Synthesis and biological evaluation of trans 6-methoxy-1,1-dimethyl-2- phenyl-3-aryl-2,3-dihydro-1H-inden-4-yloxyalkylamine derivatives against drug susceptible, non-replicating M. tuberculosis H37Rv and clinical multidrug resistant strains

Article abstract of DOI:10.1016/j.bmcl.2013.02.030

Synthesis of a library of novel trans 6-methoxy-1,1-dimethyl-2-phenyl-3- aryl-2,3-dihydro-1H-inden-4-yloxy alkyl amines and their antimycobacterial activity against drug sensitive and multidrug resistant strains of Mycobacterium tuberculosis have been reported. All the new compounds in the series exhibited MIC between 1.56 and 6.25 μg/ml. Two compounds 1i and 1j with low MIC and low cytotoxicity showed significant reduction in CFU in infected mouse macrophages at 1× MIC concentration. The compound 1i inhibited the growth of M. tuberculosis in mice at 100 mg/kg dose with 1.35 log₁₀ reduction of CFU in lungs tissue and was active against non-replicating Mycobacterium tuberculosis under anaerobic condition.

Full text of DOI:10.1016/j.bmcl.2013.02.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2404–2407
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of trans 6-methoxy-1,
1-dimethyl-2-phenyl-3-aryl-2,3-dihydro-1H-inden-4-yloxyalkyl-
amine derivatives against drug susceptible, non-replicating
M. tuberculosis H37Rv and clinical multidrug resistant strains q
Shailesh Kumar a, , Atma P. Dwivedi a, Vivek Kr. Kashyap b, A. K. Saxena a, A. K. Dwivedi c,
Ranjana Srivastava b, , Devi P. Sahu a,
⇑
⇑
a Medicinal and Process Chemistry Division, CSIR, Central Drug Research Institute, Lucknow 226001, India
b Microbiology Division, CSIR, Central Drug Research Institute, Lucknow 226001, India
c Pharmaceutics Division, CSIR, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of a library of novel trans 6-methoxy-1,1-dimethyl-2-phenyl-3-aryl-2,3-dihydro-1H-inden-4-
yloxy alkyl amines and their antimycobacterial activity against drug sensitive and multidrug resistant
strains of Mycobacterium tuberculosis have been reported. All the new compounds in the series exhibited
Received 16 October 2012
Revised 24 January 2013
Accepted 6 February 2013
Available online 15 February 2013
MIC between 1.56 and 6.25 lg/ml. Two compounds 1i and 1j with low MIC and low cytotoxicity showed
significant reduction in CFU in infected mouse macrophages at 1Â MIC concentration. The compound 1i
inhibited the growth of M. tuberculosis in mice at 100 mg/kg dose with 1.35 log10 reduction of CFU in
lungs tissue and was active against non-replicating Mycobacterium tuberculosis under anaerobic
condition.
Keywords:
Antimycobacterial
MDR
M. tuberculosis H37Rv
trans 4-Aminoalkoxy-2-phenyl-3-aryl-2
3-Dihdro-1H-inden
Ó 2013 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis (M. tb), the causative agent for
tuberculosis (TB) results in death of almost 3 million people each
year and is positioned as the leading bacterial infectious agent.1
Although an effective chemotherapy for tuberculosis is available,
the frontline anti-tubercular drugs Isoniazid, Pyrazinamide and
Rifamycin have been reported to have significant liver damaging
effects. This along with the emergence of multi-drug resistance,
reactivation of latent infection due to pathogenic synergy of the
mycobacterial infection with the HIV infection1,2 justifies the need
for the design and development of new drug which would not only
be active against multi-drug resistant TB (MDR-TB) and latent TB3
but also, should be non-toxic and shorten the duration of current
chemotherapy and be compatible with anti-HIV drugs.4
O
H
N
N
HN
O
O
OH
NH
HN
HO
O
HN
O
N
B
A
O
N
S
R1
Ar
O
n
N
R2
HO
O
Br
O
N
O
C
1
D
Figure 1.
q
CDRI Communication No.: 86/2011/RS.
⇑
Corresponding authors. Tel.: +91 522 2623411/18; fax: +91 522 2623405 (R.S.);
tel.: +91 945 2586650 (D.P.S.).
E-mail addresses: drskum10@gmail.com (S. Kumar), ranjanasrivastava5@gmail.-
com (R. Srivastava), dpsahuin@yahoo.com (D.P. Sahu).
Present address: Dept. of Applied Chemistry, Babasaheb Bhimrao Ambedkar
University, Lucknow 226025, India.
Ethambutol A (Fig. 1), a well known tuberculostatic drug has 2-
aminoethanol and 1,2-ethylenediamine as core structural features.
A number of compounds such as B, C, and D5–7 containing
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.030

S. Kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2404–2407
2405
Ar
O
CHO
OH
(ii)
(i)
O
HO
O
O
4
R1
N
n
R2
Ar
OH
Ar
O
(iii)
O
O
2
1a-j
Scheme 1. Reagent and conditions: (i) (a) PhCH2COOH, Ac2O, NEt3, 140–145 °C, 5 h, (b) (CH3)2SO4, K2CO3, CH3CN, 60 °C, 8 h, (c) Mg, CH3I, THF, 50–55 °C, then HCl (5%), 20 °C,
5 h; (ii) ArH (3), AlCl3, hexane/benzene (1:1), 0 °C, then 75–80°C, 6–10 h; (iii) substituted amino alkyl chloride, alkali, isopropanol, 50–55 °C, 3 h.
aminoalkoxy motif are shown to have anti-tubercular activity. Up
to certain limit, compounds with higher lipophilicity have higher
permeation across biological membranes (but lower aqueous solu-
bility). Ethambutol (A) has lower lipophilicity than compounds B, C
and D. It was envisaged novel 2, 3-dihydro-1H-inden, a ring isoste-
re of important class of bio-active compounds such as chroman,
flavones and isoflavone and their analogs should have drug like
properties. We ventured to explore hitherto untraversed chemical
space of 2,3-dihyro-1H-indens and here in disclose the preliminary
result of anti-mycobacterial activity of the dialkyl amino alkoxy
derivatives of trans 6-methoxy-1,1-dimethyl-2-phenyl-3-aryl-2,3-
dihydro-1H-inden 1.
The title compounds were synthesized starting from 2,2-di-
methyl-3-phenyl-7-methoxy chromene 4 as shown in Scheme 1.
Anhydrous aluminium chloride mediated tandem hydroarylation8
of 4 with an arene 3 via rearrangement9 of the intermediate chro-
man in a single pot afforded trans 6-methoxy-2-phenyl-3-aryl-4-
Table 2
In vitro activity against M. tuberculosis H37Rv, cytotoxicity and selective index of
indene derivatives 1a–j
S. No.
Compound
LogP
MIC (
lg/ml)
CC50
(l
g/ml)
SI value
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
7.66
6.62
6.11
7.19
7.24
6.57
8.11
6.83
7.82
8.53
0.14
3.71
3.125
3.125
3.125
3.125
6.25
1.56
1.56
3.125
3.125
1.56
8.11
4.59
17.48
3.74
2.96
2.41
5.52
7.49
49.16
23.98
38.51
62.46
2.6
1.4
5.6
1.19
0.47
1.54
3.53
2.39
15.73
15.37
77.02
160.1
9
10
11
12
1j
ETB
RFM
0.5
0.39
All compounds were tested in parallel for MIC and cytotoxicity.
hydroxy-2,3-dihydro-1H-inden derivative 2 exclusively in good
yield. The transformation of 4–2 was might be proceeded by tan-
dem hydroarylation of 4 with arene 3 to afford trans diarylchroman
and subsequently ring contraction to trans diaryl 2,3-dihydro-1H-
inden 2. The exclusive formation of trans isomer is probably due
to steric and thermodynamic factors. The chromene 4 could be
conveniently synthesized from commercially available 2,4-dihy-
doxybenzaldehyde in three steps with following improved litera-
ture procedure.10 Alkylation of 2 with appropriately substituted
amino alkyl chloride in presence of an alkali (LiOH, KOH or NaOH)
in a protic solvent followed by chromatographic purification fur-
nished the titled compounds 1 in above 90% yield. All the synthe-
sized compounds were characterized by IR, 1H NMR, 13C NMR and
mass spectral data and were supported by satisfactory micro-ana-
lytical data.
A total of 10 novel compounds were synthesized (Table 1) and
evaluated for their anti-TB activity by BACTEC radiometric meth-
od11 against M. tuberculosis H37Rv. Table 2 summarizes the anti-
mycobacterial activity (minimum inhibitory concentration, MIC)
of the synthesized compounds. The MIC of the compounds ranged
from 1.56 to 6.25 lg/ml (Table 2). The substituents on the amin-
oalkoxy side chain as well as the length of the spacer have limited
effect on the in vitro activity.
Table 1
trans-1-[2-(6-Methoxy-1,1-dimethyl-2-phenyl-3-aryl-2,3-dihydro-1H-inden-4-yloxy
alkyl amine 1a–j
Entry
Ar
n =
R1R2N– =
1a
1b
1c
Ph
Ph
Ph
2
3
2
N
(CH3)2-N–
O
N
1d
1e
Ph
Ph
2
2
N
(CH3)2-N–
1f
Ph
2
2
2
2
N
N
1g
1h
1i
O
N
N
Ph
Ph
The MIC of the compounds 1f, 1g and 1j having piperidine or
azepine substituent were found to be active at 1.56 lg/ml whereas
the MIC for the compounds having morpholine (1c), pyrrolidine
(1h), dimethylamino (1e) substituent were found to be active at
N
1j
2
O
higher value (3.125 lg/ml).

2406
S. Kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2404–2407
The in vitro activity of compound having two carbons spacer
was found to be favorable over those having three carbons spacer
(Table 1).
Table 2 summarizes the anti-mycobacterial activity, cytotoxic-
ity and lipophilicity of all the compounds. The compounds 1i and
1j with low MIC and low cytotoxicity are better with respect to
lipophilicity.
All the compounds were tested for in vitro cytotoxicity. The
cytotoxicity was determined with Vero monkey kidney cells (ATCC
CCL-81) using Resazurin assay.11a The indene derivatives 1 with
low MIC exhibited moderate to high degree of cytotoxicity. The
compounds 1i and 1j attained selective index above 15.5 which
is the unique feature proving the higher efficacy and low toxicity
of the above two compounds. Similar to in vitro activity, the cyto-
toxicity of the compounds were marginally affected by the substit-
uents on nitrogen of aminoalkoxy side chain. The compound with
piperidine sustistuent has moderate whereas the compound con-
taining azepane, morpholine have lower cytototoxicity. Two com-
pounds 1i and 1j with SI value >15 were selected for in vitro
activity against MDR strains of M. tuberculosis and their efficacy
in mouse macrophages. In vitro activity of compounds 1i and 1j
were evaluated against MDR (multidrug resistant) strains of M.
tuberculosis (clinical isolates from patients) which exhibited resis-
tance to Rifampicin and Isoniazid.11a The activity was evaluated by
agar dilution method and confirmed by BACTEC radiometric meth-
od. Both compounds 1i and 1j were active against MDR strains of
Figure 2. Efficacy of selected compounds in macrophages. Macrophages were lysed
and viable counts were determined at 0 and 6 day time points at 1Â and 2Â MIC
concentration.
Table 4
Tissue bacillary load (CFU) in lungs of BALB/c mice infected with M. tuberculosis
H37Rv after 30-day drug treatment
Treatment regimen
Dose (mg/kg)
log10 CFU SE in lungs
Untreated
INH
1i
7.61 0.37
4.83 0.07
6.25 0.31
25
100
TB at 6.25 and 3.125 lg/ml (Table 3).
The compounds 1i and 1j were tested for ex vivo efficacy in
mouse macrophage cell line J774A1 at 1Â and 2Â MIC concentra-
tions as described in Supplementary data section. Lysates from 0
and 6 days of control and test samples were plated on MB7H10
agar containing OADC and CFU/ml were determined (Fig. 2).
As compared to control compounds 1i and 1j caused effective
inhibition of intracellular replication of M. tuberculosis within
mouse macrophages at 1Â and 2Â MIC concentrations. However,
in case of compound 1i, as compared to control, more than 90%
inhibition of growth of M. tuberculosis H37Rv was obtained follow-
ing 6 day treatment at 1Â MIC concentration (Fig. 2). The com-
pound 1i was selected for in vivo activity evaluation in mice and
in vitro against non-replicating M. tb.
The in vivo efficacy of the selected compound 1i was evaluated
in murine TB model for acute infection.11b The mouse (18–20 g)
were infected iv with M. tuberculosis H37Rv (106 CFU) and their
chemotherapy was initiated 2 days later and continued for 30 days
(6 days/week). The mice treated with INH (25 mg/kg) were positive
control. Infected untreated mouse served as negative control. The
polyvinylpyrrolidone (PVP) complex of the compound was pre-
pared which was soluble in water and was administered by oral
gavage at a dose of 100 mg/kg. By the end of therapy, six mice
for every group were sacrificed, and 10-fold dilutions of lung
homogenates were plated on MB7H10-OADC agar for CFU determi-
nation. The experimental protocols were approved by the animal
ethics committee of the Institute.
Figure 3. Comparison of growth of hypoxic M. tb bacilli treated with INH (Isonizid),
MTZ (Metronidazole) and test compound. Viability was determined as log10 CFU/ml
as described in Supplementary data. Concentration of compound is given as lg/ml.
As shown in Table 4, a reduction of bacterial CFU in lungs of
mouse as a result of chemotherapy using compound 1i was
observed. In the murine model of TB infection, the 1i showed
1.35 log10 reductions of the CFUs in the lungs of treated mouse
at 100 mg/kg body weight dose compared to 2.8 log10 reduc-
tions of the same by INH which is indicative of anti-TB activity
Table 3
In vitro activity of compounds 1i and 1j against clinical drug susceptible and MDR strains of M. tuberculosis
M. tuberculosis strains
Susceptibility or resistance to antitubercular drugs
Rifr INHr
MIC (lg/ml)
Strain
2643
(MDR)
6.25 (1i)
3.125 (1j)
a
Strain
Rifr INHr
6.25 (1i)
3.125 (1j)
1678/05a
(MDR)
Strain
Drug sensitive
3.125 (1i)
1.56 (1j)
2280a
a
The strains were obtained from Professor Sarnam Singh, AIIMS, New Delhi.

S. Kumar et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2404–2407
2407
(tuberculosis antimicrobial acquisition and coordinating facility,
References and notes
http://www.tacf.org).
1. (a) Tuberculosis, HIV/Aids and Malaria, The Status and Impact of the Three
Diseases. WHO, Geneva, 2005.; (b) Raviglione, M. C. Tuberculosis 2003, 83, 4; (c)
Espinal, M. A. Tuberculosis 2003, 83, 44; (d) Girling, D. J. Tubercle 1978, 59, 13.
2. (a) World Health Organization, The Global MDR-TB & XDR-TB Response Plan
2007–2008 Geneva, Switzerland, 2007.; (b) Young, D. B.; Duncan, K. Ann. Rev.
Microbiol. 1995, 49, 641; (c) Schaeffer, M. L.; Khoo, K. H.; Besra, G. S.; Chatterjee,
D.; Brennan, P. J.; Belisle, J. T.; Inamine, J. M. J. Biol. Chem. 1999, 274, 31625; (d)
Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004; (e)
Saito, H.; Tomioka, H.; Sato, K.; Emori, M.; Yamane, T.; Yamashita, K.; Hosoe, K.;
Hidaka, T. Antimicrob. Agents Chemother. 1991, 35, 542.
For evaluation of activity against non-replicating M. tb, cells
were grown in hypoxia, that is, oxygen depletion condition.12a,b,c
Depletion of oxygen triggers the shift down of M. tuberculosis to
a state of dormancy. Metronidazole (active only on anaerobically
grown organisms) and isoniazid (acting only under aerobic condi-
tions) were used as controls. Cell viability in control and after drug/
compound addition was determined by CFU determination as de-
scribed in Supplementary data. The compound 1i was found to
be active against non-replicating hypoxic M. tuberculosis at
6.25 lg/ml which was twofold higher than MIC reported in repli-
cating phase (Fig. 3).
In conclusion, the preliminary in vitro antimycobacterial
activity evaluation of all the compounds except 1e, library of novel
4-aminoalkoxy indane 1 exhibited MIC below 6.25 lg/ml. The
compound 1i was selected as lead compound on basis of MIC,
selective index >15, susceptibility to drug sensitive, non-replicat-
ing M. tuberculosis H37Rv and clinical MDR strains of M. tuberculo-
sis. In the mouse model of TB infection, the compound 1i showed
1.35 log10 reduction of the CFUs in the lungs of treated mice which
is indicative of anti-TB activity. Further lead search is in progress.
3. Pedro, E.; Silva, A. D.; Ramosa, D. F.; Bonacorso, H. G.; Iglesia, A. I.; Oliveira, M.
R.; Coelho, T.; Navarini, J.; Morbidoni, H. R.; Zanatta, N.; Martins, M. A. P. Int. J.
Antimicrob. Agents 2008, 32, 139.
4. Duncan, K. Tuberculosis 2003, 83, 201.
5. Jefferson, E. A.; Seth, P. P.; Robinson, D. E.; Winter, D. K.; Miyaji, A.; Risen, L. M.;
Osgood, S. A.; Bertrand, M.; Swayze, E. E. Bioorg. Med. Chem. Lett. 2004, 14, 5257.
6. Parai, M. K.; Panda, G.; Chaturvedi, V.; Manju, Y. K.; Sinha, S. Bioorg. Med. Chem.
Lett. 2008, 18, 289.
7. (a) Nayyar, A.; Malde, A.; Coutinho, E.; Jain, R. Bioorg. Med. Chem. 2006, 14,
7302; (b) Prado, S. C.; Janin, Y. L.; Saint-Joanis, B.; Brodin, P.; Michel, S.; Koch,
M.; Cole, S. T.; Tillequin, F.; Bost, P. Bioorg. Med. Chem. 2007, 15, 2177.
8. (a) Rueping, M.; Nachtsheim, B. J.; Scheidt, T. Org. Lett. 2006, 8, 3717; (b)
Kischel, J.; Jovel, I.; Mertins, K.; Zapf, A.; Beller, M. Org. Lett. 2006, 8, 19.
9. Loudon, J. D.; Razdan, R. K. J. Chem. Soc. 1954, 4299.
10. Xiao, S.; Wang, X.; Yan, J. J. Chin. Pharm. Sci. 1998, 7, 69.
11. (a) Saquib, M.; Husain, I.; Sharma, S.; Yadav, G.; Singh, V. K.; Sharma, S. K.;
Shah, P.; Siddiqi, M. I.; Kumar, B.; Lal, J.; Jain, G. K.; Srivastava, B. S.; Srivastava,
R.; Shaw, A. K. Eur. J. Med. Chem. 2011, 46, 2217; (b) Kelly, B. P.; Furney, S. K.;
Jessen, M. T.; Orme, I. M. Antimicrob. Agents Chemother. 1996, 40, 2809.
12. (a) Wayne, L. G.; Hayes, L. G. Infect. Immun. 1996, 64, 206; (b) Lenaerts, A. J.;
Gruppo, V.; Marietta, K. S.; Johnson, C. M.; Driscoll, K.; Tompkins, M.; Jerry, D.
R.; Reynolds, R. C.; Orme, I. M. Antimicrob. Agents Chemother. 2005, 49, 2294; (c)
Saxena, A.; Srivastava, V.; Srivastava, R.; Srivastava, B. S. Tuberculosis 2008, 88,
518.
Acknowledgments
We thank Director, CDRI for support and facilities. S.K. and
V.K.K. are thankful to the University Grants Commission, New Del-
hi for the financial support and to the Sophisticated Analytical
Instrument Facility, CDRI, and Lucknow for providing spectroscopic
data.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.02.
030.