Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity

Article abstract of DOI:10.1016/j.bmcl.2009.08.073

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked w

Full text of DOI:10.1016/j.bmcl.2009.08.073

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5884–5886
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of alkanediamide-linked bisbenzamidines
with anti-trypanosomal and anti-pneumocystis activity
a
a
b
Tien L. Huang a, , Jean Jacques Vanden Eynde , Annie Mayence , Margaret S. Collins ,
Melanie T. Cushion b, Donna Rattendi c, Indira Londono c, Lakshman Mazumder c,
Cyrus J. Bacchi c,d, Nigel Yarlett c,e
*
a Xavier University of Louisiana, College of Pharmacy, 1 Drexel Drive, New Orleans, LA 70125, USA
b University of Cincinnati, Dept. of Internal Medicine, Division of Infectious Diseases, Cincinnati, OH 45267, USA
c Pace University, Haskins Laboratories, 1 Pace Plaza, New York, NY 10038, USA
d Dept. of Biological and Health Sciences, 1 Pace Plaza, New York, NY 10038, USA
e Dept. of Chemistry and Physical Sciences, 1 Pace Plaza, New York, NY 10038, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-
sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense
and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibi-
tors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were
the most potent compounds against P. carinii. N,N0-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4
displayed potent inhibition (IC50 = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the
A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the ami-
dine groups in 4 were moved from the para to the meta positions or replaced with amides.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 June 2009
Revised 18 August 2009
Accepted 20 August 2009
Available online 23 August 2009
Keywords:
Benzamidine
Pentamidine
DNA binding
Human African Trypanosomiasis
Trypanosoma brucei
Pneumocystis carinii
The benzamidine scaffold is an important structural motif
found in many bioactive compounds exhibiting antifungal,1,2 anti-
parasitic,3,4 anticoagulant,5 antitumor6 or antiviral7 properties. The
two benzamidine units in the antimicrobial drug, pentamidine, are
attached with a flexible pentyldioxy chain (Fig. 1). Pentamidine is
clinically used in the treatment of pneumonia caused by the oppor-
tunistic fungus, Pneumocystis jirovecii, early stage human African
trypanosomiasis (HAT) and antimony-resistant Leishmaniasis.
However, its clinical use is hampered by lack of oral bioavailability,
and serious adverse effects including hypotension, nephrotoxicity,
abdominal pain, pancreatic complications, cardiotoxicity, and
hypoglycemia which may progress to insulin-dependent diabetes
mellitus.4,8 In recent years, a large number of pentamidine analogs
have been synthesized with the goals of enhancing its useful ther-
apeutic properties, improving its oral bioavailability, and reducing
the undesirable adverse effects.1,3,4,9–13 The design strategy in
many of these studies has focused on modification of the central
pentyldioxy chain, the two aromatic moieties, or the terminal ami-
dine groups.
There is clearly an urgent need to develop more effective and
safer drugs for the treatment of P. jirovecii pneumonia, HAT and
Leishmaniasis. The current chemotherapeutic agents suffer from
drawbacks such as severe toxicities, limited efficacy, poor oral bio-
availability or emergence of microbial resistance. These infectious
diseases threaten millions of people worldwide, and are the major
cause of human suffering and economic burden, especially in
developing countries where these diseases are more prevalent. In
NH
HN
O
O
NH2
H2N
Pentamidine
O
O
NH
HN
N
H
N
H
(CH2)n
: n = 3
NH2
H2N
2
4 : n = 4
* Corresponding author. Tel.: +1 504 520 7603; fax: +1 504 520 7954.
E-mail address: thuang@xula.edu (T.L. Huang).
Figure 1. Structures of pentamidine and lead compounds 2 and 4.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.08.073

T. L. Huang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5884–5886
5885
recent years, the cases of patients dually infected with HIV and
NH.HCl
NH2
1, 2, 4, 6,
8-13
parasitic infections, such as Leishmaniasis, have been growing
worldwide.14 This is of concern since coinfections of HIV with
Leishmania exert a synergistic detrimental effect on the host cellu-
lar immune response, reduce the effectiveness of therapies, greatly
C
NH2
a
14
increases the risk of relapse and mortality rate.
We previously reported on the potent anti-Pneumocystis carinii
activity of a series of bisbenzamidines linked by various linkers.6,15
From these studies, bisbenzamidines linked by a pentanediamide 2
or hexanediamide 4 chain (Fig. 1), emerged as exceptionally potent
agents with limited cytotoxicity to mammalian cells. Screening of
these compounds against various protozoal parasities showed
excellent in vitro activity against Trypanosoma brucei. Conse-
quently, compounds 2 and 4 served as lead compounds for the syn-
thesis of the 11 analogs reported in this Letter. These compounds
are structurally related to pentamidine, in which the strong elec-
tron-donating ether functions of the pentyldioxy chain in pentam-
idine has been replaced by poor electron-donating amide functions
separated by alkanes of increasing chain length (Fig. 1). The effect
of moving the terminal bisamidine functions in 4 from the para to
the meta positions of the phenyl rings on bioactivity was also
investigated.
The synthesized compounds were tested in vitro against a pent-
amidine-sensitive strain of Trypanosoma brucei brucei LAB 110 EA-
TRO16,17, a drug-resistant strain (melarsoprol, pentamidine, and
berenil resistant) of Trypanosoma brucei rhodesiense KETRI
24316,17 and P. carinii.18 The cytotoxicity of the compounds was
evaluated with the A549 human lung carcinoma cell line.18 The
biological data is reported in Table 1.
H2N
NH2
CN
O
C
Cl
O
b
3
(CH2)n C
Cl
c
O
C
NH2
5, 7
Scheme 1. General procedures for the synthesis of compounds 1–13. Reagents and
conditions: (a) DMF, pyridine, reflux, 30 min to 2 h; (b) and (c) Dioxane, rt, stirred
overnight.
was verified by elemental analyses. Representative synthesis and
characterization of compounds 2, 3, and 5 are described.19
The alkanediamide-linked bisbenzamidines were generally
more active against T. brucei than P. carinii with the exception of
compounds 1, 2, and 4. Four of the bisbenzamidines, which are
linked with a hexanediamide 4, heptanediamide 9, octanediamide
10 or decanediamide 12 demonstrated potent activity (IC50
0.001–0.004 M) against both the sensitive and resistant strains
of T. brucei. The potency of these compounds approached that of
pentamidine (IC50 = 0.002 M). The most active compounds gener-
ally showed similar potency against both strains of T. brucei with
the exception of compound 2 which was about 11-fold less active
against the resistant strain. The results indicate that bisbenzami-
dines linked with longer alkanediamide chains were more active
as antitrypanosomal agents. However, those compounds with
shorter alkanediamide chains, namely 1, 2, and 4 were the most
active agents against P. carinii. The potency of compound 1
=
l
l
The synthesis of these compounds is accomplished as shown in
Scheme 1. Starting with the appropriate diacid chlorides and
aminobenzamidine or aminobenzonitrile or aminobenzamide, the
resultant final compounds 1–13 were readily obtained in fair
yields. The average yields for the bisbenzamidines (1, 2, 4, 6, 8–
13) and bisbenzamides (5, 7) were 40% and 90%, respectively.
The chemical structures of the synthesized compounds were con-
firmed by their spectral data (1H NMR and FTIR) and their purity
Table 1
Structure, cytototoxicity, and in vitro inhibitory bioactivity of compounds 1–13 against Trypanosoma brucei and Pneumocystis carinii
O
O
NH
NH2
IC50
N
H
N
H
(CH2)n
Am=
R'
R
Compd
n
R
R0
(l
M)a
T.b. bruceib
T.b. rhodesiensec
P. cariniid
Cytotox. A549e
1
2
3
4
5
6
7
8
9
10
11
12
2
3
3
4
4
4
4
4
5
6
7
8
10
p-Am
p-Am
p-CN
p-Am
p-CO-NH2
m-Am
m-CO-NH2
p-Am
p-Am
p-Am
p-Am
p-Am
p-CN
p-Am
p-CO-NH2
m-Am
m-CO-NH2
m-Am
p-Am
p-Am
p-Am
p-Am
p-Am
9.0
0.009
6.50
0.003
2.80
0.041
NT
0.012
0.002
0.003
0.400
0.002
0.008
0.002
2.19
0.096
7.90
0.002
1.10
0.021
1.97
0.007
0.002
0.001
0.240
0.004
0.007
0.002
0.442
0.003f
NTg
>44.2
2276
NT
1193
NT
0.002f
>100
33.7
>100
2.27
2.37
1.95
2.44
4.28
3.07
NT
>261
>227
>237
176
2.87
249
18
p-Am
p-Am
p-Am
13
Pentamidine
0.50f
24.0f
a
Each value is the average of at least two determinations.
b,c
The Trypanosoma brucei brucei strain was Lab 110 and the Trypanosoma brucei rhodesiense strain was KETRI 243 (see Refs. 16 and 17).
d
e
f
Pneumocytis carinii was obtained from rat lungs (see Ref. 18).
Cytotoxicity with A549 epithelial lung cell monolayers from human carcinoma (see Ref. 18).
Data obtained from Ref. 15.
g
NT, not tested.

5886
T. L. Huang et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5884–5886
(IC50 = 0.442
whereas, compounds 2 (IC50 = 0.003
l
M) was equivalent to pentamidine (IC50 = 0.50
l
l
M),
M)
References and notes
lM) and 4 (IC50 = 0.002
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were 167–250-fold more potent than pentamidine. The remaining
compounds were significantly less active than pentamidine. The
bioactivity against T. brucei or P. carinii was reduced by at least
10-fold or greater if the bisamidine groups in 4 were moved from
the para to the meta positions as in 6 or replaced with amides as
in 5.
In addition to high potency against T. brucei or P. carinii, several
bisbenzamidines exhibited very low cytotoxicity against the A549
cell line, namely, 2, 4, 8, 9, 10, and 12. The selectivity index of these
compounds for T. brucei (SIT.b) or P. carinii (SIP.c) can be defined as
the ratio of the cytotoxic IC50 to the T. brucei or P. carinii IC50 values.
Using this definition, the most selective compounds in this group
5. Phillips, G.; Guilford, W. J.; Buckman, B. O.; Davey, D. D.; Eagen, K. A.;
Koovakkat; S; Liang, A.; McCarrick, M.; Mohan, R.; Ng, H. P.; Pinkerton, M.;
Subramanyam; B; Ho, E.; Trinh, L.; Whitlow, M.; Wu; S; Xu, W.; Morrissey, M.
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against P. carinii were compounds
2
(SIP.c = 758,667) and
4
6. Vanden Eynde, J. J.; Mayence, A.; Johnson, M. T.; Huang, T. L.; Collins, M. S.;
Rebholz, S.; Walzer, P. D.; Cushion, M. T.; Donkor, I. O. Med. Chem. Res. 2005, 14,
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(SIP.c = 596,500), respectively. The selectivity of compounds 2, 4,
8, 9, 10, and 12 for T. brucei ranged from 18,917 to 596,500. The
selectivity indexes of pentamidine for P. carinii and T. brucei were
48 and 12,000, respectively. This indicates that several of the com-
pounds in this series are highly selective for the pathogenic cells (P.
carinii and T. brucei) than for the mammalian cells, and their selec-
tivity profiles are superior to that of pentamidine. Because of their
high potency against P. carinii and low cytotoxicity, compounds 2
and 4 were further evaluated in an immunosuppressed mouse
model of Pneumocystosis.1 When evaluated at different doses (5,
10, 20, and 40 mg/kg), compound 2 emerged as the most promising
anti-Pneumocystis drug. This compound was highly efficacious
against the infection at 20 mg/kg and 40 mg/kg doses, with
>1000-fold reductions in organism burden, and resulted in im-
proved survival curves versus those for pentamidine-treated mice
at the same doses.
9. Huang, T. L.; Vanden Eynde, J. J.; Mayence, A.; Donkor, I. O.; Khan, S. I.; Tekwani,
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L.; Vanden Eynde, J. J. Bioorg. Med. Chem. Lett. 2008, 18, 2658.
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Med. Chem. 2008, 43, 174.
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L.; Ter Horst, R.; Lopez-Velez, R.; Moreno, J. Clin. Micro. Rev. 2008, 21, 334.
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19. The compounds reported in this study were synthesized in the laboratory of
Professor Tien Huang at Xavier University of Louisiana (New Orleans, LA). N,N0-
Bis[4-(aminoiminomethyl)phenyl] pentanediamide, dihydrochloride salt, (2)
The mechanism of action of bisbenzamidines has been attrib-
uted to strong binding to DNA at AT rich sites followed by subse-
quent inhibition of DNA-dependent enzymes and/or direct
inhibition of transcription.8,20–22 In this series of compounds, the
introduction of poor electron-donating amide groups in the central
linker resulted in significant reduction in DNA binding compared
was synthesized by heating under reflux for 30 min
a mixture of 4-
to pentamidine. The binding (DTm) of pentamidine to poly (dA-
aminobenzamidine monohydrochloride (2.08 g, 10 mmol), glutaryl chloride
(0.64 mL, 5 mmol), and pyridine (8 mL, 100 mmol) in N,N-dimethyl formamide
(40 mL). The precipitate was filtered and thoroughly washed successively with
water and acetone. Yield: 50%. Mp: >300 °C (decomp.). 1H NMR (DMSO-d6):
10.5 (s, 2H); 9.1 (br s, 8H); 7.8 (dd, 8H); 2.5 (t, 4H); 1.9 (q, 2H) ppm. IR: 3094;
1923; 1659; 1604; 1351 cmꢀ1. Anal. Calcd for C19H22N6O2ꢁ2HCl (438.13): C,
51.94; H, 5.51; N, 19.13. Found: C, 51.79; H, 5.31; N, 18.89. (M-H-W
Laboratories, Phoenix, AZ).N,N0-Bis(4-cyanophenyl)pentanediamide, (3) was
dT) was 21.6 °C whereas the values ranged from 7.5 °C to 14.4 °C
for the other bisbenzamidines reported here (data not shown).
Although compounds that demonstrated strong potency against
P. carinii were strong binders to DNA (
pounds 1, 2 and 4), several other poor inhibitors of P. carinii (e.g.,
6 and 10) also showed strong binding to DNA ( Tm = 13.8 °C and
13.2 °C, respectively). These results are consistent with our previ-
ous reports3,6,17,18,23,24 which indicated that the anti-P. carinii or
antitrypanosomal activity of a large number of bisbenzamidines
synthesized in our laboratory do not correlate directly with their
DNA binding affinity.
In conclusion, we have synthesized and evaluated the struc-
ture–activity relationships of a series of alkanediamide-linked bis-
benzamidines as potential new agents against T. brucei and P.
carinii. The high selectivity indexes of these compounds against
both pathogens warrant further investigations especially in animal
models of trypanosomiasis. We will report the findings of such
investigations in the near future.
DTm = 11.4–14.4 for com-
D
synthesized by stirring at room temperature overnight
a mixture of 4-
aminobenzonitrile (9.25 g, 80 mmol), and glutaryl chloride (3.38 g, 20 mmol)
in dioxane (200 mL). The precipitate was filtered and washed successively with
water, ethanol and ether. Yield: 68%. Mp: 238–239 °C. 1H NMR (DMSO-d6):
10.3 (s, 2H); 7.7 (2d, 8H, J = 8 Hz); 2.4 (t, 4H); 1.9 (q, 2H) ppm. IR: 3264; 2221;
1667; 1592; 1506; 1442 cmꢀ1. Anal. Calcd for C19H16N4O2 (332.36): C, 68.66;
H, 4.85; N, 16.86. Found: C, 68.83; H, 5.00; N, 16.69. (M-H-W Laboratories,
Phoenix, AZ).N,N0-Bis[4-(aminocarbonyl)phenyl]hexanediamide, (5) was syn-
thesized by stirring at room temperature overnight
a mixture of 4-
aminobenzamide (5.44 g, 40 mmol), and adipoyl chloride (1.83 g, 10 mmol)
in dioxane (100 mL). The precipitate was filtered and washed successively with
water and ethanol. Yield: 90%. Mp: >300 °C. 1H NMR (DMSO-d6): 10.1 (s, 2H);
7.8 (d, 4H, J = 7 Hz); 7.8 (s, 2H); 7.6 (d, 4H, J = 8 Hz); 7.2 (s, 2H); 2.4 (br m, 4H);
1.6 (m, 4H) ppm. IR: 3371; 3317; 3171; 1672; 1651; 1620; 1512 cmꢀ1. Anal.
Calcd for C20H22N4O4 (382.41): C, 62.82; H, 5.80; N, 14.65. Found: C, 62.75; H,
5.76; N, 14.53. (M-H-W Laboratories, Phoenix, AZ).
20. Beerman, T. A.; McHugh, M. M.; Sigmund, R.; Lown, J. W.; Rao, K. E.; Bathini, Y.
Biochem. Biophys. Acta 1992, 1131, 53.
21. Hildebrandt, E.; Boykin, D. W.; Kumar, A.; Tidwell, R. R.; Dykstra, C. C. J.
Eukaryotic Microbiol. 1998, 45, 112.
Acknowledgments
22. Fitzgerald, D. J.; Anderson, J. N. J. Biol. Chem. 1999, 274, 27128.
23. Tao, B.; Huang, T. L.; Zhang, Q.; Jackson, L.; Queener, S. F.; Donkor, I. O. Eur. J.
Med. Chem. 1999, 34, 531.
24. Huang, T. L.; Tao, B.; Quarshie, Y.; Queener, S. F.; Donkor, I. O. Bioorg. Med.
Chem. Lett. 2001, 11, 2679.
This work was supported by Grant 2S06GM08008 from the Na-
tional Institutes of Health. The authors thank Aixa Rodriguez, Ste-
ven DeChiricho and Eric Sorrentino for technical assistance.