Riminophenazine derivatives as potential antituberculosis agents: Synthesis, biological, and electrochemical evaluations

Article abstract of DOI:10.3390/molecules26144200

A series of novel riminophenazine derivatives, having ionizable alkyl substituents at N-5 and a variety of substituents on the C-3 imino nitrogen, at C-8 and on the pendant aryl group, have been designed and synthesized. Preliminary investigations into th

Full text of DOI:10.3390/molecules26144200

molecules
Article
Riminophenazine Derivatives as Potential Antituberculosis
Agents: Synthesis, Biological, and Electrochemical Evaluations
Mpelegeng Victoria Bvumbi 1,2,3, ,†
*
, Chris van der Westhuyzen 2,† , Edwin M. Mmutlane 1 and
Andile Ngwane 4
1
2
3
4
Department of Chemistry, University of Johannesburg, Auckland Park 2006, South Africa; edwinm@uj.ac.za
CSIR, Pharmaceutical Technologies, Pretoria 0001, South Africa; cwvdwesthuyzen@csir.co.za
Department of Chemistry, University of Venda, Private X5050, Thohoyandou 0950, South Africa
Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Medicine
and Health Sciences, Stellenbosch University, Tygerberg 7505, South Africa; andilengwane@gmail.com
Correspondence: mpelegeng.bvumbi@univen.ac.za; Tel.: +27-15-962-8756
*
†
These authors contributed equally to this work.
Abstract: A series of novel riminophenazine derivatives, having ionizable alkyl substituents at N-5
and a variety of substituents on the C-3 imino nitrogen, at C-8 and on the pendant aryl group, have
been designed and synthesized. Preliminary investigations into the relationship between lipophilicity,
redox potential, and antimycobacterial activity were conducted, using the in vitro activity against
Mycobacterium tuberculosis H37Rv, mammalian cytotoxicity, and the redox potential of the compounds
determined by cyclic voltammetry as measures. Results revealed an activity “cliff” associated
with C-8 substitution (10l and 10m) that, along with defined redox activity, point to a new class of
riminophenazines as potential anti-tuberculosis agents having reasonable activity (MIC99 ~1 µM).
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Bvumbi, M.V.;
van der Westhuyzen, C.;
Mmutlane, E.M.; Ngwane, A.
Riminophenazine Derivatives as
Potential Antituberculosis Agents:
Synthesis, Biological,
Keywords: riminophenazines; alkyl substituent; drug discovery; Mycobacterium tuberculosis
and Electrochemical Evaluations.
Molecules 2021, 26, 4200. https://
doi.org/10.3390/molecules26144200
1. Introduction
Tuberculosis (TB), caused by Mycobacterium tuberculosis in man, continues to be a
serious threat to public health [
disease burden still remains high globally, mainly as a result of multidrug-resistant strains
and co-infection with HIV [ ]. Additionally, approximately one-third of the world’s
population have latent TB [ ]. The disease occurs globally, with 22 countries bearing 80%
of the total number of cases of active TB. This distribution occurs largely in the developing
world, with sub-Saharan Africa being the worst affected [ ]. An interesting class of
tricyclic heterocycles, called riminophenazines, has shown promising activity against TB,
leprosy, and cancer [ 12]. These compounds have a phenazine ring substituted on one
1]. Despite the extensive control measures in place, the
Academic Editors: Jonathan Sellars
and Alistair Brown
2,3
4
Received: 14 April 2021
Accepted: 24 May 2021
Published: 10 July 2021
5,6
7
–
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with regard to jurisdictional claims in
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iations.
of the ring nitrogens forming the central phenazine ring, as well as on one or both of the
adjacent fused benzene rings. The designation “rimino” was coined to indicate the ‘R’
substituent on the imino moiety typical of this bioactive class of compounds [8]. This
specific substituent is important in defining this bioactivity. A typical riminophenazine
is clofazimine (Figure 1), which was first discovered by Barry et al. for the treatment
of TB [13]. They proposed an intracellular redox cycling mechanism as the basis for the
activity, in effect functioning as redox ‘traps’ that unproductively deplete cells of FADH
and/or NAD(P)H. Clofazimine has also proved particularly active against M. leprae, which
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Attribution (CC BY) license (https://
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4.0/).
is the causative agent of leprosy [12,14,15]. Although the material (clofazimine) displayed
high in vitro activity against TB, animal studies using guinea pig and monkey models
failed to show in vivo activity [16]. Poor oral absorption of the drug has been implicated in
certain studies, while species-specific activity may also be part of the problem based on
considerable in vivo activity observed in hamsters and mice [17]. In vitro activity against
the M. avium complex (MAC), an opportunistic pathogen in patients with HIV/AIDS, has
Molecules 2021, 26, 4200. https://doi.org/10.3390/molecules26144200
https://www.mdpi.com/journal/molecules

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also been demonstrated [18
–
22]. A major hindrance to the use of riminophenazines in
medicine is the observed staining of the dermis and retina on prolonged use, which was
likely as the result of deposition of these lipophilic materials in fatty tissues [11].
Figure 1. Structure of clofazimine.
Previous SAR investigations using riminophenazines focused on varying the alkylim-
ino group at C-3 and the phenyl group at N-5 as handles to improve activity and phar-
macokinetic behavior [23–26]. Liu et al. proved that the phenazine nucleus is key to the
activity of the materials [24]. In addition, while a phenyl substituent at N-5 is typical of
bioactive riminophenazines, cycloalkyl groups are also well-tolerated [27]. The current
state of SAR knowledge with respect to the riminophenazine compounds is summarized
in Figure 2 [28].
Figure 2. Structure–activity relationships of the riminophenazine system. IUPAC numbering is
included for reference. Substituents targeted in this investigation are labeled R1–R4.
With these facts and the foregoing SAR in mind, an investigation was undertaken to
determine the impact of including an ionizable group at R1 on the riminophenazine skeleton
on antimycobacterial activity. This may provide a means of solubilizing the material, in so
doing diminishing accumulation in the fatty tissues of the skin. We particularly focused on
using a limited number of short tertiary aminoalkyl chains for this purpose. In an attempt
to optimize and/or modulate the impact of the amino moiety on function, a selection

Molecules 2021, 26, 4200
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of substituents at R2–R4, having distinct physicochemical properties, were included for
examination. The activity of these new compounds was assessed against the redox potential
of the materials and their inherent lipophilicity.
2. Results and Discussion
2.1. Chemistry
Several tertiary amines, in the form of N,N-diethyl- or N,N-dimethyl substituted
ethylene- or propylenediamines, as well as N,N-dimethylhydrazine were chosen as can-
didates to generate the desired ionizable group at R1. Scheme 1 shows the synthetic
sequence followed.
◦
Scheme 1. (i) R1 = Me2N(CH2)2: IPA or neat, 90–100 C, 16–24 h, 74%; R1 = Me2N(CH2)3- or Et2N(CH2)3Et2NC2H4-:
◦
◦
K2CO3, DMF, 90 C, 24 h, 52–72%; (ii) 5% Pd/C, H2NNH2, 80 C, 2–3 h; or 5% Pd/C, H2, rt, 4 h, 43–90%; (iii) 1,4-dioxane,
Et3N, 70–80 ◦C, 4 h, 71–83%; (iv) DIPEA, dry EtOH, reflux, 16 h, 50–84%; (v) (a) Zn, AcOH, 50 ◦C, 1 h; or 5% Pd/C, H2,
MeOH:THF, rt, 12 h; then (b) 5◦% Pd/C, H2, THF:MeOH, 12 h; then medical oxygen, MeOH, rt, 12 h; subdivide then
(c) R2NH2, AcOH, dioxane, 110 C, 48 h, 6–37%.
Three starting materials containing the ortho-halonitrobenzene structure
1 were com-
mercially available, namely the parent compound, 1-fluoro-2-nitrobenzene, 1,4-dichloro-2-
nitrobenzene, and 4-chloro-3-nitrotoluene. It was envisaged that the extra methyl or chloro
group’s impact on lipophilicity might have a bearing on activity while also modulating
the redox potential. Ipso-substitution of the halo groups ortho to the nitro moiety in these
materials using each of the three aliphatic amines chosen proceeded smoothly to yield
substituted 2-nitroanilines
not successful. Catalytic hydrogenation of the nitro function in
phenylenediamines in reasonable yield [25], with the exception of the chloro analogues,
for which facile protodehalogenation was noted under these conditions. This series was
subsequently abandoned from the sequence. Concurrently, compounds were synthesized
by ipso-substitution of one of the fluorine groups of 1,5-difluoro-2,4-dinitrobenzene . Three
anilines 5 featuring a variety of para-substituents with varying electronic properties were
2
[
29]. Attempts to introduce N,N-dimethylhydrazine were
2
gave substituted 1,2-
3
6
4

Molecules 2021, 26, 4200
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used for this purpose, affording desired anilines
6. Key intermediates
7
were obtained
in modest yields by similar substitutions of each of the remaining fluoro groups in ani-
lines by the primary amino groups of phenylenediamines . Reduction of the nitro
groups in under either acidic or neutral conditions, followed by immediate oxidative
cyclization without prior isolation of tetramines , afforded the desired iminophenazines
25 30]. These latter two sets of compounds were not isolated because of difficulties in
purification and questionable stability. Transimination of the unsubstituted imino moiety
in compound with commercially available isobutylamine, isopentylamine, or cyclohexy-
6
3
7
8
9
[ ,
9
lamine, was followed by column chromatography and preparative TLC to afford the target
riminophenazines 10 in low yields [25,30].
2.2. Antimycobacterial Assessment
The compounds obtained were screened in triplicate against M. tuberculosis H37Rv
using the MGIT 960 system at 1, 3, and 5
µM concentrations, with compounds showing
MIC99 M further tested in triplicate in the range 0.0625–1 µ
≈
1
µ
M to refine the values
determined. Cytotoxicity was determined using WI-38 cells (Human Fetal Lung Fibroblast)
as a model of rapidly dividing normal mammalian cells. Although 26 compounds were
synthesized, selected compounds were investigated, mainly due to the low quantities
obtained synthetically. The data are summarized in Table 1.
Lipophilicity (LogP) is an important property for riminophenazine derivatives, as the
skin discoloration side effect of clofazimine is closely related to its high lipophilicity. As
shown in Table 1, the LogP values for the most new compounds are lower than that of
clofazimine (7.50), ranging from 3.43 to 5.02. Modifications of the phenazine ring at position
N-5 with alkyl diamines provided compounds having lower LogP values compared to
clofazimine. Generally, replacement of the phenyl group at the N-5 position by an alkyl
group lowered LogP values, depending on the structure of the alkyl groups. Compounds
with a dimethylaminoethyl substituent at the N-5 position displayed significantly reduced
lipophilicity as compared to the corresponding dimethylaminopropyl/diethylaminopropyl
substituted compounds, as exemplified by compound 10o (LogP 3.43) versus compound
10b (LogP 4.21), compound 10g (LogP 3.64) versus compound 10i (LogP 3.74), and com-
pound 10c (LogP 4.31) versus compound 10f (LogP 5.02). These results are consistent with
previous observation made by Yi-li et al., who also observed that improved antibacterial
activity is dependent on increasing lipophilicity, a major factor for skin pigmentation [27].
In addition, the benzyl substituent at the C-2 position with hydrogen, methyl, and ethoxy at
the para position showed significantly reduced LogP values, with lipophilicity increasing
as R3 was changes from H to OEt to Me. This was exemplified by compounds 10o, 10n,
and 10g with LogP values of (H: 3.43 < OEt: 3.64 < Me: 3.91). The same trend was observed
for compounds 10a (LogP 3.75), 10h (LogP 3.96) and 10c (LogP 4.31). 10o could serve as an
interesting lead for identifying new analogues with further decreased lipophilicity.
In terms of cytotoxicity, there are no general trends or variances with chain length
or ring substitution. The R2 = cHex series in each case appears to be the least toxic set of
compounds (compare 10d
, 10e with 10c, 10f, or 10i, 10j, 10k with 10g, 10h). Compounds
in the R3 = OEt, R4 = H series tend to be among the least toxic members of each contiguous
series. Further substitution of the system (compare 10g and 10l, 10f and 10m) increases
the toxicity.
With regard to antimycobacterial action, most of the activity appears to be as a result
of toxicity rather than selective bactericidal action. The exceptions are 10l, 10m, and 10p
with both 10l and 10m having R3, R4 = Me as substituents, along with Me2N(CH2)2- at N-5.
Similarly, 10p has a methyl substituent at R3 which might also contribute to its activity.
Compounds 10l, 10m, and 10p exhibited potent activity comparable to that of the control
drug, isoniazid, and also the well-known riminophenazine drug, clofazimine, all with MIC
values <1
µ
M. In each case, an activity cliff (MIC99
≈
1 µM) is encountered relative to all
the other compounds, particularly conjoiners 10d and 10e (relative to 10l) and 10c and
10f (relative to 10m). The selectivity index of 10l, 10m, and 10p (defined as cytotoxicity

Molecules 2021, 26, 4200
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IC50/MIC99), SI
≥
3 is modest at best, but it does indicate that interesting activity is still
possible using the ionizable groups included in the design. Most of the rest of the materials
show an SI < 1.5 by comparison.
Table 1. Structure–activity assessment of riminophenazines 10.
Compound
R1
R2
R3
R4
LogP 1
IC50 (µM) 2
MIC99 (µM) 3
* Clofazimine
10b
4-Clphenyl
Et2N(CH2)3-
Me2N(CH2)2- iBu
Me2N(CH2)3- iBu
Et2N(CH2)3-
Me2N(CH2)2- iBu
Me2N(CH2)2- iBu
Me2N(CH2)2- iBu OEt
Me2N(CH2)3- iBu OEt
Et2N(CH2)3-
Et2N(CH2)3-
Me2N(CH2)2- cHex
Me2N(CH2)2- cHex Me
Me2N(CH2)3- cHex Me
Et2N(CH2)3-
Me2N(CH2)2- cHex Me
Me2N(CH2)2- cHex OEt
iPr
iBu
Cl
H
H
Me
Me
Me
Me
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
Me
H
7.50
4.21
3.43
4.02
4.70
4.73
3.91
3.64
3.74
4.42
4.77
3.75
4.31
4.34
5.02
4.41
3.96
68.62
4.44
2.45
2.47
1.63
2.86
3.89
12.30
4.81
5.35
3.81
9.01
5.99
5.08
4.37
3.64
11.10
<1
3
3
3
5
<1
3
5
5
3
5
3
5
<1
3
<1
5
10o
10d
10e
10l
10n
10g
10i
10j
10k
10a
10c
10p
10f
10m
10h
iBu
iBu OEt
iPent OEt
H
cHex Me
INH
<1
LogP calculated using ChemDraw version 12.0; 2 Cytotoxicity against rapidly dividing human fibroblasts (WI38
1
cell line); 3 MIC99: lowest concentration of the material inhibiting 99% of M. tuberculosis H37Rv growth in culture.
* Data taken from literature [26,27], INH = Isoniazid.
2.3. Cyclic Voltammetry Studies
Cyclic voltammetry was used to determine the redox potential of the compounds,
as a measure of the electronic impact the various substituents introduced on the rim-
inophenazine skeleton may have. The results are summarized in Table 2. The cyclic
voltammograms of the compounds each displayed a fully reversible one-electron transfer
process. For example, the cyclic voltammogram of 10n (Figure 3) showed that the redox
cycle was fully reversible: increasingly negative (reducing) applied potential through a
minimum anodic current produced reduced species on the left (10n), while an increas-
ingly positive (oxidative) potential produces a maximum cathodic current to afford the
fully oxidized species [10n(i)] to the right. All the compounds had negative half-wave
potentials, indicating that these riminophenazines are easily oxidized—as observed in the
laboratory [31].
It is interesting to note that the half-wave potential of the compounds proved to be a
modest indicator of activity. All the compounds having MIC99 < 3
µM showed E1 values
2
between 0.20 V and 0.13 V. Compounds (10b 10c 10e 10f 10j) with E1 >
−
−
,
,
,
,
−
0.22 V were
2
generally inactive. This may indicate, in conjunction with the structural requirements for
the observed activity cliff, that there is both an electronic/redox potential aspect, as well as
some form of specific steric/binding determinant operative in this series of compounds
that may be further exploited to improve the observed activity.

Molecules 2021, 26, 4200
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Table 2. Redox potentials (V vs. Ag/Ag+) of selected riminophenazines.
Compound
Epa/V
Epc/V
E1 /V
∆E
2
10b
10e
10i
10n
10j
10c
10f
10m
–0.0392
–0.215
–0.0218
–0.0021
–0.077
–0.0055
–0.0606
–0.0305
–0.41
–0.971
–0.28
–0.26
–0.38
–0.49
–0.41
–0.37
–0.22
–0.59
–0.15
–0.13
–0.23
–0.24
–0.24
–0.20
0.37
0.76
0.25
0.26
0.30
0.35
0.35
0.34
Measured in CH3CN containing 0.1 M [n-Bu4N][ClO4] at a scan rate of 100 mV s−1 and referenced to Ag/Ag+.
Epa = anodic potential peak. Epc = cathodic potential peak. E1 = Half wave potential [E1 = (Epa + Epc)/2].
2
2
∆E = Epa − Epc
.
Figure 3. Cyclic voltammogram (CV) of 10n.
3. Materials and Methods
The reactions were monitored for their completion by thin layer chromatography
(TLC) using aluminium-backed Macherey-Nagel ALUGRAM Sil G/UV254 plates pre-
coated with 0.25 mm silica gel 60Å. Column chromatography was carried out on silica gel
60 (particle size 200–300 mesh) with a silica to compound ratio of 30:1 by mass or neutral
alumina as the adsorbent for conventional preparative chromatography.
NMR spectra were recorded on a Varian INOVA 400 MHz system or a Varian Pre-
miumShield VNMRS 600 MHz system operating at 399.94 or 599.74 MHz, respectively.
Chemical shifts (δ) are reported in parts per million (ppm) relative to tetramethylsilane
(TMS) or against the residual protonated solvent signal as reference. Coupling constants (J)
are reported in Hz.
Melting points were obtained on a Reichert Hot Stage or a Mettler FP62 melting
point apparatus. High-resolution mass spectra were recorded on a Waters Acquity UPLC
coupled in tandem to a Waters photodiode array (PDA) detector and a SYNAPT G1 HDMS
mass spectrometer.
3.1. Synthesis Procedures
General Procedure for Final Compounds 10a–p
The chosen dinitro compound 7 (3 mmol) and 5% palladium on carbon (3 mmol) in 2:3
(v/v) methanol:THF was stirred at room temperature under 275 kPa hydrogen atmosphere
for 12 h in a steel pressure vessel. The orange solution turned maroon after the completion.

Molecules 2021, 26, 4200
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The catalyst was filtered off through a celite pad, and the filtrate was concentrated under
reduced pressure. The gummy maroon residue was dissolved in methanol and stirred
at room temperature under pure oxygen (99.5%) atmosphere (345 kPa) for 12 h. The
mixture was concentrated under reduced pressure to give a dark red/black product. The
crude residue was purified on silica using 5–15% methanol:chloroform as eluent and
used immediately without characterization. The material obtained was divided among a
choice of amines (isobutylamine, cyclohexylamine, or isopentylamine) (25 eq.) in dioxane
(
3–4 mL) and glacial acetic acid (0.2 eq.) and heated at 110 ◦C in a sealed or closed tube
for 48 h. After cooling to room temperature, the material was concentration in vacuo, and
the residue obtained was purified by column chromatography on silica gel [2–5% (v/v)
methanol:chloroform as eluent]. Further purification using preparative TLC was required
using the same eluent to ensure adequate purity of the isolated materials.
(E)-5-[2-(Dimethylamino)ethyl]-3-cyclohexylimino-2-phenylamino-3,5-dihydrophenazine (10a). Dark
◦
red solid (20.4 mg, 20.4%); Rf 0.46 [10% (v/v) methanol: chloroform]; mp. 94–96 C (ethanol).
1H NMR (400 MHz, CD3OD):
δ 7.33 (dd, J = 7.93, 1.03 Hz, 1H, H 9), 7.27 (dd, J = 8.51,
7.78 Hz, 2H, H 3’), 7.21–7.17 (m, H 2’), 7.15 (d, J = 7.70 Hz, 1H, H 6), 7.11 (d, J = 8.48 Hz,
1H, H 7), 6.99 (dt, J = 13.1, 7.3 Hz, 2H, H 8), 6.53 (s, 1H, H 1), 5.74 (s, 1H, H 4), 3.91 (br
s, 2H, NCH2CH2), 3.48–3.36 (m, 1H, =NCH), 2.46–2.35 (m, 2H, CH2NMe2), 2.26 (s, 6H,
NMe2), 1.93–1.75, 1.75–1.63, 1.49–1.34, and 1.33–1.24 [4
×
m, 10H, NCH(C5H10)]; 13C NMR
(101 MHz, CD3OD): 152.4 (C 3), 151.7 (C-10a), 145.1 (C 2), 141.4 (C 1’), 136.7 (C 9a), 134.7
δ
(C-4a), 131.5 (C 5a), 130.6 (C 3’), 129.7 (C 4’), 128.9 (C 9), 124.7 (C 7), 124.2 (C 8), 122.4 (C 2’),
114.0 (C 6), 99.9 (C 1), 89.0 (C 4), 59.3 (=NCH), 54.8 (CH2NMe2), 46.1 (NMe2), 44.8 (NCH2),
35.0 [CH(CH2)2], 27.2 [CH2(CH2)2], 26.3 [CH2(CH2)2]; HRMS (ESI-TOF+): m/z calculated
for C28H34N5: 440.2814; found: 440.2810 (MH+). IR (cm−1): 3209 (N-H), 2923 (Ar-C-H),
1508 (C=N), 1463 (Ar-C=C), 1241 (C-N).
(E)-5-[3-(Diethylamino)propyl]-3-isobutylimino-2-phenylamino-3,5-dihydrophenazine (10b). Dark
red solid (12.5 mg, 37%); Rf 0.42 [20% (v/v) methanol: chloroform]; mp. 102–104 ◦C
1
(ethanol). H NMR (400 MHz, CD3OD):
δ 8.19 (br d, J = 8.79 Hz, 1H, H 6), 8.03 (dd, J = 8.35,
1.42 Hz, 1H, H 9), 7.89 (ddd, J = 8.76, 7.10, 1.51 Hz, 1H, H 7), 7.70 (ddd, J = 8.18, 7.10,
0
0
0.93 Hz, 1H, H 8), 7.50–7.45 (m, 2H, H 3 ), 7.41–7.37 (m, 2H, H 2 ), 7.29 (s, 1H, H 1), 7.23 (ddt,
0
J = 7.30, 7.23, 1.17 Hz, 1H, H 4 ), 6.77 (s, 1H, H 4), 4.93 (br t, J = 7.62 Hz, 2H, NCH2CH2), 3.54
(d, J = 7.13 Hz, 2H, NCH2CH), 2.88 (t, J = 7.08 Hz, 2H, CH2NEt2), 2.73 [q, J = 7.16 Hz, 4H,
N(CH2CH3)2], 2.29 (dquin, J = 13.49, 6.78 Hz, 1H, CHMe2), 2.17 (dt, J = 15.11, 7.43 Hz, 2H, -
CH2-), 1.16 (d, J = 6.64 Hz, 6H, CHMe2) and 1.15 [t, J = 7.13 Hz, 6H, N(CH2CH3)2]; 13C NMR
0
(101 MHz, CD3OD): 154.4 (C 3), 147.2 (C 10a), 142.6 (C 2), 141.3 (C 1 ), 140.4 (C 9a), 135.5 (C
4a), 133.7 (C 7), 131.5 (C 9), 131.0 (C 30), 130.7 (C 5a), 128.5 (C 8), 126.2 (C 40), 123.7 (C 20),
117.2 (C 6), 107.7 (C 1), 90.2 (C 4), 53.9 (NCH2CH), 50.6 (CH2NEt2), 48.3 [N(CH2CH3)2], 47.6
(NCH2CH2), 29.2 (CHMe2), 25.7 (-CH2-), 21.2 (CHMe2) and 11.6 [N(CH2CH3)2]. HRMS
(ESI-TOF+): m/z calculated for C29H38N5: 456.3127; found: 456.3114 (MH+). IR (cm−1):
3381 (N-H), 2980 (Ar-C-H), 1509 (C=N), 1461 (Ar-C=C), 1250 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-cyclohexylimino-2-(4-methylphenyl)amino-3,5-dihydrophenazine
(
10c). Maroon solid (38 mg, 12% yield); Rf 0.39 [10% (v/v) methanol: chloroform]; mp.
1
110–112 ◦C (ethanol). H NMR (400 MHz, CD3OD):
δ 7.39 (dd, J = 7.91, 1.37 Hz, 1H, H
6), 7.19 (br d, J = 7.70 Hz, 1H, H 9), 7.24 (ddd, J = 8.40, 7.03, 1.37 Hz, 1H, H 7), 7.12 (d,
J = 8.49 Hz, 2H, H 2’), 7.10 (d, J = 8.89 Hz, 2H, H 3’), 7.04 (ddd, J = 8.01, 6.93, 1.07 Hz, 1H,
H 8), 6.53 (s, 1H, H 1), 5.82 (s, 1H, H 4), 4.00 (br t, J = 7.30 Hz, 2H, NCH2CH2), 3.50 (br td,
1H, =NCH), 2.50 (br t, J = 7.81 Hz, 2H, CH2NMe2), 2.34 (s, 6H, NMe2), 2.29 (3H, s, C-4’
Me), 1.93–1.75, 1.75–1.63, and 1.53–1.35 [10H, 3
×
m, NCH(C5H10)]; 13C NMR (101 MHz,
CD3OD): 152.4 (C 3), 151.6 (C-10a), 145.4 (C 2), 138.6 (C 9a), 136.8 (C 1’), 134.6 (C 4’), 134.0
δ
(C 4a), 131.3 (C 7), 131.1 (C 3’), 129.6 (C 9), 128.8 (C 5a), 124.2 (C 8), 122.6 (C 2’), 113.9 (C
6), 99.5 (C 1), 89.0 (C 4), 59.2 (=NCH), 54.8 (CH2NMe2), 46.1 (NMe2), 44.8 (NCH2), 35.0
[CH(CH2)2], 27.2 [CH2(CH2)2], 26.3 (C-4’ Me), 21.1 [CH2(CH2)2]; HRMS (ESI-TOF+): m/z

Molecules 2021, 26, 4200
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calculated for C29H36N5: 454.2971; found: 454.2956 (MH+); IR (cm−1): 3292 (N-H), 2920
(Ar-C-H), 1517 (C=N) and 1465 (Ar-C=C), 1240 (C-N).
(E)-5-[3-(Dimethylamino)propyl]-3-isobutylimino-2-(4-methylphenyl)amino-3,5-dihydrophenazine
(
10d). Dark maroon crystals (49 mg; 13%); Rf 0.30 [20% (v/v) methanol:chloroform];
1
mp. > 230 ◦C (ethanol). H NMR (600 MHz, CD3OD):
δ 8.25 (br d, J = 8.30 Hz, 1H, H 6),
8.23 (d, J = 7.90, Hz, 1H, H 9), 8.08 (t, J = 7.52 Hz, 1H, H 7), 7.90 (t, J = 7.50 Hz, 1H, H 8),
7.66 (d, J = 8.25 Hz, 2H, H 30), 7.40 (d, J = 8.40 Hz, 2H, H 20), 7.40 (s, 1H, H 1), 6.88 (s, 1H,
H 4), 5.03 (br t, J = 7.63 Hz, 2H, NCH2CH2), 3.80 (d, J = 7.04 Hz, 2H, NCH2CH), 2.93 (t,
0
J = 6.90 Hz, 2H, CH2NMe2), 2.77 (s, 6H, NMe2), 2.71 (s, C-4 3H, Me), 2.58 (quin, J = 7.04 Hz,
CHMe2), 2.44 (dt, J = 15.26, 7.34 Hz, 2H, -CH2-) and 1.52 (d, J = 6.75 Hz, 6H, CHMe2); 13
C
NMR (151 MHz, CD3OD): 154.4 (C 3), 147.0 (C 10a), 142.8 (C 2), 140.7 (C 9a), 138.4 (C 10),
136.6 (C 40), 135.6 (C 4a), 133.8 (C 7), 131.7 (C 9), 131.6 (C 30), 130.6 (C 5a), 128.8 (C 8), 124.3
(C 20), 117.4 (C 6), 107.4 (C 1), 90.3 (C 4), 57.1 (CH2NMe2), 53.4 (NCH2CH), 47.5 (NMe2),
45.6 (NCH2CH2), 29.1 (CHMe2), 26.1 (-CH2-), 21.2 (C-40 Me) and 21.1 (CHMe2); HRMS
(ESI-TOF+): m/z calculated for C28H36N5: 442.2971; found: 442.2892 (MH+). IR (cm−1):
3108 (N-H), 2954 (Ar-C-H), 1512 (C=N), 1459 (Ar-C=C), 1239 (C-N).
(E)-5-[3-(Diethylamino)propyl]-3-isobutylimino-2-(4-methylphenyl)amino-3,5-dihydrophenazine
(
10e). Dark red solid (22.4 mg, 5.8%); Rf 0.38 [10% (v/v) methanol:chloroform]; mp.
1
80–82 ◦C (ethanol). H NMR (400 MHz, CD3OD):
δ
8.11 (br d, J = 8.89 Hz, 1H, H 6), 7.99
(br d, J = 8.30 Hz, 1H, H 9),07.82 (br t, 1H, J = 7.80 Hz, H 7), 7.64 (br t, J = 7.80 Hz, 1H, H 8),
0
7.27 (q, J = 8.89 Hz, 4H, H 3 , H 2 ), 7.17 (s, 1H, H 1), 6.68 (s, 1H, H 4), 4.84 (br t, J = 8.30 Hz,
2H, NCH2CH2), 3.49 (d, J = 7.03 Hz, 2H, NCH2CH), 2.76 (t, J = 6.93 Hz, 2H, CH2NEt2), 2.64
[q, J = 7.13 Hz, 4H, N(CH2CH3)2], 2.38 (s, 3H, C-40 Me), 2.25 (dquin, J = 13.54, 6.75 Hz, 1H,
CHMe2), 2.15–2.06 (m, J = 8.50, 2H, -CH2-), 1.13 [d, J = 7.18 Hz, 6H, N(CH2CH3)2], and 1.10
(t, J = 6.64 Hz, 6H, CHMe2); 13C NMR (101 MHz, CD3OD): 154.2 (C 3), 147.9 (C 10a), 143.9
(C 2), 140.1 (C 9a), 138.5 (C 10), 136.3 0(C 40), 135.3 (C 4a), 133.1 (C 7), 131.2 (C 9), 131.5 (C
0
3 ), 130.7 (C 5a), 128.0 (C 8), 124.0 (C 2 ), 116.9 (C 6), 107.0 (C 1), 90.4 (C 4), 54.0 (NCH2CH),
51.0 (CH2NEt2), 48.3 [N(CH2CH3)2], 47.5 (NCH2CH2), 29.5 (CHMe2), 26.1 (-CH2-), 21.4
(CHMe2), 21.3 (C-40 Me) and 11.9 [N(CH2CH3)2]; HRMS (ESI-TOF+): m/z calculated for
C30H40N5: 470.3284; found: 470.3291 (MH+); IR (cm−1): 3367 (N-H), 2980 (Ar-C-H), 1517
(C=N), 1464 (Ar-C=C), 1250 (C-N).
(E)-5-[3-(Diethylamino)propyl]-3-cyclohexylimino-2-(4-methylphenyl)amino-3,5-dihydrophenazine
(
10f). Dark red solid (35.9 mg, 9.7%); Rf 0.36 [20% (v/v) methanol:chloroform]; mp.
108–110 ◦C (ethanol). 1H NMR (400 MHz, CD3OD):
δ
8.19 (br d, J = 8.79 Hz, 1H, H
6), 8.04 (dd, J = 8.40, 1.37 Hz, 1H, H 9), 7.87 (ddd, J = 8.69, 7.13, 1.57 Hz, 1H, H 7), 7.70
(ddd, J = 8.30, 7.13, 0.88 Hz, 1H, H 8), 7.29 (d, J = 8.01 Hz, 2H, H 3’), 7.26 (d, J = 9.18 Hz, 2H,
H 2’), 7.22 (s, 1H, H 1), 6.81 (s, 1H, H 4), 4.98–4.88 (m, 2H, NCH2), 4.07 (br t, J = 7.81 Hz,
1H, =NCH), 2.81 (t, J = 7.13 Hz, 2H, CH2NEt2), 2.67 [q, J = 7.10 Hz, 4H, N(CH2CH3)2],
2.38 (s, 3H, C-4’ Me), 2.23–2.17 (m, 2H, -CH2-), 1.82–1.18 [4
×
m, 10H, NCH(C5H10)], and
1.10 [t, J = 7.23 Hz, 6H, N(CH2CH3)2]; 13C NMR (101 MHz, CD3OD):
δ
152.8 (C 3), 147.3
(C-10a), 143.2 (C 2), 140.5 (C 9a), 138.4 (C 1’), 136.6 (C 4’), 135.5 (C 4a), 133.4 (C 7), 131.6
(C 3’), 131.5 (C 9), 130.6 (C 5a), 128.5 (C 8), 124.3 (C 2’), 117.2 (C 6), 106.8 (C 1), 90.1 (C
4), 55.3 (=NCH), 50.9 (CH2NEt2), 48.3 [N(CH2CH3)2], 47.6 (NCH2), 33.1 [CH(CH2)2], 26.7
(-CH2-), 26.2 [CH2(CH2)2], 21.1 (C-4’ Me) and 11.8 [N(CH2CH3)2]; HRMS (ESI-TOF+): m/z
calculated for C32H42N5: 496.3440; found: 496.3460 (MH+). IR (cm−1): 3367 (N-H), 2980
(Ar-C-H), 1511 (C=N), 1462 (Ar-C=C), 1250 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-isobutylimino-2-(4-ethoxyphenyl)amino-3,5-dihydrophenazine
(
10g). Dark red solid (193 mg, 47%); Rf 0.32 [10% (v/v) methanol:chloroform]; mp.
208–210 ◦C (ethanol). 1H NMR (400 MHz, CD3OD):
δ
8.09 (br d, J = 8.98 Hz, 1H, H
6), 8.04 (br d, J = 7.81 Hz, 1H, H 9), 7.83 (dd, J = 8.49, 6.93 Hz, 1H, H 7), 7.65 (t, J = 7.42 Hz,
1H, H 8), 7.29 (d, J = 8.79 Hz, H 2’), 7.09 (d, J = 8.89 Hz, 2H, H 3’), 7.03 (s, 1H, H 1), 6.87 (s,
1H, H 4), 4.90 (dd, J = 7.42, 6.54 Hz, 2H, NCH2CH2), 4.09 (q, J = 6.97 Hz, 2H, OCH2CH3),
3.52 (d, J = 7.0 Hz, 2H, NCH2CH), 2.89 (t, J = 7.37 Hz, 2H, CH2NMe2), 2.46 (s, NMe2), 2.26

Molecules 2021, 26, 4200
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(sep, J = 6.63 Hz, 1H, CHMe2), 1.43 (t, J = 6.98 Hz, 3H, OCH2CH3), 1.14 (d, J = 6.64 Hz, 6H,
CHMe2); 13C NMR (101 MHz, CD3OD):
δ 158.5 (C 4’), 154.1 (C 3), 147.2 (C 10a), 143.5 (C 2),
140.4 (C 9a), 135.5 (C 4a), 133.3 (C 1’), 133.1 (C 7), 131.3 (C 9), 130.4 (C 5a), 128.5 (C 8), 126.4
(C 2’), 116.9 (C 3’), 116.8 (C 6), 105.7 (C 1), 90.3 (C 4), 64.9 (OCH2CH3), 56.6 (CH2NMe2),
53.5 (NCH2CH), 47.9 (NCH2CH2), 46.1 (NMe2), 28.9 (CHMe2), 20.9 (CHMe2) and 15.2
(OCH2CH3); HRMS (ESI-TOF+): m/z calculated for C28H36N5O: 458.2920; found: 458.2891
(MH+); IR (cm−1): 3128 (N-H), 2954 (Ar-C-H), 1507 (C=N), 1464 (Ar-C=C), 1230 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-cyclohexylimino-2-(4-ethoxyphenyl)amino-3,5-dihydrophenazine
(
10h). Dark red solid (45 mg, 14%); Rf 0.42 [10% (v/v) methanol:chloroform]; mp. 222–224 ◦C
1
(ethanol). H NMR (400 MHz, CD3OD):
δ 8.04–7.96 (m, 2H, H 6, H-9), 7.82 (dd, J = 11.5,
4.2 Hz, 1H, H 7), 7.65 (br t, J = 7.45 Hz, 1H, H 8), 7.30–7.25 (m, 2H, H 2’), 7.04 (s, 1H, H
1), 7.02 (d, J = 8.80 Hz, 2H, H 3’), 6.83 (s, 1H, H 4), 4.85 (obscured, 2H, NCH2), 4.08 (q,
J = 6.85 Hz, 2H, OCH2CH3), 3.92 (m, 2H, =NCH), 2.85 (t, J = 7.63 Hz, 2H, CH2NMe2), 2.46
(s, 6H, NMe2), 2.23–2.15, 1.96–1.85, 1.83–1.75 and 1.62–1.55 [4
×
m, 10H, NCH(C5H10)],
1.40 (t, J = 6.90 Hz, 3H, OCH2CH3); 13C NMR (101 MHz, CD3OD):
δ
158.6 (C 4’), 152.7 (C
3), 147.7 (C 10a), 144.1 (C 2), 140.2 (C 9a), 135.6 (C 4a), 133.3 (C 7), 133.2 (C 1’), 131.1 (C 9),
130.6 (C 5a), 128.2 (C 8), 126.6 (C 2’), 117.0 (C 3’), 116.7 (C 6), 105.1 (C 1), 90.4 (C 4), 65.1
(OCH2CH3), 56.8 (=NCH), 55.8 (CH2NMe2), 47.7 (NCH2), 46.3 (NMe2), 33.3 [CH(CH2)2],
26.7 and 26.2 [CH2(CH2)2] and 15.3 (OCH2CH3); HRMS (ESI-TOF+): m/z calculated for
C30H38N5O: 484.3076; found: 484.3048 (MH+).
(E)-5-[3-(Dimethylamino)propyl]-3-isobutylimino-2-(4-ethoxyphenyl)amino-3,5-dihydrophenazine
(
10i). Dark red solid (41.0 mg, 15%); Rf 0.40 [20% (v/v) methanol:chloroform]; mp.
185–187 ◦C (ethanol). 1H NMR (400 MHz, CD3OD):
δ
8.21 (d, J = 8.79 Hz, 1H, H 6),
8.05 (d, J = 8.40 Hz, 1H, H 9), 7.84 (ddd, J = 9.00, 7.19, 1.27 Hz, 1H, H 7), 7.72 (t, J = 7.70 Hz,
1H, H 8), 7.30 (d, J = 8.89 Hz, 2H, H 2’), 7.11 (s, 1H, H 1), 7.03 (d, J = 8.98 Hz, 2H, H 3’), 6.87
(s, 1H, H 4), 4.98 (t, J = 7.80 Hz, 2H, NCH2CH2), 4.08 (q, J = 6.93 Hz, 2H, OCH2CH3), 3.55
(d, J = 7.03 Hz, 2H, NCH2CH), 2.61 (t, J = 6.83 Hz, 2H, CH2NMe2), 2.34 (s, 6H, NMe2), 2.27
(sep, J = 6.77 Hz, 1H, CHMe2), 2.18 (br t, J = 6.74 Hz, 2H, -CH2-), 1.41 (t, J = 6.98 Hz, 3H,
OCH2CH3), 1.15 (d, J = 6.64 Hz, 6H, CHMe2); 13C NMR (101 MHz, CD3OD):
δ 158.7 (C
4’), 154.1 (C 3), 147.2 (C 10a), 143.7 (C 2), 140.6 (C 9a), 135.5 (C 4a), 133.5 (C 1’), 133.3 (C 7),
131.5 (C 9), 130.5 (C 5a), 128.7 (C 8), 126.6 (C 3’), 117.3 (C 6), 117.0 (C 2’), 106.0 (C 1), 90.3
(C 4), 65.1 (OCH2CH3), 57.3 (CH2NMe2), 53.6 (NCH2CH), 47.6 (NCH2CH2), 46.0 (NMe2),
29.1 (CHMe2), 26.4 (-CH2-), 21.1 (CHMe2), and 15.3 (OCH2CH3); HRMS (ESI-TOF+): m/z
calculated for C29H38N5O: 472.3076; found: 472.3048 (MH+); IR (cm−1): 3085 (N-H), 2955
(Ar-C-H), 1502 (C=N), 1462 (Ar-C=C), 1232 (C-N).
(E)-5-[3-(Diethylamino)propyl]-3-isobutylimino-2-(4-ethoxyphenyl)amino-3,5-dihydrophenazine
(
10j). Dark purple solid (22.1 mg, 6.2%); Rf 0.37 [20% (v/v) methanol:chloroform]; mp.
1
205–207 ◦C (ethanol). H NMR (400 MHz, CD3OD):
δ 7.98 (d, J = 8.79 Hz, 1H, H 6), 7.84
(dd, J = 8.24, 1.28 Hz, 1H, H 9), 7.69 (ddd, J = 8.63, 7.17, 1.40 Hz, 1H, H 7), 7.52 (ddd, J = 8.20,
6.95, 0.70 Hz, 1H, H 8), 7.16 (d, J = 9.03 Hz, 2H, H 2’), 6.91 (s, 1H, H 1), 6.88 (d, J = 8.91 Hz,
2H, H 3’), 6.55 (s, 1H, H 4), 4.71 (t, J = 7.80 Hz, 2H, NCH2CH2), 3.99 (q, J = 7.00 Hz, 2H,
OCH2CH3), 3.39 (d, J = 7.08 Hz, 2H, NCH2CH), 2.66 (t, J = 6.96 Hz, 2H, CH2NEt2), 2.55
[q, J = 7.12 Hz, 4H, N(CH2CH3)2], 2.14 (sep, J = 6.76 Hz, 1H, CHMe2), 2.03–1.94 (m, 2H,
-CH2-), 1.29 (t, J = 7.02 Hz, 3H, OCH2CH3), 1.01 (d, J = 6.59 Hz, 6H, CHMe2), and 0.97 [t,
J = 7.14 Hz, 6H, N(CH2CH3)2]; 13C NMR (101 MHz, CD3OD):
δ 158.5 (C 4’), 154.0 (C 3),
148.0 (C 10a), 144.2 (C 2), 139.9 (C 9a), 135.5 (C 4a), 133.4 (C 1’), 132.7 (C 7), 130.9 (C 9),
130.6 (C 5a), 127.8 (C 8), 126.3 (C 3’), 116.9 (C 2’), 116.8 (C 6), 104.6 (C 1), 89.9 (C 4), 65.1
(OCH2CH3), 54.7 (CH2NEt2), 50.9 (NCH2CH), 48.3 [N(CH2CH3)2], 47.4 (NCH2CH2), 29.5
(CHMe2), 25.8 (-CH2-), 21.2 (CHMe2), 15.3 (OCH2CH3) and 11.9 [N(CH2CH3)2]; HRMS
(ESI-TOF+): m/z calculated for C31H42N5O: 500.3389; found: 500.3391 (MH+); IR (cm−1):
3365 (N-H), 2980 (Ar-C-H), 1509 (C=N), 1462 (Ar-C=C), 1233 (C-N).
(E)-5-[3-(Diethylamino)propyl]-3-isopentylimino-2-(4-ethoxyphenyl)amino-3,5-dihydrophenazine
(10k). Dark purple solid (19.1 mg, 6.2%) after crystallization; Rf 0.39 [20% (v/v) methanol:

Molecules 2021, 26, 4200
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◦
1
chloroform]; mp. 198–200 C (ethanol). H NMR (400 MHz, CD3OD):
δ
8.17 (d, J = 8.79 Hz,
1H, H 6), 8.02 (dd, J = 8.30, 1.37 Hz, 1H, H 9), 7.85 (ddd, J = 8.74, 7.13, 1.42 Hz, 1H, H 7),
7.69 (ddd, J = 8.30, 6.90, 0.70 Hz, 1H, H 8), 7.29 (d, J = 8.89 Hz, 2H, H 2’), 7.05 (d, J = 8.89 Hz,
2H, H 3’), 7.02 (s, 1H, H 1), 6.74 (s, 1H, H 4), 4.91 (dd, J = 8.40, 7.40 Hz, 2H, NCH2CH2),
4.07 (q, J = 7.03 Hz, 2H, OCH2CH3), 3.34–3.29 (m, 2H, =NCH2), 2.79 (t, J = 6.93 Hz, 2H,
CH2NEt2), 2.66 [q, J = 7.13 Hz, 4H, N(CH2CH3)2], 1.95–1.81 (m, 3H, CH2CHMe2), 2.15
(quin, J = 7.13 Hz, 2H, -CH2-), 1.42 (t, J = 6.98 Hz, 3H, OCH2CH3), 1.11 (d, J = 7.2 Hz,
6H, CHMe2) and 1.08 [d, J = 6.3 Hz, 6H, N(CH2CH3)2]; 13C NMR (101 MHz, CD3OD):
δ
158.6 (C 4’), 153.8 (C 3), 147.5 (C 10a), 143.8 (C 2), 140.4 (C 9a), 135.3 (C 4a), 133.3 (C 1’),
133.1 (C 7), 131.3 (C 9), 130.5 (C 5a), 128.4 (C 8), 126.4 (C 3’), 117.1 (C 6), 116.9 (C 2’), 105.3
(C 1), 89.9 (C 4), 65.1 (OCH2CH3), 50.9 (CH2NEt2), 48.3 [N(CH2CH3)2], 47.4 (NCH2CH2),
44.9 (=NCH2), 38.3 (CH2CH), 27.6 (CHMe2), 25.9 (-CH2-), 23.1 (CHMe2), 15.3 (OCH2CH3)
and 11.8 [N(CH2CH3)2]; HRMS (ESI-TOF+): m/z calculated for C32H44N5O: 514.3546;
found: 514.3546 (MH+); IR (cm−1): 3381 (N-H), 2980 (Ar-C-H), 1507 (C=N), 1462 (Ar-C=C),
1253 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-isobutylimino-8-methyl-2-(4-methylphenyl)amino-3,5-dihydrop-
henazine (10l). Dark red solid (23.0 mg, 11%); Rf 0.37 [10% (v/v) methanol: chloroform];
1
mp. 148–150 ◦C (ethanol). H NMR (600 MHz, CDCl3 + CD3OD):
δ 7.12 (br s, 1H, H 6),
7.08–7.04 (m, 3H, H 9, H-3’), 7.04–6.99 (m, 3H, H-7, H 2’), 6.46 (s, 1H, H 1), 5.68 (s, 1H, H 4),
3.96 (t, J = 7.19 Hz, 2H, NCH2CH2), 3.07 (d, J = 6.90 Hz, 2H, NCH2CH), 2.43 (t, J = 8.22 Hz,
2H, CH2NMe2), 2.25 (s, 6H, NMe2), 2.22 (s, 3H, C-8 Me), 2.18 (s, 3H, C-4’ Me), 1.92 (dquin,
J = 13.41, 6.74 Hz, 1H, CHMe2), and 0.94 (d, J = 6.75 Hz, 6H, CHMe2), 13C NMR (151 MHz,
CD3OD):
δ 153.7 (C 3), 151.2 (C 10a), 144.8 (C 2), 138.7 (C 1’), 137.0 (C 4a), 134.6 (C 9a),
134.6 (C 5a), 134.0 (C 4’), 131.2 (C 8), 131.1 (C 3’), 129.2 (C 9), 128.7 (C 7), 122.5 (C 2’), 114.1
(C 6), 99.9 (C 1), 88.5 (C 4), 58.9 (NCH2CH), 54.6 (CH2NMe2), 46.0 (NMe2), 45.1 (NCH2),
31.1 (CHMe2), 21.6 (CHMe2), 21.1 (C-4’ Me) and 20.9 (C-8 Me); HRMS (ESI-TOF+): m/z
calculated for C28H36N5: 442.2971; found: 442.2831 (MH+);IR (cm−1): 3221 (N-H), 2959
(Ar-C-H), 1518 (C=N) and 1464 (Ar-C=C), 1241 (C-N).
(E)-3-(Cyclohexylimino)-5-[2-(dimethylamino)ethyl]-8-methyl-N-p-tolyl-3,5-dihydrophenazin-2-amine
(
10m). Maroon solid (26.0 mg, 21%); Rf 0.50 [10% (v/v) methanol:chloroform]; mp.
155–157 ◦C (ethanol). 1H NMR (400 MHz, CD3OD):
δ
7.44 (br d, J = 8.80 Hz, 1H, H
0
6), 7.38 (br s, 1H, H 9), 7.30 (br dd, J = 8.80, 1.60 Hz, 3H, H 7), 7.14 (d, J = 8.01 Hz, 2H, H 3 ),
7.08 (d, J = 8.60 Hz, 2H, H 2’), 6.73 (s, H 1), 1H, 6.22 (s, 1H, H 4), 4.37 (br t, J = 7.03 Hz, 2H,
NCH2), 3.68–3.57 (m, 1H, =NCH), 2.60 (t, J = 7.60 Hz, 2H, CH2NMe2), 2.31 (s, 6H, NMe2),
2.29 (s, 3H, C-8 Me), 2.26 (s, 3H, C-4’ Me), 2.05–1.91, 1.85–1.73, 1.71–1.58 and 1.50–1.31
[4
×
m, 10H, NCH(C5H10)]; 13C NMR (101 MHz, CD3OD):
δ 152.3 (C 3), 148.8 (C 10a),
143.7 (C 2), 138.7 (C 1’), 136.9 (C 4a), 135.5 (C 9a), 134.4 (C 5a), 133.3 (C 4’), 131.3 (C 8),
131.2 (C 3’), 129.4 (C 9), 128.8 (C 7), 123.4 (C 2’), 115.3 (C 6), 103.0 (br, C 1), 89.5 (C 4),
57.1 (=NCH), 56.0 (CH2NMe2), 46.6 (NCH2), 46.1 (NMe2), 33.9 [CH(CH2)2], 26.8 and 26.2
[CH2(CH2)2], 21.0 (C-4’ Me) and 20.98 (C-8 Me); HRMS (ESI-TOF+): m/z calculated for
C30H39N5: 469.3205; found: 469.3041 (MH+). IR (cm−1): 3231 (N-H), 2922 (Ar-C-H), 1512
(C=N) and 1460 (Ar-C=C), 1242 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-(isobutylimino)-N-p-tolyl-3,5-dihydrophenazin-2-amine (10n).
Dark red solid (98 mg, 17% yield); Rf 0.50 [20% (v/v) methanol: chloroform]; mp. 140–142 ◦C
1
(ethanol). H NMR (600 MHz, CD3OD3):
δ 8.14 (d, J = 8.8 Hz, 1 H, H-6), 8.09 (d, J = 8.3 Hz,
1 H, H-7), 7.93 (t, J = 7.9 Hz, 1 H, H-9), 7.74 (t, J = 7.6 Hz, 1-H, H-8), 7.47 (d, J = 7.9 Hz, 2
H, H-30), 7.38 (d, J = 7.7 Hz, 2 H, 20), 7.37 (s, 1 H, H-1), 6.93 (s, 1 H, H-4), 5.05 (t, J = 7.3 Hz,
2 H, NCH2), 3.58 (s, 3 H, Ph-CH3) 3.56 (d, J = 7.2 Hz, 2 H, NCH2CH), 2.98–2.92 (m, 2 H,
CH2NEt2), 2.46 (s, 2.28, 6H, CH2NMe2), (t, J = 13.6, 6.7 Hz, 1 H, CHMe2), 1.14 (d, J = 6.6 Hz,
6 H, CHMe2). 13C NMR (151 MHz, CD3OD3):
δ 153.2 (C 3), 149.9 (C-10a), 143.8 (C 2), 137.4
(C 1’), 137.2 (C-4a), 133.7 (C-9a), 133.4 (C-5a), 130.1 (C 3), 129.8 (C 7), 129.4 (C 9), 128.8
(C 8), 124.0 (C-4’), 122.6 (C 2’), 112.9 (C 6), 100.2 (C 1), 87.6 (C 4), 56.8 (CH2NMe2), 54.6
(NCH2CH), 46.1 [N(CH3)2], 45.1 (C 1’), 29.6 (CHMe2), 21.2 (CHMe2), 21.1 (PhCH3); HRMS

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(ESI-TOF+): m/z calculated for C27H34N5: 428.2814; found: 428.2823 (M+H)+. IR (cm−1):
3301 (N-H), 2975 (Ar-C-H), 1510 (C=N) and 1465 (Ar-C=C), 1238 (C-N).
(E)-5-[2-(Dimethylamino)ethyl]-3-(isobutylimino)-N-phenyl-3,5-dihydrophenazin-2-amine (10o).
Dark maroon tacky compound (6.40 mg, 25%); Rf 0.39 [10% (v/v) methanol: chloroform];
1
mp. 119–121 ◦C (ethanol). H NMR (400 MHz, CDCl3):
δ
7.72 (d, J = 7.80 Hz, 1 H, H-6),
7.40–7.32 (m, 6 H, H-7, H-9, H-2’, H-3’), 7.07 (t, J = 7.22 Hz, 1 H, H 8), 6.92 (s, 1 H, H-1), 6.06
(s, 1 H, H-4), 4.29 (br s, 2 H, H 1’), 3.32 (d, J = 7.24 Hz, 2 H, NCH2CH), 2.72 (t, J = 7.80 Hz,
2-H, CH2NMe2), 2.43 [s, 6-H, N(CH3)2], 2.15 (br s, 1-H, CHMe2), 1.07 (d, J = 6.63 Hz, 6-H
N(CH2CH3)2). 13C NMR (101 MHz, CDCl3):
δ 153.1 (C 3), 150.3 (C 10a), 143.5 (C 2), 140.1
(C 1’), 137.0 (C 4a), 133.4 (C 9a), 131.1 (C 5a), 130.1 (C 7), 129.5 (C 9), 129.1 (C 3’), 129.0
(C 8), 122.2 (C 2’, C 4’), 112.8 (C 6), 100.2 (C 1), 87.6 (C 4), 57.6 (CH2NEt2), 54.5 (C 2), 46.1
[N(CH3)2], 45.0 (C 1’), 29.9 (CHMe2), 20.9 (CHMe2). HRMS (ESI-TOF+): m/z calculated for
C27H33N5: 428.2382; found: 428.2378 (M+H)+. IR (cm−1): 3289 (N-H), 2950 (Ar-C-H), 1513
(C=N), 1464 (Ar-C=C), 1239 (C-N).
(E)-3-(Cyclohexylimino)-5-[(3-dimethylamino)propyl]-N-p-tolyl-3,5-dihydrophenazin-2-amine (10p).
◦
A red solid (97.4 mg, 26%); Rf 0.43 [20% (v/v) methanol:chloroform]; mp. 212–215 C
(ethanol). 1H NMR (400 MHz, CD3OD):
δ 7.65 (d, J = 7.50 Hz, 2 H, H 6, H 9), 7.50 (t,
J = 8.30 Hz, 1 H, H 7), 7.32 (t, J = 7.60 Hz, 1 H, H 8), 7.22 (q, J = 8.44 Hz, 4 H, H 2”, H 3”),
6.81 (s, 1 H, H 1), 6.24 (s, 1 H, H 4), 4.45–4.26 (m, 2 H, H 1’), 3.83–3.63 (m, 1 H, H 1*), 2.52 (t,
J = 7.04 Hz, 2 H, H 3’), 2.35 (s, 3 H, PhCH3), 2.31 [s, 6 H, N(CH3)2], 2.07–1.94 (m, 4 H, H
2’, H 2*), 1.90 (br s, 2 H, H 2*), 1.77 (br d, J = 12.7Hz, 2 H, H 4*), 1.61–1.45 (m, 4 H, H 3*).
13C NMR (151 MHz, CD3OD):
δ 152.6 (C 3), 150.3 (C 10a), 145.0 (C 2), 138.6 (C 1”), 138.0 (C
4a), 135.4 (C 9a), 134.7 (C 5a), 131.3 (C 3”), 131.2 (C 7), 130.8 (C 4”), 129.6 (C 9), 125.6 (C 8),
123.4 (C 2”), 115.3 (C 6), 101.7 (C 1), 89.3 (C 4), 65.6 (C 1*), 57.7 (C 3’), 46.0 [N(CH3)2], 45.7
(C 1’), 34.4 (C 2*), 27.0 (C 4*), 26.3 (C 2’), 25.2 (C 3*), 21.1 (PhCH3). HRMS (ESI-TOF+): m/z
calculated for C30H38N5: 468.3726; found: 468.3102 (M+H)+. IR (cm−1): 3253 (N-H), 2922
(Ar-C-H), 1514 (C=N), 1459 (Ar-C=C), 1210 (C-N).
3.2. Method for Cyclic Voltammetry
Cyclic voltammetry was carried out using a BAS-100W electrochemical analyzer using
a one-compartment three-electrode system composed of an Ag/Ag+ reference electrode
(0.01 M AgNO3), a platinum wire as the auxiliary electrode, and a platinum disc as the
working electrode. The supporting electrolyte solution was 0.1 M aqueous tetrabutylammo-
nium perchlorate in dried acetonitrile (100 mL). All measurements were carried out at 1, 3,
or 5 µ
M of the riminophenazines, at a scan rate of 100 mV.s−1, unless otherwise stated. All
analyses were run in duplicate under an atmosphere of nitrogen at room temperature. The
solutions were deoxygenated by purging under a stream of nitrogen through the solution
for 4 min prior each run. The platinum disc electrode was polished after every run.
3.3. Antitubercular Testing (MIC99 Determination) Method
A working stock solution of 10
µ
M of the test compound in fresh DMSO was prepared
◦
and stored at
−
70 C until used. All compounds were tested at 5, 3, and 1
µM point
concentrations for the initial screens. Mycobacterium tuberculosis H37Rv inocula were
prepared from cultures grown on Lowenstein Jensen (LJ) slants. Mycobacterial suspensions
were prepared in saline, and the turbidity was adjusted to 0.5 McFarland units. Aliquots of
◦
100
µ
L were inoculated into MGIT tubes and incubated at 37 C until the inoculum became
positive (about three days). Then, 0.5 mL of positive MGIT cultures were transferred into
MGIT tubes containing the test compounds. Controls consisting of a 1:100 dilution of
the positive MGIT culture control in saline, 1.2% DMSO control in saline (concentration
equivalent to that used when introducing the drug substance), and a 0.05 µg/mL isoniazid
(INH) positive control in saline were also prepared. For mycobacterial growth evaluation,
the MGIT 960 system (Becton Dickinson, Sparks, MD, USA) was used, where M. tuberculosis
growth is observed through fluorescent changes due to oxygen consumption during

Molecules 2021, 26, 4200
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mycobacterial growth. Incubation at 37 ◦C was continued in the MGIT system, and the
growth units (GU) were recorded hourly. For MIC99 evaluations, the 1% bacterial control
culture was used; the MIC99 of the compound was determined relative to the growth
units of the control (GU_400). When the GU of the control reached 400, the results were
interpreted. If the drug-containing tube showed GU > 400, the material was defined as
inducing resistance; if it showed GU
substance was indicated at that concentration of drug substance. Compounds that showed
GU = 0 at 1 M were considered to have an MIC M, and further MIC determinations
on these materials using concentrations ranging from 0.0625–1 M were performed. All
≤
400, susceptibility of the mycobacteria to the drug
µ
≤
1 µ
µ
experiments were conducted in triplicate, with three independent experiments performed
each time and the averages are reported. Statistically, the percentage range of standard
deviation from mean showed the two concentrations (3 and 5 µM) having a close range as
compared to the lowest concentration (see Supplementary data).
3.4. Protocol for Toxicity Evaluation (IC50) Method
The WI-38 cell line—normal Human Fetal Lung Fibroblast from ECACC was routinely
◦
maintained as a monolayer cell culture at 37 C, 5% CO2, 95% air, and 100% relative
humidity in EMEM containing 10% fetal bovine serum, 2 mM L-glutamine, and 50 µg/mL
gentamicin. For screening experiment, the cells (21–50 passages) were inoculated in 96-well
microtiter plates at plating densities of 10,000 cells/well and were incubated for 24 h.
After 24 h, the cells were treated with the experimental drugs, which were previously
dissolved in DMSO and diluted in medium to produce five concentrations. The process
was performed in triplicate and the data presented are means of three repeats performed
in triplicate. Cells without drug addition served as control. The blank contains complete
medium without cells. Parthenolide was used as a standard. The plates were incubated
for 48 h after addition of the compounds. Viable cells were fixed to the bottom of each
well with cold 50% trichloroacetic acid, washed, dried, and dyed by SRB. Unbound dye
was removed, and protein-bound dye was extracted with a 10 mM Tris base for optical
density determination at the wavelength 540 nm using a multiwell spectrophotometer. Data
analysis was performed using GraphPad Prism software. 50% of cell growth inhibition
(IC50) was determined by non-linear regression.
4. Conclusions
A series of variously substituted phenazines having ionizable aminoalkyl chains
installed at N-5 of the riminophenazine system as part of the strategy to develop less
lipophilic compounds with high anti-TB activity are reported. While it is known that
lipophilicity is an important determinant of antimycobacterial activity as well as pigment
deposition in fatty tissues [14], the inclusion of aminoalkyl sidechains has generated rea-
sonably active materials with the potential to further enhance activity while still providing
a “handle” for improved compounds. Remarkably, compounds with methyl groups at
both R3 and R4 positions with Me2N(CH2)2- at N-5 position (10l and 10m) demonstrated
both lower ClogP values and were also potent against M. tuberculosis. The data obtained
give a preliminary indication that the more commonly used N-5 phenyl moieties may be
substituted without abrogating activity. Future work will focus on refining this further,
followed by pharmacokinetic testing.
Supplementary Materials: The following are available online, 1H and 13C NMR spectra of the
synthesized target compounds.
Author Contributions: M.V.B. synthesized all the target compounds, collected and analyzed the
experimental data, drafted and finalized the manuscript. C.v.d.W. conceptualized and supervised the
research, provided intellectual leadership, corrected, edited and reviewed the manuscript. E.M.M.
co-designed the research. A.N. performed the biological assays. All authors have read and agreed to
the published version of the manuscript.

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Funding: This research was funded by the National Research Foundations of South Africa (PDP
grant for Mpelegeng V. Bvumbi). This research received no other funding.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Carren Billing (Wits University) for access to cyclic voltammetry
analyses, and Paul A. Steenkamp (University of Johannesburg) for mass spectrometric analyses.
Conflicts of Interest: The authors declare no conflict of interest.
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