Synthesis and antitubercular activity evaluation of novel unsymmetrical cyclohexane-1,2-diamine derivatives

Article abstract of DOI:10.1002/ardp.201200219

A library of unsymmetrical cyclohexane-1,2-diamine derivatives were synthesized and evaluated for their activity against Mycobacterium tuberculosis H37Rv in vitro. Out of the 46 compounds synthesized, eight compounds (11h, 13a, 13e, 13f, 14a, 14c, 14d, an

Full text of DOI:10.1002/ardp.201200219

Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
1
Full Paper
Synthesis and Antitubercular Activity Evaluation of Novel
Unsymmetrical Cyclohexane-1,2-diamine Derivatives
Beena1, Seema Joshi1, Nitin Kumar1, Saqib Kidwai2, Ramandeep Singh2, and Diwan S. Rawat1
1 Department of Chemistry, University of Delhi, Delhi, India
2 Translational Health Science and Technology Institute, Vaccine and Infectious Disease Research Centre,
Gurgaon, India
A library of unsymmetrical cyclohexane-1,2-diamine derivatives were synthesized and evaluated for
their activity against Mycobacterium tuberculosis H37Rv in vitro. Out of the 46 compounds synthesized,
eight compounds (11h, 13a, 13e, 13f, 14a, 14c, 14d, and 15d) were found to be active at or below
6.25 mM concentration, with negligible toxicity to human red blood cells at a concentration much
higher than the MIC99. Compound 13a was the best active compound showing inhibition at 3.125–
6.25 mM, and was found to be non-hemolytic up to 500 mg/mL concentration.
Keywords: Bromhexine / Cyclohexane-1,2-diamine / Hemolysis / Mycobacterium tuberculosis / Tuberculosis
Received: May 22, 2012; Revised: July 13, 2012; Accepted: July 19, 2012
DOI 10.1002/ardp.201200219
Supporting information available online
:
Introduction
motif have been synthesized by Sequella Inc., out of which
compound SQ109 (N-geranyl-N0-(2-adamantyl)ethane-1,2-
diamine), was found to be the most potent with MIC 0.7–
1.56 mM (Fig. 1) and is currently undergoing phase II clinical
trials [5, 6]. In a recent study, it has been shown that SQ-109
targets the MmpL3 protein, an enzyme involved in the trans-
port of lipids into the cell wall core of M. tuberculosis [7].
Cyclohexane moiety has always drawn the attention of
chemists due to its role in many organic transformations
[8–12], and that of biologists [13] as it has been part of many
biologically active compounds such as oxaliplatin (1) [14, 15],
bromhexine (2) [16], and ambroxol (3) [17] (Fig. 2). Oxaliplatin,
According to the World Health Organization (WHO), six
infectious diseases (pneumonia, tuberculosis, diarrhoea,
malaria, measles, and HIV/AIDS) account for half of all pre-
mature deaths worldwide, killing mostly children and young
adults [1]. Tuberculosis or TB (tubercle bacillus) is one of the
bacterial diseases with the highest disease burden, mainly
caused by the bacterium Mycobacterium tuberculosis (M. tb) lead-
ing to 8.8 million new TB cases and 1.4 million deaths in the
year 2010 (WHO). From the statistics gathered each year it is
estimated that around 225 million new cases and 79 million
deaths may occur due to tuberculosis between 1998 and
2030. With the emergence of MDR-XDR-TB and HIV-TB nexus,
there is always a need for the identification of novel scaffolds
with antitubercular activity having newer drug targets [2].
One of the diamine based compounds, namely ethambutol
[(2S,20S)-2,20-(ethane-1,2-diyldiimino)dibutan-1-ol], has been
used as a drug of choice for the treatment of TB (Fig. 1) in
combination with other anti-TB drugs [3, 4]. A library of
63 238 compounds based on the ethambutol structural
{(1R,2R)-cyclohexanediamine}oxalatoplatinum(II) (1), is
a
cyclohexane-1,2-diamine based platinum complex (Fig. 2)
which has been approved by the FDA for the first line treat-
ment of colorectal carcinoma in 2004 [18].
Bromhexine [2, N-2-amino-3,5-dibromobenzyl)-N-cyclohexyl-
methylamine] is another compound based on the cyclohexane
motif, a semi-synthetic derivative of the alkaloid vasicine,
obtained from the Indian shrub Adhatoda vasica which has been
used as mucolytic agent [19]. The flowers, leaves, and roots of
A. vasica have been used for the treatment of asthma and
tuberculosis. Ambroxol [3, N-(trans-4-hydroxy-cyclohexyl)-(2-
amino-3,5-dibromo-benzyl)-amine], a metabolite of bromhexine,
has also shown secretalogic and mucolytic properties [20–22].
In a pilot study by the Medical Research Council of Ireland,
Correspondence: Diwan S. Rawat, Department of Chemistry, University
of Delhi, Delhi, India.
E-mail: dsrawat@chemistry.du.ac.in
Fax: 91 11 27667501
ß 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Gram-positive and Gram-negative bacteria with low toxicity
(Fig. 3). The antimycobacterial activity of the best active
symmetrical derivatives against M. tb H37Rv lied in the range
3.125–12.5 mM [26]. Encouraged by these observations and as a
part of our ongoing work toward the development of novel
antimicrobials [27–30], we synthesized a series of unsymmet-
rical cyclohexane-1,2-diamine compounds with þ2 charge and
hydrophobic bulk furnished by the substituted aromatic ring
and screened them for their antitubercular activity. These com-
pounds belong to the diamine class of antitubercular drugs
amongst which ethambutol and SQ109 are the representative
examples. In the present study, we also evaluated the toxicity of
these compounds toward human red blood cells (hRBCs).
OH
H
N
N
H
NH HN
SQ109
HO
Ethambutol
Figure 1. Diamine based antitubercular drugs.
OH
H2
Br
Br
N
O
O
O
O
N
H
NH2
N
Pt
NH2
N
H2
Br
Br
2
3
1
Results and discussion
Figure 2. Cyclohexane based biologically active molecules.
Chemistry
Mono-functionalized symmetrical or unsymmetrical di-
amines are important intermediates for the synthesis of
many biologically important pharmacophores [31–33].
Often, selective and efficient protecting reagents and mild
reaction conditions are required for sequential protection
and deprotection of polyfunctional molecules [34–36]. In
order to synthesize unsymmetrical cyclohexane-diamine
based compounds, one of the primary amine groups of
racemic cyclohexane-1,2-diamine (6) was protected using
(Boc)2O (Scheme 1) by literature methods [37]. The other
NH2 group of the Boc-protected compound 7 was treated with
substituted benzaldehydes to give imines (8a, R ¼ H; 8b,
R ¼ 4-Me; 8c, R ¼ 4-Et, 8d, R ¼ 4-i-Pr; 8e, R ¼ 4-Br) in quanti-
tative yields. These Schiff bases were reduced using NaBH4 in
dry MeOH to give substituted amines (9a–9e) containing one
NH2 group protected with Boc, which on reaction with 35%
aq. H3PO4 in dichloromethane leads to deprotection of the
Boc group (10a–10e) [38]. Compounds with free NH2 group
(10a, R ¼ H; 10b, R ¼ 4-Me; 10c, R ¼ 4-Et, 10d, R ¼ 4-i-Pr;
R
R
NH
NH
NH
2HCl
2HCl
NH
5
4
R
R
Figure 3. Prototype structure of antibacterial agents.
bromhexine was found to inhibit the growth of a strain of
M. tb at concentrations of 33.3 mg/mL and above, however this
effect was pH dependent [19].
We recently reported the synthesis of a series of symmetrical
cyclohexane-1,2-diamine [23, 24] and cyclohexane-1,3-diyldi-
methanamine derivatives [25] (4 and 5) and most of these
compounds have shown excellent antibacterial activity against
O
O
O
O
O
O
NH
NH
NH2
NH2
NH
a
b
c
NH2
N
NH
_
_
9a 9e
R
R
8a 8e
7
6
d
R1
NH
NH
NH2
NH
e
Scheme 1. Synthesis of unsymmetrical cyclo-
hexane-1,2-diamine derivatives. (a) (Boc)2O,
dioxane, r.t., 22 h; 92%; (b) substituted benzal-
dehydes, MeOH, r.t., 4 h; 85–95%. (c) NaBH4,
MeOH, r.t., 3 h; 80–93%; (d) 35% aq H3PO4,
CH2Cl2, r.t., 4 h; 75–85%; (e) (i) substituted
benzaldehydes, MeOH, r.t., 3–4 h, NaBH4, r.t.,
2–3 h; 60–85% (ii) CHCl3, dry HCl gas, 0.5 h.
2HCl
_
R
R
_
10a 10e
11a 11m
_
12a 12h
_
13a 13h
_
14a 14i
_
15a 15h
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antitubercular Activity of New Unsymmetrical Cyclohexane-1,2-diamine Derivatives
3
10e, R ¼ 4-Br) were then treated with different benzaldehydes
in dry MeOH and the resulting imines were reduced by NaBH4
in situ. The resulting amines were purified by column chroma-
tography using 2% MeOH–CHCl3 as eluent. All these unsym-
metrically substituted amines were converted to their
respective hydrochloride salts by passing dry HCl gas to
the solution of diamines in chloroform. A group of peaks
at 2704–2962 cmꢀ1 in the IR spectra of the amine salts (11a–
15h) and broad signals at d 9.35–10.3 in the 1H NMR spectra
confirmed the formation of salt. The salt formation was
also confirmed by conductivity measurements and all the
compounds were characterized by standard analytical and
spectroscopic methods.
aromatic rings, and a methyl group at the ortho position of
the other aromatic ring was the most potent anti-TB compound
in this series with a MIC value of 3.125–6.25 mM. Along with
compound 13a, compounds 14c, 14d, and 15d can also be
considered as leads for further exploration as these compounds
possess considerably good MIC (6.25–12.5 mM) against H37Rv.
When 4-Et of 13a was replaced with 4-i-Pr keeping the methyl
group at ortho of the other phenyl ring intact (14a,
MIC99 ¼ 6.25–12.5 mM) the activity of the compound slightly
decreased. But a dramatic loss in activity was observed when
there was no substituent present (11a) or groups such as Br (15a)
or Me (12a) were present at the para position of the analogues of
13a. Compounds 13e and 13f also exhibit promising activity
with MIC in the range of 6.25–12.5 mM. Amongst the
compounds 11a–11m in which one of the aromatic ring is
unsubstituted and the other has various substitutions, 11h
with t-butyl at the para position showed very good activity
(MIC99 ¼ 6.25 mM). In this series, when the chain length was
increased at the para position there was no change in the
activity and compounds 11d–11f exhibited the same level of
activity with MIC of 12.5 mM. When one of the aromatic
ring contained a 4-Me group and the alkyl substitution at
the para position of the other ring was varied (12a–12e) it
was observed that the activity increased with increase in
ClogP 12b < 12c < 12e (6.71, 7.24, and 7.76, respectively).
Similar trend was observed with compounds having the 4-Et
group in one of the phenyl rings and 4-Me, 4-n-Pr, 4-n-Bu in the
In vitro antitubercular activity
The significant role of diamine based compounds in anti-TB
research prompted us to screen these compounds against the
M. tb H37Rv strain. Rifampicin and ethambutol, used as
reference drugs, inhibited mycobacterial growth at a concen-
tration of <0.05 and 1.56 mM respectively. Table 1 clearly
shows that most of the synthesized compounds are moderate
to less active against M. tb. MIC99 values >50 mM, indicate low
antitubercular activity. Seventeen compounds (11d–11g, 11i,
11j, 12b–12e, 13c, 13d, 14e, 14g, 15c, 15e, and 15f) exhibit
moderate activity with MIC99 12.5–25 mM. Thirteen compounds
showed good activity with MIC99 <12.5 mM. Compound 13a
with an ethyl group at the para position of one of the
Table 1. Antimycobacterial activity of unsymmetrical cyclohexane-1,2-diamine derivatives.
Entry
R
R1
H37Rv (MIC99/mM)
ClogP
Entry
R
R1
H37Rv (MIC99/mM)
ClogP
11a
11b
11c
11d
11e
11f
11g
11h
11i
H
H
H
H
H
H
H
H
H
H
H
H
H
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Me
4-Et
4-Et
2-Me
3-Me
4-Me
4-Et
4-n-Pr
4-i-Pr
4-n-Bu
4-t-Bu
3,4-Me
4-Cl
4-Br
2-F
3-F
2-Me
4-Et
4-n-Pr
4-i-Pr
4-n-Bu
3-Cl
>50
50
50
12.5
12.5
12.5
25
6.25
25
12.5
50
50
>50
>50
25
5.633
5.683
5.683
6.212
6.741
6.611
7.270
7.010
6.132
5.897
6.047
5.327
5.327
6.132
6.711
7.240
7.110
7.769
6.396
6.396
6.546
6.661
6.711
13c
13d
13e
13f
13g
13h
14a
14b
14c
14d
14e
14f
14g
14h
14i
15a
15b
15c
15d
15e
15f
4-Et
4-Et
4-Et
4-Et
4-Et
4-Et
4-n-Pr
4-i-Pr
4-n-Bu
4-t-Bu
3,4-Me
4-Cl
2-Me
3-Me
4-n-Pr
4-n-Bu
3,4-Me
4-Cl
4-Br
2-F
3-F
2-Me
3-Me
4-n-Pr
4-n-Bu
4-t-Bu
3,4-Me
2-F
12.5
25
6.25
7.769
7.639
8.298
8.038
7.160
6.925
7.060
7.110
8.168
8.697
7.559
7.324
7.474
6.754
6.754
6.496
6.546
7.604
8.133
7.873
6.995
6.190
6.190
6.25–12.5
25–50
50
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-i-Pr
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
4-Br
6.25–12.5
12.5–25
6.25
11j
11k
11l
6.25–12.5
12.5–25
25–50
12.5–25
25–50
50
11m
12a
12b
12c
12d
12e
12f
12g
12h
13a
13b
25
25–50
50
12.5–25
12.5–25
>50
>50
50
12.5–25
6.25
25
25
50
4-Cl
4-Br
2-Me
3-Me
3.125–6.25
25–50
15g
15h
3-F
50
Ref: Rifampicin: <0.05 mM, ethambutol: 1.56 mM.
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4
et al.
Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
other 12b < 13c < 13e (ClogP values 6.71, 7.76, and 8.29, Conclusion
respectively). Compound having 4-n-butyl group in one of
the aromatic rings and the other ring unsubstituted, the result-
ing compound 11g showed poor activity. But, presence of
substituents like 4-Me (12e, MIC99 ¼ 12.5–25 mM), 4-Et (13e,
MIC99 ¼ 6.25 mM), 4-i-Pr (14d, MIC99 ¼ 6.25–12.5 mM), and
4-Br (15d, MIC99 ¼ 6.25 mM) showed better anti-TB activity.
Compounds 14a–14e with 4-i-Pr in one of the aromatic rings
showed considerably good activity with MIC99 ¼ 6.25–12.5 mM.
Presence of a fluoro group at the ortho or meta position (11l, 11m,
Herein we report the synthesis and antimycobacterial
activity evaluation of a library of 46 unsymmetrically sub-
stituted cyclohexane-1,2-diamines. Some compounds showed
moderate activity against M. tb H37Rv. Toxicity evaluation
of these compounds against hRBCs showed no toxicity of
these compounds up to a concentration higher than the
MIC, therefore making them safe as potent leads.
14h, 14i, 15g, and 15h) of one of the aromatic rings does not Experimental
contribute to the activity, irrespective of any group present in
the other ring. Compounds 14c and 14d have comparable
activity though the lipophilicity of 14d is greater than that
of 14c. The activity of 15d is much better than that of 15c and
also the ClogP of 15d is greater than that of 15c (8.13 and 7.60).
General
All of the chemicals used in the syntheses were purchased from
Sigma–Aldrich and were used as such. Thin layer chromatog-
raphy (Merck TLC silica gel 60 F254) was used to monitor the
progress of the reactions. The compounds were purified by silica
gel column (60–120 mesh). Melting points were determined on
an EZ-Melt automated melting point apparatus, Stanford
Research Systems, and are uncorrected. IR (KBr) spectra were
recorded using a Perkin-Elmer FT-IR spectrophotometer and
the values are expressed as nmax cmꢀ1. Mass spectral data were
recorded in a Thermo Finnigan LCQ Advantage max ion trap
Hemolytic activity
Toxicity of 16 compounds (11j, 12f, 13a–13c, 13f, 13g, 14b–
14d, 14f, 14h, 14h, 15d, 15g, and 15h) was investigated using
hRBCs at up to 500 mg/mL concentration (Fig. 4). Eight com-
pounds 11j, 12f, 13a, 14f, 15a, 15d, 15g, and 15h were found
to be non-hemolytic with only 6–14% hemolysis at the high-
est concentration tested. Compounds 13b, 13c, 13f, 13g, 14b,
14c, 14d, and 15a were 84, 48, 87, 77, 62, 20, 23, and 52%,
hemolytic, respectively, at 500 mg/mL. At half the concen-
tration (250 mg/mL) these compounds were either non-hemo-
lytic or showed a maximum of 23% hemolysis. Compounds
13c and 14c showed 4 and 0% hemolysis at 250 mg/mL con-
centration, which is much higher than their MIC values.
Interestingly, the most active compound 13a was non-toxic
to hRBcs and can be considered as a safe molecule.
1
mass spectrometer. H NMR spectra were recorded on a Bruker
Spectrospin spectrometer at 300 MHz and a Jeol ECX Spectrospin
400 MHz instrument while the 13C NMR spectra were recorded at
75.5 and 100 MHz, respectively, using TMS as an internal stand-
ard. The chemical shift values were recorded on the d scale and
the coupling constants (J) are in Hz. Elemental analysis was
performed on a Carlo Erba Model EA-1108 elemental analyzer
and data of C, H, and N is within ꢁ0.4% of calculated values. The
data characterizing the unsymmetrical cyclohexane-1,2-diamine
derivatives 11a–15h can be found in the Supplementary
Material provided online. ClogP values were calculated using
CambridgeSoft ChemOffice Ultra 10.0 software.
Synthesis of tert-butyl-2-aminocyclohexylcarbamate (7)
[37]
To a stirred solution of 1,2-diaminocyclohexane (6; 4.0 g,
0.035 mol) in 70 mL dioxane, a solution of di-tert-butyldicarbon-
ate (1.0 g, 0.0046 mmol) in dioxane (30 mL) was added dropwise
(Scheme 1). The mixture was stirred for 22 h and the solvent was
evaporated under reduced pressure. The solid was suspended
in H2O (50 mL) and the disubstituted product was removed by
filtration. The filtrate was extracted with CH2Cl2 (3 ꢂ 50 mL) and
the organic fractions were washed with water (3 ꢂ 30 mL). The
organic layer was dried over Na2SO4 and the solvent was then
removed to give an oily yellow liquid (7), which was dried under
vacuum (0.9 g, 92%).
General procedure for the preparation of compounds
9a–9e [38]
Figure 4. Dose–response curves of hemolytic activity of the
designed compounds against human erythrocytes. Different con-
centrations of compounds were incubated with 4% hRBC suspen-
sion for 1 h at 378C. After 1 h of incubation, hemoglobin release from
the ruptured hRBC was studied by measuring the absorbance at
414 nm. For complete lysis, 1% Triton X-100 was taken as positive
control, and for no lysis, PBS was taken as negative control.
To a stirred solution of 7 (0.93 mmol, 200 mg) in dry MeOH
(10 mL) the corresponding substituted benzaldehyde (1.1 equiv)
was added and the reaction mixture was stirred at room tempera-
ture for 4 h (Scheme 1). After completion of the reaction the
solvent was removed and product was extracted with CHCl3
(3 ꢂ 15 mL). The combined organic layer was dried over sodium
sulfate and the solvent was removed under pressure to get an oily
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Arch. Pharm. Chem. Life Sci. 2012, 000, 1–6
Antitubercular Activity of New Unsymmetrical Cyclohexane-1,2-diamine Derivatives
5
liquid. The Schiff’s base obtained (8a–8e) was dissolved in dry
MeOH (15 mL) and NaBH4 (3.0 equiv) was added and progress of
the reaction was monitored by TLC. After 3 h the solvent was
removed and the product was extracted with CHCl3 (3 ꢂ 15 mL).
The reduced product was purified by column chromatography
using CHCl3–MeOH as eluent to obtain compounds 9a–9e in
good yield.
RSIC, CDRI, Lucknow for mass. The authors are also thankful to Prof. Anil K.
Tyagi, University of Delhi for access to the BSL-3 facility.
The authors have declared no conflict of interest.
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A stock culture of M. tb H37Rv (ATCC 27294) was grown to
Abs600nm 0.2 in Middlebrook 7H9 broth (Difco) supplemented
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solutions of 50 mM. Drugs (100 mL solution) were added to
the first row of the 96-well plate at a final concentration
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1:1000 diluted cultures. Rows 6 and 12 of the 96-well plates were
no-drug controls. The plates were incubated at 378C and the
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