Parallel Solution-Phase Synthesis of Conformationally Restricted Congeners of Pentamidine and Evaluation of Their Antiplasmodial Activities

Article abstract of DOI:10.1021/jm030545e

Conformationally restricted bisbenzamidines and related congeners have been synthesized and evaluated for activity against two Plasmodium falciparum strains. The most active compounds, bisbenzamidines linked by a 1,4-piperazinediyl core, had IC₅₀

Full text of DOI:10.1021/jm030545e

2
700
J . Med. Chem. 2004, 47, 2700-2705
P a r a llel Solu tion -P h a se Syn th esis of Con for m a tion a lly Restr icted Con gen er s of
P en ta m id in e a n d Eva lu a tion of Th eir An tip la sm od ia l Activities
†
†
‡
‡
Annie Mayence, J ean J acques Vanden Eynde, Fran M. Krogstad, Donald J . Krogstad,
§
,†
Melanie T. Cushion, and Tien L. Huang*
College of Pharmacy, Division of Basic Pharmaceutical Sciences, Xavier University of Louisiana, 1 Drexel Drive,
New Orleans, Louisiana 70125, Departments of Tropical Medicine and Medicine, Center for Infectious Diseases,
Tulane University, 1430 Tulane Avenue, New Orleans, Louisiana 70112, and Department of Internal Medicine,
Division of Infectious Diseases, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0560
Received October 24, 2003
Conformationally restricted bisbenzamidines and related congeners have been synthesized and
evaluated for activity against two Plasmodium falciparum strains. The most active compounds,
bisbenzamidines linked by a 1,4-piperazinediyl core, had IC50 values between 3 and 18 nM
against both chloroquine-susceptible and -resistant parasites and IC50 values for cytotoxicity
greater than 5 µM, using the A549 human lung epithelial cell line. DNA binding affinity, as
estimated by ∆T
m
, did not correlate with either antiparasite effects or cytotoxicity. Each of the
active bisbenzamidines interfered with the formation of hemozoin in cell-free systems.
In tr od u ction
Malaria is the most common parasitic disease in the
world. Each year it affects more than 6% of the global
population (300 million cases), primarily in regions
between the Tropics of Cancer (23.5° north) and Cap-
ricorn (23.5° south). Although most people who become
ill survive after an illness of 10-20 days, malaria kills
between 1 and 2 million people annually, making it one
of the major causes of mortality in developing countries.
Many different drugs have been used to prevent and
treat malaria. The best known, safest, and one of the
1
F igu r e 1. Structures of chloroquine (1) and pentamidine (2).
most effective antimalarials is chloroquine (1; Figure
polycationic compounds to DNA (a polyanion) correlated
with either their antiplasmodial activity or their cyto-
toxicity.
1
), a drug that can be used for both treatment and
chemoprophylaxis. Unfortunately, its intensive use has
led to the development of resistant parasites.2
As part of our research program3-6 focused on the
development of novel pentamidine (2; Figure 1) conge-
ners as potential antimicrobial agents, we decided to
examine their antiplasmodial properties because aro-
matic diamidine compounds have not been studied
extensively for their inhibition of malaria parasite
growth.7
Resu lts a n d Discu ssion
To prepare the congeners of pentamidine (2) examined
in this study, we considered 2 as a molecule containing
two benzamidine groups bound by a flexible chain. In
the first step we decided to modify the nature of the
linker by shifting to more rigid moieties containing a
saturated ring (cyclopropane), a phenylene system
-10
In this paper, we describe the synthesis of a series of
novel bisbenzamidines, which differ from previously
(
meta- or para-), or a saturated heterocycle (homopi-
perazine or piperazine). The parent derivatives 3-7
were synthesized as previously reported.
9
described analogues because the aromatic moieties are
4-6,11-13
Be-
linked by a conformationally restricted structure. We
also report their activity against two Plasmodium
falciparum strains: a chloroquine-sensitive strain (Haiti
cause the piperazine-linked bisbenzamidine 7 was the
most potent antiplasmodial agent in the first set of
compounds, we decided, in a second step, to consider 7
as a lead molecule and to investigate the effect of
modifying the terminal groups of its phenylene rings
on antiplasmodial activity. This led us to prepare the
highly diverse library of 11 derivatives 8-18.
The bisbenzamidines 8-13 were synthesized as de-
scribed for the parent derivative 7 but using the
appropriate amines (R ) butyl, cyclopentyl, cyclohexyl,
isopropyl, benzyl in Scheme 1) or a diamine (1,3-
diaminopropane) instead of ammonia. Compounds 14-
16 were obtained (Scheme 2) from the dialdehyde 21
1
35) and a chloroquine-resistant strain (Indochina I).
The potential toxicity of these compounds against a
human lung epithelial cell line was also evaluated.
Binding to calf thymus DNA and poly(dA-dT) was
measured to determine whether the affinity of these
*
To whom correspondence should be addressed. Phone: 504-520-
7
603. Fax: 504-520-7954. E-mail: thuang@xula.edu.
†
Xavier University.
Tulane University.
University of Cincinnati.
‡
§
1
0.1021/jm030545e CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/14/2004

Congeners of Pentamidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2701
Sch em e 1. Synthesis of Compound 8-12a
nM) with an activity profile superior to pentamidine 2
and chloroquine 1 against both chloroquine-susceptible
and chloroquine-resistant parasites.
Plausible mechanisms of action of bioactive bisbenz-
amidines include initial binding to AT-rich sites in the
1
5
minor groove of DNA followed by inhibition of one or
more of several DNA-dependent enzymes such as topo-
isomerases or direct inhibition of the transcription
1
6,17
process.
However, other modes of action such as
disruption of polyamine metabolism cannot be ruled
1
8,19
out.
In this work we examined the binding affinity
a
Reagents: (a) K2CO3, DMF, reflux; (b) HCl (g), MeOH, CH2Cl2;
of compounds 2-7 for calf thymus DNA and poly(dA-
dT) by measuring the change in midpoint of the thermal
denaturation curves (∆Tm) for a 1:5 compound to DNA
base pair ratio. On the basis of the ∆Tm data in Table
(
c) amine, EtOH, reflux.
(
2
prepared from piperazine 20 and 4-fluorobenzaldehyde
2) and N,N′-dimethylethanediamine, 1,2-dianilinoeth-
ane, or ethylene glycol. The diester 17 was synthesized
Scheme 3) from piperazine (20) and ethyl 4-fluoroben-
1
, the most promising antiplasmodial agent 7 has a
strong binding affinity for DNA and a higher binding
affinity for AT-rich DNA (as indicated by the higher ∆Tm
values obtained for the poly(dA-dT) sample). Despite
this observation, DNA binding is not likely to be a
crucial step in the inhibition of parasite growth. This is
because the DNA binding (∆Tm values) of these com-
pounds did not correlate with their antiplasmodial
activities (IC50 values). For example, although 4 is more
potent than 3, it had a weaker binding affinity for poly-
(
zoate (23). It was then converted to 18 by reaction with
hydrazine.
Biologica l Stu d ies. The antiplasmodial activities of
the compounds synthesized were determined by their
3
ability to inhibit the incorporation of H-hypoxanthine
into nucleic acid via the parasite purine salvage path-
way, as reported previously.14 Each compound was
evaluated for activity against two Plasmodium falci-
parum strains: a cloned chloroquine-susceptible strain
from Haiti (Haiti 135) and a cloned chloroquine-
resistant strain from Indochina (Indochina I). In each
case, chloroquine was used as the control. The results
are reported as the concentrations of the test compounds
(dA-dT).
To assess the possibility that pentamidine and other
diamidines might act against malaria parasites by
binding and inhibiting hemozoin formation, as recently
1
0
proposed by Stead et al., we used infrared spectros-
3
20,21
10
necessary to inhibit the incorporation of H-hypoxan-
copy
and colorimetry to examine the interactions
thine by 50% (IC50, Tables 1 and 2).
between ferriprotoporphyrin(IX) (FPIX) and these com-
pounds in cell-free systems. On the basis of infrared
spectroscopy (monitoring the concentration of hemozoin
by observing the intensity of the band around 1710
Examination of the IC50 values for the first set of
compounds (Table 1) demonstrates the dramatic influ-
ence of the linker on the antiparasite activity of the
bisbenzamidines. Indeed, modification of the linker
permitted the synthesis of a series of bisbenzamidines
ranging in activity from 2000 to 3 nM against the
chloroquine-susceptible strain and from 500 to 4 nM
against the chloroquine-resistant strain. Interestingly,
these compounds had similar activities against both
chloroquine-susceptible (Haiti 135) and chloroquine-
resistant (Indochina I) strains. In contrast, chloroquine
was 33-fold less active against the chloroquine-resistant
Indochina I strain than against the chloroquine-
susceptible Haiti strain. The piperazine-linked bisbenz-
amidine 7 emerged as a highly potent lead (IC50 ) 3-4
-
1
cm , which represents the carbonyl groups on the
porphyrin skeleton), all the benzamidines tested inhib-
ited the formation of hemozoin in acidic acetate solu-
tions mimicking the pH of the parasitic food vacuole,
while 14-18, which lack the amidine functionality, did
not inhibit hemozoin formation. Colorimetric measure-
ments (monitoring the concentration of FPIX in solution
by measuring its absorbance at 400 nm) also demon-
strated that benzamidines formed complexes with fer-
riprotoporphyrin(IX). These results suggest that the
mechanism of diamidine action against Plasmodium
falciparum parasite may be different from their mech-
Sch em e 2. Synthesis of Compounds 14-16a
a
Reagents: (a) K2CO3, DMF, reflux; (b) for 14 N,N′-dimethylethanediamine, PTSA, benzene, Dean-Stark apparatus; for 15
,2-dianilinoethane, AcOH, EtOH reflux; for 16 ethylene glycol, PTSA, benzene, Dean-Stark apparatus.
1

2
702 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Mayence et al.
Ta ble 1. In Vitro Antiplasmodial Activity, Cytotoxicity, and DNA Binding Affinity for Compounds 1-7a
a
All biological activities resulted from the average of at least two determinations. b From the NCI database: http://dtp.nci.nih.gov/
webdata.html.
Sch em e 3. Synthesis of Compounds 17 and 18a
carboxhydrazide (18) groups abolished their antipara-
site activity. This second result suggests that the
terminal groups of biologically active derivatives may
need to contain basic, nonsterically hindered amidine
moieties.
These bisbenzamidines were also screened for toxicity
against mammalian cells in vitro, using the A549
human lung epithelial cell line (Table 2). Each of the
active compounds tested had low cytotoxicity for the
A549 cell line (IC50 values up to 1000-fold higher than
the IC50 values of these compounds for the two strains
of Plasmodium falciparum).
a
Reagents: (a) K2CO3, DMF, reflux; (b) NH2NH2‚H2O, EtOH,
Con clu sion s
H2O, reflux.
Aromatic diamidines are prescribed widely for the
treatment of fungal and protozoal infections, despite
their major side effects. However, the diamidines have
not been evaluated extensively as potential antimalari-
als. The results reported here demonstrate that pipera-
zine-linked bisbenzamidines are highly active against
both chloroquine-susceptible and chloroquine-resistant
Plasmodium falciparum and indicate that they inhibit
the formation of hemozoin in cell-free systems. The most
efficient compounds 7-13 were 3- to 11-fold more active
than pentamidine (2) and were 11- to 50-fold more active
than chloroquine (1) against the chloroquine-resistant
Indochina I strain of Plasmodium falciparum. Thus, the
1
7,22,23
anisms of action against Trypanosoma brucei
and
Leishmania donovani.1
9,23,24
The second set of derivatives is a library of 1,4-
diarylpiperazines with alkylated amidines and other
substituents in the para position on the phenylene ring.
These compounds were prepared on the basis of the
promising antiparasite properties of the lead molecule
7
. The results are tabulated in Table 2.
The bis (N-alkylbenzenecarboximidamides) 8-12 had
similar inhibitory activities (IC50 values) against both
chloroquine-susceptible and chloroquine-resistant para-
site strains. Similar findings were obtained for 13, in
which the amidine moieties are within a cyclic structure.
These observations indicate that the antiplasmodial
activity of the benzamidines is not affected by the pfcrt-
4
,4′-(piperazine-1,4-diyl)bisbenzamidines are a promis-
ing, novel class of compounds with potential antima-
larial activity.
2
5
based efflux mechanism responsible for chloroquine
resistance in Plasmodium falciparum.2 In addition,
replacement of the amidine moieties in these compounds
by imidazolidine (14, 15), dioxolane (16), ester (17), or
Exp er im en ta l Section
1H NMR spectra were obtained using a Varian Inova
instrument (500 MHz), and chemical shifts (δ) are given in
ppm using TMS as the internal reference. IR spectra were

Congeners of Pentamidine
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10 2703
Ta ble 2. In Vitro Antiplasmodial Activity, Cytotoxicity, and DNA Binding Affinity for Compounds 1, 2, and 7-18a
a
All biological activities resulted from the average of at least two determinations. b From the NCI database: http://dtp.nci.nih.gov/
webdata.html.
recorded on a Perkin-Elmer Spectrum One instrument operat-
ing in the diffuse reflectance mode. Solvents and reagents are
commercially available (Aldrich Co., Acros Organics, Fisher
Scientific, Sigma Chemical Co.) and were used without further
purification.
ate, obtained as above, was treated with cyclopentylamine (40
mmol, 4.0 mL) in refluxing ethanol (50 mL) for 90 min. After
cooling, the mixture was concentrated under reduced pressure
and the residue was successively washed with DMF and
1
ether: 30% yield; mp >300 °C; H NMR (DMSO-d
6
) δ 9.2-8.7
Compounds 3, 4,11 5,4,12,13 6,4,5 7,4,13 and 2126 have been
described in the literature. Elemental analyses were performed
by M-H-W Laboratories, Phoenix, AZ.
4
,6
(bs, 6 H), 7.6 (d, 4 H, J ) 9 Hz), 7.1 (d, 4 H, J ) 9 Hz), 4.1 (m,
2 H, J ) 7 Hz), 3.5 (s, 8 H), 2.0 (m, 4 H, J ) 7 Hz), 1.7-1.5 (m,
12 H); IR 3062, 1668, 1601, 1515, 1395, 1232 cm . Anal.
-
1
Gen er a l P r oced u r e for th e P r ep a r a tion of th e 4,4′-(1,4-
P ip er a zin ed iyl)bisben zen eca r boxim id a m id es 8-13. A
(C28
H
38
N
6
2
‚2HCl‚H O) C, H, N.
4,4′-(1,4-P ip er a zin ed iyl)bis(N-cycloh exylben zen eca r -
boxim id a m id e), Dih yd r och lor id e Sa lt 10. The crude im-
idate, obtained as above, was treated with cyclohexylamine
(20 mmol, 2.3 mL) in refluxing ethanol (50 mL) for 30 min.
After the mixture was cooled, the precipitate was filtered and
successively washed with water, ethanol, and ether: 25% yield;
mp >300 °C; H NMR (DMSO-d
2
7
mixture of 4,4′-(1,4-piperazinediyl)bisbenzonitrile (2 mmol,
0
.6 g) in dichloromethane (250 mL) and methanol (10 mL) was
saturated with HCl gas, and the reaction medium was left at
room temperature for 4 days. The precipitate (crude imidate)
was filtered, washed with acetone, and treated with the
appropriate (di)amine.
1
6
) δ 9.2 (bs, 4 H), 9.1 (bs, 2 H),
4
,4′-(1,4-P ip er a zin ed iyl)bis(N-bu tylben zen eca r boxim -
7.7 (d, 4 H, J ) 9 Hz), 7.1 (d, 4 H, J ) 9 Hz), 3.5 (m, 2 H), 3.5
(s, 8 H), 1.9 (d, 4 H, J ) 8 Hz), 1.8 (d, 4 H, J ) 8 Hz), 1.6 (d,
2 H, J ) 12 Hz), 1.4 (m, 8 H), 1.1 (m, 2 H); IR 3053, 1673,
id a m id e), Dih yd r och lor id e Sa lt 8. The crude imidate,
obtained as above, was treated with butylamine (20 mmol, 2.0
mL) in refluxing ethanol (50 mL) for 30 min. After the mixture
was cooled, the solid was filtered and successively washed with
-
1
1606, 1516, 1234 cm . Anal. (C30
42 6
H N ‚2HCl) C, H, N.
4,4′-(1,4-P ip er a zin ed iyl)bis[N-(1-m eth yleth yl)ben zen e-
ca r boxim id a m id e], Dih yd r och lor id e Sa lt 11. The crude
imidate, obtained as above, was treated with isopropylamine
(50 mmol, 4.3 mL) in refluxing ethanol (50 mL) for 90 min.
After cooling, the mixture was concentrated under reduced
pressure and the residue was successively washed with DMF
and ether: 40% yield; mp >300 °C; H NMR (DMSO-d
8.8 (bs, 6 H), 7.7 (d, 4 H, J ) 9 Hz), 7.1 (d, 4 H, J ) 9 Hz), 4.0
1
water, ethanol, and ether: 25% yield; mp >300 °C; H NMR
(
6
DMSO-d ) δ 9.2 (bs, 6 H), 7.7 (d, 4 H, J ) 9 Hz), 7.1 (d, 4 H,
J ) 9 Hz), 3.5 (s, 8 H), 3.4 (t, 4 H, J ) 8 Hz), 1.6 (m, 4 H, J )
8
1
Hz), 1.4 (m, 4 H), 0.9 (t, 6 H, J ) 8 Hz); IR 3065, 1671, 1614,
518, 1383, 1231 cm-1. Anal. (C26
‚2HCl) C, H, N.
,4′-(1,4-P ip er a zin ed iyl)bis(N-cyclop en tylben zen eca r -
boxim id a m id e), Dih yd r och lor id e Sa lt 9. The crude imid-
H
38
N
6
1
4
6
) δ 9.1-

2
704 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 10
Mayence et al.
(
1
m, 2 H), 3.5 (s, 8 H), 1.2 (d, 12 H, J ) 7 Hz); IR 3077, 1672,
602, 1516, 1232 cm-1. Anal. (C24
‚2HCl) C, H, N.
,4′-(1,4-P ip er a zin ed iyl)bis[N-(p h en ylm eth yl)ben zen e-
ester) (17, 0.4 g, 1 mmol), water (0.5 mL), ethanol (2.5 mL),
and hydrazine hydrate (5 mL) was heated under reflux for 8
h. After the mixture was cooled, the precipitate was filtered
and re-treated under the same experimental conditions. The
34 6
H N
4
ca r boxim id a m id e], Dih yd r och lor id e Sa lt 12. The crude
imidate, obtained as above, was treated with benzylamine (20
mmol, 2.2 mL) in refluxing ethanol (50 mL) for 30 min. After
the mixture was cooled, the precipitate was filtered and
successively washed with water, ethanol, and ether: 50% yield;
mp >300 °C; H NMR (DMSO-d
8
J ) 7 Hz), 7.1 (d, 4 H, J ) 8 Hz), 4.7 (s, 4 H), 3.6 (s, 8 H); IR
3
2
precipitate was filtered and washed with water: 80% yield;
1
mp >300 °C; H NMR (DMSO-d
6
) δ 9.6 (bs, 2H), 7.9 (d, 4 H,
J ) 8 Hz), 7.0 (d, 4 H, J ) 8 Hz), 4.5 (bs, 4 H), 3.5 (s, 8 H); IR
3307, 2982, 2848, 1690, 1640, 1602, 1278, 940 cm . Anal.
) C, H, N.
-
1
1
6
) δ 10.0 (s, 2 H), 9.3 (s, 2 H),
22 6 2
(C18H N O
.9 (s, 2 H), 7.8 (d, 4 H, J ) 8 Hz), 7.4 (m, 8 H), 7.3 (t, 2 H,
An tip la sm od ia l Activity. The parasite strains used for
these studies were a cloned chloroquine-susceptible parasite
-
1
099, 1668, 1607, 1518, 1384, 1235 cm . Anal. (C32
HCl) C, H, N.
H
34
N
6
‚
28,29
from Haiti (Haiti 135)
and a cloned chloroquine-resistant
29,30
parasite from Indochina (Indochina I).
In vitro antiplas-
1
,4-Bis[4-(1,4,5,6-tetr a h yd r op yr im id in -2-yl)p h en yl]p i-
modial activity was determined by the ability of a compound
p er a zin e, Dih yd r och lor id e Sa lt 13. The crude imidate,
obtained as above, was treated with 1,3-diaminopropane (30
mmol, 2.5 mL) in refluxing ethanol (50 mL) for 90 min. After
the mixture was cooled, the precipitate was filtered and
successively washed with DMF and ether: 25% yield; mp >300
°
to inhibit parasite growth, measured by 3H-hypoxanthine
incorporation and based on the parasite’s requirement for the
31
purine salvage pathway. Synchronous cultures of early ring-
stage parasites (600 µL of a 2.5% red blood cell suspension
with 0.2% parasitemia in 24-well cell culture plates; Corning
1
C; H NMR (DMSO-d
6
) δ 9.5 (bs, 4 H), 7.6 (d, 4 H, J ) 9 Hz),
32
Inc., Corning, NY) were grown in an in vitro culture system
7
.1 (d, 4 H, J ) 9 Hz), 3.5 (s, 8 H), 3.4 (t, 8 H, J ) 5 Hz), 1.9
with varying concentrations of the compounds being tested
without supplemental hypoxanthine for 48 h. After a routine
medium change using the same drug concentrations at 24 h,
-
1
(
m, 4 H, J ) 5 Hz); IR 3271, 1638, 1602, 1516, 1231 cm
Anal. (C24 ‚2HCl‚2H O) C, H, N.
,4-Bis(1,3-d im eth ylim id a zolid in e-2-yl)p ip er a zin e 14.
.
H
30
N
6
2
1
3
the medium change at 48 h included 2 µCi of H-hypoxanthine
2
6
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde (21, 2.9
g, 10 mmol), N,N′-dimethylethylenediamine (85%, 2.1 g, 20
mmol), and p-toluenesulfonic acid (100 mg) in benzene (100
mL) was heated under reflux in a Dean-Stark apparatus for
(NEN Perkin-Elmer, Boston, MA). The plates were then
incubated for an additional 12-14 h, after which they were
harvested (model 1100 Cell Harvester, Skatron, Sterling, VA)
on glass microfiber filters (934AH, Whatman, Clifton, NJ ). The
glass fiber microfilter disks were placed in scintillation vials
with 8 mL of scintillation fluid (Cytoscint, ICN, Costa Mesa,
CA) and counted in a liquid scintillation counter (Packard
TriCarb 2100 TR, Perkin-Elmer, Downers Grove, IL). The 50%
inhibitory concentration (IC50) was the concentration that
5
h. After the mixture was cooled, the solvent was evaporated
under reduced pressure to afford a solid, which was recrystal-
lized from acetonitrile: 70% yield; mp 204-206 °C; H NMR
(
4
2
1
3
CDCl ) δ 7.4 (d, 4 H, J ) 9 Hz), 7.0 (d, 4 H, J ) 9 Hz), 3.4 (m,
H), 3.3 (s, 8 H), 3.2 (s, 2 H), 2.5 (m, 4 H), 2.2 (s, 6 H); IR
940, 2826, 1611, 1515, 1449, 1227, 1043, 1032 cm-1. Anal.
) C, H, N.
,4-Bis(1,3-d ip h en ylim id a zolid in e-2-yl)p ip er a zin e 15.
3
inhibited parasite growth ( H-hypoxanthine accumulation) by
26 38 6
(C H N
5
0% in relation to the drug-free control. After a screening to
identify the biologically active range, repeat testing was
performed in duplicate to determine the actual IC50
In ter a ction w ith F er r ip r otop or p h yr in (IX) in a Cell-
1
2
6
A mixture of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde (21, 2.9
g, 10 mmol), dianilinoethane (8.5 g, 40 mmol), and acetic acid
(
h. After the mixture was cooled, the precipitate was filtered,
washed with acetone, and recrystallized from a mixture (1:1)
.
10 mL) in ethanol (100 mL) was heated under reflux for 16
F r ee System .1
0,20,21
The procedures described in the literature
were followed by using compounds 1-18 and were performed
in duplicate. A detailed study of the results will be reported
elsewhere.33
1
of ethanol and dioxane: 75% yield; mp 250-255 °C (dec); H
NMR (DMSO-d ) δ 7.4 (d, 4 H, J ) 8 Hz), 7.1 (t, 8 H, J ) 8
6
Hz), 6.8 (d, 4 H, J ) 8 Hz), 6.7 (d, 8 H, J ) 8 Hz), 6.6 (t, 4 H,
J ) 8 Hz), 6.1 (s, 2 H), 3.9 (m, 4 H), 3.7 (m, 4 H), 3.6 (s, protons
DNA Bin d in g Affin ity Mea su r em en ts. Thermal dena-
turation curves were determined by the procedure described
in the literature.3
4,35
Each ∆Tm value reported in the tables
from the dioxane of recrystallization), 3.1 (s, 8 H); IR 3071,
963, 2826, 1599, 1482, 1236, 750 cm-1. Anal. (C46
H
N
‚ /
1
2
2
46
6
represents the mean of at least two experimental determina-
tions.
C
4
H
8
O
2
) C, H, N.
1
,4-Bis[4-(1,3-d ioxola n e-2-yl)p h en yl]p ip er a zin e 16. A
Cytotoxicity Eva lu a tion . A bioluminescent ATP assay
2
6
mixture of 4,4′-(1,4-piperazinediyl)bisbenzaldehyde (21, 2.9
g, 10 mmol), ethylene glycol (10 mL, 180 mmol), and p-
toluenesulfonic acid (100 mg) in benzene (100 mL) was heated
under reflux in a Dean-Stark apparatus for 7 h. After the
mixture was cooled, the precipitate was filtered, successively
washed with benzene and dichloromethane, and recrystallized
was used to determine the cytotoxic effects of the compounds
36,37
on human lung cell monolayers.
Confluent monolayers of
lung cell carcinoma A549 (ATCC 185) were established in 24-
well plates containing 1 mL of DMEM plus High Glucose (4.5
g/L) (Fisher Scientific Inc., Cincinnati, OH) with 10% fetal
bovine serum (Fisher Scientific), 0.1 mM nonessential amino
acids, L-glutamine (0.2 mM), 1X MEM vitamins, and 1.1 µg/
mL sodium pyruvate (Fisher Scientific). Media containing
varying concentrations of each test compound were added to
individual wells in triplicate and were harvested in triplicate
at each time point tested. Medium alone was the negative
control and antimycin A was the positive control. After
incubation and appropriate treatment, 5 µL aliquots were
placed directly into the individual wells of a 96-well opaque
white plate containing 100 µL of buffer (200 mM Tris, 2.5 mM
EDTA, pH 7.75), which were then placed in a FluoSTAR
Optima plate reader (BMG Labtechnologies, Inc.). Samples
were automatically mixed with the luciferin/luciferase reagent
via an injector and immediately measured for light emission
at 562 nm.
1
from benzene: 55% yield; mp 246-249 °C; H NMR (DMSO-
d
4
1
6
) δ 7.3 (d, 4 H, J ) 8 Hz), 7.0 (d, 4 H, J ) 8 Hz), 5.6 (s, 2 H),
.0 (m, 4 H), 3.9 (m, 4 H), 3.3 (s, 8 H); IR 3045, 2960, 2879,
615, 1523, 1406, 1187, 1038, 830 cm-1. Anal. (C22
H N O )
26 2 4
C, H, N.
4
,4′-(1,4-P ip er a zin ed iyl)bis(ben zoic a cid eth yl ester )
1
4
7. A mixture of piperazine (20, 0.85 g, 10 mmol), ethyl
-fluorobenzoate (23, 3.36 g, 3.0 mL, 20 mmol), and potassium
carbonate (2.8 g, 20 mmol) in DMF (10 mL) was heated under
reflux for 8 h. After the mixture was cooled, water (20 mL)
was added to precipitate the final product that was filtered,
successively washed with water and ethanol, and recrystallized
from acetone: 40% yield; mp 199-200 °C; H NMR (CDCl
.9 (d, 4 H, J ) 8 Hz), 6.9 (d, 4 H, J ) 8 Hz), 4.4 (m, 4 H, J )
Hz), 3.5 (s, 8 H), 1.4 (t, 6 H, J ) 9 Hz); IR 2981, 2907, 2846,
710, 1688, 1601, 1519, 1446, 1402 cm-1. Anal. (C22
H N O )
26 2 4
1
3
) δ
7
9
1
Ack n ow led gm en t. The study was supported by
NIH Grants DA07970, DA13546, and 2S06GM08008 to
T.L.H. and by a Cooperative Agreement for Antimalarial
C, H, N.
,4′-(1,4-P iper azin ediyl)bisben zen ecar boxh ydr azide 18.
A mixture of 4,4′-(1,4-piperazinediyl)bis(benzoic acid ethyl
4

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