Bisbenzamidines as antifungal agents. Are both amidine functions required to observe an anti-Pneumocystis carinii activity?

Article abstract of DOI:10.3390/molecules15064283

A library of 19 novel 4-(4-phenylpiperazine-1-yl)benzamidines has been synthesized and evaluated in vitro against Pneumocystis carinii. Among these compounds, N-ethyl-and N-hexyl-4-(4-phenylpiperazine-1-yl)benzamidines emerged as the most promising compounds, with inhibition percentages at 10.0 μg/mL of 87% and 96%, respectively. Those compounds remained active at 0.1 μg/mL.

Full text of DOI:10.3390/molecules15064283

Molecules 2010, 15, 4283-4293; doi:10.3390/molecules15064283
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molecules
www.mdpi.com/journal/molecules
ISSN 1420-3049
Article
Bisbenzamidines as Antifungal Agents. Are Both Amidine
Functions Required to Observe an Anti-Pneumocystis carinii
Activity?
Julien Laurent 1, Dimitri Stanicki 1, Tien L. Huang 2, Eduardo Dei-Cas 3,4,5, Muriel Pottier 3,5,
El Mouktar Aliouat 3,5 and Jean Jacques Vanden Eynde 1,*
1
Laboratory of Organic Chemistry, Faculty of Sciences, University of Mons-UMONS, 20 place du
parc, B-7000 Mons, Belgium
2
Division of Basic Pharmaceutical Sciences, College of Pharmacy, Xavier University of Louisiana, 1
Drexel drive, New Orleans, LA 70125, USA
3
Department of Parasitology-Mycology, Faculty of Biological and Pharmaceutical Sciences,
University of Lille Nord de France, Lille, France
4
Department of Parasitology-Mycology, Faculty of Medicine, University of Lille Nord de France,
Biology-Pathology Centre, University Hospital Center, Lille, France
5
Biology and Diversity of Emergent Eukaryotic Pathogens (BDEEP) (EA3609), IFR142, Institut
Pasteur de Lille, Lille, France
*
Author to whom correspondence should be addressed; E-Mail: jjvde@umons.ac.be;
Tel.: + 32 65 373337; Fax: + 32 65 373515.
Received: 26 May 2010; in revised form: 1 June 2010 / Accepted: 7 June 2010 /
Published: 11 June 2010
Abstract: A library of 19 novel 4-(4-phenylpiperazine-1-yl)benzamidines has been
synthesized and evaluated in vitro against Pneumocystis carinii. Among these compounds,
N-ethyl- and N-hexyl-4-(4-phenylpiperazine-1-yl)benzamidines emerged as the most
promising compounds, with inhibition percentages at 10.0 µg/mL of 87% and 96%,
respectively. Those compounds remained active at 0.1 µg/mL.
Keywords: pentamidine; Pneumocystis carinii; monobenzamidines

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1. Introduction
Pneumocystis jirovecii pneumonia is a fungal disease that affects immunodeficient individuals and
remains an important cause of mortality in AIDS infected persons [1]. The parasite does not respond to
classical antifungal therapy, but it is sensitive to some antiprotozoal medicines [2]. Currently, available
anti-Pneumocystis drugs are limited by significant problems of efficacy, toxicity and emerging
resistance. Classical treatments to cure the disease include the well-known trimethoprim-
®
®
TM
sulfamethoxazole association (TMP-SMX; Bactrim , Septra ), dapsone (Avlosulfon ), atovaquone
(Mepron®), and pentamidine (NebuPent®, Pentacarinat®) (1, Figure 1). Among those medicines,
pentamidine remains the most effective drug to cure Pneumocystis pneumonia. However major side
effects [1] and a poor bioavailability limit its use.
From a structural point of view, pentamidine (1, Figure 1) can be considered as a bisbenzamidine
derivative in which both benzamidine moieties are linked by a highly flexible pentyldioxy chain. The
structural simplicity of the molecule and its efficacy have encouraged some laboratories [3–6] to
prepare original analogues with the hope to design more efficient and less toxic agents. In previous
work [7,8] we demonstrated that 4, 4’-(1,4-piperazinediyl)bisbenzenecarboximidamide (2, Figure 1) is
a promising candidate characterized by an in vitro IC50 of 2.61 µM against P. carinii (the rat-
associated specie) and exhibiting no in vitro cytotoxicity. In addition, we observed that the
introduction of alkyl substituents on the nitrogen atoms of the amidine functions of 2 could increase up
to 1,000 fold the activity of the parent compound [7,8].
Figure 1. Structure of pentamidine (1) and piperazine-1, 4-bisbenzamidine (2).
HN
NH
HN
NH
O
O
N
N
H2N
NH2
H2N
NH2
1
2
The mechanism of action of pentamidine and other bisbenzamidines analogues remains unclear.
Because bisbenzamidines were shown to bind to the minor groove of DNA [9–11], it was initially
thought that the anti-Pneumocystis properties were linked to the parasite replication. However
derivatives exhibiting high anti-P. carinii activity and a poor affinity for DNA binding have already
been identified. A mitochondrial toxicity has been postulated since it has been shown that same
bisbenzamidines, including pentamidine, could form complexes with heme [12].
In order to gain insight into Structure Activity Relationships (SAR) of bisbenzamidines, we
decided to prepare a library of monobenzamidines structurally related to compound 2 (Figure 2) and to
evaluate their biological behavior against P. carinii.
Figure 2. General structure of the monobenzamidines 5-23 prepared in this study.

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2. Results and Discussion
2.1. Chemistry
The first step in the preparation of such compounds is the nucleophilic displacement of the fluorine
atom in 4-fluorobenzonitrile by the secondary amine function of 4-phenylpiperazine in refluxing DMA
in the presence of K2CO3 as a base (Scheme 1). This step could advantageously be performed in a
laboratory microwave oven so that reaction time can be reduced from 5 hours to 60 minutes.
Conversion of the nitrile compound 3 into the amidines 5–23 was effected by the Pinner reaction
[13], whereby a solution of compound 3 in dichloromethane saturated with gaseous hydrochloric acid
was treated with methanol to afford the imidate intermediate (Scheme 1). This compound was finally
reacted with the appropriate amine to give analytically pure amidines.
Scheme 1. Synthesis of compounds 3-23.
DMA
+
N
NH
F
CN
N
N
CN
K2CO3
3
CH2Cl2 CH3OH / HCl (g)
NH.HCl
NH.HCl
CH3OH
RNH2
N
N
N
N
NHR
OCH3
4
2.2. Biological evaluation
Table 1 contains the results of the in vitro evaluation of pentamidine 1, compound 2, and the
benzamidines 5-23 against P. carinii. Analysis of the data indicated that all compounds retain an
antifungal activity at a concentration of 10 µg/mL, with inhibition percentage ranging from 56% to
96%.
At a low concentration of 0.1 µg/mL, the results are more contrasted. At that concentration, the data
suggest that the activity was dramatically dependent on the nature of the substituent on the amidine
function, as previously described in the bisbenzamidine series [3,4]. In particular, we observed that the
presence of an arylalkyl substituent on the amidine function (compounds 19-23) led to a loss of the
antifungal activity. Among the other derivatives, the most active compounds were those bearing a
linear alkyl group of 1, 2, 3, or 6 carbon atoms (compounds 6-8; 15). Whereas piperazine-1,4-
bisbenzamidine (compound 2) and most of its N-alkyl substituted congeners previously studied were at
least as efficient as pentamidine (compound 1), the situation is a little bit different in the
monobenzamidine series described in the present work. Indeed, all derivatives are less active than
pentamidine. Starting from the unsubstituted compound, introduction of an alkyl chain of increasing
length on the amidine function leads to a modulation of the anti-Pneumocystis activity, with a
maximum of activity observed in the case of ethyl and hexyl substituents. Interestingly, compounds
bearing an alkyl chain constituted by 7, 8, or 12 carbon atoms are not active against the fungus,
contrary to the bisbenzamidine series. Mention should also be made that in the bisbenzamidine series

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as well as in the monobenzamidine series, the N-hexyl substituted candidates emerged among the most
promising substances. That can tentatively be attributed to a favorable compromise between the
hydrophilic properties of the amidine function(s) and the lipophilic character of the alkyl chain(s).
Table 1. Inhibition percentages of the monobenzamidines against P. carinii.
NH
N
N
NHR
Compound
Number
Pentamidine 1
5
Inhibition % at
R
50.0 µg/mL
95.0 2.1
87.0 4.4
10.0 µg/mL
92.0 4.5
80.0 10.0
0.1 µg/mL
76.0 4.9
23.0 5.0
H
CH3
CH2
CH2
62.0 16.5
83.0 8.6
56.0 27.6
87.0 27.2
42.0 12.1
61.0 15.6
6
7
CH3
CH3
88.0 7.0
88.0 4.7
94.0 1.2
90.0 0.6
96.0 0.6
72.0 18.3
90.0 6.9
92.0 0.6
94.0 0.3
91.0 0.6
48.0 31.3
No activity
28.0 14.6
13.0 16.5
No activity
8
2
9
CH2
CH2
CH3
CH3
10
11
12
3
4
97.0 0.6
97.0 1.5
91.0 7.5
92.0 1.1
93.0 4.9
96.0 0.6
No activity
43.0 3.2
56.0 2 .6
13
14
15
CH2
CH2
CH2
CH2
CH3
CH3
CH3
CH3
5
6
7
92.0 2.6
80.0 1 6.0
85.0 12.5
90.0 4.0
93.0 4.3
79.0 9.7
No activity
No activity
No activity
16
17
18
11
93.0 4.1
95.0 2.1
91.0 1.4
No activity
No activity
19
20
94.0 1.15
95.0 2.5
73.0 4.5
96.0 2.3
70.0 9.0
No activity
No activity
21
22
F
96.0 1.5
59.0 4.5
69.0 7.6
66.0 7.5
No activity
32.0 7.0
23
2
F

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Contrary to the observation made in the bisbenzamidine series, the substitution of the amidines
group by an alkyl ramified chain led to a loss of the antifungal activity, except for the analogue bearing
a 1-ethylpropyl substituent.
3. Experimental
3.1. General
1H-NMR spectra were obtained using a Bruker AMX instrument (300 MHz), chemical shifts (δ) are
given in ppm using TMS as internal reference. The following abbreviations are used: br for broad, s
for singlet, d for doublet, t for triplet, q for quadruplet, and m for multiplet. IR spectra were recorded
on a Perkin-Elmer FTIR 1760K. Microwave synthesis were performed in a Milestone Multisynth®
oven. Solvents, reagents, and pentamidine (1) were commercially available (Aldrich, Alfa Aesar,
Acros Organics) and were used without further purification. Compounds 2 [14] and 3 [15] have been
described in the literature. Elemental analyses were performed at the Centre Wallon de Recherches
Agronomiques (Libramont-Chevigny, Belgium) or at the Laboratoire de Microanalyse Organique of
the Institut des Sciences Appliqués de Rouen (France).
3.2. General procedure for the preparation of compound 3 under microwave irradiation
A mixture of 4-fluorobenzonitrile (2.5 mmol; 0.30 g) and 1-phenylpiperazine (2.5 mmol; 0.38 mL;
0.41 gr) in DMA (2.50 mL) in the presence of K2CO3 (2.5 mmol; 0.35 g) was heated 60 minutes at 140
°C in a Multisynth® oven (Milestone) operating at 300 watts. After cooling, the solution was poured
into ice cold water and the precipitate was filtered and washed with water and ethanol.
3.3. General procedure for the preparation of compounds 4–23
A mixture of 4-(4-phenylpiperazine-1-yl)benzonitrile (3, 10 mmol, 2.66 g) in dichloromethane
(250 mL) and methanol (25 mL) was saturated with HCl gas and the reaction medium was left at room
temperature for 24 hours. The precipitate was filtered and thoroughly washed with ether. Without
further purification the crude imidate 4 (3 mmol, 1.0 g) was treated with the appropriate amine in
refluxing methanol for 1 hour. A precipitate was obtained either by cooling or by addition of ether.
4-(4-Phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (5). Prepared by treatment of
the crude imidate 4 with ammonia (15 mmol, 2.5 mL of a 7 N methanolic solution) in refluxing
ethanol (10 mL) for 1 hour. The precipitate obtained after cooling was filtered and washed with ether
1
and water. Yield: 64%. M.p.: >300 °C; H-NMR (DMSO-d6): 9.2 (br, 2H), 9.0 (br, 2H), 7.8 (d, 2H,
J = 9 Hz), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5
(t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz) ppm; IR: 3,073, 2,834, 1,658, 1,607, 1,698, 1,494 cm-1;
C17H20N4·HCl (316.15). Calc.: C, 63.68; H, 6.63; N, 17.47. Found: C, 64.03; H, 6.23; N, 17.37.
N-Methyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (6). Prepared by
treatment of the crude compound 4 with methylamine (15 mmol, 1.8 mL of an ethanolic solution at
33%) in ethanol (10 mL) at reflux for 1 hour. After cooling, the precipitate obtained by addition of
1
ether was filtered and washed with water. Yield: 57%. M.p.: >300 °C; H-NMR (DMSO-d6): 9.7 (br,

Molecules 2010, 15
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1H), 9.3 (br, 1H), 9.0 (br, 1H), 7.8 (d, 2H, J = 9 Hz), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d,
2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz), 3.0 (s, 3H) ppm; IR:
3,441, 3,056, 1,666, 1,505, 1,446, 1,367, 1,236 cm-1; C18H22N4·HCl (330.16). Calc.: C, 65.34; H, 7.01;
N, 16.93. Found: C, 65.52; H, 6.99; N, 16.96.
N-Ethyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (7). Prepared by
treatment of the crude imidate 4 with ethylamine (15 mmol, 7.5 mL of a 2 M methanolic solution) in
refluxing methanol (10 mL) for 1 hour. After cooling, the precipitate obtained by addition of ether was
1
filtered and washed with water. Yield: 47%. M.p.: 280–284 °C; H- NMR (DMSO-d6): 8.8 (br, 3H),
7.8 (d, 2H, J = 9 Hz), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H,
J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4 (q, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.2 (t, J = 7 Hz, 3H) ppm;
IR: 3,051, 2,840, 1,672, 1,604, 1,505, 1,387, 1,235 cm-1; C19H24N4·HCl (344.18). Calc.: C, 66.17; H,
7.31; N, 16.25. Found: C, 65.87; H, 7.27; N, 15.99.
N-Propyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (8). Prepared by
treatment of the crude imidate 4 with propylamine (15 mmol, 1.2 mL) in refluxing ethanol (10 mL) for
1 hour. The precipitate obtained after cooling was filtered and washed with ether and water. Yield:
1
53%. M.p.: 275–280 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8 (d, 2H, J = 9 Hz), 7.3 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4
(q, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.7 (m, 2H, J = 7 Hz), 0.9 (t, 3H, J = 7 Hz); IR: 3,052, 2,970,
2,872, 1,670, 1,505, 1,452, 1,361, 1,234 cm-1; C20H26N4·HCl (358.19). Calc.: C, 66.93; H, 7.58; N,
15.61. Found: C, 66.99; H, 7.33; N, 15.39.
N-Isopropyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (9). Prepared by
treatment of the crude imidate 4 with isopropylamine (15 mmol, 1.3 mL) in refluxing ethanol (10 mL)
for 1 hour. After cooling, the precipitate obtained by addition of ether was filtered and washed with
1
water. Yield: 67%. M.p.: >300 °C; H-NMR (DMSO-d6): 8.7 (br, 3H), 7.8 (d, 2H, J = 9 Hz), 7.3 (t,
2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 4.1 (m, 1H, J = 7 Hz),
3.5 (t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz), 1.3 (d, 6H, J = 5 Hz); IR: 3,410, 3,051, 2,971, 1,666, 1,601,
1,505, 1,385, 1,234 cm-1; C20H26N4·HCl (358.19). Calc.: C, 66.93; H, 7.58; N, 15.61. Found: C, 67.02;
H, 7.63; N, 15.57.
N-Butyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (10). Prepared by
treatment of the crude imidate 4 with butylamine (15 mmol, 1.3 mL) in refluxing ethanol (10 mL) for 1
hour. After cooling, the precipitate obtained by addition of ether was filtered and washed with water.
1
Yield: 32%. M.p.: 280–284 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8 (d, 2H, J = 9 Hz), 7.3 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4
(t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.4 (m, 2H, J = 7 Hz), 0.9 (t, 3H, J = 7
Hz); IR: 3,229, 3,091, 2,951, 1,665, 1,614, 1,520, 1,497, 1,386, 1,225 cm-1; C21H28N4·HCl (372.21).
Calc.: C, 67.63; H, 7.84; N, 15.02. Found: C, 67.59; H, 7.85; N, 15.03.
N-Pentyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (11). Prepared by
treatment of the crude imidate 4 with pentylamine (15 mmol, 1.7 mL) in refluxing ethanol (10 mL) for

Molecules 2010, 15
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1 hour. After cooling, the precipitate obtained by addition of ether was filtered and washed with water.
1
Yield: 30%. M.p.: 285–290 °C. H NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 9 Hz), 7.3 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4
(t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.3 (m, 4H), 0.9 (t, 3H, J = 7 Hz). IR:
3063, 2957, 2857, 1662, 1603, 1505, 1451, 1386, 1336, 1231 cm-1. C22H30N4·1.2 HCl (386,22). Calc.:
C, 67.02; H, 7.98; N, 14.21. Found: C, 66.88; H, 8.04; N, 14.45.
N-(3-Methylbutyl)l 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (12).
Prepared by treatment of the crude imidate with 3-methylbutylamine (15 mmol, 1.7 mL) in refluxing
ethanol (10 mL) for 1 hour. After cooling, the precipitate obtained by addition of ether was filtered and
1
washed with water. Yield: 63%. M.p.: >300 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8 (d, 2H,
J = 9 Hz), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5
(t, 4H, J = 5 Hz), 3.4 (t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.7 (m, 1H), 1.6 (m, 2H), 0.9 (d, 6H,
J = 7 Hz); IR: 3,063, 2,957, 2,857, 1,662, 1,603, 1,505, 1,451, 1,386, 1,336, 1,231 cm-1; C22H30N4·HCl
(386.22). Calc.: C, 68.28; H, 8.07; N, 14.48. Found: C, 68.08; H, 7.84; N, 14.22.
N-(2-Methylbutyl)l 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (13).
Prepared by treatment of the crude imidate 4 with 2-methylbutylamine (15 mmol, 1.8 mL) in refluxing
ethanol (10 mL) for 1 hour. The precipitate obtained after cooling was filtered and washed with ether
1
and water. Yield: 35%. M.p.: >300 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8 (d, 2H, J = 9 Hz), 7.3
(t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5
Hz), 3.4 (d, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.8 (m, 1H), 1.2 (m, 2H), 0.9 (t, 3H, J = 7 Hz), 0.9 (d,
3H,
J = 6 Hz); IR: 3,024, 2,960, 2,844, 1,671, 1,604, 1,515, 1,450, 1,387, 1,230 cm-1; C22H30N4·1.1 HCl
(386.22). Calc.: C, 67.65; H, 8.03; N, 14.34. Found: C, 67.44; H, 7.80; N, 14.11.
N-(1-Ethylpropyl) 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (14).
Prepared by treatment of the crude imidate 4 with 1-ethylpropylamine (15 mmol, 1.8 mL) in refluxing
ethanol (10 mL) for 1 hour. The precipitate obtained after cooling was filtered and washed with ether
1
and water. Yield: 61%. M.p.: >300 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8 (d, 2H, J = 9 Hz), 7.3
(t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.8 (m, 1H), 3.5 (t,
4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 4H), 0.9 (t, 6H, J = 7 Hz); IR: 3,074, 2,966, 2,935, 2,880,
1,666, 1,600, 1,505, 1,231 cm-1; C22H30N4·HCl (386.22). Calc.: C, 68.28; H, 8.07; N, 14.48. Found: C,
68.15; H, 8.08; N, 14.46.
N-Hexyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (15). The compound
was prepared by treatment of the crude imidate 4 with hexylamine (15 mmol, 2.0 mL) in refluxing
ethanol (10 mL) for 1 hour. The precipitate obtained after cooling was filtered and washed with ether
1
and water. Yield: 42%. M.p.: 285–290 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 9 Hz),
7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H,
J = 5 Hz), 3.4 (t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.3 (m, 6H), 0.9 (t, 3H,
J = 7 Hz); IR: 3,050, 2,956, 2,931, 1,669, 1,505, 1,451, 1,387, 1,232 cm-1; C23H32N4·1.1 HCl (400.24).
Calc.: C, 68.27; H, 8.25; N, 13.85. Found: C, 68.31; H, 8.19; N, 13.98.

Molecules 2010, 15
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N-Heptyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (16). Prepared by
treatment of the crude imidate 4 with heptylamine (15 mmol, 2.2 mL) in refluxing ethanol (10 mL) for
1 hour. After cooling, the precipitate obtained by addition of ether was filtered and washed with water.
1
Yield: 43%. M.p.: 290–294 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 9 Hz), 7.3 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4
(t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.3 (m, 8H), 0.9 (t, 3H, J = 7 Hz); IR:
3,078, 2,956, 2,926, 1,661, 1,505, 1,387, 1,387, 1,234 cm-1; C24H34N4·1.1 HCl (414.26). Calc.: C,
68.85; H, 8.45; N, 13.38. Found: C, 69.20; H, 8.09; N, 13.47.
N-Octyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (17). Prepared by
treatment of the crude imidate 4 with octylamine (15 mmol, 2.5 mL) in refluxing ethanol (10 mL) for 1
hour. The precipitate obtained after cooling was filtered and washed with ether and water. Yield: 43%.
M.p.: 280–284 °C; 1H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 9 Hz), 7.3 (t, 2H, J = 7 Hz), 7.1
(d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4 (t, 2H,
J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.3 (m, 10H), 0.9 (t, 3H, J = 7 Hz); IR: 3,062,
2,923, 2,853, 1,662, 1,607, 1,505, 1,386, 1,232 cm-1; C25H36N4·HCl (428.27). Calc.: C, 69.99; H, 8.69;
N, 13.06. Found: C, 69.94; H, 8.68; N, 13.04.
N-Dodecyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (18). Prepared by
treatment of the crude imidate 4 with dodecylamine (15 mmol, 3.5 mL) in refluxing ethanol (10 mL)
for 1 hour. The precipitate obtained after cooling was filtered and washed with ether and water. Yield:
1
51%. M.p.: 275–280 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 9 Hz), 7.3 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 9 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4
(t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 1.6 (m, 2H, J = 7 Hz), 1.4 (m, 18H), 0.9 (t, 3H, J = 7 Hz); IR:
3,047, 2,920, 2,850, 1,668, 1,608, 1,515, 1,466, 1,387, 1,233 cm-1; C29H44N4·HCl (484.33). Calc.: C,
71.79; H, 9.35; N, 11.55. Found: C, 71.77; H, 9.34; N, 11.57.
N-Benzyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (19). Prepared by
treatment of the crude imidate 4 with benzylamine (15 mmol, 1.6 mL) in refluxing ethanol (10 mL) for
1 hour. After cooling, the precipitate obtained by addition of ether was filtered and washed with water.
Yield: 67%. M.p.: 290–295 °C; 1H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 8 Hz), 7.4 (m, 5H),
7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 8 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 4.7 (s, 2H), 3.5
(t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz); IR: 3,033, 2,359, 1,665, 1,516, 1,497, 1,452, 1,388, 1,230 cm-1;
C24H26N4·HCl (406.19). Calc.: C, 70.83; H, 6.69; N, 13.77. Found: C, 70.77; H, 6.66; N, 13.76.
N-Phenethyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (20). Prepared
by treatment of the crude imidate 4 with phenethylamine (15 mmol, 1.9 mL) in refluxing ethanol (10
mL) at for 1 hour. After cooling, the precipitate obtained by addition of ether was filtered and washed
with water. Yield: 64%. M.p.: >300 °C; 1H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 8 Hz), 7.4
(m, 5H), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 8 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.7 (t,
2H, J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz), 3.0 (t, 2H, J = 7Hz); IR: 3,051, 1,605, 1,671,
1,498, 1,452, 1,383, 1,231 cm-1; C25H28N4·0.8 HCl (420.21). Calc.: C, 72.58; H, 7.02; N, 13.54. Found:
C, 72.71; H, 6.89; N, 13.35.

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N-(3-Phenylpropyl) 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (21).
Prepared by treatment of the crude imidate 4 with 3-phenylpropylamine (15 mmol, 2.1 mL) in reluxing
ethanol (10 mL) for 1 hour. After cooling, the precipitate obtained by addition of ether was filtered and
1
washed with water. Yield: 51%. M.p.: 250–255 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H,
J = 8 Hz), 7.4 (m, 5H), 7.3 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 8 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H,
J = 7 Hz), 3.5 (t, 4H, J = 5 Hz), 3.4 (t, 2H, J = 7 Hz), 3.3 (t, 4H, J = 5 Hz), 2.7 (m, 2H), 1.9 (m, 2H);
IR: 3,073, 1,658, 1,607, 1,505, 1,232 cm-1; C26H30N4·HCl (434.22). Calc.: C, 71.79; H, 7.18; N, 12.88.
Found: C, 71.81; H, 7.2; N, 12.86.
N-(4-Fluorobenzyl) 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt (22).
Prepared by treatment of the crude imidate 4 with 4-fluorobenzylamine (15 mmol, 1.7 mL) in
refluxing ethanol (10 mL) for 1 hour. After cooling, the precipitate obtained by addition of ether was
filtered and washed with water. Yield: 58%. M.p.: 280–285 °C; 1H-NMR (DMSO-d6): 9.3 (br, 3H), 7.8
(d, 2H, J = 8 Hz), 7.3 (m, 4H), 7.2 (t, 2H, J = 7 Hz), 7.1 (d, 2H, J = 8 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t,
1H, J = 7 Hz), 4.7 (s, 2H), 3.6 (t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz); IR: 3,039, 1,666, 1,600, 1,515,
1,385, 1,234 cm-1; C24H25FN4·HCl (424.18). Calc.: C, 67.83; H, 6.17; N, 13.18. Found: C, 67.82; H,
6.18; N, 13.17.
N-2-(4-Fluorophenyl)ethyl 4-(4-phenylpiperazine-1-yl)benzenecarboximidamide hydrochloride salt
(23). The compound was obtained by treatment of the crude imidate 4 with 2-(4-fluorophenyl)-
ethylamine (15 mmol, 2.0 mL) in reluxing ethanol (10 mL) for 1 hour. After cooling, a precipitate was
obtained by pouring ether into the solution. The solid was filtered and washed with water. Yield: 64%.
1
M.p.: >300 °C; H-NMR (DMSO-d6): 9.3 (br, 3H), 7.7 (d, 2H, J = 8 Hz), 7.3 (m, 4H), 7.2 (t, 2H,
J = 7 Hz), 7.1 (d, 2H, J = 8 Hz), 7.0 (d, 2H, J = 7 Hz), 6.8 (t, 1H, J = 7 Hz), 3.7 (t, 2H, J = 7 Hz), 3.6
(t, 4H, J = 5 Hz), 3.3 (t, 4H, J = 5 Hz), 3.2 (t, 2H, J = 7 Hz); IR: 3,040, 1,672, 1,604, 1,511, 1,463,
1,452, 1,385, 1,231, 1,206, 1,160 cm-1; C25H27FN4·HCl (438.20). Calc.: C, 68.40; H, 6.43; N, 12.76.
Found: C, 68.41; H, 6.39; N, 12.79.
3.4. Biological evaluation
To determine the in vitro drug susceptibility of P. carinii, axenic cultures of the organism were
produced as follows. All the experiments were carried out in 24-well plates with a final volume of
2 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% of fetal calf serum
containing a final inoculum of 1.0 × 106 organisms per mL. Plates with organisms were incubated for
4 days in an atmosphere of 5% CO2 at 37 °C. P. carinii was quantitated on air dried smears stained
with a rapid panoptic methanol-Giemsa stain (RAL-555), which stains trophic forms, sporocytes and
cysts of Pneumocystis. All susceptibility assays were set up in triplicate. The anti-Pneumocystis
activity of a single concentration of compound may be expressed in terms of percent inhibition,
defined as the decrease (expressed as percentage) in P. carinii forms in antifungal-treated cultures with
respect to the total microorganism count in compound-free culture.

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4. Conclusions
In summary, a library of 19 monobenzamidines linked on a 4-phenylpiperazine-1-yl scaffold has
been synthesized and evaluated in vitro against Pneumocystis carinii. As in a series of bisbenzamidine
analogues, the antifungal activity can easily be modulated by the introduction of appropriate alkyl
substituents on the amidine function. However at the lowest concentration (0.1 µg/mL) we evaluated
those monobenzamidines, only two derivatives (compounds 7 and 15) exhibited a percentage of
inhibition on the growth of P. carinii higher than 50 %. Nevertheless the monobenzamidines reported
in this work are obviously less active than pentamidine and other bisbenzamidines already tested. That
suggests that the presence of both amidine groups are required to observe a marked anti-P. carinii
effect.
Acknowledgements
The authors are grateful to F.R.I.A. (Fonds pour la formation de la Recherche dans l’Industrie et
l’Agriculture; doctoral grant, D.S.) and F.N.R.S. (Fonds National de la Recherche Scientifique;
J.J.V.E.) for financial support.
References and Notes
1. Soiero, M.N.C.; De Souza, E.M.; Stephens, C.E.; Boykin, D.W. Aromatic diamidines as
antiparasitic agents. Expert Opin. Investig. Drugs 2005, 14, 957–972.
2. Vanden Eynde, J.J.; Mayence, A.; Huang, T.L.; Collins, M.S.; Rebholz, S.; Walzer, P.D.;
Cushion, M.T. Novel bisbenzamidines as potential drug candidates for the treatment of
Pneumocystis carinii pneumonia. Bioorg. Med. Chem. Lett. 2004, 14, 4545–4548.
3. Donkor, I.O.; Queener, S.F.; Dalton, J.T. Pentamidine congeners : DNA binding affinity and anti-
Pneumocystis carinii activity of butamidine analogues. Bioorg. Med. Chem. Lett. 1996, 6,
1967–1970.
4. Delia, T.J.; Nagarajan, A.; Queener, S.F.; Bartlett, M.S. Ring-based analogues of pentamidine
versus P. carinii pneumonia in culture. Bioorg. Med. Chem. Lett 1996, 6, 2367–2370.
5. Huang, T.L.; Vanden Eynde, J.J.; Mayence, A.; Collins, M.S.; Cushion, M.T.; Rattendi, D.;
Londono, I.; Mazumder, L.; Bacchi, C.J.; Yarlett, N. Synthesis and SAR of alcanediamide-linked
bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity. Bioorg. Med. Chem.
Lett. 2009, 19, 5884–5886.
6. Wang, S.; Hall, J.E.; Tanious, F.A.; Wilson, W.D.; Patrick, D.A.; McCurdy, D.R.; Bender, B.C.;
Tidwell, R.R. Dicationic dibenzofuran derivatives as anti-Pneumocystis carinii pneumonia agents:
Synthesis, DNA binding affinity, and anti-Pneumocystis carinii activity in immunosupressed rat
model. Eur. J. Med. Chem. 1999, 34, 215–224.
7. Cushion, M.T.; Walzer, P.D.; Collins, M.S.; Rebholz, S.; Vanden Eynde, J.J.; Mayence, A.;
Huang, T.L. Highly active anti-Pneumocystis carinii compounds in a library of novel piperazine-
linked bisbenzamidines and related compounds. Antimicrob. Agents Chemother. 2004, 48,
4209–4216.

Molecules 2010, 15
4293
8. Cushion, M.T.; Walzer, P.D.; Ashbaugh, A.; Rebholz, S.; Brubaker, R.; Vanden Eynde, J.J.;
Mayence, A.; Huang, T.L. In vitro selection and in vivo efficacy of piperazine- and
alkanediamide-linked bisbenzamidines against Pneumocystis pneumonia in mice. Antimicrob.
Agents Chemother. 2006, 50, 2337–2343.
9. Simpson, I.J.; Lee, M.; Kumar, A.; Boykin, D.W.; Neidle, S. DNA minor groove interactions and
the biological activity of 2,5-bis-[4-(N-alkylamidino)phenyl]furans. Bioorg. Med. Chem. Lett.
2000, 10, 2593–2597.
10. Reddy, B.S.P.; Sondhi, S.M.; Lown, J.W. Synthetic DNA minor groove-binding drugs.
Pharmacol. Ther. 1999, 84, 1–111.
11. Cai, X.; Gray, P.J.; Von Hoff, D.D. DNA minor groove binders: back in the groove. Cancer
Treat. Rev. 2009, 35, 437–450.
12. Mayence, A.; Vanden Eynde, J.J.; Huang, T.L. Evidences for the formation of bisbenzamidine-
heme complexes in cell-free systems. Bioorg. Med. Chem. Lett. 2004, 14, 1625–1628.
13. Pinner, A. Ueber die Umwandlung der Nitrile in Imide. Ber. 1885, 18, 2845–2852.
14. Tao, B.; Huang, T.L.; Zhang, Q.; Jackson, L.; Queener, S.F.; Donkor, I.O. Synthesis and anti-
Pneumocystis carinii activity of conformationally restricted analogues of pentamidine. J. Med.
Chem. 1999, 34, 531–538.
15. von der Haar, T.; Hebecker, A.; II’ichev, Y.; Jiang, Y.B.; Kuehnle, W.; Zachariasse, K.A.
Excited-state intramolecular charge transfer in donor/acceptor-substituted aromatic hydrocarbons
and in biaryls. The significance of the redox potentials of the D/A subsystems. Recl. Trav. Chim.
Pays-Bas 1995, 114, 430–442.
Sample Availability: Samples of all compounds are available from the authors.
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(http://creativecommons.org/licenses/by/3.0/).