Systematic evaluation of structure-activity relationships of the riminophenazine class and discovery of a C2 pyridylamino series for the treatment of multidrug-resistant tuberculosis

Article abstract of DOI:10.3390/molecules17044545

Clofazimine, a member of the riminophenazine class of drugs, is the cornerstone agent for the treatment of leprosy. This agent is currently being studied in clinical trials for the treatment of multidrug-resistant tuberculosis to address the urgent need for new drugs that can overcome existing and emerging drug resistance. However, the use of clofazimine in tuberculosis treatment is hampered by its high lipophilicity and skin pigmentation side effects. To identify a new generation of riminophenazines that is less lipophilic and skin staining, while maintaining efficacy, we have performed a systematic structure-activity relationship (SAR) investigation by synthesizing a variety of analogs of clofazimine and evaluating their anti-tuberculosis activity. The study reveals that the central tricyclic phenazine system and the pendant aromatic rings are important for anti-tuberculosis activity. However, the phenyl groups attached to the C2 and N5 position of clofazimine can be replaced by a pyridyl group to provide analogs with improved physicochemical properties and pharmacokinetic characteristics. Replacement of the phenyl group attached to the C2 position by a pyridyl group has led to a promising new series of compounds with improved physicochemical properties, improved anti-tuberculosis potency, and reduced pigmentation potential.

Full text of DOI:10.3390/molecules17044545

Molecules 2012, 17, 4545-4559; doi:10.3390/molecules17044545
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Systematic Evaluation of Structure-Activity Relationships of the
Riminophenazine Class and Discovery of a C2 Pyridylamino
Series for the Treatment of Multidrug-Resistant Tuberculosis
Binna Liu 1, Kai Liu 1, Yu Lu 2, Dongfeng Zhang 1, Tianming Yang 1, Xuan Li 1, Chen Ma 1,
Meiqin Zheng 2, Bin Wang 2, Gang Zhang 1, Fei Wang 1, Zhenkun Ma 3,*, Chun Li 1,
Haihong Huang 1 and Dali Yin 1,*
1
State Key Laboratory of Bioactive Substances and Function of Natural Medicine & Beijing Key
Laboratory of Active Substance 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, China
2
Beijing Tuberculosis and Thoracic Tumor Research Institute, 97 Ma Chang Street,
Beijing 101149, China
3
Global Alliance for TB Drug Development, 40 Wall Street, New York, NY 10005, USA
* Authors to whom correspondence should be addressed; E-Mails: Zhenkun.ma@tballiance.org (Z.M.);
yindali@imm.ac.cn (D.Y.); Tel.: +1-646-616-8633 (Z.M.); Fax: +1-212-227-7541 (Z.M.);
Tel./Fax: +86-10-6303-7952 (D.Y.).
Received: 9 March 2012; in revised form: 9 April 2012 / Accepted: 10 April 2012 /
Published: 17 April 2012
Abstract: Clofazimine, a member of the riminophenazine class of drugs, is the cornerstone
agent for the treatment of leprosy. This agent is currently being studied in clinical trials for
the treatment of multidrug-resistant tuberculosis to address the urgent need for new drugs
that can overcome existing and emerging drug resistance. However, the use of clofazimine
in tuberculosis treatment is hampered by its high lipophilicity and skin pigmentation side
effects. To identify a new generation of riminophenazines that is less lipophilic and skin
staining, while maintaining efficacy, we have performed a systematic structure-activity
relationship (SAR) investigation by synthesizing a variety of analogs of clofazimine and
evaluating their anti-tuberculosis activity. The study reveals that the central tricyclic
phenazine system and the pendant aromatic rings are important for anti-tuberculosis
activity. However, the phenyl groups attached to the C2 and N5 position of clofazimine
can be replaced by a pyridyl group to provide analogs with improved physicochemical
properties and pharmacokinetic characteristics. Replacement of the phenyl group attached

Molecules 2012, 17
to the C2 position by a pyridyl group has led to a promising new series of compounds with
4546
improved physicochemical properties, improved anti-tuberculosis potency, and reduced
pigmentation potential.
Keywords: anti-tuberculosis; MDR-TB; riminophenazine; clofazimine; skin pigmentation
1. Introduction
Tuberculosis (TB) infects about some 9.2 million and kills approximately 1.8 million people
globally every year [1]. Despite the best treatment and control efforts using available vaccines and
drugs, the prevalence of TB throughout the World remains high, due in part to the emergence of
multidrug resistant Mycobacterium tuberculosis (MDR-TB) [2] and the global HIV epidemic. The
treatment of MDR-TB and TB/HIV co-infection using the currently available drug regimens is
extremely challenging [3]. New and more effective drugs are needed to address these challenges.
In an effort to identify new anti-tuberculosis agents that can be effective against MDR-TB, we
decided to revisit the riminophenazine class of drugs. Clofazimine (CFZ, Figure 1), a member of the
riminophenazine class, is the cornerstone agent for the treatment of leprosy. This agent was first
synthesized by Barry’s group [4] and has demonstrated potent in vitro activity against M. tuberculosis [5].
Recent studies indicate that clofazimine is equally potent against drug-susceptible and drug-resistant
(including MDR) M. tuberculosis strains, which suggests that it operates via a novel mechanism of
action. In addition, the frequency of resistance development for this compound is extremely low as
compared with other anti-tuberculosis drugs [6]. Clofazimine is currently in clinical trials for the
treatment of MDR-TB to address the urgent need for new drugs that can overcome existing and
emerging drug resistance in tuberculosis. However, the widespread use of clofazimine in tuberculosis
treatment is hampered by its extremely high lipophilicity and strong skin pigmentation side effects.
Although a large number of analogues of clofazimine have been prepared, they are limited in scope.
The modifications are limited to the substituents on two pendant phenyls and the imino group [7].
To identify a new generation of riminophenazines that is both less lipophilic and skin staining, while
maintaining its efficacy against tuberculosis, a systematic structure-activity and structure-liability
relationship (SLR) study is needed.
Figure 1. Structure of clofazimine.
Cl
D
6
4
C
1
5
3
2
N
B
N
N
Cl
7
8
E
A
9
N
H
10
(E)-N,5-bis(4-chlorophenyl)-3-(isopropylimino)-3,5-dihydrophenazin-2-amine

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Clofazimine is a lipophilic molecule as indicated by its high Clog P value of approximately 7.48 [8].
Its undesirable physicochemical and pharmacokinetic properties contribute to its side effects and limit
its clinical use. In particular, its extremely long half-life and propensity for tissue accumulation and
precipitation together with the dye property lead to unwelcome skin pigmentation. Herein we report
our SAR and SLR studies on the riminophenazine class with a goal of identifying a new generation of
compounds with improved physicochemical properties that are highly efficacious against both
drug-susceptible and MDR-TB and at the same time incur reduced skin pigmentation potential.
2. Results and Discussion
To improve its physicochemical properties and circumvent the skin pigmentation problem of
clofazimine, we first explored the possibility of decreasing the intrinsic colour of the molecules by
reducing the conjugation system of the riminophenazine core structure. Thus, we systematically
removed one of the phenyl rings (A, D or E) from the molecule while keeping rings B, C, and the
imino moiety intact. The syntheses of these analogs are illustrated in Schemes 1 and 2, respectively.
Scheme 1. Synthesis of A-ring deletion compound, riminoquinoxaline. a
a
Reagents and conditions: (1a) ClC6H4NH2, anhydrous K2CO3, anhydrous KF, 170 °C; (1b)
anhydrous CH3OH, 10% Pd/C, H2, r.t.; (1c) ethyl oxalylchloride, toluene, reflux; (1d) KNO3, con.
H2SO4, −5 °C ~ r.t.; (1e) ClC6H4NH2, anhydrous KF, DMSO, 140 °C; (1f) anhydrous CH3OH, Zn,
AcOH; (1g) AcOH, anhydrous CH3OH, acetone, NaBH4, r.t.; (1h) THF, LiAlH4, heated on silica plate.
The synthesis of the A-ring deletion analogs (riminoquinoxalines) was started from
2,5-difluoronitrobenzene (Scheme 1). After replacement of the ortho-fluoro group with 4-chloroaniline,
compound 1 was reduced to give diamine 2. Compound 2 was then treated with ethyl oxalyl chloride

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to form the B ring. After nitration of 3, the activated fluoro at the ortho position of the nitro adduct of 4
was replaced by 4-chloroaniline. Reduction of the nitro group in 5 produced compound 6, followed by
reductive alkylation to provide compound 7. Reduction of the carbonyl groups in 7 with LiAlH4
followed by an air auto-oxidization provided the target compound 8.
Scheme 2. Synthesis of D-ring deletion riminophenazines. a
R1
R1
R1
F
NH
NH
NH
NO2
2a
2c
2b
NO2
NO2
NH2
N
H
NO2
10a-b
11a-b
9a-b
F
R1
R1
NH
NO2
NH
NH2
2f
2d
2e
N
H
NO2
N
H
NH2
HN R2
HN R2
12a-b
13a-b
R1
R1
N
Cl
R2= CH3
9a-15a
9b-15b
R1= CH3 R2=
Cl
N
NH
N
2g
R1=
N
N R2
H
N
N R2
H
14a-b
15a-b
a
Reagents and conditions: (2a) R1NH2, anhydrous EtOH, reflux; (2b) anhydrous CH3OH, 10%
Pd/C, H2, r.t.; (2c) anhydrous CH3OH, DFDNB, Et3N; (2d) ClC6H4NH2, anhydrous KF, anhydrous
EtOH, reflux; (2e) Zn, AcOH; (2f) air, AcOH; (2g) isopropylamine, dioxane, 110 °C.
Both D-ring and E-ring deletion compounds 15a and 15b were synthesized starting from
1,5-difluoro-2,4-dinitrobenzene (DFDNB) (Scheme 2).
Thus compound 9a–b was reduced to diamine 10a–b which was then coupled with 1,5-difluoro-
2,4-dinitrobenzene to give 11a–b. The second fluorine atom was replaced by 4-chloroaniline to afford
12a–b. Reduction of both nitro groups gave 13a–b which underwent spontaneous cyclization to afford
riminophenazines 14a–b. Replacement of the imine with isopropylamine gave the target compounds
15a–b.
The A-ring deletion compound 8 exhibited considerably reduced in vitro activity against
M. tuberculosis (MIC90 = 18.89 μM, Table 1). When the appendant phenyl group D or E was replaced
by a methyl group (compounds 15a and 15b), the antimycobacterial activity was also abolished
(MIC90 ≥ 42.45 μM and MIC90 = 21.23 μM respectively, Table 1).

Molecules 2012, 17
Table 1. In vitro anti-tuberculosis activity of the riminophenazine analogues with
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systematic deletion of one of the phenyl rings against M. tuberculosis H37Rv.
MIC90
(μM)
MIC90
(μM)
Comp.
CFZ
Structure
Comp.
15a
Structure
0.25
>42.45
8
18.89
15b
21.23
Failing to identify analogues that retained antimycobacterial activity through dissecting their core
structures, we turned our attention to the modification of the D- and E-rings of the molecule to
improve its physicochemical properties and thereby change tissue distribution and tissue accumulation.
It was anticipated that the Clog P of the molecule would decrease if one or both of the phenyl groups
were replaced by a pyridyl group. Thus compounds 21a–b and 25 were synthesized following a
modified synthetic procedure (Schemes 3 and 4).
Scheme 3. Synthesis of compound 21a–b. a
a
Reagents and conditions: (a) anhydrous KF, anhydrous K2CO3, 170 °C; (b) CH2Cl2, Zn, AcOH;
(c) anhydrous THF; (d) anhydrous EtOH, reflux; (e) Zn, AcOH; (f) isopropylamine, dioxane, 110 °C.

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Scheme 4. Synthesis of compound 25. a
a Reagents and conditions: (5a) 3-aminopyridine, DIPEA, THF, reflux; (5b) Zn, AcOH, anhydrous
CH3OH, r.t.; (5c) air, anhydrous CH3OH, r.t., overnight; (5d) isopropylamine, dioxane, 110 °C.
The Clog P of the molecule decreases by 2 if one of the phenyl groups is replaced by a pyridyl
group and by 4 if both phenyl groups are replaced by pyridine. In the in vitro assay, the N5 pyridyl
analogues 21a and 21b showed reduced potency (MIC90 0.57 and 1.23 M respectively). To our
surprise, the C2 pyridylamino analogue 25 demonstrated improved potency against M tuberculosis
H37Rv (MIC90 0.07 M) as compared to clofazimine (MIC90 0.25 M) (Table 2).
Table 2. Riminophenazines with R1 and R3 variation and their anti-tuberculosis activity
against M. tuberculosis H37Rv, physicochemical property (log P).
Comp.
CFZ
R1
R2
ClogP logP MIC90 (M)
7.137 5.43
5.322
0.25
0.57
21a
21b
25
-
3.507 2.76
5.322 3.43
1.23
0.07
In addition, compound 25 demonstrated excellent in vivo efficacy in a mouse tuberculosis model as
measured by a significant drop in bacterial colony-forming units (CFU) in the lung as compared
to a no-treatment control group [9]. The mean log CFU count in the lung dropped by about 5 units

Molecules 2012, 17
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(3.83 ± 0.27 CFU/lung ) for the group treated with compound 25 as compared to the untreated group
(8.53 ± 0.32 CFU/lung), and by about 3 units as compared to rifampin (RIF)-treated positive control
groups (RIF 6.71 ± 0.13 CFU/lung, CFZ 6.33 ± 0.07 CFU/lung). Compound 25 also demonstrated a
shorter half-life (t1/2) as compared to clofazimine and reduced abdominal tissue pigmentation, which is
strikingly different from the effect on those animals treated by clofazimine (data not shown). This SAR
and SLR study illustrates that all five aromatic rings of clofazimine are important for maintaining
potent anti-tuberculosis activity. The two pendant phenyl rings attached to the C2 and N5 positions,
especially the C2 position, can be replaced by a pyridyl group, and such analogues have demonstrated
potent in vitro anti-tubercular activity, excellent in vivo efficacy, and reduced skin pigmentation potential.
3. Experimental
3.1. Reagents and Instrumentation
Unless otherwise indicated, all reagents and solvents were used as received from the suppliers.
Reactions were monitored for completion by thin layer chromatography (TLC) using silica gel GF-254
plates with detection under UV (254 nm) light. 1H-NMR spectra were recorded on a Varian 300 MHz
or 400 MHz instrument in CDCl3, CD3OD, DMSO-d6 or acetone-d6 solutions. 13C-NMR spectra were
recorded on a Varian instrument at 100 MHz or 150 MHz with CDCl3 or DMSO-d6 as solvents.
Chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane (TMS).
Coupling constants (J) are reported in Hz. High-resolution mass spectra were acquired from an Agilent
1100 series LC/MSD mass spectrometer. All MS experiments were performed using electrospray
ionization (ESI-TOF+) in positive ion mode. Column chromatography was carried out with silica gel
(200~300 mesh).
3.2. Chemistry
3.2.1. Synthesis of 2-(4-chloroanilino)-5-fluoronitrobenzene (1)
4-Chloroaniline (12.76 g, 100 mmol), 2,5-difluoronitrobenzene (8.0 g, 50 mmol), anhydrous K2CO3
(3.5 g, 25 mmol) and anhydrous KF (2.9 g, 50 mmol) were mixed and heated at 170 °C for 14 h. After
cooling to room temperature, water was added and the solid formed was filtered and washed with
water. The crude product was purified via recrystallization from 95% ethanol to give 1 as an orange
solid (11.8 g, 89%). 1H-NMR (300 MHz, CDCl3) δ 9.25 (s, 1H), 7.94–7.90 (m, 1H), 7.39 (d, J = 8.7 Hz,
2H), 7.21–7.17 (m, 4H). ESI/MS (m/z): 267 [M+H]+.
3.2.2. Synthesis of 1-(4-chlorophenyl)-6-fluoroquinoxaline-2,3(1H,4H)-dione (3)
Compound 1 above (6.45 g, 24 mmol) was suspended in anhydrous methanol (100 mL). The
mixture was hydrogenated with 10% Pd/C (1.3 g) at room temperature at 1 atmosphere pressure of
hydrogen for 8 h. After removal of catalyst, the solvent was concentrated under reduced pressure. The
residue was dissolved in toluene (150 mL) and ethyl oxalyl chloride (10 mL, 89 mmol) was added. The
mixture was refluxed for 1 h and then cooled to room temperature Approximately one half of the
solvent was removed under reduced pressure and the resulting solid was filtered and recrystallized

Molecules 2012, 17
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1
from methanol to give 3 as a white solid (4.97 g, 71%). H-NMR (300 MHz, DMSO-d6) δ 12.18
(s, 1H), 7.69 (d, J = 8.7 Hz, 2H), 7.43 (d, J = 8.7 Hz, 2H), 6.99 (dd, J = 9.0, 2.7 Hz, 1H), 6.87–9.80
(m, 1H), 6.34 (dd, J = 9.3, 5.1 Hz, 1H). ESI/MS (m/z): 291 [M+H]+.
3.2.3. Synthesis of 1-(4-chlorophenyl)-6-fluoro-7-nitroquinoxaline-2,3(1H,4H)-dione (4)
Compound 3 above (2.9 g, 10 mmol) was dissolved in concentrated sulfuric acid (30 mL) and
cooled to −5 °C. KNO3 (1.1 g, 8 mmol) was added into the solution portion-wise. After addition, the
mixture was stirred at 0 °C for 1 h and then at room temperature for 1 h. The reaction mixture was
slowly poured into ice water, and the solid formed was filtered and washed with water. The product
1
was air-dried to give a light yellow solid 3.4 g (quantitative yield). H-NMR (300 MHz, DMSO-d6)
δ 12.62 (s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 11.7 Hz, 1H), 6.96
(d, J = 6.9 Hz, 1H). ESI/MS (m/z): 336 [M+H]+.
3.2.4. Synthesis of 1-(4-chlorophenyl)-6-(4-chloroanilino)-7-nitroquinoxaline-2,3(1H,4H)-dione (5)
Compound 4 above (0.34 g, 1.0 mmol), DMSO (3 mL), anhydrous KF (0.12 g, 2.0 mmol), and
4-chloroaniline (1.27 g, 10 mmol) were heated at 140 °C for 25 h. After cooling to room temperature,
5 M HCl was added and the resulting solid was collected by filtration and washed with water. The
crude product was purified by column chromatography to give 5 as an orange solid (0.22 g, 50%).
1H-NMR (300 MHz, DMSO-d6) δ 12.09 (s, 1H), 9.35 (s, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.52–7.46
(m, 4H), 7.35 (d, J = 8.4 Hz, 2H), 7.01 (s, 1H), 6.99 (s, 1H). ESI/MS (m/z): 443 [M+H]+.
3.2.5. Synthesis of 1-(4-chlorophenyl)-6-(4-chloroanilino)-7-(isopropylamino)-quinoxaline-2,3-
(1H,4H)-dione (7)
Zinc powder (0.13 g) was added portionwise to a vigorously stirred mixture of 5 (0.08 g,
0.18 mmol) in glacial acetic acid (2 mL) and anhydrous methanol (5 mL). After the reaction was
complete, as confirmed by TLC, the reaction mixture was filtered. The filtrate was concentrated under
reduced pressure. Water was then added to the residue. After filtration, the yellow solid was dissolved
in a mixture of glacial acetic acid (4 mL) and acetone (0.3 mL) in anhydrous methanol (2 mL). The
resulting mixture was stirred at room temperature for 30 min., and then sodium borohydride (76 mg,
2.0 mmol) was added and stirred for 30 min. The reaction mixture was concentrated under reduced
pressure and water was added to the residue. The resulting solid was collected by filtration, washed
with water, and dried. The crude product was purified by column chromatography to give 7 as a solid
1
(30 mg, 37%). H-NMR (300 MHz, acetone-d6) δ 7.70 (d, J = 8.7 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H),
7.15 (d, J = 8.7 Hz, 2H), 7.12 (s, 1H), 6.75 (d, J = 8.7 Hz, 2H), 5.76 (s, 1H), 3.18 (m, 1H), 0.99
(d, J = 6.3 Hz, 6H). ESI/MS (m/z): 445 [M+H]+.
3.2.6. Synthesis of 1-(4-chlorophenyl)-6-(4-chloroanilino)-7-isopropylimino-1,7-dihydroquinoxaline (8)
Lithium aluminum hydride (8 mg, 0.21 mmol) was added into a mixture of 7 (30 mg, 0.07 mmol) in
THF (5 mL). The mixture was heated at 50 °C for 1 h under nitrogen atmosphere. After cooling to
room temperature, 3 drops of ethyl acetate were added. The mixture was filtered and washed with

Molecules 2012, 17
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ethyl acetate. The filtrate was washed with brine. The organic layer was separated and applied to
preparative TLC. The TLC plate was heated in an oven (100 °C) for 30 min. before being developed
with CHCl3/MeOH (20:1). The red band (Rf: 0.34) was collected and washed with anhydrous methanol.
The washing was concentrated, and the residue was further purified by column chromatography (HL20,
1
eluted with MeOH) to give 6 mg of 8 as a red solid (21%). H-NMR (300 MHz, CD3OD) δ 8.62 (d,
J = 3.6 Hz, 1H) 8.28 (d, J = 3.6 Hz, 1H) 7.78 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 9.0 Hz, 2H), 7.49 (s, 1H),
7.42 (d, J = 8.7 Hz, 2H), 7.31 (d, J = 9.0 Hz, 2H), 6.22 (s, 1H), 3.65–3.61 (m, 1H), 1.23 (d, J = 6.3 Hz,
13
6H). C-NMR (150 MHz, CD3OD) δ 149.3, 146.9, 142.6, 140.1, 139.5, 138.9, 134.3, 132.1, 131.2,
131.0, 129.0, 125.1, 108.6, 92.1, 46.9, 21.4. HRMS (ESI-TOF+): m/z [M+H]+ calcd. for C23H21Cl2N4:
423.1137; found: 423.1141.
3.2.7. Synthesis of 2-methylamino-nitrobenzene (9a)
2-Fluoronitrobenzene (2.82 g, 20 mmol) was dissolved in anhydrous ethanol (20 mL), and 33%
aqueous methylamine solution (4.6 mL) was added. The mixture was heated to reflux until complete
consumption of 2-fluoronitrobenzene. After cooling to room temperature, the solid formed was filtered
and washed with 95% ethanol and dried to give the title compound as a red oil (quantitative yield).
1H-NMR (400 MHz, acetone-d6) δ 8.10 (d, J = 8.0 Hz, 1H), 7.54 (t, J = 7.2, 8.4 Hz, 1H), 7.01 (d,
J = 8.4 Hz, 1H), 6.68 (t, J = 7.2, 8.0 Hz, 1H), 3.07 (d, J = 5.2 Hz, 3H). ESI/MS (m/z): 153 [M+H]+.
3.2.8. Synthesis of N-(4-chlorophenyl)-2-nitroaniline (9b)
2-Fluoronitrobenzene (14.1 g, 100 mmol), 4-chloroaniline (19.1 g, 150 mmol), anhydrous KF (5.8 g,
100 mmol) and anhydrous K2CO3 (13.8 g, 100 mmol) were heated at 160 °C for 10 h. The reaction
mixture was cooled and water and ethyl acetate were added. The aqueous layer was extracted with
ethyl acetate. The organic layer was combined and washed with 2 N HCl and dried over anhydrous
Na2SO4. The solvent was concentrated under reduced pressure and the residue was recrystallized with
anhydrous ethanol to give the title compound as red solid (20 g, 80%). 1H-NMR (300 MHz, CDCl3) δ 9.40
(s, 1H), 8.23–8.19 (d, J = 8.7 Hz, 1H), 7.41–7.37 (m, 3H), 7.23–7.16 (m, 3H), 6.78 (t, J = 8.4, 8.1 Hz, 1H).
3.2.9. Synthesis of 1-methylamino-2-(5-fluoro-2,4-dinitroanillino)-benzene (11a)
A suspension of 9a (1.1 g, 5.0 mmol) in anhydrous methanol (20 mL) was hydrogenated with 10%
Pd/C (0.06g) under atmospheric pressure until the reaction mixture turned colourless. After filtration,
the filtrate was treated with DFDNB (1.03 g, 5.0 mmol) and Et3N (0.7 mL, 5.0 mmol) and stirred at
room temperature for 1 h. The solid formed was filtered and washed with methanol to give yellow
solid (78%). 1H-NMR (300 MHz, acetone-d6) δ 9.74 (s, 1H), 9.04–9.02 (d, J = 7.8 Hz, 1H), 7.34–7.29
(t, J = 7.5 Hz, 1H), 7.20–7.17 (d, J = 7.5 Hz, 1H), 6.81–6.76 (d, J = 7.5 Hz, 1H), 6.74–6.71 (t, J = 7.8 Hz,
1H), 6.09 (d, J = 14.1 Hz, 1H), 5.13 (d, J = 3.9 Hz, 1H), 2.80–2.78 (d, J = 5.1 Hz, 3H). ESI/MS (m/z):
307 [M+H]+.

Molecules 2012, 17
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3.2.10. Synthesis of 1-(4-chloroanilino)-2-(5-fluoro-2,4-dinitroanilino)benzene (11b)
Zinc powder (9 g) was added portionwise into a mixture of 9b (2.5 g, 10 mmol) in CH2Cl2 (50 mL)
and glacial acetic acid (3 mL) and stirred at room temperature for 4 h. After filtration, the filtrate was
concentrated, and the residue was treated with DFDNB (2.04 g, 10 mmol) and Et3N (1.01 g, 10 mmol)
in anhydrous methanol (100 mL). The mixture was refluxed for 5 h. and then cooled to room
temperature. The solid formed was filtered, washed with ethanol and dried to give the title compound
1
as a red solid (2.60 g, 65%). M.p. 216–217 °C. H-NMR (300 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.87
(d, J = 8.1 Hz, 1H), 7.87 (s, 1H), 7.35–7.30 (m, 3H), 7.22–7.19 (d, J = 9.0 Hz, 2H), 7.07–7.02 (m, 1H),
6.97–6.94 (d, J = 9.0 Hz, 2H), 6.46 (d, J = 14.4 Hz, 1H). ESI/MS (m/z): 403 [M+H]+.
3.2.11. Synthesis of 1-[5-(4-chloroanilino)-2,4-dinitroanilino]-2-methylaminobenzene (12a)
A stirred suspension of 11a (1.34 g, 3.6 mmol), 4-chloroanilline (0.92 g, 7.2 mmol), and anhydrous
KF (0.21 g, 3.6 mmol) in anhydrous ethanol (30 mL) was refluxed for 4 h. After being cooled to room
temperature, the solid formed was filtered and washed with anhydrous ethanol to give the title
1
compound as an orange solid (1.52 g, 88%). H-NMR (300 MHz, DMSO-d6) δ 9.48 (brs, 2H), 9.00
(s, 1H), 7.30 (d, J = 8.7 Hz, 2H), 7.19 (d, J = 9.0 Hz, 2H), 7.08 (t, J = 7.2 Hz, 1H), 6.99 (dd, J = 7.5,
1.5 Hz, 1H), 6.70 (d, J = 7.8 Hz, 1H), 6.56 (t, J = 7.5 Hz, 1H), 6.00 (s, 1H), 4.85 (d, J = 8.7 Hz, 1H),
3.23–3.20 (m, 1H), 1.80–1.76 (m, 2H), 1.68–1.56 (m, 3H), 1.36–1.24 (m, 2H), 1.12–1.01 (m, 3H).
3.2.12. Synthesis of 1-[5-methylamino-2,4-dinitroanilino]-2-(4-chlorophenyl)benzene (12b)
A mixture of 11b (1.05 g, 2.6 mmol), 30% methylamine in ethanol (5 mL, 48.4 mmol) in anhydrous
ethanol (100 mL) was refluxed for 4 h. The reaction mixture was cooled to room temperature and
filtered. The solid obtained was washed with ethanol and dried to give the title compound as a red solid
(1.0 g, 93%). It was used directly in the next step without further purification. M.p. 239–240 °C. 1H-NMR
(300 MHz, DMSO-d6) δ 9.56 (s, 1H), 8.96 (s, 1H), 8.39 (d, J = 4.8 Hz, 1H), 7.87(s, 1H), 7.41 (d,
J = 6.9 Hz, 1H), 7.34–7.24 (m, 2H), 7.19 (d, J = 9.0 Hz, 2H), 7.08–7.02 (m, 1H), 6.95 (d, J = 9.0 Hz,
2H), 5.71 (s, 1H), 2.65 (d, J = 5.1 Hz, 3H). ESI/MS (m/z): 436 [M+Na]+.
3.2.13. Synthesis of 2-(4-chloroanilino)-5-methyl-3-isopropylimino-3,5-dihydrophenazine (15a)
Zinc powder (1.96 g) was added portionwise to a suspension of 12a (0.48 g, 1.0 mmol) in glacial
acetic acid (10 mL) and heated at 50 °C for 30 min. After filtration, the filtrate was stirred in contact
with air overnight. The reaction mixture was concentrated under reduced pressure and adjusted to
alkaline with ammonia, and then the solid formed was filtered and washed with water to give a black
solid 0.34 g. The black solid was taken up in dioxane (5 mL) and isopropylamine (2.2 mL, 25.7 mmol)
was added. The mixture was heated in a sealed bomb at 110 °C for 7 h. After being cooled to room
temperature, water was added to the mixture and the solid formed was filtered and purified by column
chromatography (eluted with P.E./ethyl acetate: 4/1 to 2/1 to 100% ethyl acetate) to give 0.13 g of the
title compound (29%). M.p. 98–102 °C. 1H-NMR (300 MHz, CDCl3) δ 7.65 (dd, J = 7.8, 1.5 Hz, 2H),
7.44 (d, J = 8.4 Hz, 1H), 7.32–7.31 (m, 1H), 7.30 (s, 4 H), 7.19–7.14 (m, 1H), 6.78 (s, 1H), 6.17
(s, 1H), 4.39 (m, 1H), 3.92–3.88 (m, 1H), 2.58–2.52 (m, 2H), 2.07–1.85 (m, 5 H), 1.56–1.51 (m, 2H),

Molecules 2012, 17
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1.35–1.34 (m, 1H), 1.31 (d, J = 6.3 Hz, 6H). 13C-NMR (100 MHz, CDCl3) δ 152.3, 150.6, 143.1, 138.8,
137.0, 133.7, 131.2, 129.2, 128.6, 127.8, 126.9, 122.5, 122.4, 114.2, 99.7, 90.5, 61.2, 49.7, 28.6, 26.5,
25.7, 23.9. HRMS (ESI-TOF+): m/z [M+H]+ calcd. for C27H30ClN4: 445.2159; found: 445.2161.
3.2.14. Synthesis of 2-methylamino-5-(4-chlorophenyl)-3-imino-3,5-dihydrophenazine (14b)
Zinc powder (4.0 g, 61.5 mmol) was added portionwise to a suspension of 12b (0.8 g, 1.9 mmol) in
glacial acetic acid (80 Ml) and stirred for 4 h. The reaction mixture was filtered and washed with
glacial acetic acid. The filtrate was stirred in contact with air overnight. The solvent was concentrated
and the residue was treated with water and adjusted to alkaline with ammonia. The solid formed was
filtered, washed with water and dried. The crude solid was purified by neutral aluminum oxide
(100–200 mesh) column chromatography eluted with ethyl acetate/methanol (10:1 to 5:1) to give the
1
title compound as a dark red solid (0.28 g, 43%). M.p. 218–220 °C. H-NMR (300 MHz, DMSO-d6)
δ 9.20 (s, 1H), 7.83 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 5.1 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.24–7.21
(m, 2H), 6.95 (brs, 1H), 6.48 (d, J = 8.4 Hz, 1H), 6.10 (s, 1H), 5.31 (s, 1H), 2.88 (d, J = 2.7 Hz, 3H).
ESI/MS (m/z): 335 [M+H]+.
3.2.15. Synthesis of 2-methylamino-3-isopropylimino-5-(4-chlorophenyl)-3,5-dihydro-phenazine (15b)
A mixture of 14b (0.15 g, 0.45 mmol), isopropylamine (1 mL, 12.0 mmol) and dioxane (20 mL)
was heated in a sealed tube at 120 °C for 10 h. After being cooled to room temperature, the reaction
mixture was concentrated under reduced pressure. The residue was purified by neutral aluminum oxide
(100–200 mesh) column chromatography eluted with hexane/ethyl acetate (2:1) to give 150 mg of 15b
1
as a red solid (88%). M.p. 235–237 °C. H-NMR (300MHz, acetone-d6) δ 7.85–7.82 (d, J = 8.7 Hz,
2H), 7.61–7.58 (d, J = 9.3 Hz, 1H), 7.54–7.51 (d, J = 8.4 Hz, 2H), 7.18–7.10 (m, 2H), 6.95 (brs, 1H),
6.48–6.45 (d, J = 9.0 Hz, 1H), 5.99 (s, 1H), 5.24 (s, 1H), 3.44–3.36 (m, 1H), 2.96 (d, J = 2.7 Hz, 3H),
0.98 (d, J = 6.0 Hz, 6H). 13C-NMR (125 MHz, CDCl3) δ 151.1, 150.8, 149.2, 136.4, 135.7, 135.5,
134.7, 131.5, 131.1, 130.5, 127.7, 126.5, 122.7, 113.7, 95.2, 88.9, 49.2, 29.2, 23.5. HRMS (ESI-TOF+):
m/z [M+H]+ calcd. for C22H22ClN4: 377.1527; found: 377.1510.
3.2.16. N1-(5-((4-chlorophenyl)amino)-2,4-dinitrophenyl)-N2-(pyridin-3-yl)benzene-1,2-diamine (19a)
Compound 17 (0.215g, 1.0 mmol) was suspended in anhydrous methanol (10 mL). Pd/C (10%,
40 mg) was added and the mixture was hydrogenated under atmospheric pressure until the reaction
mixture turned colourless. The Pd/C was filtered off. The filtrate was concentrated in vacuo to give a
light yellow oil. The oil was dissolved in THF (10 mL) and to this solution 16a (0.312 g 1.0 mmol)
and diisopropylethylamine (0.17 mL, 1.0 mmol) were added. The mixture was refluxed for 20 h and
then the reaction mixture was cooled to room temperature and concentrated in vacuo. The residue was
added with water and filtered. The crude product was purified via chromatography (PE/EA: 2/1 to 1/1)
1
to give the title compound as a yellow oil 0.243 g, yield 51%. H-NMR (300 MHz, DMSO-d6): 9.67
(brs, 1H), 9.52 (brs, 1H), 8.98 (s, 1H), 8.14 (s, 1H), 8.03–8.01 (m, 1H), 7.88 (s, 1H), 7.38 (d, J = 8.7 Hz,
2H), 7.28–7.13 (m, 7H), 7.01–6.96 (m, 1H), 6.02 (s, 1H).

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3.2.17. N1-(2,4-dinitro-5-(pyridin-3-ylamino)phenyl)-N2-(pyridin-3-yl)benzene-1,2-diamine (19b)
Compound 17 (0.215 g, 1.0 mmol) was suspended in anhydrous methanol (10 mL). Pd/C (10%, 40 mg)
was added to the suspension and the mixture was hydrogenated under atmospheric pressure until the
reaction mixture turned colourless. Then the Pd/C was filtered off. The filtrate was concentrated
in vacuo to give a light yellow oil. The oil obtained was dissolved in THF (10 mL), and the solution
was added 16b (0.278 g, 1.0 mmol) and diisopropylethylamine (0.17 mL, 1.0 mmol). The reaction
mixture was refluxed for another 20 h. and then cooled to room temperature The mixture was
concentrated in vacuo and the residues were added with distilled water and filtered. The crude product
was purified via chromatography (PE/EA: 2/1 to 1/2) to produce the title compound as a yellow solid
0.3 g, yield 68%. 1H-NMR (300 MHz, DMSO-d6): 9.72 (brs, 1H), 9.52 (brs, 1H), 8.99 (s, 1H), 8.41–8.36
(m, 1H), 8.15 (t, J = 1.5 Hz, 1H), 8.02 (t, J = 3.0 Hz, 1H), 7.85 (s, 1H), 7.64–7.60 (m, 1H), 7.35 (dd,
J = 8.1 Hz, 4.8 Hz, 1H), 7.23 (d, J = 7.8 Hz, 1H), 7.17–7.14 (m, 4H), 6.94–6.88 (m, 1H), 5.92 (s, 1H).
3.2.18. (E)-N-(4-chlorophenyl)-3-(isopropylimino)-5-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine (21a)
Compound 19a (0.243 g, 0.5 mmol) was suspended in glacial acetic acid (10 mL). The suspension
was added with zinc powder (0.66 g) portionwise in an ice bath. After addition, the mixture was stirred
at room temperature for 30 min. and then heated at 50 °C for 30 min. After being cooled to room
temperature, the reaction mixture was filtered and washed with glacial acetic acid. The filtrate was
concentrated in vacuo. The residues were added with concentrated aqueous ammonia until the mixture
became basic and then the solid was filtered out, washed with water and dried to give black solid. The
black solid was dissolved in anhydrous methanol and the solution was added with solution of ammonia
in methanol to adjust the solution to basic. The mixture was stirred in contact with air overnight and
then concentrated in vacuo. The residues were mixed with dioxane (5 mL) and isopropylamine (2 mL,
24 mmol). The mixture was heated at 110 °C in a sealed tube for 10 h. After being cooled to room
temperature, the reaction mixture was added with water and filtered to give crude product. The crude
product was purified via chromatography (PE/EA: 2/1 to EA) to produce 21 mg of the title compound
as a red solid, yield 9%. Mp: 237–239 °C. 1H-NMR (300 MHz, DMSO-d6): 8.93 (dd, J = 4.8 Hz, 1.5 Hz,
1H), 8.77 (dd, J = 1.8 Hz, 1H), 8.62 (brs, 1H), 8.15–8.11 (m, 1H), 7.87–7.83 (m, 1H), 7.67–7.63
(m, 1H), 7.48–7.41 (m, 4H), 7.24–7.21 (m, 2H), 6.73 (s, 1H), 6.44–6.41 (m, 1H), 5.12 (s, 1H), 3.39–3.30
(m, 1H), 1.03 (d, J = 6.3 Hz, 6H). 13C-NMR (100 MHz, DMSO-d6): 151.0, 150.1, 143.0, 138.8, 137.5,
135.2, 134.7, 133.9, 131.2, 129.3, 128.2, 127.9, 126.8, 126.0, 122.9, 113.9, 88.5, 48.9, 44.8, 23.3.
HRMS (m/z) calcd. for C26H23N5Cl (M+H+): 440.1636, found 440.1635.
3.2.19. (E)-3-(isopropylimino)-N,5-di(pyridin-3-yl)-3,5-dihydrophenazin-2-amine (21b)
Compound 19b (0.3 g, 0.7 mmol) was suspended in glacial acetic acid (5 mL). Zinc powder
(1.33 g) was added portionwise to the suspension in an ice bath. After addition, the mixture was heated
at 50 °C for 1 h. After being cooled to room temperature, the reaction mixture was filtered and washed
with glacial acetic acid. The filtrate was concentrated in vacuo. The residues were added with
concentrated aqueous ammonia until the mixture became basic and then the solid was filtered out,
washed with water and dried to give a dark brown solid. The solid was dissolved in anhydrous

Molecules 2012, 17
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methanol and the solution was added with ammonia in methanol to adjust the solution to basic. The
mixture was stirred in contact with air for 7 h. and then concentrated in vacuo. The residues were
mixed with dioxane (5 mL) and isopropylamine (0.036 mol, 3 mL). The mixture was heated at 110 °C
in a sealed bomb for 10 h. After being cooled to room temperature, the reaction mixture was added
with water and filtered to give crude product. The crude product was purified via chromatography
(PE/EA: 2/1 to EA) to produce 40 mg of the title compound as a red solid, yield 15%. Mp: 174–175 °C.
1H-NMR (400 MHz, DMSO-d6): 8.92 (dd, J = 4.8 Hz, 1.2 Hz, 1H), 8.76 (d, J = 2.4 Hz, 1H), 8.62 (brs,
1H), 8.60 (d, J = 2.4 Hz, 1H), 8.32 (d, J = 4.0 Hz, 1H), 8.13–8.11 (m, 1H), 7.87–7.83 (m, 1H), 7.65–7.63
(m, 1H), 7.44 (dd, J = 8.0 Hz, 4.8 Hz, 1H), 7.23–7.20 (m, 2H), 6.65 (s, 1H), 6.43–6.41 (m, 1H), 5.12
13
(s, 1H), 3.36–3.33 (m, 1H), 1.03 (d, J = 6.0 Hz, 6H). C-NMR (100 MHz, DMSO-d6): 151.0, 150.1,
150.0, 144.2, 143.9, 143.6, 137.4, 136.7, 135.2, 134.7, 133.9, 131.2, 128.5, 128.1, 127.9, 126.0, 123.9,
123.0, 113.9, 98.8, 88.5, 48.9, 23.3. HRMS (m/z) calcd. for C25H23N6 (M+H+): 407.1978, found
407.1980.
3.2.20. Synthesis of 1-[5-(3-pyridylamino)-2, 4-dinitroanilino]-2-(4-chloroanilino)benzene (22)
A mixture of compound 11b (40.3 g, 100 mmol), 3-aminopyridine (14.1 g, 150 mmol) and
triethylamine (14 mL, 100 mmol) in THF (200 mL) was refluxed under nitrogen atmosphere for 28 h.
About 150 mL of THF was evaporated at atmospheric pressure. CH2Cl2 was added and the solid
1
formed was filtered and washed with CH2Cl2 to give the title compound 22 (31.2 g, 66%). H-NMR
(300 MHz, DMSO-d6) δ 9.72 (s, 1H), 9.00 (s, 1H), 8.40–8.37 (m, 2H), 7.77 (s, 1H), 7.63–7.59 (m, 1H),
7.37–7.33 (m, 1H), 7.21–7.09 (m, 5H), 6.92–6.83 (m, 1H), 6.82–6.78 (m, 2H), 5.92 (s, 1H). ESI/MS
(m/z): 477 [M+H]+.
3.2.21. Synthesis of 2-(3-pyridylamino)-5-(4-chlorophenyl)-3-imino-3,5-dihydrophenazine (24)
Zinc powder (71 g, 1.1 mol) was added portionwise to a mixture of compound 22 (28.6 g,
60 mmol) in glacial acetic acid (150 mL) cooled in an ice water bath. The mixture was stirred until the
colour turned to light green, and then filtered, washed with glacial acetic acid and anhydrous methanol.
The filtrate was concentrated and the residue was treated with water and adjusted to alkaline with
ammonia. The solid formed was filtered, washed with water and then dissolved in anhydrous methanol.
The methanol solution was stirred in contact with air overnight. The solid formed was filtered to
produce 23.1 g of crude 24 which was used in the next step without further purification.
3.2.22. Synthesis of 5-(4-chlorophenyl)-3-isopropylimino-2-(3-pyridylamino)-3,5-dihydrophenazine (25)
A mixture of compound 24 (23.1 g), isopropylamine (100 mL, 1.2 mol) and dioxane (120 mL) was
heated in a sealed tube at 110 °C for 7 h. After cooling, the reaction mixture was treated with water
and the solid formed was filtered off and washed with water. The crude product was purified by
column chromatography (eluted with hexane/ethyl acetate: 2:1) to produce the title compound 25 as a
1
red solid (11.2 g, 44%). M.p. 194–196 °C. H-NMR (300 MHz, DMSO-d6) δ 8.63 (brs, 1H), 8.61 (d,
J = 2.4 Hz, 1H), 8.32 (dd, J = 4.8, 1.2 Hz, 1H), 7.90–7.84 (m, 3H), 7.65–7.58 (m, 3H), 7.25–7.18 (m,
2H), 6.66 (s, 1H), 6.47–6.44 (m, 1H), 5.76 (s, 1H), 3.43–3.35 (m, 1H), 1.06 (d, J = 6.3 Hz, 6H).

Molecules 2012, 17
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13C-NMR (100 MHz, DMSO-d6) δ 150.2, 149.9, 144.1, 143.8, 143.5, 136.8, 135.8, 135.3, 134.4, 131.6,
131.1, 130.9, 128.3, 128.0, 127.8, 123.9, 122.9, 114.1, 98.8, 88.3, 48.9, 23.3. HRMS (ESI-TOF+):
[M+H]+ calcd. for C26H23ClN5: 440.1641; found: 440.1643.
3.3. Biological Evaluation
In vitro and in vivo anti-tuberculosis activities were determined using our routine methods [9].
4. Conclusions
In summary, we have performed a systematic SAR and SLR investigation by synthesizing and
evaluating a variety of analogues of clofazimine with modifications at various positions. The study
revealed that the central tricyclic ring system is the pharmacophore of the molecule and is important
for anti-tuberculosis activity. Attempts to simplify this pharmacophore led to compounds with reduced
antimycobacterial activity. The two phenyl rings appended to the C2 and N5 positions can be replaced
by a pyridyl ring. Replacement of the phenyl group attached to the C2 position by a pyridyl group
leads to compounds with not only improved in vitro and in vivo anti-tuberculosis activity, but also
favourable pharmacokinetic profiles with reduced skin pigmentation potential.
Acknowledgments
The authors thank Global Alliance for TB Drug Development and National Science and
Technology Project of China (No. 2009ZX09102-054) for financial support on this project. The
authors also thank Takushi Kaneko and Christopher Cooper of TB Alliance for their suggestions and
corrections on this manuscript.
References and Notes
1. World Health Organization (WHO). Global Tuberculosis Control 2009: Epidemiology, Strategy,
Financing; WHO: Geneva, Swizerland, 2009.
2. Chan, E.D.; Iseman, M.D. Current medical treatment for tuberculosis. Br. Med. J. 2002, 325,
1282–1286.
3. Perri, G.D.; Bonora, S. Which agents should we use for the treatment of multidrug-resistant
Mycobacterium tuberculosis? J. Antimicrob. Chemother. 2004, 54, 593–602.
4. Barry, V.C.; Buggle, K. Absorption, distribution and retention of the riminocompounds in the
experimental animal. Ir. J. Med. Sci. 1960, 416, 345–352.
5. Barry, V.C.; Conalty, M.L. The antimycobacterial activity of B663. Lepr. Rev. 1965, 36, 3–7.
6. Reddy, V.M.; Nadadhur, G. Antituberculosis activities of clofazimine and its new analogs B4154
and B4157. Antimicrob. Agents Chemother. 1996, 40, 633–636.
7. Reddy, V.M.; O’Sullivan, J.F.; Gangadharam, P.R. Antimycobacterial activities of riminophenazines.
J. Antimicrob. Chemother. 1999, 43, 615–623.
8. O’Connor, R.; O’Sullivan, J.F. The pharmacology, metabolism, and chemistry of clofazimine
Drug Metab. Rev. 1995, 27, 591–614.

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9. Lu, Y.; Zheng, M.; Wang, B. Clofazimine analogs with efficacy against experimental tuberculosis
and reduced potential for accumulation. Antimicrob. Agents Chemother. 2011, 55, 5185–5193.
Sample Availability: Samples of the compounds 21a, 21b, 25 are available from the authors.
© 2012 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).