Design, synthesis, and biological testing of thiosalicylamides as a novel class of calcium channel blockers

Article abstract of DOI:10.1016/j.bmc.2005.04.012

The current research aimed to investigate the importance of the heterocyclic ring system in the structure of the cardiovascular drug diltiazem for its calcium channel blocking activity. The manuscript describes the design, synthesis, and biological testin

Full text of DOI:10.1016/j.bmc.2005.04.012

Bioorganic & Medicinal Chemistry 13 (2005) 4323–4331
Design, synthesis, and biological testing of thiosalicylamides as
a novel class of calcium channel blockers
ꢀ
*
Ahmed S. Mehanna and Jin Yung Kim
Massachusetts College of Pharmacy and Health Sciences, 179 Longwood Ave, Boston, MA 02115, USA
Received 15 April 2004; accepted 5 April 2005
Available online 28 April 2005
Abstract—The current research aimed to investigate the importance of the heterocyclic ring system in the structure of the cardio-
vascular drug diltiazem for its calcium channel blocking activity. The manuscript describes the design, synthesis, and biological test-
ing of a total of 10 S-(p-methoxybenzyl), N-substituted thiosalicylamides as a series of non-cyclic compounds derived from
diltiazemꢁs structure. The new compounds maintained all diltiazem pharmacophores except the thiazepine ring system. In vitro evalu-
ation of the new series for calcium channel blocking effects revealed moderate activities with IC50 values in the range of 4.8–
5
6.0 lM. The data suggest that the ring system is not essential for activity; however, its absence leads to a considerable drop of
activity relative to that of diltiazem (IC50 = 0.3 lM). Compounds of the current series showed optimum activity when the aliphatic
alkyl chain on the salicylamide nitrogen is part of a piperidine or piperazine ring system substituted at the terminal nitrogen with a
benzyl group.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
are those aimed at changing the thiazepine hetero-cycle
ring size of diltiazem molecule. Modifications included
both ring contraction to the five-membered benzothiaz-
Calcium channel blockers constitute an important class
of cardiovascular drugs. Members of the class are clini-
cally useful in the treatment of cardiovascular disorders
in which calcium plays a regulatory role. These disorders
include hypertension, angina, cardiac arrhythmia, con-
gestive heart failure, ischemic injury, stroke, migraine,
tumor resistance to anti-neoplastic drugs, esophageal
spasms, bronchial asthma, and dysmenorrhea. Com-
mercially available calcium channel blockers belong to
one of three chemical classes: the dihydropyridines, the
phenyl alkyl amines, and the benzothiazepines. Figure
depicts the structures of representative members of
each of the three classes. The structure–activity relation-
ships of the dihydropyridines and the phenyl alkyl
amines are well defined and previously reviewed, but
the SAR of the benzothiazepine series has not been that
extensively studied. Among the interesting structural
modifications conducted for the benzothiazepine series
7
8
ole ring or the six-membered benzothiazine ring and
ring expansion to the eight-membered naphthothiazo-
9
cine ring. Figure 2 illustrates examples of molecules
with contracted and expanded heterocyclic nuclei.
Changing the heterocyclic ring size generated derivatives
that not only retained the calcium channel blocking
activity but also resulted in several compounds that were
1
–4
7
–9
more active than diltiazem itself. To gain further in-
sight into the structure–activity relationships of diltia-
zem and related molecules, the current research
explores the importance of the heterocyclic ring for
activity. A series of molecules that lacks the ring system
but maintain all other diltiazem pharmacophores was
synthesized and tested for activity. As depicted in Figure
3, diltiazem molecule features four major pharmaco-
phores: the aromatic benzene ring fused with the hetero-
cyclic thiazepine ring (area i), the methoxy aromatic
ether (area ii), the stereo-chemical centers (area iii),
and the aliphatic basic nitrogen separated from an
amide group with a 2-carbon chain (area iv). Barrish
1
5,6
Keywords: Calcium channel blockers; Thiosalycilamides; Cardiovas-
cular agents.
1
0
et al. identified two essential pharmacophores for dilti-
azem binding to calcium channels: the aryl ether (area ii,
to act as hydrogen bond acceptor), and the basic nitro-
gen (area iv, for ionic bonding with a negative charge on
the channel). Barrish et al. also suggested that the
*
Corresponding author. Tel.: +1 617 732 2955; fax: +1 617 732
801; e-mail addresses: ahmed.mehanna@bos.mcphs.edu; jkim@
curxcel.com
2
ꢀ
Present address: Curxcel Corp., BTC, 1291 Cumberland Ave., W.
Lafayette, IN 47906.
0
968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.012

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A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
Figure 1. Examples of commercially available calcium channel blockers.
Figure 2. Examples of Diltiazem-like molecules with 5, 6 and 8-membered heterocyclic ring systems.
benzothiazepine nucleus may simply act as a spacer to
hold the two pharmacophores at the required distance
for binding to the receptors. Kimball et al. developed
a receptor-binding model identifying the benzene ring
that mimic dilatazem pharmacophoric group iv (Fig.
3) by separating the amide nitrogen from the tertiary
one with a short dimethylamino aliphatic chain. Com-
pound 11 has the aliphatic chain part of a piperidine
ring system substituted with an N-benzyl group. Com-
pounds 12–18 represent a series of derivative in which
the amide nitrogen is incorporated into a piperazine ring
system that is further substituted through the second
nitrogen with various groups including methyl, piper-
onyl, benzyl, and different p-substituted benzyl groups.
Scheme 1 depicts the synthetic route to prepare the tar-
get compounds. Reaction of thiosalicylic acid (1) with 4-
methoxybenzyl chloride in dimethylformamide (DMF)
provided S-(4-methoxybenzyl) thiosalicylic acid (2).
Reaction of (2) with thionyl chloride provided the com-
mon intermediate acid chloride (3). Reaction of (3),
without purification, with the corresponding precursor
amine (Scheme 1) gave the corresponding final com-
pounds 9–18. The precursor amines were either commer-
cially available or synthesized according to standard
1
1
(
area i) as a lipophilic group that facilitates transport
into the channel, and the absolute stereochemistry (area
iii) for the selective binding. The current series of com-
pounds were designed to lack the ring system, and
accordingly the stereo-chemical factors were eliminated.
In spite of lacking the stereochemistry, the spatial con-
formational relationships of the remaining pharmaco-
phores are expected to be maintained as suggested by
1
1
Kimball et al. for the fully extended methoxy-pheneth-
ylamines. Figure 3 illustrates different pharmacophoric
groups and the relationship of the new thiosalicylamide
derivatives (compounds 9–18) to diltiazem.
2
. Results and discussion
1
2
2
.1. Chemistry and synthesis
procedure. Commercially available amines included
N,N-dimethylaminoethylamine, N,N-dimethylamino-
Scheme 1 depicts the route to synthesize the target thio-
salicylamide derivatives (compounds 9–18, Fig. 3).
Compounds 9 and 10 are thiosalicylamide derivatives
propylamine, 1-benzyl-4-aminopiperidine, 1-methylpip-
erazine, 1-benzylpiperazine, and 1-piperonylpiperazine.
Reaction of the above listed amines with intermediate

A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
4325
Figure 3. Comparison of the pharmacophoric groups of diltiazem and the new compounds. (i) lipophilic aromatic area, (ii) p-methoxyaryl group, (iii)
stereo-chemical region and (iv) amide nitrogen linked to a tertiary amine with a spacer.
(3), gave compounds 9–14, respectively. Commercially
unavailable precursor amines required to prepare com-
pounds 15–18 of Scheme 1 were synthesized according
prepared using method A in yields ranging from 16%
to 80% as indicated in the experimental section. At-
tempts to prepare compounds 10, 11, and 16–18 under
method ꢂAꢁ reaction conditions failed. The method was
slightly modified into method B by using 10% sodium
hydroxide solution as the neutralizing base instead of
the extra equivalent of the precursor amine or the trieth-
ylamine. By applying method B, we successfully ob-
tained compounds 10, 11, and 16–18 in yields ranging
from 21% to 63% as detailed under experimental.
1
2
to a previously reported method that is outlined in
Scheme 2, and described below. Mixing of 1 equiv of
piperazine with 1 equiv of piperazine dihydrochloride
hydrate in warm absolute ethanol (65 ꢁC) gave a solu-
tion containing 1 equiv of piperazine monohydrochlo-
ride (compound 4, Scheme 2). Slow addition of 1 equiv
of an alky halide to the above solution of 4 produced
one equivalent of the corresponding monoalkylated
piperazine (compounds 5–8, Scheme 2) and 1 equiv of
piperazine dihydrochloride dihydrate. The latter was
insoluble in absolute ethanol and precipitated out of
solution by further cooling in an ice bath. Filtration of
the dihydrochloride salt followed by removal of ethanol
left the desired crude intermediates 1-alkylpiperazini-
num chlorides (compounds 5–8, Scheme 2) as either so-
lid or oily residues. Reaction of the monosubstituted
piperazine intermediates (5–8, Scheme 2) with interme-
diate 3 produced the desired final compounds 15–18
2.2. In vitro biological evaluation
All 10 compounds (9–18) were tested for inhibition of
calcium-induced contractions of potassium depolarized
isolated rat aorta strips according to a previously re-
1
3,14
ported protocol.
The in vitro testing required incu-
bation of the test compound with rat aorta strips in a
calcium-free, potassium chloride-rich, Krebs-bicarbon-
ate solution followed by addition of calcium chloride
to elicit contraction. The contractile responses were re-
corded on a Grass Polygraph (model 7H, Grass Instru-
ments, Quincy, MA), as described under experimental.
Table 1 lists the calcium channel blocking activities for
the new compounds, expressed as IC50 values, with dil-
tiazem as the reference drug. Table 1 data indicate that
in spite of lacking the heterocyclic ring system, the new
compounds are moderately active as calcium channel
(
Scheme 1), respectively. Reaction of 3 with precursor
amines shown in Scheme 1 was accomplished through
one of two general methods: A or B. According to meth-
od A, intermediate 3 reacted with either 2 equiv of the
starting amine (in cases of inexpensive ones), or 1 equiv
of the starting amine and one equivalent (or slight ex-
cess) of triethylamine. Compounds 9 and 12–15 were

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A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
Scheme 1. Synthesis of thiosalicylamide derivatives. Reagents and conditions: (i) 4-methoxybenzyl chloride, K
2 3
CO , DMF, reflux for 10 h; (ii)
SOCl , reflux for 2 h; (iii) Method A: triethylamine, benzene, stir for 24 h at room temperature. Method B: 10% NaOH, 1,4-dioxane, stir for 24 h at
2
room temperature.
Scheme 2. Synthesis of commercially unavailable mono-substituted piperazine intermediates.
blockers. However, the high IC50 values of the new com-
pounds (4.8–56.0 lM, Table 1) relative to that of dilti-
azem (0.3 lM) suggest that the absence of the
heterocyclic ring results in a considerable drop in
activity.
ing activity. The activity of the current non-cyclic
compounds clearly affirms that the heterocyclic ring sys-
tem in the diltiazem structure is not essential for the cal-
cium channel blocking activity. However, it is also
evident from the IC50 values of the new compounds
(4.8–56.2 lM, Table 1) that the absence of the ring
2
.3. Structure–activity relationships (SAR)
structure leads to a significant drop in activity relative
to that of diltizem (IC50 value of 0.3 lM). Although
the new compounds have weaker calcium channel block-
ing activities than diltiazem, data in Table 1 provide the
As the IC50 values listed in Table 1 indicate, the new ser-
ies of compounds has moderate calcium channel block-

A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
4327
Table 1. Calcium channel blocking activity of the new compounds
expressed as IC50 in lM
has an IC50 of 6.2 lM while compound 13, with the side
chain part of a piperazine ring, has an IC50 values of
7.2 lM. The piperidine and piperazine rings were chosen
to provide compounds differing in the number of atoms
separating the amide nitrogen from the basic one (three
in case of compound 11 and two in case of compound
13). The data reflect an overall 7–8 fold increase in activ-
ity over compound 12 with the N-methyl substitution
and a 2–3 fold increase in activity over the open chain
compounds 9 and 10 (IC50 values of 17.6 and
a
Compound # IC50 (lM) ± S.D. R
16.3 lM, respectively). The low IC50 values of com-
9
1
17.6 ± 8.0
16.3 ± 3.8
–NHCH
–NHCH
2
CH
CH
2
N(CH
3
)
2
pounds 11 and 13 suggest the importance of the benzyl
group for enhancing activity. The close activities of the
two compounds reinforce the previous conclusion that
the distance separating the two nitrogen atoms is not
crucial.
0
1
2
2
CH N(CH )
2
3 2
1
1
1
6.2 ± 1.5
56.2 ± 8.5
7.2 ± 3.2
2
3
4. Compounds 15–18 have the benzyl group further
substituted in the para position with an electron donat-
ing group (a methoxy group in compound 15) or elec-
tron-withdrawing groups (nitro in compound 16,
chloro in compound 17, or fluoro in compound 18).
The close IC50 value range of compounds 15–18 (4.8–
1
4
22.6 ± 5.7
6
.9 lM) and that of compound 13 with unsubstituted
1
1
1
1
5
6
7
8
6.9 ± 1.5
6.8 ± 4.1
5.4 ± 2.6
benzyl (7.2 lM) indicates that the electronic properties
of the substituent have no effect on the activity and that
lipophilicity is the only determinant factor.
5
. Compound 14, with the piperazine ring substituted
with a piperonyl group, showed an IC50 of 22.6 lM
reflecting a 3–5-fold decrease of activity relative to the
benzyl derivatives. If the piperonyl group is considered
as a di-substituted benzyl, the weak activity of com-
pound 14 relative to that of compounds 15–18 suggests
that a mono substituted benzyl group is optimum for
activity and di-substitution does not favor binding to
the channel.
4.8 ± 1.2
0.3 ± 0.1
Diltiazem
a
Using potassium depolarized rat aorta strips (n = 4–5) at 1.5 mM
CaCl
2
.
basis for the following tentative structure–activity
relationships:
In summary of the structure–activity relationships, the
calcium channel blocking activity of the new series is
optimal when the alkyl side chain is part of a piperidine
or piperazine ring structure further substituted with N-
benzyl group. Neither the spacer chain length nor the
type of substituent on the benzyl group plays role in
the activity.
1
. Compounds 9 and 10 showed moderate calcium chan-
nel blocking activity as reflected by the IC50 values of
7.6 and 16.3 lM, respectively. The two compounds
1
have short spacer aliphatic chains (compound 9 n = 2,
compound 10 n = 3) to mimic diltiazem dimethylamino-
ethyl pharmacophore. The closeness of the IC50 values
indicates that the spacer length is not a crucial factor
for activity.
3. Conclusion
The current research reports the design, synthesis, and
biological testing of 10 thiosalycilamide derivatives (9–
18) as a new series of calcium channel blockers. All
new compounds exhibited moderate activity in inhibi-
ting calcium-induced contractions of rat aorta strips.
The observed activities of the non-cyclic compounds
suggest that the presence of a heterocyclic ring system
is not essential for calcium channel blocking activity.
However, the high IC50 values of the new compounds
relative to that of diltiazem indicate that eliminating
the ring structure results in a considerable drop in activ-
ity. The structure–activity relationships of the new com-
pounds indicate optimum activity when the alkyl chain
on the thiosalycylamide nitrogen is part of a piperazine
2
. Compound 12 has the aliphatic chain separating the
two nitrogen atoms incorporated into a piperazine ring
structure with N-methyl substitution. The IC50 value
of compound 12 (56.0 lM) reflects a three-fold drop in
activity from that of compounds 9 and 10 (open chain
compounds with similar N-substitution). The data indi-
cate that piperazine substituted at the terminal nitrogen
with N-methyl group is not optimum for activity.
3
. Compounds 11 and 13 are similar to compound 12 in
having the side chain incorporated into a ring system
but with N-benzyl substitution at the terminal nitrogen.
Compound 11, with the chain part of a piperidine ring,

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A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
or piperidine ring system having a benzyl or para-substi-
tuted benzyl group at the terminal nitrogen. It is worthy
to indicate that a United States patent has been issued,
recognizing the new series of compounds as a new class
12.5 mmol of compound 4 that was used without further
isolation.
1
2
4.5. 1-(4-Methoxybenzyl) piperazine (5)
1
5
of calcium channel blockers.
To the above mixture containing 12.5 mmol of com-
pound 4, 1.95 g (12.5 mmol) of 4-methoxybenzyl chlo-
ride were added over a 5 min period with vigorous
stirring. The mixture was stirred for additional 25 min
at 65 ꢁC and was kept in an ice bath for about 30 min
before filtering the precipitated piperazine dihydrochlo-
ride. The solid residue was washed with 3 · 5 mL of
ice-cold absolute ethanol. The combined filtrates were
evapo- rated to dryness to produce a solid crude product
of 1-(4-methoxybenzyl)-4-piperazinium chloride. Heat-
ing the mixture with 20 mL absolute ethanol followed
by rapid filtration removed any residual piperazine dihy-
drochloride from the solid. Concentration of the filtrate
followed by cooling in ice produced 2.629 g (87%) of
pure 1-(4-methoxybenzyl)-4-piperazinium chloride as
white crystals having a melting point of 154–157 ꢁC.
The free base (compound 5) was released from the above
salt by dissolving it in 20 mL of water followed by alka-
linization to pH 12 (with approximately 20 mL of 5.0 M
sodium hydroxide solution). The mixture was extracted
with methylene chloride and dried over MgSO4 for
30 min. Filtration and solvent evaporation at reduced
pressure left a white solid. Crystallization of the solid
from petroleum ether gave 1.393 g (54%) of compound
4
. Experimental
4
.1. Chemistry
General information: Melting points were determined
using a MEL-TEMP apparatus and are uncorrected.
IR spectra were taken with a Perkin–Elmer 1420 Ratio
Recording infrared spectrophotometer (Model 1420,
Perkin–Elmer, Norwalk, CT) and Nicolet Impact 410
FT-IR spectrophotometers (Model: Impact, Nicolet
1
Instrument Corporation, Madison, WI). H NMR spec-
tra were recorded on a Varian T-60 NMR spectrometer
with tetramethylsilane (TMS) as an internal standard.
The values of chemical shift (d) are given in parts per
million (ppm) and coupling constants (J) in Hertz
(Hz). The reactions progress were monitored using
TLC on silica gel plate (Whatman, PE SIL G/UV). Ex-
tracts were dried over MgSO and the solvents removed
4
under reduced pressure. Reported yields are for the puri-
fied products and not optimized. Elemental analyses
were performed by Desert Analytics, Tucson, AZ.
4
.2. S-(4-Methoxybenzyl) thiosalicylic acid (2)
5
cm : 3392, 2943, 1509, 1301, 1034, 991; H NMR
as the free base. Mp: 99–101 ꢁC; IR (KBr)
ꢀ
1
1
To a solution of 15.42 g (0.1 mol) of thiosalicylic acid (1)
and 41.4 g (0.3 mol) of K CO in 150 mL of dimethyl-
(CDCl ) d ppm: 2.12 (s, 1H), 2.23–2.46 (m, 4H), 2.53–
2.98 (m, 4H), 3.40 (s, 2H), 3.73 (s, 3H), 6.9 (d, 2H,
J = 9 Hz), 7.1 (d, 2H, J = 9 Hz).
3
2
3
formamide (DMF), 15.6 g (0.1 mol) of 4-methoxybenzyl
chloride was slowly added with stirring. The mixture
was refluxed for 10 h, then cooled to room temperature.
Addition of water (150 mL) and adjusting the pH to 3.0
with 3.0 M HCl resulted in the formation of white pre-
cipitate. The precipitate was collected by filtration and
washed with acetone to give 27.364 g (99.7%) of inter-
mediate (2) as a white powder. Mp 225–227 ꢁC; IR
4.6. 1-(4-Nitrobenzyl) piperazine hydrochloride (6)
Compound 6 was obtained as a hydrochloride salt in a
26% yield using p-nitrobenzyl chloride in the same above
procedure describing the synthesis of compound 5. Mp:
ꢀ
1
209–212 ꢁC; IR (KBr) cm : 3513, 2930, 2815, 2720,
ꢀ
1
1
1
(
KBr) cm : 3200–2500, 1673, 1510, 1234, 1027;
H
1604, 1514, 1349, 1099, 865, 743; H NMR (CDCl ) d
3
NMR (CDCl + DMSO-d ) d ppm: 3.7 (s, 3H), 4.08
(s, 2H), 6.52 (s, 3H), 6.9 (d, 2H, J = 9 Hz), 7.4 (d, 2H,
J = 9 Hz), 7.5 (s, 1H), 8.1 (d, H-6, J = 8 Hz).
ppm: 2.60–2.95 (m, 4H), 3.0–3.36 (m, 4H), 3.60 (s,
2H), 7.5 (d, 2H, J = 9 Hz), 8.1 (d, 2H, J = 9 Hz).
3
6
1
6
4
.7. 1-(4-Chlorobenzyl) piperazine (7)
4
.3. S-(4-Methoxybenzyl) thiosalicyloyl chloride (3)
Compound 7 was obtained as free base in a 50% yield
using p-chlorobenzyl chloride and the same procedure
employed to prepare compound 5, mp 93–97 ꢁC; IR
Compound 3 was obtained by refluxing a mixture of
22 mg (3.0 mmol) of S-(4-methoxybenzyl) thiosalicylic
8
ꢀ1
acid (compound 2) and 10 mL thionyl chloride for two
hours. Removal of excess thionyl chloride under re-
duced pressure left compound 3 as pale yellow solid,
which was used in subsequent steps without further
purification.
(KBr) cm : 3383, 2807, 1489, 1472, 1423, 1299, 1000,
1
806; H NMR (CDCl ) d ppm: 2.15–2.45 (m, 5H),
3
2.65–2.95 (m, 4H), 3.35 (s, 2H), 7.15 (s, 4H).
4.8. 1-(4-Fluorobenzyl) piperazine hydrochloride (8)
4
.4. Piperazine monohydrochloride (4)
Compound 8 was prepared in a 91% yield, from p-fluo-
robenzyl chloride using the same procedure employed to
prepare the hydrochloride salt of compound 5. Mp 164–
A solution of 1.08 g (12.5 mmol) of piperazine in 20 mL
of absolute ethanol was heated to 65 ꢁC (with continu-
ous stirring) and 2.19 g (12.5 mmol) of piperazine dihy-
drochloride monohydrate was added while maintaining
stirring and heating. At this point, the mixture contains
ꢀ
1
167 ꢁC; IR (KBr) cm : 3501, 2926, 2810, 2709, 2473,
1
1602, 1513, 1217, 1094, 765; H NMR (CDCl ) d ppm:
3
2.4–2.72 (m, 4H), 2.8–3.24 (m, 4H), 3.45 (s, 2H), 7.7–
7.4 (m, 4H).

A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
4329
0
0
4.9. S-(4-Methoxybenzyl), N-(2-(N ,N -dimethylamino)
ethyl) thiosalicylamide (9), method A, Scheme 1
gave 0.8 g (63%) of compound 11 as white crystals.
ꢀ
1
Mp 121–122 ꢁC; IR (KBr) cm : 23294, 2934, 2788,
1
629, 1533, 1510, 1250, 1025, 705; H NMR (CDCl )
1
3
Compound 9 was prepared by slowly adding a suspen-
sion of the acid chloride 3 (878 mg, 3.0 mmol) in
d ppm: 1.34–3.00 (m, 9H), 3.44 (s, 2H), 3.66 (s, 3H),
3.96 (s, 2H), 6.62–7.78 (m, 13H); Anal. Calcd for
C H N O S: C, 72.61; H, 6.77; N, 6.27. Found: C,
1
0 mL cold benzene to a solution of 528 mg (6 mmol)
2
7
30
2
2
of N,N-dimethylethylenediamine in 10 mL ice cooled
benzene. The mixture was stirred overnight at room
temperature. Water (20 mL) was added and the aqueous
layer separated. The benzene layer was further washed
with 10% NaOH solution (10 mL) and the combined
aqueous layers were extracted with two portions of chlo-
roform (10 mL each) to recover any trapped product.
The mixture of the combined chloroform extracts and
original benzene layer were dried over anhydrous
72.28; H, 6.74; N, 6.14.
4.12. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-meth-
ylpiperazine (12)
Compound 12 was prepared using the same method de-
scribed to prepare compound 9. Reaction of 5.48 g
(0.02 mol) of S-(4-methoxybenzyl) thiosalicylic acid
and 20 mL of SOCl produced 0.02 mol of intermediate
2
MgSO . Filtration of the drying material followed by
4
3 as a yellow residue. A suspension of 3 in warm benzene
(30 mL) was slowly added to a 20 mL solution of 4.0 g
(0.04 mol) 1-methylpiperazine in 20 mL benzene. The
mixture was stirred overnight at room temperature.
After the addition of 40 mL water the aqueous layer
was extracted twice with 15 mL CH Cl . The combined
solvent removal left compound 9 as an oily residue. Dis-
solving the oil in 10 mL methanol followed by the addi-
tion of 60 mL water and stirring for 24 h at room
temperature gave white crystalline material. Filtration
of the separated crystals and washing with water fol-
lowed by petroleum ether gave 279 mg (27%) of com-
pound 9 as pale yellow crystals. Mp of 83–84.5 ꢁC; IR
2
2
methylene chloride extracts and the benzene layer
washed with 10% NaOH were dried over anhydrous
MgSO . Removal of the solvents under reduced pressure
ꢀ1
1
(
KBr) cm : 3322, 3257, 2943, 1633, 1511, 1029; H
4
NMR (CDCl ) d ppm: 2.2 (s, 6H), 2.4 (t, 2H,
left an oily product that gave a solid upon crystallization
from hot petroleum ether (10 mL). Collection of the sep-
arated solid by suction filtration followed by washing
with cold petroleum ether gave 3.4 g (47%) of compound
3
J = 6 Hz), 3.45 (q, 2H, J = 5 Hz), 3.7 (s, 3H), 4.0 (s,
2
H), 6.75 (d, 2H, J = 9 Hz), 7.15 (d, 2H, J = 9 Hz),
7
C, 66.25; H, 7.02; N, 8.13. Found: C, 66.09; H, 7.14; N,
.25 (s, 3H) 7.5 (m, 1H); Anal. Calcd for C H N O S:
1
9
24
2
2
ꢀ
1
12. Mp 77–77.8 ꢁC; IR (KBr) cm : 2943, 1633, 1512,
1
8
.14.
1440, 1251, 1001; H NMR (CDCl ) d ppm: 2.25 (s,
3
3H), 2.2–2.5 (m, 4H), 2.97–3.25 (m, 2H), 3.68 (s, 3H),
3.6–3.85 (m, 2H), 4.0 (s, 2H), 6.75 (d, 2H, J = 9 Hz),
0
0
4.10. S-(4-Methoxybenzyl) N-(3-(N ,N dimethylamino)
propyl) thiosalicylamide (10), method B, Scheme 1
7.0–7.3 (m, 6H); Anal. Calcd for C H N O S: C,
2
2
0
24
2
67.38; H, 6.79; N, 7.86; S, 9.00. Found: C, 67.15; H,
6.52; N, 7.90; S, 9.28.
A suspension of 1.45 g (5.0 mmol) of 3 in 10 mL warm
dioxane was slowly added to an ice cooled solution of
5
11 mg (5.0 mmol) of 3-(dimethylamino) propylamine
4.13. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-benz-
ylpiperazine (13)
in 20 mL 10% NaOH. The mixture was stirred overnight
at room temperature. Water (70 mL) was added and the
mixture extracted with four 10 mL portions of chloro-
form. The extracts were dried over anhydrous MgSO4.
Filtration and solvent removal left an oily product that
was dissolved in methanol (20 mL), followed by the
addition of 100 mL water and stirring overnight at room
temperature. Filtration of the separated crystals fol-
lowed by washing with water, followed by petroleum
ether gave 640 mg (35.7%) of pure compound 10
Compound 13 was prepared using same procedure de-
scribed above for compound 9 using 2.74 g (10 mmol)
of intermediate 2, 1.72 g (10 mmol) of 1-benzylpiper-
azine and 2.02 g (20 mmol) triethylamine. The oily prod-
uct was dissolved in 5 mL of methylene chloride and
purified by column chromatography on a silica gel col-
umn using ethyl acetate as an eluent to give 870 mg of
compound 13 (20% yield). The product was further puri-
fied by crystallization from hot hexane to give white
ꢀ
1
as white solid. Mp 97–98 ꢁC; IR (KBr) cm : 3254,
1
936, 1634, 1510, 1437, 1250, 1028, 758; H NMR
ꢀ1
2
crystals with mp of 84–85 ꢁC; IR (KBr) cm : 2817,
1
(
CDCl ) d ppm: 1.68 (t, 2H, J = 9 Hz), 2.22 (s, 6H),
1638, 1512, 1250, 1179, 1027; H NMR (CDCl ) d
3
3
2
2
.30 (m, 2H, J = 6 Hz) 2.90 (d, 1H, J = 6 Hz), 3.4 (q,
H, J = 6 Hz), 3.74 (s, 3H), 4.1 (s, 2H), 6.75 (d, 2H,
J = 9 Hz), 7.15–7.6 (m, 6H). Anal. Calcd for
ppm: 2.2–2.6 (m, 4H), 3.0–3.3 (m, 2H), 3.5 (s, 2H),
3.70 (s, 3H), 3.6–3.96 (m, 2H), 4.08 (s, 2H), 6.75 (d,
2H, J = 8 Hz), 7.0–7.38 (m, 11H); Anal. Calcd for
C H N O S: C, 72.19; H, 6.52; N, 6.48. Found: C,
C H N O S: C, 67.01; H, 7.31; N, 7.81. Found: C,
26
2
0
2
2
26 28
2
2
6
6.68; H, 7.47; N, 7.48.
72.11; H, 6.69; N, 6.46.
4.11. S-(4-Methoxybenzyl)-(4-amino (1-benzyl) piperidi-
nyl) thiosalicylamide (11)
4.14. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-pip-
eronylpiperazine (14)
Compound 11 was obtained as white solid by applying
method B and using of 878 mg (3 mmol) of 3 and of
A suspension of 878 mg (3 mmol) of 3 in warm benzene
(10 mL) was slowly added to an ice cooled benzene
solution (10 mL) of 661 mg (3 mmol) of 1-piperonyl-
piperazine and 455 mg (4.5 mmol) triethylamine and
570 mg (3 mmol) of 4-amino-1-benzylpiperidine. Fur-
ther purification by crystallization from ethanol/water

4330
A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
stirred overnight at room temperature. Addition of
water (20 mL) formed an aqueous layer that was ex-
4.17. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-(4-
chlorobenzyl) piperazine (17)
tracted with 2 · 15 mL portions of CHCl . The com-
3
bined chloroform extracts were combined with the
benzene layer, washed with 10% NaOH solution and
dried over anhydrous MgSO . Removal of the solvents
Compound 17 was prepared using the same procedure
described above for compound 11 using 1.37 g (5 mmol)
of 2, and 1.05 g (5 mmol) of 1-(4 chlorobenzyl) piper-
azine. The product was filtered, washed with methanol,
and further purified by column chromatography on a
silica gel column (eluent: ethyl acetate) to give 496 mg
(21%) of compound 17. Mp 109–111.5 ꢁC; IR (KBr)
4
under reduced pressure left compound 14 as an oily
product. The oil was dissolved in CHCl (5 mL) then
3
cooled in ice bath. Addition of petroleum ether precipi-
tated compound 14 as white solid. Filtration followed by
washing with acetone gave 1.144 g (80%) of the required
ꢀ
1
cm : 2917, 1623, 1581, 1511, 1179, 1427, 1237, 1025,
ꢀ
1
1
product. Mp 126–127 ꢁC; IR (KBr) cm : 2929, 2817,
846; H NMR (CDCl ) d ppm: 2.0–2.6 (m, 4H), 2.98–
3
1
1
634, 1610, 1517, 1488, 1254, 1035, 786;
H
3.28 (m, 2H), 3.38 (s, 2H), 3.68 (s, 3H), 3.6–3.96 (m,
2H), 4.0 (s, 2H), 6.7 (d, 2H, J = 9 Hz), 6.95–7.2 (m,
10H); Anal. Calcd for C H ClN O S: C, 66.87; H,
NMR (CDCl ) d ppm: 2.2–2.6 (m, 4H), 3.0–3.25 (m,
3
2
2
6
H), 3.4 (s, 2H), 3.78 (s, 3H), 3.6–3.9 (m, 2H), 4.0 (s,
H), 5.85 (s, 2H), 6.7 (s, 3H), 6.8 (s, 2H), 7.0–7.35 (m,
H); Anal. Calcd for C H N O SÆ1/2H O: C, 66.78;
2
6
27
2
2
5.83; N, 6.00. Found: C, 66.64; H, 5.89; N, 6.07.
2
7
28
2
4
2
H, 6.01; N, 5.77; S, 6.60. Found: C, 66.69; H, 6.02; N,
4.18. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-
(4-fluo- robenzyl) piperazine (18)
5
.79; S, 6.75.
4.15. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-(4-
methoxybenzyl) piperazine (15)
Compound 18 was prepared according to the same pro-
cedure described above for compound 11 using 1.36 g
(5 mmol) of S-(4-methoxybenzyl) thiosalicylic acid,
Compound 15 was prepared by the same method em-
ployed to prepare compound 14 using 823 mg (3 mmol)
of 2, 728 mg (3 mmol) 1-(4-methoxybenzyl) piperazine,
and 1.36 g (13.5 mmol) triethylamine. The compound
was obtained as a dark brown oil, which upon crystalli-
zation from acetone (5 mL)/water (20 mL) gave a solid
material. Further purification by column chromatogra-
phy on a silica gel column (ethyl acetate as eluent) gave
and 1.15 g (5 mmol) of 1-(4-fluorobenzyl) piperazine
hydrochloride. The oily product was dissolved in 5 mL
of acetone. Addition of 5 mL of 10% NaOH solution
and 40 mL of water resulted in precipitating a white
material that was collected by suction filtration to give
872 mg (39%) of the compound 18 with a melting point
of 73–84 ꢁC. Further purification by crystallization from
chloroform/petroleum ether gave compound 18 as white
ꢀ
1
2
20 mg (16%) of compound 15. Mp 99–101 ꢁC; IR
KBr) cm : 2950, 1635, 1513, 1430, 1251, 1178; H
crystals with mp of 84.2–85.8 ꢁC; IR (KBr) cm : 2917,
1623, 1581, 1511, 1179, 1427, 1237, 1025, 846; H NMR
ꢀ
1
1
1
(
NMR (CDCl ) d ppm: 2.2–2.6 (m, 4H), 3.0–3.25 (m,
(CDCl ) d ppm: 2.0–2.6 (m, 4H), 2.98–3.28 (m, 2H), 3.38
3
3
2
2
H), 3.4 (s, 2H), 3.75 (s, 3H), 3.78 (s, 3H), 3.6–3.9 (m,
H), 4.0 (s, 2H), 6.65 (d, 2H, J = 8 Hz), 6.85 (d, 2H,
(s, 2H), 3.68 (s, 3H), 3.6–3.96 (m, 2H), 4.0 (s, 2H), 6.6
7.2 (m, 12H); Anal. Calcd for C H FN O S: C,
2
6
27
2
2
J = 8 Hz), 7.0–7.3 (m, 8H); Anal. Calcd for
C H N O S: C, 70.10; H, 6.54; N, 6.06. Found: C,
69.31; H, 6.04; N, 6.22. Found: C, 68.99; H, 6.02; N,
6.36.
2
7
30
2
3
7
0.34; H, 6.80; N, 5.83.
4.19. In vitro testing for calcium channel blocking using
isolated rat aorta strips
4.16. N1-(S-(4-Methoxybenzyl) thiosalicyloyl)-N4-(4-
nitrobenzyl) piperazine (16)
The new compounds were assessed for calcium channel
blocking activities according to a previously reported
Compound 16 was prepared according to method B and
in a similar procedure to that used to prepare compound
1
3,14
protocol,
that is detailed below. Male Wistar rats
0
0
00
1
1. A suspension of 878 mg (3 mmol) of 3 in warm diox-
(Charles River Laboratory) were housed in 12 · 24
ane (10 mL), was slowly added to an ice cooled solution
of 773 mg (3 mmol) of 1-(4-nitrobenzyl) piperazine in
plastic cages in the Massachusetts College of Pharmacy
animal facility with a 12 h light and 12 h dark schedule.
Animals had access to water and food ad lib. Rats
(weighing approximately 350 g) were euthanized with
sodium pentobarbital (100 mg/kg). The thoracic aorta
was removed and placed in a 37 ꢁC Krebs-bicarbonate
solution containing NaCl, 112 mM; KCl, 5 mM;
MgSO , 1.2 mM; KH PO , 1 mM; NaHCO , 25 mM;
1
0 mL 10% NaOH solution with stirring overnight at
room temperature. The addition of water (70 mL) re-
sulted in the separation of white precipitate. Collection
of the precipitate by suction filtration followed by
washing with acetone gave 602 mg (42%) of com-
pound 16 as a white solid. Mp 122–123 ꢁC; IR
4
2
4
3
ꢀ
1
(
KBr) cm : 2913, 1622, 1508, 1439, 1179, 1350, 1241,
CaCl , 1.25 mM and glucose, 11.5 mM (pH 7.4). The
2
1
1
4
3
7
024, 837; H NMR (CDCl ) d ppm: 2.2–2.6 (m,
thoracic aorta, after removal of connective tissues, was
cut into rings that were approximately 2.5–3.0 mm in
length and immersed in a 37 ꢁC Krebs bicarbonate solu-
tion. The solution was prepared fresh on each day of an
experiment and aerated with 95% O and 5% CO . An
3
H), 2.98–3.28 (m, 2H), 3.55 (s, 2H), 3.70 (s, 3H), 3.6–
.96 (m, 2H), 4.08 (s, 2H), 6.75 (d, 2H, J = 8 Hz), 6.9–
.25 (m, 6H), 7.4 (d, 2H, J = 9 Hz), 8.1 (d, 2H,
J = 9 Hz); Anal. Calcd for C H N O S as complex
2
6
27
3
4
2
2
with acetone (CH COCH ): C, 65.03; H, 6.21; N, 7.84.
3
Found: C, 65.31; H, 6.02; N, 7.96; C, 64.94; H, 5.83;
N, 8.22.
Iso-temp Constant Temperature Circulator (Fisher Sci-
entific model 801, Pittsburgh, PA) was used to maintain
the bath temperature. Tissues were suspended between
3

A. S. Mehanna, J. Y. Kim / Bioorg. Med. Chem. 13 (2005) 4323–4331
4331
two stainless steel loops. To measure tissue contraction
and relaxation one of the loops was attached to a fixed
glass tube that provided the gas mixture to the solution
and the other loop was attached to a Grass force–dis-
placement transducer via polyester thread to a Grass
Polygraph (model 7H, Grass Instruments, Quincy,
MA). Each preparation was stretched to an optimal
resting tension of 2.0 g (previously determined by mea-
contraction. The percentage of contraction inhibition
was calculated for each tissue as follows: % Inhibi-
tion = 100 ꢀ (contraction TC/contraction CC · 100),
where contraction TC denotes the maximum response
in the presence of the test compound and contraction
CC denotes the maxi- mum response in under control
condition. Dose response curves were constructed and
IC50 values were calculated.
2
+
suring tissue contraction to Ca concentrations of
0.1–8.0 mM) that was maintained in all experiments.
All test compounds were dissolved in absolute ethanol
that was also used to prepare all other concentrations
Acknowledgements
(
0.01–10.0 mM) of the test compound. A 150 mM cal-
We as authors are grateful to Drs. L. Kelly, and T. Ma-
her for their valuable comments, Mr. N. Mahmoud for
helping in the in vitro assay. Massachusetts College of
Pharmacy and Health Sciences at Boston provided the
necessary financial support for this research.
cium chloride stock solution was prepared in de-ionized
water. A group of 4–5 animals was used to evaluate each
compound. After equilibration for 60 min with the
above solution, tissues were washed every 15 min with
fresh Krebs-bicarbonate solution. After the artery prep-
arations were equilibrated in the normal Krebs-bicar-
bonate solution, the solution was replaced with a
calcium-free, potassium-rich Krebs-bicarbonate solu-
References and notes
tion (NaCl, 17 mM; KCl, 100 mM; MgSO , 1.2 mM;
4
1. Epstein, M. Drugs 1999, 57, 1.
2. Fleckenstein, A. Circ. Res. Suppl. 1983, 52, 1.
KH PO , 1 mM; NaHCO3, 25 mM and glucose,
2
4
3
4
. Mannhold, R. Drugs Today 1984, 20, 69.
1
1.5 mM (pH 7.4)) to induce depolarization. Tissues
. Franz, D. In Remington; Genarro, A. R., Ed.; Lippincott
Williams & Wilkins: Baltimore, 2000, pp 1274–1296.
. Fossheim, R.; Savarteng, K.; Mostad, A.; Romming, C.;
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. Gualtieri, F.; Teodori, E.; Bellucci, C.; Pesce, E.; Piacenza,
G. J. Med. Chem. 1985, 28, 1621.
were washed every 15 with the calcium-free potassium-
rich Krebs-bicarbonate solution every 15 min. Follow-
ing equilibrium, 0.1 mL of absolute ethanol (vehicle
control) was added to the tissue. After 10 min incuba-
tion of ethanol, the contractile base line response was
obtained by adding 0.1 mL of a calcium chloride stock
5
6
7
. Yamamoto, K.; Fujita, M.; Tabashi, K.; Kawashima, Y.;
Kato, E.; Oya, M.; Iso, T.; Iwao, J. J. Med. Chem. 1988,
31, 919.
2
+
solution (to give 1.5 mM Ca final concentration in
the tissue bath). After obtaining the maximum responses
2
+
with 1.5 mM of Ca the tissues were washed every
5 min with Calcium-free, potassium-rich Krebs-bicar-
8. Fujita, M.; Ito, S.; Ota, A.; Kato, N.; Yamamoto, K.;
Kawashima, Y.; Yamauchi, H.; Iwao, J. J. Med. Chem.
1
1
990, 33, 1898.
. Mochacsi, E.; OꢁBrien, J.; Grove, C. U.S. Patent
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bonate solution for an hour. Once the tension had re-
mained stable for at least 15 min, a second (control)
response was obtained in the same manner. After mon-
itoring the second response, the tissues were washed
again every 15 min with calcium-free potassium-rich
Krebs bicarbonate solution for one hour. Once tension
had remained stable for at least 15 min tissues were trea-
ted (incubation period of 10 min) with 0.1 mL of the
corresponding diltiazem solution in absolute ethanol
to give the required final concentrations of 0.1–
9
4
1
1
0. Barrish, J. C.; Spergel, S. H.; Moreland, S.; Hedber, S. A.
Bioorg. Med. Chem. Lett. 1992, 2, 95.
1. Kimball, S. D.; Hunt, J. T.; Barrish, J. C.; Das, J.; Floyd,
D. M.; Lago, M. W.; Lee, V. G.; Spergel, S. H.; Morland,
S.; Hedberg, S. A.; Gougoutas, J. Z.; Malley, M. F.; Lau,
W. F. Bioorg. Med. Chem. 1993, 1, 285.
12. Mndzhoyan, A.; Afrikyan, V.; Grigoryan, M.; Sheinker,
Y.; Aleksanyan, R.; Vasilꢁyan, S.; Kaldrikyan, A.; Dzhagt-
spanyan, I. Arm. Khim. Zh. 1978, 21, 603.
1
was measured after adding 0.1 mL of CaCl stock solu-
00 lM. The contractile response at each concentration
1
1
1
3. Kelly, L. J.; Undem, B. J.; Adams, G. K. J. Appl. Phys.
993, 74, 1563.
4. Kelly, L. J.; Undem, B. J.; Adams, G. K. J. Appl. Phys.
994, 76, 916.
5. Mehanna, A. S.; Kim, J. U.S. Patent 6,541,479 B1,
003.
2
1
tion (150 mM, 1.5 mM in the tissue bath). Responses to
all test compounds (at final concentrations of 0.1–
1
1
00 lM in tissue bath) were obtained in the same man-
ner. Each tissue received only one concentration of each
test compound. The results of these experiments were
expressed as percent inhibited contraction of the initial
2
16. Ikeda, Y.; Nitta, Y.; Yamada, K. Yakugau Zasshi 1969,
89, 669.