Design, synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I, II and IX inhibitors in 5-nitroimidazole series

Article abstract of DOI:10.1080/14756366.2019.1685510

With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6 nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2 nM) and hCA IX (KI = 147.3 nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4 nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.

Full text of DOI:10.1080/14756366.2019.1685510

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Design, synthesis, in vitro inhibition and
toxicological evaluation of human carbonic
anhydrases I, II and IX inhibitors in 5-
nitroimidazole series
Ashok Aspatwar, Nanda Kumar Parvathaneni, Harlan Barker, Emilie
Anduran, Claudiu T. Supuran, Ludwig Dubois, Philippe Lambin, Seppo
Parkkila & Jean-Yves Winum
To cite this article: Ashok Aspatwar, Nanda Kumar Parvathaneni, Harlan Barker, Emilie Anduran,
Claudiu T. Supuran, Ludwig Dubois, Philippe Lambin, Seppo Parkkila & Jean-Yves Winum (2020)
Design, synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I, II
and IX inhibitors in 5-nitroimidazole series, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 109-117, DOI: 10.1080/14756366.2019.1685510
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 109–117
https://doi.org/10.1080/14756366.2019.1685510
RESEARCH PAPER
Design, synthesis, in vitro inhibition and toxicological evaluation of human
carbonic anhydrases I, II and IX inhibitors in 5-nitroimidazole series
a
a
, Nanda Kumar Parvathanenib,c , Harlan Barker , Emilie Anduranb,c, Claudiu T. Supurand
,
ꢀ
ꢀ
Ashok Aspatwar
Ludwig Duboisb , Philippe Lambinb, Seppo Parkkilaa,e
and Jean-Yves Winumc
b
aFaculty of Medicine and Health Technology, Tampere University, Tampere, Finland; Department of Precision Medicine, The M-Lab, GROW –
School for Oncology and Developmental Biology, Maastricht Comprehensive Cancer Centre, Maastricht University Medical Centre, Maastricht,
c
ꢀ
ꢀ
^
The Netherlands; Institut des Biomolecules, Max Mousseron (IBMM) UMR 5247 CNRS, ENSCM, Universite de Montpellier, Batiment de
d
ꢀ
Recherche Max Mousseron, Ecole Nationale Superieure de Montpellier, Montpellier, France; NEUROFARBA Department, Section of
Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico, Firenze, Italy; Fimlab Ltd and Tays Cancer Center, Tampere
e
University Hospital, Tampere, Finland
ABSTRACT
ARTICLE HISTORY
Received 27 September 2019
Revised 22 October 2019
Accepted 22 October 2019
With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhy-
drases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and
studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the tox-
icity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed effi-
cient inhibition against hCA II (KI ¼ 58.6 nM). Compound 10 showed moderate inhibition against hCA II
(KI ¼ 199.2 nM) and hCA IX (KI ¼ 147.3 nM), whereas it inhibited hCA I less weakly at micromolar concen-
trations (KI ¼ 6428.4 nM). All other inhibition constants for these compounds were in the submicromolar
range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study sug-
gests that these compounds are suitable for further preclinical characterisation as potential inhibitors of
hCA I, II and IX.
KEYWORDS
Carbonic anhydrases;
carbonic anhydrase
inhibitors; synthesis; toxicity
evaluation; lethal
concentration; zebra-
fish embryos
Introduction
and II proteins have been linked to retinal and cerebral oedema,
glaucoma, epilepsy and altitude sickness2,4. hCA IX, a transmem-
brane CA isoform, is overexpressed under hypoxic conditions in
Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, present
in most of the living organisms and encoded by seven evolutionarily
unrelated gene families: the a-.b-.c-.d-.f-.g-. and h-CAs1. The CAs
catalyse the basic reaction of reversible hydration of carbon dioxide
to bicarbonate and proton2. This reaction is fundamental for bio-
chemical processes in all living organisms1,3. Invertebrates and
lower organisms contain all the seven members of CA gene families
and, in some cases, multiple gene families with in the same organ-
ism1. In contrast, vertebrates and mammals contain only a-CAs1. In
humans, the CAs regulate a broad range of physiological processes,
such as respiration, transport of carbon dioxide/bicarbonate
between the lung and metabolising tissues, pH homeostasis, and
electrolyte secretion in many tissues and organs2. There are 15 CA
solid tumours and is involved in the progression of cancer8,9
.
Many CA inhibitors have been used for treating various human
diseases2. Even if the inhibitors achieve desirable therapeutic
effects, they may cause unacceptable level of toxicity in
humans10,11, most probably due to the off-target effects attribut-
able to the inhibition of CA isoforms that are not involved in the
target disease or due to general toxic effects of the chemical12,13
.
Therefore, to target particular therapeutic areas with minimal off-
target effects, inhibition of specific enzymes is necessary. Thus,
there is a particular need to develop inhibitors that are not only
selective against the human CAs but are also safe for clin-
isoforms in humans of which 12 are catalytically active. Three are ical use14.
inactive due to the lack of one or more of the three-histidine resi-
Along this line, we have previously developed numerous
dues required for enzymatic activity2–3. The human CA isoforms dif- potent CA inhibitors that are not only effective against specific
fer from one another in cellular localisation, distribution in organs, human a-CAs and mycobacterial b-CAs, but also show minimal
levels of expression in tissues and their kinetic properties4–5. The toxicity and are suitable for developing further for clinical use as
human CA isoforms have high sequence and structural similarities, drugs14–16. Recently, we reported sulphonamide and carbamate
compounds as hCA IX and mycobacterial b-CA inhibitors14–16. In
and therefore only subtle differences exist in their active sites6–7
.
In humans, increased expression of CA isoforms is sometimes addition to the synthesis and in vitro and in vivo evaluation of
associated with certain disease conditions2. The cytosolic hCA I these compounds, we also tested them for safety and toxicity
ꢀ ꢀ
Institut des Biomolecules, Max Mousseron (IBMM) UMR 5247 CNRS, ENSCM, Universite
CONTACT Jean-Yves Winum
jean-yves.winum@umontpellier.fr
^
ꢀ
de Montpellier, Batiment de Recherche Max Mousseron, Ecole Nationale Superieure de Montpellier, Montpellier, France; Ashok Aspatwar
Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland
ashok.aspatwar@tuni.fi
ꢀ
These authors contributed equally to this work.
Supplemental data for this article can be accessed here.
ß 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

110
A. ASPATWAR ET AL.
using 1–5 d post-fertilisation (dpf) zebrafish larvae. Our studies with water. The organic layer was dried over anhydrous magne-
showed that these inhibitors are safe for further preclinical charac- sium sulphate and concentrated under vacuum. The residue was
terisation using in vivo models14–16
.
purified by chromatography on silica gel using methylene chlor-
In addition, in this study, we were interested in incorporating a ide-methanol 9:1 as eluent. Yield: 43%, 1H NMR (400 MHz, DMSO-
nitro-imidazole moiety as a privileged hypoxia sensitising scaffold d6) d 9.35 (s, 1H), 8.03 (s, 1H), 7.51 (s, 2H), 7.11 (d, J ¼ 9.1, 4H),
in the structure of CA inhibitors. The rationale was to investigate a 7.01 (s, 2H), 4.41 (s, 2H), 3.81 (s, 2H), 2.68 (s, 2H), 2.36 (s, 3H). 13C
strategy of dual targeting (hypoxia and hCA IXn/hCA XII) in the NMR (101 MHz, DMSO-d6)
d 151.98, 139.08, 138.13, 133.70,
133.45–133.11, 129.40, 118.95, 55.38, 45.93, 14.30. MS (ESIþ) m/z
428.12 [M þ H]þ.
context of anticancer agents17. Previous results have shown the
validity of this approach when conjugation of 5-nitroimidazole
derivatives to CA inhibitors structures led to new radiosensitiser
agents targeting hypoxic tumours17–20. To further investigate the
effect and the influence of the 5-nitro imidazole moiety and
develop new potent and safer inhibitors, we have been continu-
ing our research on new inhibitors in the nitroimidazole series. In
the present study, we report the design, synthesis, in vitro CA
inhibition, and evaluation of toxicity of novel thiourea derivatives
3 and 4 and a sulphonamide derivative 10 using zebrafish larvae.
4-(2-(3-(2–(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl) thioureido)e-
thyl)phenyl sulphamate (4): The same protocol as for the synthesis
of compound 3 starting from 2. Yield: 60%, 1H NMR (400 MHz,
DMSO-d6) d 8.03 (s, 1H), 7.95 (s, 2H), 7.58 (d, J ¼ 38.2, 2H), 7.29 (d,
J ¼ 8.5, 2H), 7.20 (d, J ¼ 8.5, 2H), 4.42 (s, 2H), 3.82 (s, 2H), 2.77 (s,
2H), 2.36 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 151.44, 148.56,
138.56, 137.61, 133.16, 129.77, 121.97, 45.39, 30.63, 13.75. MS
(ESIþ) m/z 429.10 [M þ H]þ.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl) ethyl methanesulphonate
(6): Methane sulphonylchloride (1.1 equiv.) was added dropwise to
a stirred solution of metronidazole, 5 (1 equiv.), triethylamine (2
equiv.) and DMAP (1.1 equiv.) in anhydrous methylene chloride at
0 ꢁC. The reaction mixture was allowed to warm to room tempera-
ture and stirred for overnight. The reaction mixture was diluted
with methylene chloride and washed with water and brine. The
organic layer was dried over anhydrous sodium sulphate, filtered
and evaporated to get crude compound which was used further
without purification. Yield: quantitative. 1H NMR (DMSO-d6,
400 MHz) d 8.05 (s, 1H), 4.61 (t, J ¼ 7.3, 2H), 3.51 (t, J ¼ 7.3, 2H),
2.50 (s, 3H); 13C NMR (DMSO-d6, 101 MHz) d 151.08, 138.18,
133.14, 47.09 (s, 1H), 14.09, 2.28. MS (ESIþ) m/z 281.97 [M þ H]þ.
S-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl) ethanethioate (7):
Potassium thioacetate (2 equiv.) was added to a solution of 6 (1
equiv.) in anhydrous DMF and stirred at room temperature for
16 h. The reaction mixture was then diluted with ethyl acetate
and washed with water and brine. The organic layer was dried
over anhydrous sodium sulphate, filtered and concentrated under
vacuum to give thioacetate 7. Yield: 83%, 1H NMR (400 MHz,
DMSO-d6) d 8.01 (s, 1H), 4.45 (t, J ¼ 6.6, 2H), 3.27 (t, J ¼ 6.6, 2H),
2.46 (s, 3H), 2.29 (s, 3H). 13C NMR (101 MHz, DMSO) d 194.59,
151.25, 138.42, 132.92, 44.68, 30.32, 27.87. MS (ESIþ) m/z
230.06 [M þ H]þ.
Materials and methods
Chemistry
All reagents and solvents were of commercial quality and used
without further purification unless otherwise specified. All reac-
tions were carried out under an inert atmosphere of nitrogen. TLC
analyses were performed on silica gel 60 F254 plates (Merck Art.
no. 1.05554). Spots were visualised under 254 nm UV illumination
or by ninhydrin solution spraying. Melting points were determined
on a Buchi Melting Point 510 and are uncorrected. 1H and 13C
€
NMR spectra were recorded on Bruker DRX-400 spectrometer
using DMSO-d6 as solvent and tetramethylsilane as internal stand-
1
ard. For H NMR spectra, chemical shifts are expressed in d (ppm)
downfield from tetramethylsilane and coupling constants (J) are
expressed in Hertz. Electron ionisation mass spectra were recorded
in positive or negative mode on a Water MicroMass ZQ. All com-
pounds that were tested against purified physiological isoforms of
CAs were analysed by high-resolution ESI mass spectra (HRMS)
using on a Q-ToFI mass spectrometer fitted with an electrospray
ion source to confirm the purity of >95%.
1-(2-Isothiocyanatoethyl)-2-methyl-5-nitro-1H-imidazole
(2):
Thiophosgene (1 equiv.) and sodium hydroxide (2 equiv.) were
added to a solution of 1 (0.5 equiv.) in chloroform and water (4:1)
at 0 ꢁC, and reaction mixture was warmed to room temperature.
Thin layer Chromatography confirmed complete disappearance of
the starting material. Reaction mixture was then washed with
water and extracted with DCM. The organic layer was dried over
anhydrous sodium sulphate, filtered and evaporated to get crude
compound which was purified by chromatography on silica gel
using ethyl acetate and petroleum ether as eluent with a gradient
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethane-1-sulphonyl chloride
(9): A solution of 7 (1 equiv.) was treated with hydrogen peroxide
[35% w/w in water (6 ml)] and acetic acid (7 ml). The reaction mix-
ture was allowed to stir for 24 h at room temperature; the excess
hydrogen peroxide was quenched by addition of 10% Pd/C
(0.050 g). The mixture was filtered on celite, concentrated, and co-
evaporated with toluene. The obtained sulphonic acid was used
further without purification. The crude 2-(2-methyl-5-nitro-1H-imi-
dazol-1-yl) ethane-1-sulphonic acid 8 (1 equiv.) was suspended in
anhydrous DCM and a solution of phosgene in toluene (20% wt.,
3.5 ml) and dry DMF (1 ml) was added under argon atmosphere.
After 1 h an additional 1 ml phosgene solution was added, and
the mixture was stirred for 2 h under same conditions. After pre-
cipitation, the reaction mixture was filtered to get pure compound
1
from 5:5 to 9:1. Yield: 64%, H NMR (400 MHz, DMSO-d6) d 8.10 (s,
1H), 4.65–4.60 (m, 2H), 4.17–4.12 (m, 2H), 3.35 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) d 151.80, 138.80, 133.65, 129.62, 45.06, 14.28.
MS (ESIþ) m/z 213.04 [M þ H]þ.
4-(2-(3-(2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl)thioureido)e-
thyl)phenyl sulphamide (3): 4-(2-aminoethyl) aniline (1 equiv.) was
added to a solution of 2 (1 equiv.) in acetonitrile and allowed to
stir at room temperature overnight. Reaction mixture was washed
with water and extracted with ethyl acetate. The organic layer
was dried over anhydrous sodium sulphate, filtered and evapo-
rated under vacuum to get crude compound which was then
1
9 as solid. Yield: 66%, H NMR (400 MHz, DMSO-d6) d 8.80 (s, 1H),
4.63 (t, J ¼ 6.3, 2H), 2.92 (t, J ¼ 6.3, 2H), 2.78 (s, 3H), 13C NMR
(101 MHz, DMSO-d6) d 149.90, 138.05, 123.91, 48.67, 44.17, 12.13.
MS (ESIþ) m/z 236.00 [M þ H]þ.
2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethane-1-sulphonamide (10):
reacted as-is with sulphamoyl chloride (3 equiv.) in dimethylaceta- To a solution of compound 9 (1 equiv.) in toluene was added at
mide (DMA). After stirring for one night at room temperature, the room temperature 0.5 M solution of liq. ammonia in 1,4-dioxane
mixture was diluted with ethyl acetate, and washed three times (2 ml). After 1 h, the reaction mixture was filtered to afford pure

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
111
compound as white solid. Yield: 68%, 1H NMR (400 MHz, DMSO- Ethical statement
d6) d 8.03 (s, 1H), 7.39 (s, 2H), 4.63 (t, J ¼ 7.0, 2H), 3.49 (t, J ¼ 7.0,
2H), 2.49 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 151.43, 138.36,
133.04, 53.08, 40.85, 13.94. MS (ESIþ) m/z 235.05 [M þ H]þ.
Tampere University has an established zebrafish core facility
authorisation granted by the National Animal Experiment Board
(ESAVI/7975/04.10.05/2016). The experiments using developing
zebrafish embryos were performed according to the Provincial
Government of Eastern Finland Province Social and Health
Department Tampere Regional Service Unit protocol # LSLH-
2007–7254/Ym-23. Care was taken to ameliorate suffering by
euthanizing the 5 dpf larvae by prolonged immersion in a petri
dish containing an overdose of Tricaine (Sigma-Aldrich, St. Louis,
MO) before fixing in buffered formaldehyde for histochem-
ical analysis.
In vitro inhibition studies
An Applied Photophysics stopped-flow instrument was used for
assaying the CA-catalyzed CO2 hydration activity21. Phenol red (at
a concentration of 0.2 mM) was used as an indicator, working at
the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5)
as buffer and 20 mM Na2SO4 (for maintaining the ionic strength
constant), following the initial rates of the CA-catalysed CO2
hydration reaction for a period of 10–100 s. The CO2 concentra-
tions ranged from 1.7 to 17 mM for the determination of the kin-
etic parameters and inhibition constants. For each inhibitor, at
least six traces of the initial 5–10% of the reaction were used for
determining the initial velocity. The uncatalysed rates were deter-
mined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (0.1 mM) were pre-
pared in distilled-deionized water, and dilutions up to 0.01 nM
were done thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were pre-incubated together for 15 min at room
temperature prior to assay to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and represent the average
from at least three different determinations. CA isoforms were
recombinant ones obtained in house as reported earlier.
Determination of LC50
The LC50 values of the compounds 3, 4 and 10 were determined
using 1-dpf embryos exposed to 10–12 different inhibitor concen-
trations. For each concentration of the inhibitor, we used 30 1dpf
larvae13,22. In each group, the larvae were exposed to different
concentrations of the inhibitors that ranged from 12.5 mM to
3 mM. The dose response curve (DRC) was calculated using DRM
of the DRC R package23. The control group was constituted of an
equal number of larvae not treated with any chemical and the lar-
vae that were treated with 1% of DMSO. Toxicological evaluation
studies were performed in 24-well plates (Corning V R Co-star V R
cell culture plates). In each well, we placed 1–2 1-dpf embryos in
1 ml of embryonic medium containing a diluted inhibitor. For con-
trol groups, 1% DMSO diluted in either embryonic medium or
embryonic medium with no chemical compound. A minimum of
three sets of experiments were carried out for each inhibitor. The
mortality of the larvae was checked every 24 h until 5 d after
exposure to the inhibitors.
Toxicity evaluation
Inhibitors
The three compounds 3, 4 and 10 were designed and synthesised
with the purpose of developing them as inhibitors of human CAs.
Phenotypic analysis of control and inhibitor treated larvae
Subsequently, all the three compounds were investigated in vitro We assessed the effect of inhibitors on the zebrafish larvae after
5 d of exposure to the drugs; we analysed eight observable
phenotypic parameters: (1) mortality, (2) hatching, (3) oedema, (4)
movement pattern, (5) yolk sack utilisation, (6) heartbeat, (7) body
shape and (8) swim bladder development, using a stereo micro-
scope and recorded the observations for each inhibitor treated
and control groups. The images of the developing larvae were
taken using a Lumar V1.12 fluorescence microscope attached to a
as inhibitors of human CA I, CA II and CA IX. These compounds
were dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, St.
Louis, MO) to prepare 100 mM stock solutions. Before start of each
experiment, the series of dilatation were made from the above
stock in an embryonic medium [5.0 mM NaCl, 0.17 mM KCl,
0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% w/v Methylene Blue
(Sigma-Aldrich, Darmstadt, Germany)]. Embryos after 1-day post
fertilisation were exposed the each of the diluted inhibi-
tor solution.
€
camera with a 1.5 lens (Carl Zeiss MicroImaging GmbH, Gottingen,
Germany). The images were analysed with AxioVision software
versions 4.7 and 4.8 as described in our standard protocol for
assessment of toxicity and safety of the chemical compounds22.
Maintenance of zebrafish
AB strains of wild-type adult zebrafish were maintained at 28.5 ꢁC
in an incubator15. For collecting embryos, 3–5 pairs of male and
female fish were set up overnight for breeding22. The next morn-
ing, from the overnight breeding tanks 1–2-h post fertilisation
(hpf) embryos were collected in a sieve and rinsed with embryonic
medium [5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM
MgSO4, and 0.1% w/v Methylene Blue (Sigma-Aldrich, Darmstadt,
Germany)] and kept the collected embryos in an incubator at
28.5 ꢁC overnight. The toxicity evaluation studies of the inhibitors
were performed using the fish that were 24 hpf. All the zebrafish
experiments were performed at the zebrafish core facility,
Tampere University, Finland and according to the protocol used in
our laboratory22.
Swim pattern analysis
The movement of the zebrafish larvae was tracked from day 4 of
exposure to these inhibitors. Similarly, detailed analysis of the
swim pattern of the larvae was performed at the end of day 5
after exposure to the inhibitors. For analyses of movement pat-
tern, about 10–15 the zebrafish larvae were placed in a 35 mm ꢂ
15 mm petri dish containing embryonic medium and the larvae
were allowed to settle in the petri dish for 1 min. The movement
of the zebrafish larvae was observed under the microscope for
1 min. A short video of about 1 min was taken for each group of
larvae that were treated with 125 mM concentration. The swim pat-
terns were compared with the control group zebrafish larvae that
were not treated with any inhibitor.

112
A. ASPATWAR ET AL.
Scheme 1. Synthesis of thioureas 3 and 4. Reagents and conditions: (i) thiophosgene, NaOH, CHCl3, 0 ꢁC-rt; (ii) 4-(2-aminoethyl) aniline, CH3CN, rt; (iii) NH2SO2Cl, DMA,
rt; (iv) 4-(2-aminoethyl) phenol, CH3CN, rt; (v) NH2SO2Cl, DMA, rt.
Histological studies
then sulphamoylated with sulphamoyl chloride to give, respect-
ively, sulphamide 3 and sulphamate 4 (Scheme 1)24,25
.
The histological analyses were done to check the effect of inhibi-
tors on the internal tissues of the larvae that were treated with
different concentrations of the inhibitors used in the study and
the control group larvae not treated with any inhibitor and the
larvae treated with 1% DMSO. After 5 d of exposure to different
concentration of the inhibitors, the larvae were washed with phos-
phate buffered saline (PBS) immersed in excess amounts of
Tricaine to anaesthetize the larvae. The larvae thus prepared were
transferred to a 1.5 ml microcentrifuge tube and fixed in buffered
formaldehyde (formaldehyde solution 4%, pH 6.9) in PBS for 3 h at
room temperature or overnight at 4 ꢁC. After the fixation, the lar-
vae were transferred to 70% ethanol and stored at 4 ꢁC before
being embedded in paraffin. The paraffin-embedded samples
were sectioned into 5 mM thin slices for the histochemical staining.
The fixed sections containing samples were deparaffinised in
xylene, rehydrated in an alcohol series, and stained with Mayer’s
Haematoxylin and Eosin Y (both from Sigma-Aldrich, Darmstadt,
Germany). After dehydration, the slides were mounted with
Compound 10 was prepared in a five-step synthesis starting
from metronidazole 5. Mesylation of 5 using methane sulphonyl
chloride under basic conditions led to compound 6. Reactions
with potassium thioacetate followed by oxidation and treatment
with phosgene allowed the formation of sulphonyl chloride 9.
Finally, compound 10 was obtained by treatment with a solution
ammonia in 1,4-dioxane at room temperature (Scheme 2)26.
CA inhibition assay
All compounds reported here were assayed as inhibitors of three
physiologically relevant CA isoforms, the cytosolic hCA I and II and
the transmembrane, tumour-associated hCA IX using the CO2
hydrase assay (Table 1)17. The clinically employed sulphonamide,
acetazolamide (AAZ, 5-acetamido-1,3,4- thiadiazole-2-sulphona-
mide), has been used as a standard in these measurements.
Compound 10 showed very weak inhibition potency against
the abundant cytosolic hCA I with a KI value of 6428 nM. The thio-
urea derivatives 3 and 4 demonstrated sub-micromolar inhibition
potency against all CA isoforms. Compound 4 showed the most
efficient binding to hCA II (KI ¼ 58.6 nM) with a selectivity ratio of
0.09 for inhibiting hCA II over hCA IX, explained by the influence
of the substitution on the benzene ring. Compound 10, the sul-
phonamide derivative of the sulphamide and sulphamate ana-
logues previously reported and biologically evaluated by our
group24, showed moderate binding efficacy towards hCA II (KI ¼
199.2 nM) and hCA IX (KI ¼ 147.3 nM). The better binding efficacy
of sulphonamide 10 compared to the sulphamide and sulphamate
compounds might be explained by higher stability under acidic
conditions, i.e. sulphonamide > sulphamide > sulphamate.
R
EntellanV Neu (Merck; Darmstadt, Germany). The slides containing
the tissues were examined for morphological changes in the tis-
sues of the larvae exposed to the drugs and photographed using
a Nikon Microphot microscope (Nikon Microphot- FXA, Tokyo,
Japan). All the procedures were carried out at room temperature
unless stated otherwise.
Statistical analysis
The GraphPad Prism software (5.02) (GraphPad Software, La Jolla,
CA) was used to perform some of the statistical analyses. For stat-
istical analysis of the toxicity parameters, a two-tailed Fisher’s test
was used and p values below 0.05 were considered significant.
Evaluation of safety and toxicity of compounds 3, 4
and 10
Results and discussion
Chemistry
Determination of inhibitor half maximal lethal
concentration (LC50
)
Thiourea derivatives 3 and 4 were prepared starting from the
amino analogue of metronidazole 1 which was converted to iso-
The lethal effects of all three inhibitor compounds were concen-
tration dependent on the developing zebrafish embryos that were
exposed to the inhibitor for 5 d (Figure 1). None of the three
thiocyanate
2 using thiophosgene under basic conditions.
Reaction either with 4-(2-aminoethyl) aniline or 4-(2-aminoethyl)
phenol in acetonitrile led to the thiourea-derivatives which were inhibitors exhibited any significant mortality in up to 1.5 mM

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
113
Scheme 2. Synthesis of sulphonamide 10. Reagents and conditions: (i) CH3SO2Cl, NEt3, DMAP, DCM, 0 ꢁC – rt; (ii) KSAc, DMF, rt; (iii) 35% H2O2-CH3COOH, rt; (iv) phos-
gene, cat. dry DMF, dry DCM, rt, (v) NH3 in 1,4 dioxane, benzene, rt.
mycobacterial b-CA inhibitors showed significant effects on larval
Table 1. Inhibition data of compounds 3, 4 and 10 against hCA I, hCA II and
mortality and observable phenotypic parameters in the concentra-
hCA IX using a stopped flow CO2 hydrase assay.
tion range of 50–300 mM14,16. Therefore, our present results show
KI (nM)a
Selectivity ratios
that 3, 4 and 10 are less toxic in the zebrafish larval model.
Compound
hCA I
hCA II
hCA IX
KI hCA II/KI hCA IX
3
4
10
AAZ
152.2
127.8
6428.4
250
612.8
58.6
199.2
12.0
290.6
642.6
147.3
25
2.10
0.09
1.35
0.48
Histochemical analysis
The effects of different concentrations of CA inhibitors on hist-
ology were studied by analysing the sections of 5 dpf zebrafish
larvae stained with haematoxylin and eosin (H & E). The stained
sagittal sections of both the inhibitor treated and control larvae
were compared by light microscopy. Representative images of
zebrafish larval sections are shown in Figure 4. The histological
examination revealed no damage to the tissues of the developing
zebrafish larvae. Even high concentrations (up to 2 mM) showed
no apparent damage to the internal tissues, suggesting that these
compounds are safe and can be further developed as CA-specific
inhibitors for clinical use.
aAverage from three different assays, by a stopped flow technique (errors were
in the range of 5–10% of the reported values).
concentration at the end of 5 d of exposure to the inhibitors.
Significant mortality of zebrafish larvae emerged at higher concen-
trations (>2 mM), and the LC50 values at the end of five days
were 3 mM for 3 and 2.6 mM for 4 and 10. The half maximal lethal
concentrations of the inhibitors were higher compared to any tox-
icity and safety assessment studies that we have performed ear-
lier14–16
, suggesting that these compounds can be further
developed as inhibitors of human CA I, CA II and CA IX.
Analysis of swim pattern
Phenotypic analyses of zebrafish larvae treated with
inhibitor compounds
During development, zebrafish embryos are easily affected by
chemical compounds compared to adult zebrafish or other animal
or cell models, and therefore are suitable for assessing the subtle
toxic effects of the chemicals14,22. To assess the subtle toxic
effects of the inhibitors, the swim pattern of the larvae was exam-
ined under the microscope during treatment with the CA inhibi-
tors. The swim pattern analysis showed no abnormal or ataxic
movement pattern in the larvae exposed to the inhibitors, up to
2 mM concentration. However, a detailed analysis of the videos at
the end of 5 d of inhibitor exposure showed that the larvae of the
inhibitor treated groups (125 mM) were not as active and tended
to remain at the sides of petri dish compared to the control group
larvae (Representative video of swim pattern treated with com-
pound 3: Supplementary Appendix A). The results of the current
study reveal that the larvae treated with 3, 4 and 10 become
slower while swimming as compared to the swim pattern of the
control group larvae. However, swim pattern of larvae treated
with 3, 4 and 10 was unlike the larvae treated with nitroimida-
zole-based inhibitors DTP338 and DTP348, which showed ataxic
movement pattern due to neurotoxicity at a concentration as low
as 100 mM14. Therefore, these novel thiourea and sulphonamide
derivatives can be considered safer for further preclinical charac-
To assess toxic effects of the inhibitors on the developing zebrafish
larvae we analysed eight observable phenotypic parameters
namely: hatching, heartbeat, yolk sack utilisation, movement pat-
tern, body shape, swim bladder development, mortality and
oedema. The parameters were monitored using a stereo microscope
and observations were recorded for each group in comparison with
control embryos that were not treated with any of the compounds
or treated with 1% DMSO. The images in Figure 2 show the repre-
sentative larvae exposed to the compounds with concentrations
ranging from 75 mM to 500 mM as compared to the control group
larvae. The microscopic examination did not show any of the
observable changes in the developing zebrafish embryos at these
concentrations and the embryos showed normal phenotypes
throughout the period of embryonic development from 2 to 5 dpf.
We further assessed the toxicity of compounds 3, 4 and 10
compounds in zebrafish larvae across the concentration range
12 mM–3 mM. Figure 3 presents plots of dose-dependent effects of
inhibitors on different parameters in treated larvae. The results
indicated that none of the inhibitors show significant toxic effects
at submillimolar range. Previous toxicity studies on hCA IX and terisation and development as potential drugs.

114
A. ASPATWAR ET AL.
Figure 1. The LC50 values of the tested CA inhibitor compounds. The LC50 doses for the inhibitors were determined based on the 50% mortality of the larvae at the
end of 5 d after the exposure of embryos to different concentrations of inhibitors: compound 3 (A), 4 (B) and 10 (C). The LC50 doses were determined after three inde-
pendent experiments with similar experimental conditions (for each compound N ¼ 90).
Figure 2. The images of zebrafish larvae in control and inhibitor treated groups. Representative images of 2–5 dpf zebrafish larvae exposed to different concentrations
(75, 250 and 500 lM) of compound 3, 4 and 10. The images of control group (not treated with inhibitors) and 1% DMSO treated zebrafish larvae show normal devel-
opment (n ¼ 60).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
115
Figure 3. The effect of compounds 3, 4 and 10 CA inhibitors on the phenotypic parameters of the 5 dpf zebrafish larvae. The plot graphs of different phenotypic
ꢀ
abnormalities in (A) hatching, (B) oedema, (C) heartbeat (D) body shape and (E) yolk sac utilisation. For each concentration, n ¼ 90. p < 0.05 by two-tailed
Fisher’s test.
Figure 4. Histochemical analyses of zebrafish larvae. Representative sagittal sections of an untreated zebrafish larva (A) or larva treated with 500lM of compound
3 (B), 4 (C) or 10 (D). The images presented here are selected from three independent groups of experiments.

116
A. ASPATWAR ET AL.
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Conclusions
In conclusion, we have designed and synthesised thiourea and
sulphonamide derivative compounds with the potential to inhibit
human CA I, CA II and CA IX. These proteins which play important
pathophysiological roles in human diseases, such as well-known
role of CA IX in the progression of solid tumours, and those of CA
I and CA II in retinal and cerebral oedema, glaucoma, epilepsy
and altitude sickness. The inhibition kinetics of these inhibitors
were studied against recombinant human hCA I, hCA II and hCA
IX in vitro. Among these compounds, 4 was the most efficient
against hCA II (KI 58.6 nM). 10 inhibited human CA IX with KI
147.3 nM. The inhibition properties of these inhibitors against the
three hCA isoforms prompted us to test these drugs further for
their safety in a zebrafish larval model.
The toxicological evaluation indicated that these inhibitors
showed no adverse morphological changes nor did they cause
abnormal swim pattern at any sub-millimolar concentration. Taken
together, the results from both in vitro inhibition studies and toxi-
cological evaluation, we suggest that these inhibitors could be
developed into potential drugs targeting hCA I, hCA II and hCA IX
with no, or minimal, off-target effects.
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nazoline-24-dione as a new scaffold to develop potent and
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investigation of nimorazole and misonidazole as hypoxic
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Acknowledgements
The authors thank Aulikki Lehmus and Marianne Kuuslahti for the
histochemical analyses of the zebrafish samples. We also thank
€
Leena Makinen and Hannaleena Piippo for the assistance with
zebrafish embryos for the experiments.
12. Supuran CT. Carbonic anhydrase inhibitors and activators for
novel therapeutic applications. Future Med Chem 2011;3:
1165–80.
Disclosure statement
No potential conflict of interest was reported by the authors.
13. Gourmelon A, Delrue N. Validation in support of internation-
ally harmonised OECD Test Guidelines for assessing the
safety of chemicals. Adv Exp Med Biol 2016;856:9–32.
14. Aspatwar A, Becker HM, Parvathaneni NK, et al.
Nitroimidazole-based inhibitors DTP338 and DTP348 are safe
for zebrafish embryos and efficiently inhibit the activity of
human CA IX in Xenopus oocytes. J Enzyme Inhib Med
Chem 2018;33:1064–73.
15. Aspatwar A, Hammaren M, Koskinen S, et al. beta-CA-spe-
cific inhibitor dithiocarbamate Fc14-584B: a novel antimy-
cobacterial agent with potential to treat drug-resistant
tuberculosis. J Enzyme Inhib Med Chem 2017;32:832–40.
16. Kazokaite J, Aspatwar A, Kairys V, et al. Fluorinated benzene-
sulfonamide anticancer inhibitors of carbonic anhydrase IX
exhibit lower toxic effects on zebrafish embryonic develop-
ment than ethoxzolamide. Drug Chem Toxicol 2017;40:
309–19.
Funding
ꢀ
The work was supported by grants from Sigrid Juselius
Foundation (SP, MP), Finnish Cultural Foundation (HB, AA),
Academy of Finland (SP), and Jane & Aatos Erkko Foundation (SP).
Authors acknowledge financial support from ERC advanced grant
(ERC-ADG-2015, no. 694812 – Hypoximmuno).
ORCID
Ashok Aspatwar
Claudiu T. Supuran
Ludwig Dubois
Seppo Parkkila
http://orcid.org/0000-0002-6938-7835
http://orcid.org/0000-0003-4262-0323
http://orcid.org/0000-0002-8887-4137
http://orcid.org/0000-0001-7323-8536
http://orcid.org/0000-0003-3197-3414
17. Rami M, Dubois L, Parvathaneni NK, et al. Hypoxia-targeting
carbonic anhydrase IX inhibitors by a new series of nitroimi-
Jean-Yves Winum
dazole-sulfonamides/sulfamides/sulfamates.
J Med Chem
2013;56:8512–20.
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