Reversed isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis

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

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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 Kumar^a, Kawaljit Singh^a, Andile H. Ngwane^b, Fahreta Hamzabegovic^c, Getahun

Abate^,c, Bienyameen Baker^b, Ian Wiid^b, Daniel F. Hoft^c,d, Peter Ruminski^,f, Kelly Chibale^a,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.¹The rapid and widespread emergence of multi-drug resistant (MDR) and

extremely-drug resistant (XDR) strains have hampered TB control and its eradication.^2,3The

emergence of resistance against first line anti-TB drugs (isoniazid, rifampicin, pyrazinamide

and ethambutol) has complicated treatment both financially as well as socially.⁴Therefore,

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.^5,6A 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,⁷and (ii) cocktail use of drugs and

efflux pump inhibitors (EPIs).⁸These 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.⁹A 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.¹⁰The over expression of these

genes is known to be countered by EPIs leading to increased susceptibility of MDR strains

towards INH.¹¹However, 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.¹²In 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.^14,15When evaluated against CQ resistant Plasmodium falciparum strains, the RCQ

agents showed resistance reversal.¹⁶In 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).^17,18A 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 NaNH₂in 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 K₂CO₃in 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.¹⁹The 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).²⁰This intermediate was further reacted with

1-Boc-piperazine in DMSO in the presence of Cs₂CO₃to 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)

SI_R5401

Compounds

INH

R

CHO

Cell lines

0.73

2.5

> 10

^aND

>100

^aND

>35

^aND

1a

1.0

> 10

1b

1c

1d

1e

0.25

1.0

> 10

^bND

^aND

^bND

^aND

^bND

^aND

^bND

^aND

^bND

> 10

5.0

>10

^aND

2a

2b

> 10

^aND

10

MIC₉₉(µM)^X

H37Rv R5401 X_61 X_60

IC₅₀(µM)

SI_R5401

48.8

37

Compounds

R

CHO

Cell lines

5.0

1.0

2.5

1.0

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

5.0

> 10

^aND

296

148

^aND

236.8

59.2

^aND

> 10

^aND

6

7

5.0

2.5

5

> 10

48.1

174

19.3

139.2

11

1.25

MIC₉₉(µM)^X

H37Rv R5401 X_61 X_60

IC₅₀(µM)

SI_R5401

Compounds

R

CHO

Cell lines

1.25

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; SI_R504: Selectivity index of compounds

with respect toMIC₉₉of M. tuberculosis strain R5401 and IC₅₀against 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.^21,22The 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,^17,23and 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 IC₂₀values 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 ≤ IC₂₀except 1d and 1e (40% inhibition).

Two RINH agents 1b and 3b displayed MIC ₉₀inhibition 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).²⁰The 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

>90

0.015

13.3

INH

1a

N

24.4

>90

2.44

1b

N

C

>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 IC₂₀values 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-d₆as 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

MIC₉₉evaluations, a 1% bacterial culture was prepared in a drug free MGIT tube (GC) and

the MIC₉₉of the compound determined relative to the growth units (GU) of this tube. When

GU of the GC reached 400 the results were interpreted and MIC₉₉of 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

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

-15

WT

-15

ND

-15

ND

WT

R4965

R5401

TT149

X_3

WT

315ACC WT

WT WT

ND

516TAC 315ACC WT

1401G

94GGC WT

531TTG 315ACC 306ATC

94GGC 14CGC

94GGC Del8G

94GGC 103TAG

X_60

X_61

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 37^oC 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 37^oC. 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 37^oC. 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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29

Article Doi

Reversed isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis

DOI: 10.1016/j.bmc.2017.12.047

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