Synthesis and biological evaluation of orally active prodrugs of indomethacin

Article abstract of DOI:10.1021/jm101085j

Synthesis and biological evaluation of orally active prodrugs (1a-d) of indomethacin are described. Prodrugs 1a-c showed a similar degree of anti-inflammatory activity, and prodrug 1d was found to be less potent than the parent drug indomethacin (1). Ulcer index (UI) data indicated that 1a (UI = 19), 1c (UI = 0), and 1d (UI = 0) were substantially less ulcerogenic and 1b (UI = 62) was more ulcerogenic than parent drug 1 (UI = 47). These prodrugs demonstrated good stability at acidic and basic pH and found to be more lipophilic than parent drug compound 1, indicated by partition coefficients measured in octanol-buffer system at pH 7.4 and 3.0. On the basis of in vivo studies, 1a and 1c compounds were selected for metabolic stability in rat liver microsome (RLM) and rat plasma (RP), and both were found to be enzymatically labile. Prodrugs 1a and 1c emerged as potent anti-inflammatory agents with a lesser potential for ulcer than the parent drug indomethacin.

Full text of DOI:10.1021/jm101085j

ARTICLE
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Orally Active Prodrugs
of Indomethacin
Babasaheb P. Bandgar,*,†,‡ Rajendra Janardan Sarangdhar,† Santosh Viswakarma,† and
Fakrudeen Ali Ahamed†
†Organic Chemistry Research Laboratory, School of Chemical Sciences, Swami Ramanand Teerth Marathwada University,
Nanded-431 606, India
‡Medicinal Chemistry Research Laboratory, School of Chemical Sciences, Solapur University, Solapur-413 255, India
S Supporting Information
b
ABSTRACT: Synthesis and biological evaluation of orally
active prodrugs (1a-d) of indomethacin are described.
Prodrugs 1a-c showed a similar degree of anti-inflamma-
tory activity, and prodrug 1d was found to be less potent
than the parent drug indomethacin (1). Ulcer index (UI)
data indicated that 1a (UI = 19), 1c (UI = 0), and 1d (UI =
0) were substantially less ulcerogenic and 1b (UI = 62) was
more ulcerogenic than parent drug 1 (UI = 47). These
prodrugs demonstrated good stability at acidic and basic pH
and found to be more lipophilic than parent drug compound 1, indicated by partition coefficients measured in octanol-buffer
system at pH 7.4 and 3.0. On the basis of in vivo studies, 1a and 1c compounds were selected for metabolic stability in rat liver
microsome (RLM) and rat plasma (RP), and both were found to be enzymatically labile. Prodrugs 1a and 1c emerged as potent anti-
inflammatory agents with a lesser potential for ulcer than the parent drug indomethacin.
’ INTRODUCTION
means of reducing or abolishing the GI toxicity due to the local
action mechanism.
Indomethacin (1, Figure 1) is a well-known nonsteroidal anti-
inflammatory drug (NSAID) and is effective against severe
rheumatoid arthritis, ankylosing spondylitis, osteoarthritis of
large joints, and other inflammations.1-5 The beneficial effect
is associated with inhibition of cyclooxygenases (COX) that
convert arachidonic acid into prostaglandins in inflammatory
processes.6 The major limitation of long-term therapeutic use of
NSAIDs (COX-1 inhibitors) is their gastrotoxicity. These side
effects produced by NSAIDs are believed to involve two different
mechanisms: inhibition of prostaglandin synthesis in the stom-
ach responsible for inducing mucus production and a local
action exerted by direct contact of the drugs with gastric mucosa
due to the acidic nature of the NSAIDs.7 The most common
effects associated with NSAID therapy are upper GI irritation,
ulceration, dyspepsia, bleeding, and in some cases death.8 To
overcome the GI ulceration side effect of drug, more COX-2
selective inhibitor NSAID is preferred, which does not signifi-
cantly inhibit cyclooxygenase in the stomach and appears to be
less likely to cause GI ulceration.9 Unfortunately very effective
COX-2 selective inhibitor drugs, i.e., rofecoxib and celecoxib,
were withdrawn from the market because of the increased risk of
heart attack and stroke associated with long-term, high-dosage
use. Hence considerable attention has been focused on the
development of bioreversible derivatives, such as prodrugs, to
temporarily mask the acidic group of NSAIDs as a promising
The prodrug approach afforded compounds with better anti-
inflammatory activity, differentiated pharmacokinetic profile, and
reduced gastric ulcerogenic activity.10-14 Prodrugs are pharma-
cologically inactive derivatives of active agents, which undergo
chemical and/or enzymatic biotransformation, resulting in the
release of active drug after administration. The metabolic pro-
duct (i.e., parent drug) subsequently elicits the desired pharma-
cological response.15,16 Most prodrugs of NSAIDs have been
prepared by derivatization of the carboxylic group. The esters
have dominated prodrug research because they have the ideal
characteristic of exhibiting reasonable in vitro chemical stability
which allows them to be formulated with adequate shelf lives. In
addition, by virtue of their ability to function as esterase
substrates, esters are suitably labile in vivo.17,18 By use of the
prodrug approach, one strategy that could be useful is to
temporarily mask the carboxylic acid function of the NSAIDs
so that the prodrug hydrolyzes in vivo to release the active parent
NSAID.19-21
The present work was initiated with the aim to develop pro-
drugs of indomethacin (1), possessing a high enzymatic biocon-
version rate, favorable physicochemical properties, and fewer
ulcerogenic properties. Thus, indomethacin ester prodrugs
Received: August 21, 2010
Published: February 01, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
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Figure 1. Chemical structures of indomethacin (1) and prodrugs 1a-d.
Figure 2. Chemical structures of 1-iodomethyl pivalate (2), 1-iodomethylisopropyl carbonate (3), 2-bromoethyl acetate (4), and 4-chloromethyl-5-
methyl-1,3-dioxol-2-one (5).
(1a-d) were prepared and evaluated for their potential use as
prodrug for oral delivery. The physicochemical properties of a
drug play a major role in the development of formulation and
bioavaibility. Thus, in addition to characterization of the pro-
posed structures, physicochemical parameters like the partition
coefficient (log P), aqueous solubility, aqueous stability, in vitro
metabolic stability in rat liver microsomes and rat plasma, and
ulcerogenicity (GI toxicity) were studied.
bromide afforded 4. Commercially available 4-chloromethyl-5-
methyl-1,3-dioxol-2-one (5) was used in the preparation of
prodrug 1d (Scheme 1). The structures of all promoieties were
established by IR and 1H NMR. All promoieties were analyzed by
GC, and their purity was confirmed to be in excess of 96.5%.
’ RESULTS AND DISCUSSION
Prolonged administration of NSAIDs exhibits several unde-
sired side effects. The most important side effects are gastro-
intestinal irritation and ulceration which still represent an
unsolved therapeutic problem. With the aim of minimizing the
ulcerogenicity, a series of ester prodrugs 1a-d of 1 were
synthesized and evaluated. The potential ulcerogenic effects of
1a-d were determined after single dose administration in rats
(100 mg/kg dose) and compared to those produced by the
parent compound indomethacin (1) with identical conditions.
Lesser degree of ulcers was observed in animals treated with
prodrugs 1a-d, compared to 1 (Table 1). Animals in each group
(n = 6) treated with 1 developed an average of 41 ulcerogenic
lesions with single dose administration. Around 4.9% of the
lesions were considered to be large (higher than 3 mm diameter).
This allowed the classification of lesions, which were scored
depending upon the severity of mucosal damage. Prodrug 1a on
single dose administration led to the development of only 19 small
lesions (<1 mm diameter), and 1b on single dose administration
’ CHEMISTRY
A series of novel ester prodrugs (1a-d) were prepared as
depicted in Scheme 2 in good to excellent yields (95.5%, 96.6%,
78%, and 81.24%) by condensation of 1 with the appropriate
promoiety in the presence of organic base like TMG or sodium
carbonate in DMAc. The structures of all prodrug compounds
were established by 1H NMR, 13C NMR, and mass spectrometry.
All compounds were analyzed by HPLC, and their purity was
confirmed to be in excess of 98.0%.
Promoieties 2 and 3 (Figure 2) were prepared from their
corresponding chloro compounds for fast reaction rate and
better yield by treatment with sodium iodide in acetonitrile as
shown in Scheme 1. The synthesis of 2-bromethyl acetate (4)
was carried out according to the procedure reported in U.S. patent
number 5,155,256, issued October 13, 1992. Thus, the reaction
of ethylene glycol with acetic acid in the presence of hydrogen
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Scheme 1. Synthesis of Promoieties 2, 3, 4a
a Reagents and conditions: (i) NaI, ACN, 30 °C, 5 h; (ii) NaI, toluene, 18-crown-6, 105 °C, 2 h; (iii) CH3COOH, 48% aqueous HBr, acetic anhydride,
toluene, 110 °C, 90 min.
Scheme 2. Synthesis of Ester Prodrugs of Indomethacin (1a-d)a
a Reagents and conditions: (i) ICH2OCOC(CH3)3, TMG, DMAc, -15 °C, 30 min; (ii) ICH2OCOOCH(CH3)2, TMG, DMAc, -15 °C, 20 min; (iii)
BrCH2CH2OCOCH3, Na2CO3, DMAc, 55 °C, 10 h; (iv) Na2CO3, 5, DMAc, 35 °C, 6 h.
led to the development of around 51 lesions of level I (86.3%),
level II (5.9%), and level III (7.8%). However, 1c and 1d
developed no ulcerogenic lesions on identical dose. These
findings proved that masking of carboxylic function of indo-
methacin in prodrugs 1a, 1c, and 1d successfully decreased the
gastroulcerogenicity. All the prodrugs (except 1b) showed an
improved safety profile compared with the parent reference
compound (Table 2). The prodrug 1b (UI = 62) produced
more detectable lesions on the gastric mucosa in the group of
animals examined. 1a (UI = 19) produced fewer detectable
lesions, and 1c and 1d showed no visible lesions. This indicated
that the prodrugs 1a, 1c, and 1d were significantly less irritating
to gastric mucosa than 1 (UI = 47).
The anti-inflammatory activity of prodrugs was evaluated by
using the in vivo rat carrageenan induced paw edema method
when administered orally to rats. Prodrugs 1a, 1b, and 1c were
found to be potent anti-inflammatory compounds and produced
a significant anti-inflammatory effect (Table 3).
Partition Coefficient. The partition coefficient (log P) of
parent compound indomethacin (1) and prodrugs 1a-d were
determined at room temperature in n-octanol-phosphate buffer
at pH 7.4 and in 1-octanol-citric acid buffer at pH 3.0. The
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ARTICLE
partition coefficient of all prodrugs at pH 7.4 was higher than 1,
indicating that prodrugs are more lipophilic than the parent
drug. Partition coefficients of indomethacin and prodrugs are
summarized in Table 4, and results are in line with the literature
values.24
one reported earlier25 and could be because of the difference in
solubility study parameters. Solubility of indomethacin was pH
dependent and increased with increasing pH. Insignificant
hydrolysis of prodrug compounds to parent drug was observed
in acidic to neutral pH buffer solutions (Table 5) at 40 °C,
indicating that prodrugs 1a-d are resistant toward hydrolysis at
the pH of the stomach acidic environment, pH of the small
intestinal mucosa, and at physiological pH.
Metabolic Stability. Indomethacin ester prodrugs 1a-d
were synthesized with the aim of obtaining enzymatic lability.
Metabolic stability is an important property of drug candidates,
since it affects parameters such as clearance, half-life, and bioavail-
ability. A successful prodrug candidate is expected to undergo rapid,
complete conversion to parent compound in the plasma or micro-
somes within 1-3 h. On the basis of in vivo studies, prodrugs 1a and
1c were selected for metabolic stability study in rat liver microsomes
and rat plasma. Prodrugs 1a and 1c and reference parent drug 1
were subjected to metabolic stability in the presence of rat liver
microsomes and rat plasma. The parent drug 1 was stable up to
60 min in both rat liver microsomes and rat plasma; however,
prodrugs were highly metabolized (Table 6).
Aqueous Stability. The orally administered drug should be
stable at various pH environments encountered in the gastro-
intestinal tract to deliver the intact prodrug to the systemic
circulation. Four prodrugs candidates (1a-d) were evaluated for
solubility and stability in a series of buffer solutions ranging from
pH 1 to 9. All the prodrug compounds were found to be stable
and practically insoluble in buffer solutions (below detection
limit). Aqueous solubility of 1a-d was lower than that of parent
drug indomethacin (1), which is related to the increased lipo-
philicity of prodrugs. Limit of detection (LOD) of prodrugs
1a-d was estimated by HPLC (LOD: 1a, 0.084 μg/mL; 1b,
0.119 μg/mL; 1c, 0.111 μg/mL; 1d, 0.0638 μg/mL). The
aqueous solubility of 1 at 40 °C was 0.03, 24, 1500, and 1600
μg/mL at pH 1.0, 3.0, 5.2, 7.4, 9.0, as against that reported in the
literature25 (3.882 and 767.5 μg/mL at pH 1.2 and pH 7.2).
Experimental solubility value of 1 was slightly different from the
Formation of Parent Compound Indomethacin (1) from
Prodrugs (1a, 1c) in Rat Liver Microsomes (RLM) and Rat
Plasma (RP). The experimental findings depicted in Tables 7
and 8 proved that prodrugs 1a and 1c were enzymatically labile
and converted rapidly to parent compound 1. Indomethacin was
the only metabolite observed after biotransformation in both
RLM and RP. Release of parent drug from these ester prodrugs
upon hydrolysis was confirmed by HPLC. Prodrug peaks in
HPLC (retention time, 1a, ∼14.1 min; 1c, ∼11.1 min) disap-
peared in 30-60 min, and the peak area of the parent compound
1 (retention time, ∼9.15 min) was increased (Tables 7 and 8).
Solid State Morphology. The solid state morphology of
active pharmaceutical ingredient (API) is a key parameter for
their further utilization when several forms can coexist as crystal-
line or amorphous and/or as different polymorphs (allotropes).
Table 1. Ulcerogenic Effect of Indomethacin (1) and Pro-
drugs (1a-d) in Ratsa
level I
level II
level III
compd
no. of ulcers§
(<1 mm)
(1-3 mm)
(>3 mm)
1
41
19
51
0
37 (90.2%)
2 (4.9%)
2 (4.9%)
1a
1b
1c
1d
19 (100.0%)
0
0
44 (86.3%)
3 (5.9%)
4 (7.8%)
0
0
0
0
0
0
0
a The results were obtained with an average of six animals analyzed per
group (n = 6).
Table 2. Ulcer Index for Compounds 1a-d and Indometha-
cin (1)
Table 4. Partition Coefficients of Indomethacin (1) and
Prodrugs 1a-d in Buffer Solution
group
dose (mg/kg)
ulcer index (UI)a
normal
1
nil
47
19
62
0
partition coefficient (log P)a
100
100
100
100
100
1a
compd
pH 7.4
pH 3.0
1b
1
2.1
4.7
5.0
3.5
2.9
4.2
4.6
5.1
4.2
4.5
1c
1a
1b
1c
1d
1d
0
a Ulcer index (UI) is calculated based on the lesions developed in the
stomach on single dose administration in rats in each group (n = 6). Data
are presented as the mean ( SEM at 6 h after oral administration of the
test compound.
a Results are reported as average of three experiments (n = 3).
Table 3. Anti-Inflammatory Activities of Prodrugs 1a-d and Parent Drug (1)
group
dose (mg/kg)
paw volume (mL)
inhibition at 180 min (%)
normal
1.88 ( 0.04
3.12 ( 0.04a
2.52 ( 0.06b
2.52 ( 0.08c
2.56 ( 0.18c
2.58 ( 0.09c
2.96 ( 0.08
carrageenan (1%), 0.1 mL
1
100
100
100
100
100
48.39
48.39
45.16
44.35
12.10
1a
1b
1c
1d
a P < 0.001, compared to normal control . b P < 0.001, compared to carrageenan control. c P < 0.01, compared to carrageenan control.
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This is particularly important for API, as their morphology can
have a significant impact on their bioavailability and stability.
Hence, it was necessary to know the solid state morphology of
prodrugs (1a-d). Prodrugs 1a-d were crystalline when
screened in a Bruker AXS D8 advance diffractometer. Powder
XRD profiles of prodrugs are shown in Figures 3, 4, 5, and 6.
techniques. These prodrugs emerged as potent anti-inflamma-
tory agents with lesser potential for ulcer than parent drug
indomethacin. Low ulcer index (UI) was observed with three
prodrug compounds (except 1b). On the basis of in vivo
evaluation, prodrugs 1a and 1c were selected for metabolic
stability. Both these prodrugs were rapidly transformed enzyma-
tically to the parent drug indomethacin in both rat liver chromo-
somes and rat plasma. Thus, on the basis of in vitro and in vivo
evaluation, prodrugs 1a and 1c emerged as potent inflammatory
drugs for prevention of gastrointestinal disorders and as sub-
stitutes for parental drug.
’ CONCLUSION
Indomethacin ester prodrugs were evaluated for their
anti-inflammatory and ulcer potential by known experimental
Table 5. Aqueous Solubility and Stability of Indomethacin
(1) and Prodrugs 1a-d in Buffer Solutions at 40°C for 4 ha
’ EXPERIMENTAL SECTION
Materials and Methods. Melting points were determined on an
MR VIS (Lab India) melting point apparatus and are uncorrected. 1HNMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded on a Bruker
Advance spectrophotometer using CDCl3 and DMSO solvent. The
chemical shifts are reported in ppm downfield from zero, and coupling
constants are reported in hertz (Hz). IR spectra were acquired by using a
FTIR Perkin-Elmer model RXI or FTIR spectrophotometer Nicolet 380
(Thermo Nicolet); software was Omnic. Mass spectra were recorded on a
PE-SCIEX API-3000 LCMS/MS (Applied Biosystem) spectrophotometer.
Commercially available solvents and reagents for reactions (ethyl acetate,
DMAc, TMG, sodium carbonate) were procured from S.D.Fine-Chem
Ltd., India. HPLC analysis was performed by using Shimadzu LC 2010
CHT with UV detector and Waters Alliance 2695, with PDA 2996 detector.
All prodrug compounds were analyzed by HPLC, and their purity was
confirmed to be in excess of 98.0%. HPLC grade solvents (acetonitrile,
methanol, acetic acid, and toluene) were used in the analysis and were
procured from a local Indian supplier. GC analysis was performed on
Perkin-Elmer model Clarus 500 instrument. Promoieties were analyzed by
GC for chromatographic purity, which was confirmed to be in excess of
96.5%. UV spectra were recorded by using a Shimadzu UV-2401 PC
spectrophotometer. In vitro metabolic stability study was conducted in rat
liver microsomes and rat plasma by the HPLC method, which was
developed in-house in the laboratory. Powder XRD of prodrugs was
performed on a Bruker AXS D8 Advance diffractometer. The in vivo
anti-inflammatory and ulcer index assays were carried out by using protocol
approved by the Institutional Animal Ethics Committee (IAEC) and
compiled with National Institutes of Health (NIH) guidelines on handling
of experimental animals. Male Wistar rats weighing 150-200 g were used
for the study. Carrageenan was purchased from Sigma (St. Louis, MO, U.S.),
hydrolysis to parent drug (μg/mL)
1a 1b 1c 1d
0.0210 0.010
solubility (μg/mL)
buffer pH
1a 1b 1c 1d
1.0
3.0
5.2
7.4
9.0
0.0103 0.0191 BDL BDL BDL BDL
0.0075 0.00539 0.0092 0.0134 BDL BDL BDL BDL
0.1525 0.01379 0.0197 0.0244 BDL BDL BDL BDL
0.2940 0.01332 0.0093 0.0101 BDL BDL BDL BDL
2.187
0.24614 0.369
0.5106 BDL BDL BDL BDL
a BDL, below detection limit. Limit of detection (LOD) is estimated by
the HPLC method: 1a (0.084 μg/mL), 1b (0.119 μg/mL), 1c (0.111
μg/mL), 1d (0.0638 μg/mL).
Table 6. Metabolic Stability of 1 and Prodrugs (1a and 1c) in
RLM and RP
metabolic stability in RLM
% remaining in RLMa
metabolic stability in RP
% remaining in RPa
compd 0 min 30 min 60 min Timef
0 min
60 min
1
100
100
100
90.3
0
80.2
0
1
100
100
100
97.1
0
1a
1c
1a
1c
1.14
0
0
a The percentage of prodrug remaining after metabolism at respective
time points was calculated by ratio of peak area at the respective time
(min) to peak area found at 0 min multiplied by 100: (% remaining) =
[(peak area at respective time (min))/(peak area at 0 min)] ꢀ 100.
Table 7. Formation of Parent Compound 1 from Prodrugs 1a and 1c in RLM and RP
formation of indomethacin parent compound from prodrugs in RLM
indomethacin peak area in HPLC analysis
formation of indomethacin parent compound from prodrugs in RP
indomethacin peak area in HPLC analysis
compd
0 min
30 min
60 min
compd
0 min
60 min
1a
1c
0
0
1435186
739541
1801169
738838
1a
1c
0
0
101220
855753
Table 8. Disappearance of Prodrugs by Metabolic Transformation in Presence of RLM and RP
prodrug peak area in RLM
prodrug peak area in RP
prodrug peak area in HPLC analysis
prodrug peak area in HPLC analysis
compd
0 min
30 min
60 min
compd
0 min
60 min
1a
1c
1157146
794700
0
0
0
1a
1c
491862
748633
0
0
9082
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Figure 3. XRD diffractogram for prodrug 1a.
Figure 4. XRD diffractogram for prodrug 1b.
Indomethacin was procured from a local bulk API manufacturer.
4-Chloromethyl-5-methyl-1,3-dioxol-2-one and chloromethylisopropyl
carbonate reagents were gifted by M/s Matrix Laboratories, India, and
M/s Huaren Chemical Co. Ltd., India, respectively. NADPH was pur-
chased from Sigma (lot no. N-7785).
sodium iodide (180 g, 1.20 mol) at 30 °C for 5 h under N2 atmosphere.
Reaction progress was monitored by GC. After completion of reaction,
the reaction mixture was transferred to a mixture of dichloromethane
(1000 mL) and water (1000 mL), stirred for 10 min, and separated into
two phases. The aqueous phase was discarded, and the organic phase was
washed with 2% sodium thiosulfate (Na2S2O3, 500 mL) and concen-
trated under vacuum to give 2 as a yellowish oil (138.8 g, 86.6%). Purity
Iodomethyl Pivalate (2). A solution of chloromethyl pivalate
(100 g, 0.664 mol) in acetonitrile (200 mL) was allowed to react with
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Figure 5. XRD diffractogram for prodrug 1c.
Figure 6. XRD diffractogram for prodrug 1d.
by GC 99.01%. 1H NMR (CDCl3, 400 MHz) δ 5.93 (s, 2H, -CH2)),
1.24 (s, 9H, -C(CH3)3); IR (Nujol) cm-1 1759 (CdO), 1097
(C-O str).
1-Iodomethylisopropyl Carbonate (3). Sodium iodide (112 g,
0.747 mol) and 18-crown-6 (2.5 g, 0.009 45 mol) were added to toluene
(600 mL). The reaction mixture was stirred vigorously and heated to
110-112 °C under dry nitrogen atmosphere to remove total moisture.
The reaction mixture was cooled to 105 °C, and chloromethyl isopropyl
carbonate (50 g, 0.3276 mol) was added. The reaction mixture was
stirred at 105 °C for 2 h. The reaction progress was monitored by GC.
After completion of reaction, the reaction mixture was cooled to 5 °C
and washed with 2% sodium thiosulfate (160 mL) followed by
brine (670 mL). The organic layer was evaporated under vacuum to
afford 3 as a pale yellow oil (69.5 g, 87%). Purity by GC, 99.61%.
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1H NMR (CDCl3, 400 MHz) δ 5.93 (s, 2H, -OCH2I)), 4.95 (m, 1H,
J = 6.32 Hz, -CH(CH3)2), 1.35 (s, 6H, -CH(CH3)2); IR (KBr) cm-1
1759 (CdO), 1077 (C-O).
(s, 3H, -C-CH3), 3.76 (s, 3H, -OCH3), 3.88 (s, 2H, -CH2CO), 4.76
(h, 1H, J = 6.24 Hz, -OCH(CH3)3], 5.71 (s, 2H, -OCH2O), 6.71 (dd,
1H, J = 9.0 Hz and J = 2.5 Hz, indole Ar-H), 6.90 (d, 1H, J = 9.0 Hz,
indole Ar-H), 7.01 (d, 1H, J = 2.5 Hz, Ar-H), 7,63 - 7.68 (m, 4H,
Ar-H); 13C NMR (CDCl3,100 MHz) δ 13.53, 21.75 (2C), 30.20,
55.81, 73.32, 82.19, 101.13, 111.72, 112.07, 115.13, 129.29 (2C), 130.51,
130.89, 131.37 (2C), 133.96, 136.32, 139.45, 153.43, 156.23, 168.42,
169.47.
2-Bromoethyl Acetate (4). A solution of ethylene glycol (51.6 g,
0.8322 mol), glacial acetic acid (75 g, 1.248 mol), toluene (20 mL), and
48% hydrogen bromide solution (140.3 g, 0.832 mol) was refluxed for 90
min to distill out azeotropically 130 mL of water under nitrogen
atmosphere. The solution was cooled to 25 °C, and acetic anhydride
(23.7 g, 0.232 mol) was added dropwise by controlling the temperature
below 35 °C. Reaction was monitored by GC. After completion of
reaction, sodium metabisulfite (0.25 g) and sodium carbonate (0.3 g)
were added under stirring, and the reaction mass was maintained
overnight without agitation. Reaction mixture was subjected to fractional
distillation under vacuum, and the main fraction was collected at 40-47 °C.
The main fraction was washed with chilled brine solution (100 mL) to
remove acetic acid to afford 4 as a colorless liquid (55 g, 39.6%). Purity
by GC, 96.72%. 1H NMR (CDCl3, 400 MHz) δ 2.1 (s, 3H, -CH3CO),
3.50 (t, 2H, J = 3.96 Hz, -CH2Br), 4.4 (t, 2H, J = 6.16 Hz, -CH2O); IR
(Nujol) cm-1 1753 (CdO).
Pivaloyloxymethyl 1-(p -Chlorobenzoyl)-5-methoxy-2-
methyl-3-indolylacetate (1a). Compound 1 (10 g, 27.9 mmol)
was dissolved in DMAc (45 mL) at 20 °C and cooled to -15 °C under
nitrogen atmosphere. TMG (3.37 g, 29.1 mmol) was added once, and
the mixture was stirred for 20 min at -10 ( 2 °C. The reaction mixture
was then cooled to -20 °C. To it was added iodomethyl pivalate (6.76 g,
27.9 mmol), and the mixture was stirred for 30 min at -15 °C. Reaction
progress was monitored by HPLC. After completion of reaction,
the reaction mixture was transferred into a mixture of ethyl acetate
(120 mL), water (400 mL), and sodium thiosulfate (1 g) under vigorous
stirring. The pH of the reaction mixture was adjusted to 10.5 by addition
of sodium carbonate, and the organic layer was separated. The organic
layer was washed with brine (125 mL), decolorized with activated
charcoal (1 g), and filtered. After evaporation of solvent in vacuo, the
desired product 1a was obtained as an oil which solidified upon cooling
to an off-white solid (12.6 g, 95.5%), mp 68 °C. Chromatographic purity
(HPLC) 98.13%; MS (þESI) m/z = 489.2 (M þ NH4)þ. UV max
(ethanol): 272, 301, 320 nm (23 mM-1 cm-1). IR (KBr) cm-1 1757
(CdO), 1738 (CdO) 1673 (N-CdO); 1H NMR (DMSO, 400 MHz)
δ 1.04 (s, 9 H, -(CH3)3), 2.21 (s. 3H, -CH3), 3.76 (s, 2H, -OCH3),
3.85 (s, 2H, -CH2CO), 5.72 (s, 2H, -OCH2O), 6.71 (dd, 1H, J = 8.9
Hz and J = 2.2 Hz), 6.9 (d, 1H, J = 8.9 Hz), 7.01 (s, 1H, J = 2.0 Hz),
7.63-7.65 (m, 4H, Ar-H); 13C NMR (CDCl3, 100 MHz) δ 13.48,
26.85 (3C), 30.29, 38.81, 55.79, 79.73, 101.22, 111.84, 112.02, 115.1,
129.29 (2C), 130.49, 130.89, 131.25 (2C), 133.96, 136.25, 139.46,
156.24, 168.4, 169.61, 177.15.
[(1-Methyl)ethoxycarbonyloxy]methyl 1-(p-Chlorobenzoyl)-
5-methoxy-2- methyl-3-indolylacetate (1b). Compound 1
(2.5 g, 6.98 mmol) was dissolved in DMAc (12.5 mL) at 10 °C. The
reaction mixture was cooled to -15 °C under nitrogen atmosphere.
TMG (0.87 g, 7.51 mmol) was added, and the mixture was stirred for
20 min at -15 °C. Then 1-iodomethylisopropyl carbonate (1.7 g, 6.98
mmol) was added at -15 °C. The reaction mixture was stirred for 20
min at -15 °C. Reaction completion was monitored by HPLC. The
reaction mixture was transferred into a mixture of ethyl acetate (30 mL),
water (100 mL), and sodium thiosulfate (1 g) under vigorous stirring.
The pH of the reaction mixture was adjusted 10.6 by addition of sodium
carbonate. The organic phase was separated, washed with brine (125
mL), decolorized with activated carbon (0.5 g), and filtered. After
evaporation of solvent in vacuo, the desired product 1b was obtained
as an oil which solidified upon cooling to an off-white solid (3.2 g,
96.6%), mp 84.2 °C. Chromatographic purity (HPLC) 99.39%; MS
(þESI) m/z = 491.1(M þ NH4)þ; UV max (ethanol) 320, 272, 301.3
nm (23 mM-1 cm-1), IR (KBr) cm-1 1753 (CdO), 1686 (N-CdO);
1H NMR (DMSO, 400 MHz) δ 1.20 (d, 6H, J = 6.2 Hz, -2CH3), 2.22
Acetyloxyethyl 1-(p-Chlorobenzoyl)-5-methoxy-2-meth-
yl-3-indolylacetate (1c). Compound 1 (5 g, 13.97 mmol) was
dissolved in DMAc (20 mL) at 30 °C. Sodium carbonate (0.963 g, 9.08
mmol) was added, and the mixture was stirred for 30 min at 30 °C under
nitrogen atmosphere followed by addition of 2-bromoethyl acetate (2.917 g,
17.46 mmol). The reaction mass was heated to 55 °Candstirredfor10h, and
completion of reaction was monitored by HPLC. Then the reaction mixture
was transferred to a mixture of ethyl acetate (60 mL), water (200 mL), and
sodium thiosulfate (2 g) under vigorous stirring. The pH of the reaction
mixture was adjusted to 10.6 by addition of sodium carbonate, and the organic
phase was separated. The organic phase was then washed with brine (240
mL) and filtered. After evaporation of the solvent in vacuo, the desired
product 1c was obtained as an oil which solidified upon cooling to off-white
solid (4.8 g, 77.4%), mp 106.6 °C. Chromatographic purity (HPLC),
99.31%; MS (þESI) m/z = 444.2 (M þ H)þ; UV max (ethanol) 319.85,
271.1, 301.94 nm (21 mM-1 cm-1); IR (KBr) cm-1 1740 (CdO), 1685
(N-CdO); 1H NMR (DMSO, 400 MHz) δ 1.92 (s, 3H, -COCH3), 2.22
(s, 3H, -C-CH3), 3.76 (s, 3H, -OCH3), 3.79 (s, 2H, -CH2CO), 4.20
(dd, 2H, J=5.8HzandJ=2.5Hz, -OCH2), 4.26 (dd, 2H, J=6.8HzandJ=
3.2 Hz, -OCH2), 6.71 (dd, 1H, J = 9.0 Hz and J = 2.5 Hz, indole Ar-H),
6.92 (d, 1H, J = 2.5 Hz, Ar-H), 7,63 - 7,69 (m, 4H, Ar-H); 13C NMR
(CDCl3, 100MHz) δ13.19, 20.44, 29.26, 55.39, 61.89, 62.39, 101.65, 111.43,
112.61, 114.62, 129.1(2C), 130.22, 130.52, 131.20 (2C), 134.12, 135.51,
137.7, 155.6, 167.91, 170.25, 170.47.
(5-Methyl-2-oxo-1,3-dioxolene-4-yl)methyl 1-(p-Chloro-
benzoyl)-5-methoxy-2-methyl-3-indolylacetate (1d). Com-
pound 1 (3 g, 8.3 mmol) was dissolved in DMAc (15 mL) at 30 °C.
Sodium carbonate (0.67 g, 6.2 mmol) and 5 (1.43 g, 8.5 mmol) were
added. The reaction mass was stirred for 6 h at 35 °C under nitrogen
atmosphere. Reaction progress was monitored by HPLC. After comple-
tion of reaction, the reaction mixture was transferred into a mixture of
ethyl acetate (36 mL), water (120 mL), and sodium thiosulfate (1 g)
under vigorous stirring. The pH of the reaction mixture was adjusted to
10.6 by addition of sodium carbonate, and the organic phase was
separated. The organic phase was washed with brine (240 mL), treated
with activated carbon (1 g), and filtered. After evaporation of solvent in
vacuo, the desired product 1d was obtained which on recrystallization in
methanol gave a pale yellow solid (3.2 g, 81.24%), mp 118.2 °C.
Chromatographic purity (HPLC) 99.54%; MS (þESI) m/z = 487.2
(M þ NH4)þ; UV max (ethanol) 319.61, 268.9, 303.82 nm (29 mM-1
cm-1); IR (KBr) 1811 (OCOO), 1739 (CdO), 1678 (N-CdO); 1H
NMR (DMSO, 400 MHz) δ 2.14 (s, 3H, -CH3-C(OC=O)), 2.22 (s,
3H, -C-CH3), 3.76 (s, 3H, -OCH3), 3.85 (s, 2H, -CH2CO), 5.01 (s,
2H, -OCH2CdCCH3), 6.71 (dd, 1H, J = 9.0 Hz and J = 2.5 Hz, indole
Ar-H), 6.93 (d, 1H, J = 9.0 Hz, indole Ar-H), 7.03 (d, 1H, J = 2.5 Hz,
indole Ar-H), 7.64-7.69 (m, 4H, Ar-H); 13C NMR (CDCl3, 100
MHz) δ 9.47, 14.33, 30.12, 54.36, 55.82, 101.36, 111.71, 111.8, 115.13,
129.28(2C), 130.49, 130.89, 131.30 (2C), 133.43, 133.88, 136.27,
139.46, 140.31, 152.13, 156.19, 168.38, 170.44.
GC Analysis. Analysis Method for Promoiety 2. The following
equipment and parameters were used: instrument, Perkin-Elmer, model
Clarus 500; column, DB-624, 30 mm ꢀ 0.53 mm, 3.0 μm; oven
temperature, 40 °C; ramp rate, 10 °C/min up to 220 °C; final oven
temperature, 220 °C; injection temperature, 200 °C; detector tempera-
ture, 250 °C; flow (carrier), 5.0 mL/min; injection volume, 0.2 μL;
split, 20:1
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Journal of Medicinal Chemistry
ARTICLE
Analysis Method for Promoiety 3. The following equipment and
parameters were used: instrument, Perkin-Elmer, model Clarus 500;
column, DB-1, 30 mm ꢀ 0.53 mm, 1.5 μm; oven temperature, 75 °C;
ramp rate, 10 °C/min up to 200 °C; final oven temperature, 200 °C for
10 min; injection temperature, 150 °C; detector temperature, 250 °C;
flow (carrier), 5.0 mL/min; injection volume, 0.2 μL; split, 20:1
Analytical Method for Promoiety 4. The following equipment and
parameters were used: instrument, Perkin-Elmer, model Clarus 500;
column, DB-5, 30 mm ꢀ 0.53 mm, 1.5 μm; detector, FID; carrier gas
flow (N2), 3.5 mL/min; initial oven temperature, 50 °C; initial time, 6.0
min; rate “1”, 15 °C/min; final oven temperature “1”, 110 °C; final time
“1”, 6 min; rate “2”, 15 °C/min; final oven temperature “1”, 250 °C; final
time “1”, 10 min; injection temperature, 125 °C; detector temperature,
270 °C; flow (carrier), 5.0 mL/min; injection volume, 0.2 μL; split, 1:10;
run time, 30 min.
Table 9. Gradient Elution Program
time (min)
mobile phase A
mobile phase B
0-2
90
30
10
30
90
90
10
70
90
70
10
10
2-8
8-10
10-12
12-15
15-20
Chemicals. Carrageenan was purchased from Sigma (St. Louis, MO,
U.S.). Indomethacin (EP-grade) is gifted by R & D Division of Orchid
Chemicals and Pharmaceutical Ltd., and prodrugs 1a-d were synthe-
sized in the laboratory.
HPLC Analysis. Testing Procedure for In-Process Analysis, Aqu-
eous Solubility, Chromatographic Purity, Partition Coefficient Anal-
ysis. A reverse phase HPLC method was used for analysis of metabolic
stability, in-process analysis, and chromatographic purity of prodrugs.
In-process analysis, chromatographic purity, partition coefficient, aque-
ous solubility were performed on an HPLC Shimadzu LC 2010 CHT
with UV detector. Mobile phase A consisted of a mix of 2.0 mL of glacial
acetic acid in 1000 mL of Milli Q water. Filtration was through a 0.45 μm
filter, and the samples were degassed (pH of this solution was around
3.0). Mobile phase B involved filtered and degassed acetonitrile (HPLC
grade). Mobile phase ratio was (mobile phase A (30%))/(mobile phase
B (70%)). Chromatographic parameters were as follows: column, YMC-
Pack C 8 (100 mm ꢀ 4.6 mm, 3 μm); flow rate, 0.8 mL/min;
wavelength, 254 nm; injection volume, 20 μL; run time, 20 min.
Testing Procedure for Metabolic Stability. The procedure involved
the following: HPLC system, Waters Alliance 2695 separation module
with 2996 PDA detector; software, Empower; column C18, ODS 3V, 250
mm ꢀ 4.6 mm, 5 μm; mobile phase A of 10 mM potassium dihydrogen
phosphate; mobile phase B of acetonitrile; wavelength, 265 nm. The
gradient elution program is depicted in Table 9.
Animals. Male Wistar rats weighing 150-200 g were used for the
study. All the experiments were approved by the Institutional Animal
Ethics Committee (IAEC) and complied with the NIH guidelines on
handling of experimental animals. The animals were housed in a group of
three rats per cage under well-controlled conditions of temperature (22
( 2 °C), humidity (55 ( 5%), and 12 h/12 h light-dark cycle. Animals
had free access to diet purchased from vet care and water ad libitum.
Paw edema was induced with carrageenan 1% (0.1 mL) administered
as a single subplantar injection under light ether anesthesia in the left
paw. Animals were divided into groups, namely, normal control,
carrageenan, and carrageenan treated with indomethacin and prodrug
(100 mg/kg, po) 1 h prior to carrageenan administration. Paw volume
was measured using the plethysmograph 3 h after the carrageenan
administration. Percentage of inhibition was calculated by the following
equation.
carrageenan - test compound
% inhibition ¼
ꢀ 100
carrageenan - normal
Aqueous Solubility. The solubility of prodrugs 1a-d was deter-
mined at 40 °C in 0.1 M boric acid buffer at pH 9, 0.2 M phosphate buffer
at pH 7.4, 0.1 M phosphate buffer at pH 5.2, 0.1 M citric acid buffer at pH
3.0, and 0.2 M hydrochloric acid buffer at pH 1.0. Test compound (5 mg
each) in buffer solution (10 mL) was incubated at 40 °C for 4 h at 400
rpm on a mechanical shaker. The solution was filtered through 0.20 μm
membrane filter, and the filtrate was analyzed quantitatively by HPLC at
a wavelength of 254 nm for its solubility and hydrolysis.
Determination of Partition Coefficients. The partition coef-
ficients of indomethacin ester prodrugs 1a-d were determined in
octanol-buffer system by HPLC method at 25 °C. The aqueous phase
was 0.2 M phosphate buffer of pH 7.4 and 0.1 M citric acid buffer of pH
3.0. Before use, the 1-octanol and buffer solution were mutually
saturated for 24 h at 400 rpm on a mechanical shaker at 25 °C. A known
concentration of compounds in 1-octanol (5 mL) and buffer solution
(5 mL) was shaken on a mechanical shaker for 60 min at 400 rpm at 25 °C
and centrifuged at 3500 rpm for 5 min. The concentration of the
compound (solute) in both phases were analyzed quantitatively by
HPLC at 254 nm. Each experiment was repeated in triplicate. The
partition coefficient was calculated by following equation.
Acute Ulcerogenic Assay. The test compounds 1a-d and the
reference drug indomethacin were evaluated for ulcerogenic side effect
by using distension ulcer model reported previously.23
Animals (n = 6/group) were fasted for 18 h with free access to water
before administration of test compounds. Ulcerogenic activity was
evaluated after 100 mg/kg single dose oral administration of indometha-
cin and prodrugs 1a-d. Animals were sacrificed under ether anesthesia
after a 6 h dosing of drug compounds. Stomach was removed, opened
along the greater curvature, washed, mounted on a thermostat sheet, and
examined for ulcers. Ulcerative lesions were scored as follows. (1)
Length of all lesions was measured using a Vernier caliper. (2) The ulcers
were classified as level I (ulcer area if <1 mm diameter), level II (ulcer
area of 1-3 mm diameter), level III (ulcer area of >3 mm diameter). (3)
Ulcerative lesion index (UI) was calculated as as 1(number of ulcer
level I) þ 2(number of ulcer level II) þ 3(number of ulcer level III).
Mean value of six animal readings was reported as ulcer index (UI).
Rat Plasma. Rat plasma was harvested from in-house rats. Fresh
blood was collected from the male rat using the retro-orbital bleeding
method in the tube containing heparin (100 IU/mL blood). After the
collection of blood, plasma was separated from the blood by centrifuga-
tion at 9000 rpm for 5 min. The supernatant plasma was separated and
utilized for further experiments.
!
½soluteꢁoctanol
In Vitro Physiological Stability of Prodrugs 1a-d and
Indomethacin (1) in Rat Plasma. The test compound solution
(5 μL of 5 mM) was dissolved in rat plasma (495 μL). Immediately after
addition (0 min), aliquots (100 μL) were removed and added to ice-cold
acetonitrile (100 μL) and mixed well by vortexing for 2 min. The mixture
was centrifuged at 14 000 rpm for 10 min. The supernatant was diluted
with acetonitrile and analyzed by HPLC. After 0 min, the remaining
logPoct=wat ¼ log
un-ionized
½soluteꢁwater
Anti-Inflammatory Assay. The test compounds 1a-d and the
reference drug indomethacin were evaluated by using the in vivo rat
carrageenan-induced foot paw edema model reported previously.22
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Journal of Medicinal Chemistry
ARTICLE
sample was incubated at 37 °C for 60 min. After 30 and 60 min, the
sample (100 μL) was treated with ice cold acetonitrile (100 μL)
and centrifuged at 14 000 rpm for 10 min. The supernatant was diluted
with acetonitrile and injected into the HPLC instrument. The percen-
tage of prodrug remaining was calculated according to the following
equation.
authors gratefully acknowledge the support extended by
Dr. Sivakumar B., Dr. Narayanan Surendran, Gopal Kandawamy,
and Fruthous Khan.
’ ABBREVIATIONS USED
NSAID, nonsteroidal anti-inflammatory drug; Tris-HCl, tris-
(hydroxymethyl)aminomethane hydrochloride; NADPH, nico-
tinamide adenine dinucleotide phosphate; HPLC, high performance
liquid chromatography; GC, gas chromatography; RLM, rat liver
microsomes; RP, rat plasma; API, active pharmaceutical ingredi-
ent; UI, ulcer index; GI, gastrointestinal; DMAc, dimethylaceta-
mide; PXRD, powder X-ray diffraction;RT,roomtemperature;TMG,
1,1,3,3-tetramethylguanidine; 18-crown-6, 1,4,7,10,13,16-hex-
aoxacyclooctadecane; EP, European Pharmacopoeia; Ar, aro-
matic; BDL, below detection limit
peak area at respective time ðminÞ
% remaining ¼
ꢀ 100
peak area at 0 min
Rat Liver Microsomes. Rat liver microsomes were prepared in-
house by a previously published method and used immediately in the
experiments. Protein concentrations were determined by the Biorad
protein assay (Bio-Rad, Hercules).
Microsomal Stability. Materials. Materials used were as follows:
NADPH (Sigma, lot no. N 6674); acetonitrile (HPLC grade, Merck,
India); Tris-HCl (S.D.Fine-Chem, India); rat liver chromosomes (in-
house); prodrugs; indomethacin reference compound.
Requirement. Required items were as follows: rat liver microsomes
(10 mg/mL protein concentration), NADPH (10 mM solution), Tris-
HCl buffer (pH 7.4), Eppendorf tubes, test compound solutions
(5 mM).
Study Conditions. Study conditions were as follows: incubation
period, 60 min; incubation conditions, 37 °C with 60 rpm shaking;
protein precipitation solvent, acetonitrile.
Assay Procedure. Tris-HCl buffer (395 μL), 10 mM NADPH
solution (50 μL), and 50 μL of rat liver microsomes were mixed and
vortexed for 10 s. To this mixture, 5 mM drug solution (5 μL) was
injected and vortexed well. The sample (75 μL) was immediately taken
out (0 min) and transferred to the centrifuge tube containing ice cold
acetonitrile (75 μL), vortexed, and centrifuged at 14 000 rpm for 10 min.
Aliquots of the supernatant were separated and used for analysis by
HPLC. Then the assay mixture was incubated in a water bath at 37 °C for
60 min. At specific time points (30 min, 60 min), the assay mixture (75
μL) was taken out and added to the centrifuge tube containing an equal
volume of cold acetonitrile. Then the tubes were vortexed and centri-
fuged at 14 000 rpm for 10 min. Aliquots of the supernatant were
separated and used for analysis by HPLC.
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’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, IR, and chromato-
S
b
graphic purity (GC) data for promoieties 2, 3, 4; 1H NMR, 13
C
NMR, IR, chromatographic purity (HPLC), UV, and mass data
for prodrugs. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(13) Ribeiro, L.; Silva, N.; Iley, J.; Rautio, J.; Jarvinene, T.; Mota-
Filipe, H.; Moreira, R.; Mendes, E. Aminocarbonyloxymethyl ester
prodrugs of flufenamic acid and diclofenac: suppressing the rearrange
pathway in aqueous media. Arch. Pharm. 2007, 340, 32–40.
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Synthesis and pharmacological evaluation of some dusl-acting amino-
alcohol ester derivatives of flurbiprofen and 2-[1,10-biphenyl-4-yl]acetic
acid: a potential approach to reduce local gastrointestinal toxicity. Chem.
Biodiversity 2006, 3, 1238–1248.
Corresponding Author
*Phone: (91)-217-9423138703. Fax: (91)-217-2351300. E-mail:
bandgar_bp@yahoo.com.
’ ACKNOWLEDGMENT
Spectral studies and biological evaluation were carried out at
Orchid Chemicals and Pharmaceuticals Ltd., Chennai, India. The
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