Synthesis of related substances of cilostazol

Article abstract of DOI:10.3987/COM-08-11529

The impurities in API of cilostazol were detected by LC/MS during the process development. The structures of two impurities 6 and 7 and the related formation mechanisms were proposed. Synthesis of 6 and 7 was conducted for confirmation of the speculated structures.

Full text of DOI:10.3987/COM-08-11529

HETEROCYCLES, Vol. 78, No. 1, 2009
189
HETEROCYCLES, Vol. 78, No. 1, 2009, pp. 189 - 195. © The Japan Institute of Heterocyclic Chemistry
Received, 19th August, 2008, Accepted, 18th September, 2008, Published online, 22nd September, 2008.
DOI: 10.3987/COM-08-11529
SYNTHESIS OF RELATED SUBSTANCES OF CILOSTAZOL
Jin Zheng,† Zheng Liu,† Yiru Dai,‡ Qingjie Zhao,† and Jingshan Shen*
†
† Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, P.R.China
‡ Topharman Shanghai Co., Ltd., Shanghai 201209, P.R. China
*E-mail: jsshen@mail.shcnc.ac.cn
Abstract – The impurities in API of cilostazol were detected by LC/MS during
the process development. The structures of two impurities 6 and 7 and the related
formation mechanisms were proposed. Synthesis of 6 and 7 was conducted for
confirmation of the speculated structures.
INTRODUCTION
As a potent and reversible phosphodiesterase III inhibitor, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-
3,4-dihydro-2(1H)-quinolinone (1, cilostazol), is used in clinic for the treatment of intermittent
claudication and the control of its impurities is important to clinical safety.1 The two key steps for the
preparation of cilostazol 1 (Scheme 1) involved Friedel-Crafts intramolecular cyclization2 of
3-chloro-N-(4-methoxyphenyl)propanamide 2 to give the corresponding 3, 4-dihydrocarbostyril 3, which
was then condensed with 5-(4-chlorobutyl)-1-cyclohexyl-1H-tetrazole 4 in the presence of potassium
hydroxide in DMF to give cilostazol 1.3 Besides known impurities, we detected two unreported impurities
in the final crude product during our process development of cilostazol 1 (Scheme 1).
Cl
OMe
OH
150oC
AlCl3
O
N
O
N
H
H
O
N
2
3
KOH
DMF
N
N N
O
N
H
1, Cilostazol
N
Cl
N
N N
4
Scheme 1

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HETEROCYCLES, Vol. 78, No. 1, 2009
RESULTS AND DISCUSSION
Four major impurities detected by LC-MS were shown in Table 1. By indication of molecular ion peak
and possible formation mechanisms, structures of unknown impurities were suggested. Compound 55
(M+161) was a known impurity of cilostazol, resulting from the trace amount of 5-(4,4-dichlorobutyl)-1-
cyclohexyl-1H-tetrazole in the starting material 4. The unknown impurity 6 (M-2), derived from 9, was
identical to 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-1H-quinolin-2-one (OPC-13015, the known
active metabolite of cilostazol). The impurity 7 (M+220) was formed by the reaction of compound 11
with two equivalents of 4. Another known impurity 8 (M+206) was formed due to double linking of
compound 3 with two equivalents of 4 in the alkaline condition.
Table 1. Structure and LC/MS analysis of cilostazol and its related impurities
Compound
RRT
0.85
Area (%)
0.12
ESI (MH)+
Structure a
O
O
5
531.4
R
O
N
N
O
H
H
OCH2R
OCH2R
OCH2R
6
1
0.97
1.00
0.51
368.3
370.2
O
O
O
N
H
97.02
N
H
7
8
1.53
1.57
0.35
0.50
590.3
576.5
N
OCH2R
OCH2R
O
N
CH2R
a) R represents (1-cyclohexyl-1H-tetrazol-5-yl)propyl
In the Friedel-Crafts reaction of preparing 3, the possible coordination of the carbonyl with AlCl3 led to
the conversion of the amide group on 3, from an electron donating substituent into an electron
withdrawing group, and reduced the activity of the aromatic ring.4 Excess of AlCl3 (3 equivalents) and
high temperature (150 °C) were required to improve the yield of 3.2
While it remains unclear how 9 was formed during the Friedel-Crafts reaction. Two pathways can be
postulated as the formation mechanisms of 9. Dehydrogenation of 3,4-dicarbostyril was well-documented
in the literatures.7 Lewis acids such as AlCl3 and SbCl5 sometimes were used as the powerful

HETEROCYCLES, Vol. 78, No. 1, 2009
191
dehydrogenating agents.8-10 One possible mechanism was AlCl3-mediated dehydrogenation of 3 (Scheme
2). The other possible mechanism was plausible via the auto-oxidation of 3 at high temperature due to the
presence of adventitious O2 or other oxidants (Scheme 3).
HAlCl3
H
H
H
H
HO
HO
HO
H
-H2
AlCl3
AlCl3
OH
N
O
N
H
O
H
N
AlCl3
3
HO
HO
-H+
-AlCl3, +H+
O
AlCl3
N
N
H
O
9
Scheme 2
HO
HO
[O]
-H2O
N
O
O
N
H
OH
3
HO
HO
H
O
HO
O
N
N
OH
N
H
9
Scheme 3
The LC-MS analysis showed that no N-Oxide existed in the starting material of 4-methyoxyaniline. The
literatures11 indicated that direct oxidation of 2-quinolinone by m-CPBA at room temperature led to 1%
hydroxamic acid derivative. We postulated that N-O bond of 11 was formed by auto-oxidation of 9.
Coordination of the amide carbonyl with Lewis acid leads to a polarized complex 10. The polarization
facilitated the oxidation on N atom of quinolinol segment, by radical oxygen, to hydroxamic acid 11
(Scheme 4).
HO
HO
HO
AlCl3
δ
AlCl3
N
H
O
O
H
AlCl3
N
N
O
H
δ
9
10
HO
HO
-AlCl3
[O]
AlCl3
O
H
N
O
O
N
OH
11
Scheme 4

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Based on the above speculation, compounds 6 and 7 were synthesized. Compound 6 was conveniently
obtained by reaction of 6-hydroxy-1H-quinolin-2-one 912 with 4 (Scheme 5).
RO
HO
N N
80 oC, 7 h
N
CH2
N
N
H
O
R=
N
H
O
4, K2CO3, DMF
6
9
Scheme 5
Attempts13 to synthesis of hydroxamic acid structure 11 were achieved by oxidation of quinoline, with
H2O2 in acetic acid, to its N-oxide and further oxidation of the resulted N-oxide with lead tetraacetate
(Scheme 6). Though the oxidation of 12 with H2O214 in acetic acid led to 6-hydroxyl-1-oxyquinoline in
good yield, the following oxidation of the resulted oxyquinoline 14 (R=H) with lead tetraacetate 15 did not
give satisfactory result according to the literature procedure, due to the presence of the unprotected
hydroxyl group on 14 (R=H) . We then modified the strategy, and firstly protected the hydroxyl group on
6-quinolinol by using 4. The resulted 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]quinoline 13 was
oxidized with m-CPBA16 in chloroform at 0 °C to give 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-1-
oxyquinoline 14 in good yield. 6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-1-hydroxyquinolin-2-one 15
was subsequently obtained by oxidation of the oxy-quinoline derivative 14 with lead tetraacetate15 in
presence of CaCO3 in refluxing benzene. Following the procedure similar to preparation of 6, the target
compound 7 was obtained in 75% yield by the treatment of 15 with 4 in the presence of potassium
carbonate.
RO
HO
RO
80 oC, 7 h
o
0 C, 14 h
+
N
4, K2CO3, DMF
N
m-CPBA, CHCl3
N
O-
13
12
14
N N
RO
RO
N
CH2
o
benzene, reflux, 6 h
Pb(OAc)4, CaCO3
80 C, 7 h
N
R=
N
O
N
O
4, K2CO3, DMF
OR
OH
15
7
Scheme 6
Finally, the LC-MS analysis showed that compound 6 and 7 respectively had the same molecular ion
peak and relative retention time as the unknown byproducts. Thus, the two unknown byproducts were
identical with the speculated structures.

HETEROCYCLES, Vol. 78, No. 1, 2009
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EXPERIMENTAL
Melting points were determined on a WRR melting point apparatus and are uncorrected. NMR spectra
were determined using a Mercury 300 MHz, FT NMR spectrometer. Elemental analyses were performed
on Elementar Vario EL. Reaction solvents were purchased without further purification. LC-MS analysis
was carried out with Perkin-Elmer triple quadrupole mass spectrometer coupled with Agilent 1100 with
VWD UV-vis detector. The ion spray voltage of LC-MS spectrum was set at 4.5 kv and temperature was
kept at 500 °C and spectra data were acquired from m/z 60 to 800. A YMC C8, 250 mm×4.6 mm 5 μm
particle diameter column, and water-acetonitril (60:40, v: v) and acetonitril as gradient eluents were used
for the separation analysis. Detection was carried out at 254 nm and flow rate was maintained at 1.0
mL/min.
6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-1H-quinolin-2-one (6)
A mixture of 6-hydroxy-1H-quinolin-2-one 9 (207 mmol, 33 g), 5-(4-chlorobutyl)-1-cyclohexyl-1H-
tetrazole 4 (228 mmol, 55 g) and potassium carbonate (414 mmol, 57 g) in DMF (300 mL) was stirred at
80 °C under nitrogen for 7 h. The reaction mixture was cooled to 25 °C and diluted with water (1.5 L),
resulting in the formation of a thick brown precipitate. The precipitate was isolated by filtration, washed
with H2O (3×100 mL), and recrystallized from EtOAc to give 6 (64 g, 85%) as off-white solid, mp
181-183 °C (lit.,17 177.5-178.5 °C). 1H NMR (DMSO-d6): δ 1.14-2.02 (m, 14H), 2.98 (t, 2H, J = 6.3 Hz),
4.04 (t, 2H, J = 6.0 Hz), 4.40 (m, 1H), 6.48 (d, 1H, J = 9.8 Hz), 7.10-7.27 (m, 3H), 7.82 (d, 1H, J = 9.4
13
Hz), 11.63 (s, 1H). C NMR (CDCl3): δ 22.93, 23.82, 24.70, 25.22, 28.36, 32.81, 57.53, 67.56, 109.37,
114.71, 119.52, 120.60, 121.17, 131.49, 136.90, 154.61, 156.94. ESI m/z 368.2 (M+ + H). Anal. Calcd for
C20H25N5O2: C, 65.37; H, 6.86; N, 19.06. Found: C, 65.29; H, 6.99; N, 18.82.
6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]quinoline (13)
13 was obtained in 80% yield as a white solid by the same procedure as described for 6, mp 106-108 °C.
1H NMR (DMSO-d6): δ 1.20-2.16 (m, 14H), 2.95 (t, 2H, J = 7.4 Hz), 4.14 (t, 2H, J = 6.0 Hz), 4.05-4.20
(m, 1H), 7.06 (d, 1H, J = 2.9 Hz), 7.33 (dd, 1H, J1 = 6.3 Hz, J 2 = 2.9 Hz), 7.36 (dd, 1H, J1 = 6.3 Hz, J2 =
4.1 Hz), 8.01 (d, 1H, J = 9.3 Hz), 8.05 (dd, 1H, J1 = 8.4 Hz, J2 = 1.5 Hz), 8.76 (dd, 1H, J1 = 4.1 Hz, J2 =
13
1.5 Hz). C NMR (CDCl3): δ 22.89, 23.86, 24.65, 25.16, 28.27, 32.77, 57.46, 67.24, 105.73, 121.31,
122.22, 129.17, 130.73, 134.71, 144.22, 147.87, 153.40, 156.71. ESI m/z 352.2 (M+ + H). Anal. Calcd for
C20H25N5O: C, 68.35; H, 7.17; N, 19.93. Found: C, 68.33; H, 7.18; N, 20.22.
6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-1-oxyquinoline (14)
To a stirred solution of 13 (35.0 g, 0.1 mol) in CHCl3 (80 mL) was added dropwise a solution of m-CPBA

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HETEROCYCLES, Vol. 78, No. 1, 2009
(17.3 g, 0.1 mol) in CHCl3 (40 mL) at 0 °C for 1.5 h. After further stirred for 4 h, the reaction mixture
was washed sequently with 10% aqueous sodium sulfite, saturated aqueous sodium bicarbonate solution
and water. After removal of the chloroform in vacuo, the crude product was crystallized from EtOAc to
give the pure product 14 (238 g, 65%) as a white solid, mp 175-176 °C. 1H NMR δ 1.28-2.15 (m, 14H),
2.94 (t, 2H, J = 7.4 Hz), 4.14 (t, 2H, J = 5.8 Hz), 4.04-4.20 (m, 1H), 7.10 (d, 1H, J = 2.6 Hz), 7.25 (dd,
1H, J1 = 8.6 Hz, J2 = 6.3 Hz), 7.34 (dd, 1H, J1 = 9.3 Hz, J1 = 2.6 Hz), 7.62 (d, 1H, J = 8.6 Hz), 8.38 (d, 1H,
13
J = 6.3 Hz), 8.65 (d, 1H, J = 9.3 Hz). C NMR (DMSO-d6): δ 21.80, 23.17, 24.59, 27.79, 32.45, 56.06,
67.61, 107.32, 120.61, 122.34, 122.38, 124.06, 131.86, 133.29, 136.38, 154.08, 158.06. ESI m/z 368.3
(M+ + H). Anal. Calcd for C20H25N5O2: C, 65.37; H, 6.86; N, 19.06. Found: C, 65.37; H, 6.89; N, 19.10.
6-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)butoxy]-1-hydroxyquinolin-2-one (15)
Lead tetraacetate (15.0 g, wet with acetic acid) and calcium carbonate (0.8 g) were added portionwise to a
solution of quinoline N-oxide 14 (5.5 g, 1.7 mmol) in benzene (150 mL) at 0 °C for 10 min. The mixture
was heated at reflux for 6 h. After cooling, the reaction mixture was filtered to remove inorganic salts.
The filtrate was concentrated in vacuo to dryness. 3% Dilute HCl (50 mL) was added to the residue, and
the mixture heated to 80 °C for 2 h, resulting in the formation of a brown solid. After filtration, the crude
product was recrystallized from EtOAc/CHCl3 to yield 15 (4.1 g, 72%) as a white solid, mp 135-137 °C.
1H NMR (CDCl3): δ 1.22-2.15 (m, 14H), 2.94 (t, 2H, J = 7.5 Hz), 4.08 (t, 2H, J = 6.0 Hz), 4.13 (m, 1H),
6.84 (d, 1H, J = 9.3 Hz), 7.04 (d, 1H, J = 2.7 Hz), 7.34 (dd, 1H, J1 = 9.3 Hz, J2 = 2.7 Hz), 7.68 (d, 1H, J =
13
9.6 Hz), 7.76 (d, 1H, J = 9.6 Hz). C NMR (CDCl3): δ 22.93, 23.82, 24.70, 25.22, 28.36, 32.81, 57.53,
67.56, 109.37, 114.71, 119.52, 120.60, 121.17, 131.49, 136.90, 154.61, 156.94. ESI m/z 384.3 (M+ + H).
Anal. Calcd for C20H25N5O3: C, 62.65; H, 6.57; N, 18.26. Found: C, 62.62; H, 6.61; N, 18.31.
1,6-Bis[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-1H-quinolin-2-one (7)
A mixture of 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-1-hydroxyquinolin-2-one 15 (4 g 10.4 mmol),
5-(4-chlorobutyl)-1-cyclohexyl- 1H-tetrazole 4 (2.8 g, 11.5 mmol) and potassium carbonate (2.9 g, 20.8
mmol) in DMF (25 mL) was stirred at 80 °C under nitrogen for 7 h. The reaction mixture was cooled to
25 °C and diluted with water (200 mL), resulting in the formation of a brown precipitate. The precipitate
was isolated by filtration, washed with H2O (3×50 mL), and recrystallized from EtOAc to give 7 (4.6 g,
1
75%) as a white solid, mp 98-99 °C. H NMR (CDCl3): δ 1.20-2.23 (m, 28H), 2.93 (t, 2H, J = 7.5 Hz),
3.11 (t, 2H, J = 7.5 Hz), 4.06 (t, 2H, J = 6.0 Hz), 4.13 (m, 1H), 4.29 (t, 2H, J = 6.0 Hz), 4.34 (m, 1H),
6.73 (d, 1H, J = 9.3 Hz), 7.01 (d, 1H, J = 2.7 Hz), 7.19 (dd, 1H, J1 = 9.3 Hz, J2 = 2.7 Hz), 7.47 (d, 1H, J =
9.6 Hz), 7.60 (d, 1H, J = 9.6 Hz). 13C NMR (DMSO-d6): δ 21.67, 21.83, 24.59, 26.69, 27.94, 32.46, 32.51,
55.98, 56.06, 67.48, 74.03, 111.15, 112.91, 120.39, 122.66, 132.57, 138.23, 154.09, 156.32. ESI m/z

HETEROCYCLES, Vol. 78, No. 1, 2009
195
590.3 (M+ + H). Anal. Calcd for C31H43N9O3: C, 63.14; H, 7.35; N, 21.38. Found: C, 63.30; H, 7.33; N,
21.61.
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