Design, synthesis, 99mTc labeling, and biological evaluation of a novel pyrrolizine derivative as potential anti-inflammatory agent

Article abstract of DOI:10.1134/S10663622170600121

The design and synthesis of ethyl 4-(6-amino-7-cyano-2,3-dihydro-1H-pyrrolizine-5-carboxamido)-benzoate (KH16) were discussed, and its structure was determined. The anti-inflammatory activity of a new compound was evaluated using in vitro cyclooxygenase (

Full text of DOI:10.1134/S10663622170600121

ISSN 1066-3622, Radiochemistry, 2017, Vol. 59, No. 6, pp. 630–638. © Pleiades Publishing, Inc., 2017.
Published in Russian in Radiokhimiya, 2017, Vol. 59, No. 6, pp. 554–561.
Design, Synthesis, 99mTc Labeling, and Biological Evaluation
of a Novel Pyrrolizine Derivative
as Potential Anti-Inflammatory Agent
K. M. Attallah*a,b, A. M. Goudac,d, I. T. Ibrahimb, and L. Abouzeide
a Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, 21955 Saudi Arabia
b Labeled Compounds Department, Hot Laboratories Center, Atomic Energy Authority,
P.O. Box 13759, Cairo, Egypt
c Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62514 Egypt
d Department of Pharmaceutical Chemistry, College of Pharmacy, Umm Al-Qura University, Makkah,
21955 Saudi Arabia
e Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, 35516 Egypt
*e-mail: Kmhassan@uqu.edu.sa
Received March 20, 2017
Abstract—The design and synthesis of ethyl 4-(6-amino-7-cyano-2,3-dihydro-1H-pyrrolizine-5-carboxamido)-
benzoate (KH16) were discussed, and its structure was determined. The anti-inflammatory activity of a new
compound was evaluated using in vitro cyclooxygenase (COX) inhibitory assay. KH16 exhibits higher selectiv-
ity to COX-2 than to COX-1 with the selectivity index of 3.46. KH16 was labeled with 99mTc with the maxi-
mum radiochemical yield of 99mTc-KH16 of 90.5 ± 1.5%. Biodistribution of 99mTc-KH16 in normal, infected,
and inflamed mice was studied. The uptake in inflamed muscle was higher than that in normal muscle through-
out the examined time interval. This work is a step ahead in the direction of using pyrrolizine derivatives for
site-specific delivery to the inflamed tissue.
Keywords: pyrrolizine, anti-inflammatory drugs, COX, technetium-99m, inflammation, infection
DOI: 10.1134/S10663622170600121
Nonsteroid anti-inflammatory drugs (NSAIDs) are
among the most widely used drugs in the world [1].
The wide use of NSAIDs in treatment of inflamma-
tions, pain, and hyperthermia is often accompanied by
a wide range of side effects [2, 3], of which gastroin-
testinal erosions and bleeding are the most common
[4]. Most of NSAIDs exert their actions through
cyclooxygenase (COX) inhibition [5]. Along with be-
ing a vital key element in the normal physiological
function of the stomach, COX enzymes plays a crucial
role in inflammation response and cell proliferation
[6, 7].
tion, with little or no damage of the gastric mucosa [9,
10]. The structure–activity relationship for licofelone
showed that the removal of the 2,2-dimethyl groups
and removal/masking of the free carboxy group re-
sulted in active anti-inflammatory agents (compounds
2–5 [11–14]; IC50 is the half-maximum inhibition con-
centration, μM, and IP is the inhibition percent).
Several pyrrolizine-based anti-inflammatory agents
have been reported [8–16]. Of these pyrrolizines, li-
cofelone 1 displayed anti-inflammatory, analgesic, and
antipyretic activities mediated by COX/5-LOX inhibi-
Licofelone (1)
2
1 The text was submitted by the authors in English.
IC50 = 2 (COX), 1.6 (5-LOX)
IC50 = 0.21 (COX), 0.18 (5-LOX)
630

DESIGN, SYNTHESIS, 99mTc LABELING
631
ly 60% of the anti-inflammatory activity of ketorolac
[23]. In this work, a potential anti-inflammatory agent,
KH16, was designed and synthetized by masking the
free carboxy group in compound 7.
3
IP = 10% (COX-1), IC50 = 3.6 (COX-2)
4
↓Masking of COOH group
IC50 = 0.5 (COX-1), 7.5 (COX-2)
5
The upregulated COX-2 in inflammation and can-
cer provides useful target in designing COX-2 selec-
tive inhibitors and in imaging of inflammation and
cancer using radiolabeled COX-2 inhibitors. This
study was also aimed at investigating the mechanism
of action of KH16, its tagging with 99mTc, the factors
affecting the labeling yield, tissue localization, and
efficacy of the compound as anti-inflammatory agent
in model septic and aseptic inflammations.
IP > 20% (COX-1), > 10% (COX-2)
(R,S)-Ketorolac (6)
IC50 = 1.23 (hCOX-1), 3.50 (hCOX-2)
EXPERIMENTAL
Ketorolac 6 displayed potent anti-inflammatory and
analgesic activity mediated by inhibition of COX en-
zymes [15]. The (S) enantiomer of ketorolac showed
60 timed more potent anti-inflammatory activity than
the (R) enantiomer did [16].
Chemicals and Instruments
Chemical reagents and solvents were obtained from
Sigma–Aldrich (commercial sources in Saudi Arabia).
Solvents were dried by standard methods when neces-
sary. The melting points (mp) were uncorrected and
were determined by the open capillary tube method
using IA 9100MK-Digital Melting Point Apparatus.
Microanalyses were carried out at the Microanalytical
Center, Faculty of Science, Cairo University. The IR
spectra were taken with a Bruker Tensor 37 spectro-
The conventional anatomical imaging techniques
such as computerized tomography (CT), radiology, and
nuclear magnetic resonance (NMR) were unable to
differentiate between inflammatory and infectious
processes [17]. The development of 99mTc-based radio-
pharmaceutical provides a better tool to differentiate
between inflammatory and infectious lesions [18]. The
99mTc-labeled selective and nonselective NSAIDs have
been used in inflammation imaging [19, 20]. On the
other hand, 99mTc-labeled antibacterial agents were
used in imaging of infection sites [21, 22].
1
photometer using KBr pellets. The H NMR spectra
were recorded on a Bruker Avance II spectrometer at
the faculty of pharmacy, Umm Al-Qura University at
500 MHz in CDCl3. The chemical shifts are presented
on the δ scale using the residual solvent signal as inter-
nal reference, and the J values are given in Hz. The
13C NMR and DEPT135 spectra were recorded on the
same spectrometer at 125 MHz. The mass spectra were
Previously we have reported compound 7 as an an-
algesic anti-inflammatory agent which displayed near-
RADIOCHEMISTRY Vol. 59 No. 6 2017

632
ATTALLAH et al.
dure described in [25] with some modifications. Ethyl
4-aminobenzoate (54 mmol) was reacted with chloro-
acetyl chloride in glacial acetic acid. Saturated aqueous
solution of sodium acetate (3 mL) was added dropwise
after complete addition of chloroacetyl chloride. The
heavy precipitate formed was filtered off, washed with
water, and recrystallized from ethanol.
recorded on a Shimadzu GCMS QP5050A spectrome-
ter at 70 eV (EI) at the Regional Center for Mycology
and Biotechnology, Al-Azhar University. Thin layer
chromatography was done using Macherey–Nagel
AlugramSil G/UV254 silica gel plates and benzene–
ethanol (9.5 : 0.5) as the eluting system.
Synthesis of Organic Compounds
Preparation of ethyl 4-(6-amino-7-cyano-2,3-
dihydro-1H-pyrrolizine-5-carboxamido)benzoate
(KH16). A mixture of 9 (1.81 g, 7.5 mmol), 8 (1 g,
7.5 mmol), and anhydrous K2CO3 (1.04 g, 7.5 mmol)
in dry acetone (50 mL) was stirred for 24 h under re-
flux. The reaction mixture was filtered while hot, con-
centrated, and left to cool. The crystals formed were
collected, dried, and recrystallized from ethanol–
acetone mixture. Compound KH16 was obtained as
yellowish white crystals, mp 228–230°C, yield 62%.
IR, ν, cm–1: 3448, 3340, 3300 (NH), 3038 (C–H aro-
matic), 2927 (C–H aliphatic), 2210 (CN), 1699, 1638
Preparation of 2-pyrrolidin-2-ylidenemalononi-
trile 8. Compound 8 was synthesized as described pre-
viously [24]. A solution of 2-pyrrolidinone (5 g,
58.8 mmol) in dry benzene (15 mL) was treated with
dimethyl sulfate (7.4 g, 58.8 mmol, see scheme).
The reaction mixture was refluxed for 3 h and then
allowed to cool. A concentrated NaOH solution (3 mL,
600 g L–1) was added dropwise. The organic phase was
separated, dried over anhydrous sodium sulfate, and
filtered. Malononitrile (2.5 g, 38.2 mmol) was added to
the benzene solution, whereby white crystals were
formed. The crystals were collected, dried (yield 5.9 g,
75%), and recrystallized from ethanol, mp 159–161°C
(published data: 158–159°C).
1
(C=O). H NMR (CDCl3, 500 MHz), δ, ppm: 1.42 t
(3H, J = 7.0 Hz, CH3), 2.56 m (2H, CH2-2), 3.02 t (2H,
J = 7.5 Hz, CH2-1), 3.60 s (2H, NH2), 4.37–4.44 m
(4H, OCH2 + CH2-3), 7.68 d (2H, J = 10 Hz, aromatic
CH-2', CH-6'), 8.05 d (2H, J = 10 Hz, aromatic CH-3',
CH-5'), 9.84 s (H, NH). 13C NMR (CDCl3), δ, ppm:
14.37 (CH3), 24.79 (CH2-1), 25.43 (CH2-2), 49.75
(CH2-3), 60.81 (OCH3), 83.58 (C-7), 114.25 (C≡N),
114.59 (phenyl C-4), 118.71 (phenyl C-2 + C-6),
125.56 (C-5), 130.85 (phenyl C-3 + C-5), 137.66
(C-7a), 142.35 (phenyl C-1), 145.58 (C-6), 158.70
(CONH), 166.19 (COO). MS (EI), m/z (I, %) 339
(M+ + 1, 3), 338 (M+, 16), 324 (2), 293 (2), 265 (1),
237 (1), 174 (100), 146 (41), 120 (21), 105 (3), 92
(27), 77 (2). Analytical data: Calculated for
C18H18N4O3 (M = 338.36), %: C 63.89, H 5.36, N 6.56.
Found, %: C 63.47, H 5.20, N 16.72.
Preparation of ethyl 4-(2-chloroacetamido)ben-
zoate 9. Compound 9 was synthesized by the proce-
Pharmacological Screening
In vitro COX-1/2 inhibitory assay. The ability of
the tested compound KH16 to inhibit COX-1 (ovine)
and COX-2 (human recombinant) was measured using
COX colorimetric inhibitor screening assay kit (Cay-
man Chemical, Ann Arbor, MI, the United States, cata-
log no. 560131) according to the manufacturer’s instruc-
tions. The procedure was described previously [26].
Preparation and Analysis of the Labeled Compound,
Evaluation of Its Stability
Scheme of the synthesis of KH16. (I) (CH3)2SO4, benzene,
CH2(CN)2; (II) ClCH2COCl, glacial acetic acid, CH3·
COONa; (III) acetone, K2CO3, reflux, 24 h.
Labeling process. The required amount of the
ligand (KH16) was transferred to a clean evacuated
RADIOCHEMISTRY Vol. 59 No. 6 2017

DESIGN, SYNTHESIS, 99mTc LABELING
633
10-mL penicillin vial and then was dissolved in 1 mL
of DMSO. The vial was closed under positive pressure
of nitrogen gas, and then the required amount of tin(II)
chloride was added. 1 mL of 99mTc eluate (2 mCi mL–1)
was added to the above mixture, and pH 5 was ad-
justed with a few drops of phosphate buffer (0.5 M).
The reaction volume was completed to 2.2 mL with
normal saline and incubated for the recommended time
before assay of the 99mTc–ligand complex.
Paper chromatography. 99mTc-KH16 was sepa-
rated from free pertechnetate and reduced hydrolyzed
species using paper chromatography with two develop-
ing systems. Acetone was used to determine free
pertechnetate, which moved to the top of the chroma-
togram (Rf = 1), whereas reduced hydrolyzed species
and 99mTc-KH16 remained at the point of spotting (Rf =
0). 5 N NaOH was used as the mobile phase to deter-
mine reduced hydrolyzed species, which remained at
the origin (Rf = 0), whereas free pertechnetate and
99mTc-KH16 moved with the solvent front (Rf = 1). The
radiochemical yield was determined by subtracting the
relative content of free pertechnetate and reduced hy-
drolyzed species from 100% [27]. The radiochemical
yield is the mean value of three experiments.
KH16 (0.1 mL) in 0.9% NaCl, prepared as described
above, was diluted with 0.9% NaCl to a volume of
2 mL and incubated at 37 ± 1°C. PC was performed at
different time intervals (1, 2, 4, 6 and 8 h). The degree
of degradation was judged from a decrease in the per-
centage of 99mTc-KH16.
In vitro stability in human serum was studied
similarly, except that human serum (1.9 mL) was used
instead of 0.9% NaCl for diluting the 99mTc-KH16 so-
lution.
Biological Characterisitcs of 99mTc-KH16
The study was approved by the animal ethics com-
mittee, Labeled Compounds Department, and was in
accordance with the guidelines set out by the Egyptian
Atomic Energy Authority.
Induction of septic and aseptic inflammation.
Three groups of Swiss Albino mice (weighing 25 g),
each of 25 mice, were used. In the first group, septic
inflammation was induced in the right thigh of each
mouse by intramuscular injection of 1 × 105 E. coli
cells. Inflammation developed within 7 days [29]. In
the second group, aseptic inflammation was induced in
the right thigh of each mouse by intramuscular injec-
tion of sterile turpentine oil (0.1 mL/mouse) [30]. Two
days later, swelling appeared [31]. The third group
consisted of normal mice.
Electrophoresis was done with a EC-3000 p-series
(E.C. Apparatus Corporation) programmable power
and chamber supply units using cellulose acetate strips.
The strips were moistened with 0.05 M phosphate
buffer (pH 7.2 ± 0.2) and then were introduced into the
chamber. The 99mTc-KH16 solution was passed
through a Millipore filter (0.22 μm) to separate col-
loids, if present. Samples (5 µL) were applied at a dis-
tance of 10 cm from the cathode. The experiment was
performed for 1.5 h at a voltage of 300 V. The strips
were dried, cut into 1-cm segments, and counted with a
well-type γ-scintillation counter. The radiochemical
yield was calculated as the ratio of the radioactivity of
the labeled product to the total radioactivity [28].
In vivo biodistribution. Each animal was injected
with 100 µL of a solution containing 3.7 MBq
(100 µCi, 0.148 µmol) of 99mTc-KH16 via the tail vein.
The mice were kept in metabolic cages for the required
time, weighed, anaesthetized with chloroform, and sac-
rificed by cervical dislocation at 5, 15, 30, 60, and
120 min post injection. Five mice were taken for each
time point. Organs or tissues of interest were removed,
washed with saline, and weighed; their activity was
measured in a shielded well-type γ-scintillation counter
using a sample containing 1% of the injected dose as a
reference. The results were calculated as percent of the
injected dose per gram of tissue or organ (% ID/g).
The weights of blood, bones, and muscles were as-
sumed to be 7, 10 and 40% of the total body weight,
respectively [32]. The ratio of the uptake in the in-
fected (inflamed) thigh muscle to that in normal
(control) thigh muscle was also calculated [33].
HPLC analysis was done by injecting a 10 µL
sample after filtration through a 0.22-µm Millipore
filter into the column (RP-18, 300 × 3.9 mm, Alpha
bond). We used a UV detector (SPD-6A) adjusted to
the 260 nm wavelength. The column was eluted with a
mixture of 60% acetonitrile and 40% water. The flow
rate was 1 mL min–1. Fractions of 1 mL volume were
collected separately using a fraction collector (up to 30
fractions) and counted using a well-type NaI(Tl) detec-
tor connected to a single-channel analyzer.
Docking Study
Comparative molecular modeling studies of the
binding mode of compound KH16 in both COX-1 and
In vitro stability in saline. A solution of 99mTc-
RADIOCHEMISTRY Vol. 59 No. 6 2017

634
ATTALLAH et al.
Table 1. In vitro inhibition of COX-1/2 enzymes with KH16
RESULTS AND DISCUSSION
COX-1
IC50, μM
5.33
0.73
15.6
COX-2
Synthesis and Properties of KH16
Compound
KH16
Indomethacin
Celecoxib
SI
1.54
32.6
0.32
3.46
0.02
48.75
The KH16 synthesis procedure is described above.
In the IR spectrum of KH16, the absorption bands of
the geminal CN groups disappear, and a single CN
stretching band appears at 2210 cm–1. In addition, NH
stretching bands appear at 3448, 3340, and 3300 cm–1,
and a carbonyl absorption band appears at 1699 cm–1.
COX-2 pockets were performed to delineate the inter-
action features. The starting coordinates of the X-ray
crystal structure of the COX-2 enzyme in the complex
with SC-558 (1CX2) and COX-1 (PDB code 2OYE)
were obtained from RCSB protein databank (Brook-
haven National Laboratory) [34, 35]. The hydrogen
atoms were added, and the enzyme structure was sub-
jected to the refinement protocol in which the con-
straints on the enzyme were gradually removed and
minimized until the rms gradient became
0.01 kcal mol–1 Å–1. The energy minimization was car-
ried out by the method of molecular mechanics with
AMBER force field.
1
In the H NMR spectrum of KH16, a singlet at
1.42 ppm belongs to CH3 protons; a singlet at 3.60 ppm,
to NH2 protons; two doublets at 7.68 and 8.04 ppm,
to the p-substituted phenyl ring; and a singlet at
9.84 ppm, to the amide proton. The 13C NMR spectrum
consists of 16 signals in the range 14.37–166.19 ppm,
of which the highest-field signal belongs to the CH3
group (14.37 ppm), and the lowest-field signals, to the
carbonyl groups (158.70 and 166.19 ppm). The DEPT-
135 spectrum was used to differentiate between differ-
ent types of carbon atoms. The mass spectrum of
KH16 contains the molecular ion peak at m/z 338 with
the relative abundance of 16%.
The newly designed compound KH16 was con-
structed from fragment libraries using ChemDraw pro-
gram. The partial atomic charges for each analog were
calculated by AM1 semiempirical method imple-
mented in MOE program package. The docking was
carried out on both COX-1 and COX-2 enzyme. The
lowest-energy conformer (global minimum) was pre-
positioned using the crystal structure of SC-558 ligand
as a template. Compound KH16 was optimally docked
in the enzyme binding pocket.
In vitro COX inhibitory assay. For the majority of
NSAIDs, the COX inhibition is the main mechanism
of action [36]. The ability of KH16 to inhibit COX
enzymes was compared to that of indomethacin
(nonselective COX inhibitor) and celecoxib (selective
COX-2 inhibitor). The results were expressed in terms
of IC50 values and COX-1/COX-2 selectivity index
(SI) (Table 1). It was noteworthy that KH16 has cer-
tain COX-2 selectivity over COX-1 with the selectivity
index of 3.46.
Statistical Analysis
The results are expressed as mean ± SD for at least
three experiments in the case of labeling procedures
and five experiments in the case of biological study.
For all analyses, the significance level P < 0.05 was
set, and unpaired Student’s t-test was used.
Analysis of Labeled KH16
Electrophoresis revealed that 99mTc-KH16 moved
toward the anode (suggesting anionic nature of the
complex), and 99mTcO4– moved toward the anode to
a considerably longer distance (Fig. 1).
HPLC analysis (Fig. 2) showed that the reten-
tion time of 99mTc-KH16 was approximately 16 min,
whereas the retention time of KH16 was approxi-
mately 14 min (UV detector).
Factors Influencing the Labeling Yield
Tin chloride content. The majority of 99mTc-ra-
diopharmaceuticals are prepared using SnCl2·2H2O
[Sn(II)] for reducing 99mTc from heptavalent to lower
valence state, which facilitates its chelation by com-
Fig. 1. Electrophoretic pattern of 99mTc-KH16.
RADIOCHEMISTRY Vol. 59 No. 6 2017

DESIGN, SYNTHESIS, 99mTc LABELING
635
Fig. 2. HPLC of (1) free 99mTcO–4 and (2) 99mTc-KH16.
Fig. 4. Influence of the KH16 amount on the radiochemical
yield of 99mTc-KH16. Conditions: 50 μg of Sn(II),
20 mCi mL–1 99mTcO4– solution, pH 5, room temperature,
10 min.
Fig. 3. Influence of the Sn(II) content on the radiochemical
yield of 99mTc-KH16. Conditions:
1 mg of KH16,
20mCi mL–1 99mTcO4– solution, pH 5, room temperature,
10 min. (1) 99mTc-KH16, (2) free pertechnetate, and
(3) colloid; the same for Figs. 4–6.
Fig. 5. Influence of pH of the reaction medium on the ra-
diochemical yield of 99mTc-KH16. Conditions: 1 mg of
KH16, 50 μg of Sn(II), 20 mCi mL–1 99mTcO4– solution,
room temperature, 10 min.
pounds of diagnostic importance. The effect of tin
chloride as a reducing agent on the labeling of KH16
with99mTc is illustrated in Fig. 3. As can be seen, the
radiochemical yield significantly increased as the
Sn(II) amount was increased from 5 (0.042 µmol) to
50 µg (0.421 µmol), reaching a maximum of 90.3%.
Further increase in the Sn(II) amount above 50 µg
leads to a decrease in the labeling yield (75.5% at
200 μg, 1.684 µmol) owing to colloid formation
(15.1%).
Substrate amount. The influence of the KH16
amount on the labeling yield was studied with the
Sn(II) amount fixed at 50 μg. The results are shown in
Fig. 4. Increasing the amount of KH16 was accompa-
nied by a significant increase in the labeling yield:
from 81.5% at 200 µg (0.59 µmol) to 90.5% at 1 mg
(2.95 µmol), which is associated with the presence of a
minimum limit for the volume used [37]. The 1-mg
amount of KH16 is optimum. At larger amounts of
KH16, the labeling yield decreases because of in-
creased formation of colloids and, probably, increased
generation of free radicals.
Fig. 6. Influence of reaction time on the radiochemical
yield of 99mTc-KH16. Conditions: 1 mg of KH16, 50 μg of
Sn(II), 20 mCi mL–1 99mTcO4– solution, pH 5, room tem-
perature.
from 1 to 6 (Fig. 5) with 1 mg of KH16 and 0.5 mL of
each buffer. The reaction time was 30 min. pH 5 is
optimum (90.5%). At pH > 7, the compound precipi-
tated.
Reaction time. The relationship between the reac-
tion time and the yield of 99mTc-KH16 is shown in
Fig. 6. The radiochemical yield was increased from
73.5 to 90.5% with increasing reaction time from 1 to
pH. Experiments were performed at pH ranging
RADIOCHEMISTRY Vol. 59 No. 6 2017

636
ATTALLAH et al.
Table 2. Stability of 99mTc-KH16 (relative content, %) in
showed a significant decrease in the 99mTc-KH16 up-
take.
normal saline and human serum (n = 3)a
Bacterially infected mice. The 99mTc-KH16 biodis-
tribution in bacterially inflamed mice (Table 4) was,
on the whole, similar to that in normal mice. The up-
take in the bacterially inflamed leg only slightly ex-
ceeded that in the normal leg. Thus, the complex is not
specific to bacterial infections.
Sterile inflamed mice. The 99mTc-KH16 biodis-
tribution in sterile inflammed mice (Table 5) is also, on
the whole, similar to that in normal mice, but the up-
take in the sterile inflamed muscle considerably ex-
ceeded that in the normal and infected muscles. Thus,
99mTc-KH16 can be used as a model to distinguish be-
tween sterile and bacterial inflammations.
Time, h
In saline
In human serum
90.5 ± 1.3
1
2
4
6
8
91.5 ± 1.7
90.7 ± 1.3
90.5 ± 1.5
85.3 ± 1.9*
75.5 ± 1.2*
89.7 ± 1.2
89.5 ± 1.4
83.3 ± 1.7*
73.5 ± 1.3*
a
Here and in Tables 3–5, the values significantly differing from
the previous value (P < 0.05) are marked with an asterisk.
10 min. Extending the reaction time to 120 min led to
a slight decrease in the radiochemical yield [38].
In vitro Stability of 99mTc-KH16
As shown in Table 2, incubation of the solution
containing 99mTc-KH16 for 8 h at 37°C in saline and
human serum (five experiments in each case) resulted
in a small release of the radioactivity from 99mTc-
KH16, as determined by paper chromatography. The
relative content of 99mTc-KH16 decreased in 8 h from
91.5 to 75.5% and from 90.5 to 73.5%, respectively.
The complex is practically stable for 4 h.
Docking Study
The degree of recognition for KH16 at binding
pockets of the COX-1 and COX-2 enzymes was evalu-
ated using Molecular Operating Environment 10.2008
(MOE) software (Chemical Computing Group, Can-
ada). The calculation results (Fig. 7) show that KH16
exhibits affinity for the amino acid residues of COX-2
pocket, namely, Phe518 and Ala527. Strong hydrogen
bonds are formed between the ester carbonyl oxygen
and Phe518 at the end cleft of the merged pocket and
the free Ala527 amino group at the terminal end of the
groove.
Biodistribution of 99mTc-KH16
Normal mice. The data on the 99mTc-KH16 biodis-
tribution in normal mice are given in Table 3. 99mTc-
KH16 was rapidly (in 5 min after injection) distributed
in blood, kidneys, heart, liver, and urine. After 30 min,
the 99mTc-KH16 uptake significantly decreased in
blood and heart but increased in kidneys and stomach.
At 2 h post injection, the majority of tissues and organs
On the other hand, KH16 was poorly recognized at
the narrow binding pocket, as it formed only one hy-
drogen bond with Tyr355; therefore, the ability to in-
hibit COX-1 is lower.
Table 3. Biodistribution of 99mTc-KH16 in normal mice (% ID/g ± SD, n = 5) at different times post injection
Organs and body fluids
Blood
Bones
Muscles
Brain
Lungs
5 min
15 min
30 min
60 min
120 min
15.50 ± 0.14
1.20 ± 0.01
1.20 ± 0.01
0.40 ± 0.01
1.70 ± 0.01
4.5 ± 0.5
3.30 ± 0.14
5.28 ± 0.16
1.10 ± 0.01
1.90 ± 0.01
1.10 ± 0.01
0.95 ± 0.01
13.80 ± 0.23*
1.60 ± 0.01*
1.50 ± 0.03*
0.90 ± 0.02*
1.90 ± 0.02*
3.50 ± 0.11*
4.10 ± 0.18*
6.90 ± 0.41*
1.70 ± 0.01*
3.90 ± 0.22*
3.95 ± 0.03*
0.70 ± 0.02*
7.10 ± 0.18*
1.41 ± 0.03
1.80 ± 0.04*
1.23 ± 0.03*
2.40 ± 0.03*
3.1 ± 0.3
3.40 ± 0.24*
6.11 ± 0.02
2.30 ± 0.22*
4.90 ± 0.01*
4.80 ± 0.01*
0.80 ± 0.01
4.70 ± 0.01*
1.10 ± 0.01*
1.90 ± 0.01
0.60 ± 0.01*
1.70 ± 0.03٭
2.7 ± 0.4
3.11 ± 0.11
12.50 ± 0.18*
0.95 ± 0.03*
5.40 ± 0.01*
3.85 ± 0.01*
1.10 ± 0.03
1.50 ± 0.02*
0.88 ± 0.02*
1.30 ± 0.01*
0.50 ± 0.03
0.90 ± 0.02*
1.80 ± 0.15*
1.20 ± 0.11*
18.0 ± 0.13*
0.70 ± 0.01*
3.50 ± 0.15*
3.80 ± 0.02
1.11 ± 0.01
Heart
Liver
Kidneys
Spleen
Intestine
Stomach
Thyroid
RADIOCHEMISTRY Vol. 59 No. 6 2017

DESIGN, SYNTHESIS, 99mTc LABELING
637
Table 4. Biodistribution of 99mTc-KH16 in bacterially infected mice (% ID/g ± SD, n = 5) at different times post injection
Organs and body fluids
Blood
Bones
Brain
Lungs
Heart
Liver
Kidneys
Spleen
Intestine
Stomach
Thyroid
Normal muscle
Infected muscle
5 min
15 min
30 min
60 min
120 min
14.90 ± 0.11
1.20 ± 0.01
0.30 ± 0.01
1.60 ± 0.01
4.1 ± 0.5
4.30 ± 0.14
5.60 ± 0.16
1.13 ± 0.01
1.85 ± 0.01
0.95 ± 0.01
0.95 ± 0.01
1.30 ± 0.01
1.50 ± 0.25
12.6 ± 0.23*
1.70 ± 0.01*
0.80 ± 0.02*
1.70 ± 0.02
3.50 ± 0.11*
4.40 ± 0.18
6.7 ± 0.4*
1.80 ± 0.01*
3.50 ± 0.22*
2.95 ± 0.03*
0.77 ± 0.02*
1.30 ± 0.01
1.6 ± 0.3
11.2 ± 0.18*
1.60 ± 0.03
1.20 ± 0.03*
2.30 ± 0.03*
3.1 ± 0.3
4.40 ± 0.24
6.40 ± 0.02
2.40 ± 0.22*
4.20 ± 0.01*
3.80 ± 0.01*
0.85 ± 0.01*
1.20 ± 0.01
1.7 ± 0.3
5.50 ± 0.01*
1.30 ± 0.01*
0.60 ± 0.01*
2.80 ± 0.03*
2.7 ± 0.4
3.40 ± 0.11*
10.90 ± 0.18*
0.85 ± 0.03*
5.10 ± 0.01*
3.65 ± 0.01*
1.10 ± 0.03*
1.30 ± 0.01
1.40 ± 0.20
2.40 ± 0.02*
0.90 ± 0.02*
0.65 ± 0.03
1.90 ± 0.02*
1.80 ± 0.15*
1.30 ± 0.11*
16.8 ± 1.3*
0.40 ± 0.01*
3.90 ± 0.15*
3.30 ± 0.02*
1.14 ± 0.01
1.20 ± 0.01
1.3 ± 0.3
Table 5. Biodistribution of 99mTc-KH16 in sterile inflamed mice (% ID/g ± SD, n = 5) at different times post injection
Organs and body fluids
Blood
Bones
Brain
Lungs
Heart
Liver
Kidneys
Spleen
Intestine
Stomach
Thyroid
Normal muscle
Inflamed muscle
T/NT
5 min
15 min
30 min
60 min
120 min
2.50 ± 0.02*
0.80 ± 0.02*
0.65 ± 0.03
2.10 ± 0.02*
1.70 ± 0.15*
1.30 ± 0.11*
15.80 ± 0.30*
0.60 ± 0.01*
3.90 ± 0.15*
3.70 ± 0.02
1.13 ± 0.01
1.20 ± 0.01
4.30 ± 0.51
3.58 ± 0.30
14.5 ± 0.11
1.10 ± 0.01
0.35 ± 0.01
1.50 ± 0.01
3.8 ± 0.5
4.50 ± 0.14
5.40 ± 0.16
1.10 ± 0.01
1.80 ± 0.01
1.05 ± 0.01
0.93 ± 0.01
1.30 ± 0.01
2.5 ± 0.4
12.8 ± 0.23*
1.50 ± 0.01*
0.75 ± 0.02*
1.60 ± 0.02
3.60 ± 0.11
4.60 ± 0.18
6.10 ± 0.4*
1.60 ± 0.01*
3.60 ± 0.22*
3.80 ± 0.03*
0.72 ± 0.02*
1.30 ± 0.01
5.6 ± 0.5*
11.1 ± 0.18*
1.60 ± 0.03
1.10 ± 0.03*
2.20 ± 0.03*
2.90 ± 0.33*
4.60 ± 0.24
5.95 ± 0.02
2.20 ± 0.22*
4.50 ± 0.01*
4.60 ± 0.01*
0.83 ± 0.01
1.20 ± 0.01
6.10 ± 0.51
5.08 ± 0.40
5.70 ± 0.01*
1.20 ± 0.01*
0.70 ± 0.01*
2.60 ± 0.03*
2.70 ± 0.44
3.30 ± 0.11*
11.80 ± 0.18*
0.95 ± 0.03*
5.20 ± 0.01*
3.65 ± 0.01*
1.10 ± 0.03*
1.30 ± 0.01
5.20 ± 0.51
4.0 ± 0.20
1.92 ± 0.3
4.3 ± 0.4
(a)
(b)
Fig. 7. Docking of KH16 in the active sites of (a) COX-1 (one H-bond between NH2 and Tyr355) and (b) COX-2 (two H-bonds
between ester carbonyl oxygen and NH2 of Phe518 and Ala527).
RADIOCHEMISTRY Vol. 59 No. 6 2017

638
ATTALLAH et al.
macol. Exp. Ther., 1999, vol. 288, pp. 1288–1297.
Thus, KH16 demonstrated good inhibitory activity
16. Guzman, A., Yuste, F., Toscano, R.A., et al., J. Med.
for COX-1 and COX-2 enzymes with IC50 values of
5.33 and 1.54 µM, respectively (selectivity index
3.46). A docking study revealed the formation of two
H-bonds between KH16 and amino acid residues in
COX-2, whereas only one H-bond is formed with the
active site of COX-1 enzyme. KH16 was labeled with
99mTc with a yield of about 90%. The labeled com-
pound allows revealing sterile inflammation but is
practically nonspecific to bacterial inflammation.
KH16 is a promising scaffold for the development of
potential anti-inflammatory agents.
Chem., 1986, vol. 29, pp. 589–591.
17. Simone, O.F.D., Cristiano, F.S., David, L.N., et al.,
Braz. Arch. Boil. Technol., 2005, vol. 48, pp. 89–96.
18. Beiki, D., Yousefi, G., Fallahi, B., et al., Iran. J. Pharm.
Res., 2013, vol. 12, pp. 347–353.
19. Sanad, M.H. and Amin, A.M., Radiochemistry, 2013,
vol. 55, no. 5, pp. 521–526.
20. Oliveira Pereira, M., Souza Rocha, G., Medeiros, A.C.,
et al., Med. Chem. Res., 2011, vol. 21, pp. 1433–1438.
21. El-Ghany, E.A., Amin, A.M., El-Kawy, O.A., and
Amin, M., J. Label. Compd. Radiopharm., 2007,
vol. 50, pp. 25–31.
ACKNOWLEDGMENTS
22. Welling, M.M., Lupetti, A., Balter, H.S., et al., J. Nucl.
Med., 2001, vol. 42, pp. 788–794.
The authors are grateful to the Institute of Scientific
Research and Revival of Islamic Heritage, Umm Al-
Qura University, Makkah, Saudi Arabia, for financial
support of the study (project no. 43310014).
23. Abbas, S.E., Awadallah, F.M., Ibrahim, N.A., and
Gouda, A.M., Eur. J. Med. Chem., 2010, vol. 45,
pp. 482–491.
24. Etienne, A. and Correia, Y., Bull. Soc. Chim. Fr., 1969,
vol. 10, pp. 3704–3712.
REFERENCES
25. Jacobs, W.A. and Heidelberger, M., J. Am. Chem. Soc.,
1. Roberts, L.J. and Morrow, J.D., The Pharmacological
Basis of Therapeutics, Goodman, L.S., Gilman, A.G.,
Hardman, J.G., and Limbird, A.E., Eds., New York:
McGraw-Hill, 2001, 10th ed., pp. 687–733.
2. Schneider, V., Levesque, L.E., Zhang, B., et al., Am. J.
Epidemiol., 2006, vol. 164, pp. 881–889.
3. Cryer, B., Am. J. Gastroenterol., 2005, vol. 100,
pp. 1694–1695.
1917, vol. 39, pp. 1435–1439.
26. Praveen Rao, P.N., Amini, M., Li, H., et al., J. Med.
Chem., 2003, vol. 46, pp. 4872–4882.
27. Motaleb, M.A., El-Said, H., Atef, M., and Abd-Allah, M.,
Radiochemistry, 2012, vol. 54, no. 3, pp. 274–278.
28. Moustapha, M.E., Motaleb, M.A., and Ibrahim, I.T.,
J. Radioanal. Nucl. Chem., 2011, vol. 287, pp. 35–40.
29. Van Der Laken, C.J., Boerman, O.C., and Oyen, W.J.G.,
4. Singh, G., Am. J. Med., 1998, vol. 105, pp. 31S–38S.
5. Vane, J.R. and Botting, R.M., Scand. J. Rheumatol.
Suppl., 1996, vol. 102, pp. 9–21.
J. Nucl. Med., 2000, vol. 41, pp. 463–469.
30. Oyen, W.J.G., Boerman, O.C., and Corstens, F.H.M.,
J. Microbiol. Meth., 2001, vol. 47, pp. 151–157.
6. Smith, C.J., Zhang, Y., Koboldt, C.M., et al., Proc. Nat.
31. Asikoglu, M., Yurt, F., Cagliyan, O., et al., Appl. Ra-
Acad. Sci., 1998, vol. 95, pp. 133–138.
diat. Isot., 2000, vol. 53, pp. 411–413.
7. Sarkar, F.H., Adsule, S., Li, Y., and Padhye, S., Mini
Rev. Med. Chem., 2007, vol. 7, pp. 599–608.
8. Gouda, A.M. and Abdelazeem, A.H., Eur. J. Med.
32. Johannsen, B. and Spies, H., Chemistry and radiophar-
macology of technetium complexes, Workshop on Gen-
erator and Cyclotron Produced Radiopharmaceuticals,
Riyad (Saudi Arabia), Oct. 13–31, 1991.
33. Sanad, M.H., Borai, E.H., and Fawzy, A.S.M., IOSR J.
Environ. Sci., Toxicol. Food Technol., 2014, vol. 8,
pp. 10–17.
Chem., 2016, vol. 114, pp. 257–292.
9. Laufer, S.A., Augustin, J., Dannhardt, G., and Kiefer, W.,
J. Med. Chem., 1994, vol. 37, pp. 1894–1897.
10. Bias, P., Buchner, A., Klesser, B., and Laufer, S., Am. J.
Gastroenterol., 2004, vol. 99, pp. 611–618.
34. Kurumbail, R.G., Stevens, A.M., Gierse, J.K., et al.,
11. Ulbrich, H., Fiebich, B., and Dannhardt, G., Eur. J.
Med. Chem., 2002, vol. 37, pp. 953–959.
12. Laufer, S., Tollmann, K., and Striegel, H.G., US Patent
Nature, 1996, vol. 384, pp. 644–648.
35. Harman, C.A., Turman, M.V., Kozak, K.R., et al.,
J. Biol. Chem., 2007, vol. 282, pp. 28096–28105.
6 878 738 B1, 2005.
36. Warner, T.D., Giuliano, F., Vojnovic, I., et al., Proc.
13. Liu, W., Zhou, J., Bensdorf, K., et al., Eur. J. Med.
Chem., 2011, vol. 46, pp. 907–913.
14. Liedtke, A.J., Keck, P.R., Lehmann, F., et al., J. Med.
Natl. Acad. Sci. USA, 1999, vol. 96, pp. 7563–7568.
37. Sankha, C., Sujata, S.D., Susmita, C., et al., Appl. Ra-
diat. Isot., 2010, vol. 68, pp. 314–316.
Chem., 2009, vol. 52, pp. 4968–4972.
38. Sarda, L., Cremieux, A.C., Lebellec, Y., et al., J. Nucl.
15. Jett, M.F., Ramesha, C.S., Brown, C.D., et al., J. Phar-
Med., 2003, vol. 44, pp. 920–926.
RADIOCHEMISTRY Vol. 59 No. 6 2017