Synthesis and synergistic antimycobacterial screening of chlorpromazine and its metabolites

Article abstract of DOI:10.1039/c3md00387f

The antimycobacterial activities of chlorpromazine and its metabolites were evaluated alone and in combination with antitubercular drugs. Although associated with limited antimycobacterial activity when tested individually, chlorpromazine and its metabolites exhibited clear synergy when tested in combination with a number of aminoglycosides as well as the active metabolite of rifampicin, 25-desaceteylrifampicin. The combination of chlorpromazine and spectinomycin was associated with the greatest synergy, yielding a fractional inhibitory concentration index (FICI) of 0.31. Synergistic interactions were also observed for combinations of 7-hydroxychlorpromazine or nor-chlorpromazine with kanamycin, streptomycin, spectinomycin and 25-desacetylrifampicin (FICI 0.19-0.5).

Full text of DOI:10.1039/c3md00387f

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Synthesis and synergistic antimycobacterial
screening of chlorpromazine and its metabolites†
Cite this: Med. Chem. Commun., 2014,
, 502
5
a
a
a
a
Elizabeth M. Kigondu, Mathew Njoroge, Kawaljit Singh, Nicholas Njuguna,
bc
abd
Digby F. Warner and Kelly Chibale*
The antimycobacterial activities of chlorpromazine and its metabolites were evaluated alone and in
combination with antitubercular drugs. Although associated with limited antimycobacterial activity when
tested individually, chlorpromazine and its metabolites exhibited clear synergy when tested in
combination with a number of aminoglycosides as well as the active metabolite of rifampicin, 25-
desaceteylrifampicin. The combination of chlorpromazine and spectinomycin was associated with the
greatest synergy, yielding a fractional inhibitory concentration index (FICI) of 0.31. Synergistic interactions
were also observed for combinations of 7-hydroxychlorpromazine or nor-chlorpromazine with
kanamycin, streptomycin, spectinomycin and 25-desacetylrifampicin (FICI 0.19–0.5).
Received 17th December 2013
Accepted 28th January 2014
DOI: 10.1039/c3md00387f
www.rsc.org/medchemcomm
6
,10
of the fractional inhibitory concentration index (FICI).
A
Introduction
key goal of any drug discovery programme is to synthesise
Every year, approximately 9 million people develop active metabolically stable analogues of a lead compound, and this
tuberculosis (TB), 30% of which reect co-infection with applies equally to antimycobacterial drugs. However, it has
1
–3
HIV. However, the number of new TB drugs that make it been recognized that pharmacologically active metabolites
into the market is very low, which is of great concern for a have in some instances been successfully developed as drugs,
disease whose global incidence remains elevated, resulting in which oen possess superior physicochemical, pharmacody-
4
almost 1.4 million deaths per annum. This problem is namic and pharmacokinetic properties compared to the
1
2
further compounded by the continued emergence of drug parent drugs. Accordingly, we have become interested in
resistance, which severely limits the utility of existing drugs. studying the relative contribution of metabolites to anti-
Clinically, anti-TB drugs are administered in combination to mycobacterial activity.
maximize their efficacy and prevent resistance. However,
Phenothiazines have been used for many years in the clinical
most antimycobacterial drug discovery efforts are based on management of psychosis. However, they have also been
screening of single agents. An alternative and potentially reported to have in vitro antimycobacterial activity specically
5
–8
more relevant strategy is the use of synergistic screening inhibiting NADH:menaquinone oxidoreductase which is
6
,9,10
13
(
the so-called “checkerboard assays”)
to investigate the responsible for aerobic respiration. Studies have demon-
activity of two agents in combination. Synergy is dened as strated that thioridazine, a phenothiazine, has activity in mice
the biological activity achieved when a combination of two against multidrug resistant (MDR) and extensively drug resis-
14–20
drugs against a given microorganism is greater than the sum tant (XDR) strains of Mycobacterium tuberculosis.
Thiorida-
of the individual activities of each member of that combina- zine in combination with several antibiotics causes synergy.
1
1
tion. Practically, this is determined through the calculation Phenothiazines inhibit the synthesis of proteins necessary for
21
bacterial cell walls leading to death of the mycobacteria.
Chlorpromazine (CPZ), another phenothiazine, was selected
as a proof-of-concept for this study. Previously, CPZ was
a
Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa.
E-mail: Kelly.Chibale@uct.ac.za; Tel: +27 21 650 2553. Fax: +27 21 650 5195
reported to exhibit a 4-fold reduction of efflux pump activity in
b
18
Institute of Infectious Disease & Molecular Medicine, University of Cape Town, M. avium. In another study, CPZ in combination with some
Rondebosch 7701, South Africa
22
anti-TB drugs was shown to exhibit synergism. This makes
c
MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of
CPZ a potential partner for combination studies with anti-TB
drugs. The fast replicating non-pathogenic M. smegmatis was
used as a mycobacterial model in this study. This is consistent
Excellence for Biomedical Tuberculosis Research, Department of Clinical Laboratory
Sciences, Faculty of Health Sciences, University of Cape Town, Rondebosch 7701,
South Africa
d
South African Medical Research Council Drug Discovery and Development Research with other reports which have successfully applied M. smegmatis
23
Unit, University of Cape Town, Rondebosch 7701, South Africa
for the preliminary identication of hit compounds as well as
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3md00387f
6
promising drug combinations.
1
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For further investigation of the contribution of drug
metabolites to the biological activity, we included 25-desace-
tylrifampicin in these experiments since it is the major active
28
metabolite of rifampicin, a frontline TB drug.
Results and discussion
Determination of MIC99 of chlorpromazine and its
metabolites
The minimum inhibitory concentrations (MICs99) of CPZ and
its metabolites are shown in Table 1. The antimycobacterial
activity of CPZ and its metabolites was generally low. Interest-
ingly, the activity of CPZ metabolites, 7-hydroxyCPZ (M2) and
nor-CPZ (M5), was comparable to that of chlorpromazine. CPZ-
N-oxide (M3) has been reported to revert to CPZ in solution and
Fig. 1 Chlorpromazine and its metabolites.
29
this may contribute to some of its activity. No anti-
mycobacterial activity was observed for the other metabolites
Because of the relevance of metabolites to the activity
1
2
of known clinical compounds, the synergistic combination
screening was also performed with CPZ metabolites. CPZ
is metabolized to chlorpromazine sulfoxide (M1), 7-hydroxy-
chlorpromazine (M2), chlorpromazine-N-oxide (M3), chlor-
(M1, M4, M6) at the highest concentration tested.
Synergistic/matrix screening
promazine-N-S-dioxide (M4), nor-chlorpromazine (M5) Tables 2 and 3 summarize the MIC99 of the individual
and nor-chlorpromazine sulfoxide (M6) (Fig. 1), among compounds, the lowest MIC99 achieved in the various combi-
2
4–26
others.
nations of CPZ and its metabolites with known anti-TB drugs,
and the fractional inhibitory concentration indices (FICIs).
Synergy is assigned where the FICI # 0.5; a FICI $ 4 is
considered an antagonistic interaction, while any value falling
in between indicates no interaction. Generally, combinations
of CPZ with known anti-TB drugs exhibited improved activity
against M. smegmatis.
A combination of CPZ with spectinomycin exhibited a
synergistic effect with a FICI of 0.31. Combinations of 7-
hydroxyCPZ (M2) resulted in synergistic effects with kanamycin
and spectinomycin (FICI 0.50 and 0.19 respectively).
Interestingly, nor-CPZ (M5) was able to augment the anti-
mycobacterial activity of anti-TB drugs to a greater extent
compared to CPZ and M2. Its synergistic effect was observed for
combinations with rifampicin and its metabolite, 25-desace-
tylrifampicin, kanamycin, and streptomycin – all of which
yielded a FICI value of 0.5, with the best interaction observed
with spectinomycin (FICI 0.31). It is worth noting that even in
combinations that exhibited a FICI > 0.5, a clear drop in the
MIC99 of several of the anti-TB drugs was observed. For
instance, CPZ and its metabolites were able to cause a 4–8 fold
drop in the MIC99 of rifampicin and its metabolite 25-desace-
tylrifampicin (Table 2). As per the FICI denition, combinations
Synthesis
7
CPZ sulfoxide (M1), CPZ-N-oxide (M3), CPZ-N-S-dioxide (M4),
nor-CPZ (M5) and nor-CPZ sulfoxide (M6) were synthesized
from CPZ via a non-classical Polonovski reaction (Scheme 1),
which involves oxidation of CPZ with m-chloroperbenzoic acid
m-CPBA) to afford the N-oxide derivative M3 as a major product
and CPZ sulfoxide (M1) as a minor product. Subsequent
demethylation of M3 with ferrous sulphate in methanol yielded
nor-CPZ (M5). CPZ-N-S-dioxide (M4) was obtained by
reacting M1 with m-CPBA. Likewise, reaction of nor-CPZ (M5)
with m-CPBA yielded nor-chlorpromazine sulfoxide (M6).
27
(
7
-HydroxyCPZ (M2) was purchased from Sigma-Aldrich (SA).
We conrmed the identities of the major CPZ metabolites by
LC-MS analysis following exposure of CPZ to liver microsomes
see ESI†).
(
Table 1 MIC99 of chlorpromazine and its metabolites
Compound
MIC99 (mM)
CPZ
117.26
>1990.89
124.44
CPZ sulfoxide (M1)
7-HydroxyCPZ (M2)
CPZ-N-oxide (M3)
CPZ-N-S-dioxide (M4)
Nor-CPZ (M5)
995.43
>1900.10
136.70
Scheme 1 Synthesis of chlorpromazine metabolites.
Nor-CPZ sulfoxide (M6)
>2077.89
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a
Table 2 MIC99 & FICI of CPZ and its active metabolites in combination with anti-TB drugs, in M. smegmatis
MIC99 (mM)
MIC99 (mM)
MIC99 (mM)
MIC99 (mM)
Compound
singly
combination
FICI
0.63
0.50
0.50
—
Compound
singly
combination
FICI
0.19
0.75
1.00
0.75
–
Rifampicin
CPZ
Rifampicin
2.53
117.27
1.26
2.66
5.33
117.27
0.76
117.27
7.16
117.27
0.07
58.62
84.12
117.27
0.05
117.27
1435.28
117.27
0.63
117.27
0.58
117.27
2.53
62.21
1.33
124.44
0.76
124.44
3.57
124.44
0.07
124.44
0.32
58.62
0.63
1.33
1.33
Spectinomycin
7-HydroxyCPZ (M2)
Chlorpromazine
7-HydroxyCPZ (M2)
Rifampicin
CPZ-N-oxide (M3)
25-Desacetylrifampicin
CPZ-N-oxide (M3)
Ethambutol
CPZ-N-oxide (M3)
Kanamycin
CPZ-N-oxide (M3)
Streptomycin
CPZ-N-oxide (M3)
Spectinomycin
CPZ-N-oxide (M3)
CPZ
CPZ-N-oxide (M3)
Rifampicin
Nor-CPZ (M5)
25-Desacetylrifampicin
Nor-CPZ (M5)
Ethambutol
Nor-CPZ (M5)
Kanamycin
84.12
124.44
117.27
124.44
1.26
995.43
2.66
995.43
0.76
995.43
1.79
995.43
0.29
995.43
168.22
995.43
117.27
995.43
1.26
136.70
2.66
136.70
1.51
63.33
3.57
136.70
0.29
136.70
84.12
136.70
117.27
136.70
5.25
15.56
29.32
62.21
0.63
497.73
0.67
497.73
IAE
2
2
5-Desacetylrifampicin
5-Desacetylrifampicin
CPZ
Ethambutol
CPZ
Kanamycin
CPZ
Streptomycin
CPZ
Spectinomycin
CPZ
TMC207
CPZ
Nalidixic acid
CPZ
Ciprooxacin
CPZ
29.32
NC
3.57
58.62
0.02
29.32
5.25
29.32
0.01
58.62
358.81
58.62
0.30
1.00
0.79
0.31
0.70
—
0.89
497.73
0.07
497.73
84.12
124.44
58.62
248.85
0.32
34.18
0.67
34.18
IAE
1.00
0.74
0.63
0.75
0.50
0.50
—
0.73
—
29.32
NC
Levooxacin
CPZ
Rifampicin
0.32
31.12
0.33
62.21
IAE
0.63
0.75
—
7
2
7
-HydroxyCPZ (M2)
5-Desacetylrifampicin
-HydroxyCPZ (M2)
0.89
34.18
0.07
34.18
5.25
34.18
29.32
68.33
0.50
0.49
0.31
0.75
Nor-CPZ (M5)
Streptomycin
Nor-CPZ (M5)
Spectinomycin
Nor-CPZ (M5)
CPZ
Ethambutol
-HydroxyCPZ (M2)
Kanamycin
-HydroxyCPZ (M2)
Streptomycin
7
0.89
31.12
0.02
0.50
0.79
7
7
-HydroxyCPZ (M2)
62.21
Nor-CPZ (M5)
a
IAE – inconsistent antagonistic effect; NC – no change; note: MIC99 of two compounds in combination is less than the MIC99 of the individual
compounds because in combination the compounds potentiate each other's activity.
that yield FICI > 0.5 but #4.0 indicate no interaction. its metabolites did not exhibit synergism but antagonism.
2
2,30
However, for some of these (for example, CPZ plus This effect has been reported in previous studies.
The
rifampicin and CPZ-N-oxide plus streptomycin), the change results clearly indicate that CPZ and its metabolites are able
in MIC99 of the known anti-TB drug suggests the potential to increase M. smegmatis susceptibility to anti-TB drugs.
to identify compounds and/or metabolites which can Spectinomycin, which hardly has any antimycobacterial
potentiate activity. Notably, for CPZ-N-oxide (M3) this effect activity, exhibited the highest drop in MIC99. Similar inter-
is observed even where the metabolite itself is only very actions were observed for spectinomycin and other drugs in a
6
weakly active on its own. CPZ sulfoxide (M1), CPZ-N-S-dioxide recent study by Ram ´o n-Garc ´ı a et al. The basis for the
(
M4) and nor-CPZ sulfoxide (M6) were all inactive propensity of spectinomycin to interact synergistically with a
hence FICI could not be calculated for the various variety of different compound classes requires further
combinations.
investigation.
Nevertheless, as seen in Table 3, inactive metabolites
Aminoglycosides appeared to interact mostly with CPZ and
were still able to augment the antimycobacterial activity of its metabolites. It has been reported that aminoglycosides,
some of the anti-TB drugs used in this study. For example, which are known to target ribosomes leading to inhibition of
CPZ sulfoxide (M1) and nor-CPZ sulfoxide (M6) decreased the protein synthesis, tend to display synergistic effects when used
MIC99 of spectinomycin 16-fold and 4-fold, respectively. At in combination with other drugs such as cell wall synthesis
least a 2-fold drop in MIC99 was observed for the other inhibitors, which help to increase accumulation of the drug
31
combinations.
within the mycobacterial cell. Elucidation of the molecular
Combinations of the parents (CPZ and rifampicin) with mechanism underlying those interactions that yielded FICI
their metabolites yielded a FICI of ꢀ1.00 which is expected of # 0.5 would contribute signicantly to the interpretation of
an additive interaction (Table 2). Ethambutol with CPZ and these ndings.
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Table 3 MIC99 of inactive metabolites in combination with anti-TB
drugs, in M. smegmatis
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MIC99 (mM)
singly
MIC99 (mM)
combination
4
Drugs/compound
Rifampicin
CPZ sulfoxide (M1)
Ethambutol
CPZ sulfoxide (M1)
Kanamycin
CPZ sulfoxide (M1)
Streptomycin
CPZ sulfoxide (M1)
Spectinomycin
CPZ sulfoxide (M1)
Rifampicin
CPZ-N-S-dioxide (M4)
Ethambutol
CPZ-N-S-dioxide (M4)
Kanamycin
CPZ-N-S-dioxide (M4)
Streptomycin
CPZ-N-S-dioxide (M4)
Spectinomycin
CPZ-N-S-dioxide (M4)
Rifampicin
Nor-CPZ sulfoxide (M6)
Ethambutol
Nor-CPZ sulfoxide (M6)
Kanamycin
Nor-CPZ sulfoxide (M6)
Streptomycin
Nor-CPZ sulfoxide (M6)
Spectinomycin
Nor-CPZ sulfoxide (M6)
2.53
>1990.89
0.76
>1990.89
1.79
>1990.89
0.29
>1990.89
168.22
>1990.89
2.53
>1900.10
0.76
>1900.10
1.76
>1900.10
0.29
>1900.10
84.12
>1900.10
1.26
>2077.89
0.76
>2077.89
1.79
>2077.89
0.14
NC
AE
0.89
1990.89
0.14
62.21
10.52
1990.89
NC
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475.03
0.14
237.50
42.05
1900.10
NC
1
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1
5
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the investigation of these agents as potential antimycobacterial
drugs.
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the Department of Science and Technology administered
through the National Research Foundation for funding support.
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