Novel bisbenzamidines as potential drug candidates for the treatment of Pneumocystis carinii pneumonia

Article abstract of DOI:10.1016/j.bmcl.2004.06.034

A series of pentamidine congeners has been synthesized and screened for their in vitro activity against Pneumocystis carinii. Among the tested compounds, bisbenzamidines linked by a flexible pentanediamide or hexanediamide chain (7 and 9) emerged as excep

Full text of DOI:10.1016/j.bmcl.2004.06.034

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4545–4548
Novel bisbenzamidines as potential drug candidates for the
treatment of Pneumocystis carinii pneumonia
Jean Jacques Vanden Eynde,a Annie Mayence,a Tien L. Huang,a,* Margaret S. Collins,b,c
Sandra Rebholz,b,c Peter D. Walzerb,c and Melanie T. Cushionb,c
aXavier University of Louisiana, College of Pharmacy, Division of Basic Pharmaceutical Sciences, 1 Drexel Drive, New Orleans,
LA70125, USA
bDivision of Infectious Diseases, Department of Internal Medicine, University of Cincinnati, Cincinnati, OH 45267-0560, USA
cResearch Service, Veterans Affairs Medical Center, Cincinnati, OH 45220, USA
Received 19 April 2004; revised 10 June 2004; accepted 10 June 2004
Available online 2 July 2004
Abstract—A series of pentamidine congeners has been synthesized and screened for their in vitro activity against Pneumocystis
carinii. Among the tested compounds, bisbenzamidines linked by a flexible pentanediamide or hexanediamide chain (7 and 9)
emerged as exceptionally potent agents that were more effective and less toxic than pentamidine in the assays described in this
study.
Ó 2004 Elsevier Ltd. All rights reserved.
Pneumocystis carinii pneumonia (PCP) is an opportu-
nistic infection that occurs in individuals with weakened
immune systems due to cancer, HIV/AIDS, inherited
immune deficiencies, or organ transplants as well as in
patients receiving corticosteroids.1 In humans, the
infection is caused by the species, Pneumocystis jirovecii.
PCP does not respond to standard antifungal therapy,
but shows pliancy to some antiprotozoal therapies, for
example, pentamidine. The most effective treatment for
PCP is the well-known trimethoprim–sulfamethoxazole
(TMP–SMX; Bactrime, Septraâ) drugs combination.
Unfortunately almost half of the HIV-positive patients
who take it develop a sulfa allergic reaction necessitating
a switch to other less effective medications2;3 including
pentamidine (NebuPentâ, Pentacarinatâ, 1, Fig. 1),
dapsone (Avlosulfonâ), and atovaquone (Mepronâ).
However, deleterious side effects, limited efficacies, and
emerging resistance are associated with these drugs, thus
justifying the search for more effective and less toxic
agents.
Our groups are interested in the design4–6 and evalua-
tion7–11 of novel bisbenzamidines and other analogs
that are more efficient and less toxic than the parent
compound, pentamidine (1). With that goal in mind
and based on previous works,12–15 we considered 1 as a
bisbenzamidine in which both benzamidine moieties are
linked by a flexible pentamethylene chain and activated
by electron-donating ether functions. Starting from that
simple representation, we first decided to study the
influence of the linking chain by reducing its flexibility as
in compounds 2–4 (Fig. 2). Among these compounds,
2 can be considered to be an analog of pentamidine in
which the degrees of freedom of the linker are reduced
by incorporating three (of five) carbon atoms into a 1,3-
phenylene ring. Derivative 3 is the para analog of 2 and
is characterized by a sequence of six carbon atoms
between the activating ether functions. On the other
hand, 4 is the ortho analog of 2 and in that molecule,
there are only four carbon atoms between the ether
functions. Interestingly, such simple structural changes
have not been reported as factors that could modulate
the anti-Pneumocystis carinii effects of bisbenzamidines.
NH
HN
O
O
NH2
H2N
Pentamidine (1)
Figure 1. Structure of pentamidine.
Keywords: Bisbenzamidine; Pentamidine; Pneumocystis carinii pneu-
monia; Linker; Conformation; Electronic effect.
* Corresponding author. Tel.: +1-504-520-7603; fax: +1-504-520-7954;
e-mail: thuang@xula.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.034

4546
J. J. Vanden Eynde et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4545–4548
HN
NH
LINKER
H2N
NH2
2 - 11
LINKER:
O
O
O
O
O
O
O
O
O
O
O
N
H
N
H
N
H
N
H
N
H
N
H
2
5
6
7
O
N
H
N
H
O
O
NH
NH
O
O
O
O
3
8
9
10
O
O
O
O
4
11
Figure 2. Compounds used in this study.
Pentamidine 1 is thought to exert its anti-P. carinii
effects through binding interactions between the amidine
moieties and macromolecular targets such as DNA.16
The strength of the binding interactions may depend on
the chemical nature (e.g., basicity) of the amidine moi-
eties. The nature of the heteroatoms present in the linker
may influence the chemical properties of the amidine
moieties in the para position by conjugation. To assess
the electronic effects of the heteroatoms in the linker on
the biological activity, we replaced the strong electron-
donating ether functions present in 1 with poor electron-
donating groups, namely amides as in 5–9, ester as in 10,
or an oxymethylene as in 11.
Typical procedures5;11;12;17–19 for the synthesis of 2–11 are
represented in the Scheme 1. Preparation of 2–4, and 11
CN
1)
CH2Br
a
b, c
O
O
+
HO
CN
4
CH2Br
12
13
14
CN
2 and 3 were prepared in a similar way
CN
2)
O
H
N
f
d, e
b, c
COCl
CO2Et
N
H
ClOC
EtO2C
5
O
NC
15
16
17
3)
O
Cl
NH.HCl
NH2
g
HO
+
10
O
Cl
18
19
NH.HCl
NH2
H2N
6 - 9 were prepared in a similar way but starting from
20
4)
CN
O
O
a
OH
OH
b,c
+
BrH2C
CN
11
CN
21
22
23
Scheme 1. Typical procedures for the preparation of the compounds used in this study. Reagents, solvent, temperature, time: (a) tetrabutylam-
monium hydrogen sulfate, NaOH 5 M, dioxane, reflux, 1 h; (b) HCl(g), EtOH, CH2Cl2, rt, 4 d; (c) NH3 (g), EtOH, reflux, 2 h; (d) NaOH 2 M, reflux,
1 h; (e) SOCl2, 60 °C, 30 min; (f) 4-aminobenzonitrile, N-diisopropylethylamine, THF, 0 °C, 16 h; (g) pyridine, dioxane, reflux, 8 h.

J. J. Vanden Eynde et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4545–4548
4547
Table 1. Anti-P. carinii and cytotoxicity of compounds 1–11a
P. carinii. This large selectivity index was not evident for
the other compounds in the study including pentam-
idine.
Compound Anti-P. carinii
activity10;22
P. carinii IC50 in A549 IC50
lM (in lg/mL)
(lM)
1
Markedb
Moderate
Marked
0.5 (0.3)
2.6 (1.2)
1.3 (0.6)
24.0
12.1
In conclusion, the compounds evaluated here suggest
that both conformational and electronic effects of the
linker play an important role in influencing the biolog-
ical properties of the bisbenzamidines. This study
allowed us to identify the simple small molecules 7 and 9
as two very promising therapeutic leads, which deserve
further pre-clinical and clinical testing. We are also
actively pursuing the synthesis and testing of additional
analogs of 7 and 9 for a comprehensive study of their
structure–activity relationships.
2
3
30.2
33.1
4
Marked
Moderate
Marked
1.4 (0.8)
5.3 (2.3)
1.2 (0.6)
5
>526
>120
>0.3
>2280
>0.2
121
6
7
Highly active
Slight
0.003 (0.0013)
22.8 (10.8)
0.002 (0.0009)
13.4 (6.4)
1.3 (0.6)
8
9
Highly active
Moderate
Marked
10
11
44.1
a All biological activities resulted from the average of at least two
determinations.
b Drug activity scale: highly active, <0.01 lg/mL; very marked,
<0.1 lg/mL; marked, 0.1–0.9 lg/mL; moderate, 1.0–9.9 lg/mL; slight,
10.0–49.9 lg/mL.
Acknowledgements
The study was supported by NIH grants DA13546
(T.L.H.), 2S06GM08008 (T.L.H), NO1 AI75319
(P.D.W. and M.T.C.), RO1 AI050450 (M.T.C.) and by
the Medical Research Service, Department of Veterans
Affairs.
was straightforward and involved a nucleophilic sub-
stitution of bromine atoms by phenolate anions fol-
lowed by a Pinner reaction.20 The key step to obtain the
other derivatives is the formation of the diamides 5–9,
or diester 10 from the appropriate diacid chlorides
and substituted anilines or phenol.
References and notes
Compounds 1–11 were evaluated21 against P. carinii in
an ATP detection assay based on the release of bio-
luminescence driven by ATP in a luciferin–luciferase
mediated reaction.10 The results shown in the Table 1
indicated that the compounds exhibited variable anti-P.
carinii activity. However partial immobilization of the
flexible pentyldioxy linker between the benzamidine
functions in 1 was accompanied by a decrease in the
activity, irrespective of whether the substituents on the
phenylene ring are in the ortho, meta, or para positions
(4, 2, or 3, respectively). Interestingly, the meta deriva-
tive (2), which is the closest conformationally restricted
congener of pentamidine, was less active than the para
isomer (3). The situation was reversed when the ether
groups are replaced with amides (2 and 3 vs 6 and 8) and
we noticed that derivatives of terephthalic acid (8 and
10) were poor inhibitors of P. carinii. Remarkably, relief
of any conformational constraint in the diamides series
(5–9) yielded the exceptionally effective lead compounds
7 and 9. These compounds were characterized by IC50
values of 3 and 2 nM, respectively, whereas the value
measured for pentamidine reached 500 nM. It is to be
noted that replacement of the rigid 1,4-phenylene ring
linker in 8 with a flexible butyl chain in 9 led to an
increase in potency by 4 orders of magnitude.
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All compounds were subsequently tested for toxicity to
mammalian cells before consideration for in vivo testing
in the mouse model. The ATP assay was used to eval-
uate the effects of these compounds on the viability of
A549 epithelial lung cell monolayers derived from a
human carcinoma.15 It is noteworthy, from the values
gathered in the Table 1, that all diamides derivatives (5–
9), including the most potent agents 7 and 9, showed no
cytotoxicity at 100 times the IC50 concentration against
16. Gambari, R.; Nastruzzi, C. Biochem. Pharmacol. 1994, 47,
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4548
J. J. Vanden Eynde et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4545–4548
17. Caine, B. F.; Atwell, G. J.; Seelye, R. N. J. Med. Chem.
1969, 12, 199.
(475.32): C, 55.59; H, 4.24; N, 11.79. Found: C,
55.34; H, 4.42; N, 11.53. (M-H-W Laboratories, Phoenix,
AZ).
18. N,N0-Bis[4-(aminoiminomethyl)phenyl]
hexane-1,6-di-
carboxamide, dihydrochloride salt, (9) was obtained by
heating under reflux for 1 h a mixture of 4-aminobenz-
amidine monohydrochloride (20, 0.86 g, 5 mmol), adipoyl
dichloride (0.37 mL, 2.5 mmol), and pyridine (4 mL,
50 mmol) in N,N-dimethyl formamide (20 mL). The pre-
cipitate was filtered and thoroughly washed successively
with water and acetone. Yield: 40%. Mp: 294–295 °C
(decomp.). 1H NMR (DMSO-d6): 10.6 (s, 2H); 9.2 (s, 4H);
9.0 (s, 4H); 7.8 (dd, 8H); 2.4 (br m, 4H); 1.7 (br m, 4H)
ppm. IR: 3096, 1671, 1611, 1523, 1483, 1256 cmÀ1. Anal.
Calcd for C20 H24N6O2Æ2HCl (452.15): C, 52.98; H, 5.78;
N, 18.54. Found: C, 52.91; H, 5.65; N, 18.31. (M-H-W
Laboratories, Phoenix, AZ).
20. Roger, R.; Neilson, D. G. Chem. Rev. 1961, 61, 179.
21. Pneumocystis carinii were obtained from chronically
immunosuppressed Long Evans and Brown Norway rats
housed under conventional conditions at the Cincinnati
VA medical Center (VAMC) or from CD rats (Charles
River Laboratories, Hollister, CA) inoculated intratrach-
eally with P. carinii and maintained under barrier condi-
tions at the University of Cincinnati Laboratory Animal
Medicine Unit (Cincinnati, OH). P. carinii were extracted
and purified from the lungs of rats after 8–12 weeks of
immunosuppression, enumerated, cryopreserved and
stored in liquid nitrogen. Typically, infected rat lungs
yield up to 2 ꢀ 1010 organism nuclei with the vast majority
(about 95%) of the life cycle forms present as trophic
forms with the remainder (about 5%) being composed of
cysts. P. carinii preparations were evaluated for microbial
contamination, ATP content, karyotype, and host cell
content prior to use in the ATP assay. Each concentration
of every compound is assayed in triplicate wells and the
results expressed as the average relative light units.
Triplicate runs for each compound concentration are run
using different organism isolation batches.
19. Bis(4-aminoiminomethylphenyl) benzene-1,4-dicarboxyl-
ate, dihydrochloride salt, (10) was obtained by heating
under reflux for 8 h a mixture of 4-hydroxybenzamidine
hydrochloride (19, 1.72 g, 10 mmol), terephthaloyl chloride
(18, 1.01 g, 5 mmol), and pyridine (1.6 mL, 20 mmol) in
dioxane (50 mL). The precipitate was filtered and washed
successively with water and ethanol. Yield: 45%. Mp: 273–
277 °C. 1H NMR (DMSO-d6): 9.5 (s, 4 H); 9.3 (s, 4 H); 8.4
(s, 4 H); 8.0 (d, 4H, J ¼ 8 Hz); 7.6 (d, 4H, J ¼ 8 Hz) ppm.
IR: 3600–2400 (br), 3030, 1743, 1721, 1668, 1607, 1487,
22. Walzer, P. D.; Ashbaugh, A.; Collins, M.; Cushion, M. T.
Antimicrob. Agents Chemother. 2001, 45, 3234.
1176, 1071 cmÀ1
. Anal. Calcd for C22H18N4O4Æ2HCl