Identification of less lipophilic riminophenazine derivatives for the treatment of drug-resistant tuberculosis

Article abstract of DOI:10.1021/jm300828h

Clofazimine (CFZ), a member of the riminophenazine class, has been studied in clinical trials for the treatment of multidrug-resistant tuberculosis (MDR-TB). CFZ has several side effects which can be attributed to its extremely high lipophilicity. A series of novel riminophenazine analogues bearing a C-2 pyridyl substituent was designed and synthesized with the goal of maintaining potent activity against Mycobacterium tuberculosis (M. tuberculosis) while improving upon its safety profile by lowering the lipophilicity. All compounds were evaluated for their in vitro activity and cytotoxicity. The results demonstrated that many new compounds had potent activity against M. tuberculosis with MICs of less than 0.03 μg/mL and low cytotoxicity with IC₅₀ values greater than 64 μg/mL. Some compounds were tested for in vivo efficacy against MDR-TB in an experimental mouse infection model. Two compounds demonstrated equivalent or better efficacy than CFZ in this model with significantly reduced skin discoloration potential.

Full text of DOI:10.1021/jm300828h

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Article
Identification of Less Lipophilic Riminophenazine
Derivatives for the Treatment of Drug-Resistant Tuberculosis
Dongfeng Zhang, Yu Lu, Kai Liu, Binna Liu, Jingbin Wang, Gang Zhang, Hao Zhang,
Yang Liu, Bin Wang, Meiqin Zheng, Lei Fu, Yanyan Hou, Ning-Bo Gong, Yang Lv, Chun
Li, Christopher B Cooper, Anna M Upton, Dali Yin, Zhenkun Ma, and Haihong Huang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm300828h • Publication Date (Web): 29 Aug 2012
Downloaded from http://pubs.acs.org on August 31, 2012
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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of their duties.

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of Natural Medicine & Beijing Key Laboratory of Active Substances
Discovery and Druggability Evaluation, Institute of Materia Medica, Peking
Union Medical College & Chinese Academy of Medical Sciences,
Gong, Ning-Bo; State Key Laboratory of Bioactive Substances and Function
of Natural Medicine & Beijing Key Laboratory of Active Substances
Discovery and Druggability Evaluation, Institute of Materia Medica, Peking
Union Medical College & Chinese Academy of Medical Sciences,
Lv, Yang; State Key Laboratory of Bioactive Substances and Function of
Natural Medicine & Beijing Key Laboratory of Active Substances Discovery
and Druggability Evaluation, Institute of Materia Medica, Peking Union
Medical College & Chinese Academy of Medical Sciences,
Li, Chun; State Key Laboratory of Bioactive Substances and Function of
Natural Medicine & Beijing Key Laboratory of Active Substances Discovery
and Druggability Evaluation, Institute of Materia Medica, Peking Union
Medical College & Chinese Academy of Medical Sciences,
Cooper, Christopher; Global Alliance for TB Drug Development,
Upton, Anna; Global Alliance for TB Drug Development,
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Yin, Dali; State Key Laboratory of Bioactive Substances and Function of
Natural Medicine & Beijing Key Laboratory of Active Substances Discovery
and Druggability Evaluation, Institute of Materia Medica, Peking Union
Medical College & Chinese Academy of Medical Sciences,
Ma, Zhenkun; Global Alliance for TB Drug Development,
Huang, Haihong; State Key Laboratory of Bioactive Substances and
Function of Natural Medicine & Beijing Key Laboratory of Active Substances
Discovery and Druggability Evaluation, Institute of Materia Medica, Peking
Union Medical College & Chinese Academy of Medical Sciences,
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Identification of Less Lipophilic
Riminophenazine Derivatives for the
Treatment of Drug-Resistant Tuberculosis
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Dongfeng Zhang#,† Yu Lu#, ‡ Kai Liu,† Binna Liu,† Jingbin Wang,† Gang Zhang,† Hao
Zhang,† Yang Liu,† Bin Wang,‡ Meiqin Zheng,‡ Lei Fu,‡ Yanyan Hou,† Ningbo Gong,†
Yang Lv,†Chun Li,† Christopher B. Cooper,§ Anna M. Upton,§ Dali Yin,† Zhenkun
,†
Ma*,§ and Haihong Huang*
†State Key Laboratory of Bioactive Substances and Function of Natural Medicine &
Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation,
Institute of Materia Medica, Peking Union Medical College and Chinese Academy of
Medical Sciences, 1 Xian Nong Tan Street, Beijing 100050, P. R. China
‡Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research
Institute, Beijing Chest Hospital, Capital Medical University, 97 Ma Chang Street,
Beijing 101149, P. R. China
§Global Alliance for TB Drug Development, 40 Wall Street, New York, NY 10005,
USA
# These authors contributed equally to this work
*Correspondence should be addressed to:
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Haihong Huang, phone: (8610) 63165244, fax: (8610) 63165244, email:
joyce@imm.ac.cn
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Zhenkun
Ma,
phone:
646-616-8633,
fax:
212-227-7541,
email:
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Zhenkun.ma@tballiance.org
Abstract:
Clofazimine (CFZ), a member of the riminophenazine class, has been studied in
clinical trials for the treatment of multidrug-resistant tuberculosis (MDR-TB). CFZ
has several side effects which can be attributed to its extremely high lipophilicity. A
series of novel riminophenazine analogues bearing a C-2 pyridyl substituent was
designed and synthesized with the goal of maintaining potent activity against
Mycobacterium tuberculosis (M. tuberculosis) while improving upon its safety profile
by lowering the lipophilicity. All compounds were evaluated for their in vitro activity
and cytotoxicity. The results demonstrated that many new compounds had potent
activity against M. tuberculosis with MICs of less than 0.03 ꢀg/mL, and low
cytotoxicity with IC50 values greater than 64 ꢀg/mL. Some compounds were tested for
in vivo efficacy against MDR-TB in an experimental mouse infection model. Two
compounds demonstrated equivalent or better efficacy than CFZ in this model with
significantly reduced skin discoloration potential.
Key words: Clofazimine, riminophenazine, MDR-TB, skin discoloration
Introduction
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The spread of multidrug-resistant tuberculosis (MDR-TB) and the emergence of
extensively drug-resistant tuberculosis (XDR-TB) pose a challenge for the treatment
and control of this devastating disease, one of the leading causes of death in the
world.1 The treatment of MDR-TB is lengthy, expensive and extremely difficult,
requiring daily administration of a combination of 5-7 drugs, some of them injectable,
for a period of 18-24 months. Some of these existing drugs have shown significant
toxicities.2 As a consequence, fewer than 3% of MDR-TB patients worldwide are
adequately treated, which contributes to the further spread of MDR-TB and the
emergence of XDR-TB.3 Therefore, there is a significant unmet medical need for
safer, more effective therapy against MDR-TB.
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Despite the increased effort over the past decade in searching for new drugs against
TB, compounds with a novel mode of action against M. tuberculosis are still scarce,
which presents a significant barrier for the development of a completely new regimen
for the treatment of MDR-TB. It has been recognized that repurposing or optimizing
existing antibiotics could be an effective approach in identifying new TB drugs.4
Clofazimine (CFZ, Figure 1), an antimycobacterial agent first introduced in the 1960s
for the treatment of leprosy,5 has demonstrated excellent activity against various
drug-resistant M. tuberculosis strains and is currently in clinical trials as a component
of new regimens for the treatment of MDR-TB.6 CFZ is a member of the
riminophenazine structural family.7,8 Its unique mechanism of action (MOA)9
precludes it from rapid development of drug resistance. However, this compound is
highly lipophilic (logP 5.39)10 and accumulates extensively in skin and fat tissues,
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leading to an extremely long terminal half-life and undesirable side effects such as
skin discoloration.11 The undesirable properties of CFZ may preclude its widespread
use in the future for the treatment of TB. Structural modification of CFZ to address
the above concerns is therefore an important approach to fully utilizing the novel
mode of action that this drug class could offer. The goal of our ongoing effort in this
area is to identify less lipophilic derivatives of CFZ that are at least as potent as CFZ
in vitro and in vivo, but which exhibit improved pharmacokinetic (PK) profiles and
reduced potential for skin discoloration.7,12
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We have previously conducted extensive structural modification on CFZ to address
the skin discoloration problem, a major obstacle for its clinical use. Replacement of
the N-5 phenyl ring with an alkyl substituent (e.g., compound 1, Figure 1)13 and the
C-2 phenyl ring with pyridyl (e.g., compound 2, Figure 1)14,15 led to analogues with
reduced lipophilicity. Compound 2 demonstrated potent activity against M.
tuberculosis both in vitro and in vivo and an improved PK profile as compared to
CFZ. However, the safety profile of compound 2 as measured by its acute toxicity in
mice appeared less desirable as compared to CFZ (LD50 422 mg/kg vs. > 3000
mg/kg). In addition to addressing the skin discoloration issue, the present work aimed
to identify additional C-2 pyridyl analogues with improved therapeutic windows
while maintaining a favorable PK and efficacy profile.
Because of the high lipophilicity and redox nature of CFZ (redox potential -0.18 V
at pH 7), Vincent C. Barry et al. have hypothesized intracellular redox cycling as a
potential mechanism of CFZ-mediated antimicrobial activity.5 This hypothesis was
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further supported by the work conducted by Yano et al.16 Based on these findings, we
hypothesized that the substituents at the C-2, C-3 and N-5 positions in a
riminophenazine molecule might modulate the redox potential of the phenazine
pharmacophore and at the same time influence the physicochemical properties of the
molecule. Hence we designed the following three target compound series to further
study the impact of each of the three substituent patterns:
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• In the first series, we focused on the N-5 modification, while keeping the C-3
isopropylimino and C-2 pyridyl group intact. When the 4-chloro group in
compound 2 was replaced by various substituents, a series of new N-5
derivatives 10-16 was obtained.
• In the second series, we focused on the C-3 modification by replacing the
isopropyl group at this position with a nitrogen or oxygen-containing alkyl
group. Compounds in this series (17-38) contain one of the following
substitutions: either the 4-tetrahydropyranyl, 4-methoxycyclohexyl,
N-methylpiperidyl, or N-isobutylpiperidyl group.
• In the third series, we focused on the C-2 modification by introducing
various
substituents,
including
both
electron-donating
and
electron-withdrawing groups, to the pyridyl ring (compound 39-78).
This report details our findings regarding the structure-activity relationships of
riminophenazine derivatives bearing a C-2 pyridyl substituent. We further report
additional in vitro and in vivo data for selected compounds in the series to illustrate
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the potential of this novel riminophenazine series for the treatment of MDR- and
XDR-TB.
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Chemistry
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The synthetic route for target compounds 10-78 is outlined in Scheme 1.
Aromatic nucleophilic substitution of commercially available 2-fluoronitrobenzene
(3) with aryl amines afforded compounds 4a-h.17,18 The nitro group in compounds
4a-f was reduced via catalytic hydrogenation. For compounds 4g and 4h, zinc powder
and glacial acetic acid were used to reduce the nitro group, as the halogen on the
phenyl ring could be cleaved under catalytic hydrogenation conditions. Compounds
5a-h were subsequently reacted with 1,5-difluoro-2,4-dinitrobenzene (DFDNB) under
ambient temperature to give the corresponding 6a-h as red or orange solids in good
yields. The remaining fluorine group in compounds 6a-h was substituted by
conventional aromatic nucleophilic displacement with unsubstituted or substituted
3-aminopyridines, such as 3-amino-2-methylpyridine, 3-amino-6-methylpridine,
3-amino-2-methoxylpyridine and 3-amino-5-bromopyridine, in ethanol, to give key
intermediates 7a-v. Reduction of both nitro groups in 7a-f and 7p-v by catalytic
hydrogenation gave tetra-amines 8a-f and 8p-v, which underwent spontaneous
oxidative cyclization to afford riminophenazines 9a-f and 9p-v. Accordingly, 7g-o
were subjected to reduction using zinc powder and glacial acetic acid similar to the
conditions used for the reduction of compounds 4g and 4h. The crude products 8g-o
were washed with water or dilute ammonia to remove any remaining acetic acid for
spontaneous oxidative cyclization to afford imine products 9g-o.19 Finally,
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replacement of the imine (9a-v) with commercially available amines or amines
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(N-isobutyl-4-aminopypiperidine,
N-methyl-4-aminopiperidine,
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4-aminotetrahydropyrane, and 4-methoxycyclohexylamine) 20-22 prepared by known
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23,24
methods provided target compounds (10-78),
which were further purified via
column chromatography or recrystallization. We found that a catalytic amount of
glacial acetic acid could facilitate the final reaction and shorten the reaction time
significantly. For example, for target compound 10, the reaction was complete in
about 13 hours in the presence of a catalytic amount glacial acetic acid, while it took
20 hours in the absence of acetic acid.
1
All target compounds were characterized by H NMR, 13C NMR and HRMS
spectra. 1D NOE study performed on compound 50 showed that the imino double
bond in this molecule had an E configuration. Moreover, the Py-4 H on the pyridyl
ring is in close proximity to 1-H on the phenazine ring, deduced from the fact that a
positive NOE was observed at Py-4 H upon irradiation 1-H (Figure 2). This
interesting NOE phenomenon led us to further study the configuration of this novel
C-2 pyridyl substituted riminophenazine series. The single-crystal X-ray structure of
CFZ was reported by Hodgson in 1985.25 The literature data showed that the value of
the bond angle α at N(3) [defined by C(3)-N(3)-C(21) in CFZ] may be indicative of in
vitro antileprosy activity in these systems (Figure 3). Intramolecular hydrogen
bonding involves N(3)-H⋅⋅⋅⋅N(2) interaction, leading to a new five-membered ring
approximately coplanar with the phenazine core. The authors hypothesized that
compounds with an expanded α angle (131±1º) induced by an intramolecular
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hydrogen bonding would be active in vitro. We also hypothesized that the
five-membered ring formed through this intramolecular hydrogen bonding would
increase the planarity of the molecules and promote intermolecular π-π stacking
interactions, which might be partially responsible for CFZ’s extremely low solubility,
causing crystallization in fat tissues and skin discoloration.
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Single crystals of compound 50 were obtained from a solution of acetone and
characterized by X-ray diffraction. The crystal structure of compound 50 is depicted
in Figure 4. The lattice parameters were a=8.031(3), b=10.835(4), c=13.656(1) Å and
the lattice volume was 2404.3(1) Å3. There were 4 molecules in one lattice. The
dihedral
angles
between
rings
1.0º,
0.3º,
were
A
(C1-C2-C3-C4-C5-C6)/B
(N1-C1-C6-N2-C7-C12)/C
(N1-C1-C6-N2-C7-C12)/D
(C11-C12-C7-C8-C9-C10)/E
(N1-C1-C6-N2-C7-C12):
(C11-C12-C7-C8-C9-C10):
B
B
(C13-C14-C15-C16-C17-C18): 85.6º, and
C
(C19-C20-N4-C21-C22-C23): 18.4º. The configuration of the imine double bond is an
E configuration, as already indicated by the 1D NOE data. Interestingly, the only
intramolecular hydrogen bonding formed in this molecule is by N(3)-H⋅⋅⋅⋅O(1) within
the pyridyl group. This hydrogen bonding prevents the formation of the same
intramolecular hydrogen bonding between N(3)-H⋅⋅⋅⋅N(2) as observed in CFZ (Figure
3), and the important α angle C(9)-N(3)-C(19) is 132.8 º, which was very similar to
the α angle of 131±1º as observed in CFZ. Although the phenazine core in this new
series of compounds is the same as that in CFZ, the absence of the five-membered
ring formed by the intramolecular hydrogen bonding between N(3)-H…N(2) could
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potentially reduce the extent of the molecular planarity, suggesting that the
intermolecular π-π stacking might be interrupted in this new series of
riminophenazine derivatives. We hoped that this may translate into better solubility
and reduced potential for skin discoloration. In fact, we found that the solubility of
compound 50 in pH 1 buffer solution is 0.70 g/100 mL, but CFZ is practically
insoluble.15
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Results and Discussion
All new compounds synthesized through this program were tested for their activity
against M. tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA) in
96-well plate format; the minimum inhibitory concentration (MIC) effecting >90%
growth inhibition was recorded. The compounds were further tested for mammalian
cell cytotoxicity using Vero cells measured as a concentration inhibiting 50% growth
(IC50) as compared to a no treatment control.15 Table 1 summarizes the biological data
for 69 new riminophenazine derivatives. CFZ and compound 2 were used as reference
compounds. The lipophilicity of these compounds was estimated using their ClogP
values, calculated using the MedChem Studio prediction software (ADMET Predictor
5.5, Simulations Plus Inc.). Generally, all compounds bearing a C-2 pyridyl group
have lower ClogP values as compared to CFZ. Additional replacement of the
isopropyl group on the imino group at the C-3 position by an alkyl group with a
heteroatom such as N or O, with the exception of the 4-methoxycyclohexyl and
N-isobutyl-4-piperidyl groups, further reduced ClogP values as exemplified by
compound 17 (△ClogP = -0.41), 19 (△ClogP = -0.04) and 21 (△ClogP = -0.72) as
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compared with compound 2. However, all compounds with a substituent on the C-2
pyridyl group, regardless the nature of the substituent (2-OCH3, 2-CH3, 6-CH3 or
5-Br), showed slightly increased ClogP as compared with their parents.
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Interestingly, most of the compounds bearing a substituted pyridyl group exhibited
potent in vitro activity against M. tuberculosis with MIC values less than 0.03 ꢀg/mL
as compared with compound 2. This observation seems consistent with general SAR
knowledge for the riminophenazine class, that lipophilic compounds tend to be more
potent.11 M. tuberculosis cells have an exceptionally lipophilic cell wall and require
lipophilic molecules to penetrate through passive uptake.26 Therefore, striking a
balance between reducing lipophilicity and retaining potent antimicrobial activity has
been one of the major challenges in searching for new riminophenazine derivatives.
The effect of replacing the chloro atom on the N-5 phenyl ring with an
electron-donating or electron-withdrawing group was first examined as shown in
Table 1. Compounds 11 and 12, with an electron-donating substituent, showed
slightly weaker activity than compound 2, whereas compounds 13, 14, 15 and 16,
with a F, CF3, OCF3 and Br substituent, retained potent activity.
Compounds 17 to 38 possess a heterocyclic R2 group attached to the imino nitrogen
at the C-3 position. All compounds with a nitrogen-containing R2 group showed
relatively weaker potency as compared with compound 2, with MICs ranging from
0.06 to 0.5 ꢀg/mL. Compounds with an oxygen-containing group at this position
displayed potent activity. This observation is consistent with the results obtained from
our previous study.13 4-Tetrahydropyranyl and 4-methoxycyclohexyl substituents at
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this position provide both potent activity and reduce lipophilicity as measured by
ClogP.
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Although most compounds with an unsubstituted pyridyl group at the C-2 position
had potent antimycobacterial activity, many compounds (10 to 38) showed a certain
level of cytotoxicity, with the exception of compounds 14, 23, 27, 32 and 33. We
decided to explore the impact of a substituent R3 on the potency and cytotoxicity of
this promising series by keeping an electron-withdrawing substituent attached to the
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phenyl
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isopropyl,
tetrahydropyranyl
or
trans-4-methoxylcyclohexyl group at R2. A larger set of substituted pyridyl
analogues, such as 2-OCH3, 2-CH3, 6-CH3 and 5-Br, were synthesized and evaluated.
The majority of the compounds bearing a substituted pyridyl group showed potent
antimycobacterial activity with MIC values less than 0.03 ꢀg/mL. More importantly,
many compounds in the series had low cytotoxicity, with IC50 values greater than 64
ꢀg/mL.
Prior to further conducting in vivo efficacy studies, a preliminary in vivo tolerability
study was carried out in mice for 16 compounds which demonstrated potent activity
against M. tuberculosis while maintaining low cytotoxicity. Based on the previous
data obtained for compound 2 (LD50 = 420 mg/kg), all compounds were screened in
vivo with a single dose at 500 mg/kg. Table 2 summarizes the tolerability data for
selected compounds by recording the number of mice which survived after an oral
administration of a single dose of compounds at 500 mg/kg followed by a 7-day
observation. We found that compounds with a 2-methylpyridyl group displayed
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relatively high acute lethal toxicity in mice. On the other hand, compounds with a
2-methoxypyridyl group showed good tolerance in mice at 500 mg/kg.
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Based on the acute toxicity results in mice, complemented by measured logP and
pharmacokinetic data (Table 3),15 we selected 6 compounds that represent significant
structural diversity to evaluate in vivo efficacy in a mouse model of MDR-TB
infection. Compounds were given orally at a dose of 20 mg/kg, once daily for 5 days a
week for a total of 20 days in BALB/c mice, as described in published protocols.15
Table 4 indicates that all tested compounds, including the positive control CFZ,
demonstrated excellent efficacy in mice by reducing lung colony forming units (CFU)
by between 3.5 logs and 5.1 log units; the log CFU counts following treatment with
these compounds were significantly lower than those for the untreated control group
(P< 0.01). During the efficacy study we observed discoloration of the ears, internal
organs and fat tissues of the mice that were treated with riminophenazines. The test
compounds caused a wide range of discolorations, with color intensity appearing in
the following descending order: CFZ = 50 = 52 > 47 > 41 = 40 > 78. This study
illustrates that the tissue discoloration observed following treatment with compounds
of the riminophenazine class could be significantly reduced while maintaining potent
efficacy in vivo. Further evaluation of this promising compound series is currently
under way to fully assess its potential for further development for the treatment of
MDR- and XDR-TB.
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Conclusions
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A series of novel riminophenazine derivatives bearing a C-2 pyridyl substituent was
designed and synthesized. Many compounds in the series exhibited potent in vitro
activity against M. tuberculosis, with improved physicochemical properties as
compared to CFZ. Electron-withdrawing substituents at the N-5 phenyl position
appear important for antimycobacterial activity. 4-Tetrahydropyranyl and
4-methoxycyclohexyl are the preferred substituents at the C-3 imino nitrogen position
for providing both potent antimycobacterial activity and excellent pharmacokinetic
profiles. The majority of compounds with a substituted pyridyl group at the C-2
position demonstrate potent activity against M. tuberculosis with low mammalian cell
cytotoxicity. A preliminary acute toxicity study demonstrated that compounds with a
2’-OCH3 pyridyl group at the C-2 position were well tolerated in mice, whereas
compounds with a 2-methylpridyl appeared less tolerated. As lipophilic compounds
tend to be more active against M. tuberculosis, one of the major challenges in
identifying an improved riminophenazine compound is to balance the needs for
reducing lipophilicity and retaining potent activity. Information obtained from the
X-ray crystal structure of compound 50 suggested a potential solution to addressing
the problem of skin discoloration which has been one of the key limiting factors for
widespread clinical use of CFZ. Compounds with a 2-methoxy substituent attached to
the C-2 pyridyl group demonstrated reduced potential for skin discoloration in mice,
which could be attributed to the unique intramolecular hydrogen bonding and reduced
intermolecular π-π stacking of this structural series.
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Six compounds with potent activity against M. tuberculosis in vitro, low acute
toxicity in mice, and excellent pharmacokinetic profiles were selected and evaluated
in a mouse model of acute MDR-TB infection. All compounds showed significant
efficacy, producing 3-5 logs of CFU reduction in the lungs as compared with the
untreated group after 20 days of treatment. Compounds 41 and 47 exhibited equal or
better in vivo efficacy against MDR-TB than CFZ with reduced discoloration
potential in fat tissues and organs. Further evaluation of these promising compounds
is under way to fully assess their potential for further development.
Experimental Section
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1. General information and methods
1.1 Chemistry
1H NMR spectra were obtained on Varian mercury-300 at 300 MHz; 13C NMR
spectra were obtained on Varian-400 at 100 MHz or Inova-500 at 125 MHz in CDCl3;
DMSO-d6 and acetone-d6, chemicals and coupling constants are recorded in units of
ppm and Hz, respectively. Melting points were determined on Yanaco MP-J3 melting
point apparatus. High-resolution mass spectra were measured on an Agilent 1100
series LC/MSD trap mass spectrometer (ESI-TOF). All target compounds were
purified by chromatography or recrystalization and have purity of ≥ 95% as
determined by HPLC/MS analysis conducted on an Agilent 1100 system, using a
reversed-phase C18 column with 75% CH3CN in water (0.1% HCOOH), with a flow
rate of 0.4 mL·min−1.
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All reagents and solvents were purchased from commercial sources unless
otherwise indicated. Thin layer chromatography was carried out on silica gel plates
(GF254) with visualization of components by UV light (254 nm) or exposure to I2.
Column chromatography was carried out on silica gel (200-300 mesh) or neutral
alumina (100-200 mesh).
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1.2 Minimum Inhibitory Concentration and Cytotoxicity Assays
These were carried out according to published protocols.15
1.3 In Vivo Acute MDR-TB Infection Assay
SPF BALB/c male mice weighing 18-20 g were used in this study. Mice were
infected via aerosol with a suspension of 5×106 CFU/mL Multidrug-Resistant
Mycobacterium tuberculosis (the MDR clinical isolate (040), known to be resistant to
INH, RFP, PTO, RPT, OFX, and LVFX) using a Glas-Col inhalation system, to
deposit 50-100 bacilli into the lungs of each animal. The course of infection was
followed by plating homogenates of harvested organs [n = 3] on 7H11agar plates
(7H11 plates containing 10% oleic acid-albumin-dextrose-catalase (OADC)
enrichment and 50 µg/mL cycloheximide, 200 U/mL polymyxin B, 50 µg/mL
carbenicillin and 20 µg/mL trimethoprim) and determining CFU on Day 3, Day 10
and Day 30 post-infection. Drugs and compounds were dissolved or suspended in
0.5% CMC and administered by oral gavage in a maximum volume of 200 ꢀL such
that a dose of 20 mg/kg body weight was achieved. Mice were treated 5/7 days per
week during the acute phase of infection, from Day 10 until Day 30. Each treated
group was composed of 5 or 6 mice, while the control group, which received only
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CMC, was composed of 7 to 10 mice. Mice were sacrificed the day after the last day
of treatment, organ weights determined, lungs removed, homogenized, and serially
diluted in 10-fold steps in HBSS. One hundred microliters were spread on 7H11 agar
in duplicate. The plates were incubated at 37°C for 2-3 weeks. Data are expressed as
the log10 (and as log10 reduction) provided by a given dose of the compound against
the growth of the organism in the untreated control group. Mean log10 values were
calculated from bacterial burden counts. Student’s t test was used to compare means
between test and control groups. A P value of ≤ 0.05 was considered significant.
2. Procedure A for synthesis of the intermediate 5g and 5h.
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2-(4-Chloroanilino)-aniline. (5h). Zinc powder (9 g, 13.8 mol) was added
portion-wise into a mixture of 4h (2.5 g, 10 mmol) in 50 mL of CH2Cl2 and 3 mL of
glacial acetic acid and stirred at room temperature for 4 h. After filtration, the filtrate
was concentrated to dryness. Water was added, filtered, and the resulting material was
washed with water to give a brown solid. This solid was used directly in the next step
without further purification.
In a similar manner, 5g were synthesized according to Procedure A.
3. Procedure B for synthesis of the intermediate 9g-o.
5-(4-Chlorophenyl)-3-imino-2-(3-pyridylamino)-3,5-dihydrophenazine
(9h):
Zinc powder (71 g, 1.09 mol) was added portion-wise into a mixture of 7h (28.6 g, 60
mmol) in 150 mL of glacial acetic acid cooled with an ice water bath. The mixture
was stirred until the color turned to light green, and then filtered and washed with
glacial acetic acid and anhydrous methanol. The filtrate was concentrated and the
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residue was treated with water and made alkaline with ammonia. The solid was
filtered, washed with water and then dissolved in anhydrous methanol. The solution
was stirred under air overnight. The solid formed was filtered to give crude product
23.1 g, which was taken to the next step without further purification.
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In a similar manner, 9g, and 9i-o were synthesized according to Procedure B.
4. Procedure C for synthesis of the target compounds.
2-Phenylanilino-nitrobenzene (4a). A mixture of 2-fluoronitrobenzene (21.2 g,
150 mmol), aniline (16.8 g, 180 mmol), and anhydrous potassium fluoride (8.7 g, 150
mmol) was stirred at 160℃ for 8 hours. The mixture was then cooled, water and ethyl
acetate were added, the water layer was extracted with ethyl acetate, and the organic
layer was washed with 2N HCl and dried with anhydrous sodium sulfate. After
filtration, the filtrate was concentrated in vacuo, and 32 g crude product was
recrystallized in 100 mL 95% ethanol to give 24.6 g 4a as red solid in 76.6% yield.
Mp: 69-70 ℃. 1H NMR (300 MHz, acetone-d6) δ: 9.43 (s, 1 H), 8.16 (d, J = 7.2 Hz, 1
H), 7.35-7.51 (m, 5 H), 7.21-7.27 (m, 2 H), 6.86 (t, J = 7.2 Hz, 1 H).
1-[2-(Anilino)anilino]-3-fluoro-4,6-dinitrobenzene (6a). A mixture of 4a (24.6 g,
115 mmol) and 10% Pd/C (2.4 g) in ethanol was shaken at room temperature under a
hydrogen atmosphere (40 psi) for 1.5 hours. After filtration, DFDNB (23.4 g, 115
mmol) and triethylamine (11.6 g, 115 mmol) were added to the filtrate (5a); the
mixture was stirred at room temperature for 3hours, filtered, and washed with ethanol
to give 38.1 g 6a as a red solid in 89.9% yield. Mp: 165-167 ℃. 1H NMR (300 MHz,
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acetone-d6) δ: 9.93 (s, 1 H), 9.00 (d, J = 8.1 Hz, 1 H), 7.32-7.42 (m, 3 H), 7.22 (t, J =
7.5 Hz, 2 H), 7.02-7.08 (m, 3 H), 6.91 (t, J = 7.2 Hz, 1 H), 6.69 (d, J = 14.1 Hz, 1 H).
1-[2-(Anilino)anilino]-3-(3-pyridyl)amino-4,6-dinitrobenzene (7a). A mixture of
6a (14.73 g, 40 mmol), 3-aminopyridine (3.76 g, 40 mmol), triethylamine (4.04 g, 40
mmol) and THF was refluxed for 18 hours. After being cooled to room temperature,
the mixture was concentrated in vacuo, CH2Cl2 was added to the residue, and the solid
obtained was filtered to give 15.95 g of product as a red solid in 90.1% yield. Mp:
212-213 ℃. 1H NMR (300 MHz, DMSO-d6 ) δ: 9.71 (s, 1 H), 9.49 (s, 1 H), 9.00 (s, 1
H), 8.37-8.41 (m, 2 H), 7.60-7.63 (m, 2 H), 7.32-7.36 (m, 1 H), 7.08-7.20 (m, 5 H),
6.82-6.85 (m, 4 H), 5.96 (s, 1 H).
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5-Phenyl-3-imino-2-(3-pyridyl)amino-3, 5-dihydrophenazine (9a). A mixture of
7a (15 g, 34 mmol) and 10% Pd/C in methanol and THF was shaken at room
temperature under a hydrogen atmosphere (40 psi) for 10 hours. After filtration, the
filtrate was concentrated in vacuo and the residue (8a) was dissolved in methanol, and
the solution stirred at room temperature under air for 10 hours. The mixture was
1
filtered to give 7.69 g of product as a red solid in 62.5% yield. Mp: 216-218 ℃. H
NMR (300 MHz, DMSO-d6) δ: 8.54 (d, J = 2.1 Hz, 1 H), 8.25 (d, J = 5.7 Hz, 1 H),
7.76-7.81 (m, 3 H), 7.71 (d, J = 7.5 Hz, 1 H), 7.60-7.63 (m, 1 H), 7.50-7.52 (m, 2 H),
7.38-7.42 (m, 1 H), 7.15-7.23 (m, 2 H), 6.63 (s, 1 H), 6.42 (d, J = 7.2 Hz, 1 H), 5.40
(s, 1 H). ESI/MS: m/z [M+H]+ 364.1534.
5-Phenyl-3-isopropylimino-2-(3-pyridyl)amino-3,5-dihydrophenazine (10).
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To a solution of 9a (0.182 g, 0.5 mmol) and isopropylamine (1 mL, 11.7 mmol) in
dioxane was added 0.06 mL glacial acetic acid, and the mixture was stirred and heated
at 110℃ in a sealed tube for 13 hours. The mixture was concentrated in vacuo and the
residue was purified by column chromatography on silica gel, using ethyl acetate; 170
mg black oil was obtained, which was purified by column chromatography on neutral
alumina, using petro ether: ethyl acetate = 2:1 to give 125 mg of product as a red solid
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in 62.5% yield. Mp: 183-185 ℃. H NMR (300 MHz, CDCl3) δ: 8.59 (d, J = 2.4 Hz,
1 H), 8.32 (d, J = 3.9 Hz, 1 H), 7.62-7.80 (m, 5 H), 7.29-7.35 (m, 3 H), 7.10-7.19 (m,
2 H), 6.85 (s, 1 H), 6.49 (d, J = 7.5 Hz, 1 H), 5.28 (s, 1 H), 3.38-3.46 (m, J = 6.0 Hz, 1
13
H), 1.07 (d, J = 6.0 Hz, 6 H). C NMR (100 MHz, CDCl3) δ: 150.9, 150.6, 144.2,
143.9, 143.7, 137.6, 136.9, 135.6, 135.1, 131.7, 131.3, 129.7, 128.8, 128.2, 127.8,
127.6, 123.6, 122.8, 114.2, 99.4, 89.1, 49.3, 23.5. HRMS (ESI-TOF+): m/z [M+H]+
calcd for C26H24N5: 406.2031; found: 406.2041.
In a similar manner, other target compounds (11-78) were synthesized according to
Procedure C.
Acknowledgments: The authors wish to thank the Global Alliance for TB Drug
Development and the National Science and Technology Project of China (No.
2009ZX09102-054) for financial support of this project.
Supporting Information Available: Additional characterizations for compounds in
Tables 1; NOE spectra and crystal data of compound 50. This material is available
free of charge via the internet at http://pubs.acs.org.
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Abbreviations: CFZ, clofazimine; TB, tuberculosis; M. tuberculosis,
mycobacterium tuberculosis; MDR-TB, multidrug-resistant tuberculosis; XDR-TB,
extensively drug-resistant tuberculosis; MOA, mechanism of action; LD50, dose that is
lethal in 50% of test subjects; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; NMR,
nuclear magnetic resonance; HRMS, high-resolution mass spectrometry; NOE,
nuclear overhauser effect; MABA, microplate alamar blue assay; MIC, minimum
inhibitory concentration; IC50, half-maximum inhibitory concentration; PK,
pharmacokinetic; CFU, colony forming unit; INH, isoniazid; RFP, rifampicin; PTO,
protionamid; RPT, rifapentine; OFX, ofloxacin; LVFX, levofloxacin; HBSS, hank's
balanced salt solution; SI, selectivity index; T1/2, half-life; Cmax, maximum plasma
concentration; AUC, area under curve.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: (8610) 63165244. Fax: (8610) 63165244. E-mail: joyce@imm.ac.cn;
*Phone: 646-616-8633. Fax: 212-227-7541. E-mail: Zhenkun.ma@tballiance.org.
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(16) Yano, T.; Kassovska-Bratinova, S.; Teh, J. S.; Winkler, J.; Sullivan, K.;
Isaacs, A.; Schechter, N. M.; Rubin, H. Reduction of clofazimine by mycobacterial
type 2 NADH: quinone oxidoreductase: a pathway for the generation of bactericidal
levels of reactive oxygen species. J. Biol. Chem. 2011, 286, 10276-10287.
(17) Zhu, Y. F.; Lin, G. Q.; Chen, Y. Q. New modified preparation of
2-nitrodiarylamines. Synthesis 1990, 430.
(18) Xu, Z. B.; Lu, Y.; Guo, Z. R. An efficient and fast procedure for the
preparation of 2-nitrophenylamines under microwave conditions. SynLett 2003, 4,
564-566.
(19) Vivian, D. L.; Greenberg, G. Y.; Hartwell, J. L. Direct ring closure through the
nitro group. Isomer formation in the synthesis of unsymmetrical phenazines, and
some general observations on the phenazine syntheses. J. Org. Chem, 1951, 16, 1-7.
(20) Iwakubo, M.; Takami, A.; Okada, Y.; Kawata, T.; Tagami, Y.; Sato, M.;
Sugiyama, T.; Fukushima, K.; Taya, S.; Amano, M.; Kaibuchi, K.; Iijima, H. Design
and synthesis of rho kinase inhibitors (III). Bioorg. Med. Chem. 2007, 15, 1022-1033.
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(21) Marcello, A.; Valerio, B.; M. Candida, C.; Roberto, C.; Luca, N.; Alessandra,
T. One-pot, new stereoselective synthesis of endo-tropanamine. Tetrahedron Lett.
2001, 42, 4257-4259.
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(22) Mortensen, D. S.; Mederos, M. M. D.; Sapienza, J. J.; Albers, R. J.; Lee B. G.;
Harris, R. L. III; Shevlin, G. I.; Huang, D. H.; Schwarz, K. L.; Packard, G. K.; Parnes,
J. S.; Papa, P. W.; Tehrani, L. R.; Perrin-ninkovic, S. Heteroaryl compounds,
compositions thereof, and methods of treatment therewith. Patent WO 2008051493
A2, 2008.
(23) Sharma, H. L.; Sharma, V. N.; Mital, R. L. Studies on Nitrophenothiazines I.
their synthesis and an explanation for the peculiar behaviour of halonitrobenzenes
with o-aminothiophenol. Aust. J. Chem. 1968, 21, 3081-3086.
(24) Andre, G.; Andre, R. Novel phenazine compounds and process. Patent US
3499899 A, 1970.
(25) Rychlewska, U.; Broom, M. B. H.; Eggleston, D. S.; Hodgson, D. J.
Antileprosy dihydrophenazines. Structural characterization of two crystal forms of
clofazimine and of isoclofazimine, B.3857. J. Am. Chem. Soc. 1985, 107, 4768-4772.
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FIGURES
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6
7
Figure 1. Structures of CFZ and its analogues
8
9
Cl
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11
12
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4
6
9
5
N
N
N
3
Cl
7
8
2
N
H
1
10
CFZ
OMe
Cl
N
N
N
N
N
N
Cl
N
N
H
N
H
Compound 2
Compound 1
Figure 2. Structure of compound 50
F
3'
2'
1'
D
H
4
N
B
N
N
A
C
H
NH
O
N
1
H
E
Py-4 H
50
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Figure 3. X-ray crystallographic structure of CFZ
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60
Figure 4. X-ray crystallographic structure of compound 50
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SCHEMES
Scheme 1. Synthesis of the target compounds 10-78a
7
8
9
R1
10
11
12
13
14
15
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20
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60
O2N
NH
H
N
H
N
H
N
F
ii
iii
i
NO2
NO2
NO2
R1
NH2
R1
F
3
4a-h
5a-h
6a-h
R1
R1
O2N
H2N
NH
NH
H
N
iv
H
N
v
NO2
NH2
R3
R3
HN
HN
N
N
8a-v
7a-v
R1
R1
R2
N
vii
N
NH
vi
N
R3
R3
N
N
N
N
H
N
N
H
9a-v
10-78
7q-9q: R1=F, R3=6-Me;
7r-9r: R1=F, R3=2-OMe;
7s-9s: R1=CF3, R3=2-Me;
7t-9t: R1=CF3, R3=6-Me;
7u-9u: R1=CF3, R3=2-OMe;
7v-9v: R1=OCF3, R3=2-OMe
4a-6a: R1=H;
7i-9i: R1=Cl, R3=2-Me;
7j-9j: R1=Cl, R3=6-Me;
7k-9k: R1=Cl, R3=2-OMe;
7l-9l: R1=Cl, R3=5-Br;
7m-9m: R1=Br, R3=2-Me;
7n-9n: R1=Br, R3=6-Me;
7o-9o: R1=Br, R3=2-OMe;
7p-9p: R1=F, R3=2-Me;
7a-9a: R1=H, R3=H;
4b-6b: R1=Me;
4c-6c: R1=SMe;
4d-6d: R1=F;
4e-6e: R1=CF3;
4f-6f: R1=OCF3;
4g-6g: R1=Br;
4h-6h: R1=Cl;
7b-9b: R1=Me, R3=H;
7c-9c: R1=SMe, R3=H;
7d-9d: R1=F, R3=H;
7e-9e: R1=CF3, R3=H;
7f-9f: R1=OCF3, R3=H;
7g-9g: R1=Br, R3=H;
7h-9h: R1=Cl, R3=H;
aReagents and conditions: (i) KF, R1-aniline, 160℃; (ii) 10% Pd/C, H2 or
Zn/CH3COOH, rt; (iii) DFDNB, Et3N, ethanol, rt; (iv) 3-aminopyridine or substituted
3-aminopyridine, Et3N, THF, reflux; (v) 10% Pd/C, H2 or Zn/CH3COOH, rt; (vi) air,
methanol, rt; (vii) R2NH2, CH3COOH, dioxane, reflux or in sealed tube, 110℃.
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TABLES
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9
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22
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36
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39
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Table 1. Physicochemical property (ClogP), antimycobacterial potency (MIC),
cytotoxicity (VERO IC50) and selectivity index (SI) values for target compounds.
R1
R2=
O
C:
O
N
B:
A:
*
*
*
R2
N
5'
N
N
6'
R3
4'
O
E:
N
F:
N
D:
N
1'
*
*
N
H
*
3'
2'
Compd.
R1
R2
R3
ClogPa MIC
IC50(Vero) SIb
(ꢀg/mL)
0.12
0.03
0.06
0.06
0.05
0.03
0.03
0.03
0.02
0.03
0.03
0.125
0.25
0.25
0.06
(ꢀg/mL)
6.80
5.12
4.64
5.04
5.41
4.74
5.18
4.79
5.22
4.71
5.46
5.08
6.15
4.40
4.33
68.6
8.2
572
273
66
CFZ
2
Cl
H
A
A
A
A
A
A
A
A
B
C
D
E
H
H
H
H
H
H
H
H
H
H
H
H
H
H
3.93
2.71
1.83
6.25
19.66
4.93
2.3
10
11
12
13
14
15
16
17
18
19
20
21
22
CH3
45
SCH3
F
37
208
655
164
115
83
CF3
OCF3
Br
Cl
2.5
Cl
12.03
5.58
15.06
4.02
7.36
401
45
Cl
Cl
60
Cl
F
16
F
B
123
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