Novel diphenylalkyl piperazine derivatives with dual calcium antagonistic and antioxidative activities

Article abstract of DOI:10.1016/S0960-894X(02)00322-0

Two types of novel diphenylalkyl piperazine derivatives containing the thio or aminopropanol moiety substituted by phenyl or benzyl group were synthesized, and evaluated for their calcium antagonistic and antioxidative activities. These compounds showed apparent inhibitions against KCl-induced contractions in isolated rat aorta. Among them, phenylamino compound 9 and benzylamino compound 13 also possessed potent inhibitory activities against auto-oxidative lipid peroxidations in canine brain homogenates. Two representative compounds 3a and 9 were evaluated for their inhibitory activities against KCl-induced contractions in isolated canine arteries (basilar, coronary, mesenteric, and renal). Both compounds showed the most potent inhibitions to basilar artery.

Full text of DOI:10.1016/S0960-894X(02)00322-0

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1947–1950
Novel Diphenylalkyl Piperazine Derivatives with Dual Calcium
Antagonistic and Antioxidative Activities
Makoto Kimura,* Tomoko Masuda, Koji Yamada, Nobuo Kubota,
Nobuyuki Kawakatsu, Masaki Mitani, Kenichi Kishii, Masato Inazuy and
Takayuki Namiki*
Pharmaceutical R & D Laboratories, POLA Chemical Industries Inc., 560, Kashio-cho, Totsuka-ku, Yokohama,
Kanagawa, 244-0812, Japan
Received 30 January 2002; accepted 6 May 2002
Abstract—Two types of novel diphenylalkyl piperazine derivatives containing the thio or aminopropanol moiety substituted by
phenyl or benzyl group were synthesized, and evaluated for their calcium antagonistic and antioxidative activities. These com-
pounds showed apparent inhibitions against KCl-induced contractions in isolated rat aorta. Among them, phenylamino compound
9 and benzylamino compound 13 also possessed potent inhibitory activities against auto-oxidative lipid peroxidations in canine
brain homogenates. Two representative compounds 3a and 9 were evaluated for their inhibitory activities against KCl-induced
contractions in isolated canine arteries (basilar, coronary, mesenteric, and renal). Both compounds showed the most potent inhi-
bitions to basilar artery. # 2002 Elsevier Science Ltd. All rights reserved.
In spite of the therapeutic progress for cerebrovascular
disease, stroke is one of the major causes of death in the
developed countries, and has presented both health and
economic problems. Until now, thrombolytic reagents1
and neuroprotectants such as calcium antagonists,2,3
NMDA receptor antagonists,4 AMPA receptor antago-
nists,5 5-HT1A agonists,6 and antioxidants7 have been
used or developed in clinical trials for the treatment of
patients with cerebral ischemia and traumatic injury,
but their therapeutic estimations have not been estab-
lished as yet.8 The priority for the treatment of cerebral
ischemia and traumatic injury is an improvement of
lowered blood flow in ischemic and penumbra regions.
However, after reperfusion of them, the destruction of
the cell membrane and the death of nerve cells occur.9
These are caused by a pathological increase of intra-
increase in the generation of reactive oxygen species to
cause irreversible damage to cell membrane through
extensive lipid peroxidation.11 The consequences of
these enzyme perturbations and the production of reac-
tive oxygen species induce widespread dysfunctions to
lead to the death of nerve cells, including disruptions of
neuronal membrane and cytoskeletal integrity, and
damage to mitochondrial function.
Recently, it has been demonstrated that the use of a
neuroprotectant acting on multiple target molecules at
the same time or a combination therapy with neuro-
protectant having different mechanisms of action for
preventing cell death exhibits an obvious therapeutic
effect in ischemic animal models.12,13 For these findings,
the neuroprotectant with multiple mechanisms of action
is expected to provide a possibility of exhibiting the
clinically significant efficacy. In pathological events of
ischemic cascade described above, we took notice of
calcium antagonistic and antioxidative activities to find
a neuroprotectant having dual actions for the treatment
of cerebral ischemia and traumatic injury. In this paper,
we will describe the synthesis of two types of novel
diphenylalkyl piperazine derivatives containing the thio
and aminopropanol moiety substituted by phenyl or
benzyl group, and their evaluation for calcium
antagonistic and antioxidative activities.
cellular Ca2+ concentration due to
a significant
diminution of cellular energy reserves, following the
failure of intracellular energy-dependent ion homeo-
stasis.10 The calcium overloading activates a large num-
ber of intracellular enzymes, which induce a marked
*Corresponding authors. Tel.: +81-45-826-7264; fax: +81-45-826-
7249; e-mail: m-kimura@pola.co.jp (M. Kimura); t-namiki@pola.co.
jp (T. Namiki).
yPresent address: Department of Pharmacology, and Intractable Dis-
ease Research Center, Tokyo Medical University, 6-1-1 Shinjyuku,
Shinjyuku-ku, Tokyo, 160-8402, Japan.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00322-0

1948
M. Kimura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1947–1950
as a base in DMF, followed by ring opening of the
resulting epoxides 7a–c with 1, afforded N-substituted
phenylamino compounds 8a–c (Scheme 3). Deprotec-
tion of the tert-butoxycarbonyl (Boc) group in 8a with
HCl in EtOH gave 9 (Scheme 4). Alkylation of 8a with
iodomethane using sodium hydride in DMF, followed
by deprotection of the Boc group with HCl in EtOH,
afforded a methoxide 10 (Scheme 4). Acetylation of 8a
with acetic anhydride in pyridine, followed by depro-
tection of the Boc group, afforded an acetate 11 (Scheme
4). Ring opening of epichlorohydrin with benzylamine
provided a chlorohydrin derivative 12. Subsequently,
alkylation of 1 with 12 using potassium carbonate and
potassium iodide in EtOH gave the benzylamino
derivative 13 (Scheme 5).
The diphenylmethyl piperazine derivatives including
flunarizine,14 tamolarizine,15 and lifarizine16 have been
well-known as calcium antagonists with calcium over-
load inhibitory activity, and flunarizine has been repor-
ted to show an antioxidative activity in addition to
them.17 However, flunarizine has also been known to
have a clinical risk of extrapyramidal side effects caused
through the binding to D2 receptor.18 In order to
increase the calcium antagonistic and antioxidative
activities and avoid the side effect of flunarizine, we
attempted to find new compounds having the diphenyl-
alkyl piperazine moiety. And we used commercially
available 1-[4,4-bis(4-fluorophenyl)butyl]piperazine in
place of the diphenylmethyl piperazine moiety of
flunarizine.
Scheme 3. Synthesis of 8a–c: (a) NaH, epibromohydrin, DMF, 7a–c,
90–100%; (b) 1, EtOH, 8a–c, 18–74%.
The syntheses of phenylthiopropanol derivatives 3a, 4,
and 5 and benzylthiopropanol derivative 3b are shown
in Schemes 1 and 2. Alkylation of 1-[4,4-bis(4-fluoro-
phenyl)butyl]piperazine 1 with epibromohydrin using
tetrabutylammonium bromide in CH2Cl2 under alkaline
conditions, followed by ring opening of epoxide 2 with
the corresponding mercaptans in EtOH, afforded 3a and
3b (Scheme 1). Alkylation of 3a with iodomethane in
the presence of sodium hydride in N,N-dimethylform-
amide (DMF) gave a methoxide 4 (Scheme 2). Acetyla-
tion of 3a with acetic anhydride in pyridine gave an
acetate 5 (Scheme 2).
Scheme 4. Synthesis of 9–11: (a) HCl/EtOH, 9, 98%; (b) NaH, MeI,
DMF, 10, 29%; (c) Ac2O, pyridine, 11, 89%.
Scheme 1. Synthesis of 3a and 3b: (a) Bu4NBr, NaOH (aq), epi-
bromohydrin, CH2Cl2, 3a: 65%, 3b: 54%; (b) mercaptans, EtOH,
62%.
Scheme 5. Synthesis of 13: (a) epichlorohydrin, EtOH, 46%; (b) 1,
K2CO3, KI, EtOH, 62%.
Two types of novel diphenylalkyl piperazine derivatives
synthesized as described above were evaluated for the
inhibitory activities against KCl-induced contractile
responses in isolated rat aorta.19 The contractile
response by KCl has been considered to be associated
with an influx of Ca2+ into vascular smooth muscle
through voltage-dependent calcium channels. The results
are shown in Tables 1 and 2. Both types of diphenyl-
alkyl piperazine derivatives showed apparent inhibitions
against KCl-induced contractions. A typical phe-
nylthiopropanol derivative 3a showed a more potent
inhibition than flunarizine with PD02 value of 6.79. The
Scheme 2. Synthesis of 4 and 5: (a) NaH, MeI, DMF, 38%; (b) Ac2O,
pyridine, 77%.
The syntheses of phenylaminopropanol derivatives 8b,
8c, 9–11, and benzylaminopropanol derivative 13 are
exemplified in Schemes 3–5. Alkylation of N-substituted
aniline 6a–c with epibromohydrin using sodium hydride

M. Kimura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1947–1950
1949
insertion of methylene group between sulfur atom and
phenyl group 3b showed similar potency to 3a, indicat-
ing a 10-fold potency in relation to that of flunarizine.
Since hydroxy group has often been important for the
molecular recognition through the hydrophilic or
hydrogen bonding interaction, the modifications of the
hydroxy group were attempted. The modification of the
hydroxy group with methoxy group 4 had equal efficacy
and that with acetoxy group 5 showed a slightly
decreased activity, as compared with 3a. Concerning
aminopropanol derivatives, phenylamino compound 9,
showed an obvious calcium antagonistic activity with
PD02 value of 6.70, exhibiting efficacy similar to that of
the phenylthio compound 3a. The insertion of methyl-
ene group between nitrogen atom and phenyl group 13
considerably decreased the activity as compared with 9,
in contrast with benzylthio derivative 3b, but it still
maintained potent efficacy comparable to flunarizine.
The compound 11 modified at the hydroxy group with
acetoxy group was somewhat less potent than com-
pound 9. The introductions of methyl group 8b and
acetyl group 8c into nitrogen atom in compound 9
apparently decreased the activities, but they also kept
similar potency to flunarizine.
Table 2. Biological activities of phenylamino and benzylaminopro-
panol derivatives
Compd
n
R1 R2 Ca antagonistic
activitya PD02
Antioxidative
activityb IC50 (mM)
8b
8c
9
10
11
13
0
0
0
0
0
1
H
H
H
Me
Ac
H
Me
Ac
H
H
H
5.83
5.98
6.70
N.T.c
6.38
5.85
19
390
6.5
15
13
6.9
H
Flunarizine
a-Tocopherol
5.98
—
15
1.5
aSee ref 20.
bSee ref 21.
cN.T., not tested.
On the other hand, the phenylamino compound 9
showed a more potent inhibitory activity with IC50
value of 6.5 mM than flunarizine, although it was less
potent than a-tocopherol. This finding prompted us to
investigate further modifications at the 2-hydroxy-3-
phenylaminopropyl moiety. The insertion of methylene
group between nitrogen atom and phenyl group 13
showed a potent activity comparable to compound 9 on
antioxidative activity. The modifications of the hydroxy
group with methoxy group 10 and acetoxy group 11
decreased the activities as compared with compound 9,
but their activities were equipotent to that of fluna-
rizine. The introduction of methyl group 8b into nitro-
gen atom in compound 9 similarly decreased its activity,
while that of acetyl group 8c dramatically decreased it.
Table 1. Biological activities of phenylthio and benzylthiopropanol
derivatives
Compd
n
R
Ca antagonistic Antioxidative activityb
activitya PD02
IC50 (mM)
Thus, on both calcium antagonistic and antioxidative
activities, the modifications of the thiopropanol moiety
had a slight influence, whereas those of the aminopropanol
moiety had an apparent influence.
3a
3b
4
0
1
0
0
H
H
Me
Ac
6.79
6.93
6.69
6.55
15
28
20
20
5
Flunarizine
a-Tocopherol
5.98
—
15
1.5
As described above, we found that some diphenylalkyl
piperazine derivatives containing the thio or aminopro-
panol moiety substituted by phenyl or benzyl group
showed calcium antagonistic and antioxidative activities
equal to or higher than flunarizine. These results suggest
that the replacement of the diphenylmethyl moiety of
flunarizine with the diphenylbutyl moiety and the
introductions of heteroatoms such as sulfur or nitrogen
atom and functional groups into the cinnamyl moiety of
flunarizine can be allowed to exhibit both biological
activities. In particular, it is suggested that the presence
of nitrogen atom at the 3-position of the propanol
moiety plays an important role in exhibiting a potent
antioxidative activity.
aSee ref 20.
bSee ref 21.
Next, two types of diphenylalkyl piperazine derivatives
were evaluated for the inhibitory activities against
auto-oxidative lipid peroxidations in canine brain
homogenates.22 The results are shown in Tables 1 and 2.
The phenylthio compound 3a showed an apparent inhi-
bition with IC50 value of 15 mM and it was equivalent in
activity to flunarizine, but it was 10-fold less potent than
a-tocopherol as a well-known antioxidant. The insertion
of methylene group between sulfur atom and phenyl
group 3b resulted in a decrease of activity as compared
with 3a or flunarizine. The modifications of the hydroxy
group with methoxy group 4 and acetoxy group 5 also
showed slightly decreased potencies.
In order to examine the selectivity to various arteries,
two types of representative diphenylalkyl piperazine
derivatives 3a and 9 were evaluated for inhibitory
activities against KCl-induced contractile responses in
isolated canine arteries (basilar, coronary, mesenteric,
and renal).19 The results are shown in Table 3. The
phenylthio compound 3a showed potent inhibitions
against all KCl-induced contractile responses in the iso-
lated arteries. The PD02 values of 3a were 7.00, 6.38,

1950
M. Kimura et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1947–1950
Table 3. Vasodilatation effects of 3a and 9
4. Mueller, A. M.; Artman, L. D.; Balandrin, E. B.; Chien,
Y. E.; Delmar, E. G.; Gerge, K.; Kierstead, A.; Marriott, T. B.;
Moe, S. T.; Kewman, M. K.; Raszkiewicz, J. R.; Sanguinetti,
E. L.; VanWagebnen, B. C. Ann. N.Y. Acad. Sci. 1999, 450.
5. Kawasaki-Yatsugi, S.; Yatsugi, S.; Koshiya, K.; Shimizu-
Sasamata, M. Jpn. J. Pharmacol. 1997, 74, 253.
6. Semkova, I.; Wolz, P.; Krieglstein, J. Eur. J. Pharmacol.
1998, 359, 251.
PD02
a
Compd
Basilar
Coronary
Mesenteric
Renal
3a
9
7.00
6.76
6.38
6.32
5.80
6.10
5.73
6.27
aSee ref 20.
7. Hampson, A. J.; Grimalidi, M.; Lolic, M.; Wink, D.;
Rosenthal, R.; Axelord, J. Ann. N.Y. Acad. Sci. 2000, 899,
274.
5.80, and 5.73, respectively, and the inhibitory activity
to basilar artery was the most potent of those to four
8. (a) Martinez-Vila, E.; Sieira, P. I. Cerebrovasc. Dis. 2001,
11, 60. (b) Hickenbottom, S. L.; Grotta, J. Semin. Neuro.
1998, 18, 485.
9. Farber, J. L. Life Sci. 1989, 29, 1289.
10. Kristian, T.; Siesjo, B. K. Stroke 1998, 29, 705.
11. Halliwell, B. Drug Aging 2001, 18, 685.
12. Stuiver, B. T.; Douma, B. R. K.; Bakker, R.; Nyakas, C.;
Luiten, P. G. M. Neurodegeneration 1996, 5, 153.
13. Hicks, C. A.; Ward, M. A.; Swettenham, J. B.; O’Neill,
M. J. Eur. J. Pharmacol. 1999, 381, 113.
14. Van Zwieeten, P. A. Eur. Neurol. 1986, 25, 57.
15. Shanklin, J.; Jonson, C.; Proakis, A. J. Med. Chem. 1991,
34, 3011.
16. Ito, C.; Im, W. B.; Takagi, H.; Takahashi, M.; Tsuzuki,
K.; Liou, S.-Y.; Kunihara, M. Eur. J. Pharmacol. 1994, 203.
17. Goncalves, T.; Carvalho, A. P.; Oliverira, C. R. Eur. J.
Pharmacol. 1991, 12, 315.
18. Daniel, J. R.; Mauro, V. F. Ann. Pharmacother. 1995, 29,
73.
arteries. The phenylamino compound
9 similarly
showed potent inhibitions, exhibiting the most potent
inhibition to basilar artery with PD02 value of 6.76, but
its selectivity was lower than that of 3a.
In conclusion, we synthesized two types of novel diphe-
nylalkyl piperazine derivatives containing the thio or
aminopropanol moiety substituted by phenyl or benzyl
group, and evaluated for their calcium antagonistic and
antioxidative activities. Two compounds 9 and 13 pos-
sessed potent antioxidative activities along with potent
calcium antagonistic activities. Two representative
compounds 3a and 9 were evaluated for inhibitory
activities against KCl-induced contractile responses in
isolated canine arteries (basilar, coronary, mesenteric,
and renal). Both compounds 3a and 9 showed the most
potent inhibitions to basilar artery, although the selec-
tivity of 9 was lower than that of 3a. Further chemical
modification and biological study of the diphenylalkyl
piperazine derivatives containing the aminopropanol
moiety have been continued to find a calcium antagonist
with antioxidative activity.
19. Sundberg, R. J.; Biswas, S.; Murthi, K. K.; Donna, R. J.
Med. Chem. 1998, 41, 4317.
20. The evaluation of calcium antagonistic activity was car-
ried out by the modification of the method described in ref 19.
In brief, each smooth muscle of rat aorta cut into ring segment
was mounted in organ bath filled with Krebs–Henseleit solu-
tion saturated with 95% O2 and 5% CO2 mixture. After the
equilibration at 37 ꢀC, the test compounds were added, subse-
quently KCl (10–60 mM) was added cumulatively, and the
changes of isometric tension were recorded. Taking the con-
traction at 60 mM KCl as 100%, a concentration–response
curve was drawn from 4–5 separate experiments. PD02 value
was calculated as ꢁlog value of the concentration of the test
compound required to inhibit the 60 mM KCl-induced con-
tractions to 50%.
21. The evaluation of an antioxidative activity was carried out
according to the method described in ref 22. A concentration–
response curve was obtained from the three separate experi-
ments and the IC50 value was calculated as the concentration
of the test compound required to inhibit the auto-oxidative
lipid peroxidations in canine brain homogenates by 50%.
22. Yao, K.; Ina, Y.; Nagashima, K.; Ohmori, K.; Ohno, T.
Biol. Pharm. Bull. 2000, 23, 766.
References and Notes
1. Hacke, W.; Kaste, M.; Fieschi, C.; Toni, D.; Lesaffre, E.;
Kummer, R. V.; Boysen, G.; Bluhmki, E.; Hoxter, G.;
Mahagne, M. H.; Hennerici, M. JAMA 1995, 274, 1017.
2. Annoura, H.; Nakanishi, K.; Toba, T.; Takemoto, N.;
Imajyo, S.; Miyajima, A.; Tamura-Horikawa, Y.; Tamura, S.
J. Med. Chem. 2000, 43, 3372.
3. Hu, L. Y.; Ryder, T. R.; Rafferty, M. F.; Feng, M. R.;
Lotarrski, S. M.; Lotarrski, D. M.; Rock, D. M.; Sinz, M.;
Stoehr, S. J.; Taylor, C. P.; Weber, M. L.; Bowersox, S. S.;
Miljanich, E. M.; Wang, Y. X.; Szoke, B. G. J. Med. Chem.
1999, 42, 4239.