6-Substituted nicotinic acid analogues, potent inhibitors of CAIII, used as therapeutic candidates in hyperlipidemia and cancer

Article abstract of DOI:10.1007/s00044-017-1825-x

Nicotinic acid has been reported as a potential inhibitor of carbonic anhydrase III enzyme. Carbonic anhydrase III (CAIII) is an emerging new pharmacological target for the management of dyslipidemia and cancer progression. The activity of 6-substituted n

Full text of DOI:10.1007/s00044-017-1825-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1825-x
ORIGINAL RESEARCH
6-Substituted nicotinic acid analogues, potent inhibitors of CAIII,
used as therapeutic candidates in hyperlipidemia and cancer
Haneen K. Mohammad1 Muhammed H. Alzweiri1 Mohammad A. Khanfar1
Yusuf M. Al-Hiari1
●
●
●
Received: 22 February 2016 / Accepted: 28 February 2017
© Springer Science+Business Media New York 2017
Abstract Nicotinic acid has been reported as a potential
inhibitor of carbonic anhydrase III enzyme. Carbonic
anhydrase III (CAIII) is an emerging new pharmacological
target for the management of dyslipidemia and cancer
progression. The activity of 6-substituted nicotinic acid
analogs against carbonic anhydrase III was studied using a
size-exclusion chromatography. The appearance of
concentration-dependent vacancy peak was indicative of
binding with CAIII. Chromatographic and docking studies
revealed that the carboxylic acid of ligand is essential for
binding via coordinate bond formation with Zn+2 ion in the
enzyme active site. Moreover, the presence of a hydro-
phobic group, containing a hydrogen bond acceptor, at
position 6 of the pyridine improves activity, e.g., 6-(hex-
yloxy) pyridine-3-carboxylic acid (Ki = 41.6 µM). Utilizing
the weak esterase activity of CAIII, the inhibitory mode of
6-substituted nicotinic acid was confirmed.
catalyze the hydration of carbon dioxide to bicarbonate.
In mammals, there are 16 different CA that differ in
their amino acid sequences, sites of expression and
enzymatic activity (Hassan et al. 2013). In addition to their
CO2-hydration activity, CA displays weak esterase activity
and involves in various physiological processes, such as
acid–base balance, lipogenesis, and cell growth (Krishna-
murthy et al. 2008, Mitterberger et al. 2012).
However, carbonic anhydrase III (CAIII) isoenzyme has
a very low catalytic activity in hydrating CO2 to bicarbonate
compared to other CA isoenzymes (only 2% of activity of
CAI and CAII (Mitterberger et al. 2012), and weak esterase
hydrolytic activity (Lindskog and Silverman 2000, Cabiscol
and Levine 1996). On the other hand, CAIII isoenzyme is
highly expressed in adipose tissue and involved in lipo-
genesis (Mitterberger et al. 2012). Therefore, the inhibition
of CAIII can be regarded as a promising drug target for the
management of hyperlipidemia (Alzweiri and Al-Hiari
2013).
●
●
●
Keywords Nicotinic acid QSAR Carbonic anhydrase
Esterase
Furthermore, CAIII is involved in certain cancerous
diseases such as acute myeloid leukemia and liver carci-
noma. Overexpression of CAIII increases the resistance to
anti-cancer medication such as paclitaxel (Roy et al. 2010).
Moreover, inhibition of CAIII can lead to H2O2-induced
apoptosis. These activities suggest a potential therapeutic
role of CAIII inhibitors in combination therapy with some
anticancer medication (Roy et al. 2010, Dai et al. 2008,
Mahon and Mckenna 2015). Unlike other CA enzymes,
CAIII is not susceptible to traditional CA inhibitors such as
sulfonamides (Supuran 2008, Alzweiri and Al-Hiari 2013).
Overall, the involvement of CAIII in the pathology of
hyperlipidemia and cancer necessitates the development of
potent inhibitors against this isoenzyme.
Introduction
Carbonic anhydrase (CA) enzymes are a family of zinc-
containing metalloproteins, which reversibly and efficiently
* Muhammed H. Alzweiri
m.alzweiri@ju.edu.jo
1
Benzoic acid derivatives were reported earlier by our
group as ligands of CAIII (Alzweiri et al. 2015). The
Department of Pharmaceutical Sciences, Faculty of Pharmacy, The
University of Jordan, Amman 11942, Jordan

Med Chem Res
preliminary structure activity relationship (SAR) revealed
that the benzoate moiety is essential for binding by coor-
dinating to Zn+2 ion (Alzweiri et al. 2015). Nicotinic acid is
a water-soluble vitamin that has been known for decades as
a lipid-modifying drug (Ginsberg 2006), and was recently
reported as CAIII ligand by our research group (Jarrar et al.
2015). The carboxylate group of nicotinic acid forms a
coordinate bond with the zinc atom in CAIII active site
(Jarrar et al. 2015). The binding was determined by the size-
exclusion chromatography. This method has the advantage
of preserving the equilibrium during the analysis and also it
does not involve additional separation steps (e.g., pre-
cipitation and filtration), which might lead to disturbances
in the equilibrium (Sebille et al. 1990, Busch et al. 1997,
Hummel and Dreyer 1962, Berger and Girault 2003).
Moreover, this method can provide information on the
binding ratio and the association constants. It can be used as
a screening tool of various compounds against a known
enzyme, providing an attractive, cheap and accurate alter-
native to the currently used enzymatic assays irrespective of
their mode of action (Alzweiri and Al-Hiari 2013).
According to our earlier work, 6-bromonicotinic acid and
6-hydroxynicotinic acid analogs have the highest binding
affinity for CAIII (Jarrar et al. 2015). This prompted us to
provide more insight into the SAR of nicotinic acid deri-
vatives by designing and synthesizing of new 6-substituted
analogs of nicotinic acid. Chromatographic and docking
studies were conducted to study the binding affinity of these
compounds and CAIII and to determine the specific inter-
actions that contribute to the binding process. Additionally,
a less sensitive enzymatic assay, utilizing the weak esterase
character of the enzyme, was implemented to reveal whe-
ther the binding is inhibitory or not.
phenoxynicotinic acid, 6-morpholinonicotinic acid, 6-
(2,2,2-trifluoroethoxy) nicotinic acid, 6-thien-2ylnicotinic
acid, 6-[4-(tert-butoxycarbonyl) piperazin-1-yl] nicotinic
acid and 6-(2-furyl) nicotinic acid were obtained from
Acros Organics (USA). Additionally, a group of chemicals
were synthesized and fully characterized, including 6-
ethoxypyridine-3-carboxylic acid, 6-butoxypyridine-3-car-
boxylic, 6-(hexyloxy) pyridine-3-carboxylic acid and 6-
(octyloxy) pyridine-3-carboxylic acid. Also, the following
chemicals were purchased to conduct the assay of esterase
activity of CAIII enzyme: boric acid (MERCK, Germany),
sodium hydroxide (Lonver, UK), nicotinic acid ethyl ester
and 4-nitrophenyl acetate (Sigma-Aldrich, USA).
Instrumentation
Thermo Scientific high performance liquid chromatography
(HPLC) system equipped with Spectra system P1000 pump,
Spectra system UV1000 detector and SN 4000 controller
were used. ChromQuest 3.1.6 software was used for
acquiring and processing the chromatographic results. A
Phenomenex, BioSep-SEC-S2000, 300 × 7.8 mm column
was used for incubation in a thermal chamber at ambient
temperature. The column properties: exclusive range:
1000–300,000 Da, silica phase, 5-µm particle size, 145 Å
pore size, efficiency ≥ 70,000 plates/m, and suitable for low
molecular weight proteins. A fixed flow rate of 1 ml/min
was maintained during the run time. A wavelength of 260
nm was selected for the analysis of 6-phenoxynicotinic acid,
6-(2,2,2-trifluoroethoxy) nicotinic acid, 6-(tetrahydropyran-
4-yloxy) nicotinic acid, 6-ethoxypyridine-3-carboxylic acid,
6-butoxypyridine-3-carboxylic acid, 6-(hexyloxy) pyridine-
3-carboxylic acid and 6-(octyloxy) pyridine-3-carboxylic
acid, whereas a wavelength of 230 nm was selected for the
analysis of the rest of analogs.
Experimental
The synthesized compounds were characterized by
melting point measurements and spectroscopic measure-
ments such as nuclear magnetic resonance (1H/13C-NMR),
mass spectroscopy (MS) and infrared (IR) spectroscopy.
Melting point was measured using Stuart™ melting point
apparatus SMP3. 1H NMR and 13C NMR spectra were
conducted on a Burker Ultra shield-300 (300 MHZ) spec-
trometer NMR at Al Al-Bayt University. NMR data are
reported as follows: chemical shift (p.p.m.), multiplicity,
coupling constant, number of protons, the corresponding
proton; (s) stands for singlet, (d) for duplet, (t) for triplet and
(m) for multiplet.
Mass data were obtained via a high-resolution mass
spectrometer, Bruker Apex IV, at The Faculty of Science,
University of Jordan. Electro spray ionization (ESI) tech-
nique was used in negative or positive ion mode of analysis.
IR spectra were obtained via Shimadzu FTIR-8400S using
Chemicals and material
Bovine CA isozyme III (BIO-VAR Ltd, Armenia) was
stored at 0 °C in refrigerator until use. HPLC-grade acet-
onitrile and methanol (Fisher Scientific, UK) were used
without further purification. Potassium dihydrogen phos-
phate monohydrate (Fisher Scientific, UK), sodium hydro-
xide (Lonver, UK), and deionized water were used in the
mobile-phase preparation. Dichloromethane and octanol
were obtained from MERCK, Germany, 6-chloronicotinic
acid from Acros Organics, USA, ethanol and butanol from
Scharlau, Spain, hexanol and dimethyl sulfoxide (DMSO)
from BDH, UK, potassium hydroxide from Pharma-Cos
Ltd, UK, hydrochloric acid from Carlo Erba, Italy, acetic
acid from AZ Chem, USA, were used in synthesis. 6-
Phenylnicotinic acid, 6-piperidinonicotinic acid, 6-

Med Chem Res
KBr disks at Faculty of Pharmacy, The University of
Jordan.
CH3), 1.42 (m, 2H, CH2), 1.70 (m, 2H, CH2), 4.32 (t, J =
6.49, 2H, OCH2), 6.87 (d, J = 8.6 Hz, 1H, H-5), 8.13 (dd, J
= 1.6, 8.37, 1H, H-4), 8.7 (s, 1H, H-2), 13.0 (brs, 1H,
COOH). 13C-NMR (75 mhz, DMSO-d6): δ = 13.60 (CH3),
18.64 (CH2), 30.39 (CH2), 65.90 (OCH2), 110.52 (C-5),
120.14 (C-3), 139.79 (C-4), 149.44 (C-2), 165.97 (C-6),
166.07 (COOH). MS (ESI, negative mode): m/z [M–H]–
194.08180 (C10H12NO3 requires 194.08172). IR ν =
3456.55 (OH carboxylic), 2970.48, 2546.12, 1697.40
(C=O bond), 1612.45, 1419.66, 1365.65, 1303.92, 1141,
1064.74, 1026.16, 964.4, 840.4, 786.98, 671.25,
Preparation of mobile phase and samples of HPLC
The method was applied as described by Alzweiri and Al-
Hiari (Alzweiri and Al-Hiari 2013) and modified to suit this
work. Briefly, the mobile phase was composed mainly of
phosphate buffer system with pH around 6 that aids in the
analytes solubilization and partial ionization. Acetonitrile
was used in the mobile phase in a small percentage (10%) as
an organic modifier; it aids in solubilizing of hydrophobic
analytes and in elution of analytes from the column. The
inhibition constant was calculated by preparing different
mobile phases; each contained one of the analytes dissolved
in a mixture of 10% acetonitrile and 90% phosphate buffer
pH = 6 to get an analyte concentration of 0.24 mM. Mobile
phases were filtered and degassed. After baseline stabili-
zation, CAIII solution (dissolved in deionized water) with
concentration 1.7 mM was injected.
547.80 cm−1
.
6-(Hexyloxy) pyridine-3-carboxylic acid
Yield 80% cream-colored crystals, m.p. 95–97 °C. 1H NMR
(300 MHz, DMSO-d6): δ = 0.84 (m, 3H, CH3), 1.25–1.68
(m, 8H, 4CH2), 4.27(m, 2H, OCH2), 6.85 (d, J = 8.1 Hz,
1H, H-5), 8.11 (d, J = 6.2 Hz, 1H, H-4), 8.68 (d, 1H, H-2),
13 (brs, IH, COOH). 13C-NMR (75 MHz, DMSO-d6) δ =
13.81 (CH3), 22.0, 25.08, 8.27, 30.95 (4CH2), 66.16
(OCH2), 110.49 (C-5), 120.08 (C-3), 139.75 (C-4), 149.43
(C-2), 165.93(C-6), 166.09(COOH). MS (ESI, negative
mode): m/z [M–H]– 222.11682 (C12H16NO3 requires
222.11302). IR ν = 3456.55 (OH carboxylic), 3093.92,
2955.04, 2862.46, 2553.84, 1697.40 (C=O bond), 1604.83,
1419.66, 1365.64, 1303.92, 1141.10, 1064.74, 1018.45,
Preparation of synthetic compounds
6-Chloronicotinic acid (0.5 g, 3.2 mmol), potassium
hydroxide powder (1 g, 17.8 mmol) and 1 ml of one of the
following alcohol (ethanol, butanol, hexanol or octanol)
were added to DMSO (12 ml) at room temperature. The
reaction mixture was heated at 70–80 °C for 22 h, then
cooled to room temperature and poured over 100 ml of
water and crushed ice. An aqueous solution of HCl (2 M)
was added to acidify the solution to pH = 2. The resulting
precipitate was collected by filtration, washed with water
and dried under vacuum. Structures of the products were
confirmed by NMR, MS and IR as follows.
949.00, 848.71, 786.98, 725.26, 671.25, 555.51 cm−1
.
6-(Octyloxy) pyridine-3-carboxylic acid
1
Yield 80% White powder, m.p. 90–93 °C. H NMR (300
MHz, DMSO-d6): δ = 0.60–1.63 (3 m, 15H, 6 CH2 and 1
CH3), 4.06 (m, 2H, OCH2), 6.6 (d, J = 8.2 Hz, 1H, H-5),
7.88 (d, J = 7.6 Hz, 1H, H-4), 8.46 (brs, 1H, H-2), 13 (brs,
IH, COOH). 13C-NMR (75 MHz, DMSO-d6): δ = 13.86
(CH3), 22.04, 25.41, 28.32, 28.59, 28.68, 31.16 (6CH2),
66.15 (OCH2), 110.47 (C-5), 120.26 (C-3), 139.75 (C-4),
149.41 (C-2), 165.91 (C-6), 166.13 (COOH). MS (ESI,
negative mode): m/z [M–H]– 250.14861 (C14H20NO3
requires 250.14432). IR ν = 3456.55 (OH carboxylic),
3101.64, 2924.18, 2854.74, 2553.84, 1697.40 (C=O bond),
1604.83, 1419.66, 1365.64, 1311.64, 1141.80, 941, 848.71,
6-Ethoxypyridine-3-carboxylic acid
1
White powder yield 57.39%, m.p. 190–193 °C. H NMR
(300 MHz, DMSO-d6): δ = 1.3 (t, J = 6.9 Hz, 3H, CH3),
4.3 (q, J = 7.0 Hz, 2H, OCH2), 6.8 (d, J = 8.6 Hz, 1H, H-5),
8.1 (dd, J = 2.2, 8.6 Hz, 1H, H-4), 8.7 (s, 1H, H-2), 13 (brs,
1H, COOH). 13C-NMR (75 MHz, DMSO-d6): δ = 14.31
(CH3), 62.00 (OCH2), 110.53 (C-5), 120.127 (C-3), 139.77
(C-4), 149.45 (C-2), 165.80 (C-6), 166.08 (COOH). MS
(ESI, negative mode): m/z [M–H]– 166.05044 (C8H8NO3
requires 166.05042). IR ν = 3471(OH carboxylic), 2985.9,
2561.5, 1689 (C=O bond), 1604.8, 1504.5, 1427.3, 1296.2,
786.98, 725.26, 671.25, 555.52 cm−1
.
Docking experiment
1141.9, 1026.2, 949.0, 856.4, 786.9, 555.5 cm−1
.
CAIII Apo-protein crystal structure with 2.1 Å resolution
(PDB code1Z93) was used. The water molecules presented
on the surface were removed. Ligand fit docking engine was
used to conduct the docking experiments; it considers the
rigidity of the receptor and the ligand to be flexible.
6-Butoxypyridine-3-carboxylic
1
Yield 72.9% cream-colored crystals, m.p. 102–105 °C. H
NMR (300 mHz, DMSO-d6): δ = 0.94 (t, J = 7.3 Hz, 3H,

Med Chem Res
Additionally, Discovery Studio 2.5 was used to determine
CAIII binding site with the following configurations:
Monte Carlo search algorithm with 30,000 trials; search
step for torsions with polar hydrogens = 30.0°. The RMS
threshold for ligand-to-binding-site shape matching was set
to 2.0 Å, employing a maximum of 1.0 binding-site parti-
tions. The interaction energies were assessed employing the
consistent force field (v.1.02) with a non-bonded cutoff
distance of 10.0 Å and distance-dependent dielectric mea-
sure. An energy grid extending 5.0 Å from the binding site
was also implemented. The interaction energy was esti-
mated with a trilinear interpolation value using soft poten-
tial energy approximations. Rigid body ligand minimization
parameters: 40 steepest descent iterations followed by 80
BFGS-minimization iterations were applied to every
orientation of the docked ligand. The proposed inhibitors
were further energy minimized within the binding site by
implementing the “Smart Minimization” option for a max-
imum of 1000 iterations.
steric hindrance inside its active pocket (Ren et al. 1991).
Because of that, the inhibitor size is quite important for the
binding of a ligand within CAIII and must be studied as a
determinant factor for the activity. Therefore, most of our
selected compounds were substituted with groups of various
sizes, specifically at position 6 of the nicotinic acid. This
position was selected based on our previous work that
showed that most of the active nicotinic acid analogs pre-
viously studied were 6-substituted ligands (Jarrar et al.
2015).
The synthetic work was focused mainly on carbon chain
homologation at position 6 of the nicotinic acid. Nearly all
the synthesized compounds have an ether linkage due to the
ease of preparation and potential binding effect on CAIII.
Several 6-substituted nicotinic acid derivatives with diverse
steric, electrostatic, and hydrogen bond donor and acceptor
properties were prepared (1–14, Table 1).
The activity of these compounds in binding with CAIII
enzyme was tested by a size exclusion HPLC method, in
which the analytes were dissolved in a mobile phase com-
posed mainly of phosphate buffer (pH = 6.0 0.2) to sup-
port analyte dissolution and ionization. A small percentage
of acetonitrile (10%) was used in the mobile phase as an
organic modifier; it participates in dissolving hydrophobic
analytes and accelerating the analyte elution from the col-
umn. The concentration of the inhibitor in the mobile phase
was 0.24 mM; it was selected in order to get a 1:1 binding
ratio (Alzweiri and Al-Hiari 2013). The typical chromato-
graphic response for the injection of CAIII solution is
shown in Fig. 1.
The CAIII that was used in docking study is a human
CAIII, while the one that was used in the biological eva-
luation was bovine. However, the CAIII (human, sheep and
bovine), which closely resemble each other in catalytic
properties, have very similar active site regions. All active
site’s amino acids (within 8 Å from the Zn atom) are iden-
tical in human and bovine CAIII (Henderson et al. 1976).
Sample preparation in evaluation of CAIII esterase
activity
The following reagents were prepared for the assay,
includes borate buffer (pH = 8, 10 mM), sodium hydroxide
(0.01 N), CAIII enzyme solution (2.13 mM) and porcine
liver esterase solution (3 mg/ml).
The appearance of a concentration-dependent negative
peak is indicative for binding. The negative peak reflects the
amount of ligand drawn from the mobile phase at the
expense of binding to CAIII enzyme.
The procedure is based on “Enzymatic Assay of Esterase”
protocol provided by Sigma-Aldrich. Briefly, 25 ml borate
buffer was pipetted into a suitable titration vessel and the
temperature was equilibrated at 25 °C using water bath.
Hundred milligrams of the following esters (4-nitrophenyl
acetate or nicotinic acid ethyl ester) was added to the buffer
each at once with stirring; then the pH was adjusted to
8.1–8.2 using 0.01 N sodium hydroxide. Hundred micro-
liters of bovine CAIII was added with stirring and the time
was recorded until pH reaches 8.0. Another 0.1 ml aliquot
of 0.01 N sodium hydroxide was added and the time elapsed
was recorded. This operation was repeated for a minimum
of 10 time points.
Since the chromatographic testing was done at equimolar
binding ratio, the inhibition constant (Ki) was calculated as
the reciprocal value of the affinity constant Kf. The latter
was measured as described by Alzweiri et al. (2015). Ki
values for all analytes were calculated and listed in Table 1
from lowest to highest along with their chemical structures.
All synthesized compounds (except 13 and 14) were more
active with higher binding affinity than nicotinic acid.
Analog 1 substituted with 6-hexyloxy moiety has the
highest binding affinity, which implies the importance of
lengthy substitution at this position. Shorter substitutions
were less active; however, octyloxy substitution (as in 9)
decreases binding affinity. 6-Phenoxy substitution, as in
analog 2, was the second most active, which highlights the
role of hydrophobic interactions within CAIII binding site,
as will be described in the docking studies. Although the
most active nicotinic acid analog (1) has a binding affinity
of 41.6 µM, we believe that these compounds are much
more active than they appear to be, primarily because of the
Results and discussion
The weak inhibition of CAIII by sulfonamides was attrib-
uted in a part to the rigid structure of the enzyme and to the

Med Chem Res
Table 1 The calculated Ki values for tested compounds with their chemical structures
No. Ligand name
Ligand structure
Ki (µM) No. Ligand name
Ligand structure
Ki (µM)
1
6-(Hexyloxy) pyridine-3-
carboxylic acid
41.6
2
6-Phenoxynicotinic acid
43.6
O
O
OH
OH
O
N
O
N
3
6-Butoxypyridine-3-carboxylic
acid
62.9
4
6-Ethoxypyridine-3-
carboxylic acid
63.3
76.9
O
O
OH
OH
O
N
O
N
5
7
6-(2-Furyl)nicotinic acid
6-Phenylnicotinic acid
64.0
78.1
6
8
6-Thien-2-ylnicotinic acid
6-Chloro nicotinic acid
O
O
OH
OH
N
N
S
O
91.5
O
O
OH
OH
N
Cl
N
9
6-(Octyloxy) pyridine-3-
carboxylic acid
142.7
10 6-Morpholinonicotinic acid
214.2
O
O
OH
OH
N
N
O
N
O
11 6-(2,2,2-Trifluoroethoxy)
221.9
454.1
12 Nicotinic acid
248.7
760.5
O
O
nicotinic acid
OH
OH
O
F
O
N
F
N
F
13 6-[4-(tert-Butoxycarbonyl)
14 6-Piperidinonicotinic acid
O
piperazin-1-yl]nicotinic acid
OH
OH
N
N
N
N
O
N
O
dilution effect of the mobile phase in size exclusion chro-
matography. Moreover, there is no CAIII ligand reported in
literature to compare and normalize our binding affinity
results.
In order to understand the SAR of synthesized com-
pounds and to describe the binding interactions within the
binding pocket of CAIII, in silico docking studies were
conducted.
(PHE198) (Fig. 2). On the other hand, the phenolic ether
oxygen forms a hydrogen bond interaction with guanidine
group of Arginine 67 (ARG67), while the carboxylate
moiety strongly coordinates with the zinc atom with addi-
tional hydrogen bonding interaction with hydroxyl side
chain of Threonine 199 (THR199). These interactions
potentiate the binding and increase the affinity of compound
2 within CAIII active site.
Docking of 6-phenoxy nicotinic acid (2) showed π-π
stacking of the phenoxy ring with the Phenylalanine 198
Interestingly, a hydrophobic pocket was found within
CAIII active site. This pocket is composed of the

Med Chem Res
hydrophobic amino acid residues, namely, phenylalanine
131 (PHE131), leucine 135 (LEU135), proline 202
(PRO202) and phenylalanine (PHE198). This explains the
high binding affinity of the analogs containing aliphatic
hydrophobic substituents in close proximity to this hydro-
phobic pocket and may also explain the pattern of binding
of nicotinic acid analogs substituted at position 6.
pocket. Rather, it is protruded out and became unfavorably
exposed to hydrophilic residues (ARG67 and ARG91)
within the active site of CAIII (Fig. 3b).
All active compounds shared both a hydrogen bonding
and electrostatically enforced coordinate interactions with
ARG67 and zinc atom, respectively, which highlight the
importance of ether linkage (at position 6) and the
Aliphatic substitution of nicotinic acid at position 6,
facilitated with free rotation of ether linkage, provides
favorable alignment into the hydrophobic pocket. However,
this was only accessible with chain length of 2–6 carbons
with a maximum affinity with the hexyl derivative (1)
(Fig. 3a). Yet, such hydrophobic binding was not possible
with the eight-carbon chain, as in the octyl derivative (9),
mainly due to size hindrance within this hydrophobic
Fig. 1 Chromatograms for a mobile phase containing 0.24 mM 6-
phenylnicotinic acid resulted after the injection of different con-
centration of CAIII solutions. a 1.7 mM CAIII solution, b 0.85 mM
CAIII solution, c 0.21 mM CAIII solution
Fig. 2 Schematic illustration for 6-phenoxynicotinic acid binding in
CAIII binding site showing the zinc ion as a blue circle
Fig. 3 Schematic illustration for 6-(hexyloxy) pyridine-3-carboxylic acid a and 6-(octyloxy) pyridine-3-carboxylic acid b binding in CAIII
binding site

Med Chem Res
analogs as the most active inhibitors to act as prodrugs that
can be activated by the esterase activity of CAIII enzyme.
Acknowledgements Part of this research was conducted during the
sabbatical year granted to Dr. Muhammed Alzweiri by the University
of Jordan in the academic year 2015/2016.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Fig. 4 A plot representing time of degradation of nicotinic acid ethyl
ester a and 4-nitrophenyl acetate b by CAIII vs the cumulative amount
of NaOH
References
Alzweiri M, Al-Balas Q, Al-Hiari Y (2015) Chromatographic eva-
luation and QSAR optimization for benzoic acid analogues
against carbonic anhydrase III. J Enzyme Inhib Med Chem
30:420–429
Alzweiri M, Al-Hiari Y (2013) Evaluation of vanillic acid as inhibitor
of carbonic anhydrase isozyme III by using a modified Hummel-
Dreyer method: approach for drug discovery. Biomed Chroma-
togr 27:1157–1161
Berger G, Girault G (2003) Macromolecule–ligand binding studied by
the Hummel and Dreyer method: current state of the methodol-
ogy. J Chromatogr B 797:51–61
Busch M, Carels L, Boelens H, Kraak J, Poppe H (1997) Comparison
of five methods for the study of drug–protein binding in affinity
capillary electrophoresis. J Chromatogr A 777:311–328
Cabiscol E, Levine RL (1996) The phosphatase activity of carbonic
anhydrase III is reversibly regulated by glutathiolation. Proc Natl
Acad Sci 93:4170–4174
Dai HY, Hong CC, Liang SC, Yan MD, Lai GM, Cheng AL, Chuang
SE (2008) Carbonic anhydrase III promotes transformation and
invasion capability in hepatoma cells through FAK signaling
pathway. Mol Carcinogen 47:956–963
Ginsberg HN (2006) Niacin in the metabolic syndrome: more risk than
benefit? Nat Clin Pract Endocrinol Metab 2:300–301
Hassan MI, Shajee B, Waheed A, Ahmad F, Sly WS (2013) Structure,
function and applications of carbonic anhydrase isozymes.
Bioorgan Med Chem 21:1570–1582
Henderson LE, Henriksson D, Nyman PO (1976) Primary structure of
human carbonic anhydrase C. J Biol Chem 251:5457–5463
Hummel JP, Dreyer W (1962) Measurement of protein-binding phe-
nomena by gel filtration. Biochim Biophys Acta 63:530–532
Jarrar N, Alzweiri M, Al-Hiari Y, Farah S, A Khanfar M (2015)
Modified hummel–dreyer method and molecular modeling stu-
dies identified nicotinic acid analogues as carbonic anhydrase III
ligands. Lett Drug Des Discov 12:401–410
carboxylate moieties for binding within the active site of
CAIII.
In order to verify the inhibitory binding of the mentioned
ligands, the reported weak esterase activity of CAIII
(Lindskog and Silverman 2000) was measured against two
esteric compounds, 4-nitrophenyl acetate and nicotinic acid
ethyl ester. The ability of CAIII enzyme to hydrolyze 4-
nitrophenyl acetate was reported (Tu et al. 1986). However,
its phenolic degradant has a negligible binding affinity to
CAIII (Alzweiri and Al-Hiari 2013), whereas the nicotinic
acid, as a degradant of nicotinic acid ethyl ester, has a
strong binding affinity to CAIII (Jarrar et al. 2015).
As depicted in Fig. 4 and in comparison with the
hydrolysis rate of 4-nitrophenyl acetate, the reduction in the
hydrolysis rate of nicotinic acid ethyl ester (the last part of
curve A) is suggested to be due to the inhibitory action of
the released nicotinic acid. Such reduction was not observed
with the hydrolysis rate of the non-binder 4-nitrophenyl
acetate. This in the first place suggests that binding of
nicotinic acid with CAIII enzyme is inhibitory binding.
Conclusion
The ligand binding to CAIII is affected by several factors.
The determinant factor was the presence of carboxylic acid
group, which forms a coordinate bond with the zinc atom.
The presence of a flexible hydrophobic carbon chain (2–6
carbon length) increases the activity. This was attributed to
the presence of a small hydrophobic pocket inside the active
site. Additionally, a hydrogen bond acceptor such as oxy-
gen atom substituted at position-6 connected to aromatic
moieties increases the affinity of binding. CAIII was able to
hydrolyze nicotinic acid ethyl ester and nicotinic acid was
proved to act as an inhibitor to CAIII enzyme. These results
open the door for future work on the development of ester
Krishnamurthy VM, Kaufman GK, Urbach AR, Gitlin I, Gudiksen
KL, Weibel DB, Whitesides GM (2008) Carbonic anhydrase as a
model for biophysical and physical-organic studies of proteins
and protein-ligand binding. Chem Rev 108:946–1051
Lindskog S, Silverman D (2000) The catalytic mechanism of mam-
malian carbonic anhydrases. In: Chegwidden WR, Carter N,
Edwards Y (eds) The Carbonic Anhydrases. Birkhäuser Basel,
Berlin, pp 175–195
Mahon BP, Mckenna R (2015) Chapter 5—carbonic anhydrase III. In:
Simone CTSD (ed) Carbonic Anhydrases as Biocatalysts. Else-
vier, Amsterdam, pp 91–108
Mitterberger MC, Kim G, Rostek U, Levine RL, Zwerschke W (2012)
Carbonic anhydrase III regulates peroxisome proliferator-
activated receptor-γ2. Exp Cell Res 318:877–886

Med Chem Res
Ren X, Jonsson B-H, Lindskog S (1991) Some properties of site-
specific mutants of human carbonic anhydrase II having active-
site residues characterizing carbonic anhydrase III. Eur J Biochem
201:417–420
Sebille B, Zini R, Madjar C-V, Thuaud N, Tillement J-P (1990)
Separation procedures used to reveal and follow drug—protein
binding. J Chromatogr B Biomed Sci Appl 531:51–77
Supuran CT (2008) Carbonic anhydrases: novel therapeutic applica-
tions for inhibitors and activators. Nat Rev Drug Discov
7:168–181
Roy P, Reavey E, Rayne M, Roy S, Abed El Baky M, Ishii Y,
Bartholomew
C (2010) Enhanced sensitivity to hydrogen
peroxide‐induced apoptosis in Evi1 transformed Rat1
fibroblasts due to repression of carbonic anhydrase III. FEBS J
277:441–452
Tu C, Thomas HG, Wynns GC, Silverman DN (1986) Hydrolysis of 4-
nitrophenyl acetate catalyzed by carbonic anhydrase III from
bovine skeletal muscle. J Biol Chem 261:10100–10103