The antibiotic furagin and its derivatives are isoform-selective human carbonic anhydrase inhibitors

Article abstract of DOI:10.1080/14756366.2020.1752201

The clinically used antibiotic Furagin and its derivatives possess inhibitory activity on human (h) carbonic anhydrases (CA, EC 4.2.1.1), some of which are highly expressed in various tissues and malignancies (hCA IX/XII). Furagin exhibited good hCA IX and XII inhibition with K_Is of 260 and 57 nM, respectively. It does not inhibit off-target CA I and poorly inhibited CA II (K_I = 9.6 μM). Some synthesised Furagin derivatives with aminohydantoin moieties as zinc binding group exhibited weak inhibition of CA I/II, and good inhibition of CA IX/XII with K_Is ranging from 350 to 7400 and 150 to 5600 nM, respectively. Docking and molecular dynamics simulations suggest that selectivity for the cancer-associated CA IX/XII over CA II is due to strong H-bond interactions in CA IX/XII, involving the tail orientated towards hydrophobic area of the active site. These results suggest a possible drug repurposing of Furagin as anti-cancer agent.

Full text of DOI:10.1080/14756366.2020.1752201

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
The antibiotic furagin and its derivatives are
isoform-selective human carbonic anhydrase
inhibitors
Aleksandrs Pustenko, Alessio Nocentini, Paola Gratteri, Alessandro Bonardi,
Igor Vozny, Raivis Žalubovskis & Claudiu T. Supuran
To cite this article: Aleksandrs Pustenko, Alessio Nocentini, Paola Gratteri, Alessandro Bonardi,
Igor Vozny, Raivis Žalubovskis & Claudiu T. Supuran (2020) The antibiotic furagin and its
derivatives are isoform-selective human carbonic anhydrase inhibitors, Journal of Enzyme Inhibition
and Medicinal Chemistry, 35:1, 1011-1020, DOI: 10.1080/14756366.2020.1752201
To link to this article: https://doi.org/10.1080/14756366.2020.1752201
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1011–1020
https://doi.org/10.1080/14756366.2020.1752201
RESEARCH PAPER
The antibiotic furagin and its derivatives are isoform-selective human carbonic
anhydrase inhibitors
Aleksandrs Pustenkoa,b, Alessio Nocentinic,d, Paola Gratterid, Alessandro Bonardic,d, Igor Voznya,
a,b
Raivis Zalubovskis and Claudiu T. Supuranc
ꢀ
b
aLatvian Institute of Organic Synthesis, Riga, Latvia; Institute of Technology of Organic Chemistry, Faculty of Materials Science and Applied
c
Chemistry, Riga Technical University, Riga, Latvia; Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences,
d
University of Florence, Firenze, Italy; Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, Laboratory of
Molecular Modeling Cheminformatics & QSAR, University of Florence, Firenze, Italy
ABSTRACT
ARTICLE HISTORY
Received 6 March 2020
Revised 31 March 2020
Accepted 1 April 2020
The clinically used antibiotic Furagin and its derivatives possess inhibitory activity on human (h) carbonic
anhydrases (CA, EC 4.2.1.1), some of which are highly expressed in various tissues and malignancies (hCA
IX/XII). Furagin exhibited good hCA IX and XII inhibition with KIs of 260 and 57 nM, respectively. It does
not inhibit off-target CA I and poorly inhibited CA II (KI ¼ 9.6 lM). Some synthesised Furagin derivatives
with aminohydantoin moieties as zinc binding group exhibited weak inhibition of CA I/II, and good inhib-
ition of CA IX/XII with KIs ranging from 350 to 7400 and 150 to 5600 nM, respectively. Docking and
molecular dynamics simulations suggest that selectivity for the cancer-associated CA IX/XII over CA II is
due to strong H-bond interactions in CA IX/XII, involving the tail orientated towards hydrophobic area of
the active site. These results suggest a possible drug repurposing of Furagin as anti-cancer agent.
KEYWORDS
Carbonic anhydrase
inhibitors; molecular
dynamics; furagin;
hydantoin; synthesis
1. Introduction
selectivity, were the coumarins27, the sulfocoumarins23–26 and
their congeners, homosulfocoumarins (3H-1,2-benzoxathiepine 2,2-
dioxides)31, and saccharin derivatives32–34. Considering the fact
that this last chemotype was somewhat chemically similar to
hydantoin (imidazolidine-2,4-dione) that may serve as zinc binding
group (ZBG) we investigated clinically used antibiotic Furagin
(Figure 1), also known under names Furazidine, Furamags or
Furazidin35, that contains hydantoin moiety, as well as newly pre-
pared its derivatives.
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloen-
zymes, being encoded by at least eight different genetic families,
which have been found in organisms all over the phylogenetic
tree1–10. CAs catalyse a crucial physiologic reaction, where by
hydratation of CO2 is formed a weak base (bicarbonate) and a
strong acid (hydronium ions). These enzymes are involved in a
multitude of physiologic processes, starting with pH regulation
and ending with metabolism1–3,7–10,11–22
.
CAs are also involved in various pathological processes and
therefore are drug targets for decades, with their inhibitors having
pharmacological applications in many fields1–3,7–19. The primary 2. Materials and methods
sulphonamides were discovered as CA inhibitors (CAIs) already in
the 40 s, and most of the drugs that were launched in the next
2.1. Chemical syntheses – general
decades as diuretics, antiepileptics, or antiglaucoma agents target- Reagents, starting materials and solvents were obtained from
ing CAs belonged to this class of compounds1–3,7–19. Although
commercial sources and used as received. Thin-layer chromatog-
highly potent as CAIs1–3, the sulphonamides generally non-select-
raphy was performed on silica gel, spots were visualised with UV
light (254 and 365 nm). Melting points were determined on an
OptiMelt automated melting point system. IR spectra were
ively inhibit most a-CA isoforms present in humans and mammals
in general1–3 as well as CAs from the other genetic families (b-, c-,
d-, f-, g-, h- and i-CAs)4–19, therefore alternative, isoform selective
recorded on Shimadzu FTIR IR Prestige-21 spectrometer. NMR
CAI classes were searched. A multitude of new chemotypes as
spectra were recorded on Bruker Avance Neo (400 MHz) spectrom-
well as novel CA inhibition mechanisms were reported in the last
eter with chemical shifts values (d) in ppm relative to TMS using
decade1–3,11–14,23–30
.
the residual DMSO-d6 signal (1H 2.50; 13C 39.52) or CDCl3 signal
(1H 7.26; 13C 77.16) as an internal standard, or D2O signal and
dioxane (1H 4.79; 13C 67.19). High-resolution mass spectra (HRMS)
That has highly enriched our understanding of these enzymes
and also allowed obtaining of isoform-selective CAIs targeting
physiologically relevant isoforms11–14,23–27. Among the new che-
motypes, which also exhibited the highest levels of isoform were recorded on a mass spectrometer with a Q-TOF micro mass
ꢀ
CONTACT Raivis Zalubovskis
raivis@osi.lv
Riga Technical University, Riga, Latvia; Claudiu T. Supuran
claudiu.supuran@unifi.it
University of Florence,
Firenze, Italy
Supplemental data for this article can be accessed here.
ß 2020 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.

1012
A. PUSTENKO ET AL.
from compound 1 (0.5 g; 3.30 mmol) and 4-nitrobenzaldehyde
(0.52 g; 3.46 mmol) as yellowish solid (0.68 g; 82%). Mp 280 ꢀC dec.
IR (film, cmꢁ1) ꢀmax¼ 1780 (C¼O), 1714 (C¼O); 1H NMR (400 MHz,
DMSO-d6) d ¼ 4.38 (s, 2H), 7.90–7.96 (m, 3H), 8.28–8.33 (m, 2H),
11.39 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d6) d ¼ 49.1, 124.2,
127.7, 140.6, 140.7, 147.6, 153.4, 168.9 ppm HRMS (ESI) [M þ H]þ:
m/z calcd for (C10H9N4O4) 249.0624. Found 249.0616.
Figure 1. Structure of Furagin.
analyser using the ESI technique. Examples of spectral data are
furnished in the Supporting Information to the aricle.
2.2.4. Methyl 4-(((2,4-dioxoimidazolidin-1-yl)imino)methyl)ben-
zoate (5)
2.2. General procedure for compound 2–17 synthesis
To a solution of 1-aminoimidazolidine-2,4-dione hydrochloride (1)
(1.0 eq.) in EtOH (15 ml per 1 mmol of compound 1) appropriate
aldehyde (1.05 eq.) was added. The resulting mixture was stirred
at room temperature overnight.
The solvent was removed under vacuum and the crude prod-
uct was re-crystallized form EtOH to afford product.
Compound 5 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and methyl 4-formylbenzoate
(0.57 g; 3.46 mmol) as white solid (0.82 g; 95%). Mp 280 ꢀC dec. IR
(film, cmꢁ1) ꢀmax¼ 1763 (C¼O), 1717 (C¼O); 1H NMR (400 MHz,
DMSO-d6) d ¼ 3.86 (s, 3H), 4.37 (s, 2H), 7.80–7.87 (m, 3H), 8.00–8.05
(m, 2H), 11.33 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d6) d ¼ 49.0,
52.2, 127.0, 129.7, 130.2, 138.8, 141.6, 153.4, 165.9, 168.9 ppm
HRMS (ESI) [M þ H]þ: m/z calcd for (C12H12N3O4) 262.0828.
Found 262.0834.
2.2.1. 1-(Benzylideneamino)-imidazolidine-2,4-dione (2)36
2.2.5. 1,1’-((Pentane-1,5-diylidene)bis(azaneylylidene))bis(imidazoli-
Compound 2 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and benzaldeyde (0.35 ml;
3.46 mmol) as white solid (0.60 g; 90%). Mp 252 – 253 ꢀC. IR (film,
cmꢁ1) ꢀmax¼ 1778 (C¼O), 1717 (C¼O); 1H NMR (400 MHz, DMSO-
d6) d ¼ 4.36 (s, 2H), 7.38–7.48 (m, 3H), 7.68–7.72 (m, 2H), 7.80 (s,
1H), 11.25 (s, 1H) ppm 13C NMR (100 MHz, DMSO-d6) d ¼ 48.9,
126.8, 128.8, 129.8, 134.3, 143.0, 153.4, 169.0 ppm HRMS (ESI)
[M þ H]þ: m/z calcd for (C10H10N3O2) 204.0773. Found 204.0783.
dine-2,4-dione) (6)
Compound 6 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and glutaraldehyde 50 wt %
solution in H2O (0.31 ml; 3.46 mmol) as white solid (0.49 g; 50%).
2.2.2. 1-((4-Methoxybenzylidene)amino)imidazolidine-2,4-dione (3)
1
Mp 237 ꢀC dec. IR (film, cmꢁ1) ꢀmax¼ 1768 (C¼O), 1734 (C¼O); H
NMR (400 MHz, DMSO-d6) d ¼ 1.72 (p, 2H, J ¼ 7.4 Hz), 2.28–2.36 (m,
4H), 4.17 (s, 4H), 7.06 (t, 2H, J ¼ 5.2 Hz), 11.07 (s, 2H) ppm 13C NMR
(100 MHz, DMSO-d6) d ¼ 23.1, 31.3, 48.5, 146.7, 153.4, 169.1 ppm
HRMS (ESI) [M þ Na]þ: m/z calcd for (C11H14N6O4Na) 317.0974.
Found 317.0978.
Compound 3 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 4-methoxybenzaldehyde
(0.42 ml; 3.46 mmol) as white solid (0.62 g; 80%). Mp 242 – 244 ꢀC.
IR (film, cmꢁ1) ꢀmax¼ 1768 (C¼O), 1718 (C¼O); 1H NMR (400 MHz,
DMSO-d6) d ¼ 3.80 (s, 3H), 4.33 (s, 2H), 6.99–7.04(m, 2H), 7.62–7.66
(m, 2H), 7.75 (s, 1H), 11.18 (s, 1H) ppm 13C NMR (100 MHz, DMSO-
d6) d ¼ 48.9, 55.3, 114.3, 126.9, 128.4, 142.9, 153.4, 160.6,
169.1 ppm HRMS (ESI) [M þ H]þ: m/z calcd for (C11H12N3O3)
234.0879. Found 234.0885.
2.2.6. 1-((Furan-3-ylmethylene)amino)imidazolidine-2,4-dione (7)
Compound 7 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 3-furaldehyde (0.33 g;
3.46 mmol) as yellowish solid (0.57 g; 89%). Mp 235 ꢀC dec. IR
(film, cmꢁ1) ꢀmax¼ 1780 (C¼O), 1714 (C¼O);
2.2.3. 1-((4-Nitrobenzylidene)amino)imidazolidine-2,4-dione (4)37
1H NMR (400 MHz, DMSO-d6) d ¼ 4.30 (s, 2H), 6.74–6.76 (m, 1H),
7.73–7.77 (m, 2H), 8.05–8.07 (m, 1H), 11.18 (s, 1H) ppm 13C NMR
(100 MHz, DMSO-d6) d ¼ 48.8, 107.0, 122.5, 136.1, 144.8, 144.9,
153.3, 169.1 ppm HRMS (ESI) [M þ H]þ: m/z calcd for (C8H8N3O3)
Compound 4 was prepared according to the general procedure 194.0566. Found 194.0570.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1013
2.2.7. 1-((4-(Benzyloxy)benzylidene)amino)imidazolidine-2,4-dione (8)
trans-4-methoxycinnamaldehyde (0.56 g; 3.46 mmol) as white solid
(0.61 g; 71%). Mp 250 ꢀC dec. IR (film, cmꢁ1) ꢀmax¼ 1770 (C¼O),
1731 (C¼O); 1H NMR (400 MHz, DMSO-d6) d ¼ 3.78 (s, 3H), 4.29 (s,
2H), 6.85–7.00 (m, 4H), 7.51–7.59 (m, 3H), 11.18 (s, 1H) ppm 13C
NMR (100 MHz, DMSO-d6) d ¼ 48.8, 55.2, 114.3, 123.1, 128.5, 128.6,
138.5, 145.5, 153.3, 159.9, 169.1 ppm HRMS (ESI) [M þ H]þ: m/z
calcd for (C13H14N3O3) 260.1035. Found 260.1047.
Compound 8 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 4-benzyloxybenzalde-
hyde (0.73 g; 3.46 mmol) as white solid (0.92 g; 90%). Mp
258–260 ꢀC. IR (film, cmꢁ1) ꢀmax¼ 1790 (C¼O), 1730 (C¼O); 1H
NMR (400 MHz, DMSO-d6) d ¼ 4.33 (s, 2H), 5.15 (s, 2H), 7.07–7.12
(m, 2H), 7.31–7.36 (m, 1H), 7.37–7.43 (m, 2H), 7.44–7.49 (m, 2H),
7.62–7.67 (m, 2H), 7.75 (s, 1H), 11.19 (s, 1H) ppm 13C NMR
(100 MHz, DMSO-d6) d ¼ 48.9, 69.4, 115.1, 127.1, 127.8, 127.9,
128.4, 128.5, 136.8, 142.8, 153.4, 159.7, 169.1 ppm HRMS (ESI)
[M þ H]þ: m/z calcd for (C17H16N3O3) 310.1192. Found 310.1194.
2.2.11. 1-((2,4-Dihydroxybenzylidene)amino)imidazolidine-2,4-dione (12)
Compound 12 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 2,4-dihydroxybenzalde-
hyde (0.48 g; 3.46 mmol) as white solid (0.72 g; 93%). Mp >300 ꢀC.
IR (film, cmꢁ1) ꢀmax¼ 3260 (OH), 3188 (OH), 1780 (C¼O), 1717
(C¼O); 1H NMR (400 MHz, DMSO-d6) d ¼ 4.34 (s, 2H), 6.31 (d, 1H,
J ¼ 2.3 Hz), 6.35 (dd, 1H, J ¼ 8.5, 2.3 Hz), 7.33 (d, 1H, J ¼ 8.5 Hz),
7.90 (s, 1H), 9.90 (br s, 1H), 10.73 (s, 1H), 11.23 (br s, 1H) ppm 13C
NMR (100 MHz, DMSO-d6) d ¼ 48.5, 102.6, 107.8, 110.7, 130.5,
144.0, 153.3, 158.6, 160.5, 169.1 ppm HRMS (ESI) [M þ H]þ: m/z
calcd for (C10H10N3O4) 236.0671. Found 236.0677.
2.2.8. Ethyl (2E)-4-((2,4-dioxoimidazolidin-1-yl)imino)but-2-enoate (9)
Compound 9 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and ethyl trans-4-oxo-2-bute-
noate (0.42 ml; 3.46 mmol) as white solid (0.60 g; 81%). Mp
210–211 ꢀC. IR (film, cmꢁ1) ꢀmax¼ 1772 (C¼O), 1721 (C¼O); 1H
NMR (400 MHz, DMSO-d6) d ¼ 1.24 (t, 3H, J ¼ 7.1 Hz), 4.17 (q, 2H,
J ¼ 7.1 Hz), 4.27 (s, 2H), 7.37 (d, 1H, J ¼ 15.6 Hz), 7.16–7.24 (m, 1H),
7.60 (d, 1H, J ¼ 9.3 Hz), 11.39 (s, 1H) ppm 13C NMR (100 MHz,
DMSO-d6) d ¼ 14.1, 49.0, 60.4, 126.5, 140.3, 141.7, 153.2, 165.4,
168.7 ppm HRMS (ESI) [M þ H]þ: m/z calcd for (C9H12N3O4)
226.0828. Found 226.0834.
2.2.12. 4-(((2,4-Dioxoimidazolidin-1-yl)imino)methyl)phenyl)boronic
acid (13)
Compound 13 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 4-formylphenylboronic
acid (0.52 g; 3.46 mmol) as white solid (0.72 g; 88%). Mp >300 ꢀC.
IR (film, cmꢁ1) ꢀmax¼ 3349 (OH), 3173 (OH), 1780 (C¼O), 1716
2.2.9. 1-((3-Methylbut-2-en-1-ylidene)amino)imidazolidine-2,4-dione (10)
1
(C¼O); H NMR (400 MHz, DMSO-d6) d ¼ 4.37 (s, 2H), 7.64–7.68 (m,
2H), 7.79 (s, 1H), 7.83–7.87 (m, 2H), 8.12 (s, 2H), 11.26 (s, 1H) ppm
13C NMR (100 MHz, DMSO-d6) d ¼ 48.9, 125.8, 134.5, 135.7, 136.0
(br) 143.0, 153.4, 169.1 ppm HRMS (ESI) [M þ H]þ: m/z calcd for
(C10H11BN3O4) 248.0843. Found 248.0847.
Compound 10 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 3-methyl-2-butenal
(0.33 ml; 3.46 mmol) as white solid (0.43 g; 72%). Mp 186–187 ꢀC.
IR (film, cmꢁ1) ꢀmax¼ 1768 (C¼O), 1717 (C¼O); 1H NMR (400 MHz,
DMSO-d6) d ¼ 1.84–1.89 (m, 6H), 4.28 (s, 2H), 5.93–5.99 (m, 1H),
7.57 (d, 1H, J ¼ 9.5 Hz), 11.11 (s, 1H) ppm 13C NMR (100 MHz,
DMSO-d6) d ¼ 18.7, 26.2, 48.9, 121.9, 142.4, 144.3, 153.3, 169.2 ppm
HRMS (ESI) [M þ H]þ: m/z calcd for (C8H12N3O2) 182.0930.
Found 182.0938.
2.2.13. 1-((Pyridin-2-ylmethylene)amino)imidazolidine-2,4-dione (14)
2.2.10 1-(((2e)-3–(4-methoxyphenyl)allylidene)amino)imidazolidine-
2,4-dione (11)
Compound 14 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and pyridine-2-carbaldehyde
(0.33 ml; 3.46 mmol) as white solid (0.64 g; 95%). Mp 280 ꢀC dec. IR
(film, cmꢁ1) ꢀmax¼ 1770 (C¼O), 1730 (C¼O); 1H NMR (400 MHz,
DMSO-d6) d ¼ 4.43 (s, 2H), 7.61–7.66 (m, 1H), 7.89 (s, 1H), 8.02–8.06
(m, 1H), 8.16 (dt, 1H, J ¼ 7.7, 1.4 Hz), 8.68–8.72 (m, 1H), 11.50 (s,
1H) ppm 13C NMR (100 MHz, DMSO-d6) d ¼ 49.0, 121.6, 125.2,
139.3, 140.6, 146.8, 150.5, 153.3, 168.7 ppm HRMS (ESI) [M þ H]þ:
Compound 11 was prepared according to the general
procedure from compound
1
(0.5 g; 3.30 mmol) and m/z calcd for (C9H9N4O2) 205.0726. Found 205.0732.

1014
A. PUSTENKO ET AL.
2.2.14. 1-((Pyridin-3-ylmethylene)amino)imidazolidine-2,4-dione (15)
three different determinations. The four tested CA isoforms were
recombinant ones obtained in-house as reported earlier41–43
.
2.4. Computational studies
The crystal structure of CA II (pdb 5LJT)43, CA IX (pdb 5FL4)44 and
CA XII (pdb JLD0)45 were prepared using the Protein Preparation
Compound 15 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and pyridine-3-carbaldehyde
(0.33 ml; 3.46 mmol) as white solid (0.60 g; 90%). Mp 280 ꢀC dec. IR
(film, cmꢁ1) ꢀmax¼ 1764 (C¼O), 1722 (C¼O); 1H NMR (400 MHz,
D2O þ NaOH þ dioxane) d ¼ 7.46–7.51 (m, 1H), 7.56 (s, 1H), 8.18
(td, 1H, J ¼ 8.0, 1.8 Hz), 8.48 (dd, 1H, J ¼ 4.9, 1.6 Hz), 8.74 (d, 1H,
J ¼ 1.8 Hz) ppm 13C NMR (100 MHz, D2O þ NaOH þ dioxane)
d ¼ 49.3 (br), 125.1, 131.6, 135.1, 138.9, 148.1, 149.7, 170.0,
186.3 ppm HRMS (ESI) [M þ H]þ: m/z calcd for (C9H9N4O2)
205.0726. Found 205.0731.
€
Wizard tool implemented in Maestro - Schrodinger suite, assigning
bond orders, adding hydrogens, deleting water molecules, and
optimising H-bonding networks46. Energy minimisation protocol
with a root mean square deviation (RMSD) value of 0.30 was
applied using an Optimised Potentials for Liquid Simulation
(OPLS3e) force field. 3D ligand structures were prepared by
Maestro46a and evaluated for their ionisation states at pH 7.4 0.5
with Epik46b. Additionally, the imidic nitrogen of the hydantoin
nucleus was negatively charged in simulations. OPLS3e force field
in Macromodel46e was used for energy minimisation for a max-
imum number of 2500 conjugate gradient iteration and setting a
convergence criterion of 0.05 kcal molꢁ1 Åꢁ1. The docking grid
was centred on the centre of mass of the co-crystallized ligands
and Glide used with default settings. Ligands were docked with
the standard precision mode (SP) of Glide46e and the best 5 poses
of each molecule retained as output. The best pose for each com-
pound, evaluated in terms of coordination, hydrogen bond inter-
actions and hydrophobic contacts, was refined with Prime46d with
a VSGB solvation model considering the target flexible within 3 Å
2.2.15. 1-((Pyridin-4-ylmethylene)amino)imidazolidine-2,4-dione (16)
around the ligand47–49
.
Compound 16 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and pyridine-4-carbaldehyde
(0.33 ml; 3.46 mmol) as white solid (0.61 g; 91%). Mp 280 ꢀC dec. IR
(film, cmꢁ1) ꢀmax¼ 1750 (C¼O), 1723 (C¼O); 1H NMR (400 MHz,
D2O þ NaOH þ dioxane) d ¼ 7.46 (s, 1H), 7.62–7.66 (m, 2H),
8.47–8.51 (m, 2H) ppm 13C NMR (100 MHz, D2O þ NaOH þ dioxane)
d ¼ 49.3 (br), 121.9, 139.2, 143.5, 149.7, 170.0, 186.4 ppm HRMS
The best poses of Furagin and 12 to CA II, CA IX and CA XII
were submitted to a MD simulation using Desmond50 and the
OPL3e force field. Specifically, the system was solvated in an
orthorhombic box using TIP4PEW water molecules, extended 15 Å
away from any protein atom. It was neutralised adding chlorine
and sodium ions. The simulation protocol included a starting
relaxation step followed by a final production phase of 100 ns. In
particular, the relaxation step comprised the following: (a) a stage
of 100 ps at 10 K retaining the harmonic restraints on the solute
heavy atoms (force constant of 50.0 kcal molꢁ1 Åꢁ2) using the NPT
ensemble with Brownian dynamics; (b) a stage of 12 ps at 10 K
with harmonic restraints on the solute heavy atoms (force con-
stant of 50.0 kcal molꢁ1 Åꢁ2), using the NVT ensemble and
Berendsen thermostat; (c) a stage of 12 ps at 10 K and 1 atm,
retaining the harmonic restraints and using the NPT ensemble
and Berendsen thermostat and barostat; (f) a stage of 12 ps at
300 K and 1 atm, retaining the harmonic restraints and using the
NPT ensemble and Berendsen thermostat and barostat; (g) a final
24 ps stage at 300 K and 1 atm without harmonic restraints, using
the NPT Berendsen thermostat and barostat. The final production
phase of MD was run using a canonical NPT Berendsen ensemble
(ESI)
Found 205.0730.
[M þ H]þ:
m/z
calcd
for
(C9H9N4O2)
205.0726.
2.2.16. 1-(((1 h-Imidazol-5-yl)methylene)amino)imidazolidine-2,4-
dione (17)
Compound 17 was prepared according to the general procedure
from compound 1 (0.5 g; 3.30 mmol) and 1H-imidazole-5-carbalde-
hyde (0.33 g; 3.46 mmol) as white solid (0.62 g; 97%). Mp 270 ꢀC
dec. IR (film, cmꢁ1) ꢀmax¼ 1764 (C¼O), 1715 (C¼O); 1H NMR at temperature 300 K. During the MD simulation, a time step of 2
fs was used while constraining the bond lengths of hydrogen
(400 MHz, D2O þ NaOH þ dioxane) d ¼ 7.45–7.48 (m, 1H), 7.67 (s,
1H),
7.72–7.76
(m,
1H)
ppm
13C
NMR
(100 MHz,
atoms with the M-SHAKE algorithm. The atomic coordinates of the
system were saved every 100 ps along the MD trajectory. Protein
and ligand RMSD values, ligand torsions evolution and occupancy
of intermolecular hydrogen bonds and hydrophobic contacts were
computed along the production phase of the MD simulation with
the Simulation Interaction Diagram tools implemented in Maestro.
D2O þ NaOH þ dioxane) d ¼ 49.5 (br), 125.1, 134.2, 136.5, 140.8,
170.3, 186.7 ppm HRMS (ESI) [M þ H]þ: m/z calcd for (C7H8N5O2)
194.0678. Found 194.0687
2.3. Ca inhibitory assay
An Applied Photophysics stopped-flow instrument has been used
for assaying the CA catalysed CO2 hydration activity, as reported
earlier38,39. The inhibition constants were obtained by non-linear
least-squares methods using PRISM 3 and the Cheng-Prusoff equa-
3. Results and discussion
3.1. Chemistry
A series of Furagin derivatives 2–17 were prepared in reaction
tion as reported earlier40 and represent the mean from at least between 1-aminohydantoin hydrochloride (1) and various

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1015
Scheme 1. Reagents and conditions: i. RCHO, EtOH, RT, 16 h
3.2. Biological evaluation
Table 1. Inhibition data of human CA isoforms CA I, II, IX, and XII with aminohydan-
toines (2–17, Furagin) using AAZ as a standard inhibitor.
The CA inhibitory profiles of Furagin and synthesised aminohydan-
toin derivatives were evaluated by applying a stopped flow car-
bon dioxide hydrase assay51, in comparison to acetazolamide
(AAZ) as a standard CAI against four physiologically significant iso-
forms CA I, II, IX, and XII. The following structure–activity relation-
ship (SAR) can be concluded from the inhibition data presented in
Table 1.
ꢂ
KI (nM)
a. All the tested aminohydantoins exhibited weak inhibitory
effect on the slow cytosolic isoform, hCA I, where the binding
affinity constant (KI) values fluctuating in the thousands nM
range (KI 16 800->100 000 nM).
Comp.
R
CA I
CA II
900
6400
11 100
8300
4000
710
540
23 600
16 500
3100
59 900
14 200
4200
620
3300
12 400
9600
CA IX
CA XII
2
3
4
5
6
7
8
9
C6H5
39 600
57 600
>100 000
>100 000
19 100
16 800
>100 000
45 900
3500
1200
7400
4900
1100
850
350
810
2900
400
5800
7300
4500
2300
1600
560
5600
4700
2800
930
160
1700
910
440
880
360
150
230
1300
3200
810
350
57
4-OCH3-C6H4
4-NO2-C6H4
4-(CO2CH3)-C6H4
-
b. The physiologically relevant isoform, hCA II, was better inhib-
ited by most of the tested compounds (KIs: 620-59 000 nM). It
is observed that, the aminohydantoin compounds (2, 7, 8
and 15) were more potent hCA II inhibitors with KIs in range
from 540-900 nM. These compounds have unsubstituted Ph
or hetaryl moieties. Rest of the compounds showed weaker
inhibitory effect of CA II with KIs in range from 3100–59
900 nM. It is interesting to note, that compound 12 having
dihydroxyphenyl substituent stood out by nearly three times
weaker inhibition compare to the second weakest inhibitor 9.
c. The tumour associated isoform hCA IX was inhibited in nano-
molar range by compounds 7-9, 11, 17 and Furagin (KIs:
260–850 nM), where the strongest inhibition was observed
for Furagin. Rest of the aminohydantoin derivatives showed
one order weaker inhibition with KIs in range from 1100-7
300 nM. Certain pattern can be observed, where better CA IX
inhibition can be observed for compounds with vinyl sub-
stituents (9, 11, 17 and Furagin) or small hetaryl substituents
(7 and 17), with exception in case of compound 8, contain-
ing ester moiety on phenyl ring.
3-furanyl
4-(OCH2C6H5)-C6H4
CHCH(CO2C2H5)
CHC(CH3)2
CHCH(4-OCH3-C6H4)
2,4-(OH)2-C6H3
4-(B(OH)2)-C6H4
2-pyridyl
10
11
12
13
14
15
16
17
Furagin
AAZ
ꢂ
28 800
>100 000
>100 000
90 700
51 800
45 600
26 600
9600
>100 000
250
3-pyridyl
4-pyridyl
5-imidazolyl
-
260
25
-
12
6
Mean from 3 different assays, by a stopped flow technique (errors were in the
range of 5–10% of the reported values).
aldehydes (Scheme 1). Compounds 2–17 were isolated in good to
excellent yields, all new structure were proven by H and 13C NMR
1
and IS spectroscopy as well as high-resolution mass spectra. The
purity of all compounds was greater than 95% according
UPLC analysis.

1016
A. PUSTENKO ET AL.
Figure 2. Predicted docking orientations of 7 (green) and Furagin (pink) to (A) CA II, (B) CA IX and (C) CA XII.
Figure 3. RMSD analysis of Furagin heavy atoms and (A) CA II, (B) CAIX and (C) CA XII backbone over the 100 ns MD simulation. The ligand colour darkens over the
dynamic simulation.
d. The other tumour associated isoform hCA XII was best inhib-
3.3. Computational studies
ited among all isoforms studied. The best compound of this
Docking studies were used to investigate the binding mode of
series was Furagin with KI ¼ 57 nM. It was followed by vinyl
Furagin and aminohydantoines 2-17 within the active site of CA II
(pdb 5LJT)44, IX (pdb 5FL4)43 and XII (pdb JLD0)45. Similarly to
benzenesulfonamides (pKa 10.1) which binds to the CA Zn ion in
the deprotonated form, the imidic nitrogen of the hydantoin
nucleus as well was considered negatively charged (pKa 9.16)52 in
the docking experiments and resulted to coordinate the zinc ion
in all the obtained poses with CAs II, IX and XII. Furthermore, the
oxygen atom of the CO in position 4 of the hydantoin core acts
as a bifurcated acceptor establishing two H-bonds with T199, that
substituted aminohydantoin derivatives 6, 9 and 10 with KIs
160, 360 and 880 nM, respectively. One order weaker CA XII
inhibition compare to Furagin was also observed for aryl (5,
8 and 12) and hetaryl (16 and 17) derivatives ranging KIs
from 150 to 930 nM.
In general good selectivity against cancer associated CA iso-
forms (CA IX and CA XII) compare to off-target ones (CA I and CA
II) was observed for three compounds Furagin, 9 and 12.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1017
Figure 4. Dynamics evolution of the binding mode of Furagin to (A) CA II, (B) CA IX and (C) CA XII over the course of 100ns. Water molecules are represented as red
spheres. The ligand colour darkens over the dynamic simulation.
Figure 5. RMSD analysis of 12 heavy atoms and (A) CA II, (B) CAIX and (C) CA XII backbone over the 100ns MD simulation. The ligand colour darkens over the
dynamic simulation.
is, O… (H-N, HG1-O), whereas overall the heterocycle forms VdW simulations on the predicted binding conformations of Furagin
contacts with residues H94, H96, H119, L198, T200 and W209 and 12, the most potent CA XII inhibitors also showing significant
(Figure 2).
In CA II and CA IX, the N1 pendants of all ligands are oriented
CA XII over CA II selectivity. The structure of the three investigated
CA isoforms was stable during the computation with the back-
bone atom RMSDs exhibiting small fluctuations over the 100 ns
(Figures 3 and 5). Additionally, the ZBG of the ligands remains sta-
bly anchored to the metal ion all over the MD, with the hydantoin
core receiving H-bonds by the amidic NH and side chain OH of
Thr199 (Figures 4 and 6).
towards a hydrophilic cleft defined by H4, W5, N62, N67 and H64,
except 8 and 9, whose N1 tails are housed, in CA II, into a hydro-
phobic pocket formed by I91, V121 and F131 (Figure 2(A–B)).
Amino acids T91, Q92, A131, S132 and S135 are instead targeted
by the pendants on the aminohydantoin of the ligands in all
docking solutions with CA XII (Figure 2(C)). The docking procedure
After an initial equilibration, mainly occurring in CA II and IX,
was complemented with 100 ns long molecular dynamic (MD) the molecular tail of Furagin undergoes minor conformational

1018
A. PUSTENKO ET AL.
Figure 6. Dynamics evolution of the binding mode of 12 to (A) CA II, (B) CA IX and (C) CA XII over the course of 100 ns. Water molecules are represented as red
spheres. The ligand colour darkens over the dynamic simulation.
fluctuations during simulation approaching to stable binding con- 910 nM). The rest of the compounds exhibited slightly weaker
formations within the three CA isoforms (Figures 3 and 4). In CA
IX and XII, the ligand accommodates the N1-pendant in the
hydrophilic half of the active sites where it makes VdW contacts
and both direct and water mediated H-bond interactions with the
enzymes (Figure 4(B,C)). In the CA II, the ligand-bound conform-
ation of Furagin orients the tail towards the hydrophobic area of
the target and does not form persistent H-bond interactions over
the 100 ns (Figure 4(A)). The hydrogen bond persistence within
the three CA isoforms is in good agreement with the inhibitory
profile of the ligand (CAXII > CA IX > CA II).
An ensemble of few conformations is representative of the
binding of 12 within CA II and IX (Figures 5 and 6). Here, the lig-
and approaches the hydrophobic regions of the enzymes and,
coming next to the end of the simulation, the N1 tails lose direct
or water-bridged H-bonds with glutamine and asparagine resi-
dues, progressively moving towards T199 or T200, that is, the area
of the enzyme that undergoes to the greatest residue displace-
ment. In CA XII, the docked pose of 12 remains firmly anchored
to the residues of the hydrophilic portion of the enzyme through-
out the dynamic. A wide network of direct and water mediated H-
bonds stabilise the binding of the ligand. This is consistent with
the inhibition profile exhibited by 12 in CA XII as compared with
the other two CA isoforms.
inhibition with KIs ranging from 1300 to 5600 nM. Docking and
molecular dynamics simulations shed light on the ligands selectiv-
ity for the cancer-associated CAs over ubiquitous CA II. The signifi-
cant inhibition activity and especially selectivity of Furagin against
hCA IX and XII was attributed due to the strong H-bond interac-
tions, whereas in case of hCA II no persistent H-bond interactions
are formed due to Furagin’s tails orientation towards hydrophobic
area of the enzyme.
The knowledge obtained gives the solid base for both – inves-
tigation of drug repurposing of clinically used antibiotic Furagin
for anti-cancer therapy and further studies of new chemotype of
inhibitors of CAs.
Disclosure statement
The authors have no relevant affiliations of financial involvement
with any organisation or entity with a financial interest in or finan-
cial conflict with the subject matter or materials discussed in
the manuscript.
ORCID
Claudiu T. Supuran
http://orcid.org/0000-0003-4262-0323
4. Conclusions
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Furagin and all newly synthesised hydantoin derivative were
examined for their inhibitory activities towards hCA I, II, IX and XII.
The four studied hCA isoforms were inhibited by Furagin and its
derivatives at various degrees. In particular, Furagin and prepared
compounds 2-17 did not inhibit or poorly inhibited off-target hCA
I with KIs ranging from >100 lM (compounds 4, 5, 8, 11, 12 and
Furagin) to 9.6 lM. Ubiquitous hCA II was poorly inhibited by
compounds 3-6, 9-14, 16, 17 and Furagin (KIs from 59.9 to
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rest of compounds exhibited slightly weaker inhibition of hCA IX
with KIs ranging from 1100 to 7400 nM. Other cancer associated
isoform – hCA XII also was significantly inhibited by Furagin
(KI¼57 nM) and compounds 5, 6, 8-13, 16 and 17 (KIs from 160 to
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