Synthesis and antibacterial activity of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives

Article abstract of DOI:10.1016/j.bmcl.2009.12.074

A series of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives with different substitution pattern on the aromatic ring have been prepared and evaluated for their antibacterial activity against Gram-positive and Gram-negative bacterial strains. Most of the compounds exhibit potent activity against Pseudomonas aeruginosa and Staphylococcus epidermidis while compounds 6l and 6m showed antibacterial activity against all the four bacterial strains with MIC values ranging from 0.002 to 0.016 μg/mL and no hemolytic activity up to 512 μg/mL in mammalian erythrocytes was observed.

Full text of DOI:10.1016/j.bmcl.2009.12.074

Bioorganic & Medicinal Chemistry Letters 20 (2010) 893–895
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial activity
of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives
Deepak Kumar a, Seema Joshi a, Rajesh K. Rohilla b, Nilanjan Roy b, Diwan S. Rawat a,
*
a Department of Chemistry, University of Delhi, Delhi 110 007, India
b Department of Biotechnology, National Institute of Pharmaceutical Education and Research, SAS Nagar, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives with different substi-
tution pattern on the aromatic ring have been prepared and evaluated for their antibacterial activity
against Gram-positive and Gram-negative bacterial strains. Most of the compounds exhibit potent activ-
ity against Pseudomonas aeruginosa and Staphylococcus epidermidis while compounds 6l and 6m showed
Received 3 December 2009
Revised 15 December 2009
Accepted 17 December 2009
Available online 24 December 2009
antibacterial activity against all the four bacterial strains with MIC values ranging from 0.002 to 0.016
mL and no hemolytic activity up to 512 g/mL in mammalian erythrocytes was observed.
lg/
l
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Antibacterials
Multi drug resistance
Bromhexine
Most of the antibacterial drugs in the clinical use have been dis-
covered by the middle of last century and no new class of antibac-
terial was introduced in the market after 1962 until the discovery
of linezolid in 2000.1–3 Interestingly bacteria have developed resis-
tance against most of these drugs soon after or even before their
introduction in the market. For example linezolid was introduced
in the market in 2000 and resistance of this compound was re-
ported in 1999.4–6 The multi-drug resistant has reached to the
alarming stage7–13 and situation has worsen due to the increasing
incidences of methicillin resistant Staphylococcus aureus (MRSA),
Vancomycin-resistant enterococci (VRE) and other antibiotic resis-
tant human pathogenic microorganisms.14–17
To overcome the antibiotic resistant problem, there is an urgent
need for the development of new antibacterial class that are not af-
fected by resistance mechanisms already present in the bacterial
population.18,19 As a part of our ongoing efforts towards the syn-
thesis of novel antibacterial agents,20–22 we became interested to
modify the bromhexine molecule (1). During the course of this
study, we observed that simple structural manipulation led to
the discovery of potent antibacterial agents (2) that exhibit antimi-
crobial activity at very low concentration without any hemolytic
activity at very high concentration.23,24
Br
R
NH
N
Br
2HCl
NH
2
NH
1
2
R
Encouraged by this observation, we decided to synthesize struc-
turally diverse compounds using 3-(aminomethyl-cyclohexyl)-
methylamine as a starting material and to study their antibacterial
activity.
Simple two step synthetic protocol was used for the preparation
of title compounds (6a–6u). The first step involves treatment of
diamine (3) with different substituted benzaldehydes (4) in dry
methanol to give Schiff bases (5a–5u) in quantitative yield in the
form of viscous liquid or solid. In the next step Schiff bases were
reduced with sodium borohydride in dry methanol to give the
substituted diamines (6a–6u) in quantitative yields. In most of
the cases Schiff bases were soluble in methanol but in some cases
dry THF was used as a co-solvent. All the synthesized amines were
converted into their HCl salt by passing dry HCl gas in the solution
of diamines in chloroform (Scheme 1). The formation of HCl salt of
these diamines was confirmed by conductivity experiments. All
these compounds were characterized spectroscopically (Table 1).25
In vitro antibacterial activity: Antimicrobial susceptibility testing
was carried out using National Committee for Clinical Laboratory
* Corresponding author. Tel.: +91 11 27667465, fax: +91 11 27667501.
E-mail address: dsrawat@chemistry.du.ac.in (D.S. Rawat).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.12.074

894
D. Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 893–895
R
R
CHO
N
NH
NH
NH
2HCl
2
2
b, c
a
R
R
N
NH
R
3
4
5a-5u
6a-6u
Scheme 1. Reagents and conditions: (a) MeOH, molecular sieves, 3–4 h, rt; (b) NaBH4, MeOH, 2–3 h, rt; (c) CHCl3, HCl gas.
Table 1
Table 2
Synthesis of benzyl-[3-(benzylamino-methyl)-cyclohexylmethyl]-amine derivatives
Antibacterial activity of benzyl-[3 (benzylamino-methyl)-cyclohexylmethyl]-amine
derivatives
Compd No.
R
Yield (%)
Mp (°C)
Compd No.
MIC (
l
g/mL)
6a
6b
6c
6d
6e
6f
6g
6h
6i
H
70
62
70
68
71
65
70
80
55
60
65
80
90
85
85
90
75
65
67
75
78
110–112
114–116
198–200
152
240
196–198
150
198
218
228
264
202
278
252
168
120–122
202
182
110–113
150–152
210
4-Me
3-Me
3,5-Me
3,4-Me
2-Me
2,5-Me
2,4-Me
4-Et
4-n-Pr
4-iso-Pr
4-n-Bu
4-t-Bu
4-Cl
E. coli
P. aeruginosa
S. aureus
S. epidermidis
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
0.5
1.00
1.00
1.00
1.00
0.25
*
0.125
*
*
*
*
*
*
6j
6k
6l
0.125
0.016
0.125
0.002
0.004
0.032
0.25
0.016
0.016
0.125
0.004
0.008
0.065
6m
6n
6o
6p
6q
6r
6s
6t
*
0.016
0.004
0.016
3-Cl
2,6-Cl
4-Br
3-Br
4-F
0.002
*
0.025
*
*
*
*
*
*
*
*
0.125
0.25
*
*
*
*
*
*
*
0.032
0.125
3-F
3-NO2
0.125
0.125
6u
*
*
6s
6t
6u
Tet
*
*
*
*
0.02
0.01
0.001
0.01
0.02
Standards (NCCLS) microdilution assay. Briefly, the bacterial
strains were grown in standard media until exponential growth
was achieved. Tests were performed in a 96-well microtiter plate
Tet
Tetracycline was used as a reference compound.
g/mL.
*
MIC >250
l
in a final volume of 100
5% DMSO at an initial concentration of 500
l
L. Test compounds were dissolved in
g and serially diluted
l
in plate. Each well was then inoculated with ꢀ2–5 ꢁ 105 bacterial
cells and incubated at 37 °C for 24 h with shaking at 200 rpm. One
well containing cells and 5% DMSO without any test compound
(growth control), and one well containing only growth medium
(sterility control) were used as controls. Growth of bacteria was
determined using Power wave200 microplate scanning spectro-
photometer (Bio-Tek Instruments, Winooski, VT, USA). Percent sur-
vival was calculated using growth without any compound as 100%
survival. The MIC values are calculated using Grafit 4.0 software
(Erithacus Software Ltd., Horley, Surrey, UK).
(Escherichia coli, P. aeruginosa, S. aureus, S. epidermidis) with MIC
values ranging from 0.002 to 0.016 g/mL, while compound with
n-propyl group (entry 6j) exhibit activity against E. coli, P. aerugin-
osa and S. epidermidis with MIC values 0.016 g/mL. Interestingly
compounds with ethyl and iso-propyl groups at para-position of
the phenyl ring (entry 6i and 6k) showed good activity against P.
aeruginosa and S. epidermidis only. Compounds with Cl, Br or NO2
l
l
30
The compounds were evaluated against Gram-positive and
Gram-negative bacterial strains. The minimum inhibitory concen-
tration (MIC) values of each compound are shown in Table 2.
Compound 6a in which both of the benzene rings are unsubsti-
tuted, showed no activity against any of the bacterial strains. Com-
pounds with methyl group in the aromatic ring at different
positions exhibit potent antibacterial activity against Pseudomonas
aeruginosa, Staphylococcus epidermidis with MIC values ranging
6j
25
6k
20
6l
15
10
5
6m
from 0.125 to 1.00 lg/mL, with exception of compound 6f in which
0
methyl group is at ortho-position. Compound 6f was found to be
totally inactive against all the strains. Introduction of ethyl, n-pro-
pyl, iso-propyl, n-butyl or t-butyl groups at para-position of the
phenyl ring have marked effect on the antibacterial activity of
these compounds (entry 6i–6m).
0
100
200
300
400
500
Concentration (µg/ml)
Figure 1. Hemolytic activity of the compounds. Fresh hRBC suspension (4 % v/v in
35 mM phosphate buffer with 150 mM NaCl) was used for the assay. After
incubation of the test sample in the erythrocyte solution for 1 h at 37 °C, the
solution was centrifuged and the supernatant absorbance was determined at
414 nm. Hemolysis affected by 0.1% Triton X-100 was considered as 100%.
For example compounds having n-butyl or tert-butyl (entry 6l,
6m) exhibit antibacterial activity against all the four strains

D. Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 893–895
895
15. Mitscher, L. A.; Pillai, S. P.; Gentry, E. J.; Shankel, D. M. Med. Res. Rev. 1999, 19,
477.
16. Berber, I.; Cokmus, C.; Atalan, E. Microbiology 2003, 72, 54–59.
17. Viksveen, P. Homeopathy 2003, 92, 99.
18. Terzulli, L. S.; Croft, C. A.; D‘Antoni, V. A. Med. Sci. Monit. 2007, 13, 103.
19. Alanis, J. A. Arch. Med. Res. 2005, 36, 697.
at meta- or para-position exhibit good activity against P. aeruginosa
and S. epidermidis (entry 6n, 6o, 6q, 6r, 6u), while other compounds
with fluoro group exhibit no activity against any of the bacterial
strain (entry 6s, 6t). Other compounds having substitution at
ortho-position of the phenyl ring were devoid of antibacterial
activity (entry 6f, 6g, 6h, and 6p).
20. Beena, Kumar, N.; Rohilla, R. K.; Roy, N.; Rawat, D. S. Bioorg. Med. Chem. Lett.
2009, 19, 1396.
21. Bisht, G. S.; Rawat, D. S.; Kumar, A.; Kumar, R.; Pasha, S. Bioorg. Med. Chem. Lett.
2007, 17, 4343.
22. Joshi, M. C.; Bisht, G. S.; Rawat, D. S. Bioorg. Med. Chem. Lett. 2007, 17, 3226.
23. Rawat, D. S.; Sharma, M.; Roy, N.; Rohilla, R. K. Indian Patent Application No.:
1462/DEL/2008.
Toxicity of these compounds were investigated using human
red blood cells (hRBC) the results showed that the concentration
up to 512 lg/mL of compounds 6b–6f, 6n–6r did not show any
toxicity while compounds 6j–6m showed hemolysis between 4%
24. Sharma, M.; Joshi, P.; Rohilla, R. K.; Roy, N.; Rawat, D. S., unpublished work.
25. (4-Ethyl-benzyl)-{3-[(4-ethyl-benzylamino)-methyl]-cyclohexylmethyl}-amine
(6i): Yield: 55%; mp: 218 °C; IR (Nujol, cmꢂ1): 2923, 2852, 2723, 2403, 1461,
1377, 1021, 828, 722; 1H NMR (300 MHz, DMSO-d6): 0.57–0.65 (m, 1H), 0.76–
0.83 (m, 2H), 1.13–1.18 (t, 6H), 1.32–1.36 (m, 1H), 1.74–1.96 (m, 6H), 2.55–
2.77 (m, 8H), 4.03 (s, 4H, 2CH2Ph), 7.21–7.24 (d, J = 9 Hz, 4H, Ar), 7.46–7.49 (d,
J = 9 Hz, 4H, Ar), 9.23 (br s, 4H, 2NH2þ); 13C NMR (75.5 MHz, DMSO-d6): 15.53,
24.31, 27.87, 29.60, 30.67, 33.86 50.01, 51.83, 127.85, 129.23, 130.28, 144.43;
ESI-HRMS (m/z) calculated for C26H38N2: 378.3035, found: 378.3487 (M+).
(4-n-Propyl-benzyl)-{3-[(4-n-propyl-benzylamino)-methyl]-cyclohexyl methyl}-
amine (6j): Yield: 60%; mp: 228 °C; IR (Nujol, cmꢂ1): 2924, 2845, 2751, 2599,
1584, 1462, 1027, 802; 1H NMR (300 MHz, DMSO-d6): 0.57–0.65 (m, 1H), 0.76–
0.83 (m, 2H), 0.84–0.89 (t, 6H), 1.15–1.26 (m, 1H), 1.52–1.56 (m, 4H),
1.69–1.83 (m, 6H), 2.52–2.56 (t, 4H), 2.63–2.66 (d, 4H), 4.04 (s, 4H, 2CH2Ph),
7.20–7.23 (d, J = 9 Hz, 4H, Ar), 7.44–7.47 (d, J = 9 Hz, 4H, Ar), 9.18 (br s,
4H, 2NH2þ); ESI-HRMS (m/z) calculated for C28H42N2: 406.3348, found:
406.3150 (M+). (4-Isopropyl-benzyl)-{3-[(4-isopropyl-benzyl amino)-methyl]-
cyclohexylmethyl}-amine (6k): Yield: 65%; mp: 264 °C; IR (Nujol, cmꢂ1): 2924,
2854, 2754, 2379, 1584, 1459, 1022, 833; 1H NMR (300 MHz, DMSO-d6): 0.57–
0.65 (m, 1H), 0.76–0.83 (m, 2H), 1.17–1.19 (d, 13H), 1.70–1.83 (m, 6H), 2.66–
2.68 (t, 4H), 2.86–2.91 (m, 2H), 4.05 (s, 4H, 2CH2Ph), 7.26–7.29 (d, J = 9 Hz, 4H,
Ar), 7.45–7.48 (d, J = 9 Hz, 4H, Ar), 9.11 (br s, 4H, 2NH2þ); 13C NMR (75.5 MHz,
DMSO-d6): 23.76, 26.43, 29.57, 30.68, 33.17, 33.80, 50.01, 51.89, 126.38,
129.28, 130.34, 151.23; ESI-HRMS (m/z) calculated for C28H42N2: 406.3348,
found: 406.3291 (M+). (4-n-Butyl-benzyl)-{3-[(4-n-butylbenzyl-amino)-methyl]-
cyclohexylmethyl}-amine (6l): Yield: 80%; mp: 202 °C; IR (Nujol, cmꢂ1): 2923,
2854, 2739, 1585, 1461, 1377, 1026, 833;1H NMR (300 MHz, DMSO-d6): 0.57–
0.67 (m, 1H), 0.76–0.80 (m, 2H), 0.84–0.89 (t, 6H), 1.24–1.31 (m, 5H), 1.47–1.57
(m, 4H), 1.73–1.82 (m, 6H), 2.54–2.59 (t, 4H), 2.64–2.68 (d, 4H), 4.04 (s, 4H,
2CH2Ph), 7.22–7.23 (d, J = 9 Hz, 4H, Ar), 7.44–7.47 (d, J = 9 Hz, 4H, Ar), 9.12 (br
s, 4H, 2NH2þ); 13C NMR (75.5 MHz, DMSO-d6): 13.65, 21.60, 24.21, 29.49,
32.95, 33.73, 34.14, 34.40, 49.91, 51.74, 128.31, 129.04, 130.15, 142.96;
ESI-HRMS (m/z) calculated for: C30H46N2: 434.3661, found: 434.69 (M+).
(4-t-Butyl-benzyl)-{3-[(4-t-butyl-benzylamino)-methyl]-cyclohexylmethyl}-amine
(6m): Yield: 90%; mp: 278 °C; IR (Nujol, cmꢂ1): 2924, 2854, 2475, 1586, 1458,
1377, 1023, 834;1H NMR (300 MHz, DMSO-d6): 0.56–0.68 (m, 1H), 0.76–0.81
(m, 2H), 1.15–1.2 (m, 1H), 1.26 (s, 18H), 1.74–1.85 (m, 6H), 2.63–2.80 (d, 2H),
4.04 (s, 4H, CH2Ph), 7.39–7.42 (d, J = 9 Hz, 4H, Ar), 7.50–7.53 (d, J = 9 Hz, 4H,
Ar), 9.34 (br s, 4H, NH2þ); 13C NMR (75.5 MHz, DMSO-d6): 24.49, 29.40, 31.51,
30.05, 34.09, 34.51, 50.85, 51.50, 125.86, 127.05, 130.22, 152.20; ESI-HRMS
(m/z) calculated for: C30H46N2: 434.3661, found: 434.3556 (M+).
and 10% at same concentration (Fig. 1). Compound 6i lysed only
0.5% of the mammalian erythrocytes at 512 lg/mL concentration.
Out of 21 compounds, 14 compounds showed impressive anti-
bacterial activity against P. aeruginosa and S. epidermidis, while
two compounds exhibit potent antibacterial activity against all
the bacterial strains with no hemolysis up to 512 lg/mL. Further
chemical modification of selected compounds is under progress
and results will be published in due course of time.
Acknowledgments
D.S.R. thanks Department of Science and Technology (SR/S1/OC-
08/2008), New Delhi and University of Delhi, Delhi, India for finan-
cial support. D.K. and S.J. are thankful to CSIR for the award of ju-
nior research fellowship.
References and notes
1. Payne, R. D.; Gwynn, N. M.; Holmes, J. D. L.; Pompliano, D. Nat. Rev. 2007, 6, 29.
2. Talbot, H. G. Expert Rev. Anti-Infect. 2008, 6, 39.
3. Stevens, D. L.; Dotter, B.; Madaras-Kelly, K. Expert Rev. Anti-Infect. Ther. 2004, 2,
51.
4. Clemett, D.; Markham, A. Drugs 2000, 59, 815.
5. Bozdogan, B.; Appelbaum, P. C. Int. J. Antimicrob. Agents 2004, 23, 113.
6. Bush, K. Clin. Microbiol. Infect. 2004, 10, 10.
7. Harbart, S.; Albrich, W.; Goldman, D. A.; Huebner, J. Lancet Infect. Dis. 2001, 1,
251.
8. Mitscher, L. A.; Pillai, S. P.; Gentry, E. J.; Shankel, D. M. Med. Res. Rev. 1999, 19,
477.
9. Berber, I.; Cokmus, C.; Atalan, E. Microbiology 2003, 72, 54.
10. Levy, S. B.; Marshall, B. Nat. Med. 2004, 10, S122.
11. Lode, H. Clin. Microbiol. Infect. 2005, 11, 778.
12. Rice, L. B. Biochem. Pharmacol. 2006, 71, 991.
13. Alckshun, M. N. Expert Opin. Invest. Drugs 2005, 14, 117.
14. Harbart, S.; Albrich, W.; Goldman, D. A.; Huebner, J. Lancet Infect. Dis. 2001, 1,
251.