Design, synthesis, bioconversion, and pharmacokinetics evaluation of new ester prodrugs of olmesartan

Article abstract of DOI:10.1016/j.ejmech.2011.05.019

Synthesis of new ester prodrugs of olmesartan is described. Their in vitro stabilities in simulated gastric juice, rat plasma, and rat liver microsomes were tested. And the pharmacokinetic parameters for olmesartan after their oral administration were also estimated and compared with those in case of olmesartan medoxomil. Compounds 13 and 14 demonstrated high stability in simulated gastric juice and were rapidly metabolized to olmesartan in rat liver microsomes and rat plasma in vitro. In addition, C_max and AUC_last parameters were significantly increased in case of compounds 13 and 14 compared with olmesartan medoxomil. These results indicate that compounds 13 and 14 with cyclohexylcarboxyethyl and adamantylcarboxymethyl promoieties, respectively, are promising prodrugs of olmesartan with markedly increased oral bioavailability.

Full text of DOI:10.1016/j.ejmech.2011.05.019

European Journal of Medicinal Chemistry 46 (2011) 3564e3569
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, bioconversion, and pharmacokinetics evaluation of new ester
prodrugs of olmesartan
,c,
Jeong-Soo Chang a, Mohammed I. El-Gamal b d, Woong San Lee b, Hanan S. Anbar b, Hye Jin Chung b,
Hyun-Il Kim e, Young-Jin Cho e, Bong Sang Lee e, Sun Ahe Lee e, Ji Yun Moon e, Dong Jin Lee e,
**
,
b,c,
*
Hong-Ryeol Jeon e, Jaehwi Lee a, Young Wook Choi a , Chang-Hyun Oh
a College of Pharmacy, Chung-Ang University, 221 Heuksuk-dong, Dongjak-gu, Seoul 156-756, Republic of Korea
b Life Sciences Division, Korea Institute of Science and Technology, PO Box 131,Cheongryang, Seoul 130-650, Republic of Korea
c Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno, Yuseong-gu, Daejeon 305-333, Republic of Korea
d Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
e CTCBIO Inc. 450-34, Noha-ri, Paltan-myeon, Hwaseong-si, Gyeonggi-do 445-913, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis of new ester prodrugs of olmesartan is described. Their in vitro stabilities in simulated gastric
juice, rat plasma, and rat liver microsomes were tested. And the pharmacokinetic parameters for
olmesartan after their oral administration were also estimated and compared with those in case of
olmesartan medoxomil. Compounds 13 and 14 demonstrated high stability in simulated gastric juice and
were rapidly metabolized to olmesartan in rat liver microsomes and rat plasma in vitro. In addition, Cmax
and AUClast parameters were significantly increased in case of compounds 13 and 14 compared with
olmesartan medoxomil. These results indicate that compounds 13 and 14 with cyclohexylcarboxyethyl
and adamantylcarboxymethyl promoieties, respectively, are promising prodrugs of olmesartan with
markedly increased oral bioavailability.
Received 12 April 2011
Received in revised form
4 May 2011
Accepted 8 May 2011
Available online 13 May 2011
Keywords:
Antihypertensive
Olmesartan
Olmesartan medoxomil
Prodrug
Ó 2011 Elsevier Masson SAS. All rights reserved.
Ester
Pharmacokinetics
1. Introduction
There are many antihypertensive drugs available, many of which
act on the RAAS system. Angiotensin receptor blockers (ARBs) are
Hypertension is a disease which affects an estimated one billion
people worldwide [1]. It is a serious disease with a momentous
impact on health and life expectancy. Controlling blood pressure
and prevention of its complications such as coronary heart disease,
renal failure, and cerebral vascular disease are the main objectives
for the treatment of hypertension [2].
The renin-angiotensin-aldosterone system (RAAS) is a very
complex system that plays an important role in the regulation of
blood pressure. Angiotensin II, the primary effector hormone of
RAAS, affects the cardiovascular system by influencing the vascular
tone, fluid volume, and electrolyte balance [3,4].
a class of antihypertensive agents that are growing in popularity
due to their excellent blood pressure control potential, low adverse
event profile, and high patient tolerability [5]. Olmesartan is an
example of ARBs which acts by blocking type 1 angiotensin II
receptors (AT1-R), leading to blocking of vasoconstriction, reduc-
tion of sodium and water retention, and decrease of cellular
proliferation and hypertrophy [6]. Besides AT1-R blockade, olme-
sartan is assumed to exhibit an angiotensin-converting enzyme
(ACE) inhibitory action, prevent an increase in angiotensin II level,
and protect cardiovascular remodeling through an increase in
cardiac nitric oxide production and endogenous angiotensin-(1e7)
via over-expression of ACE2 [7].
The once daily dosing interval of most ARBs helps enhance
patient compliance which may lead to better patient outcomes [5].
Olmesartan medoxomil is an ester prodrug of olmesartan which
has shown potent and long-lasting antihypertensive activity by oral
administration [8]. Olmesartan medoxomil is rapidly de-esterified
by an enzyme, arylesterase, which is located in both the intestine
* Corresponding author. Biomaterials Center, Korea Institute of Science and
Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea. Tel.: þ82 2
958 5160; fax: þ82 2 958 5189.
** Corresponding author. Tel.: þ82 2 820 5609; fax: þ82 2 826 3781.
E-mail addresses: ywchoi@cau.ac.kr (Y.W. Choi), choh@kist.re.kr (C.-H. Oh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.019

J.-S. Chang et al. / European Journal of Medicinal Chemistry 46 (2011) 3564e3569
3565
was important at the beginning to prepare the key carboxylic acid
compound, trityl olmesartan 3. N-Alkylation of ethyl 4-(1-hydroxy-
1-methylethyl)-2-propylimidazole-5-carboxylate (1) with 5-(40-
bromomethyl-biphenyl-2-yl)-1-trityl-1H-tetrazole afforded the
ethyl ester of trityl olmesartan 2. Alkaline hydrolysis of the ethyl
ester moiety of 2 followed by acidification of the formed potassium
salt furnished trityl olmesartan 3 [11]. The target ester compounds
10e15 were synthesized by esterification of the carboxylic acid
group of 3 with the appropriate alkyl halide, and subsequent
detritylation using conc. HCl.
N
N
OH
O
OH
+
N
N
H2O
CO2
O
OH
O
O
O
O
N
N
N
N
O
Enzymatic Hydrolysis
N
N
N
H
N
H
O
Diacetyl
Olmesartan medoxomil
Ester Prodrug
Olmesartan
Active metabolite
Fig. 1. Hydrolysis of olmesartan medoxomil to olmesartan.
2.2. Biological evaluation
and plasma [6]. Fig. 1 illustrates the enzymatic hydrolysis of
olmesartan medoxomil into the active metabolite, olmesartan [9].
We have reported the synthesis, bioconversion, and pharma-
cokinetic (PK) evaluation of a new derivative of olmesartan
medoxomil with higher lipophilicity. The new ester prodrug of
olmesartan showed improved PK parameters for olmesartan,
compared with olmesartan medoxomil [10]. These results showed
that lipophilic ester prodrugs of olmesartan can improve the oral
bioavailability and PK properties of olmesartan. In the present
investigation, we report the design, synthesis, in vitro bioconver-
sion, and PK evaluation of new ester prodrugs of olmesartan. The
medoxomil moiety of olmesartan medoxomil was replaced by more
lipophilic promoieties, such as myristoyl, cyclohexylcarboxyethyl,
adamantylcarboxymethyl, and benzoyloxyethyl, in order to
increase the lipophilicity of olmesartan ester prodrugs, and hence,
olmesartan bioavailability. Our goal is to improve the pharmaco-
kinetic properties of olmesartan, and hence, the antihypertensive
outcomes. Moreover, we have reported synthesis and in vitro
stability in rat plasma of octyl and uracil esters of olmesartan [10].
Herein, we report their in vivo PK properties also. The synthetic and
screening protocols are illustrated in details.
2.2.1. In vitro stability evaluation
The stabilities of all compounds were determined in rat plasma
in vitro. The remaining percentages of the compounds in rat plasma
after the incubation are summarized in Table 1. The results of
compounds 10 and 12 have been previously reported [10]. After the
30 min incubation period of 11 and 13, the prodrug peak dis-
appeared, and the olmesartan peak was increased. The results
showed that compounds 11 and 13 were rapidly hydrolyzed to
olmesartan, active metabolite, in plasma.
The prodrugs should be stable in acidic condition encountered
in the stomach following oral dosing and be hydrolyzed to active
metabolites in the systemic circulation after absorption. The
stabilities of compounds 13 and 14, as representative examples of
these new ester prodrugs, were determined in simulated gastric
juice, rat plasma, and rat hepatic microsomes in vitro. The chem-
ical or enzymatic stabilities of compounds after the incubation are
presented (Table 2). The half-lives of both compounds in simulated
gastric juice were more than 300 min. In addition, the half-lives of
13 and 14 in rat hepatic microsomes and rat plasma were ꢀ10 min.
The results showed that these compounds were rapidly hydro-
lyzed to olmesartan, the active metabolite, in rat liver microsomes
and rat plasma, while the hydrolysis rates of the compounds in
simulated gastric juice were slow. These results suggested that
compounds 13 and 14 can pass through the stomach with slow
degradation and the absorbed molecules from the gastrointestinal
tract can be rapidly converted into the active form in liver and
plasma.
2. Results and discussion
2.1. Chemistry
Synthesis of the target compounds 10e15 was carried out
according to the sequence of reactions illustrated in Scheme 1. It
N
N
N
OH
OH
OH
OH
N
N
N
O
O
N
b
a
c
R
O
O
O
HN
OH
O
N
N
N
N
N
N
N
N
N
O
N
N
N
Tr
Tr
Tr
1
2
3
4-9
N
O
OH
-(CH2)7CH3
4,10: R =
5,11: R =
7,13: R =
N
O
CH3
O
R
O
(CH2)12CH3
O
O
d
O
O
8,14: R =
9,15: R =
N
N
H
N
O
N
O
N
H
6,12: R =
NH
O
CH3
O
10-15
Scheme 1. Reagents and conditions: (a) 5-(40-bromomethyl-biphenyl-2-yl)-1-trityl-1H-tetrazole, K2CO3, acetone, DMAc, reflux, 10 h; (b) (i) KOH, isopropanol, 60 ꢁC, 4 h; (ii) HCl,
workup; (c) R-X, K2CO3, KI, DMAc, 70 ꢁC, 2 h; (d) conc. HCl, acetone, H2O, rt, 2 h.

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J.-S. Chang et al. / European Journal of Medicinal Chemistry 46 (2011) 3564e3569
Table 1
In vitro stabilities of compounds 10e15 in rat plasma.
Structure
Compound
no.
R
Stability
(%remaining)
10
e(CH2)7CH3
90.2
O
O
(CH2)12CH3
11
12
NAa
H
O
N
O
96.3
N
NH
OH
N
O
R
O
NAa
O
13
CH3
O
O
N
N
N
N
H
14
15
79.8
O
O
85.8
NAa
CH3
O
Olmesartan
medoxomil
a
NA, not applicable.
2.2.2. In vivo pharmacokinetic (PK) studies
concentrations of olmesartan (Cmax) were significantly higher
(290% and 52% increase for 13 and 14, respectively) than those
observed after administration of olmesartan medoxomil. The
AUClast values of prodrugs were also significantly greater (157% and
46% of olmesartan for 13 and 14, respectively) than that of olme-
sartan medoxomil. These effects are possibly due to increases in
lipophilicity of 13 and 14 induced by cyclohexylcarbonyloxyethyl
and adamantylcarbonyloxymethyl moieties, respectively, over
olmesartan medoxomil. The results indicated that introduction of
these lipophilic moieties enhanced the oral absorption and
systemic exposure level of olmesartan.
Pharmacokinetic studies were conducted to determine whether
the new prodrugs of olmesartan were converted into olmesartan
in vivo. Olmesartan medoxomil and compounds 10e15 were
administered orally using a feeding tube to male SpragueeDawley
rats at a dose of 20 mg/kg as olmesartan. The plasma concentra-
tions of olmesartan were determined by a slight modification of the
reported liquid chromatography-tandem mass spectrometric
method (LC-MS/MS) [12]. Pharmacokinetic parameters were
determined by a non-compartmental analysis. The total area under
the plasma concentration-time curve from time zero to the last
measured time (AUClast) was calculated by the linear trapezoidal
rule method [13]. The mean arterial plasma concentration-time
profiles of olmesartan after oral administration of the newly
synthesized ester compounds and olmesartan medoxomil in rats
are shown in Fig. 2, and some relevant pharmacokinetic parameters
of olmesartan are summarized in Table 3. Olmesartan was detect-
able in plasma from the first blood sampling time, 30 min, after oral
administration of compounds 13e15 and olmesartan medoxomil.
This result suggested that these compounds were well absorbed
from rat gastrointestinal tract and rapidly converted into the active
form. After administration of prodrugs, the peak plasma
Table 2
Half-lives of olmesartan medoxomil and compounds 13 and 14 in simulated gastric
juice, rat plasma, and rat liver microsomesa
Compd. no.
Half-lives (min)
Simulated gastric juice
Rat plasma
0.956
Rat liver microsomes
4.95
Olmesartan
medoxomil
13
>1000
Fig. 2. Mean arterial plasma concentration-time profiles of olmesartan after oral
administration of olmesartan medoxomil (A; n ¼ 7), 10 (C; n ¼ 4), 11 (,; n ¼ 4),
;
928
312
9.68
10.00
2.70
2.28
14
12 (B; n ¼ 4), 13 ( ; n ¼ 6), 14 (-; n ¼ 4), and 15 (V; n ¼ 4) and at a dose of 20 mg/kg
a
Data represent the mean of duplicated experiments.
as olmesartan in rats.

J.-S. Chang et al. / European Journal of Medicinal Chemistry 46 (2011) 3564e3569
3567
Table 3
mixture of DMAc (25 mL) and acetone (250 mL) was heated under
reflux for 10 h. The mixture was cooled to room temperature and
filtered to remove the insoluble material. The filtered inorganic
solid material was washed with acetone (25 mL). The washing
solution was combined with the filtrate and the solvent was
evaporated under reduced pressure to afford the desired product 2
(22.37 g, 75%). LC-MS m/z: 717.40 [M þ 1]þ.
Pharmacokinetic parameters (mean ꢃ standard deviation) of olmesartan after oral
administration of prodrugs (20 mg/kg as olmesartan) to male rats.
Compd. administered
Cmax (ng/mL)a
Tmax (h)b
AUClast (ng h/mL)c
Olmesartan medoxomil
(n ¼ 7)
891.2 ꢃ 1136.5 2.9 (0.5e6.0) 3385.9 ꢃ 261.6
10 (n ¼ 4)
17.9 ꢃ 22.1
32.6 ꢃ 13.7
8.0 ꢃ 3.0
0.8 (0.5e1.0)
0.8 (0.5e1.0)
1.8 (0.5e4.0)
32.8 ꢃ 29.2
100.4 ꢃ 11.9
21.7 ꢃ 12.7
11 (n ¼ 4)
12 (n ¼ 4)
4.3. 4-(1-Hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(trityltetrazol-
5-yl)phenyl]phenyl-methyl}imidazole-5-carboxylic acid (3) [11]
13 (n ¼ 6)
3474.4 ꢃ 1779.3 0.6 (0.5e1.0) 8711.5 ꢃ 2155.8
14 (n ¼ 4)
1355.2 ꢃ 235.8
398.3 ꢃ 239.0
2.8 (0.5e6.0) 4952.7 ꢃ 334.9
1.1 (0.5e3.0) 1511.4 ꢃ 482.8
15 (n ¼ 4)
a
Cmax: peak plasma concentration.
Tmax: time to reach Cmax, expressed as median (range).
AUClast: total area under the plasma concentration-time curve from time zero to
To a solution of compound 2 (10 g, 13.95 mmol) in isopropanol
(100 mL), 10% aqueous potassium hydroxide solution (15 mL) was
slowly added at room temperature. The reaction mixture was
heated at 60 ꢁC for 4 h. The organic solvent was evaporated under
reduced pressure. The residue was dissolved in water (40 mL) and
the resulting solution was washed with ethyl acetate (3 ꢂ 30 mL).
The aqueous layer was acidified using HCl and the target compound
was precipitated. The precipitate was filtered, washed with water,
and dried to give compound 3 in a pure form (8.65 g, 90%).
b
c
last measured time.
3. Conclusion
New ester prodrugs of olmesartan were designed and synthe-
sized. Their in vitro stabilities in rat plasma were examined. And the
half-lives of compounds 13 and 14 in simulated gastric juice, rat
plasma, and rat hepatic microsomes were determined. In addition,
the in vivo pharmacokinetic parameters of olmesartan after
oral administration of compounds 10e15 were evaluated and
compared with those after administration of olmesartan medox-
omil. The newly synthesized ester prodrugs 13 and 14 with cyclo-
1H NMR (300 MHz, DMSO-d6)
(m, 2H), 1.54 (s, 6H), 2.41 (t, J ¼ 6.6 Hz, 2H), 5.58 (s, 2H), 6.86 (d,
J ¼ 6.9 Hz, 8H), 7.03 (d, J ¼ 8.1 Hz, 2H), 7.27e7.40 (m, 10H),
7.43e7.45 (m, 1H), 7.51e7.63 (m, 2H), 7.76e7.78 (m, 1H).
d
0.73 (t, J ¼ 7.3 Hz, 3H), 1.44e1.49
4.4. General procedure for synthesis of compounds 4e9
hexylcarboxyethyl
and
adamantylcarboxymethyl moieties,
respectively, showed high stability in simulated gastric juice and
rapid hydrolysis in rat plasma and rat liver microsomes. Both
compounds demonstrated improved pharmacokinetic profiles
compared with olmesartan medoxomil. These new prodrugs are
proposed to be effective prodrugs of olmesartan with markedly
increased oral bioavailability.
A mixture of compound 3 (5 g, 7.259 mmol), potassium
carbonate (0.502 g, 3.63 mmol), and potassium iodide (0.3 g,
1.807 mmol) in DMAc (30 mL) was stirred at room temperature for
30 min. To the reaction mixture, a solution of the appropriate alkyl
halide (8.71 mmol) in DMAC (10 mL) was slowly added at room
temperature. The reaction mixture was heated at 70 ꢁC for 2 h. The
reaction mixture was cooled to room temperature and partitioned
between water and ethyl acetate. The organic layer was separated
and the aqueous layer was extracted with ethyl acetate (3 ꢂ 50 mL).
The combined organic extracts were washed with brine and dried
over anhydrous Na2SO4. After evaporation of the organic solvent,
the residue was purified by column chromatography (silica gel,
hexane: ethyl acetate 10:1 v/v then switching to hexane: ethyl
acetate 4:1 v/v) to give the desired product 4e9.
4. Experimental
4.1. General
All the reagents and solvents were purchased from Sigma
Aldrich Chemical Co. and used without purification. The purity of
all final compounds was over 95% on the basis of HPLC analysis. The
purity was confirmed by Waters LC-MS system: Waters 2998
photodiode array detector, Waters 3100 mass detector, Waters
SFO system fluidics organizer, Waters 2545 binary gradient
module, Waters reagent manager, Waters 2767 sample manager,
4.4.1. Octyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tritylte-
trazol-5-yl)phenyl]phenylmethyl}imidazole-5-carboxylate (4)
Yield: 72%; LC-MS m/z: 802.06 [M þ 1]þ.
SunfireÔ C18 column (4.6 ꢂ 50 mm, 5
mm particle size); solvent
gradient ¼ 95% A at 0 min, 1% A at 5 min; solvent A: 0.035% tri-
fluoroacetic acid (TFA) in water; solvent B: 0.035% TFA in MeOH;
flow rate ¼ 3.0 mL/min. The AUC was calculated using Waters
MassLynx 4.1 software. Melting points were obtained on a Walden
Precision Apparatus Electrothermal 9300 apparatus and are
uncorrected. 1H NMR and 13C NMR spectra were obtained using
a Bruker 300 MHz FT-NMR and a Bruker 400 MHz FT-NMR spec-
trometers. The animal experiments were approved by the pertinent
committees of our institutions and performed in compliance with
institutional guidelines for the conduct of animal experimentation.
4.4.2. Tetradecanoyloxymethyl 4-(1-hydroxy-1-methylethyl)-2-
propyl-1-{4-[2-(trityltetrazol-5-yl)phenyl]phenylmethyl}imidazole-
5-carboxylate (5)
Yield: 75%; LC-MS m/z: 930.00 [M þ 1]þ.
4.4.3. (1,2,3,4-Tetrahydro-2,4-dioxopyrimidin-5-yl)methyl 4-(1-
hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(trityltetrazol-5-yl)
phenyl]phenylmethyl}imidazole-5-carboxylate (6)
Yield: 68.3%; 1H NMR (300 MHz, DMSO-d6)
d
0.87 (t, J ¼ 7.4 Hz,
3H), 1.57 (q, J ¼ 7.5 Hz, 2H), 2.59 (t, J ¼ 7.5 Hz, 2H), 4.85 (s, 2H), 5.43
(d, J ¼ 6.5 Hz, 3H), 6.87e6.91 (m, 8H), 7.01 (d, J ¼ 8.1 Hz, 2H),
7.32e7.51 (m, 9H), 7.63e7.69 (m, 3H), 7.76 (d, J ¼ 7.8 Hz, 1H), 11.05
(d, J ¼ 13.0 Hz, 2H).
4.2. Ethyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-
(trityltetrazol-5-yl)phenyl]phenylmethyl}imidazole-5-carboxylate
(2) [11]
4.4.4. [1-(Cyclohexylcarbonyloxy)]ethyl 4-(1-hydroxy-1-
methylethyl)-2-propyl-1-{4-[2-(trityltetrazol-5-yl)phenyl]
phenylmethyl}imidazole-5-carboxylate (7)
A mixture of ethyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1H-
imidazole-5-carboxylate (1, 10 g, 41.61 mmol), 5-(40-bromomethyl-
biphenyl-2-yl)-1-trityl-1H-tetrazole (25.516 g, 45.77 mmol), and
anhydrous potassium carbonate (2.876 g, 20.81 mmol) in a solvent
Yield: 88.2%; 1H NMR (300 MHz, DMSO-d6)
d
0.75 (t, J ¼ 7.8 Hz,
3H), 1.15e1.27 (m, 5H), 1.47 (s, 6H), 1.54 (t, J ¼ 7.1 Hz, 6H), 1.69e1.74

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J.-S. Chang et al. / European Journal of Medicinal Chemistry 46 (2011) 3564e3569
(m, 2H), 2.18e2.21 (m, 2H), 2.45 (t, J ¼ 7.8 Hz, 2H), 4.25e4.29 (m,
1H), 5.17 (s, 1H), 5.39 (s, 2H), 6.85 (q, J ¼ 10.9 Hz, 1H), 6.85e6.90 (m,
8H), 7.07 (d, J ¼ 4.1 Hz, 2H), 7.29e7.43 (m, 9H), 7.51 (t, J ¼ 7.4 Hz,
1H), 7.63 (t, J ¼ 7.4 Hz, 2H), 7.75 (d, J ¼ 7.5 Hz, 1H).
(d, J ¼ 8.1 Hz, 2H), 7.56 (t, J ¼ 7.5 Hz, 2H), 7.63e7.69 (m, 2H), 11.07 (d,
J ¼ 13.1 Hz, 2H); 13C NMR (100 MHz, DMSO-d6)
d 14.1, 21.0, 28.7,
30.3, 48.3, 61.9, 70.4, 99.3, 116.8, 124.1, 126.0, 128.2, 129.5, 131.0,
131.1, 131.4, 137.0, 138.7, 141.5, 150.2, 151.5, 151.7, 155.6, 157.7, 161.1,
164.4.
4.4.5. (1-Adamantyl)carbonyloxymethyl 4-(1-hydroxy-1-
methylethyl)-2-propyl-1-{4-[2-(trityltetrazol-5-yl)phenyl]
phenylmethyl}imidazole-5-carboxylate (8)
4.5.4. [1-(Cyclohexylcarbonyloxy)]ethyl 4-(1-hydroxy-1-
methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)phenyl]phenylmethyl}
imidazole-5-carboxylate (13)
Yield: 95.4%; 1H NMR (300 MHz, DMSO-d6)
d
0.74 (t, J ¼ 7.8 Hz,
3H), 1.44 (s, 6H), 1.46e1.70 (m, 14H), 1.87 (s, 3H), 2.45 (t, J ¼ 7.8 Hz,
2H), 4.91 (s, 1H), 5.33 (s, 2H), 5.75 (s, 2H), 6.78e6.85 (m, 8H), 7.05
(d, J ¼ 4.1 Hz, 2H), 7.27e7.41 (m, 9H), 7.52 (t, J ¼ 7.5 Hz, 1H), 7.62 (t,
J ¼ 7.5 Hz, 2H), 7.73 (d, J ¼ 7.3 Hz, 1H).
Yield: 90.9%; mp: 108e110 ꢁC; 1H NMR (300 MHz, DMSO-d6)
d
0.87 (t, J ¼ 7.4 Hz, 3H), 1.11e1.32 (m, 8H), 1.48 (s, 6H), 1.58
(t, J ¼ 7.3 Hz, 4H), 1.68e1.75 (m, 2H), 2.21e2.28 (m, 1H), 2.59
(t, J ¼ 7.4 Hz, 2H), 5.16 (s, 1H), 5.43 (d, J ¼ 5.8 Hz, 2H), 6.86 (q,
J ¼ 10.9 Hz, 1H), 6.91 (d, J ¼ 8.0 Hz, 2H), 7.09 (d, J ¼ 8.0 Hz, 2H),
4.4.6. [1-(Benzoyloxy)]ethyl 4-(1-hydroxy-1-methylethyl)-2-
propyl-1-{4-[2-(trityltetrazol-5-yl)phenyl]phenylmethyl}imidazole-
5-carboxylate (9)
7.50e7.70 (m, 4H); 13C NMR (75 MHz, DMSO-d6)
d 14.1, 19.4, 20.9,
24.9, 25.0, 25.6, 28.5, 28.8, 30.1, 30.2, 42.2, 48.4, 70.1, 89.1, 116.4,
124.1, 126.1, 128.2, 129.5, 131.1, 131.4, 136.9, 138.7, 141.5, 151.7, 158.4,
159.8, 173.5; LC-MS m/z: 602.22 [M þ 1]þ.
Yield: 88.9%; 1H NMR (300 MHz, DMSO-d6)
d
0.74 (t, J ¼ 7.8 Hz,
3H), 1.44e1.46 (m, 9H), 1.49 (q, J ¼ 7.6 Hz, 2H), 2.44 (t, J ¼ 7.8 Hz,
2H), 5.15 (s, 1H), 5.36 (s, 2H), 6.88 (d, J ¼ 7.9 Hz, 1H), 6.92e7.01 (m,
8H), 7.28e7.41 (m, 9H), 7.65e7.70 (m, 6H), 7.81 (d, J ¼ 7.4 Hz, 2H),
7.90 (d, J ¼ 7.4 Hz, 2H).
4.5.5. (1-Adamantyl)carbonyloxymethyl 4-(1-hydroxy-1-
methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)phenyl]phenylmethyl}
imidazole-5-carboxylate (14)
Yield: 92.3%; mp: 119e122 ꢁC; 1H NMR (300 MHz, DMSO-d6)
4.5. General procedure for synthesis of the target compounds
10e15
d
0.86 (t, J ¼ 7.3 Hz, 3H), 1.47 (s, 6H), 1.52e1.70 (m, 14H), 1.90 (s, 3H),
2.57 (t, J ¼ 7.5 Hz, 2H), 5.03 (s, 1H), 5.44 (s, 2H), 5.82 (s, 2H), 6.91 (d,
J ¼ 8.2 Hz, 2H), 7.06 (d, J ¼ 8.1 Hz, 2H), 7.49e7.69 (m, 4H); 13C NMR
To a solution of the trityl compound 4e9 (6.25 mmol) in acetone
(30 mL), conc. HCl (30 mL) and water (20 mL) were added. The
reaction mixture was stirred at room temperature for 2 h. The
organic solvent was evaporated under reduced pressure and the pH
of the remained aqueous solution was adjusted to 4e5 by addition
of aqueous potassium carbonate solution. The aqueous mixture was
extracted with ethyl acetate (3 ꢂ 50 mL) and the combined organic
extracts were washed with brine and dried over anhydrous Na2SO4.
After evaporation of the organic solvent, the residue was purified
by column chromatography (silica gel, hexane: ethyl acetate 5:1 v/v
then switching to ethyl acetate) to give the target product 10e15.
(100 MHz, DMSO-d6) d 14.1, 20.9, 27.6, 28.7, 30.1, 36.2, 38.3, 48.2, 70.1,
79.9, 116.2, 124.0, 126.3, 128.3, 129.6, 130.9, 131.1, 131.4, 136.9, 138.7,
141.5, 151.8, 155.5, 158.3, 160.0, 175.7; LC-MS m/z: 640.00 [M þ 1]þ.
4.5.6. [1-(Benzoyloxy)]ethyl 4-(1-hydroxy-1-methylethyl)-2-propyl-
1-{4-[2-(tetrazol-5-yl)phenyl]phenylmethyl}imidazole-5-
carboxylate (15)
Yield: 91.9%; mp 98e101 ꢁC; 1H NMR (300 MHz, DMSO-d6)
0.87 (t, J ¼ 7.4 Hz, 3H), 1.46e1.48 (m, 9H), 1.58 (q, J ¼ 7.4 Hz, 2H),
d
2.59 (t, J ¼ 7.5 Hz, 2H), 5.17 (s, 1H), 5.42 (d, J ¼ 4.5 Hz, 2H), 6.88
(d, J ¼ 8.0 Hz, 2H), 7.01 (d, J ¼ 7.9 Hz, 2H), 7.10 (q, J ¼ 7.6 Hz,1H), 7.42
(d, J ¼ 7.5 Hz, 1H), 7.49 (t, J ¼ 7.6 Hz, 2H), 7.54e7.66 (m, 4H), 7.89 (d,
4.5.1. Octyl 4-(1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-
5-yl)phenyl]phenyl-methyl}imidazole-5-carboxylate (10)
J ¼ 7.9 Hz, 2H); 13C NMR (75 MHz, DMSO-d6)
d 14.1, 19.6, 20.9, 28.7,
30.1, 48.4, 70.1, 89.9, 116.5, 126.0, 128.2, 129.0, 129.3, 129.5, 129.9,
131.0, 131.4, 134.4, 136.9, 138.7, 141.5, 151.7, 158.5, 159.8, 164.3; LC-
MS m/z: 595.50 [M þ 1]þ.
Yield: 91%; mp: 209e212 ꢁC (dec.); 1H NMR (400 MHz, DMSO-
d6)
d
0.81 (t, J ¼ 6.1 Hz, 3H), 0.91 (t, J ¼ 7.3 Hz, 3H), 1.17 (brs, 10H),
1.49 (brs, 7H), 1.63 (q, J ¼ 7.4 Hz, 2H), 2.60 (t, J ¼ 7.5 Hz, 2H), 3.99
(brs, 2H), 4.13 (t, J ¼ 6.3 Hz, 2H), 5.43 (brs, 2H), 6.80 (d, J ¼ 7.8 Hz,
2H), 7.09 (d, J ¼ 7.9 Hz, 2H), 7.31 (d, J ¼ 6.9 Hz, 1H), 7.35e7.41 (m,
4.6. Stability studies of prodrugs
2H), 7.55 (d, J ¼ 7.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d
14.1,
Stabilities of the newly synthesized ester prodrugs and olme-
sartan medoxomil were determined in rat plasma. To determine
14.4, 21.0, 22.5, 25.8, 28.3, 28.8, 29.0, 30.2, 31.7, 48.5, 65.4, 70.1,
117.3, 125.2, 127.3, 128.0, 128.2, 129.7, 130.5, 131.0, 131.7, 135.9, 140.5,
140.9, 151.1, 157.5, 160.5, 162.2.
their plasma stability, 50
mg/mL compound in rat plasma was
incubated for 30 min at 37 ꢁC. After that, the samples were analyzed
immediately by LC-MS/MS.
4.5.2. Tetradecanoyloxymethyl 4-(1-hydroxy-1-methylethyl)-2-
propyl-1-{4-[2-(tetrazol-5-yl)phenyl]phenylmethyl}imidazole-5-
carboxylate (11)
In addition, the half-lives of compounds 13, 14, and olmesartan
medoxomil were measured in simulated gastric juice, rat plasma,
and rat liver microsomes. To determine the microsomal stability of
prodrugs, prodrugs were incubated with rat liver microsomes in
the presence of NADPH. The microsomal reaction mixtures were
containing 1.2 mM NADPH, 0.5 mg/mL (total protein) microsomes,
100 mM phosphate buffer (pH 7.4). Solutions of the prodrugs in
Yield: 92%; 1H NMR (300 MHz, DMSO-d6)
d 0.82e0.89 (m, 6H),
1.16e1.22 (m, 22H), 1.47 (brs, 6H), 1.53e1.61 (m, 2H), 2.25 (t,
J ¼ 7.3 Hz, 2H), 2.58 (t, J ¼ 7.4 Hz, 2H), 5.05 (brs, 1H), 5.44 (brs, 2H),
5.80 (brs, 2H), 6.91 (d, J ¼ 8.0 Hz, 2H), 7.06 (d, J ¼ 8.0 Hz, 2H),
7.50e7.63 (m, 2H), 7.67 (d, J ¼ 7.4 Hz, 2H).
acetonitrile (100
plasma, or rat liver microsomes reaction mixture with a final
concentration of 1
M. The reaction solutions were kept at 37 ꢁC
and sampled at 0, 15, 30, 60, and 120 min. The 50 L aliquot of the
mM) were added to simulated gastric juice, rat
4.5.3. (1,2,3,4-Tetrahydro-2,4-dioxopyrimidin-5-yl)methyl 4-(1-
hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)phenyl]
phenylmethyl}imidazole-5-carboxylate (12)
m
m
mixtures was terminated at the above time points by addition of
two-fold volume of cold acetonitrile containing internal standard.
After centrifugation, the supernatant was collected and analyzed
immediately by LC-MS/MS.
Yield: 93.7%; mp: 210e213 ꢁC (dec.); 1H NMR (300 MHz, DMSO-
d6)
d
0.87 (t, J ¼ 7.4 Hz, 3H), 1.57 (q, J ¼ 7.5 Hz, 2H), 2.59 (t, J ¼ 7.5 Hz,
2H), 4.85 (s, 2H), 5.43 (d, J ¼ 6.5 Hz, 3H), 6.87 (d, J ¼ 8.2 Hz, 2H), 7.02

J.-S. Chang et al. / European Journal of Medicinal Chemistry 46 (2011) 3564e3569
3569
4.7. Pharmacokinetic studies
Milford, MA) at a flow rate of 0.25 mL/min. The lower limit of
quantitation of olmesartan in rat plasma was 5 ng/mL.
Each compound was administered orally using a feeding tube to
male SpragueeDawley rats at a dose of 20 mg/kg as olmesartan.
Blood samples were collected via carotid artery at 0 (to serve as
a control), 0.5, 1, 2, 3, 4, 6, 8, and 10 h after oral administration
of each compound. After centrifugation at 3000 rpm for 10 min,
Acknowledgments
This work was supported by Seoul R&BD program (grant
number PA100015). We would like to thank CTCBIO Inc., Republic of
Korea, for kind contribution to the biological experiments.
a 200-
m
L aliquot of plasma samples were stored at ꢄ80 ꢁC until
analysis. Pharmacokinetic parameters were determined by
a
non-compartmental analysis using WinNonlinÒ (Pharsight
References
Corporation, Mountain View, CA) program. The AUClast values were
calculated by the trapezoidal rule-extrapolation method.
[1] A. Ferro, R. Gilbert, H. Krum, Int. J. Clin. Pract. 60 (2006) 577e581.
[2] M. Fukuda, T. Yamanaka, M. Mizuno, M. Motokawa, Y. Shirasawa, S. Miyagi,
T. Nishio, A. Yoshida, G. Kimura, J. Hypertens. 26 (2008) 583e588.
[3] M.A. Weber, Am. J. Hypertens. 12 (1999) 189Se194S.
4.8. Quantitative analysis
[4] T. Unger, Am. Heart J. 139 (2000) S2eS8.
[5] A.M. Bell, D. Nykamp, Therapeutics 1 (2009) 1e9.
[6] D.E. Mire,T.N.Silfani,M.K. Pugsley,J.Cardiovasc,Pharmacology46(2005)585e593.
[7] J. Agata, N. Ura, H. Yoshida, Y. Shinshi, H. Sasaki, M. Hyakkoku, S. Taniguchi,
K. Shimamoto, Hypertens. Res. 29 (2006) 865e874.
[8] H. Yanagisawa, Y. Amemiya, T. Kanazaki, Y. Shimoji, K. Fujimoto, Y. Kitahara,
T. Sada, M. Mizuno, M. Ikeda, S. Miyamoto, Y. Furukawa, H. Koike, J. Med.
Chem. 39 (1996) 323e338.
Concentrations of olmesartan in the plasma samples were
analyzed using LC-MS/MS. To 200-
150 L of methanol containing internal standard (telmisartan) and
0.1 N HCl 200 L was added. After mixing, olmesartan was extracted
mL aliquot of plasma sample,
m
m
by liquid extraction using diethyl ether: dichloromethane (3:2 v/v),
and the upper layer was transferred and dried under nitrogen
evaporation. The LC-MS/MS system consisted of a HP1100Ò HPLC
system (Agilent, Santa Clara, CA) and API3200Ò triple-quadrupole
mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada).
The HPLC mobile phases consisted of 0.1% formic acid in acetonitrile
and 0.1% formic acid in 10 mM ammonium acetate (85:15 v/v).
Chromatographic separation was achieved on a reversed-phase
[9] S.-F. Ma, M. Anraku, Y. Iwao, K. Yamasaki, U. Kragh-Hansen, N. Yamaotsu,
S. Hirono, T. Ikeda, M. Otagiri, Drug Metab. Dispos. 33 (2005) 1911e1919.
[10] J.-H. Park, J.-S. Chang, M.I. El-Gamal, W.-K. Choi, W.S. Lee, H.J. Chung, H.-I. Kim,
Y.-J. Cho, B.S. Lee, H.-R. Jeon, Y.S. Lee, Y.W. Choi, J. Lee, C.-H. Oh, Bioorg. Med.
Chem. Lett. 20 (2010) 5895e5899.
[11] G.S. Ramanjaneyulu, B. Mohan, P.C. Ray, M.K. Sethi, V.S. Rawat, Y.R. Krishna,
V. Lakshminarayana, M. Srinivas, PCT Int. Appl. (December 27, 2007) WO
2007/148344 A2.
[12] B.S. Lee, M.J. Kang, W.S. Choi, Y.B. Choi, H.S. Kim, S.K. Lee, J. Lee, Y.W. Choi,
Arch. Pharm. Res. 32 (2009) 1629e1635.
[13] K. Yeh, K. Kwan, J. Pharmacokinet. Biopharm. 6 (1978) 79e98.
XterraÒ C18 column (50 ꢂ 2.1 mm, 3.5
mm, Waters Corporation,