New 2-hydroxyethyl substituted N-Heterocyclic carbene precursors: Synthesis, characterization, crystal structure and inhibitory properties against carbonic anhydrase and xanthine oxidase

Article abstract of DOI:10.1016/j.molstruc.2019.02.063

Here, the synthesis, spectral and the structural studies of 2-hydroxyethyl substituted N-heterocyclic carbene (NHC) precursors and the enzyme inhibition activities of the NHC precursors were investigated against the cytosolic carbonic anhydrase I and II i

Full text of DOI:10.1016/j.molstruc.2019.02.063

Accepted Manuscript
New 2-hydroxyethyl substituted N-Heterocyclic carbene precursors: Synthesis,
characterization, crystal structure and inhibitory properties against carbonic
anhydrase and xanthine oxidase
Aydın Aktaş, Samir Abbas Ali Noma, Duygu Barut Celepci, Fatoş Erdemir, Yetkin
Gök, Burhan Ateş
PII:
S0022-2860(19)30193-0
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.063
MOLSTR 26220
Reference:
To appear in: Journal of Molecular Structure
Received Date: 8 December 2018
Revised Date: 1 February 2019
Accepted Date: 15 February 2019
Please cite this article as: Aydı. Aktaş, S.A. Ali Noma, D.B. Celepci, Fatoş. Erdemir, Y. Gök, B. Ateş,
New 2-hydroxyethyl substituted N-Heterocyclic carbene precursors: Synthesis, characterization,
crystal structure and inhibitory properties against carbonic anhydrase and xanthine oxidase, Journal of
Molecular Structure (2019), doi: https://doi.org/10.1016/j.molstruc.2019.02.063.
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ACCEPTED MANUSCRIPT
New 2-Hydroxyethyl Substituted N-Heterocyclic Carbene Precursors:
Synthesis, Characterization, Crystal Structure and Inhibitory Properties
Against Carbonic Anhydrase and Xanthine Oxidase
Aydın Aktaşa*, Samir Abbas Ali Nomaa, Duygu Barut Celepcib, Fatoş Erdemira, Yetkin Göka,
Burhan Ateşa
1Department of Chemistry, Faculty of Arts and Sciences, Inönü University, 44280, Malatya, Turkey
2Dokuz Eylül University, Faculty of Sciences, Department of Physics, 35160-Buca,
İzmir, Turkey
*Corresponding Author: Aydın Akta
Address: Department of Chemistry
ş
Inonu University, Malatya 44280, Turkey
Phone: 90-5356891975
Email: aydinaktash@hotmail.com
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Abstract
Here, the synthesis, spectral and the structural studies of 2-hydroxyethyl substituted N-
heterocyclic carbene (NHC) precursors and the enzyme inhibition activities of the NHC
precursors were investigated against the cytosolic carbonic anhydrase I and II isoenzymes (hCA I
and hCA II), and xanthine oxidase (XO). The IC50 values of NHC precursors against these
enzymes were determined by spectrophotometric method. The spectra of new NHC precursors
1
have been obtained by using H NMR, 13C NMR, FTIR spectroscopy and elemental analysis
techniques. The structure of a new NHC precursor was established by using single-crystal X-ray
diffraction method. The results of inhibition experiment indicated that all 2-hydroxyethyl
substituted NHC derivatives showed remarkable inhibition activity toward hCA I, hCA II and
XO. The range of IC50 values for hCA I, hCA II and XO inhibition was determined as 0.1565-
0.5127, 0.1524-0.5368 and 1.253-5.342 µM. Especially, trimethylbenzyl derivative of 2-
hydroxyethyl substituted NHC precursor has demonstrated high inhibition effect on all studied
enzymes due to steric bulk of this substituent.
Keywords: Carbonic anhydrase; Crystal structure; Enzyme inhibition; N-heterocyclic carbene
precursor; Xanthine oxidase.
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1. Introduction
The first complex with the carbene ligand which is heteroatom and stabilized was
prepared by Tschugajeff (English transcription, Chugaev) in 1925 [1]. The reactivity and stability
of N-heterocyclic carbenes (NHC) were investigated by Wanzlick in the early 1960s [2].
Wanzlick and Öfele independently synthesized stable NHC transition metal complexes in 1968
[3]. Arduengo et al. discovered extraordinary stability, isolation, and storability of crystalline
NHC IAd in 1991 [4]. Hence, NHC precursors have remained in the shadow of metal-NHC
complexes for three decades. Numerous studies on NHCs were published after the stable NHC
synthesis of Arduengo et al.
NHCs are neutral ligands that can be altered electronically and sterically. These ligands
have strong σ-donor and weak π-acceptor properties [5-8]. In addition, NHC ligands that are
resistant to the air and moisture can form stable complexes with most of the d-block and f-block
metals. [9-14] Due to these unique properties, the NHC ligands are still alternative ligands to the
phosphine ligands, which are often used in the industry [15].
NHC precursors and their metal complexes are of great interest in the fields of organic
and organometallic synthesis. Numerous studies on the catalytic activity of the transition metal
complexes of NHC ligands have been carried out for many years [16-22]. Several studies on the
biological activities and medical applications of these complexes have been carried out in the last
two decades [23-26]. Also, studies on the biological applications of NHC ligands have been
carried out. From these studies, enzyme inhibition studies of NHC precursors are noteworthy [27-
32].
Carbonic anhydrase (CA, carbonate hydrolase, E.C. 4.2.1.1) is a metalloenzyme that is
very important for biological systems and contains a zinc (Zn+2) ion in its active site [33-36]. CAs
are important enzymes that have attracted considerable interest [37, 38]. CA inhibitors can serve
as useful therapeutic agents for treating numerous diseases, including glaucoma, cancer, and
obesity because of the roles of CAs in many physiological and pathophysiological processes,
[39,40]. CA basically ensures that the CO2 emerging from respiration is dissolved in water,
transported and driven out of body as well as plays an important role in many physiological
events such as acid-base balance, ion exchange, regulation of the cardiovascular system [41,42].
CA catalyzes the rapid and reversible hydration of carbon dioxide (CO2) and water (H2O) in
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-
twostep reaction to yield bicarbonate (HCO3 ) and a proton (H+) to maintain acid-base balance in
blood and other tissues, and to help transport CO2 out of tissues in biological systems [43,44].
Xanthine oxidase (XO, EC 1.17.3.2) is a critical, rate-limiting enzyme in purine
metabolism [45]. XO, a highly versatile flavoprotein enzyme, is ubiquitous among species (from
bacteria to human) and within various tissues in mammals [46]. It catalyzes the last two steps of
purine catabolism in humans, i.e., hydroxylation of hypoxanthine to xanthine and xanthine to uric
acid [47]. In parallel with the hydroxylation process, two kinds of reactive oxygen species (ROS),
-
superoxide anion (O2 ) and hydrogen peroxide (H2O2), are produced [48]. XO is therefore a
critical source of uric acid and ROS. Over-production of uric acid can lead to hyperuricemia,
which is the key cause of gout. XO is considered the most promising target for treating
hyperuricemia and gout [49]. An increase in the excess of ROS production cause various
pathological states including inflammation, metabolic disorders, atherosclerosis, cancer and
chronic obstructive pulmonary disease [50, 51]. The inhibition of this enzyme would be
beneficial to reduce the formation of ROS, knowing that its serum levels are significantly
increased in various pathological states, like inflammation, ischemia-reperfusion, vascular
diseases, and carcinogenesis [52]. Allopurinol is a prototypical XO inhibitor and has been widely
used in the treatment of hyperuricemia and gout for several decades.
Previously, the synthesis of different 2-hydroxyethyl substituted metal-NHC complexes
and their catalytic activities were investigated by our study group [53-55]. In this study,
inhibition effects of a new series of 2-hydroxyethyl substituted NHC precursors on carbonic
anhydrase and xanthine oxidase enzymes were investigated. The structure of a compound from
the NHC precursors was established through the single-crystal X-ray diffraction method.
2. Experimental
All synthesis involving 2-hydroxyethyl substituted NHC precursors 1a-f and 2a-c were carried out
under an inert atmosphere in flame-dried glassware using standard Schlenk techniques. The
solvents were commercial products and were used without purification. All other reagents were
purchased from Aldrich, Merck and Alfa Aesar Chemical Co, and used without further
purification. Melting points were recorded in glass capillaries under air with an Electrothermal-
9200 melting point apparatus. The UV-Vis absorption was obtained from a ethyl alcohol
(C2H5OH) solution and recorded with a Shimadzu UV-1601 instrument UV-Vis
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spectrophotometer. FTIR spectra were recorded in the range 400-4000 cm-1 on Perkin Elmer
1
13
Spectrum 100 FT-IR spectrometer. H and C NMR spectra were recorded using a Bruker AS
400 Merkur spectrometer operating at 400 MHz (1H) or 100 MHz (13C) in CDCl3 and DMSO-d6
with tetramethylsilane as an internal reference. Elemental analyses were performed by İnönü
University Scientific and Technological Research Center (Malatya, TURKEY).
Single-crystal X-ray diffraction data set of the complex 2a was collected at room temperature on a
Rigaku-Oxford Xcalibur diffractometer with an Eos-CCD detector using graphite-monochromated
Mo-Kα radiation (λ = 0.71073 Å). Data collection and reduction along with absorption correction
was performed using CrysAlisPro software package [56]. Structure solution was performed using
Intrinsic Phasing method with SHELXT [57] package program embedded in the Olex2 [58].
Refinement of coordinates and anisotropic thermal parameters of non-hydrogen atoms were
carried out by the full-matrix least-squares method in SHELXL [59]. All non-hydrogen atoms
were refined anisotropically. Except the hydrogen of the hydroxyethyl oxygen atom, all hydrogen
atom positions were calculated geometrically and refined using the riding model. The OH
hydrogen was located in a difference Fourier map and refined freely. The details of the crystal
data, data collection and structure refinement of the compound are summarized in Table 1.
Table 1. Crystal data and experimental details for the complex 2a.
Empirical Formula
Formula Weight
C10H13IN2O
304.12
Temperature (K)
Crystal System, space group
a, b, c (Å)
293(2)
Monoclinic, P21/n
9.3224(5), 8.8270(5), 14.4183(7)
93.289(5)
β (⁰)
V (Å3)
1184.51(11)
Z
4
Density (calculated) (g/cm3)
Absorbtion coefficient (µ, mm-1)
F(000)
1.705
2.676
592
Crystal size (mm3)
0.416 × 0.354 × 0.290
Radiation
MoKα (λ=0.71073)
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6.362 to 51.348
2θ range for data collection (⁰)
Index ranges
-11
≤
h
≤11, -5
≤
k
≤
10, -11
≤
l
≤17
Reflections collected
Independent reflections
Parameters
4287
2256 [Rint = 0.019, Rsigma = 0.033]
129
Goodness-of-fit on F2
1.033
Final R indices [ I
R indices
Largest diff. peak/hole (eÅ-3)
≥
2
σ
(I) ]
R1 = 0.033, wR2 = 0.071
R1 = 0.047, wR2 = 0.078
0.45/-0.58
2.1. Synthesis of 1-(2-hydroxyethyl)-3-(2-methylbenzyl)benzimidazolium chloride, 1a
For the synthesis of 1a as described in the literature [30], 2-methylbenzyl chloride (1.41 g, 10
mmol) and 1-(2-tetrahydro-pyran-2-yloxyethyl)benzimidazole (2.46 g, 10 mmol) was added in
dry DMF (4 mL). Yield: 85 %; (2.57 g); m.p.: 147-149 °C; ν(CN): 1557 cm-1; ν(O-H): 3175 cm-1.
1
Anal. Calc. for C17H19ClN2O: C: 67.43; H: 6.32; N: 9.25. Found: C: 67.65; H: 6.40; N: 9.18. H
NMR (400 MHz, CDCl3); δ 2.38 (s, 3H, -NCH2(C6H4(CH3)); 4.09 (s, 2H, -NCH2CH2OH); 4.78
(s, 2H, -NCH2CH2OH); 5.80 (s, 2H, -NCH2(C6H4(CH3)); 5.96 (s, 1H, -NCH2CH2OH); 7.16-7.84
( m, 4H, -N(C6H4)N- Ar-H); 10.68 (s, 1H, 2-CH). 13C NMR (100 MHz, CDCl3); δ 21.4 (-
NCH2(C6H4(CH3)); 49.7 (-NCH2CH2OH); 51.6 (-NCH2(C6H4(CH3)); 59.0 (-NCH2CH2OH);
113.6, 113.8, 126.9, 127.0, 128.7, 128.8, 129.2, 130.0, 131.2, 131.4, 131.9, 132.5, 132.7 and
139.3 (Ar-C); 143.4 and 143.9 (2-CH).
2.2. Synthesis of 1-(2-hydroxyethyl)-3-(3-methylbenzyl)benzimidazolium chloride, 1b
The synthesis of 1b was carried out in the same method as that described for 1a, but 3-
methylbenzyl chloride (1.41 g, 10 mmol) was used instead of 2-methylbenzyl chloride. Yield: 91
% (2.75 g); m.p.: 110-112 °C; ν(CN): 1559 cm-1; ν(O-H): 3237 cm-1. Anal. Calc. for C17H19ClN2O:
1
C: 67.43; H: 6.32; N: 9.25. Found: C: 67.58; H: 6.41; N: 9.21. H NMR (400 MHz, CDCl3); δ
2.24 (s, 3H, -NCH2(C6H4(CH3)); 4.08 (s, 2H, -NCH2CH2OH); 4.70 (s, 2H, -NCH2CH2OH); 5.64
(s, 2H, -NCH2(C6H4(CH3)); 5.77 (s, 1H, -NCH2CH2OH); 7.04-7.77 ( m, 4H, -N(C6H4)N- Ar-H);
10.76 (s, 1H, 2-CH). 13C NMR (100 MHz, CDCl3); δ 21.2 (-NCH2(C6H4(CH3)); 49.6 (-
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NCH2CH2OH); 51.5 (-NCH2(C6H4(CH3)); 58.7 (-NCH2CH2OH); 113.0, 113.7, 113.8, 127.0,
128.2, 128.3, 129.5, 129.7, 130.1, 131.2, 131.9 and 139.3 (Ar-C); 143.4 (2-CH).
2.3. Synthesis of 1-(2-hydroxyethyl)-3-(4-methylbenzyl)benzimidazolium chloride, 1c
The synthesis of 1c was carried out in the same method as that described for 1a, but 4-
methylbenzyl chloride (1.41 g, 10 mmol) was used instead of 2-methylbenzyl chloride. Yield: 88
% (2.66 g); m.p.: 135-137 °C; ν(CN): 1562 cm-1; ν(O-H): 3305 cm-1. Anal. Calc. for C17H19ClN2O:
1
C: 67.43; H: 6.32; N: 9.25. Found: C: 67.59; H: 6.42; N: 9.19. H NMR (400 MHz, CDCl3); δ
2.24 (s, 3H, -NCH2(C6H4(CH3)); 4.08 (s, 2H, -NCH2CH2OH); 4.70 (s, 2H, -NCH2CH2OH); 5.64
(s, 2H, -NCH2(C6H4(CH3)); 5.77 (s, 1H, -NCH2CH2OH); 7.09-7.68 ( m, 4H, -N(C6H4)N- Ar-H);
10.89 (s, 1H, 2-CH). 13C NMR (100 MHz, CDCl3); δ 21.2 (-NCH2(C6H4(CH3)); 49.6 (-
NCH2CH2OH); 51.5 (-NCH2(C6H4(CH3)); 58.7 (-NCH2CH2OH); 113.0, 113.7, 127.0, 128.2,
128.3, 129.4, 130.1, 131.2, 131.9, 132.6 and 139.3 (Ar-C); 143.4 (2-CH).
2.4. Synthesis of 1-(2-hydroxyethyl)-3-(2,4,6-trimethylbenzyl)benzimidazolium chloride, 1d
The synthesis of 1d was carried out in the same method as that described for 1a, but 2,4,6-
trimethylbenzyl chloride (1.69 g, 10 mmol) was used instead of 2-methylbenzyl chloride. Yield:
83 % (2.74 g); m.p.: 181-183 °C; ν(CN): 1557 cm-1; ν(O-H): 3241 cm-1. Anal. Calc. for
1
C19H23ClN2O: C: 68.97; H: 7.01; N: 8.47. Found: C: 69.09; H: 7.11; N: 8.51. H NMR (400
MHz, CDCl3); δ 2.20-2.23 (s, 9H, -NCH2(C6H2(CH3)3); 3.94 (s, 2H, -NCH2CH2OH); 4.67 (s, 2H,
-NCH2CH2OH); 5.63 (s, 2H, –NCH2(C6H2(CH3)3); 5.78 (s, 1H, -NCH2CH2OH); 6.86 (s, 2H, –
NCH2(C6H2(CH3)3); 7.38-7.86 ( m, 6H, Ar-H); 9.93 (s, 1H, 2-CH). 13C NMR (100 MHz, CDCl3);
δ 20.1-21.1 (-NCH2(C6H2(CH3)3); 46.7 (-NCH2CH2OH); 49.9 (-NCH2(C6H2(CH3)3); 59.1 (-
NCH2CH2OH); 113.4, 113.6, 124.8, 126.2, 126.3, 127.1, 128.8, 129.2, 130.1, 131.1, 131.4,
131.9, 138.0, 138.1 and 139.8 (Ar-C); 142.3 (2-CH).
2.5. Synthesis1-ethyl-3-(2-hydroxyethyl)benzimidazolium bromide, 1e
The synthesis of 1e was carried out in the same method as that described for 1a, but ethyl
bromide (1.1 g, 10 mmol) was used instead of 2-methylbenzyl chloride. Yield: 88 % (2.38 g);
m.p.: 96-98 °C ν(CN): 1560 cm-1; ν(O-H): 3325 cm-1. Anal. Calc. for C11H15BrN2O: C: 48.72; H:
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5.58; N: 10.33. Found: C: 48.79; H: 5.62; N: 10.31. 1H NMR (400 MHz, CDCl3); δ 1.65 (t, 3H, J
= 8 Hz -NCH2CH3); 3.90 (s, 1H, -NCH2CH2OH); 4.02 (t, 2H, J = 6 Hz -NCH2CH2OH); 4.54 (t,
2H, J = 6.7 Hz -NCH2CH3); 4.72 (t, 2H, J = 4 Hz -NCH2CH2OH); 7.52-7.85 ( m, 4H, -
N(C6H4)N- Ar-H); 10.42 (s, 1H, 2-CH). 13C NMR (100 MHz, CDCl3); δ 14.7 (–NCH2CH3); 43.0
(-NCH2CH2OH); 59.5 (-NCH2CH2OH); 49.8 (-NCH2CH3); 113.0, 114.0, 127.0, 127.1, 127.8,
131.0, 131.2 and 131.9. (Ar-C); 141.7 (2-CH).
2.6. Synthesis of 1-(2-hydroxyethyl)-3-isopropylbenzimidazolium bromide, 1f
The synthesis of 1f was carried out in the same method as that described for 1a, but isopropyl
bromide (1.23 g, 10mmol) was used instead of 2-methylbenzyl chloride. Yield: 79 % (2.25 g);
m.p.: 146-147 °C; ν(CN): 1548 cm-1; ν(O-H): 3323 cm-1. Anal. Calc. for C12H17BrN2O: C: 50.54; H:
6.01; N: 9.82. Found: C: 50.61; H: 6.06; N: 9.79. 1H NMR (400 MHz, CDCl3); δ 1.78 (d, 6H, J =
4 Hz -NCH(CH3)2); 4.08 (s, 2H, -NCH2CH2OH); 4.10 (s, 1H, -NCH2CH2OH); 4.80 (s, 2H, -
NCH2CH2OH); 4.92 (m, 1H,-NCH((CH3)2); 7.57-7.81 ( m, 4H, -N(C6H4)N- Ar-H); 10.36 (s, 1H,
2-CH). 13C NMR (100 MHz, CDCl3); δ 22.4 (–NCH(CH3)2); 49.6 (-NCH2CH2OH); 51.9 (–
NCH(CH3)2); 59.1 (-NCH2CH2OH); 113.4, 113.8, 127.1, 127.3, 130.6 and 132.1. (Ar-C); 140.4
(2-CH).
2.7. Synthesis of 1-(2-hydroxyethyl)-3-methylbenzimidazolium iodide, 2a
For the synthesis of 2a as described in the literature [30], 1-methylbenzimidazole (1.3 gr, 10
mmol) and 2-iodoethanol (1.25 g, 10 mmol) was added in DMF (4 mL). Yield: 83 % (2 g); m.p.:
0
148-150 C, v(CN): 1568 cm-1; ν(O-H): 3326 cm-1. Anal. Calc. for C10H13IN2O: C: 39.49; H: 4.31;
1
N: 9.21. Found: C: 39.41; H: 4.27; N: 9.15. H NMR (400 MHz, DMSO-d6), δ 4.12 (s, 3H,
NCH3); 4.57 (t, 2H, J: 4, NCH2CH2OH); 3.83 (m, 2H, J: 6.6, NCH2CH2OH); 5.19 (s, 1H,
NCH2CH2OH); 7.72-8.10 (m, 4H, Ar-H); 9.69 (2-CH). 13C NMR (100 MHz, DMSO-d6), δ 33.7
(NCH3); 49.9 and 5.1 (NCH2CH2OH); 114.0, 114.2, 126.8, 126.9, 131.6 and 132.3 (Ar-C); 143.4
(2-CH).
2.8. Synthesis of 1-ethyl-3-(2-hydroxyethyl)benzimidazolium iodide, 2b
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The synthesis of 2b was carried out in the same method as that described for 2a, but 1-
ethylbenzimidazole (1.5 g, 0.6 mmol) was used instead of 1-methylbenzimidazole. Yield: 80 %
(2.25 g); m.p.: 135-136 °C; ν(CN): 1566 cm-1; ν(O-H): 3341 cm-1. Anal. Calc. for C11H15IN2O: C:
1
41.53; H: 4.75; N: 8.81. Found: C: 41.49; H: 4.70; N: 8.76. H NMR (400 MHz, DMSO-d6), δ
1.53 (t, 3H, J=7,3 Hz, -NCH2CH3); 5.37 (s, 1H, -NCH2CH2OH); 4.50 (m, 2H, -NCH2CH2OH);
4.53 (t, 2H, J=5.0 Hz, NCH2CH2OH); 2.51 (m, 2H, J:1.6, NCH2CH3); 7.66-8.03 (m, 4H, Ar-H);
9.59 (2-CH). 13C NMR (100 MHz, DMSO-d6), δ 14.6 (NCH2CH3); 42.6 and 49.9
(NCH2CH2OH); 59.1 (NCH2CH3); 113.9, 114.2,127.1, 131.3 and 131.7 (Ar-C); 142.1 (2-CH).
2.9. Synthesis of 1-(2-hydroxyethyl)-3-isopropylbenzimidazolium iodide, 2c
The synthesis of 2c was carried out in the same method as that described for 2a, but 1-
isopropylbenzimidazole (1.5 g, 0.6 mmol) was used instead of 1-methylbenzimidazole. Yield: 70
0
% (2.1 g); m.p.: 135-137 C, v(CN): 1557 cm-1. ν(O-H): 3306 cm-1. Anal. Calc. for C12H17IN2O: C:
43.39; H: 5.16; N: 8.43. Found: C: 43.35; H: 5.13; N: 8.39. 1H NMR (400 MHz, CDCl3), δ 1.77
(d, 6H, J=6.7 Hz, NCH(CH3)2); 4.63 (s, 1H, NCH2CH2OH); 4.09 (t, 2H, J=4.8 Hz,
NCH2CH2OH); 4.79 (t, 2H, J=4.0 Hz, NCH2CH2OH); 4.92 (m, 1H, J:6.6 Hz, NCH(CH3)2); 7.57-
7.82 (m, 4H, Ar-H); 10.32 (2-CH). 13C NMR (100 MHz, CDCl3), δ 22.4 (NCH(CH3)2); 49.6 and
51.9 (NCH2CH2OH); 59.1 (NCH2(CH3)2); 113.4, 113.8, 127.1, 127.3, 130.6 and 132.1 (Ar-C);
140.4 (2-CH).
2.10. Purification of hCA isoenzymes and esterase activity assay
The purification and inhibition studies hCA I and II isoenzymes were purified according to
methods described by Gocer et al [60]. The protein quantity in the purification step was also
determined spectrophotometrically at 280 nm. For visualizing of both CA isoenzymes purity,
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The
esterase activity of CA isoenzymes was measured according to the method described by
Verpoorte et al [61]. The change in absorbance at 348 nm of 4-nitrophenylacetate (NPA) to 4-
nitrophenylate was recorded spectrophotometrically for 3 min at room temperature. NPA was
used as the substrate (Figure 1). The reaction mix contained 50 mM Tris–SO4 buffer (pH 7.4), 3.0
mM 4-NPA, distilled water, and purified enzyme. The inhibition studies of the esterase’s were
carried out by adding of inhibitor substance at different concentrations to the medium instead of
9

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part of the distilled water. Inhibitory activity was expressed as IC50 values (the concentration at which
50 % of the enzyme activity inhibited), which were calculated using Graphpad Prism 5.0 from a dose-
response curve. Each experiment was carried out as triplicates.
Figure 1. The mechanism in the conversion of 4-nitrophenylacetate to 4-nitrophenol by Carbonic
anhydrases.
2.11. Xanthine oxidase activity assay
XO activity was determined by quantifying the amount of uric acid produced from xanthine in
the reaction mixture. Inhibition of XO catalyzed reaction was measured by observing the
decrease in the uric acid formation at 294 nm. The reaction mixtures contain 0.2 U XO, 50 mM
phosphate buffer (pH 7.4), different concentration of tested compounds and 1 mM xanthine
prepared freshly was added to initiate the reaction. The reaction mixture was incubated for 10
min at 37 °C before added xanthine, then xanthine was added to start the reaction and the activity
was measured at wavelength 294 nm. All the experiments were performed in triplicates, then the
average of three reading was taken. Allopurinol was used as a positive control. Then inhibition
percentage (IC50) of XO was founded in terms to decrease in uric acid formation as compared to
the product formation in absence of inhibitor. Supporting information includes sample graph that
enzyme inhibition activities are monitored by the absorption pattern as function of time for XO.
The percent inhibition (IC50) of XO activity was calculated as the following:
Xanthine oxidase inhibition (%) = (Acontrol – Asample / Acontrol )100
3. Results and discussion
3.1. Synthesis of 2-hydroxyethyl substituted NHC Precursors (1a-f and 2a-c)
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The 2-hydroxyethyl substituted NHC precursors were synthesized successfully that have been
illustrated in Scheme 1 and Scheme 2. The compounds 1a–f have been synthesized from the 1-(2-
tetrahydro-pyran-2-yloxyethyl)benzimidazole and alkyl/aryl halide (2-methylbenzyl chloride, 3-
methylbenzyl chloride, 4-methylbenzyl chloride, 2,4,6-trimethylbenzyl chloride, ethyl bromide,
isopropyl bromide). The compounds 2a–c have been synthesized from the N-alkylbenzimidazole
(Alkyl: methyl, ethyl and isopropyl) and 2-iodoethanol. The chemical structures of all
1
13
compounds were confirmed by H NMR, C NMR, FTIR spectroscopy and elemental analysis
techniques. The air and moisture stable NHC precursors are soluble both in polar solvents such as
water, dimethylsulfoxide and in halogenated solvents such as dichloromethane and chloroform.
The NHC precursors (chlorinated and brominated salts) 1a-f were obtained as a white solid in
between 79 % and 91 % yield. The NHC precursors (iodized salts) 2a-c were obtained as a
yellow-brown solid in between 70 % and 83 % yield. The formation of the 2-hydroxyethyl
1
13
substituted NHC precursors 1a–f and 2a–c was confirmed by FT-IR, H NMR and C NMR
spectroscopic methods and elemental analysis techniques. These spectra are consistent with the
1
proposed formulate. In the H NMR spectra, the 2-hydroxyethyl substituted NHC precursors
were identified by a characteristic proton peak at the 2-position (NCHN) of the NHC precursors,
which appeared highly downfield shifted singleds at δ 10.68, 10.76, 10.81, 9.93, 10.42, 10.36,
9.69, 9.59 and 10.32 ppm for 1a-f and 2a-c, respectively. The NCHN carbon resonances of these
13
NHC precursors in the C NMR spectra appeared highly downfield shifted at δ 143.9, 143.4,
143.4, 142.3, 141.7, 140.4 143.4, 142.1 and 140.4 ppm for (1a-f and 2a-c), respectively. The
results of the elemental analysis were evaluated and it was observed that the calculated values
were very close to the found values. The FTIR data clearly indicated the presence of ν(CN) 1557,
1559, 1562, 1557, 1560, 1548, 1568, 1566 and 1557 cm-1 for the NHC precursors 1a–f and 2a-c,
respectively. The FTIR data clearly indicated the presence of ν(OH) 3175, 3237, 3305, 3241, 3325,
3323, 3326, 3341 and 3306 cm-1 for the NHC precursors 1a–f and 2a-c, respectively. All
spectroscopic data are consistent with the literature [29-31]. In this study, we obtained a single
crystal for NHC precursor 2a by using the X-ray diffraction method.
11

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Scheme 1. Synthesis of 2-hydroxyethyl substituted NHC precursor 1a-f
Scheme 2. Synthesis of 2-hydroxyethyl substituted NHC precursor 2a-c
3.2. Enzyme inhibition studies
Novel synthesized derivatives have recently continued to attract attention, as many enzyme
inhibitors. In this study both CA isoforms (hCA I & II) were firstly purified by affinity
chromatography sepharose-4B-L-tyrosine-sulfanilamide in a single purification step. The eluates
protein from the column were measured spectrophotometrically at 280 nm. CA isoforms
activities were measured according to a procedure recorded by Verpoorte et al.. The changes in
12

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the absorbance at 348 nm for 4-nitrophenylacetate to 4-nitrophenolate was fallowed kinetically
for 3 min by using a spectrophotometer. CA isoenzymes have attracted this type of interest for
the design of inhibitors or activators with pharmaceutical and physiological biomedical
applications. In this study, inhibition properties of some novel 2-hydroxyethyl substituted NHC
precursors against hCA I and II isoenzymes were determined. For both isoenzymes, esterase
activity method was used for determination of IC50 values as inhibition parameter. The range of
IC50 value for hCA I inhibition was determined from 0.1565±0.178 to 0.5127±0.043 ꢀM as
shown in Table 2 and Fig. 2a. Compound 2c showed the lowest IC50 value found as
0.1565±0.178 ꢀM, on the other side compound 1b showed the highest IC50 value as
0.5127±0.043 ꢀM, on the other side the range of IC50 value for hCA II inhibition was determined
from 0.1524±0.013 to 0.5368±0.042 ꢀM. Compound 1d showed the lowest IC50 value as
0.1524±0.013 ꢀM, on the other side compound 1a showed the highest IC50 value as
0.5368±0.042 ꢀM. Cytosolic hCA I and II are expressed in red blood cells and are necessary for
-
maintaining the physiological pH of the blood through production of HCO3 . Abnormal levels of
CA I in the blood are used as a marker for hemolytic anemia. Behçet at al. found that 2-(4-
hydroxyphenyl)ethyl and 2-(4-nitrophenyl)ethyl substituted benzimidazolium salts were inhibited
the cytosolic isoform hCA I and hCA II, with IC50 values in the range from 40.81 to 79.92ꢁnM
and from 44.91 to 107.02, respectively [31]. In this study, the 2c compound contains the iodide
anion. Therefore, the solubility of this compound in the water system is higher than 1a-f. The 2c
compound has also more steric bulk due to isopropyl group compared with 2a-2b (containing
methyl and ethyl groups). This steric bulk provides interaction between salt, and active center of
the enzymes. Thus, the decrease in the IC50 values of the compound 2c has observed. These
results are similar with the inhibition level of our some 2-hydroxyethyl substituted NHC
precursors [30].
The XO inhibitory activities of 2-hydroxyethyl substituted NHC precursors are given in Table 2
and Fig. 2b, as half maximal inhibitory micromolar concentration (IC50) values. The experimental
results indicated that all 2-hydroxyethyl substituted NHC precursors showed remarkable
inhibition activity toward XO as compared with the standard allopurinol. The range of IC50 value
for XO inhibition was determined from 1.253 ± 0.084 to 5.342 ± 0.168 ꢀM as shown in Table 2
13

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and Fig. 2. Compound 1d showed the lowest IC50 value found as 1.253 ± 0.084 ꢀM, on the other
side compound 1f showed the highest IC50 value as 5.342 ꢀM. The IC50 for allopurinol was
determined as 4.2 ꢀM as a positive control. Compound 1d which includes trimethyl benzyl
substituent has demonstrated high inhibition effect on hCA I, hCA II and XO compared to other
complexes due to trimethylbenzyl substituent compound 1d has steric bulk that has more Van
Der Walls interaction between enzyme and compound. In addition, iodide salts (2b and 2c) have
demonstrated more inhibition effect on all enzymes compared to chloride salts (1e and 1f) due to
iodide salts have a higher solubility than chloride salts. This property of iodide salts might
contribute in higher inhibition activity of complexes. In a previous study, the range of IC50 value
for XO inhibition was determined from 4.608 to 7.084 ꢀM for some pyrrole carboxamide
derivatives by Kıbrız et al. [59] Zhang et al. founded the range from 6.7 to 45 ꢀM for 17
compounds of benzonitrile derivatives as IC50 value for XO inhibition [62]. In another study,
inhibitory effect of natural plant flavonoids on XO enzyme was found that IC50 was in between
4.5 and 21.3 ꢀg/mL [63]. Our results showed that 2-hydroxyethyl substituted NHC precursors
have potential inhibition activity against hCA I, hCA II and XO. When examined the Table 2 and
Fig. 2, there are some compounds exhibited more XO inhibition activity than allopurinol.
Table 2. The IC50 value of 2-hydroxyethyl substituted NHC precursors on both human carbonic
anhydrase isoenzymes (hCA I and II) and xanthine oxidase enzyme (XO).
IC50 (µM)
hCA I
r2
hCA II
r2
XO
r2
Compounds
0.4875 ± 0.039 0.9895 0.5368 ± 0.042 0.9742 2.445 ± 0.140 0.9997
0.5127 ± 0.043 0.9867 0.4898 ± 0.039 0.9986 4.219 ± 0.079 0.9951
0.3957 ± 0.069 0.9897 0.3186 ± 0.079 0.9768 1.914 ± 0.072 0.9800
0.1746 ± 0.096 0.9876 0.1524 ± 0.013 0.9784 1.253 ± 0.084 0.9771
0.3955 ± 0.072 0.9786 0.3257 ± 0.059 0.9879 5.342 ± 0.168 0.9617
0.4236 ± 0.125 0.9985 0.3646 ± 0.086 0.9897 4.992 ± 0.113 0.9915
0.3568 ± 0.079 0.9879 0.3246 ± 0.178 0.9658 2.877 ± 0.042 0.9888
1a
1b
1c
1d
1e
1f
2a
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0.2351 ± 0.057 0.9968 0.1937 ± 0.084 0.9764 1.515 ± 0.013 0.9615
0.1565 ± 0.178 0.9875 0.1859 ± 0.197 0.9876 2.318 ± 0.056 0.9798
2b
2c
4.2 ± 0.035
0.9796
Allopurinol
Figure 2. The IC50 values of NHC precursors on a) hCAI and hCAII, b) XO enzymes.
15

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3.3. Structural description of 2a
The molecular structure of 2a with displacement ellipsoids drawn at 30% probability level is
as shown in the Fig. 3. The asymmetric unit contains an iodide anion and a cation features
hydroxyethyl and methyl groups substituted to N-benzimidazole ring. The single crystal X-ray
diffraction studies indicate that the N-benzimidazole ring system together with C8 and C9 atoms
is essentially coplanar with a mean deviation of 0.013 Å. The hydroxyethyl moiety is tilted
towards to the iodide anion with the torsion angle of N2–C9–C10–O1 = -65.4(6)°. Bond
parameters such as bond lengths and angles are comparable with our previously published
structures of NHC salts consisting iodide ions [30].
Crystal packing of 2a is consolidated by intramolecular hydrogen bonds and
intermolecular weak interactions involving the iodide anion. The I– ions behave as a H-bond
acceptor in the crystal structure, resulting in the formation of two-dimensional supramolecular
array (see Table 3 and Fig. 4.). Iodide anions connect the cation molecules with C1–H1A···I1i
and C6–H6···I1ii intermolecular interactions along the a axis, while connecting the molecules via
O1–H1···I1 hydrogen bond and C6-H6···I1ii interactions along the b axis. In the view of this
result, molecules form one-dimensional polymeric infinite zigzag chains along the a and b axes
with the C(7) and C(8) motif, respectively.
Table 3. Intramolecular hydrogen bond and intermolecular weak interactions [Å,°] for 2a.
Compound
2a
D–H···
A
D–H
0.90
0.93
0.93
H
···
A
D
···
A
D–H···
A
O1–H1ꢂꢂꢂI1
C1–H1AꢂꢂꢂI1i
C6–H6ꢂꢂꢂI1ii
2.56
2.88
3.01
3.453(4)
3.768(5)
3.926(5)
172
160
167
Symmetry codes: (i) 3/2-x, 1/2+y, 1/2-z; (ii) 1/2-x, 1/2+y, 1/2-z
16

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Figure 3. Molecular structure of 2a, anisotropic displacement parameters depicting 30% probability.
Selected bond parameters (Å,°): O1–C10 1.400(6), N1–C1 1.336(5), N1–C2 1.376(5), N1–C8 1.469(5),
N2–C1 1.328(6), N2–C7 1.383(5), N2–C9 1.470(5); N1–C1–N2 109.6(4), C1–N1–C8 124.4(4), C1–N2–
C9 124.8(4), N2–C9–C10 111.6(3), C9–C10–O1 112.2(4), C8–N1–C2 127.3(4), C9–N2–C7 126.3(4);
N1–C1–N2–C9 -177.7(3), N2–C1–N1–C8 179.2(4), C1–N2–C9–C10 86.6(5), N2–C9–C10–O1 -65.4(6),
C3–C2–N1–C8 2.0(7), C7–N2–C9–C10 -89.0(5), C6–C7–N2–C9 -4.2(6)
Figure 4. Packing of the cation molecules via the intramolecular hydrogen bonds and intermolecular
interactions, bridged by the iodide anions, which lead to the infinite chain along the a and b axes. All
17

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hydrogen atoms except those participating in the hydrogen bonds were omitted for clarity. Cation
molecules are shown in stick drawing style.
4. Conclusions
Consequently, we have reported the synthesis of the 2-hydroxyethyl substituted NHC
1
precursors. They have been characterized by H NMR, 13C NMR, FTIR spectroscopy and
elemental analysis techniques. Molecular and crystal structure of 2a has been determined by
single-crystal X-ray diffraction method. Structural analysis has shown that the iodide ion acts as a
H-bond acceptor and plays on important role for the stabilization of crystal structure, considering
its contribution to the formation of the 2D supramolecular architecture. All the synthesized 2-
hydroxyethyl substituted NHC precursors inhibited metabolic enzymes hCA I, hCA II and XO
effectively as compared with the standards.
Conflicts of interests
The authors declare that no conflicts of interests.
Supplementary
Crystallographic data as .cif files for the structures reported in this paper have been deposited at
the Cambridge Crystallographic Data Center with CCDC 1878826 for 2a. Copies of the data can
be obtained free of charge at http://www.ccdc.cam.ac.uk/conts/retrieving.html or from the
Cambridge Crystallographic Data Center, 12, Union Road, Cambridge CB2 1EZ, UK. Fax: (+44)
1223-336-033, email: deposit@ccdc.cam.ac.uk.
Acknowledgments
This work was financially supported by Inonu University Research Fund (IUBAP FOA-
2018-1342). The authors acknowledge Inonu University Scientific and Technology Center for the
elemental analyses of the compounds and the authors acknowledge the İnönü University Faculty
of Science Department of Chemistry for the characterization of compounds. The authors
acknowledge Dokuz Eylül University for the use of the Oxford Rigaku Xcalibur Eos
Diffractometer (purchased under University Research Grant No: 2010.KB.FEN.13).
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Highlight
1. The 2-hydroxyethyl substituted NHC precursors have been prepared.
2. The 2-hydroxyethyl substituted NHC precursors have been characterized by using
spectroscopy and elemental analysis techniques.
3. The 2-hydroxyethyl substituted NHC precursors were investigated against the cytosolic
carbonic anhydrase I and II isoenzymes (hCA I and hCA II), and xanthine oxidase (XO).
4. The structure of a new 2-hydroxyethyl substituted NHC precursor was established by using
single-crystal X-ray diffraction method.