Synthesis and biological evaluation of novel 2-methoxypyridylamino- substituted riminophenazine derivatives as antituberculosis agents

Article abstract of DOI:10.3390/molecules19044380

Clofazimine, a member of the riminophenazine class, is one of the few antibiotics that are still active against multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis). However, the clinical utility of this agent is limited by its undesirable physicochemical properties and skin pigmentation potential. With the goal of maintaining potent antituberculosis activity while improving physicochemical properties and lowering skin pigmentation potential, a series of novel riminophenazine derivatives containing a 2-methoxypyridylamino substituent at the C-2 position of the phenazine nucleus were designed and synthesized. These compounds were evaluated for antituberculosis activity against M. tuberculosis H37Rv and screened for cytotoxicity. Riminophenazines bearing a 3-halogen- or 3,4-dihalogen-substituted phenyl group at the N-5 position exhibited potent antituberculosis activity, with MICs ranging from 0.250.01 μg/mL. The 3,4-dihalogensubstituted compounds displayed low cytotoxicity, with IC50 values greater than 64 μg/mL. Among these riminophenazines, compound 15 exhibited equivalent in vivo efficacy against M. tuberculosis infection and reduced skin discoloration potential in an experimental mouse infection model as compared to clofazimine. Compound 15, as compared to clofazimine, also demonstrated improved physicochemical properties and pharmacokinetic profiles with a short half-life and less drug tissue accumulation. This compound is being evaluated as a potential drug candidate for the treatment of multidrug resistant tuberculosis.

Full text of DOI:10.3390/molecules19044380

Molecules 2014, 19, 4380-4394; doi: 10.3390/molecules19044380
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Novel
2-Methoxypyridylamino-Substituted Riminophenazine
Derivatives as Antituberculosis Agents
Dongfeng Zhang 1, Yang Liu 1, Chunlin Zhang 1, Hao Zhang 1, Bin Wang 2, Jian Xu 2, Lei Fu 2,
Dali Yin 1, Christopher B. Cooper 3, Zhenkun Ma 3,*, Yu Lu 2,* and Haihong Huang 1,*
1
State Key Laboratory of Bioactive Substance and Function of Natural Medicines & Beijing Key
Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica,
Peking Union Medical College & Chinese Academy of Medical Sciences, 1 Xian Nong Tan Street,
Beijing 100050, China; E-Mails: zdf@imm.ac.cn (D.Z.); liuyang3923@163.com (Y.L.);
chunlin.zhang@hotmail.com (C.Z.); hubert365@163.com (H.Z.); yindali@imm.ac.cn (D.Y.)
2
Department of Pharmacology, Beijing Tuberculosis and Thoracic Tumor Research Institute, Beijing
Chest Hospital, Capital Medical University, 97 Ma Chang Street, Beijing 101149, China;
E-Mails: binbin69823233@126.com (B.W.); xujian4614@gmail.com (J.X.);
fl2000x@163.com (L.F.)
3
Global Alliance for TB Drug Development, 40 Wall Street, New York, NY 10005, USA;
E-Mail: christopher.cooper@tballiance.org
* Authors to whom correspondence should be addressed; E-Mails: joyce@imm.ac.cn (H.H.);
luyu4876@hotmail.com (Y.L.); zhenkun.ma@tennorx.com (Z.M.);
Tel./Fax: +86-10-6316-5244 (H.H.); Tel./Fax: +86-10-8050-5770 (Y.L.);
Tel.: +86-512-8686-1980 (Z.M.); Fax: +86-512-8686-1986 (Z.M.).
Received: 26 February 2014; in revised form: 31 March 2014 / Accepted: 2 April 2014 /
Published: 9 April 2014
Abstract: Clofazimine, a member of the riminophenazine class, is one of the few
antibiotics that are still active against multidrug-resistant Mycobacterium tuberculosis
(M. tuberculosis). However, the clinical utility of this agent is limited by its undesirable
physicochemical properties and skin pigmentation potential. With the goal of maintaining
potent antituberculosis activity while improving physicochemical properties and lowering
skin pigmentation potential, a series of novel riminophenazine derivatives containing a
2-methoxypyridylamino substituent at the C-2 position of the phenazine nucleus were
designed and synthesized. These compounds were evaluated for antituberculosis activity
against M. tuberculosis H37Rv and screened for cytotoxicity. Riminophenazines bearing a

Molecules 2014, 19
3-halogen- or 3,4-dihalogen-substituted phenyl group at the N-5 position exhibited potent
4381
antituberculosis activity, with MICs ranging from 0.25~0.01 μg/mL. The 3,4-dihalogen-
substituted compounds displayed low cytotoxicity, with IC50 values greater than 64 μg/mL.
Among these riminophenazines, compound 15 exhibited equivalent in vivo efficacy against
M. tuberculosis infection and reduced skin discoloration potential in an experimental
mouse infection model as compared to clofazimine. Compound 15, as compared to
clofazimine, also demonstrated improved physicochemical properties and pharmacokinetic
profiles with a short half-life and less drug tissue accumulation. This compound is being
evaluated as a potential drug candidate for the treatment of multidrug resistant tuberculosis.
Keywords: clofazimine; antituberculosis activity; 2-methoxypyridylamino-substituted
riminophenazines; skin discoloration
1. Introduction
Tuberculosis (TB) has become a global public health emergency due to the rapid development of
multidrug-resistant tuberculosis (MDR-TB). According to the Global Tuberculosis Report released by
World Health Organization in 2013, an estimated 8.6 million people developed TB and 1.3 million
people died from TB in 2012, including 320,000 deaths among HIV-positive people [1]. Some
progress has been made to develop new TB drugs. Currently, the global TB drug pipeline consists of
about 10 new or repurposed drugs in Phase II or Phase III clinical trials. Bedaquiline, a new TB drug
with a novel mechanism of action has become the first drug approved by the US Food and Drug
Administration (FDA) for the treatment MDR-TB in more than 40 years [1–3]. Despite the progress,
there is still an urgent need for new and more effective therapy for the treatment MDR-TB and
repurposing or optimizing existing antibiotics has been proved an effective approach in identifying
new TB drugs [2,4].
Clofazimine (CFZ, Figure 1) was first introduced in 1960s for the treatment of leprosy [5]. This
agent has demonstrated excellent activity against MDR-TB both in vitro and in vivo, and has been used
in clinical trials for the treatment of MDR-TB [6]. We have conducted a systematic structural
modification on CFZ to address the skin discoloration problem, which is one of the major obstacles for
its clinical use [7–9]. We have identified a novel series of riminophenazine derivatives containing a
2-methoxypyridylamino substituent at the C-2 position of the phenazine nucleus. This series of
compounds, exemplified by TBI-1004 (Figure 1), displayed potent antituberculosis activity, reduced
lipophilicity, and improved pharmacokinetic profiles as compared to CFZ [8]. We also concluded that
an electron-withdrawing group at the para-position of the phenyl ring at the N-5 position was
beneficial to antituberculosis activity [9]. Previously, O’Sullivan and coworkers found that compounds
bearing a 3,4-dichlorophenyl or 3,4,5-trichlorophenyl group at the N-5 position and a
tetramethylpiperidyl group at the C-3 position, such as compound B4100 (Figure 1), possessed
improved antituberculosis activity as compared to CFZ [10,11]. These work prompted us to further
investigate the effect of the halogen atoms on the phenyl ring at the N-5 position for antituberculosis
activity based on our 2-methoxypyridylamino-substituted riminophenazine system.

Molecules 2014, 19
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Figure 1. Structures of CFZ and its analogues.
The present work aimed to identify novel riminophenazine derivatives with potent antituberculosis
activity, improved physicochemical property and pharmacokinetic profiles and low skin pigmentation
potential. Hence, we kept the phenazine nucleus with the 2-methoxypyridylamino group at the C-2
position intact. Our previous work indicated that the 2-methoxypyridylamino group is a privileged
moiety [9]. We focused our investigation on the halogen effect of the N-5 phenyl group. In addition,
we introduced cyclic alkyl group including O-containing heterocyclic group to the imino nitrogen of
CFZ and evaluated their impact on antituberculosis activity and lipophilicity (ClogP).
2. Results and Discussion
2.1. Chemistry
The synthetic route for target compounds 8–25 is illustrated in Scheme 1. The most compounds
were prepared based on the previously published protocols [9,12]. Nitro compounds 2a–d were
synthesized from commercially available 2-fluoronitrobenzene (1) and aryl amines via aromatic
nucleophilic substitution. The nitro group in compounds 2a–d was reduced to give amine compounds
3a–d, and followed by substitution with 1,5-difluoro-2,4-dinitrobenzene (DFDNB) to afford
compounds 4a–d. The formation of the key intermediates 5a–d was achieved by aromatic nucleophilic
displacement in good yields. Reduction of nitro groups in 5a–d was carried out using different
methods depending on the structure of the substrate. For examples, compounds 5a and 5c were
reduced by using catalytic hydrogenation, while compounds 5b and 5d were reduced by using zinc
powder and glacial acetic acid. Compounds 6a–d underwent spontaneous cyclization to afford
riminophenazines 7a–d. Final target compounds 8–25 were prepared by replacing the imines
compounds 7a–d with different amine side chains in the presence of glacial acetic acid.
2.2. Biological Results and Discussion
Table 1 summarizes the structure and biological data for 18 new riminophenazines. CFZ and
TBI-1004 were included as reference compounds. All new compounds were screened for in vitro
activity against M. tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA) in 96-well
plate format. The antibacterial activity of the compounds is indicated by minimum inhibitory
concentration (MIC) values. The target compounds were also tested for cytotoxicity using Vero cells
measured as a concentration inhibiting 50% growth (IC50) as compared to a no treatment control [8].
The lipophilicity is estimated by ClogP, calculated by ChemOffice 2004 software.

Molecules 2014, 19
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Scheme 1. Synthesis of the target compounds 8–25.
Reagents and conditions: (i) KF, R1NH2, 160 °C, 24%–76%; (ii) 10% Pd/C, H2, ethanol or Zn/CH3COOH,
THF, rt; (iii) DFDNB, Et3N, ethanol, rt, 30%–94% (two steps from 2a–d); (iv) 3-amino-2-methoxypyridine,
Et3N, THF, reflux, 51%–88%; (v) 10% Pd/C, methanol, THF, H2 or Zn/CH3COOH, CH2Cl2, rt; (vi) air,
methanol, rt, 24%–94% (two steps from 5a–d); (vii) R2NH2, CH3COOH, dioxane, sealed tube, 110 °C,
33%–86%.
Lipophilicity (ClogP) is an important property for riminophenazine derivatives, as skin
discoloration side effect of CFZ is closely related to its high lipophilicity [13]. As shown in Table 1,
the ClogP values for the most new compounds are lower than that of CFZ (7.50), ranging from 4.70 to
7.17 with the exception of compound 13. Compounds with a 3-fluoro- or 3,4-difluorophenyl group at
the N-5 position displayed significantly reduced lipophilicity as compared to the corresponding chloro
substituted compounds, as exemplified by compound 8 (ClogP 5.90) versus compound 10 (ClogP
6.47), compound 15 (ClogP 5.98) versus compound 21 (ClogP 7.07). These results are consistent with
previous observation that chlorination of CFZ and its analogues improves antibacterial activity and
increases lipophilicity, which is a major factor for skin pigmentation [14]. In addition, the
tetrahydropyranyl substituent on the imino nitrogen at the C-3 position, as compared to the other
groups, significantly reduces ClogP value, as exemplified by compound 14 (ClogP 5.27) versus
compound 10 (ClogP 6.47), compound 19 (ClogP 4.77) versus compound 15 (ClogP 5.98). This
substituent could serve as an interesting lead for identifying new analogues with further decreased
lipophilicity [15].

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Table 1. Physicochemical property (ClogP), antituberculosis activity (MIC), cytotoxicity
(VERO IC50), and selectivity index (SI) values for target compounds.
Compound
R1
R2
ClogP a MIC(μg/mL) IC50(μg/mL) SI b
CFZ
7.50
0.12
68.6
572
F
TBI-1004
5.90
0.038
>64
1684
*
*
F
F
8
5.90
4.70
0.06
2.65
9.21
44
*
*
O
9
0.061
151
*
*
Cl
Cl
10
11
12
13
6.47
5.99
6.55
7.67
5.27
0.250
0.030
0.060
0.057
0.025
5.10
50.88
>64
20
*
*
1696
1067
934
*
*
Cl
Cl
*
*
53.26
23.40
*
*
Cl
O
14
15
936
*
*
F
5.98
0.066
>64
970
*
F
*
F
16
17
18
19
20
21
5.50
6.06
7.17
4.77
5.80
7.07
0.014
0.040
0.030
0.067
0.056
0.099
25.51
>64
>64
>64
>64
>64
1822
1600
2133
955
F
F
*
*
F
*
*
F
F
*
*
*
F
O
O
F
*
F
*
1143
646
F
*
Cl
*
Cl
*
Cl
Cl
*
22
6.59
0.025
>64
2560
Cl
*
23
24
25
7.15
5.86
6.89
0.06
0.080
0.059
>64
>64
>64
1067
800
Cl
Cl
Cl
*
*
Cl
Cl
O
*
*
O
*
1085
*
a ClogP values, calculated using ChemOffice 2004 software. b SI = selectivity index IC50/MIC (For IC50 values
of >64 μg/mL, a value of 64 μg/mL was used for the SI calculation).

Molecules 2014, 19
4385
Against M. tuberculosis H37Rv, the majority of compounds exhibited potent activity equal or better
than CFZ and TBI-1004 with MIC values ranging from 0.01 to 0.1 μg/mL. It appears that the number,
type and position of the halogen atoms on the phenyl ring at the N-5 position have no significant
impact on the antituberculosis activity. However, compounds with a 3-F or 3-Cl substituted phenyl
group on the N-5 position have showed higher cytotoxicity as compared to its corresponding 4- and
3,4-disubstituted compounds, as exemplified by compound 8 (IC50 2.65 μg/mL) versus TBI-1004
(IC50 > 64 μg/mL). Interestingly, all compounds with a 3,4-dihalogen-substituted phenyl groups displayed
very low cytotoxicity, with IC50 > 64 μg/mL, with the exception of compound 16 (IC50 25.51 μg/mL).
Based on our systematic evaluation of lipophilicity, antituberculosis activity and cytotoxicity, two
compounds 15 and 22 were selected for a multiple doses in vivo toxicity study in mice. Table 2
summarizes the tolerability data by recording the number of mice which survived after an oral
administration at a dose of 500 mg/kg, once daily for 4 days, followed by a 7-day observation. As
shown in Table 2, both compounds 15 and 22 demonstrated good safety in mice. Therefore, the in vivo
efficacy evaluation of compounds 15 and 22 were conducted in a mouse model infected by M.
tuberculosis H37Rv. The in vivo efficacy results were summarized in Table 3. Compound 15
demonstrated excellent efficacy in mice, the bacterial burden in the lungs was reduced by 3.8 logs
colony forming units (CFU) as compared to the untreated control group and equivalent to CFZ.
Furthermore, during the in vivo study, we also observed that the discoloration of the ears, internal
organs and fat tissues of the mice treated with compound 15 appeared less intense as compared to
CFZ. However, compound 22 failed to display efficacy in mouse model. The inconsistency between
the in vivo and in vitro results might be explained by poor pharmacokinetics [16]. Finally, preliminary
PK in mice was evaluated for compound 15. As shown in Table 4, compound 15 demonstrated a
shorter plasma half-life (t1/2), higher Cmax and AUC as compared to CFZ, which could explain its
excellent efficacy and reduced skin discoloration potential compared to CFZ.
Table 2. Preliminary multidose toxicity study of compounds 15 and 22 a.
Number of animals that
Compound
survived/Total number of animals
15
22
5/5
6/6
a oral administration at a dose of 500 mg/kg, once daily for 4 days, followed by 7-day observation.
Table 3. Efficacy of CFZ, compounds 15 and 22 after 30 days of treatment of BALB/c
mice infected with M. tuberculosis H37Rv. (mean ± SD) a.
Groups
Control (D3)
Control (D10)
Control (D30)
CFZ
Dose (mg/kg)
logCFU/lung
1.94 ± 028
3.72 ± 0.46
8.32 ± 0.19
4.29 ± 0.58
4.56 ± 0.38
8.06 ± 0.13
20
20
20
15
22
a 5 mice were used in every group (n = 5).

Molecules 2014, 19
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Table 4. Preliminary pharmacokinetic parameters of CFZ and 15 dosed orally in mice at 20 mg/kg.
Compound
CFZ
T1/2 (h) Tmax (h) Cmax (mg/L) AUC0~24 h (mg/L*h)
27.11
13.80
2
2
0.47
8.22
15
1.835
21.156
3. Experimental
3.1. General Information
3.1.1. Chemistry
All reagents and solvents were purchased from commercial sources unless otherwise indicated.
Melting points were determined on Yanaco MP-J3 melting point apparatus. Thin-layer
chromatography was performed with fluorescent silica gel plates GF254, which were checked under
UV (254 nm) light. 1H-NMR spectra were recorded on Varian mercury-300 MHz or Varian-400 MHz
NMR spectrometer in CDCl3 or DMSO-d6. 13C-NMR spectra were obtained on Varian-400 at 100 MHz
in CDCl3. High-resolution mass spectra were measured on an Agilent 1100 series LC/MSD trap mass
spectrometer (ESI-TOF).
3.1.2. Minimum Inhibitory Concentration and Cytotoxicity Assays
These were carried out according to our published protocols [8].
3.1.3. In vivo Acute M. tuberculosis H37Rv Infection Assay and Mouse Pharmacokinetic Study
These were carried out according to our published protocols [8].
3.2. General Procedure for Preparation of Compounds 2a–d
N-(3-Fluorophenyl)-2-nitroaniline (2a). A mixture of 2-fluoronitrobenzene (21.2 g, 150 mmol),
3-fluoroaniline (20.0 g, 180 mmol) and anhydrous potassium fluoride (8.7 g, 150 mmol) was stirred at
160 °C for 36 h. The mixture was then cooled, water and ethyl acetate were added, the aqueous layer
was extracted with ethyl acetate, and the combined organic layer was washed with 2 N HCl and dried
over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under vacuum, and the
crude product was recrystallized in 60 mL of 95% ethanol to give 17.4 g of 2a as a red solid in 50%
1
yield. Mp: 73–74 °C. H-NMR (300 MHz, CDCl3) δ: 6.82–6.94 (2 H, m), 6.99–7.07 (2 H, m),
7.29–7.45 (3 H, m), 8.21 (1 H, d, J = 8.4 Hz), 9.43 (1 H, s).
N-(3-Chlorophenyl)-2-nitroaniline (2b). Compound 2b was synthesized in a similar manner as
1
described for 2a to provide 18.7 g of a red solid, yield 76%. Mp: 88–90 °C. H-NMR (300 MHz,
DMSO-d6) δ: 6.98 (1 H, m), 7.19 (1 H, m), 7.28 (2 H, m), 7.37 (2 H, m), 7.56 (1 H, m), 8.12 (1 H, dd,
J = 8.1, 1.5 Hz), 9.28 (1 H, brs).
N-(3,4-Difluorophenyl)-2-nitroaniline (2c). Compound 2c was synthesized in a similar manner as
described for 2a to give 23.1 g of a red solid, yield 61%. Mp: 95–97 °C.1H-NMR (300 MHz, CDCl3)

Molecules 2014, 19
4387
δ: 6.84 (1 H, t, J = 7.5 Hz), 6.99–7.03 (1 H, m), 7.10–7.17 (2 H, m), 7.22 (1 H, d, J = 9.0 Hz), 7.41
(1 H, m), 8.22 (1 H, dd, J = 8.4, 1.2 Hz), 9.35 (1 H, s).
N-(3,4-Dichlorophenyl)-2-nitroaniline (2d). Compound 2d was synthesized in a similar manner as
described for 2a to afford 20.0 g of a red solid, yield 24%. Mp: 78–79 °C. 1H-NMR (300 MHz, CDCl3)
δ: 6.87 (1 H, d, J = 6.9 Hz), 7.12 (1 H, d, J = 8.4 Hz), 7.22 (1 H, s), 7.41 (1 H, d, J = 6.9 Hz), 7.44–7.48
(2 H, m), 8.21 (1 H, d, J = 8.4 Hz), 9.47 (1 H, s).
3.3. General Procedure for Preparation of Compounds 4a–d
1-[2-(3-Fluoroanilino)anilino]-3-fluoro-4,6-dinitrobenzene (4a). Zinc powder (40 g, 615 mmol) was
added portionwise into a mixture of 2a (17 g, 73 mmol) in THF (250 mL) and glacial acetic acid
(18 mL, 315 mmol) at room temperature. After filtration, DFDNB (14.9 g, 73 mmol) and triethylamine
(7.4 g, 73 mmol) were added to the filtrate (3a); the mixture was stirred at room temperature for 9 h,
and was concentrated to dryness. The residue was washed with ethanol and filtered to give 19.81 g of
1
4a in 70% yield as a red solid. Mp: 170–171 °C. H-NMR (300 MHz, DMSO-d6) δ: 6.50 (1 H, d,
J = 14.1 Hz), 6.60 (1 H, t, J = 8.7 Hz), 6.69–6.78 (2 H, m), 7.08–7.22 (2 H, m), 7.32–7.38 (3 H, m),
7.99 (1 H, s), 8.88 (1 H, d, J = 7.5 Hz), 9.99 (1 H, s).
1-[2-(3-Chloroanilino)anilino]-3-fluoro-4,6-dinitrobenzene (4b). Compound 4b was synthesized in a
similar manner as described for 4a to provide 1.21 g of an orange solid, yield 30%. Mp: 145–147 °C.
1H-NMR (300 MHz, DMSO-d6) δ: 6.49 (1 H, d, J = 14.1 Hz), 6.82 (1 H, d, J = 7.5 Hz), 6.90 (2 H, m),
7.12 (2 H, m), 7.34 (3 H, m), 7.97 (1 H, s), 8.87 (1 H, d, J = 8.1 Hz), 9.97 (1 H, s).
1-[2-(3,4-Difluoroanilino)anilino]-3-fluoro-4,6-dinitrobenzene (4c). A mixture of 2c (23 g, 91.9 mmol)
and 10% Pd/C (2.3 g) in ethanol was shaken at room temperature under a hydrogen atmosphere (40 psi)
for 2 h. After filtration, DFDNB (18.75 g, 91.9 mmol) and triethylamine (9.29 g, 91.9 mmol) were
added to the filtrate (3c); the mixture was stirred at room temperature for 15 h, filtered, and washed
with ethanol to give 34.82 g of 4c in 94% yield as an orange solid. Mp: 185–186 °C. 1H-NMR (300 MHz,
CDCl3) δ: 5.59 (1 H, s), 6.65 (1 H, d, J = 12.9 Hz), 6.72 (1 H, d, J = 9.0 Hz), 6.83–6.90 (1 H, m),
7.04–7.13 (2 H, m), 7.29–7.37 (3 H, m), 9.17 (1 H, d, J = 7.5 Hz), 9.60 (1 H, s).
1-[2-(3,4-Dichloroanilino)anilino]-3-fluoro-4,6-dinitrobenzene (4d). Compound 4d was synthesized
in a similar manner as described for 4a to give 5.9 g of an orange solid, yield 65%. Mp: 210–211 °C.
1H-NMR (300 MHz, CDCl3) δ: 5.60 (1 H, s), 6.47 (1 H, d, J = 13.2 Hz), 6.83 (1 H, dd, J = 8.7,
2.4 Hz), 7.07 (1 H, d, J = 2.7 Hz), 7.12–7.17 (1 H, m), 7.29–7.37 (4 H, m), 9.14 (1 H, d, J = 8.1 Hz),
9.61 (1 H, s).
3.4. General Procedure for Preparation of Compounds 5a–d
1-[2-(3-Fluoroanilino)anilino]-3-(2-methoxy-3-pyridyl)amino-4,6-dinitrobenzene (5a). A mixture of
4a (3.09 g, 8 mmol), 3-amino-2-methoxypyridine (0.99 g, 8 mmol), triethylamine (0.81 g, 8 mmol)
and THF (50 mL) was refluxed for 31 h. After being cooled to room temperature, the mixture was
concentrated in vacuo, CH3OH was added to the residue, and the solid obtained was filtered to give 3 g

Molecules 2014, 19
4388
of 5a as a red solid in 77% yield. Mp: 180–182 °C. 1H-NMR (400 MHz, CDCl3) δ: 3.92 (3 H, s), 5.78
(1 H, s), 6.19 (1 H, s), 6.62 (3 H, m,), 6.70–6.85 (1 H, m), 6.99 (1 H, t, J = 7.4 Hz), 7.09–7.27 (3 H,
m), 7.47–7.29 (2 H, m), 7.98 (1 H, d, J = 4.6 Hz), 9.30 (1 H, s), 9.40 (1 H, s), 9.68 (1 H, s).
1-[2-(3-Chloroanilino)anilino]-3-(2-methoxy-3-pyridyl)amino-4,6-dinitrobenzene (5b). Compound 5b was
synthesized in a similar manner as described for 5a to give 2.59 g of a red solid, yield 51%.
1
Mp: 151–153 °C. H-NMR (300 MHz, CDCl3) δ: 3.80 (3 H, s), 5.82 (1 H, s), 6.68 (1 H, dd, J = 8.1,
1.5 Hz), 6.77 (1 H, brs), 6.81 (1 H, dd, J = 8.1, 1.5 Hz), 6.98 (2 H, m), 7.14 (1 H, t, J = 8.1 Hz), 7.22
(3 H, m), 7.53 (1 H, dd, J = 7.8, 1.5 Hz), 7.88 (1 H, brs), 8.03 (1 H, dd, J = 8.1, 1.5 Hz), 9.01 (1 H, s),
9.51 (1 H, brs), 9.56 (1 H, brs).
1-[2-(3,4-Difluoroanilino)anilino]-3-(2-methoxy-3-pyridyl)amino-4,6-dinitrobenzene (5c). Compound
5c was synthesized in a similar manner as described for 5a to afford 8.92 g of an orange solid, yield
1
88%. Mp: 201–203 °C. H-NMR (300 MHz, DMSO-d6) δ: 3.78 (3 H, s), 5.76 (1 H, s), 6.53 (1 H, d,
J = 8.7 Hz), 6.66–6.73 (1 H, m), 6.92–6.97 (2 H, m), 7.13–7.23 (4 H, m), 7.52 (1 H, d, J = 7.5 Hz),
7.78 (1 H, s), 8.01 (1 H, d, J = 5.1 Hz), 9.00 (1 H, s), 9.49 (1 H, s), 9.54 (1 H, s).
1-[2-(3,4-Dichloroanilino)anilino]-3-(2-methoxy-3-pyridyl)amino-4,6-dinitrobenzene (5d). Compound
5d was synthesized in a similar manner as described for 5a to give 4.0 g of a yellow solid, yield 74%.
Mp: 220–221 °C. 1H-NMR (300 MHz, DMSO-d6) δ: 3.83 (3 H, s), 5.76 (1 H, s), 6.66 (1 H, d, J = 7.8 Hz),
6.93 (1 H, d, J = 5.1 Hz), 6.99–7.03 (2 H, m), 7.23–7.32 (4 H, m), 7.51 (1 H, d, J = 6.6 Hz), 7.95 (1 H, s),
8.01 (1 H, d, J = 6.6 Hz), 8.99 (1 H, s), 9.49 (1 H, s), 9.54 (1 H, s).
3.5. General Procedure for Preparation of Compounds 7a–d
5-(3-Fluorophenyl)-3-imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (7a). A mixture of
5a (2.99 g, 6.1 mmol) and 10% Pd/C (0.3 g) in methanol and THF was shaken at room temperature
under a hydrogen atmosphere (40 psi) for 15 h. After filtration, the filtrate was concentrated under
vacuum and the residue 6a was dissolved in methanol. The result solution stirred at room temperature
under air for 18 h. The mixture was filtered to give 1.72 g crude 7a as a red solid in 69% yield.
Compound 7a was taken to the next step without further purification.
5-(3-Chlorophenyl)-3-imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (7b). Zinc powder
(1.63 g, 25 mmol) was added portionwise into a mixture of 5b (2.53 g, 5 mmol) and glacial acetic acid
(0.86 mL, 15 mmol) in CH2Cl2 (300 mL) at room temperature. The mixture was stirred until the color
turned to light green and then filtered and washed with CH2Cl2. The filtrate was concentrated, and the
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 2.01 g crude 7b in 94% yield, which was taken to the next step
without further purification.
5-(3,4-Difluorophenyl)-3-imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (7c). Compound
7c was synthesized in a similar manner as described for 7a to give 6.46 g of a red solid, yield 86%.
Compound 7c was taken to the next step without further purification.

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5-(3,4-Dichlorophenyl)-3-imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (7d). Compound
7d was synthesized in a similar manner as described for 7b to afford 0.8 g of a red solid, yield 24%.
Compound 7d was taken to the next step without further purification.
3.6. General Procedure for Preparation of Compounds 8–25
To a solution of 7a–d (1.0 equiv.) and amine (2.0 equiv.) in dioxane was added glacial acetic acid
(0.2 equiv.), then the mixture was stirred and heated at 110 °C in a sealed tube for 10–20 h. The
mixture was concentrated under vacuum, and the residue was purified by column chromatography on
silica gel, using petroleum ether/ethyl acetate to afford target products 8–25.
5-(3-Fluorophenyl)-3-isopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (8). Red solid.
Yield 83 mg (37%). Mp: 198–199 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.11 (6 H, brs), 3.42–3.50 (1 H,
m), 4.04 (3 H, s), 5.29 (1 H, s), 6.47 (1 H, d, J = 8.1 Hz), 6.93 (2 H, brs), 7.10–7.20 (4 H, m), 7.37
13
(1 H, t, J = 8.1 Hz), 7.69–7.76 (2 H, m), 7.81–7.86 (2 H, m). C-NMR (100 MHz, CDCl3) δ: 23.5,
49.4, 53.7, 89.3, 100.1, 113.8, 116.5 (d, J = 22.3 Hz), 116.8, 117.0 (d, J = 20.6 Hz), 122.9, 124.8,
124.9, 127.6, 128.2, 131.4, 132.6, 132.7, 134.6, 135.5, 138.8, 138.9, 142.9, 150.6, 151.3, 155.5, 165.2
(d, J = 249.3 Hz). HRMS (ESI-TOF+): m/z [M + H]+ calcd for C27H25FN5O: 454.2038; found: 454.2043.
5-(3-Fluorophenyl)-3-(4-tetrahydropyranyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydro-
phenazine (9). Red solid. Yield 100 mg (41%). Mp: 190–191 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.67
(4 H, brs), 3.41–3.50 (3 H, m), 4.01–4.04 (5 H, m), 5.26 (1 H, s), 6.50 (1 H, d, J = 8.1 Hz),
6.90–6.94 (1 H, m), 6.97 (1 H, s), 7.09–7.22 (4 H, m), 7.38 (1 H, t, J = 8.7 Hz), 7.69–7.76 (2 H, m),
13
7.81–7.86 (2 H, m), 9.06 (1 H, s). C-NMR (100 MHz, CDCl3) δ: 33.3, 33.4, 53.5, 53.7, 65.7, 89.0,
100.3, 113.9, 116.5 (d, J = 22.3 Hz), 116.8, 117.1(d, J = 20.6 Hz), 123.1, 124.4, 124.8, 127.8, 128.3,
131.2, 132.6, 132.7, 134.8, 135.6, 138.7, 138.9, 142.6, 150.9, 151.2, 155.3, 165.2 (d, J = 249.3 Hz).
HRMS (ESI-TOF+): m/z [M + H]+ calcd for C29H27FN5O2: 496.2143; found: 496.2152.
5-(3-Chlorophenyl)-3-isopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (10). Red
solid. Yield 110 mg (33%). Mp: 215–217 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.11 (6 H, d, J = 6.0 Hz),
3.46 (1 H, m), 4.04 (3 H, s), 5.27 (1 H, s), 6.45 (1 H, m), 6.91 (2 H, m), 7.15 (2 H, m), 7.27 (1 H, m),
7.38 (1 H, s), 7.64 (2 H, m), 7.68 (1 H, m), 7.83 (2 H, m), 8.90 (1 H, brs). 13C-NMR (100 MHz,
CDCl3) δ: 23.5, 49.4, 53.7, 89.4, 100.1, 113.8, 116.8, 122.9, 124.8, 124.9, 127.3, 127.6, 128.2, 129.4,
130.1, 131.4, 132.3, 134.6, 135.5, 136.7, 138.7, 138.8, 142.9, 150.6, 151.2, 155.5. HRMS (ESI-TOF+):
m/z [M + H]+ calcd for C27H25ClN5O: 470.1712; found: 470.1696.
5-(3-Chlorophenyl)-3-cyclopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (11). Red
solid. Yield 149 mg (46%). Mp: 211–214 °C. 1H-NMR (300 MHz, CDCl3) δ: 0.84 (4 H, m), 2.72 (1 H,
m), 4.03 (3 H, s), 5.54 (1 H, s),6.44 (1 H, m), 6.90 (2 H, m), 7.17 (3 H, m), 7.38 (1 H, s), 7.66 (2 H,
m), 7.74 (1 H, m), 7.80 (2 H, m). 13C-NMR (100 MHz, CDCl3) δ: 10.1, 32.9, 53.7, 89.8, 100.2, 113.9,
116.8, 124.4, 124.7, 127.1, 127.2, 127.8, 128.3, 129.3, 130.1, 132.3, 134.6, 136.7, 138.6, 138.9, 142.2,
142.7, 150.9, 153.5, 155.4, 161.0. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C27H23ClN5O:
468.1586; found: 468.1573.

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5-(3-Chlorophenyl)-3-cyclobutylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (12). Red
solid. Yield 142 mg (42%). Mp: 205–208 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.75 (2 H, m), 2.07 (2 H,
m), 2.20 (2 H, m), 3.93 (1 H, m), 4.04 (3 H, s), 5.08 (1 H, s), 6.50 (1 H, m), 6.93 (2 H, m), 7.17 (3 H,
m), 7.38 (1 H, s), 7.69 (3 H, m), 7.84 (2 H, m). 13C-NMR (100 MHz, CDCl3) δ: 16.1, 32.0, 53.7, 54.9,
90.9, 100.3, 113.9, 116.8, 123.1, 124.8, 127.4, 127.8, 128.3, 129.4, 130.1, 131.2, 132.2, 134.2, 135.6,
136.7, 138.7, 138.9, 142.6, 142.8, 151.2, 151.4, 155.4. HRMS (ESI-TOF+): m/z [M + H]+ calcd for
C28H25ClN5O: 482.1742; found: 482.1703.
5-(3-Chlorophenyl)-3-cyclohexylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (13). Red
solid. Yield 62 mg (49%). Mp: 197–201 °C.1H-NMR (300 MHz, CDCl3) δ: 1.10 (3 H, m), 1.28 (2 H,
m), 1.53 (3 H, m), 1.68 (2 H, m), 2.98 (1 H, m), 3.94 (3 H, s), 5.12 (1 H, s), 6.55 (2 H, m), 6.81 (1 H,
13
s), 7.11 (3 H, m), 7.55 (2 H, m), 7.81 (5 H, m), 9.00 (1 H, s). C-NMR (100 MHz, CDCl3) δ: 23.8,
25.4, 33.0, 53.6, 56.9, 88.7, 100.1, 113.9, 117.4, 122.8, 124.0, 124.1, 127.8, 127.9, 128.0, 128.2, 129.0,
130.0, 131.0, 132.9, 134.2, 135.1, 135.3, 138.2, 138.5, 141.5, 150.1, 150.3. HRMS (ESI-TOF+): m/z
[M + H]+ calcd for C30H29ClN5O: 510.2061; found: 510.2059.
5-(3-Chlorophenyl)-3-(4-tetrahydropyranyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine
(14). Red solid. Yield 308 mg (86%). Mp: 203–206 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.75 (4 H, m),
2.85 (1 H, m), 3.43 (2 H, m), 3.96 (2 H, m), 4.04 (3 H, s), 5.27 (1 H, s), 6.53 (1 H, m), 6.90 (2 H, m),
7.17 (3 H, m), 7.38 (1 H, s), 7.64 (2 H, m), 7.74 (1 H, m), 7.84 (2 H, m), 9.08 (1 H, s). 13C-NMR (100 MHz,
CDCl3) δ: 34.2, 53.5, 53.8, 66.9, 89.1, 100.4, 113.9, 116.7, 124.4, 124.7, 127.1, 127.2, 127.8, 128.3,
129.3, 130.1, 132.3, 134.6, 136.7, 138.6, 138.9, 142.2, 142.7, 150.9, 153.5, 155.4, 161.0. HRMS (ESI-
TOF+): m/z [M + H]+ calcd for C29H27ClN5O2: 512.1848; found: 512.1838.
5-(3,4-Difluorophenyl)-3-isopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (15).
1
Red solid. Yield 270 mg (80%). Mp: 229–231 °C. H-NMR (300 MHz, CDCl3) δ: 1.13 (6 H, d,
J = 6.9 Hz), 3.45–3.53 (1 H, m, J = 6.3 Hz), 4.04 (3 H, s), 5.28 (1 H, s), 6.43 (1 H, d, J = 7.5 Hz),
6.89–6.93 (2 H, m), 7.13–7.25 (4 H, m), 7.49–7.59 (1 H, m), 7.68 (1 H, d, J = 7.2 Hz), 7.81–7.84 (2 H,
13
m), 8.92 (1 H, s). C-NMR (100 MHz, CDCl3) δ: 23.5, 23.6, 49.5, 53.7, 89.2, 100.1, 113.5, 116.8,
118.8 (d, J = 17.8 Hz), 120.0 (d, J = 18.5 Hz), 123.1, 124.8, 124.9, 125.8, 127.7, 128.3, 131.4, 133.7,
134.7, 135.5, 138.9, 142.9, 151.0 (dd, J = 251.8, 12.0 Hz), 151.2, 152.0 (dd, J = 252.6, 13.2 Hz),
155.5. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C27H24F2N5O: 472.1943; found: 472.1937.
5-(3,4-Difluorophenyl)-3-cyclopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (16).
1
Red solid. Yield 233 mg (yield 71%). Mp: 213–214 °C. H-NMR (300 MHz, CDCl3) δ: 0.86 (2 H, d,
J = 2.7 Hz), 0.93 (2 H, d, J = 6.0 Hz), 2.77 (1 H, m), 4.01 (3 H, s), 5.54 (1 H, s), 6.42 (1 H, d, J = 8.4 Hz),
6.86 (1 H, s), 6.99 (1 H, m), 7.12 (4 H, m), 7.52 (1 H, m), 7.66 (1 H, d, J = 8.1 Hz), 7.82 (2 H, m), 8.58
(1 H, s). 13C-NMR (100 MHz, CDCl3): δ: 10.2, 33.0, 53.7, 89.7, 100.1, 113.5, 116.8, 118.9 (d, J = 17.9 Hz),
120.0 (d, J = 18.3 Hz), 123.1, 124.7, 125.1, 125.9, 127.7, 128.2, 131.6, 133.7, 134.5, 135.7, 139.0,
142.7, 151.0 (dd, J = 254.5, 13.1 Hz), 151.4, 151.9 (dd, J = 252.9, 13.5 Hz), 155.5. HRMS (ESI-TOF+):
m/z [M + H]+ calcd for C27H22F2N5O: 470.1787; found: 470.1762.

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5-(3,4-Difluorophenyl)-3-cyclobutylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (17).
Red solid. Yield 300 mg (78%). Mp: 200–201 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.70–1.82 (2 H, m),
2.03–2.20 (4 H, m), 3.81–4.04 (1 H, m), 4.04 (3 H, s), 5.08 (1 H, s), 6.46 (1 H, d, J = 7.5 Hz),
6.89–6.93 (2 H, m), 7.12–7.23 (4 H, m), 7.50–7.59 (1 H, m), 7.69 (1 H, d, J = 7.5 Hz), 7.82–7.84 (2 H, m).
13C-NMR (100 MHz, CDCl3) δ: 16.1, 32.0, 53.7, 54.9, 90.7, 100.3, 113.6, 116.8, 118.8 (d, J = 17.7 Hz),
119.9 (d, J = 18.1 Hz), 123.2, 124.7, 124.9, 125.8, 127.8, 128.4, 131.3, 133.6, 134.2, 135.6, 138.9,
142.7, 151.0 (dd, J = 251.1, 11.8 Hz), 151.1, 151.3, 151.9 (dd, J = 253.4, 13.7 Hz), 155.4. HRMS
(ESI-TOF+): m/z [M + H]+ calcd for C28H24F2N5O: 484.1943; found: 484.1933.
5-(3,4-Difluorophenyl)-3-cyclohexylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (18).
1
Red solid. Yield 330 mg (81%). Mp: 218–219 °C. H-NMR (300 MHz, CDCl3) δ: 1.23–1.79 (10 H,
m), 3.14–3.20 (1 H, m), 4.03 (3 H, s), 5.26 (1 H, s), 6.45 (1 H, d, J = 7.5 Hz), 6.88–6.93 (2 H, m),
7.11–7.25 (4 H, m), 7.49–7.58 (1 H, m), 7.69 (1 H, d, J = 7.5 Hz), 7.80–7.84 (2 H, m). 13C-NMR (100 MHz,
CDCl3) δ: 24.2, 25.9, 33.5, 33.6, 53.7, 57.4, 89.4, 100.1, 113.5, 116.8, 118.8 (d, J = 17.8 Hz), 119.8 (d,
J = 18.1 Hz), 123.0, 124.5, 124.9, 125.8, 127.7, 128.3, 131.4, 133.7, 134.6, 135.6, 138.7, 142.8, 150.4,
151.0 (dd, J = 251.5, 11.9 Hz), 151.3, 151.9 (dd, J = 252.6, 13.5 Hz), 155.4. HRMS (ESI-TOF+): m/z
[M + H]+ calcd for C30H28F2N5O: 512.2256; found: 512.2263.
5-(3,4-Difluorophenyl)-3-(4-tetrahydropyranyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydro-
1
phenazine (19). Red solid. Yield 240 mg (59%). Mp: 255–256 °C. H-NMR (300 MHz, CDCl3) δ:
1.65–1.68 (4 H, m), 3.46–3.55 (3 H, m), 4.05 (5 H, m), 5.25 (1 H, s), 6.46 (1 H, d, J = 9.0 Hz),
6.90–6.95 (2 H, m), 7.13–7.24 (4 H, m), 7.51–7.60 (1 H, m), 7.71 (1 H, d, J = 7.5 Hz), 7.81–7.85 (2 H,
m), 9.08 (1 H, s).13C-NMR (100 MHz, CDCl3) δ: 33.2, 33.4, 53.3, 53.8, 65.5, 88.9, 100.3, 113.7,
116.8, 118.7 (d, J = 19.7 Hz), 119.9 (d, J = 18.1 Hz), 123.3, 124.3, 124.8, 125.7, 127.9, 128.4, 131.2,
133.5, 134.9, 135.6, 138.8, 142.8, 150.9, 151.0 (dd, J = 251.4, 13.6 Hz), 151.9 (dd, J = 252.8, 13.4
Hz), 155.3. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C29H26F2N5O2: 514.2049; found: 514.2042.
5-(3,4-Difluorophenyl)-3-(4-methoxycyclohexyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydro-
phenazine (20). Red solid. Yield 432 mg (84%). Mp: 214–215 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.26
(2 H, m), 1.40 (2 H, m), 1.73 (2 H, m), 2.05 (2 H, m), 3.16 (1 H, m), 3.27 (1 H, m), 3.36 (3 H, s), 4.03
(3 H, s), 5.26 (1 H, s), 6.46 (1 H, d, J = 7.5 Hz), 6.92 (2 H, m), 7.15 (3 H, m), 7.22 (1 H, m), 7.53 (1 H,
13
m), 7.69 (1 H, d, J = 8.1 Hz), 7.82 (2 H, m), 8.94 (1 H, brs). C-NMR (100 MHz, CDCl3) δ: 29.3,
29.5, 30.8, 53.7, 55.8, 56.9, 78.2, 89.2, 100.2, 113.6, 116.8, 118.7 (d, J = 18.0 Hz), 119.9 (d, J = 18.9 Hz),
123.2, 124.7, 125.7, 127.8, 128.4, 131.4, 133.5, 134.7, 135.6, 138.8, 142.7, 151.0 (dd, J = 252.5, 13.7 Hz),
151.1, 151.9 (dd, J = 252.2, 13.5 Hz), 155.4. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C31H30F2N5O2:
542.2362; found: 542.2338.
5-(3,4-Dichlorophenyl)-3-isopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (21).
Red solid. Yield 130 mg (68%). Mp: 245–247 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.13 (6 H, d, J = 6.3 Hz),
3.50 (1 H, m), 4.03 (3 H, s), 5.29 (1 H, s), 6.43 (1 H, d, J = 6.3 Hz), 6.89 (1 H, s), 6.93 (1 H, m),
7.10–7.23 (3 H, m), 7.49 (1 H, d, J = 2.4 Hz), 7.68 (1 H, m), 7.80–7.84 (3 H, m). 13C-NMR (100 MHz,
CDCl3) δ: 23.6, 49.5, 53.7, 89.3, 100.1, 113.6, 116.8, 123.1, 124.8, 125.0, 127.7, 128.3, 128.6, 131.3,

Molecules 2014, 19
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133.1, 134.3, 134.5, 135.3, 135.5, 136.8, 138.9, 142.9, 150.4, 151.2, 155.5. HRMS (ESI-TOF+): m/z
[M + H]+ calcd for C27H24Cl2N5O: 504.1853; found: 504.1852.
5-(3,4-Dichlorophenyl)-3-cyclopropylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (22).
Red solid. Yield 120 mg (65%). Mp: 215–217 °C. 1H-NMR (300 MHz, CDCl3) δ: 0.86–0.96 (4 H, m),
2.80 (1 H, m), 4.00 (3 H, s), 5.54 (1 H, s), 6.41 (1 H, d, J = 8.7 Hz), 6.86 (1 H, s), 6.91 (1 H, m),
7.10–7.28 (3 H, m), 7.51 (1 H, d, J = 2.1 Hz), 7.65 (1 H, m), 7.80–7.83 (3 H, m). 13C-NMR (100 MHz,
CDCl3) δ: 10.3, 33.1, 53.7, 89.8, 100.0, 113.6, 116.8, 123.1, 124.6, 125.2, 127.7, 128.2, 128.8, 131.4,
133.2, 134.3, 135.4, 135.7, 136.8, 139.0, 142.7, 151.4, 152.2, 155.4. HRMS (ESI-TOF+): m/z [M + H]+
calcd for C27H22Cl2N5O: 502.1317; found: 502.1316.
5-(3,4-Dichlorophenyl)-3-cyclobutylimino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydrophenazine (23).
Red solid. Yield 167 mg (65%). Mp: 216–219 °C. 1H-NMR (300 MHz, CDCl3) δ: 1.81 (2 H, m), 2.08
(2 H, m), 2.22 (2 H, m), 3.95 (1 H, m), 4.05 (3 H, s), 5.11 (1 H, s), 6.47 (1 H, d, J = 7.5 Hz), 6.91 (2 H,
m), 7.17 (2 H, m), 7.23 (1 H, m), 7.50 (1 H, d, J = 2.1 Hz), 7.69 (1 H, d, J = 7.5 Hz), 7.84 (3 H, m),
8.79 (1 H, brs). 13C-NMR (100 MHz, CDCl3) δ: 16.1, 32.0, 53.7, 54.8, 90.8, 100.3, 113.7, 116.8,
123.2, 124.7, 124.9, 127.8, 128.4, 128.6, 131.1, 131.3, 133.0, 134.0, 134.3, 135.3, 135.6, 136.7, 139.0,
142.8, 151.1, 151.2, 155.4. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C28H24Cl2N5O: 516.1352;
found: 516.1322.
5-(3,4-Dichlorophenyl)-3-(4-tetrahydropyranyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydro-
1
phenazine (24). Red solid. Yield 140 mg (67%). Mp: 232–233 °C. H-NMR (300 MHz, CDCl3) δ:
1.61–1.72 (4 H, m), 3.48–3.52 (3 H, m), 4.04 (5 H, m), 5.26 (1 H, s), 6.47 (1 H, d, J = 7.8 Hz),
6.90–6.94 (1 H, m), 6.95 (1 H, s), 7.13–7.23 (3 H, m), 7.49 (1 H, s), 7.70 (1 H, d, J = 7.2 Hz),
13
7.81–7.85 (3 H, m). C-NMR (100 MHz, CDCl3) δ: 33.3, 53.3, 53.8, 65.5, 89.1, 100.3, 113.7, 116.8,
123.3, 124.4, 124.8, 127.9, 128.4, 128.5, 131.0, 131.2, 133.1, 134.4, 134.7, 135.4, 135.6, 136.7, 138.8,
142.6, 150.9, 155.3. HRMS (ESI-TOF+): m/z [M + H]+ calcd for C29H26Cl2N5O2: 546.1875;
found: 546.1874.
5-(3,4-Dichlorophenyl)-3-(4-methoxycyclohexyl)imino-2-(2-methoxy-3-pyridyl)amino-3,5-dihydro-
1
phenazine (25). Red solid. Yield 100 mg (45%). Mp: 215–217 °C. H-NMR (300 MHz, CDCl3) δ:
1.23–1.51 (4 H, m), 1.73–2.10 (4 H, m), 3.15–3.29 (2 H, m), 3.37 (3 H, s), 3.78 (3 H, s), 5.27 (1 H, s),
6.48 (1 H, d, J = 8.4 Hz), 6.90–6.93 (2 H, m), 7.12–7.24 (3 H, m), 7.49 (1 H, d, J = 2.1 Hz), 7.70 (1 H,
d, J = 7.2 Hz), 7.79–7.84 (3 H, m). 13C-NMR (100 MHz, CDCl3) δ: 29.3, 29.4, 30.7, 53.7, 55.8, 56.8,
89.3, 100.2, 113.7, 116.8, 123.2, 124.7, 124.8, 127.8, 128.4, 128.5, 131.1, 131.2, 133.0, 134.4, 134.5,
135.3, 135.6, 136.7, 138.8, 142.7, 151.0, 151.1, 155.4. HRMS (ESI-TOF+): m/z [M + H]+ calcd for
C31H30Cl2N5O2: 574.2129; found: 574.2128.
4. Conclusions
A systematic structure-activity relationship study was conducted based on riminophenazine
pharmacophore containing a 2-methoxypyridylamino group at the C-2 position. This side chain was
identified as having potent antituberculosis activity and less lipophilicity in our previous study.

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Although the number, type and position of the halogen atoms on the phenyl ring at the N-5 position
have no significant impact on the antituberculosis activity, the halogen substitution pattern appears
important for cytotoxicity. The 3,4-dihalogen substitution pattern displayed significantly lower
cytotoxicity. Compound 15 exhibited good pharmacokinetic properties, low skin pigmentation
potential and excellent efficacy in vivo and warrants further evaluation. Our observations indicate that
novel riminophenazine derivatives with a 2-methoxypyridylamino moiety as the promising pharmacophore
have the potential to deliver a new drug candidate for the treatment of TB and MDR-TB.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/4380/s1.
Acknowledgments
We 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.
Author Contributions
Haihong Huang, Dali Yin, Zhenkun Ma, Yu Lu and Christopher B. Cooper conceived and designed
the study. Dongfeng Zhang, Yang Liu, Chunlin Zhang and Hao Zhang performed the chemical
experiments. Bin Wang, Jian Xu and Lei Fu performed the biological experiments. The manuscript
was written and revised by Dongfeng Zhang, Haihong Huang and Zhenkun Ma.
Conflicts of Interest
The authors declare no conflict of interest.
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