.1, 52.8 (2C), 50.0, 31.1
(2C) and 24.5; MS (EI+) m/z calculated for C26H28ClN5OS: 493.17; found, 494.1 (M+1),
495.1 (M+2) and 496.1 (M+3), HPLC purity 99.5%.
5.1.4.6. N'-(3-(4-(9H-thioxanthen-9-ylidene)piperidin-1 yl)propyl)isonicotinohydrazide (4a).
1
Yield (70 mg, 37%) as a white solid; m.p. 105-107 °C; H-NMR (400 MHz, DMSO-d6) δ
8.76 (bs, 2H), 7.67 (d, J = 5.6 Hz, 2H), 7.49 (dd, J = 7.6 and 1.2 Hz, 2H), 7.29 (dd, J = 7.6
and 1.6 Hz, 2H), 7.25 (td, J = 7.6 and 1.6 Hz, 2H), 7.18 (td, J = 7.6 and 1.6 Hz, 2H), 3.02 (d,
J = 5.6 Hz, 2H), 2.92 (m, 2H), 2.82-2.60 (m, 4H), 2.55 (t, J = 5.6 Hz, 2H), 2.18 (m, 2H), 1.80
(quin, J = 6.4 Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 164.2, 150.6 (2C), 140.5, 136.6 (2C),
135.3 (2C), 134.1, 130.9, 128.7 (2C), 127.3 (2C), 126.3 (2C), 125.9 (2C), 121.0 (2C), 56.6,
22

54.7 (2C), 51.1, 30.1 (2C) and 25.5; MS (EI+) m/z calculated for C27H28N4OS: 456.20; found,
457.2 (M+1); HPLC purity 96.5%.
5.1.4.7. N'-(3-(4-(2-chloro-9H-thioxanthen-9-ylidene)piperidin-1-
yl)propyl)isonicotinohydrazide (4b). Yield (0.06 g, 32%) as a white solid; m.p. 99-102 °C;
1H-NMR (400 MHz, CDCl3) δ 8.50 (d, J = 5.2 Hz, 2H), 7.70 (d, J = 5.6 Hz, 2H), δ 7.48 (d, J
= 8.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.30 (m, H-5’, 3H), 7.21 (m, 1H), 7.28 (dd, J = 8.4
and 2.0 Hz, 1H), 3.02 (t, J = 6.0 Hz, 2H), 2.97 (m, 2H), 2.76 (m, 4H), 2.62 (t, J = 6.4 Hz,
2H), 2.23 (m, 1H), 1.82 (quin, J = 6.4 Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 164.5, 150.6
(2C), 140.3, 138.2, 136.0, 135.0, 134.6, 133.9, 131.9, 130.4, 128.8, 128.5, 128.4, 127.3,
126.7, 126.4, 126.2, 121.0 (2C), 56.3, 54.6, 54.4, 52.0, 50.7, 29.8 and 22.0; MS (EI+) m/z
calculated for C27H27ClN4OS: 490.2; found, 491.2 (M+1), 492.2 (M+2) and 493.2 (M+3);
HPLC purity 96.8%.
5.1.4.8. N'-(3-(4-(5H-dibenzo[a,d][7]annulen-5-ylidene)piperidin-1-
yl)propyl)isonicotinohydrazide (4c). Yield (90 mg, 40%) as a white solid; m.p. 120-122 °C;
1H-NMR (400 MHz, CD3OD) δ 8.70 (dd, J = 6.0 and 1.6 Hz, 2H), 7.66 (dd, J = 6.4 and 1.6
Hz, 2H), 7.34-7.39 (m, 4H), 7.27-7.30 (m, 2H), 7.22 (dd, J = 8.0 and 1.2 Hz, 2H), 6.95 (s,
2H), 2.99 (t, J = 6.8 Hz, 2H), 2.80 (m, 2H), 2.59 (t, J = 6.8 Hz, 2H), 2.45 (m, 4H), 2.23 (m,
1H), 1.82 (quin, J = 6.8Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 164.2, 150.5 (2C), 140.3,
138.7 (2C), 134.7 (2C), 134.3, 134.1, 131.0 (2C), 128.3 (4C), 127.6 (2C), 126.4 (2C), 121.0
(2C), 56.4, 55.0 (2C), 50.8, 29.4 (2C) and 25.3; MS (EI+) m/z calculated for C29H30N4O:
450.24; found, 451.2 (M+1); HPLC purity 95.5%.
5.1.4.9. Isonicotinic
acid
N'-{3-[4-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)-
piperazin-1-yl]-propyl}-hydrazide (5). Yield (0.2 g, 42%) as a white solid; m.p. 155-157 °C;
1H-NMR (400 MHz, CDCl3) δ 8.68 (d, J = 5.6 Hz, 2H), 7.67 (d, J = 5.6 Hz, 2H), 7.02 (m,
8H), 3.87 (s, 1H), 3.85 (m, 2H), 2.91 (t, J = 5.6 Hz, 2H), 2.68 (m, 2H), 2.65-2.49 (two br s,
8H), 2.65 (t, J = 6.4 Hz, 2H), 1.76 (quin, J = 6.4 Hz, 1H); 13C-NMR (101 MHz, CDCl3) δ
164.3, 150.5 (2C), 140.5, 139.6, 138.5, 130.9 (2C), 130.8 (3C), 128.10 (3C), 125.7 (2C),
121.2 (2C), 56.1, 53.3 (2C), 50.6 (2C), 50.2, 31.8 (2C), 29.6 and 25.0; MS (EI+) m/z
calculated for C28H33N5O: 455.27; found, 456.20 (M+1); HPLC purity 99.8%.
23

5.1.4.10. Isonicotinic acid N'-[3-(4-benzhydryl-piperazin-1-yl)-propyl]-hydrazide (6). Yield
1
(0.2 g, 42%) as a white solid; m.p. 55-57 °C; H-NMR (400 MHz, CDCl3) δ 8.75 (d, J = 4.0
Hz, 2H), 7.65 (d, J = 5.6 Hz, 2H), 7.35 (d, J = 7.6 Hz, 4H), 7.30 (t, J = 7.2 Hz, 4H), 7.11 (t, J
= 7.2 Hz, 2H), 4.21 (s, 1H), 3.01 (t, J = 6.0 Hz, 2H), 2.64 (bs, 4H), 2.47 (bs, 4H), 2.65 (t, J =
6.4 Hz, 2H), 1.83 (quin, J = 6.4 Hz, 2H); 13C-NMR (101 MHz, CDCl3) δ 164.1, 150.5 (2C),
142.3 (2C), 140.5, 128.5 (4C), 127.9 (4C), 127.1 (2C), 121.1 (2C), 76.0, 56.4, 53.6(2C), 51.0
(2C), 50.07, and 25.6; MS (EI+) m/z calculated for C26H31N5O: 429.25; found, 430.20 (M+1);
HPLC purity 98.8%.
5.1.4.11. N'-(3-(4-(2-hydroxy-2,2-diphenylethyl)piperazin-1-yl)propyl)isonicotinohydrazide
(7). Yield (0.1 g, 35%) as a white solid; m.p.115-117 °C; 1H-NMR (400 MHz, CDCl3) δ 8.75
(2H, dd, J = 6.0 and 1.6 Hz, H-11), 7.52 (2H, dd, J = 6.4 and 1.6 Hz, H-10), 7.50 (4H, dd, J =
7.2 and 1.2 Hz, H-1), 7.32 (4H, td, J = 6.4 and 1.2 Hz, H-2), 7.23 (2H, tt, J = 7.6 and 1.2 Hz,
H-3), 5.14 (1H, s, OH), 3.29 (2H, s, H-4), 2.99 (2H, t, J = 6.0 Hz, H-9), 2.52 (2H, t, J = 6.8
13
Hz, H-7), 2.40-2.54 (8H, m, H-5 and H-6) 1.76 (2H, quin, J = 6.4 Hz, H-8); C-NMR (101
MHz, CDCl3) δ 164.3, 150.6 (2C), 146.8 (2C), 140.3, 128.0 (4C), 126.7 (2C), 125.3 (4C),
120.9 (2C), 74.3, 67.5, 56.6, 54.0 (2C), 53.1 (2C), 51.0 and 25.2; MS (EI+) m/z calculated for
C27H33N5O2: 459.26; found, 460.3 (M+1); HPLC purity 99.8%.
5.1.4.12. N'-(3-(4-tritylpiperazin-1-yl)propyl)isonicotinohydrazide (8). Yield (0.2 g, 40%) as
1
a white solid; m.p. 199-200 °C; H-NMR (400 MHz, CDCl3) δ 8.65 (dd, J = 6.0 and 1.6 Hz,
2H), 7.52 (m, 8H), 7.27 (t, J = 7.2 Hz, 6H), 7.16 (t, J = 7.2 Hz, 3H), 3.37 – 2.27 (bm, 8H),
2.96 (t, J = 6.0 Hz, 2H), 2.64 (t, J = 6.4 Hz, 2H), 1.82 (quin, J = 6.4 Hz, 2H), 13C-NMR (101
MHz, CDCl3) δ 163.8, 150.4 (3C), 148.7, 140.3 (3C), 129.2 (6C), 127.6 (6C), 126.4 (2C),
120.9 (2C), 76.8, 57.1 (2C), 53.9 51.0 (2C), 47.0 and 25.6; MS (EI+) m/z calculated for
C32H35N5O: 505.28; found, 505.07 (M)+; HPLC purity 96.3%.
5.2. Biological assays
5.2.1. In vitro anti-mycobacterial testing
All compounds were dissolved in 100% dimethyl sulfoxide (DMSO) and stored as frozen
stocks at a concentration of 10 mM. Inocula were prepared from cultures of all strains grown
in Lowestein Jensen (LJ) slants that were 2 to 5 weeks old. Stock mycobacterial cultures were
prepared by inoculating frozen stocks into 10 ml 7H9 mycobacterial culture medium
supplemented with Oleic Albumin Dextrose Catalase (OADC) (1%) final concentration,
(BBL Microbiology Systems, Cockeysville, Maryland) and 0.05% Tween 80 For growth
24

evaluation in the (MGIT) 960 instrument (Becton Dickinson, Maryland, USA) was used.24
Mycobacterial growth is monitored through changes in oxygen consumption which in turn
changes fluorescence. Serially two-fold diluted compounds (0.1 ml in DMSO) were added to
the 7H9 culture medium contained in Mycobacteria Growth Inhibition Tube (MGIT) tube
with the final DMSO concentration not exceeding 1.2%. Five hundred microliters of a
mycobacterial culture, previously cultured in a MGIT tube, was also added to the MGIT test
tube (Becton Dickinson and Company. 1999. Bactec MGIT 960 system user’s manual,
catalog number 445876. Becton Dickinson and Company, Franklin Lakes, NJ). Incubation at
37⁰C was continued in the MGIT system and the growth units were monitored daily. For
MIC99 evaluations, a 1% bacterial culture was prepared in a drug free MGIT tube (GC) and
the MIC99 of the compound determined relative to the growth units (GU) of this tube. When
GU of the GC reached 400 the results were interpreted and MIC99 of compounds was
determined as GU equal or less than the GU of the GC.
25

Table 3: M. tuberculosis clinical isolates classification, genotyping and sequencing of strains used.
Clinical Isolate Genotype
Resistance Profile
Susceptibility
Status
R73
LCC/X
H
Mono-resistant
Mono-resistant
Mono-resistant
XDR
R4965
R5401
TT149
X_3
LAM3
H
Beijing
H
Atypical Beijing
Beijing
H;R;ET;S;A;C;O;M;
H;R;E;K;S;C;O;
XDR
X_60
X_61
Beijing
H;R;E;A;ET;O;S;C;K XDR
Beijing
H;R;ET;A;O;K;S;
XDR
Resistance confering mutations
Clinical
Isolate
rpoB
katG
embB
rrs
inhA-
gyrA
pncA
promoter
R73
531TTG 315ACA 306ATC
ND
WT
WT
-15
WT
-15
ND
-15
ND
ND
ND
ND
WT
WT
R4965
R5401
TT149
X_3
WT
WT
315ACC WT
WT WT
ND
ND
516TAC 315ACC WT
1401G
1401G
1401G
1401G
94GGC WT
531TTG 315ACC 306ATC
94GGC 14CGC
94GGC Del8G
94GGC 103TAG
X_60
X_61
531TTG WT
531TTG WT
306GTG
WT
ND: Not determined; WT: wild type; H: isoniazid; R: rifampicin; E: ethionamide; ET:
ethambutol; S: streptomycin; A: amikacin; C: capreomycin; O: ofloxacin; M: moxifloxacin; Z:
pyrazinamide
5.2.2. Cytotoxicity assay:
THP-1 cells were cultured for 24 hours at a concentration of 1.5 x 104/well in a 96 well plate
in the presence of different concentrations of HEPI. After 24 hours, MTT was added to the
cells and the plate was incubated at 37oC for 4 hours. Solubilisation buffer was then
added and the cells were incubated for an additional 30 minutes. Formazan formation was
measured at an absorbance of 570 nm using an SLT Rainbow Plate reader (Tecan, U.S., Inc).
26

The cytotoxicity percentage was calculated using the following formula: 100 - [100 x (optical
density (OD) with drug/OD without drug)].25
5.2.3. Intracellular drug Inhibition Assays:
Peripheral blood mononuclear cell (PBMCs) were plated in round bottom 96 well plates at a
concentration of 1.5x104/well. After 24 hours, non-adherent cells were gently washed away
using Roswell Park Memorial Institute (RPMI) media. Adherent monocytes were cultured for
6 days and then infected with BCG for 4 hours using a multiplicity of infection (MOI) of 3.
Extracellular bacteria were removed by washing three times with RPMI media. The infected
monocytes were then cultured in the presence or absence of different concentrations of drugs
for 72 hours. Monocytes were then lysed with a 0.2% solution of saponin in RPMI. The
bacteria were quantified using an [3H]-uridine incorporation assay or by culturing onto 7H10
media and counting colony forming units (CFU) weekly for 4 weeks. The [3H]-uridine
incorporation assay was performed by adding 1 µCi [3H]-uridine in 7H9 Middlebrook broth
per well and incubating for another 72 hours at 37oC. The plate was then harvested onto
a filter mat using a Tomtec Mach III cell harvester (Tomtec Inc., Hamden CT). The filter mat
was air dried and placed into a pouch with scintillation fluid and read using a
Microbeta scintillation counter (PerkinElmer Wallac Inc.) BCG percent inhibition was
− [100 × (
calculated with the following formula: percentage inhibition = 100
disintegrations
per min (dpm) in the drug-treated cultures/dpm in the untreated cultures)].25
5.2.4. Ethidium Bromide Assay:
Efflux pump activity was measured using BCG. BCG aliquots of 225ul (BCG culture
turbidity in 7H9 Middlebrook broth was adjusted to the standard turbidity of McFarland 0.5)
were added into 2ml tubes in the presence of ethidium bromide and verapamil at
concentrations of 2.5 µM and 250 µM respectively. The bacteria were then incubated at room
temperature for 1 hour. The bacteria were then centrifuged and washed with 1mL PBS and
then re-suspended in PBS containing 0.4% glucose in the presence or absence of RINHs. The
cultures were then incubated for an additional one hour at 37oC. Aliquots were transferred
into a 96 well plate in triplicates. A minimum of 5000 events were collected via flow-
cytometric acquisition using Guava easyCyte (Guava Technology, Hayward, CA). Analyses
were performed using FlowJo (version 6.2.1; Tree Star) software.25
27

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