Synthesis and antimycobacterial activity of novel camphane-based agents

Article abstract of DOI:10.1016/j.bmcl.2013.11.050

A series of six new amidoalcohols was designed and synthesized on the base of the camphor scaffold. Natural amino acids were transformed into their α-hydroxy analogues with retention of configuration, and attached to isobornylamine. The compounds were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv. Some of the new compounds show 25 times higher activity than the classical anti-TB drug ethambutol. The activity shifts from micromolar to nanomolar inhibitory concentrations depending on the α-hydroxy acid moiety. Two of the most potent compounds exert low level of cytotoxic activity. These camphane-based amido-alcohols present promising potential lead compounds for further elaboration of antimycobacterial agents.

Full text of DOI:10.1016/j.bmcl.2013.11.050

Accepted Manuscript
Synthesis and antimycobacterial activity of novel camphane-based agents
Georgi Stavrakov, Irena Philipova, Violeta Valcheva, Georgi Momekov
PII:
S0960-894X(13)01341-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.11.050
BMCL 21077
Reference:
Bioorganic & Medicinal Chemistry Letters
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11 October 2013
18 November 2013
20 November 2013
Please cite this article as: Stavrakov, G., Philipova, I., Valcheva, V., Momekov, G., Synthesis and antimycobacterial
activity of novel camphane-based agents, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/
10.1016/j.bmcl.2013.11.050
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Synthesis and antimycobacterial activity of novel camphane-based agents
Georgi Stavrakova, *, Irena Philipovab, Violeta Valchevac, Georgi Momekova
aFaculty of Pharmacy, Medical University of Sofia, str. Dunav 2, Sofia 1000, Bulgaria
bInstitute of Organic Chemistry, Bulgarian Academy of Sciences, Acad. Bonchev 9, Sofia 1113,
Bulgaria
cInstitute of Microbiology, Bulgarian Academy of Sciences, Akad. Bonchev 26, Sofia 1113,
Bulgaria
*Corresponding author. Faculty of Pharmacy, str. Dunav 2, Sofia, BG-1000; tel +3599236516
E-mail address: gstavrakov@pharmfac.net
Abstract
A series of six new amidoalcohols was designed and synthesized on the base of the camphor
scaffold. Natural amino acids were transformed into their -hydroxy analogues with retention of
configuration, and attached to isobornylamine. The compounds were evaluated for their in vitro
activity against M. tuberculosis H37Rv. Some of the new compounds show 25 times higher
activity than the classical anti-TB drug ethambutol. The activity shifts from micromolar to
nanomolar inhibitory concentrations depending on the -hydroxy acid moiety. Two of the most
potent compounds exert low level of cytotoxic activity. These camphane-based amido-alcohols
present promising potential lead compounds for further elaboration of antimycobacterial agents.
Keywords: camphane, isobornylamine,-hydroxy acids, antimycobacterial activity, M.
tuberculosis H37Rv.
Mycobacterium tuberculosis (MTB) infects latently one-third of the world’s population
causing approximately 9 million cases of active disease each year.1 This, coupled with the rise
of multidrug resistant and extensively drug resistant strains, necessitates the search for novel
antimycobacterial agents. Wilkinson and coworkers first reported the synthesis and activity of
ethambutol (EMB) (Fig. 1. I).2 Based on structure-activity relationship (SAR) studies it
appeared that the distance between the two nitrogens, the presence of -aminoalcohols, and the
small side chains were critical for determining activity. The configuration of the molecule is

decisively important for the activity, since EMB (with S,S -configuration) is approx. 200-500
fold more potent than its (R,R)-enantiomer. Removal or significant alteration of the basicity of
either amino group resulted in a loss of potency, with the exception that the corresponding
amides retained activity in some analogues (Fig. 1. II).3
Lately, the occurrence of a ‘better ethambutol’ has been systematically investigated through
virtual screening or a combinatorial approach.3,4 Several 1,2-diamines, such as SQ 109 (Fig. 1.
III), displaying improved antimycobacterial potencies and promising pharmacokinetic
properties have thus been reported.5 It is very likely that the highly lipophilic adamantane
structure was integral for the antitubercular activity. Adamantyl ureas (Fig. 1. IV and V) were
also identified as a group of compounds active against MTB.6,7 These compounds have been
shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate and to
inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases.7,8
Figure 1. Structures of ethambutol (I), amide modified ethambutol (II), SQ 109 (III),
adamantyl ureas (IV and V), 3-exo-aminoisoborneol (VI) and isobornylamine (VII) based
amidoalcohols.

Inspired by the -aminoalcohol fragment in the molecule of EMB and the retention of the
activity on its amide analogues, we dedicated our efforts towards the development of camphane
based amido-alcohol structures. Another aspect was the analogy between the bulky lipophilic
camphane fragment and the adamantyl. Among the numerous monoterpenoids, camphor is of
particular importance as a starting compound for the synthesis of various multifunctional
enantiopure building blocks for organic synthesis.9,10 Although a number of biologically active
camphor derivatives are isolated from plants, almost no efforts are dedicated to the development
of synthetic analogues.11-16
Recently, we have accomplished practical synthesis of a small number of new
amidoalcohols and amidodiols on the base of 3-exo-aminoisoborneol (Fig. 1. VI).17 These were
screened for antimycobacterial activities against two MTB strains (H37Rv and MDR strain 43).
Some of the compounds show 25 times higher activity than ethambutol. Small structural
changes in the side chain shift the activity from micromolar to nanomolar inhibitory
concentrations.
Encouraged by these observations, we expanded the approach to the synthesis of
isobornylamine analogues (Fig. 1. VII). Thus, we pursue the exploration of the camphane based
structures as a novel class of anti-TB compounds.
Since peptides and peptide-related structures have a wide variety of physiological and
pharmacological actions, the concept of peptidomimetics was aimed by the design of -amino
acids derived amido-alcohols. The key step was the stereoselective preparation of a series of
chiral -hydroxy acids as starting building blocks. The natural amino acids were used as easily
available and cheap chiral auxiliaries. Reaction of L-phenylalanine 1a with nitrous acid is
known to give -hydroxy acid 2a with retention of configuration due to the participation of the
neighbouring carboxyl group.18 Applying the reaction to a set of -amino acids 1b-e afforded
the corresponding -hydroxy analogues 2b-e (Scheme 1).
Scheme 1. Synthesis of -hydroxy acids.

Readily available isobornylamine 3 was selected as the key (+)-camphor-derived starting
compound.19 Our aim was to obtain the target structures using simple and effective synthetic
transformations (Scheme 2).
Scheme 2. Synthesis of isobornylamine based amidoalcohols.

The amide linkage between the -hydroxy acids and isobornylamine 3 was accomplished by
procedures developed for peptide synthesis. The reaction was optimized for the commercially
available mandelic acid 5 in the presence of N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDCI) and 1-Hydroxybenzotriazole hydrate (HOBT) as coupling reagents to
yield 4a. Following the same protocol for -hydroxy acids 2a-e we synthesized the
corresponding amido-alcohols 4b-f (Scheme 2). The products were obtained in good yields and
excellent purity after flash column chromatography.
All compounds were identified by elementary analysis, 1H NMR and 13C NMR. The spectral
analyses were in accordance with the assigned structures.
The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis
H37Rv using the method of Canetti (Table 1). EMB and Rifampicin were used as reference
antituberculous agents. The amidoalcohols 4a-b exhibited excellent activities against M.
tuberculosis H37Rv. The mandelic acid derived amido-alcohol 4a with MIC of 0.17 M and the
phenylalanine derived 4b with MIC of 0.16 M differ only in a methylene group, which
obviously does not has an essential influence on the activities. The change of the aromatic
fragments with aliphatic in the cases of leucine, isoleucine and valine derived amido-alcohols
4c-e revealed MICs comparable with the one of the reference ethambutol. Interestingly, the
aliphatic but sulfur containing methionine derived camphane 4f exhibited very high MIC of 0.17
M (Table 1).
Noteworthy, although that the carbon atom at the nitrogen in the camphane scaffold
possesses (R)-configuration, three of the molecules are extremely active. This is opposite to the
fact that (S,S)-EMB is approximately 500 fold more active than (R,R)-EMB.2 Therefore, the
direct comparison of the structure presented in this paper with EMB should be handled with
care. Moreover, this is an exploratory study and it is not known do the compounds share the
target with EMB.
Evident from the calculated LogP values the presented series of compounds are lipophilic
and thus have low water solubility, a feature which conditions limited bioavailability and
presumably suboptimal pharmacokinetic behavior. Nevertheless, the nanomolar
antimycobacterial activity of some of the agents within the series with their generally low
cytotoxic properties, give us reason to consider these camphane-modified amido-alcohols as
perspective leads for further exploration.

Table 1. In vitro screening data for antimycobacterial activity and cytotoxicity of synthesized compounds.
Compound
Antimycobacterial activity
towards reference strain of
Mycobacterium tuberculosis
H37Rv, MIC (M)
In vitro cytotoxicity towards Selectivity index,
LogP
human embryonal kidney
SI = IC50/MIC.
cell line 293T, IC50 (M)a
0.17
0.16
7.48
7.48
7.89
0.17
7.22
0.06
105.2
52.8
111.8
106.6
> 200
171.5
-
618.8
330.0
14.9
13.6
> 25.3
1008.8
-
3.81
4.14
3.80
3.80
3.27
3.20
-0.12
3.85
4a
4b
4c
4d
4e
4f
EMB.2HCl
Rifampicin
-
-
LogP values, octanol-water partitioning coefficient of the new compounds, were calculated using
ACDLabs/ChemSketch; LogP values of EMB.2HCl and Rifampicin are known in the literature: [DrugBank].
In order to examine the selectivity of the antiproliferative effects, the cytotoxicity of the
presented compounds, was assessed against the human embryonal kidney cell line 293T, after
72 h exposure. Evident from the IC50 values summarized in Table 1, the compounds were
generally of low-to-moderate cytotoxicity against the human cells; with the only exception of
4b. All other compounds induced 50% inhibition of cellular viability at concentrations
exceeding 100 µM. Compound 4e failed to induce 50% inhibition of cellular viability within the
tested range of concentrations (12.5 – 200 µM). The results are extremely in favour of structures
4a and 4f. They exhibit both high antimycobacterial activity and low cytotoxicity, which is
represented by their selectivity indexes: 618.8 for 4a, and 1008.8 for 4f.
In summary, a series of six new amido-alcohols was synthesized by condensation of
isobornylamine with -hydroxy acids. The compounds were screened for antimycobacterial
activities against M. tuberculosis H37Rv . The activity shifts from micromolar to nanomolar
inhibitory concentrations depending on the -hydroxy acid moiety. Three of the new
compounds show 25 times higher activity than the classical anti-TB drug ethambutol. The other
three structures demonstrate activities comparable with ethambutol. Interestingly, two of the
most potent compounds proved to exert low level of cytotoxic activity against a human

embryonal kidney cell line 293T. On this basis, the camphane-based amides could be considered
as promising lead compounds for elaboration of antimycobacterial agents. Further optimizations
of these scaffolds in order to improve their water solubility, which is crucial for their
bioavailability and pharmacokinetics, are in progress.
Acknowledgements
Financial support of National Science Fund, Bulgaria (DMU 02/3 – 2009) is gratefully
acknowledged.
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