Site specific chemical delivery of NSAIDs to inflamed joints: Synthesis, biological activity and γ-imaging studies of quaternary ammonium salts of tropinol esters of some NSAIDs or their active metabolites

Article abstract of DOI:10.1016/j.bmc.2008.09.050

Quaternized tropinol ester derivatives of some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) or their active metabolites, were prepared and studied for their anti-inflammatory activity in a chronic inflammation model and for inflamed tissue tropism. The quaternized esters were radiolabeled with 99^mTechnetium (^99mTc) and their selective localization in the inflamed tissue was traced using scintigraphy. In the chronic arthritis rodent model, most of the quaternized esters exhibited anti-inflammatory effect comparable to their respective parent drugs. In the γ-imaging studies only the quaternary derivatives exhibited selective accumulation into the inflamed tissue unlike the parent NSAIDs or the unquaternized tropinol esters. This work is a step ahead in the direction of use of quaternary ammonium ester derivatives for site specific chemical delivery of commonly used NSAIDs to the inflamed tissues to minimize their GIT side effect or other systemic toxicities.

Full text of DOI:10.1016/j.bmc.2008.09.050

Bioorganic & Medicinal Chemistry 16 (2008) 9443–9449
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Site specific chemical delivery of NSAIDs to inflamed joints: Synthesis,
biological activity and
c-imaging studies of quaternary ammonium salts
of tropinol esters of some NSAIDs or their active metabolites
a
a
a
Mange Ram Yadav a, , Vijay P. Pawar , Sejal M. Marvaniya , Parmeshwari K. Halen ,
*
Rajani Giridhar a, Anil K. Mishra b
a Pharmacy Department, Faculty of Technology & Engineering, Kalabhavan, The M.S. University of Baroda, Vadodara 390 001, India
b Institute of Nuclear Medicine and Allied Sciences, Brig. S.K. Mazumdar Road, New Delhi 110 054, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Quaternized tropinol ester derivatives of some commonly used non-steroidal anti-inflammatory drugs
(NSAIDs) or their active metabolites, were prepared and studied for their anti-inflammatory activity in
a chronic inflammation model and for inflamed tissue tropism. The quaternized esters were radiolabeled
with 99mTechnetium (99mTc) and their selective localization in the inflamed tissue was traced using scin-
tigraphy. In the chronic arthritis rodent model, most of the quaternized esters exhibited anti-inflamma-
Received 2 August 2008
Revised 16 September 2008
Accepted 17 September 2008
Available online 20 September 2008
tory effect comparable to their respective parent drugs. In the
c-imaging studies only the quaternary
Keywords:
Tropinol
NSAIDs
Anti-inflammatory activity
derivatives exhibited selective accumulation into the inflamed tissue unlike the parent NSAIDs or the
unquaternized tropinol esters. This work is a step ahead in the direction of use of quaternary ammonium
ester derivatives for site specific chemical delivery of commonly used NSAIDs to the inflamed tissues to
minimize their GIT side effect or other systemic toxicities.
c
-Imaging studies
Ó 2008 Elsevier Ltd. All rights reserved.
Site specific delivery
1. Introduction
circumstances COX-2 may be important to homeostasis.10 Rofecox-
ib was withdrawn11 worldwide because of evidence of increased
NSAIDs are among the most widely used prescription drugs, for
the treatment of pain and inflammation, particularly arthritis.1 As
inhibitors of COX isozymes, traditional NSAIDs relieve the signs
and symptoms of inflammation by decreasing prostaglandin (PG)
production but may cause serious gastrointestinal (GI) and renal
damage, especially with the long term use.2,3 To improve the GI
tolerance of NSAIDs by masking the free carboxylic group tempo-
rarily, numerous ester and amide prodrugs of NSAIDs have been
designed and evaluated in the past.4–6 However, it is still not pos-
sible to avoid the systemic side effects of the parent drugs by the
prodrug approach.
Classical NSAIDs inhibit COX-1 and COX-2 isozymes to varying
extent.7 The differential tissue distribution of COX-1 and COX-2
provided a rationale for the development of selective COX-2 inhib-
itors as anti-inflammatory and analgesic agents that lacked the GI
and hematological liabilities exhibited by currently marketed NSA-
IDs.8 Highly selective COX-2 inhibitors have been developed and
marketed as promising gastroprotective agents.9 Despite the initial
enthusiasm surrounding selective COX-2 inhibitors, questions
remain regarding their ultimate benefit and safety, since in certain
cardiovascular risk in selected, high dose patients that may reflect
rofecoxib’s greater COX-2 selectivity.12 This voluntary withdrawal
of rofecoxib from the market by Merck and Co. raised the question
of cardiovascular safety of the entire class of COX-2 inhibitors.13
An alternative way to reduce the adverse reactions is to carry
the drugs selectively to their target tissue. Pharmacokinetic studies
on 14C-labeled acetylcholinesterase reactivators containing a qua-
ternary ammonium (QA) moiety demonstrated a rapid and inten-
sive concentration of these compounds in cartilaginous tissues
after intramuscular injection.14,15 It was demonstrated on cultured
chondrocytes that high specificity for cartilage resulted for QA
compounds from ionic interactions between the QA entity of the
reactivator and the anionic sites (carboxylate and sulfate ester
groups) of proteoglycans, a major component of cartilage.16 This
property has been used to target NSAIDs toward cartilage. New
potential anti-inflammatory drugs have been synthesized by bind-
ing pharmacologically active structures to QA salts. These new
molecules could concentrate in the cartilaginous tissue and thus
allow a significant decrease of the effective dose and therefore an
attenuation of adverse effects such as GI toxicity.17 A study on
quaternization of oxicams suggested that a QA group linked to an
NSAID could help to increase the drug concentration in joints
and permit a decrease in the administered dose to diminish the
* Corresponding author. Tel.: +91 0265 2434187; fax: +91 0265 2418927.
E-mail address: mryadav11@yahoo.co.in (M.R. Yadav).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.09.050

9444
M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
side effects of these drugs.17 In a study on biodistribution of 14C-
labeled QA–glucosamine conjugates it was shown that introduc-
tion of QA-moiety on D-glucosamine allowed the molecule to be
plexation of the compounds (16–22) with 99mTc, experiments were
performed under different set of conditions by varying parameters
like quantity of stannous chloride used for reduction at pH 4.0,
change of pH and incubation period of the ligands and the metal
ions in the medium. pH 7.0 and incubation period of 30 min were
found out to be optimum to afford highest radiolabeling efficiency
(Tables 1 and 2). All the radiolabeled complexes (99mTc–QA) were
found to be stable enough for 6 h in serum and normal saline
(Table 3).
carried more selectively and intensively to cartilaginous tissues
soon after injection.18
To optimize the therapeutic efficacy of the existing NSAIDs, a
program was initiated in this Department. Under this program
aminoalcohol ester derivatives of carboxyl group-bearing NSAIDs
were prepared and evaluated for their potency and GI protecting
efficiency19–24 in comparison to the parent drugs. All the ester
derivatives exhibited equal or higher anti-inflammatory activity
than the parent NSAIDs except for the tropinol esters, in the acute
model of inflammation. Another important observation made dur-
ing these studies, regarding the tropinol ester derivatives, was
their resistance toward enzymatic hydrolysis in simulated
experiments.
c-Imaging studies were planned to be carried out to study the
localization pattern of the 99mTc–QA conjugates in inflamed tis-
sues of the animal model. Carrageenan was used as a phlogistic
agent for producing inflammation in one of the hind paws of
rat. The conjugates (99mTc–QA) were administered intravenously
and imaging studies were performed at different time intervals.
The parent NSAIDs (1–7) were also labeled with 99mTc and used
as one set of controls in the imaging studies. Two tertiary amine
derivatives (11, 12) were also labeled with 99mTc in similar way
and used as another set of controls. These studies clearly
revealed significant differences between the 99mTc–QA conjugates
and the parent drugs (1–7) for their localization behavior in the
inflamed tissue (Fig. 1). None of the parent NSAIDs (1–7) showed
affinity for the inflamed joint tissue whereas, all the quaternized
compounds (16–22) selectively localized in the inflamed areas.
The two tertiary amines (11 and 12) also did not show any pref-
erential localization behavior. The images also revealed a prefer-
ential distribution of the 99mTc–QA conjugates in liver and
kidney.
The observation14–18 that QA compounds are selectively and
intensivey concentrated in the cartilaginous tissue prompted us
to study the tropinol esters of some NSAIDs after quaternization,
for their localization into the inflamed tissues and their anti-
inflammatory activity in a chronic model. Tropinol esters specifi-
cally were chosen for the study due to their higher stability in
the in vitro experiments. Although, these derivatives did not exhi-
bit anti-inflammatory activity in the acute model for a period of 3 h
but, it was hypothesized that these derivatives would be slowly
hydrolyzed chemically in the blood, releasing the parent NSAIDs
for exhibiting their inherent anti-inflammatory activity.
In this paper we report the synthesis of QA derivatives of trop-
inol esters of indomethacin (1), ketoprofen (2), biphenylacetic acid
(3, an active metabolite of fenbufen), flurbiprofen (4), ibuprofen
(5), 6-methoxy-2-napthylacetic acid (6) (6-MNA, an active metab-
olite of nabumetone) and naproxen (7); their tagging to techne-
2.3. Anti-inflammatory activity
The tropinol esters (9–15) did not exhibit any anti-inflamma-
tory activity in the carrageenan-induced rat paw edema
model20–24 which is considered to be a model for acute inflamma-
tory conditions. In the kinetic studies, these esters were found to
be resistant to hydrolysis in plasma and buffers (pH 2.0 and 7.4)
up to 8 h. The absence of anti-inflammatory activity in the acute
inflammation model could be due to the non-conversion of these
esters (9–15) into their parent NSAIDs in a duration of 3 h (the time
period for which% inhibition of inflammation was recorded in the
carrageenan-induced inflamed model). Considering that the ester
derivatives (16–22) ultimately would be chemically hydrolyzed
in due course of time, liberating the parent NSAIDs, it was planned
to evaluate their anti-inflammatory potential in a chronic inflam-
mation model. Adjuvant arthritis, an accepted and well established
standard model25 was chosen for this purpose. The parent NSAIDs
(1–7) were used as standards for comparing the anti-inflammatory
activity of the quaternized ester derivatives (16–22), which were
administered on equivalent molar doses. Animals were dosed daily
with parent drugs (1–7) and the derivatives (16–22) separately
from day 1 to day 12. The phlogistic agent (Mycobacterium butyri-
cum) suspended in heavy paraffin oil was injected into the sub-
plantar region of the hind paws of the rats only on day 1. Paw vol-
umes of all the test animals and controls were measured on days 3,
7, 14 and 21.
On days 3 and 7, the derivatives (16–22) showed lesser inhibi-
tion than the parent NSAIDs (1–7). On day 14 all the derivatives
exhibited comparable anti-inflammatory activity to the parent
NSAIDs except for derivatives (17 and 18). On day 21, all the qua-
ternary ammonium ester derivatives exhibited significantly higher
anti-inflammatory activity than the parent drugs except for deriv-
ative 17 (Table 4). The percent inhibition of inflammation exhib-
ited by the parent drugs (1–7) declined on 21st day as compared
to that on 14th day whereas, the percent inhibition of inflamma-
tion by the derivatives increased on 21st day in comparison to that
observed on 14th day. This could be due to the slow chemical
tium-99m
(
99mTc) and tissue localization studies, and their
efficacy as anti-inflammatory agents in a chronic inflammation
model, adjuvant arthritis.
2. Results and discussion
2.1. Syntheses
Indomethacin (1) was reacted with tropinol (8) in presence of
dicyclohexylcarbodiimide (DCC) and dimethylaminopyridine
(DMAP) to obtain the ester (9) as an oily product. The ester (9)
was quaternized with methyl iodide to obtain the desired quater-
nary derivative (16). The other products (17–22) (Scheme 1) were
obtained in a similar way by reacting their respective esters21,22,24
(10–15) with methyl iodide. All the compounds were characterized
by their spectral and elemental data.
2.2.
c-Imaging studies
In order to check the site specific concentration of these quater-
nary derivatives (16–22) it was planned to tag these derivatives
with radioactive 99mTc and study their site specific localization ten-
dency in the inflamed tissue. For such a study to give some mean-
ingful output it is essential that the compounds should have high
binding affinity for the radioactive complexing agent and the com-
plexed derivatives (99mTc–QA) should be stable enough in serum to
last for a period of about 4 hours or more.
Radiolabeling of the compounds (16–22) with 99mTc was carried
out by direct labeling method.25 Technetium, available as pertech-
netate (TcO4À) with +7 oxidation state does not complex with
ligands by direct addition. For this to occur, it has to be reduced
to +4 oxidation state using stannous chloride in acidic medium.
For determining the ideal conditions for highest degree of com-

M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
9445
Me
Me
N
N
DCC, DMAP
R-COOH
+
H
H
R
(8)
(9-15)
O
OH
(1-7)
O
MeI
Me
Me
N
+
I
.
H
(16-22)
O
R
R
O
(1, 9, 16)
Me
(4, 12, 19)
MeO
Me
F
N
Me
O
(5, 13, 20)
(6, 14, 21)
Me
Me
Cl
(2, 10, 17)
(3, 11, 18)
O
Me
MeO
MeO
Me
(7, 15, 22)
Scheme 1.
Table 1
Effect of pH on the percent binding of parent NSAIDs and their derivatives with 99mTc
Table 2
Effect of incubation time on the percent binding of compounds
Compound
pH
Compound
Time (min)
20
5.5
6.0
6.5
7.0
7.5
8.0
0
5
10
30
40
% Radiolabeling
% Radiolabeling
1
2
3
4
5
77.15
79.96
75.71
75.96
71.45
70.05
69.05
67.34
61.58
79.06
76.90
62.21
69.70
83.23
89.56
91.50
82.56
79.96
79.66
75.74
78.50
72.26
89.34
86.56
74.50
77.70
96.01
90.97
93.50
93.06
94.76
95.06
90.08
90.79
85.30
95.98
96.59
92.01
91.40
96.37
91.48
96.78
95.22
95.71
95.05
93.95
92.90
86.42
96.24
97.61
93.86
91.56
95.87
91.30
94.67
94.12
94.96
94.12
92.98
91.44
86.01
96.66
95.46
91.76
90.23
93.12
90.56
91.75
90.11
91.07
92.12
87.50
87.16
84.36
90.40
91.47
87.63
86.67
1
2
3
4
5
66.56
60.66
66.12
68.51
71.56
63.61
70.07
62.16
60.05
66.15
61.01
61.32
61.34
62.57
87.15
81.56
84.23
86.16
86.55
81.56
87.85
82.12
82.15
83.70
81.56
68.36
80.66
80.61
93.15
86.79
89.73
90.97
88.93
91.50
90.26
85.24
85.26
88.20
87.56
85.23
87.77
84.96
96.12
91.37
96.72
95.20
94.98
94.52
94.01
92.60
87.57
94.57
97.46
93.06
91.96
92.98
96.38
91.93
97.28
96.01
95.0
94.59
94.30
92.71
88.05
94.63
97.63
93.10
92.05
92.99
96.40
91.95
97.15
96.20
95.96
94.56
94.60
92.70
87.99
94.12
97.58
93.08
92.70
92.95
6
7
6
7
16
17
18
19
20
21
16
17
18
19
20
21
22
hydrolysis of the ester derivatives (16–22) in the body to release
the parent NSAIDs (1–7) which exhibited their normal anti-inflam-
matory activity. Concentration of the parent NSAIDs (1–7) must
have fallen down on day 21 due to their metabolic inactivation
as the last dosing was performed on day 12.
3. Conclusions
Cartilage targeting strategy for the conventional NSAIDs has
been explored for reducing their local and systemic side effects.

9446
M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
Table 3
Stability studies of radiolabeled complexes in normal saline and human serum
Compound
% Radiolabeling efficiency (saline)
% Radiolabeling efficiency (serum)
0.25 h
02 h
04 h
06 h
24 h
0.25 h
02 h
04 h
06 h
24 h
1
2
3
4
5
96.37
93.95
96.78
91.48
95.7
95.05
95.22
92.90
86.42
96.24
97.61
93.86
91.56
93.18
95.60
92.38
96.44
89.61
94.04
94.66
94.27
92.01
85.22
94.92
97.35
93.38
89.69
93.24
93.4
89.37
85.47
92.82
81.65
87.89
90.90
88.23
83.54
76.11
86.18
95.16
88.52
85.65
91.30
84.55
81.95
84.27
81.18
81.19
82.65
82.52
81.56
76.19
82.97
93.17
86.75
81.08
85.53
95.39
92.95
95.89
90.48
94.70
94.55
93.22
91.90
87.42
93.24
96.61
92.81
90.56
92.08
94.60
92.38
92.44
89.61
93.04
91.66
93.20
91.01
85.22
92.92
96.35
92.38
89.09
92.00
90.40
85.95
89.07
88.82
90.56
88.09
91.89
88.94
80.88
91.81
96.35
91.70
88.95
91.78
89.32
83.47
86.89
86.65
86.89
87.96
88.23
83.54
76.19
86.18
95.16
86.52
85.65
91.30
74.50
74.95
74.27
72.18
78.19
72.69
79.52
80.51
75.19
82.03
91.37
84.75
80.08
85.53
85.95
96.07
88.82
90.56
94.09
91.89
86.94
81.88
92.81
97.35
92.70
89.95
91.78
6
7
16
17
18
19
20
21
22
The carboxylic group-bearing NSAIDs were derivatized into qua-
ternary tropinol esters. These esters showed the tendency for
selective accumulation into the inflamed tissues and exhibited
anti-inflammatory activity comparable to their parent drugs in
chronic inflammation model. Selective concentration of these ester
derivatives into the tissue in demand could also allow reduction in
their doses. It could be concluded that site specific delivery of these
quaternary ester derivatives could prove to be an effective strategy
for long term therapy of inflammatory conditions like arthritis
with these well known time tested NSAIDs inflicting GIT and sys-
temic side effects to the minimum extent.
nia solution (2 Â 10 ml) to remove any unreacted acid and then
washed with chilled water. The organic layer was separated, dried,
and evaporated to get the ester (8-dimethyl-8-azabicy-
clo[3,2,1]octan-3-yl) 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1H-indol-3-acetate (9) as an oily product.
4.1.2. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl 1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-acetate
methiodide (16)
The oily tropinol ester (9) obtained above was refluxed with
methyl iodide (0.8 ml) in diisopropyl ether (20 ml) for half an hour.
Ether was removed under vacuum and the residue triturated with
ethyl acetate to remove impurities. The crude product was dried
and crystallized from methanol to afford the quaternized product
(16); 71% yield, mp 228–30 °C; IR(KBr, cmÀ1): 1728, 1686, 1313,
1225, 1151, 1070, 1010 and 752; PMR (DMSO-d6): d (ppm) 7.65–
7.67 (d, 2H), 7.52–7.55 (d, 2H), 6.94–6.95 (d, 1H), 6.85–6.87 (d,
1H), 6.66–6.69 (dd, 1H), 5.13–5.16 (t, 1H), 4.02 (s, 2H), 3.83 (s,
3H), 3.75 (s, 2H), 3.30 (s, 3H), 3.17 (s, 3H), 2.59–2.68 (m, 2H),
2.40 (s, 3H), 2.16–2.18 (m, 2H), 1.84–1.99 (m, 4H). Anal. Calcd for
C28H32N2O4ClI (622.92): C 53.98, H 5.17, N 4.49; found: N 4.36%.
4. Experimental
Parent drug indomethacin (1) was obtained as gift sample from
M/s Ranbaxy Laboratories, Gurgaon, India. Tropinol was obtained
by the basic hydrolysis (methanol/potassium hydroxide) of atro-
pine. Lambda (Type IV) carrageenan, Freund’s adjuvant and all
the other chemicals were purchased from Sigma–Aldrich. Distilled
water was used in preparation of buffer solutions. Anhydrous
sodium sulfate was used as drying agent. Melting points were
taken in open capillaries and are uncorrected. 1H NMR spectra
were recorded on a 300/400 MHz instrument. The IR spectra were
recorded on Shimadzu-8300 FT-IR using KBr disc. Absorbance was
measured on a Shimadzu UV-1601 spectrophotometer. Plethys-
mometer (Eugo-Basil, Italy) was used for measuring the rat paw
volume. Scintigraphic studies were performed using SPECT Gamma
camera (LC-75-005, Diacam, Siemens, USA). The protocol for the
animal experiments performed was approved by the IAEC (Institu-
tional Animal Ethics Committee) as registered under CPCSEA
(Committee for the Purpose of Control and Supervision of Experi-
ments on Animals), Govt. of India.
4.1.3. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl
2-(3-benzoylphenyl)propionate methiodide (17)
The ester (10)24 was reacted with methyl iodide as described
under compound (16) to obtain the methiodide (17), 85% yield,
mp 211–13 °C; IR(KBr, cmÀ1): 1728, 1661, 1576, 1448, 1319,
1286, 1207, 117, 1159, 1043, 719, and 704; PMR (DMSO-d6): d
(ppm) 7.44–7.81 (m, 9H), 5.05–5.08 (t, 1H), 4.01–4.05 (m, 2H),
3.73–3.81 (m, 1H), 3.28 (s, 3H), 3.14 (s, 3H), 2.52–2.60 (m, 2H),
1.97–2.07 (m, 2H), 1.65–1.87 (m, 3H), 1.54–1.56 (d, 3H), 1.39–
1.45 (m, 1H). Anal. Calcd for C25H30NO3I (519.40): C 57.80, H
5.82, N 2.69; found: C 57.62, H 5.76, N 2.48%.
4.1. Synthesis
4.1.4. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl
4-biphenylacetate methiodide (18)
4.1.1. Synthesis of tropinol ester (9) of indomethacin (1)
Prepared from ester (11)23 as described under compound (16):
90% yield, mp 248–53 °C; IR(KBr, cmÀ1): 1730, 1489, 1175, 1156,
1045, 1005, 750 and 650; PMR (DMSO-d6): d (ppm) 7.31–7.60
(m, 9H), 5.13–5.16 (t, 1H), 3.69 (s, 2H), 3.28 (s, 3H), 3.12 (s, 3H),
2.72–2.81 (m, 2H), 2.23–2.31 (m, 2H), 1.66–2.17 (m, 4H). Anal.
Calcd for C23H28NO2I (477.38): C 57.86, H 5.91, N 2.93; found: C
57.55, H 6.22, N 2.76%.
Parent drug (1) (3.91 mmol) was dissolved in anhydrous
dichloromethane (25 ml). The solution was cooled in ice bath, dicy-
clohexylcarbodiimide (DCC, 0.9 g, 4.36 mmol) and dimethylamino-
pyridine (DMAP, 0.06 g, 0.49 mmol) was added to the above
solution and the reaction mixture stirred for 30 min. A solution
of tropine (0.55 g, 3.91 mmol) in anhydrous dichloromethane
(10 ml) was added drop-wise with stirring to the above chilled
reaction mixture. The stirring was continued for 16 h at RT. The
reaction mixture was filtered to remove DCU. The filtrate was
diluted with dichloromethane (50 ml) and stirred with dilute ace-
tic acid (10%, 10 ml) to convert unreacted DCC to dicyclohexylurea
(DCU). Organic layer was separated and washed with dilute ammo-
4.1.5. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl 2-(2-fluoro-4-
biphenyl)propionate methiodide (19)
Prepared form ester (12)23 as described under compound (16).
87% yield, mp 235–38 °C; IR(KBr, cmÀ1): 1728, 1661, 1574, 1485,

M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
9447
Parent NSAID 1
Compound 16
Parent NSAID 2
Compound 17
Parent NSAID 3
Parent NSAID 5
Parent NSAID 7
Compound18
Compound 20
Compound 22
Parent NSAID 4
Compound 19
Parent NSAID 6
Compound 21
Tertiary amines 11 and 12
Figure 1. Gamma images of compounds 1–7 and 16–22 in arthritis model in rats.
1456, 1418, 1159, 1043, 1006, 935, 914, 768 and 698; PMR (DMSO-
d6): d (ppm) 7.08–715 (m, 2H), 7.39–7.54 (m, 6H), 5.13–5.16
(t, 1H), 4.18–4.28 (m, 2H), 3.70–3.79 (m, 1H), 3.32 (s, 3H), 3.21
(s, 3H), 2.75–2.82 (m, 2H), 2.25–2.30 (m, 2H), 1.78–2.09 (m, 4H),
1.53–1.58 (d, 3H). Anal. Calcd for C24H29NO2FI (509.39): C 56.59,
H 5.73, N 2.74; found: C 56.44, H 5.96, N 2.51%.
(m, 2H), 1.69–1.90 (m, 4H), 1.48–1.50 (d, 3H), 1.36–1.44 (m, 1H),
0.88 (d, 6H). Anal. Calcd for C22H34NO2I (471.42): C 56.05, H 7.26,
N 2.97; found: C 56.27, H 7.06, N 3.22%.
4.1.7. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl 6-methoxy-2-
naphthylacetate methiodide (21)
The ester (14)21 was reacted as described under compound (16)
to obtain the product (21). 78% yield, mp 276–78 °C; IR(KBr, cmÀ1):
1732, 1632, 1607, 1481, 1429, 1391, 1337, 1267, 1205, 1159, 1132,
1030, 851 and 820; PMR (DMSO-d6): d (ppm) 7.63–7.72 (m, 2H),
7.32–7.35 (dd, 1H), 7.12–7.18 (m, 2H), 5.10–5.15 (t, 1H), 4.03–
4.06 (m, 1H), 3.92 (s, 3H), 3.82–3.86 (m, 3H), 3.28 (s, 3H), 3.11 (s,
3H), 2.49–2.65 (m, 2H), 1.98–2.08 (m, 2H), 1.76–1.90 (m, 2H),
1.63–1.68 (m, 1H), 1.32–1.40 (m, 1H). Anal. Calcd for C22H28NO3I
(481.36): C 54.89, H 5.86, N 2.90; found: N 2.48%.
4.1.6. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl
2-(4-isobutylphenyl)propionate methiodide (20)
The ester (13)24 was reacted as described under compound (16)
to obtain the desired product (20). 83% yield, mp 270–72 °C;
IR(KBr, cmÀ1): 1732, 1508, 1456, 1202, 1184, 1163, 1050, 1005;
PMR (CDCl3): d (ppm) 7.10–7.15 (dd, 4H), 5.12–5.15 (t, 1H),
4.26–4.28 (m, 1H), 4.09–4.11 (m, 1H), 3.64–3.70 (q, 1H), 3.49 (s,
3H), 3.32 (s, 3H), 2.58–2.74 (m, 2H), 2.44–2.46 (d, 2H), 1.99–2.21

9448
M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
Table 4
Percent anti-inflammatory activity of compounds (Freund’s adjuvant arthritis model)
Compound (parent derivative)
Dose (mg/kg)
% Inhibition of paw edema on days
3rd
37.4 1.56
7th
14th
21st
1
16
0.81
1.56
46.50 1.45
40.54 1.41
54.60 3.13
56.35 2.26
49.2 2.05
23.9 1.79
55.97 2.78*
2
17
0.85
1.80
35.10 3.30
17.03 1.62
43.9 3.20
29.96 1.10
54.4 2.26
39.75 2.10
55.03 2.56
44.82 1.54*
3
18
4
0.85
2.00
2.59
5.61
37.40 1.56
22.31 3.50
36.52 1.98
25.53 2.33
46.47 1.44
36.86 1.92
44.22 1.82
40.96 2.73
54.60 3.78
47.71 1.58
55.57 2.58
53.62 1.37
48.5 2.36
53.51 2.11*
53.07 2.49
60.41 1.40*
19
5
20
7.20
16.44
35.10 2.93
22.52 3.72
44.1 2.39
41.5 2.06
53.82 0.95
53.81 3.00
53.21 2.42
60.12 2.81*
6
21
4.27
10.01
35.6 3.48
23.05 1.98
45.5 2.77
40.86 2.08
56.4 1.75
55.75 2.09
52.51 1.85
71.29 1.19*
7
22
5.13
11.60
36.30 1.67
23.04 2.48
46.6 2.51
39.70 1.16
54.7 2.66
53.46 1.42
50.20 2.78
58.74 2.82*
Each value represents the mean SD (n = 6). Significance levels *p < 0.05 in comparison to respective parent drug.
4.1.8. 8-Methyl-8-azabicyclo[3,2,1]octan-3-yl 2-(6-methoxy-2-
naphthyl)propionate methiodide (22)
the incubation time period (5–40 min) of the reaction mixture.
All the compounds were radiolabeled by this procedure.
The ester (15)21 was reacted with methyl iodide as described
under compound (16) to obtain the quaternized product (22).
82% yield, mp 260–62 °C; IR(KBr, cmÀ1): 1728, 1634, 1609, 1464,
1265, 1178, 1157, 1080, 1043, 1025 and 800; PMR (DMSO-d6-
CDCl3): d (ppm) 7.65–7.75 (m, 3H), 7.33–7.35 (dd, 1H), 7.13–7.17
(m, 2H), 5.09–5.19 (t, 1H), 4.05–4.07 (m, 1H), 3.93 (s, 3H), 3.84–
3.89 (m, 2H), 3.29 (s, 3H), 3.13 (s, 3H), 2.53–2.70 (m, 2H), 2.01–
2.12 (m, 2H), 1.91–1.95 (m, 1H), 1.75–1.85 (m, 1H), 1.65–1.70
(m, 1H), 1.59–1.60 (d, 3H), 1.36–1.46 (m, 1H). Anal. Calcd for
C23H30NO3I (495.39): C 55.76, H 6.10, N 2.82; found: N 2.48%.
4.3. In vitro stability studies of 99mTc-labeled complexes in
saline
The in vitro stability of radiolabeled complexes was determined
in sodium chloride (0.9%) by ascending thin layer chromatography.
The 99mTc-labeled compound solution (0.1 ml) prepared in saline
(0.9%) as described above was mixed with normal saline (1.9 ml)
and incubated (37 1 °C). ITLC was performed at different time
intervals (0, 2, 4, 6 and 24 h) as described above, in acetone to assess
the stability of the complex. Any decrease in percentage of 99mTc-
labeled complex was considered as its degree of degradation.
4.2. Radiolabeling of the compounds
Radiolabeling of the compounds with reduced 99mTc was car-
ried out as per the direct labeling method.25 Sodium pertechnetate
(
4.4. In vitro stability studies of 99mTc-labeled complexes in
human serum
99mTc) (1.0 ml, 2.0 mci/ml) was mixed with stannous chloride
solution (0.1 ml, 1 mg/ml in acetic acid (10%)) and mixed well.
The pH was adjusted to 7.0 using sodium bicarbonate solution
(0.5 M) and to this mixture, solution of the compounds (1.0 ml, dis-
solved in DMSO/water (9:1)) was added and incubated for 15 min
(37 1 °C). The labeling efficiency/percent labeling was deter-
mined by performing ascending instant thin layer chromatography
(ITLC) using acetone as the mobile phase. Radiolabeled complex
The in vitro stability of radiolabelled complexes was tested in
human serum. The study was accomplished by incubating an ali-
quot of 0.1 ml of labeled complex prepared in saline (0.9%) and
mixed with human serum (1.9 ml) and incubated (37 1 °C). ITLC
was performed at different time intervals (0, 2.0, 4.0, 6.0, and
24 h) as described above, in acetone to assess the stability of the
complex. Any decrease in percentage of 99mTc-labeled complex
was considered as its degree of degradation.
(2–3 ll) was applied at a point 1.0 cm away from one end of an
ITLC-SG strip. The strip was developed in acetone and the solvent
front was allowed to rise up to 8 cm from the origin. The strip
was cut one cm below the solvent front and the radioactivity in
each segment was determined by scintillation counting. The free
pertechnetate which moved with the solvent (Rf = 0.9) and the
reduced/hydrolyzed (R/H) technetium along with the labeled com-
plex remaining at the point of application were determined. ITLC
was also run in pyridine-acetic acid- water (3:5:1.5 v/v, PAW)
system to determine the amount of reduced/hydrolyzed (R/H)
99mTc (radio-colloids). The R/H 99mTc remained at the point of
application while both the free pertechnetate and the labeled com-
plex moved away with the solvent front in this solvent system
(PAW). The difference between the activity for the spots which
moved along with the solvent front using acetone from that
obtained in the PAW system (at the point of application) gave
the net amount of 99mTc-labeled complex.
4.5.
c-Imaging studies
Sprague–Dawley rats (three in a group) were used for the stud-
ies. Imaging was performed using a Single Photon Emission Com-
puterized Tomography gamma camera. Inflammation was
induced in each rat by subcutaneous injection of carrageenan
(0.1 ml, 1% w/v in normal saline) into the sub-plantar region of
the right paw. Three hours later when maximum inflammation
was achieved, 99mTc-labeled complex (0.2 ml) of the QA (9–22)
was administered through the tail vein. The animals were anaes-
thetized (ketamine, im), fixed on a board using the adhesive tapes
and c-imaging photographs (Fig. 1) were taken 3 h after injecting
the complex through the tail vein.
4.6. Anti-inflammatory activity (Arthritis model)
The experiment was repeated by varying the conditions one at a
time, that is, using different moles of stannous chloride (0.05–
0.2 ml), changing the pH of the medium (5.5 and 8) and varying
Sprague–Dawley male rats with an initial body weight of 150–
200 g were used. Rats were divided into groups of six each for test

M. R. Yadav et al. / Bioorg. Med. Chem. 16 (2008) 9443–9449
9449
compounds and control group.26 On day 1, they were injected into
the sub-plantar region of the left hind paw with 0.1 ml of complete
Freund’s adjuvant (6 mg/ml Mycobacterium butyricum suspended
in heavy paraffin oil). Once a day dosing with the test compounds
or the standard (on equivalent molar doses) was started on the
same day and continued for 12 days. Paw volumes of both sides
was measured plethysmographically. The paw volumes were mea-
sured on days 3, 7, 14 and 21. From day 13 to 21, the animals were
not dosed with the test compounds or the standards. Parent NSA-
IDs (1–7) as standards and QA (16–22) dissolved in aqueous-DMSO
were administered to the animals through tail vein in the doses as
given in Table 4. Results (Table 4) are expressed as percentage
inhibition of edema formation, calculated by the formula, % Inhibi-
tion of paw edema = (1 À Edtest/Edcontrol) Â 100 where Edtest and
Edcontrol are the edema volumes in compound treated and control
groups, respectively. The data obtained in the pharmacological
experiment was subjected to statistical analysis using the Stu-
dent’s t test and the chosen level of significance was p < 0.05.
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Acknowledgments
19. Yadav, M. R.; Halen, P. K.; Hemalatha, B.; Chagti, K. K.; Giridhar, R. ARS Pharm.
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The authors gratefully acknowledge the help provided by Mrs.
Krishna Chuttani, Mr. Ankur Kaul and Mr. Anil K. Babbar in suc-
cessful completion of this work.
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