Quinolone-isoniazid hybrids: Synthesis and preliminary: In vitro cytotoxicity and anti-tuberculosis evaluation

Article abstract of DOI:10.1039/c8md00480c

Herein, we propose novel quinolones incorporating an INH moiety as potential drug templates against TB. The quinolone-based compounds bearing an INH moiety attached via a hydrazide-hydrazone bond were synthesised and evaluated against Mycobacterium tuberculosis H37Rv (MTB). The compounds were also evaluated for cytotoxicity against HeLa cell lines. These compounds showed significant activity (MIC₉₀) against MTB in the range of 0.2-8 μM without any cytotoxic effects. Compounds 10 (MIC₉₀; 0.9 μM), 11 (MIC₉₀; 0.2 μM), 12 (MIC₉₀; 0.8 μM) and compound 15 (MIC₉₀; 0.8 μM), the most active compounds in this series, demonstrate activities on par with INH and superior to those reported for the fluoroquinolones. The SAR analysis suggests that the nature of substituents at positions -1 and -3 of the quinolone nucleus influences anti-MTB activity. Aqueous solubility evaluation and in vitro metabolic stability of compound 12 highlights favourable drug-like properties for this compound class.

Full text of DOI:10.1039/c8md00480c

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DOI: 10.1039/C8MD00480C
Journal Name
ARTICLE
Quinolone-isoniazid hybrids: Synthesis and preliminary in vitro
cytotoxicity and anti-tuberculosis evaluation
Richard M. Beteck,* Ronnett Seldon , Audrey Jordaan , Digby F. Warner c,d,e, Heinrich C. Hoppe f,g,
a
b
c
Received 00th January 20xx,
Accepted 00th January 20xx
g
h
a,g,i
Dustin Laming , Lesetja J. Legoabe , and Setshaba D. Khanye *
DOI: 10.1039/x0xx00000x
Herein, we propose novel quinolones incorporating an INH moiety as potential drug templates against TB. The quinolone-
based compounds bearing an INH moiety attached via a hydrazide-hydrazone bond were synthesised and evaluated against
Mycobacterium tuberculosis H37Rv (MTB). The compounds were also evaluated for cytotoxicity against HeLa cell lines. These
compounds showed significant activity (MIC90) against MTB in the range of 0.2 – 8 µM without any cytotoxic effects.
Compounds 10 (MIC90; 0.9 µM), 11 (MIC90; 0.2 µM), 12 (MIC90; 0.8 µM) and compound 15 (MIC90; 0.8 µM), the most active
compounds in this series, demonstrate activities on par with INH and superior to those reported for the fluoroquinolones.
The SAR analysis suggests that the nature of substituents at positions -1 and -3 of the quinolone nucleus influences anti-
MTB activity. Aqueous solubility evaluation and in vitro metabolic stability of compound 12 highlights favourable drug-like
properties for this compound class.
www.rsc.org/
recorded in 2017. In the same year, an additional 300 000
9
Introduction
Tuberculosis (TB), an air borne infectious disease, has been
a scourge to the human race for thousands of years. The
people died from TB and HIV co-infections. The recommended
treatment regimen for TB consists of a cocktail of four drugs;
isoniazid (INH), ethambutol, pyrazinamide, and rifampicin,
which must be administered daily at a total dose of
approximately 5 grams for two months followed by isoniazid
and rifampicin daily for another four months. These drugs act
on different targets within MTB. Despite this combination
therapy approach, the present scenario of TB treatment has
been aggravated by the emergence and spread of several forms
of drug-resistant MTB. Three resistance types, classified based
on drug resistance profiles, includes; multi-drug resistant (MDR)
strain, which is resistant to isoniazid and rifampicin but
susceptible to fluoroquinolones, extensively drug resistant
XDR) strain, which is resistant to isoniazid, rifampicin, fluoro-
quinolones and at least one of the three second line injectable
anti-TB drugs (amikacin, kanamycin and capreomycin), and
totally drug resistant (XXDR or TDR) strain, which is resistant to
all anti-TB agents except newly approved agents such as
bedaquiline. In 2017, there were 457,560 cases of MDR-TB
and 6-11% of these cases were XDR-TB. There is low treatment
success rate for drug(s) resistant TB, reported to be 55%
globally. Poor patient adherence, partly due to the cocktail of
drugs and lengthy period required to take them, has been
attributed to the drug resistance crisis. Also suggested to be
implicated in the emergence of resistant strains is the issue of
mis-matched pharmacokinetics(PK) and pharmacodynamic(PD)
properties of the various drugs employed in the first line
regimen, creating a scenario wherein bacilli in different
compartments and/or stages are often not exposed to lethal
1
1
0
2
disease is caused by the bacterium, Mycobacterium tuberculosis
3
(
MTB), with infections resulting in both latent TB and active
1
1
4
pulmonary TB. Approximately 2-3 billion people worldwide
1
2
5
carry the latent form of the disease, and about 10% of this
group will eventually develop active pulmonary TB in their life
6
time. Risk factors associated with conversion of latent to the
1
3
active form of the disease include HIV infection, diabetes,
7
8
smoking, malnutrition, and rheumatoid arthritis. It is
important to note that the latent form of TB is void of signs and
symptoms of the disease.
An estimated 10.0 million people worldwide suffered from
active pulmonary TB, and about 1.3 million deaths were
1
4
(
1
5
a. Faculty of Science, Department of Chemistry, Rhodes University, Grahamstown
1
6
6
140, South Africa.
b.
c.
d.
e.
f.
Drug Discovery and Development Centre (H3-D), Department of Chemistry,
University of Cape Town, Rondebosch 7701, South Africa.
SAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of
Pathology, University of Cape Town, Observatory 7925, South Africa.
Institute of Infectious Diseases and Molecular Medicine, University of Cape Town,
Observatory 7952, South Africa.
Wellcome Centre for Clinical Infectious Diseases Research in Africa, University of
Cape Town, Observatory 7925, South Africa.
Faculty of Science, Department of Biochemistry and Microbiology, Rhodes
University, Grahamstown 6140, South Africa
Centre for Chemico- and Biomedicinal Research, Rhodes University, Grahamstown
6
Centre of Excellence for Pharmaceutical Science, North-West University,
Potchefstroom 2520, South Africa
Faculty of Pharmacy, Rhodes University, Grahamstown 6140, South Africa.
Footnotes relating to the title and/or authors should appear here.
9
17
1
8
g.
h.
140, South Africa.
i.
19
concentrations of some drugs.
†
In addition, the first line treatment regimen is not very safe:
INH and rifampicin- the main drugs in the regimen - have been
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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reported to cause hepatotoxicity and other serious side topoisomerase IV.33 Hong et al demonstrated that
View Article Online
effects.20
DOI: 10.1039/C8MD00480C
incorporating lipophilic units at position -2 of the quinolone ring
The cumbersome first line treatment and the growing leads to compounds that target the QcrB subunit of the
resistance necessitate the adoption and implementation of new cytochrome bc complex in the mycobacterial electron
1
measures to treat TB effectively and to limit the rising transport chain.3 Moreover, another set of quinolone
4
3
5
prevalence of drug-resistant disease. To this effect, several molecules (2), with potent anti-TB activity has been reported.
2
1
strategies, including drug repurposing, derivatization of This set differs structurally from fluoroquinolones and
existing drugs,22 mechanism and structure based designed, quinolones reported by Hong et al. and, as such, may present us
high-throughput screening, all aimed particularly at the with a novel mode of action not yet reported with other
development of new molecules exhibiting unique and novel quinolones. To exploit the hybridisation strategy in TB
2
3
2
4
modes of action, have been exploited. This has culminated in chemotherapy, our approach is to imprint the hydrazide-
new drug candidates (Bedaquiline, Pretomanid, Sutezolid, hydrazone structural motif of compound 1 (Figure 1) onto a
Q203, SQ109, PBTZ169) entering preclinical and clinical reputable anti-biotic class of compound, the quinolone, with
studies.25 Bedaquiline acts on ATP synthase and interferes with intent to generate quinolone-isoniazid hybrid molecules 3 with
energy production leading to death of both dormant and potential antimycobacterial activity against MTB H37Rv strain.
replicating bacilli.26 SQ109 target MmpL3 and prevent the
export of trehalose monomycolate which is required for mycolic identify novel anti-TB agents that interfere with multiple
Considering the above observations and our efforts to
3
5
acid synthesis. This ultimately leads to disruption of cell wall biochemical pathways, a series of quinolone aroylhydrazone
2
7
synthesis. Although novel in their mode of actions, these new conjugates was synthesised based on quinolone
compounds will eventually enter into combinations wherein pharmacophore, and the resulting compounds screened in vitro
mis-matched PK/PD is likely to be an issue. Another strategy against MTB to determine their anti-TB potential. In addition,
worth exploiting is hybridizing chemical motifs with these compounds were also screened against a HeLa cell lines
experimentally proven anti-TB molecules to generate new to investigate potential cytotoxicity.
molecular entities with the hope of interfering with multiple
biochemical pathways within MTB; this might also slow the range of biological properties, which are usually attributed to
emergence of drug-resistance. Compounds containing a their ability to chelate copper or iron. Iron chelators have been
hydrazide-hydrazone structural motif (1, Figure 1) have been shown to retard or completely halt MTB growth. The
Aroylhydrazones have been established to exhibit a wide
2
8
3
6
3
7
reported to exhibit anti-TB activity, as well as activities against quinolone scaffold is a notable compound class that can be
2
9
other infectious diseases like malaria and trypanosomiasis,
chemically tailored to probe different cellular targets within
thereby demonstrating an inherent ability to interfere with MTB. By imprinting the aroylhydrazones fragment of compound
essential biochemical pathways within infectious agents.
1 on to new quinolone structures, we hoped to generate novel
compounds capable of inhibiting the growth of MTB. The aroyl
hydrazine utilised for synthesis of target aroylhydrazones is INH,
O
O
N
H
O
N
R2
9
4
N
a known anti-TB agent. Besides forming a structural motif
O
O
N
OH
capable of eliciting an additional pharmacological response,
functionalisation of the hydrazine unit in INH into
aroylhydrazone was predicted to avoid the fast acetylation of
Molecular Hybridisation
R1
2
1
3
8
INH which has been implicated in its toxicity and, in addition,
incorporates INH in to a molecular framework of higher
lipophilicity. Higher lipohilicity is very much desirable to
N
O
O
H
N
N
R2
3
8,39
O
effectively target MBT within lung granulomas.
N
R1
3
Results and discussion
Chemistry
Figure 1. Structural design strategy. (1) Iron chelating hydrazide-hydrazone containing
compound36, (2) quinolones exhibiting anti-TB activity35, and (3) target hybrid.
Target compounds were achieved following the synthetic
routes presented in Schemes 1 and 2. The p-nitroacetophenone
Compound 1 has been reported to exhibit anti-TB activity by
inhibiting a novel target, methionine aminopeptidase.3
On the other hand, the quinolone nucleus (2, Figure 1) is a
versatile and efficacious anti-TB agent. Previous literature
highlights this compound class as a prominent source of anti-TB
agents that can be tailored via structural modification to
generate novel compounds capable of targeting different
0
(
4) was reduced to p-aminoacetophenone (5) using reduced
iron powder and acetic acid under ultra sound conditions.
Treatment of in acetonitrile with diethyl
3
1
5
ethoxymethylenemalonate under reflux generated condensed
methylenemalonate ester, which was isolated upon in vacuo
evaporation of solvents. Cyclisation of methylenemalonate
esters was carried out in boiling diphenylether at 245-250 C for
five minutes to afford quinolone (6) in 55 % yield (Scheme 1).
biochemical
pathways
within
the
bacillus.
The
fluoroquinolones, exemplified by ciprofloxacin, are an
important second-line treatment option for treating multidrug-
resistant MTB32 and work by inhibiting DNA gyrase and
2
| J. Name., 2012, 00, 1-3
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O
O
O
O
O
O
O
O
required for further functionalisation with aroylhydrazine
(isoniazid). The ethylester functionality in 7 underwent
aminolysis in the presence of respective amines and DBU as a
base under refluxing conditions to generate amide containing
compounds exemplified by 8 and 9 in 40-60 % yields.
View Article Online
DOI: 10.1039/C8MD00480C
a
b
c
O
d
O
NO2
NH2
N
H
6
N
R1
4
5
7
e
R2
O
O
O
O
Treatment of compounds 7 and 8 in ethanol with INH and
catalytic amount of acetic acid under reflux overnight resulted
in compounds 10-18 in 30-70 % yields, the compounds
precipitated out of the reaction mixture and were isolated by
A
B
N
H
H
N
R2
N
H
OH
N
R1
8
-9
; R1= Ph, R2= B
; R1= 2,4-diCl- Ph, R2= A
8
9
1
filtration (Scheme 2). All compounds were confirmed using H
1
3
1
and C NMR, and HRMS. The H NMR spectra of all compounds
Scheme 1 aReagents and conditions: (a) Reduced Fe powder, AcOH; (b) Acetonitrile, show a singlet at ca 2.5 ppm, which is assigned to the -CH - of
3
diethyl ethoxylmethylenemalonate, reflux 12 h; (c) Diphenyl ether, 245 - 250 °C, 5 mins;
methylketone or methylimine unit. Apart from compounds 10
(
d) K
CHCl
2
CO
3 3
, CHCl /THF (2:1), alkylhalide (1.2 eqv), 12 h; (e) Amine (5 eqv), DBU (1.2 eqv),
1
and 11, having an ethylester at position -3, the H NMR of all
3
, reflux 12 h.
compounds 10-18 show a singlet at ca 11 ppm on their
1
respective H NMR spectra, and this signal suggests the
The temperatures and reaction times needed to be carefully
successful formation of hydrazone-hydrazide bond (-C(=O)NH-
N-).
controlled during the cyclisation process. We observed that
cyclisation failed to occur at temperatures below 240 C, and
temperatures above 255 C led to decarboxylation of the ester
group. Also heating at 245-250 C for more than five minutes
produced a mixture of two compounds; one having the ester
group intact and a decarboxylated compound. Treatment of
Comparing the 13C NMR spectra of compounds 8 and 9
against compounds 10-18 further confirmed this observation.
1
3
The C NMR spectra of compounds 8 and 9 showed a peak at
ca 197 ppm which was assigned to the -C=O unit of the
methylketone, and its disappearance and the appearance of a
new peak at ca 111 ppm in compounds 10-18 further confirmed
the transformation of a carbonyl unit into an imine. The purity
of all compounds was determined to be greater than 95 % using
preparative HPLC.
2 3
compound 6 with K CO and alkyl/arylhalides under reflux
overnight resulted in N-alkylation of the secondary amine. After
reaction completion as indicated by TLC, the reaction mixture
was filtered and the filtrate evaporated to dryness in vacuo and
later washed with petroleum ether to afford intermediate 7 (in
5
0 % yield), the quinolone fragment containing a ketone
O
O
O
N
O
O
H
R2
N
a
N
R2
N
O
N
R1
R1
8
-9
1
2-18
O
1
2: R = Ethyl, R = A
1 2
O
O
O
NH2
13: R = Ethyl, R = C
N
1 2
O
H
14: R = Ph, R = B
1 2
N
Isoniazid
15: R = 4-Br-Ph, R = B
1 2
N
1
1
1
6: R = 4-Br-Ph, R = D
1 2
R1
7: R = 4-CF -Ph, R = B
1
3
2
b
7
8: R = 2,4-diCl-Ph, R = A
1
2
R
2
O
A
N
H
H
N
N
O
N
O
H
N
OH
N
B
C
H
N
O
N
H
N
O
N
D
R1
O
N
H
OH
10-11
1
1
0: R = Methyl
1
1: R = Ethyl
1
a
Scheme 2 Reagents and conditions: (a) and (b) Isoniazid, AcOH (cat), EtOH, reflux overnight.
Evaluation of cytotoxicity
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Target compounds were screened in vitro against human within the bacterium, to this effect, our library waVsiedweAsritgicnleeOdnlitnoe
cervix adenocarcinoma (HeLa) cell lines to investigate potential incorporate target compounds bearing dDifOfeI:r1e0n.1t0s3u9/bCs8tMitDu0e0n4t8s0aCt
cytotoxicity effects at 20 µM. Emetine (which induces cell positions -1 and -3 of the quinolone ring. SAR analysis of this
apoptosis) was used as positive control drug and yielded an IC50 series suggested that an ethylester at position -3 of the
value of 0.015 µM. Compounds exhibiting ˂50 % cell viability quinolone nucleus promoted activity better than amides, this
were considered to pose cytotoxicity risk, while compounds was evident when comparing compound 11 (MIC90; 0.2 µM)
exhibiting ˃50 % cell viability were assumed to have IC50 values bearing an ethylester with compound 12 (MIC90; 0.8 µM) and
≥
20 µM and as such pose little cytotoxicity risk. None of the compound 13 (MIC90; 7.8 µM)- both having an amide at position
compounds in this study reduced HeLa cell viability to below -3 of the quinolone ring. Furthermore, comparing compounds
5% during a 24-hour incubation (Figure 2), which suggested 12 (MIC90; 0.8 µM) and 18 (MIC90; 4.8 µM), which differ only on
they possessed little overt cytotoxicity risk at the the nature of substituent at position -1 of the quinolone ring,
7
concentrations used for anti-MTB evaluation.
suggested that the ethyl unit at this position was favoured for
anti-TB activity compared to a phenyl ring. It is, however,
important to note that compound 15 which bears a substituted
phenyl ring and an amide at position -1 and -3, respectively of
the quinolone ring demonstrated potent activity (MIC90; 0.8
µM) comparable to that of INH and superior to that reported
for most fluoroquinolones. The substitution pattern on the
phenyl ring also seemed to greatly influence activity, with para
bromo substituents giving the best activity. This was evident
when comparing the structure and activity of 15 (MIC90; 0.8 µM)
against compounds 14 (MIC90; 2.0 µM) and 17 (MIC90; 1.8 µM).
Table 1 In vitro antimycobacterial activity, cytotoxicity and calculated clogP of target
compounds in comparison with standard drugs.
MIC90 (µM)
Compound
clogPa
Figure 2 Percentage HeLa cell viability after incubation with test compounds at 20
µM for 24 hours. The values are the mean  SD of experiments performed in duplicate,
and none of the compounds showed any cytotoxicity effects. The SD values were
calculated using the expression .40
H37Rv
8
9
43.4
125
0.9
0.5
2.6
0.8
1
0
Antitubercular activity
against Mtb H37Rv
1
1
1
1
1
2
3
4
0.2
0.8
7.8
2.0
1.3
0.1
0.2
0.5
Target compounds 10-18 and quinolone fragments 8 and 9
were evaluated for antimycobacterial activity in vitro. The
compounds were screened in a broth microdilution assay
against Mycobacterium tuberculosis H37Rv, drug-susceptible
laboratory strain, with rifampicin and INH included as
standards. The antimycobacterial activities are reported as
minimum inhibitory concentration (MIC90) required to inhibit
1
5
0.8
1.3
1
1
1
6
7
8
2.7
1.8
4.8
1.5
1.1
2.6
9
0% of mycobacterial growth. Antimycobacterial activities of
INH
Rif
0.2
-0.8
ND
target compounds are summarised in Table 1. All tested
compounds exhibited antimycobacterial activity, with most of
the compounds exhibiting potent activity, having MIC90 values
less 3 µM. This activity profile is comparable to those of INH and
fluoroquinolones currently deployed in the treatment of TB.
As has been noted earlier that the nature of substituents
around the quinolone ring determines activity as well as target
0.06
aClogP values are calculated from ACD Chemsketch freeware version
2.0. INH = Isonazid, Rif = Rifampicin, ND = not determined,
1
4
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DOI: 10.1039/C8MD00480C
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ARTICLE
Table 2 Aqueous solubility (S
Compound
w
) and in vitro metabolic stability evaluation of compound 12 in human, rat and mouse liver microsomes.
aHLM/RLM/MLM
bS
(µM)
w
MIC90 (µM)
pH 6.5
% remaining
CLint μg/min/mg protein
t1/2 (min)
c
EH
Table 3
0
.8
95
90.6/87/77
13.3/18.7/35.3
>150/>150/79.7
<0.42/<0.3/<0.49
a
b
c
HLM/RLM/MLM are human, rat and mouse liver microsomes. Aqueous solubility estimated by nephelometry. Predicted hepatic extraction (E
H
) ratio based on in vitro
intrinsic clearance (CLint).
Comparison of the structure and activity of compound 8 advantage over INH in that they have higher lipophilicity and
(MIC90; 43.4 µM) against the structure and activity of compound lack the free hydrazine unit – the feature to which INH toxicity
1
4 (MIC90; 2.0 µM) suggested that anti-TB activity increased has been attributed. In summary, these observations support
significantly with the incorporation of the hydrazone-hydrazide the design strategy adopted and suggest that the approach is
unit. This was also evident when comparing compound 9 (MIC90
;
worth further exploitation in search of more potent and safe
1
25 µM) against compound 18 (MIC90; 4.8 µM). Comparing anti-TB agents.
compounds 12 (MIC90; 0.8 µM) and 13 (MIC90; 7.8 µM)
suggested that the nature of the amide moiety at position -3
greatly affected anti-TB activity, an observation also evident
Conflicts of interest
when comparing compounds 15 (MIC90; 0.8 µM) and 16 (MIC90
;
There are no conflicts to declare.
2
.7 µM), which differ only in their amide moiety at position -3.
Aqueous solubility and in vitro microsomal stability
Acknowledgements
Compound 12 having an MIC90 value of 0.8 µM and clogP of
The authors acknowledge the financial support of the
National Research Foundation of South Africa (SDK), Rhodes
University for a Postdoctoral Fellowship and RC grant (RMB) as
well as Rhodes University Sandisa Imbewu (SDK, HCH) towards
this research. The cytotoxicity component of the project was
funded by the South African Medical Research Council (SAMRC)
with funds from National Treasury under its Economic
Competitiveness and Support Package awarded to HCH. All TB
screening work was conducted in the MMRU (UCT) with the
support of the SAMRC through the Strategic Health Innovation
Partnerships (SHIP) initiative (to DFW). Aqueous solubility
studies and in vitro metabolic stability studies were performed
at H3D, University of Cape Town.
0
.1 was selected for aqueous solubility evaluation in phosphate
buffered saline (PBS) at pH 6.5. The compound showed
moderate aqueous solubility and was then subjected to
metabolic turnover stability studies in human, rat, and mouse
liver microsomes (Table 2). The data suggested that compound
1
2 was stable across the three species, with more than 70% of
the compounds remaining after 80 – 150 minutes (t1/2 ˃150
min). This good stability profile was further confirmed by a low
hepatic extraction (E ) ratio of ˂0.5 across the three species.
H
Conclusions
This work presents novel quinolones incorporating an INH
moiety through a hydrazide-hydrazone functional unit. Target
compounds were realised through a simple hybridisation
approach and were tested against the HeLa cell line and H37Rv
strain of MTB to evaluate for cytotoxicity and anti-TB potentials,
respectively. While none of the compounds showed a potential
cytotoxicity risk, they all exhibited potent anti-TB activity (MIC90
in the range of 0.2 - 8 µM). An ethylester at position -3 seems to
promote activity better than amides, while an aliphatic unit at
position -1 seems to favour anti-TB activity compared to having
a benzyl unit at this position. Besides having an in vitro activity
profile comparable to INH, our hit compounds present an
Notes and references
1
2
3
4
5
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DOI: 10.1039/C8MD00480C
Graphical Abstract
O
O
O
N
O
N
O
O
O
NH2
H
N
cat. AcOH
O
N
H
N
N
EtOH, reflux
O
N
INH
7
1
1
MIC90: 0.2 M
cLogP: -0.89
MIC90: 0.2 M
cLogP: 1.36
A new series of quinolone-isoniazid hybrid compounds were designed, synthesised and studied for
their potential anti-mycobacterial tuberculosis activity in vitro.