Synthesis and carbonic anhydrase inhibitory properties of novel uracil derivatives

Article abstract of DOI:10.1016/j.bmcl.2015.05.073

Abstract Carbonic anhydrase (CA) inhibitors are valuable molecules based on several therapeutic applications, including antiglaucoma activity. In the present study, inhibition of two human cytosolic carbonic anhydrase isozymes I and II with some uracil derivatives (3-9) were investigated. Compounds 3-9 showed K_I values in the range of 10.83-464 μM for hCA I and of 28.88-778.5 μM against hCA II, respectively. Kinetic investigations showed that similarly to classical CA inhibitors, all investigated natural compounds act as competitive inhibitors with 4-NPA as substrate. Uracil derivatives investigated here are promising agents which may be used as lead molecules in order to derivative novel carbonic anhydrase inhibitors that might be useful in medical applications.

Full text of DOI:10.1016/j.bmcl.2015.05.073

Accepted Manuscript
Synthesis and carbonic anhydrase inhibitory properties of novel uracil deriva-
tives
Murat Güney, Hüseyin Çavdar, Murat Şentürk, Deniz Ekinci
PII:
S0960-894X(15)00547-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.05.073
BMCL 22763
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Bioorganic & Medicinal Chemistry Letters
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Please cite this article as: Güney, M., Çavdar, H., Şentürk, M., Ekinci, D., Synthesis and carbonic anhydrase
inhibitory properties of novel uracil derivatives, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.05.073
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Synthesis and carbonic anhydrase inhibitory properties of novel uracil derivatives
Murat Güneya,*, Hüseyin Çavdarb, Murat Şentürka,*, Deniz Ekincic
aAğrı İbrahim Çeçen University, Department of Chemistry, 04100 Ağrı, Turkey
bDumlupinar University, Education Faculty, 43100 Kutahya, Turkey
cOndokuz Mayıs University, Department of Agricultural Biotechnology, 55139 Samsun,
Turkey
ABSTRACT
Carbonic anhydrase (CA) inhibitors are precious molecules based on several therapeutic
applications, including antiglaucoma activity. In the present study, inhibition of two human
cytosolic carbonic anhydrase isozymes I and II with some uracil derivatives (3-9) were
investigated. Compounds 3-9 showed KI values in the range of 10.83 - 464 µM for hCA I and
of 28.88 - 778.5 µM against hCA II, respectively. Kinetic investigations showed that similarly
to classical CA inhibitors, all investigated natural compounds act as competitive inhibitors
with 4-NPA as substrate. Uracil derivatives investigated here are promising agents which
may be used as lead molecules in order to derivative novel carbonic anhydrase inhibitors that
might be useful in medical applications.
Keywords: Carbonic anhydrase; hydroxyl; uracil; enzyme inhibitor.
* Corresponding authors: Phone: +90472 215 9994; Fax: +904722156554; E-mail:
mguney@agri.edu.tr (MG) and Phone: +90472 215 9994; Fax: +904722156554; E-mail:
msenturk@agri.edu.tr (MS)

Uracil (U) which is a common and naturally occurring pyrimidine derivative is one of
the four nucleobases in the nucleic acid of RNA.1 Uracil undergoes two tautomeric forms at
pH 7.0. This amide-imidic acid tautomeric shifts from lactam which is the amide tautomer to
the imidic acid tautomer is referred to as the lactim structure. (Fig. 1). Uracil is a weak acid.2
Figure 1 here
In drug discovery studies, uracils are very important structures because of wide range
of biological activities and synthetic accessibility and ability to confer drug like properties to
the compound libraries appended on them at N1, N3, C5 and C6 positions.3a As well as
antiviral and anti-tumour properties which is two most reported activities of uracil
analogues;3b,c they also possess herbicidal, insecticidal and bactericidal actions.4
5-Fluorouracil (5-FU) is antimetabolite of the pyrimidine analogue type and a well-
known anti-tumour agent which has been widely used in the treatment of solid tumours such
as colon or breast cancers.5 Because 5-FU is similar in shape to uracil, but does not perform
the same chemistry as uracil, the drug inhibits RNA replication enzymes, thereby eliminating
RNA synthesis and stopping the growth of cancerous cells. Although 5-FU has had clinical
success as a single agent, it has been modified by different ways to synthesize its derivatives
which may improve its therapeutic index because of its well-known side effects such as short
half-life, wide distribution, low selectivity, and various toxic side effects.2
Some researcher prepared N1-mono and N1, N3-bis-substituted 5-fluoro, 5-bromo, 5-
iodouracils.6 Uracils were converted to the corresponding benzyl halides in the presence of
K2CO3 the target pyrimidines as mixtures of mono- and bis-derivatives that were separated by
treatment with KOH solution. Some biologically activities as toxicity, anti-tumour, and
antibacterial properties of synthesized compounds were investigated. Chemotherapy tests
showed that the a large number of the studied compounds had a statistically important
antitumour activity. In addition to 5-fluoro- and 5-iodouracils exhibited more pronounced
anti-tumour properties than 5-bromouracil derivatives.6
The carbonic anhydrases (CA; carbonate hydrolyase, EC 4.2.1.1) are a ubiquitous
family of zinc-containing enzymes, which classically participate in the maintenance of pH
homeostasis in the human body, catalyzing the reversible hydration of carbon dioxide in a

two-step reaction to yield bicarbonate and protons.7 Isoforms of carbonic anhydrase are found
in several of tissues where they participate in many important biological processes such as
acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis,
gluconeogenesis, lipogenesis and electrolyte secretion. Many CA isozymes involved in these
processes are important therapeutic targets with the potential to be inhibited / activated for the
treatment of a range of disorders such as edema, glaucoma, obesity, cancer, epilepsy and
osteoporosis.2-4
Some research groups recently investigated the interaction of 12 mammalian CA
isozymes with phenol and several types of phenolic compounds and determined the inhibitory
action of the phenols.6 In this study we extend these researches to series of phenolic
compounds, some of which are widely used as antioxidant food additives or as drugs and
prodrugs. Among the various natural or unnatural phenolic compounds with antioxidant
properties, these compounds are very active in quenching reactive oxygen species.9c They are
reported to possess anticancer, anti-carcinogenic, antimutagenic, antibacterial, antiviral or
anti-inflammatory activities.10 Phenol, phenolic compounds and hydroxybenzoic acid
derivatives are widely used as prodrugs or drugs. Salicylic acid is known for its ability to ease
aches, pains, and reduce fevers. These medicinal properties, particularly fever relief, have
been known since ancient times, and it was used as an anti-inflammatory drug.9,10c
In the present study we have purified CA I and II (hCA I and hCA II) from human
erythrocytes and examined the in vitro inhibition effects of above mentioned uracil
derivatives 3-9 on these enzymes (Table 1), using the esterase activity of hCA I and II, with 4-
NPA as substrate. Uracil derivatives 3-513 were synthesized from commercially available
uracil (1). For this purpose uracil 1 was converted to the brom uracil 214a by using 1.1 Eq
o
bromine in DMF at 120 C. Condensation of brom uracil 2 with methanesulfonyl chloride, p-
toluenesulfonyl chloride and acetic anhydryde to afford uracil derivatives 3 which has
antitomour activity14a, 414b and 514c in comparable yield, respectively. Also uracil (1) was
converted to uracil derivative 614d by using pyrdine and acetic anhydryde. The other tested
compounds 7-12 were purchased from sigma aldrich.
Scheme 1, Figure 2 and Table 1 here
(i) Against the slow cytosolic isozyme hCA I, compounds 3, 4, and 8 behave as weak
inhibitors, with Ki values in the range of 316.2-464 µM, similarly to the structurally related
compounds 10 and 11 (KIs of 795 and 4003 µM). It is interesting that compound 5 was much

better hCA I inhibitor as compared to the corresponding compounds 7 and 9 from which it
was derivated. Kinetic investigations (Lineweaver-Burk plots, data not shown) indicate that
similarly to sulfonamides and inorganic anions,9-12 all the investigated natural compounds act
as competitive inhibitors with 4-NPA as substrate.12a
(ii) A better inhibitory activity has been observed with compounds 10-12 investigated
here for the inhibition of the rapid cytosolic isozyme hCA II (Table 1). Structure-activity
relationship (SAR) is thus quite sharp for this small series of hydroxylic compounds:
compounds 10, 11 and 12 are effective leads, with two mono or di hidroxy moieties is already
a submicromolar hCA II inhibitor. This effect is maintained when different groups are present
in the meta position to the phenol OH moiety, such as in resorcinol. The best hCA II inhibitor
in this series of derivatives were compound 5 with a KI of 28.88 µM.11
In a recent study it was reported that catechol and resorcinol10a act as a CAI inhibitor,
and could represent the starting point for a new class of inhibitors that may have advantages
for patients with sulfonamide allergies.11 The sulfonamide zinc-binding group is thus superior
to the thiol one (from the thioxolone hydrolysis product) for generating CA inhibitors with a
varied and sometimes isozyme-selective inhibition profile against the mammalian enzymes.
However, it is still important to explore further classes of potent CAIs in order to detect
compounds with different inhibition profiles.
Compounds 3-12 used in this study affect the activity of CA isozymes due to the
presence of the different functional groups (OH, Br, mesityl, tosyl, and acetate) present in
their scaffold. Therefore, our findings indicate another class of possible CAIs of interest, in
addition to the well-known inhibitors, the phenols/biphenyl diphenols bearing bulky ortho
moieties in their molecules. Some hyroxylic compounds investigated here exhibited effective
hCA I and II inhibitory activity, in the low micromolar range, by the esterase method which
usually gives KI-s an order of magnitude higher as compared to the CO2 hydrase assay.15
These findings point out that substituted hdroxylic compounds may be used as leads for
generating potent CAIs eventually targeting other isoforms.
Declarations of interest
The authors report no declarations of interest

Acknowledgments
We are grateful to Prof. Dr. Claudiu T. Supuran, University of Florence, for various valuable
studies on carbonic anhydrase inhibitors. This study was financed by Agri Ibrahim Cecen
University Scientific Research Council, (project no: Agri BAP-2010/K-09).
References and Notes
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Fidan, I.; Durdagi, S.; Kaban, S.; Supuran, C. T. J. Enzyme Inhib. Med. Chem. 2013,
28, 370 (d) Koz, O.; Ekinci, D.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O.;
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7. (a) Maren, T. H. Annu. Rev. Physiol. 1988, 50, 695; (b) Supuran, C. T. Nat. Rev. Drug
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2010, 18, 3559; (b) Alp, C.; Ekinci, D.; Gultekin, M. S.; Senturk, M.; Sahin, E.;
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Abbas, G.; Senturk, M.; Supuran, C. T. J. Enz. Inhib. Med. Chem. 2012, 27, 744.
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Am. Chem. Soc. 1934, 57, 685.
13. Detailed synthetic procedures for the preparation of all derivatives can be found in:
Synthesis of 5-Bromo-1H-pyrimidine-2,4-dione (2): To a magnetiacally stirred suspension
of 1H-Pyrimidine-2,4-dione (1) (1.00 g, 8.92 mmol) in 5 ml of DMF at 120 oC was added
dropwise bromine (1.57 g, 9.81 mmol) over a period of 5 min. After being stirred for 4 h,
reaction mixture was cooled to room temperature. CH2Cl2 was added into the reaction
mixture and the obtained solid was filtered and identifyed as 5-Bromo-1H-pyrimidine-
o
1
2,4-dione (2) (1.50 g, 88%). Colorless crystals, m.p. > 300 C. H-NMR (400 MHz,
DMSO-d6): δ 11.48 (bs, 1H), 11.20 (bs, 1H), 7.88 (d, J= 6.2 Hz, 1H). 13C-NMR (100
MHz, DMSO-d6): δ 160.80, 151.53, 142.88, 95.14.
5-Bromo-1-methanesulfonyl-1H-pyrimidine-2,4-dione (3): A 2.5 M solution of n-BuLi in
hexanes (0.63 mL, 1.57 mmol) was added dropwise to a solution of monobromide 2 (300

mg, 1.57 mmol) in dry THF (10 mL) at 0 ºC and the resulting mixture was stirred 30 min.
at the same temperature. Methanesulfonyl chloride (0.12 mL, 1.57 mmol) was added
portion wise. The mixture was stirred 10 min at 0 ºC and was stirred at room temperature 3
h. The resulting suspension was treated with CH2Cl2, filtered and product 3 was obtained
1
(240 mg, 57%). White crystals, m.p. 237-239 oC. H-NMR (400 MHz, DMSO-d6): δ 12.32
(s, 1H, NH), 8.07 (s, 1H), 3.68 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): δ 159.75, 148.52,
137.59, 98.69, 42.18.
1-Acetyl-5-bromo-1H-pyrimidine-2,4-dione (4): To the monobromide 2 (100 mg, 0.52
mmol) ) a solution of acetic anhydride (190 mg, 1.86 mmol) and pyridine (3 mL) was
added. The reaction mixture was kept under nitrogen at room temperature for 24 h. The
1
excess acetic anhydride and the solvent were removed under vacuum and the H NMR
analysis of residue indicated the formation of the acetate 4 (122 mg, 100%).
1
Recrystallization from CH2Cl2/n-hexane (1:3), white crystals, m.p. 174-175 ºC. H-NMR
(400 MHz, Acetone-d6): δ 10.74 (bs, 1H, NH), 8.48 (s, 1H, CH), 2.68 (s, 3H, CH3) 13C-
NMR (100 MHz, Acetone-d6): δ 169.24, 158.70, 149.15, 137.01, 100.22, 26.18. IR (KBr,
cm-1): 3070, 2857, 1708, 1619, 1406, 1362, 1263, 1171, 1037, 976, 855.
5-Bromo-1-(toluene-4-sulfonyl)-1H-pyrimidine-2,4-dione (5): A 2.5 M solution of n-BuLi
in hexanes (0.21 mL, 0.52 mmol) was added dropwise to a solution of monobromide 2
(100 mg, 0.52 mmol) in dry THF (10 mL) at 0 ºC and the resulting mixture was stirred 30
min. at the same temperature and p-toluenesulfonyl chloride (100 mg, 0.52 mmol) was
added portion wise. The mixture was stirred 10 min at 0 ºC and was stirred at room
temperature 20 h. The crude was quenched with water (50 mL), extracted with EtOAc
(3x30 mL). The combined organic extracts were dried over MgSO4, concentrated in vacuo.
The crude product was purified by crystallization from hot methanol to give 5-Bromo-1-
(toluene-4-sulfonyl)-1H-pyrimidine-2,4-dione (5) (120 mg, % 66). White crystals, m.p.
1
252-254 ºC. H-NMR (400 MHz, DMSO-d6): δ 12.16 (bs, 1H, NH), 8.38 (d, J= 0.7, 1H,
CH), 7.96 (d, J= 7.9 Hz, 2H, TsH) 7.47 (d, J= 7.9 Hz, 2H, TsH), 2.41 (s, 3H, CH3). 13C-
NMR (100 MHz, DMSO-d6): δ 159.63, 147.37, 147.24, 137.46, 133.51, 130.54, 129.92,
99.51, 21.90. IR (KBr, cm-1): 3148, 3053, 2913, 2845,1756, 1682, 1376, 1253, 1173, 1065,
998, 811.

1-Acetyl-1H-pyrimidine-2,4-dione (6): The reaction was carried out according to the above
mentioned procedure by using 100 mg (0.89 mmol) of 1, pyridine (3 mL), and acetic
1
anhydride (320 mg, 3.13 mmol). After 48 h, the solvent was evaporated and the H NMR
analysis of a residue indicated the formation of the acetate 6 ( 137 mg, 100%).
1
Recrystallization from CH2Cl2/n-hexane (1:3), white crystals, m.p. 190-192 ºC. H-NMR
(400 MHz, CDCl3): δ 8.70 (bs, 1H, NH), 8.23 (d, J= 8.4, 1H, H6), 5.89 (dd, J= 8.4, J= 1.83
Hz, 1H, H5), 2.76 (s, 3H, CH3). 13C-NMR (100 MHz, CDCl3): δ 169.56, 162.51, 149.36,
-1
137.60, 104.98, 27.27. IR (KBr, cm ): 3014, 2823, 1730, 1683, 1630, 1440, 1397, 1222,
1107, 830.
14. (a) Glavasˇ-Obrovac, L.; Karner, I.; Pavlak, M.; Radacˇic, M.; Kasˇnar-Sˇamprec, J.;
Zˇinic´ B. Nucleos Nucleot Nucl. 2005, 24, 557; (b) Fytelson, M.; Johnson, B. T.; J. Am.
Chem. Soc., 1942, 64, 306; (c) Višnjevac, A.; Žinić, M.; Luić, M.; Žiher, D.; Kajfež
Novak, T.; Žinić, B. Tetrahedron, 2007, 63, 86; (d) Spector, B. L.; Keller, B. E. J. Biol.
Chem. 1958, 232, 185.
15. Detailed procedures for enzyme purification can be found in: (a) Ekinci, D.; Cavdar, H.;
Durdagi, S.; Talaz, O.; Senturk, M.; Supuran, C. T. Eur. J. Med. Chem. 2012, 49, 68; (b)
Ekinci, D.; Ceyhun, S. B.; Senturk, M.; Erdem, D.; Kufrevioglu, O. I.; Supuran, C. T.
Bioorg. Med. Chem. 2011, 19, 744; (c) Ekinci, D.; Kurbanoglu, N. I.; Salamci, E.; Senturk,
M.; Supuran, C. T. J. Enzym. Inhib. Med. Chem. 2012, 27, 845.

Figure 1. Tautomeric forms of Uracil: lactam-lactim
Scheme 1. Compounds 1-6 sythesis scheme.

Figure 2. Structure of tested compounds.
Table 1: hCA I and II inhibition data some compounds, by an esterase assay with 4-
nitrophenylacetate as substrate.12b
KI (µM)*
Compound
hCA I
428 2.42
464 3.24
10.83 0.76
57.76 0.28
316.2 5.32
49.51 0.35
795
hCA II
645 3
778.5 5.3
28.88 0.92
NE
3
4
5
7
166.4 2.46
NE
8
9
10a
7.7
11a
12a
4003
9.9
10.2
5.5
Acetazolamideb
36.2
0.37
a From Ref 6b. b From Ref 14d. NE: No-effect.
* Mean from at least three determinations. Errors in the range of 3- 8 % of the reported value
(data not shown).

Synthesis and carbonic anhydrase inhibitory properties of novel uracil derivatives
Murat Güney*, Hüseyin Çavdar, Murat Şentürk*, Deniz Ekinci,