Synthesis and pharmacokinetic profile of rhein- boswellic acid conjugate

Article abstract of DOI:10.1016/j.bmcl.2012.10.017

Rhein, an active metabolite of diacerein, down-regulates the gene-expression and production of pro-matrix metalloproteinases and up-regulates the tissue inhibitors of metalloproteinase-1 production. The therapeutic effects of diacerein on osteoarthritis are, at least in part, due to the chondroprotective effect of rhein. Boswellic acid is a specific, non-redox inhibitor of leukotriene synthesis. It is claimed to possess good anti-inflammatory, anti-arthritic, analgesic, and anti-ulcer activities. It prevents the destruction of articular cartilage by decreasing degradation of glycosaminoglycans. Therefore, rhein and boswellic acid were linked chemically through a bioreversible ester linkage to synthesize their mutual prodrug by reported procedure. In vitro release profile of this prodrug was extensively studied in aqueous buffers of varied pH, upper GIT homogenates and 80% human plasma. In vivo release studies were undertaken in blood, urine and feces of rats. The prodrug was stable in HCl buffer (pH 1.2) and stomach homogenates of rats. However; in phosphate buffer (pH 7.4) and in intestinal homogenates the prodrug exhibited 91% and 96% release of rhein and 27.5% and 38% release of boswellic acid respectively over a period of 6 h following first order kinetics. In 80% human plasma (in vitro) and rat blood (in vivo) also 96.35% and 91% release of rhein and 78% and 86.41% release of boswellic acid respectively was observed. The 24 h pooled samples of rat urine revealed presence of 6.2% intact prodrug, 7.1% of rhein and 8.9% of boswellic acid indicating their renal excretion. Samples of rat feces pooled over a period of 24 h showed absence of rhein and presence of 3.1% of intact boswellic acid and 4.6% of boswellic acid emphasizing their intestinal excretion. The in vivo release kinetics of prodrug in rat clearly indicated activation of prodrug to be occurring in blood, being catalyzed by the weak alkaline pH of blood (7.4) in combination with esterases present therein.

Full text of DOI:10.1016/j.bmcl.2012.10.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7582–7587
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacokinetic profile of rhein- boswellic acid conjugate
⇑
Dhaneshwar Suneela , Patil Dipmala
Department of Pharmaceutical Chemistry, Bharati Vidyapeeth Deemed University, Poona College of Pharmacy, Pune 411038, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Rhein, an active metabolite of diacerein, down-regulates the gene-expression and production of pro-
matrix metalloproteinases and up-regulates the tissue inhibitors of metalloproteinase-1 production.
The therapeutic effects of diacerein on osteoarthritis are, at least in part, due to the chondroprotective
effect of rhein. Boswellic acid is a specific, non-redox inhibitor of leukotriene synthesis. It is claimed to
possess good anti-inflammatory, anti-arthritic, analgesic, and anti-ulcer activities. It prevents the
destruction of articular cartilage by decreasing degradation of glycosaminoglycans. Therefore, rhein
and boswellic acid were linked chemically through a bioreversible ester linkage to synthesize their
mutual prodrug by reported procedure. In vitro release profile of this prodrug was extensively studied
in aqueous buffers of varied pH, upper GIT homogenates and 80% human plasma. In vivo release studies
were undertaken in blood, urine and feces of rats. The prodrug was stable in HCl buffer (pH 1.2) and
stomach homogenates of rats. However; in phosphate buffer (pH 7.4) and in intestinal homogenates
the prodrug exhibited 91% and 96% release of rhein and 27.5% and 38% release of boswellic acid respec-
tively over a period of 6 h following first order kinetics. In 80% human plasma (in vitro) and rat blood
Received 18 August 2012
Revised 21 September 2012
Accepted 2 October 2012
Available online 11 October 2012
Keywords:
Diacerein
Inflammation
In vitro/vivo kinetics
Mutual prodrug
Osteoarthritis
(in vivo) also 96.35% and 91% release of rhein and 78% and 86.41% release of boswellic acid respectively
was observed. The 24 h pooled samples of rat urine revealed presence of 6.2% intact prodrug, 7.1% of
rhein and 8.9% of boswellic acid indicating their renal excretion. Samples of rat feces pooled over a period
of 24 h showed absence of rhein and presence of 3.1% of intact boswellic acid and 4.6% of boswellic acid
emphasizing their intestinal excretion. The in vivo release kinetics of prodrug in rat clearly indicated acti-
vation of prodrug to be occurring in blood, being catalyzed by the weak alkaline pH of blood (7.4) in com-
bination with esterases present therein.
Ó 2012 Elsevier Ltd. All rights reserved.
Osteoarthritis (OA) is a multifactorial, complex disease of syno-
vial joints. It is characterized by loss of articular cartilage due to
(NSAIDs) but steroidal anti-inflammatory agents and immunosup-
pressants are also equally important components of OA therapy.
1
10
degradation resulting in reduced joint space, remodeling of the sub-
chondral bone, formation of osteophytes, cyst formation, joint mis-
alignment and inflammation of the synovial membrane; the later
being the main cause of chronic pain which is the major symptom
Long-term administration of NSAIDs has deleterious effects on the
vital organs like gastrointestinal tract (GIT), kidney, liver, central
1
1–13
nervous system and immune system.
Intra-articular, long act-
ing corticosteroid injections although used in the management of
knee OA, can be given only three to four times in year as repeated
injections lead to progressive cartilage damage in weight-bearing
2
–4
of OA.
Ligament laxity, meniscal degradation and bone marrow
edema are other important characteristics which lead to joint
impairment and disability. All of these are responsible more or less
for the generation of local, chronic pain with insidious onset char-
acterized by aching with episodic stabbing.5 OA generally occurs
when the normal cartilage matrix is overloaded or when cartilage
matrix is vulnerable. Interleukin- 1 (IL-1) is principally involved
in degradation while transforming growth factor (TGF-b) is impli-
cated in the excessive repair of synovium and chondrocytes. Degra-
1
4–17
joints.
Chondro-protective or disease-modifying drugs pre-
serve normal joint function by slowing down the rate of anatomical
–8
18
progression of OA. Chondroitin sulfate, glucosamine, diacerein,
doxycycline and minocycline are the frontline disease modifying
1
9
anti-osteoarthritic drugs (DMOADs).
Diacerein [4,5-bis(acetyloxy)-9, 10-dioxo-2-anthracene carbox-
ylic acid], a low molecular weight anthraquinone derivative has
been introduced as an effective chondroprotective for the manage-
ment of OA. Since then it has emerged as a better and safer alter-
native for the treatment of the OA, which provides symptomatic
treatment along with modification of underlying pathological pro-
9
dation and repair are simultaneously occurring phenomenon.
A large number of palliative treatment modalities are available
for OA which mainly use non-steroidal anti-inflammatory drugs
2
0,21
cess.
It has demonstrated efficacy on the functional and struc-
⇑
Corresponding author. Tel.: +91 20 25437237; fax: +91 20 25439383.
E-mail address: suneeladhaneshwar@rediffmail.com (D. Suneela).
tural manifestations of OA by inhibition of IL-1 which is mainly
involved in cartilage destruction and acting on the synthesis of
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.10.017

S. Dhaneshwar, D. Patil / Bioorg. Med. Chem. Lett. 22 (2012) 7582–7587
7583
principal components of cartilage like proteoglycans and hyalu-
Panjab University, Chandigarh, India. The elemental analysis of syn-
thesized prodrug was performed on Elemental Analyzer (Vario MI-
CRO CUBE, Germany) at Department of Pharmaceutical Chemistry,
Poona College of Pharmacy, Pune (India). The absorbance maxima
(kmax) of synthesized compound in various solvents were deter-
mined on Jasco V-530 UV–Visible double-beam spectrophotometer.
Partition coefficient was determined in n-octanol/phosphate buffer
(pH 7.4) whereas the aqueous solubility was determined in distilled
water at room temperature 25 ± 1 °C using Jasco V-530 UV–Visible
double-beam spectrophotometer. For release kinetic studies, a
new HPLC method was developed for simultaneous estimation of
prodrugandits active metabolites(rheinand boswellicacid). Invitro
release was studied in aqueous buffers of varied pH, rat’s upper GIT
homogenates and 80% human plasma. In vivo release was studied in
rat blood, feces and urine. Human plasma was procured from Bharati
Vidyapeeth Medical College, Dhankavadi, Pune. The HPLC system
used for this purpose consisted of a pump (Jasco PU model 2080),
2
2
ronic acid. Diacerein inhibits interleukin-1b (IL-1b) and down-
regulates the IL-1b-induced inflammatory pathways and cartilage
breakdown in OA as opposed to inhibiting the cyclooxygenase
(
COX) pathway as NSAIDs do. In addition, it down-regulates the
gene-expression and production of pro-matrix metalloproteinases
proMMPs) that are involved in cartilage degradation and up-reg-
ulates the tissue inhibitor of metalloproteinase-1 (TIMP-1) produc-
(
1
0,23–25
tion.
Orally administered diacerein is completely converted
into its active metabolite rhein by deacetylation before reaching
2
6
systemic circulation. Rhein being an IL-1 inhibitor, reduces colla-
genase production in articular cartilage, dose-dependently inhibits
superoxide anion production, chemotaxix and phagocytic activity
2
7
of neutrophils, macrophage migration and phagocytosis. The
therapeutic effects of diacerein on OA may be due, at least in part,
to the chondroprotective effect of rhein.28
Therefore in the present work, rhein was chosen as potential
candidate for development of a mutual prodrug which would have
quick onset of action with improved absorption and bioavailability
than diacerein. Boswellic acid was selected as the promoiety in this
mutual prodrug design owing to its diverse pharmacological activ-
ities that would potentiate the chondroprotetive, disease modify-
ing effect of rhein.
with autosampler programmed at 20 lL capacity per injection and
a UV/VIS detector (Jasco UV 2075). Data was integrated using Jasco
Borwin version 1.5, LC-net-II/ADC system. XTerra RP18 column
(4.6 ꢀ 150 mm; 3.5
lM) in the reversed phase partition chromato-
graphic condition was used for HPLC analysis. The system was used
in an air-conditioned HPLC laboratory atmosphere (20 ± 1 °C). Be-
fore analysis, the mobile phase was degassed using sonicator and fil-
Boswellic acid and its acetates are ursane-type pentacyclic triter-
pene acids isolated from the gummy exudates of Boswellia serrata
Roxb and Boswellia carterri Birdw.29 The resin of Boswellia serrata is
used in India for the treatment of chronic inflammatory arthritis.
Ethanolic extract of the resin contains boswellic acid as the main ac-
tered through a 0.45 lM Millipore filter. Sample solutions were also
filtered through the same. The system was equilibrated before mak-
ing an injection. The column was monitored for UV absorbance at a
detection wavelength selected after taking the overlay spectra of
all the components.
3
0,31
tive constituent.
Gum resin extracted from Boswellia serrata
Roxb possesses good anti-inflammatory, anti-arthritic and analgesic
Rhein 1 was synthesized from diacerein by standard proce-
3
2
41
activities. Boswellic acid is reported to possess anti-ulcer property
dure. The mutual ester prodrug 3 of rhein 1 with boswellic acid
3
3
0
as well. It also has protective effect on articular cartilage by the vir-
2 was synthesized by using N,N -dicyclohexylcarbodiimide (DCC)
3
4
tue of its inhibitory effect on degradation of glycosaminoglycans.
We have reported the synthesis and the chondroproctective ef-
coupling (Fig. 1) and the structure was confirmed by spectral anal-
4
2–44
ysis.
3
5,36
fect of mutual prodrugs of diacerein with glucosamine
and
In vitro hydrolysis kinetics of prodrug was studied in aqueous
buffers pH 1.2 and 7.4. A reversed-phase HPLC method was devel-
oped for the simultaneous estimation of prodrug and its hydrolytic
metabolites rhein and boswellic acid. The mobile phase composed
of a phosphate buffer: acetonitrile (55:45 v/v, pH 3.0 ± 0.1) at a flow
rate of 1.0 mL/min and the column effluent was monitored at
211 nm. The retention times for rhein, boswellic acid and prodrug
were found to be 7.1, 5.4 and 8.2 min respectively. Aqueous buffers
of pH 1.2 (simulating the pH of stomach) and pH 7.4 (corresponding
to physiological pH of small intestine and blood) were prepared to
study the release profile of prodrug. The prodrug was incubated in
these media at 37 ± 0.5 °C with continuous stirring at 100 rpm.
Prodrug (10 mg) was introduced in the medium. Aliquots (5 mL)
were withdrawn and replaced with an equal volume of fresh incu-
bation medium by a sampler at different time intervals up to 7 h.
The release studies were performed in triplicate.
essential amino acids in monosodium iodoacetate-induced osteo-
arthritis in Wistar rats.37 Two hydrolytically activated antraqui-
none-diclofenac prodrugs have been designed and synthesized by
Daan et al. (2009) for bone targeting.38 Synthesis and pharmacolog-
ical activities of anthraquinone-ibuprofen prodrugs targeting osse-
ous tissues were also reported by Duan et al. (2009).39 Singh et al.
(
2007) have revealed the synergistic effect of mixture of boswellic
acid and glucosamine for anti-inflammatory and anti-arthritic
4
0
activities in rats.
There are no reports of any prodrugs of rhein in the literature so
far. The present work reports synthesis, in vitro and in vivo release
kinetics of mutual prodrug of rhein with boswellic acid with aims
of decreasing local irritant effect, potentiating anti-inflammatory
activity, anti-arthritic effect and increasing lipophilicity of rhein
in order to increase its bioavailability.
Diacerein was generously gifted by Glenmark Pharmaceuticals
Ltd., Mumbai, India. Rhein was synthesized in our lab from diacerein
by reported procedure. Boswellic acid was purchased from Jai Rhade
Sales, Ahmedabad, Gujrat, India. All chemicals and solvents used in
the study were purchased from Merck Chemical Corporation, USA
and were of analytical reagent grade (AR) or higher purity and were
used as it is. Thin layer chromatography was performed on pre-
coated silica gel plates-60 F264 (Merck) for purity check and moni-
toring of reactions. The IR spectrum of the synthesized compound
was recorded on Jasco V-530 FTIR in anhydrous IR grade potassium
In vitro release of rhein from the prodrug was further studied in
stomach and small intestinal homogenates of rats. A solution of pro-
drug was prepared in respective buffers (250 lg/mL) and 0.8 mL of it
was added to 0.2 g of stomach or small intestinal homogenate placed
in 1 mL centrifuge tubes that were pre-equilibrated at 37 ± 0.5 °C.
Samples were withdrawn at appropriate time intervals, centrifuged
at 10,000 rpm for 10 min. The supernatants were filtered through
membrane filter (0.45 lM) and estimated by HPLC for the amount
of prodrug remaining and also rhein and/or boswellic acid that
might have released after hydrolysis of prodrug. Rate constants for
the hydrolysis of prodrug were determined from the slopes of linear
plots of the logarithm of residual prodrug concentration versus time.
These studies were performed in triplicate and the mean of the rate
constant was calculated.
1
3
bromide. Proton and C NMR spectra of the synthesized prodrug
were recorded in DMSO-d using Bruker Avance II 400 NMR spec-
6
trometer at Sophisticated Analytical Instrumentation Facility (SAIF),
Panjab University, Chandigarh, India. Chemical shifts are reported in
ppm downfield on d scale. The mass spectrum was recorded employ-
ing Waters Q-Tof Macromass spectrometer, LC–MS system at SAIF,
In vitro release kinetic studies were performed in human plas-
ma also. For this purpose 80% human plasma was prepared in

7
584
S. Dhaneshwar, D. Patil / Bioorg. Med. Chem. Lett. 22 (2012) 7582–7587
OH
O
OH
H
H
2
COOH
O
(
1)
HO
H
HOOC
(2)
2
4
D
h
,
C
r
C
o
,
o
P
m
T
t
S
e
A
m
H
H
p
.
H
H
HO
OH
H
H
C
O
C
O
O
O
O
COOH
O
(3)
Figure 1. Scheme of synthesis.
0
.05 M phosphate buffer (pH 7.4). Stock solution of prodrug was
prepared in methanol (250 g/mL) and 100 L of it was added to
00 L of 80% plasma placed in 1 mL eppendorf tubes. The tubes
of Control and Supervision of Experiments on Animals (CPCSEA).
The protocols for the same were approved by Institutional Animal
Ethics Committee of Poona College of Pharmacy, Pune. Wistar rats
(200–250 g; n = 3) were housed in metabolic cages individually un-
der normal conditions (at 27 ± 0.5 °C and a relative humidity of
70 ± 0.5% under natural light/dark conditions). The rats were fasted
overnight before the experiment but had free access to water.
Prodrug (2.5 mg) was dissolved in saline (0.5 mL) and was adminis-
tered to rats by oral route. Blood was drawn by retro-orbital
puncture from both the eyes of the rats and collected in eppendorf
tubes over a period of 8 h. Same procedure as described for 80% hu-
man plasma was followed to study the fate of the prodrug in terms
of its absorption/distribution in the body. Urine and feces of the rats
treated with the prodrug were collected at different time intervals
from the metabolic cages over a period of 24 h and pooled together
and diluted with phosphate buffer (pH 7.4) by 10 and 100-fold
respectively. Same procedure as described for 80% human plasma
was followed to study the excretion profile of the prodrug.
l
l
1
l
were kept in incubator at 37 ± 0.5 °C and removed at appropriate
time intervals and then centrifuged at 5000 rpm at 0–5 °C for
1
tube (1 mL capacity) and 0.9 mL methanol was added to it for
immediate plasma protein precipitation. All the solutions were
then vortexed for 2 min and again centrifuged at 10,000 rpm for
0 min. The supernatant (0.1 mL) was added to another eppendorf
5
min at 0–5 °C in order to precipitate other impurities or solid
matter present in the biological samples. The clear supernatant
20 L) was filtered through a membrane filter (0.45 M) and ana-
(
l
l
lyzed by HPLC for presence of residual prodrug and released rhein
and/or boswellic acid. These studies were performed in triplicate
and the mean of the rate constant was calculated.
In vivo behavior of orally administered prodrug was investigated
in Wistar rats at animal facilities of Poona College of Pharmacy,
Pune, India which were approved by Committee for the Purpose

S. Dhaneshwar, D. Patil / Bioorg. Med. Chem. Lett. 22 (2012) 7582–7587
7585
In this study, a novel covalently bound conjugate of rhein and
boswellic acid was synthesized and structurally characterized by
100
9
8
0
0
1
13
spectral analysis wherein satisfactory FTIR, H and C NMR spec-
tra were obtained. The IR spectrum showed characteristic peaks
ꢁ
1
ꢁ1
at 1721 cm for C@O stretching ester, broad peak at 3300 cm
70
ꢁ1
RH-I
BA-I
RH-II
BA-II
RH-III
BA-III
for carboxylic OH stretching (rhein backbone) and 3538 cm for
alcoholic OH stretching (boswellic acid backbone). The chemical
shifts observed in H NMR of prodrug were in accordance with
6
5
0
0
1
the anticipated structure. 13C NMR exhibited all the relevant chem-
ical shifts as per the anticipated number of carbon atoms present in
prodrug. Elemental analysis, 13C NMR and mass spectroscopy re-
sults confirmed the formation of prodrug by reaction of one mole-
cule of rhein with two molecules of boswellic acid. The molecular
weight of prodrug was calculated from the results of elemental
analysis and mass spectroscopy and it was in accordance with
the anticipated molecular weight. The lipophilicity of rhein (LogP
40
30
2
1
0
0
0
0
60 120 180 240 300 360 420
Time (min)
0
.2402) was markedly enhanced in prodrug (LogP 2.568) due to
Figure 2. In vitro percent release of rhein and boswellic acid from prodrug in
different incubation media. RH: Rhein; BA: Boswellic acid; I-Phosphate buffer pH-
its conjugation with boswellic acid. This would improve absorption
and bioavailability of rhein.
7.4; II-Intestinal homogenates; III-80% human plasma.
The results of in vitro kinetics studies are compiled in Table 1.
The prodrug resisted acid-catalyzed hydrolysis on incubation with
HCl buffer and stomach homogenates. However, the prodrug was
susceptible to weak alkaline hydrolysis in phosphate buffer (pH
95
75
55
35
15
-5
7
.4) accomplishing 91% release of rhein and 27.5% release of
boswellic acid over a period of 6 h following first order kinetics
with a half life of 192 min. The result of release kinetics of prodrug
in small intestinal homogenates was slightly better (96% of rhein
and 38% of boswellic acid) than in phosphate buffer (pH 7.4). The
release kinetics was further studied in 80% human plasma over a
period of 7 h offering 96.35% of rhein and 77.31% of boswellic acid
Rhein
Boswellic acid
(Fig. 2).
In vivo behavior of the orally administered prodrug was studied
in Wistar rats over a period of 7 h in blood. The concentrations of
prodrug remaining after hydrolysis and released rhein and boswel-
lic acid were determined in blood which was withdrawn at various
time intervals by the retro-orbital puncture and also in 24 h pooled
samples of urine and feces collected from the metabolic cages. First
appearance of prodrug in blood was noted at 2 h (93.6%) indicating
its absorption in intact form from the stomach, which decreased
consistently at 3 h (70.43%), 4 h (34.5%), 5 h (22%) and 6 h (12%)
eventually reaching a negligible level at the end of 7 h (3.4%) owing
to its hydrolysis (activation) into rhein and boswellic acid. Pres-
ence of rhein and boswellic acid (approximately 29%) was first ob-
served in blood at 3 h. A constant increase in the concentration of
rhein and boswellic acid was observed from 4 h onwards reaching
the highest concentration at the end of 7 h. Approximately 91% of
the administered dose of prodrug was recovered in blood as rhein
and 86.41% as boswellic acid in 7 h (Fig. 3). The in vivo release pro-
file of prodrug in rat clearly indicates that its activation is mediated
by hydrolysis which in turn is catalyzed by the weak alkaline pH of
blood (7.4) in combination with esterases present therein (Fig. 4).
The results of in vitro release kinetics of prodrug in small intestinal
homogenates and 80% human plasma correlate well with its in vivo
0
60
120
180
240
300
360
420
Time (min)
Figure 3. In vivo percent release of rhein and boswellic acid from orally
administered prodrug in rat blood.
behavior because of the similarity in their enzymatic set up (ester-
ases) and pH of the environment (7.4). The 24 h pooled samples of
rat urine revealed presence of 6.2% of intact prodrug, 7.1% of rhein
and 8.9% of boswellic acid indicating their renal excretion. The ur-
ine also indicated presence of negligible concentration of unknown
metabolites. Samples of rat feces collected and pooled over a per-
iod of 24 h showed absence of rhein and presence of 3.1% of intact
prodrug and 4.59% of boswellic acid emphasizing their intestinal
excretion.
In conclusion, the release kinetics has supported the biorevers-
ible nature of the ester linkage which upon alkaline and enzymatic
(esterases in small intestine and blood) hydrolysis is almost com-
pletely releasing rhein and boswellic acid at the end of 7 h. Markedly
enhanced lipophilicity of prodrug as compared to rhein and its sta-
bility in stomach might be responsible for its quick absorption from
Table 1
In vitro release kinetics data of prodrug at 37 °C
K ± S.D. (min 1
ꢁ
*
)
t1/2 (min)
Av % release
Boswellic acid
S.N.
Incubation medium
Order of reaction
Rhein
1
2
3
4
5
Hydrochloric acid buffer (pH 1.2)
Phosphate buffer (pH 7.4)
Stomach homogenates
Small intestinal homogenates
80% Human plasma
Stable
First
Stable
First
—
—
192.3
—
160.3
186.1
—
91
—
96
—
27.5
—
38
77.31
ꢁ
ꢁ
4
0.003 ± 3ꢀ10
—
3
0.005 ± 2ꢀ10
0.004 ± 3ꢀ10ꢁ
4
96.35
First
*
Average of three readings.

7
586
S. Dhaneshwar, D. Patil / Bioorg. Med. Chem. Lett. 22 (2012) 7582–7587
H
H
H
H
OH
HO
H
H
C
C
O
O
O
O
O
COOH
O
(
3)
Esterases
OH
O
O
OH
H
H
2
COOH
HO
(
1)
H
HOOC
(
2)
Figure 4. Proposed scheme of activation of prodrug.
the gastric mucosa which is characterized by appearance of 93.6% of
intact prodrug in blood during first 2 h of its oral administration. The
in vivo release kinetics of prodrug in rat clearly indicates that the
activation of prodrug is occurring in blood and is being catalyzed
by the weak alkaline pH of blood (7.4) in combination with esterases
present therein. The results of evaluation of disease-modifying ef-
fect of prodrug in collagenase (type-II)-induced OA in Wistar rats
are very promising and will be published separately.
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Acknowledgment
Authors thank the UGC for providing financial assistance and
Glenmark Pharmaceutical Pvt. Ltd, Mumbai, India, for providing
the gift sample of diacerein.

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39. Duan, Y.; Yu, J.; Liu, S.; Ji, M. Med. Chem. 2007, 5, 577.
40. Singh, S.; Khajuria, A.; Taneja, S. C.; Khajuria, R. K.; Singh, J.; Qazi, G. N. Bioorg.
Med. Chem. Lett. 2007, 17(13), 3706.
41. Synthesis of rhein from diacerein: Diacerein (0.012 M, 4.5 g) was dispersed in
80 ml of 10% aqueous sodium hydroxide solution. The solution was heated on a
boiling water bath for 30 min and then poured into 120 mL of 10% HCl. Rhein
was obtained in the form of yellow flakes which were recrystallized twice with
1. Jieyun, J.; Qiang, X. J. Ethnopharmacology 2003, 85, 53.
2. Lichtenstein, D. R.; Syngle, S.; Wolfe, M. Arthritis Rheum. 1995, 38, 5.
3. Lester, R. S.; Konwles, S. R.; Shear, N. H. Dermatol. Clin. 1998, 16, 277.
4. Patino, F. G.; Olivieri, J.; Allison, J. J. J. Rheumatol. 2003, 30, 2680.
5. http://www.rheumatology.org/publications/guidelines/oa-mgmt/oa-mgmt.asp?
aud=mem.
1
6. Jordan, K. M.; Arden, N. K.; Doherty, M.; Bannwarth, B. Ann. Rheum. Discov.
2
003, 62, 1145.
pyridine. Rhein. mp 318–319 °C, R
yield 60, LogP 0.2402. IR (m
f
0.58 (chloroform: methanol; 3:0.5 v/v),%
, cm , KBr) 3566 (Phenolic OH stretch.), 3100, 2850
(Broad OH stretch. of COOH), 1682 (C@O stretch. of –COOH), 1193 (C–O
stretch. of phenol), 1H NMR (d, ppm, DMSO-d
) 8.57 carboxylic OH [s; 1H], 8.26
benzene CH [s; 2H], 8.25 benzene CH [s; 2H], 7.32–7.83 benzene CH [t; 3H],
3.79 phenolic OH [d; 2H].
42. Holmberg, K.; Hansen, B. Acta Chem. Scand. 1979, B33, 410.
ꢁ
1
1
1
1
7. Schnitzer, T. J. Am. Coll. Rheumatol. 2002, 24, 24.
8. http://www.bly.org/journal/2002_4404_oct/gps_670.htm.
9. Altman, R. D.; Hochberg, M. C.; Moskowitz, R. W.; Schnitzer, T. J. Arthritis Rheum
6
1
905, 2000, 43.
2
2
2
2
0. http://www.realage.com/joint-pain/disease-modifying-drugs-for-osteoarthritis-
dmoads.
2
2
2
1. Spencer, C. M.; Wilde, M. I. Drugs 1997, 53, 98.
2. Nicolas, P.; Tod, M.; Padoin, C.; Petitjean, O. Clin. Pharmacokinet. 1998, 35, 347.
3. http://www.cipladoc.com/therapeutic/admin.php?mode=prod&action=disp&id=
43. Fieser, L. F.; Fieser, M. In Reagents for organic synthesis; Wiley: New York, 1967;
vol. 1,
44. Synthesis of prodrug DSRB (3): Rhein (1) (0.002 M, 0.57 g) and boswellic acid (2)
(0.008 M, 3.65 g) were dissolved in 30 mL of DMF. p-Toluene sulfonic acid
(PTSA) 0.004 g was added to above mixture and stirred for 15 min. DCC
(0.004 M, 1.15 g) was added to above solution and stirred for 30 h at room
temperature. Then 20 mL glacial acetic acid was added and reaction mixture
was kept at 0–5 °C overnight. The mixture was filtered and the crystals were
washed with pyridine (2–4 mL). Mixture of ethyl acetate: ether: ice (30 mL:
30 mL: 30 g) was added to the filtrate followed by slow addition of 5 M HCl with
stirring to adjust the pH to 2.5. Then the organic phase was washed first with
water, then 0.5 M aqueous potassium carbonate solution and again water. The
solvent was evaporated under vacuum and the product was recrystallized with
5
51.
2
4. Pelletier, J. P.; Yaron, M.; Haraoui, B.; Cohen, P.; Nahir, M. A.; Choquette, D.;
Wigler, I.; Rosner, I. A.; Beaulieu, A. D. Arthritis Rheum. 2000, 43(10),
2
339.
2
5. Tamura, T.; Kosaka, N.; Ishiwa, J.; Sato, T.; Nagase, H.; Ito, A. Osteoarthritis
Cartilage 2001, 9, 257.
6. Nicholas, P.; Tod, M.; Padoin, C.; Petitijean, O. Clin. Pharmacokinet. 1998, 35(5),
2
3
47.
2
2
2
7. Spencer, C. M.; Wilde, M. I. Drugs 1997, 53, 98. Discussion 107.
8. Tamura, T.; Ohmori, K. Jpn. J. Pharmacol. 2001, 85, 101.
9. Krohn, K.; Rao, M. S.; Raman, N. V.; Khalilullah, M. Phytochem. Anal. 2001, 12,
ethyl acetate. DSRB. mp 380 °C (d, uncorrected), R
f
0.76, chloroform: methanol:
g/ml, LogP
, cm , KBr) 3538 (OH stretch.), 3300 (broad OH-stretch. of –COOH),
1721 (C@O stretch. ester), 1628, 1451(aromatic C@C stretch.), 1267 (C–O
stretch. ester), 1249 (C–O stretch. COOH), 1081 (C–O stretch alcoholic OH), 1
NMR (d, ppm, DMSO-d ) rhein backbone: 11.8 carboxylic OH [s; 1H], 8.59
benzene CH [s; 2H], 8.26 benzene CH [s; 2H], 7.37–7.88 benzene CH [t; 3H];
3
74.
acetic acid (4:1:3 drops v/v/v),% yield 46.15, aqueous solubility 92 l
ꢁ
1
3
0. Majeed, M.; Badmaev, V.; Gopinathan, S.; Rajendran, R.; Norton, T.; Braly, J.
2.568, IR (m
Anti-inflammatory Phytonutrient; Nutriscience Publishers Inc.: Piscataway, NJ,
1
996.
H
3
3
1. Henderson, W. R. Ann. Intern. Med. 1994, 121, 684.
6
2. Selected medicinal plants of India- A Monograph of identity, safety and clinical
usage. Compiled by Swami Prakashanand Ayurved Reserch Centre (SPARC) for
Chemixil. Tata Press: India, 1992, 65–66.
3. Singha, S.; Khajuriaa, A.; Tanejab, S. C.; Khajuriab, R. K.; Singha, J.; Johria, R. K.;
Qazi, G. N. Phytomedicine 2008, 15, 408.
2
2
2
boswellic acid backbone: 5.22 cyclohexene 2 ꢀ CH [t; 2H], 3.77–3.79
cyclohexanol 2 ꢀ CH [t; 2H], 2.01–2.05 cyclohexanol 2 ꢀ OH [d; 2H], 1.44–
3
3
3
2.11 cyclohexane 2 ꢀ CH [m; 6H], 1.24–1.93 cyclohexane 2 ꢀ CH
2
[m; 40H],
[m; 46H]. 13C NMR (d, ppm, DMSO-d
) rhein backbone:
3 6
cyclohexane 2 ꢀ CH
4. Reddy, G. K.; Chandrakashan, G.; Dhar, S. C. Biochem. Pharmacol. 1989, 38,
124.9, 125.1, 125.2, 127.6, 133.8, 134.2, 135.2, 149.5, 149.7, 164.9, 180.0;
boswellic acid backbone: 20.6, 38.9, 39.1, 39.3, 39.5, 39.7, 39.9, 40.1, 78.3, 78.6,
3
527.
5. Dhaneshwar, S. S.; Patil, D.; Mengi, S.; Mulay, G.; Lahane, J. J. Drug Del. Sci. Tech.
009, 19, 25.
78.8, 124.9, 149.5, 187.0, 187.6. MS, m/z 1161.5901 (C75
100 10
H O , calc.
2
1161.5905). Elemental Anal. calcd: C, 77.55; H, 8.68. Found: C, 77.72; H, 8.65.