N,N'-bis[4-(N-alkylamidino)phenyl]homopiperazines as anti-Pneumocystis carinii agents.

Article abstract of DOI:10.1016/S0960-894X(01)00541-8

The synthesis, anti-Pneumocystis carinii activity and DNA binding properties of eight new N,N'-bis[4-(N-alkylamidino)phenyl]homopiperazines are reported. Compounds 2 and 8 were the most potent and caused about 70% inhibition of Pneumocystis carinii growth

Full text of DOI:10.1016/S0960-894X(01)00541-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2679–2681
N,N0-Bis[4-(N-alkylamidino)phenyl]homopiperazines as
Anti-Pneumocystis carinii Agents
Tien L. Huang,a,* Bin Tao,a Yvonne Quarshie,a Sherry F. Queenerb and
Isaac O. Donkorc
aXavier University of Louisiana, College of Pharmacy, New Orleans, LA 70125, USA
bIndiana University, School of Medicine, Indianapolis, IN 46202, USA
cThe University of Tennessee, College of Pharmacy, Memphis, TN 38163, USA
Received 23 July 2001; accepted 27 July 2001
Abstract—The synthesis, anti-Pneumocystis carinii activity and DNA binding properties of eight new N,N0-bis[4-(N-alkylamidi-
no)phenyl]homopiperazines are reported. Compounds 2 and 8 were the most potent and caused about 70% inhibition of Pneumo-
cystis carinii growth in a cell culture model at 1 mM concentrations. # 2001 Elsevier Science Ltd. All rights reserved.
There is considerable interest in the development of
novel aromatic bis-amidines, based on the prototype
agent pentamidine, as potential agents for the treatment
of Pneumocystis carinii pneumonia (PCP).1 These com-
pounds have been shown to exhibit a wide range of
biological activities including antimicrobial, anticancer
and antiviral effects.1,2 The current hypothesis for the
mechanism of action of aromatic bis-amidines is that
they first bind to the minor groove of DNA at AT-rich
sites and subsequently inhibit the normal functions of
DNA-dependent enzymes (e.g., topoisomerases, nuclea-
ses) or they may cause direct inhibition of transcription.3
Pentamidine is the only agent in this chemical class that
is currently used clinically to treat PCP. The major fac-
tor hampering its clinical usage is due to its vast array of
serious adverse reactions especially in patients with
AIDS. There is therefore a critical need for more effica-
cious and less toxic agents to treat AIDS-related PCP.
We have recently reported on the promising anti-P.
carinii activity of several novel conformationally
restricted analogues of pentamidine including N,N0-
bis(4-amidinophenyl)homopiperazine 1.1d In an effort
to enhance the anti-P. carinii activity of this compound,
we have synthesized and evaluated a series of analogues
of 1 in which various alkyl groups have been attached to
the bis-amidinium nitrogens. The rationale for making
*Corresponding author. Tel.: +1-504-483-7603; fax: +1-504-485-
7930; e-mail: thuang@xula.edu
Scheme 1. Reagents used for the synthesis of compounds 2–9: (a) HCl
(g)/EtOH/CHCl3; (b) 1,3-propyldiamine/EtOH; (c) R-NH2/EtOH.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00541-8

2680
T. L. Huang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2679–2681
this structural modification was based on the observa-
tion that substitution of various alkyl groups at the
amidinium nitrogens of 2,5-bis(4-amidinophenyl)furan
resulted in compounds with improved efficacy against
PCP and reduced toxicity.1c The resultant analogues
also showed significantly stronger binding to DNA, and
this has been attributed to increased van der Waals
interactions between the N-alkyl groups and the walls of
the minor groove.1c
obtained from an earlier reported study1d and included
here for comparison purposes.
Compounds 2–9 were evaluated for anti-P. carinii
activity at 1 mM concentrations. Pentamidine, TMP/
SMX and compound 1 were highly effective and caused
total inhibition of P. carinii growth. However, none of
the newly synthesized analogues of 1 were more potent
than 1 or the positive controls. Among this series, com-
pounds 2 and 8 were the most potent and caused about
70% inhibition of P. carinii growth. Compounds 5, 6,
and 7 were less active with inhibition of P. carinii
growth ranging from 35 to 42%. Compounds 3, 4, and 9
were inactive. With the exception of 6, the other com-
pounds exhibited strong binding affinity for both calf
thymus DNA and Poly(dA-dT). A direct correlation
between the DNA binding affinity and the anti-P. carinii
activity was not evident for this series of compounds. It
is interesting to note that the cyclopentyl analogue 9 has
the strongest binding for DNA but was inactive when
tested against P. carinii in culture. However, the cyclo-
pentyl analogue of 2,5-bis(4-amidinophenyl)furan in the
diaryl furan series was not only the strongest DNA
binder but was also the most potent.1c This analogue
was found to be about 100 times more effective than
pentamidine in an animal model.1c These studies clearly
indicate that the nature of the central linker between the
benzamidine groups strongly influences the biological
properties of aromatic bis-amidines. Furthermore, alkyl
substituents on the amidino nitrogens may or may not
The synthesis of compound 1 was reported earlier1d
whereas compounds 2–9 were synthesized using N,N0-
bis(4-cyanophenyl)homopiperazine1d as the key inter-
mediate, as outlined in Scheme 1. The classical Pinner
reaction was employed to convert the bis-nitrile to the
corresponding bis-imidate ester, which was then reacted
with the appropriate alkylamine to form the desired
products. All final compounds exhibited satisfactory
1H NMR and elemental analysis data.4
The anti-P. carinii activity and DNA binding affinities
of the synthesized compounds 2–9 are shown in Table 1.
The usefulness of the P. carinii cell culture model as a
rapid screen in the identification of promising anti-P.
carinii agents has been discussed earlier.1b,d In this
study, the untreated control was used as the negative
control whereas pentamidine (1 mM) and trimethoprim/
sulfamethoxazole (TMP/SMX 50/250 mg/mL) were used
as the positive controls. The data for compound 1 and
pentamidine conducted under experiment
1 were
Table 1. Anti-P. carinii activity and DNA binding affinity of diaryl homopiperazines
Expt, Compd, Concn
Change in numbers of
P. carinii trophozoites from
day 1 through day 7 in
% of control growth
DNA binding (ÁTm, ꢂC)
Calf thymus DNAb
Poly(dA-dT)c
20.6
culture (ꢀ10ꢁ5 a
)
Expt 1d, Control
Pentamidine 1.0 mM
Expt 2, Control
TMP/SMX 50/250 mg/mL
Expt 1,d Compd 1
1.0 mM
Expt 2, Compd 2
1.0 mM
Expt 2, Compd 3
1.0 mM
Expt 2, Compd 4
24.10ꢃ4.02
ꢁ3.98ꢃ1.64
33.23ꢃ3.20
ꢁ0.31ꢃ0.74
< 0
< 0
< 0
32
11.1
15.0
15.5
11.1
17.4
11.8
4.1
23.1
21.3
14.7
23.6
13.3
7.4
ꢁ3.32ꢃ1.60
10.73ꢃ0.51
33.62ꢃ0.39
34.79ꢃ0.66
19.19ꢃ0.59
21.57ꢃ0.8265
20.20ꢃ0.51
10.92ꢃ1.21
34.32ꢃ0.74
101
105
58
1.0 mM
Expt 2, Compd 5
1.0 mM
Expt 2, Compd 6
1.0 mM
Expt 2, Compd 7
1.0 mM
Expt 2, Compd 8
1.0 mM
Expt 2, Compd 9
1.0 mM
13.8
15.3
17.9
20.0
23.6
26.5
61
33
103
aValues are reported as meansꢃSEMs. Values are calculated by subtracting the numbers of organisms on day 1 of culture from the numbers
detected on day 7; thus, positive values denote growth, numbers near zero denote little change, and negative numbers denote decreasing organisms in
the culture.
b,cIncrease in thermal melting of calf thymus DNA and Poly(dA-dT), respectively.
dData for pentamidine and compound 1 are taken from ref 1d in which the change in numbers of P. carinii trophozoites conducted in experiment 1
was from day 1 through day 5 in culture.

T. L. Huang et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2679–2681
2681
enhance the anti-P. carinii activity depending on the
nature of the central linker.
d6) d 1.93 (m, 2H), 3.44 (t, J=4.6 Hz, 4H), 3.51 (m, 4H), 3.61
(t, J=4.6 Hz, 4H), 3.74 (s, 4H), 5.16 (bs, 2H), 6.90 (d, J=8.7
Hz, 4H), 7.65 (d, J=8.0 Hz, 4H), 8.58 (s, 2H), 8.90 (s, 2H),
.
.
9.23 (s, 2H). Anal. calcd C23H32N6O2 2HCl 0.5H2O: C, 54.54;
H, 6.97; N, 16.59; found: C, 54.35; H, 6.80; N, 16.50.
N,N0 -Bis[4-(N-3-hydroxypropylamidino)phenyl]homopiper-
Acknowledgements
ꢂ
1
azine (4): HCl salt, 59% yield, mp 284 C; H NMR (DMSO-
d6) d 1.76 (m, 4H), 1.94 (m, 2H), 3.40 (m, 4H), 3.49 (m, 8H),
3.74 (s, 4H), 4.73 (bs, 2H), 6.91 (d, J=8.8 Hz, 4H), 7.63 (d,
J=8.8 Hz, 4H), 8.57 (s, 2H), 9.04 (s, 2H), 9.30 (s, 2H). Anal.
This study was supported by grants NIDA DA07970,
DA13546 and 2S06GM08008 from the National Insti-
tutes of Health.
.
.
calcd C25H36N6O2 2HCl 2H2O: C, 53.47; H, 7.54; N, 14.97;
found: C, 53.65; H, 7.28; N, 14.94.
N,N0-Bis[4-(N-n-butylamidino)phenyl]homopiperazine (5):
HCl salt, 81% yield, mp >300 ꢂC; 1H NMR (DMSO-d6) d
0.82–0.87 (m, 6H), 1.27–1.32 (m, 4H), 1.51–1.55 (m, 4H), 1.88
(m, 2H), 3.26–3.28 (m, 4H), 3.45 (s, 4H), 3.68 (s, 4H), 6.82 (d,
J=8.7 Hz, 4H), 7.50 (d, J=8.7 Hz, 4H), 8.46 (s, 2H), 8.84 (s,
References and Notes
1. (a) Boykin, D. W.; Kumar, A.; Spychala, J.; Zhou, M.;
Lombardy, R. J.; Wilson, W. D.; Dykstra, C.; Jones, S. K.;
Hall, J. E.; Tidwell, R. R.; Laughton, C.; Nunn, C. M.; Nei-
dle, S. J. Med. Chem. 1995, 38, 912. (b) Huang, T. L.; Zhang,
Q.; White, A. T.; Queener, S. F.; Bartlett, M. S.; Smith, J. W.;
Donkor, I. O. Bioorg. Med. Chem. Lett. 1996, 6, 2087. (c)
Boykin, D. W.; Kumar, A.; Xiao, G.; Wilson, W. D.; Bender,
B. C.; McCardy, D. R.; Hall, J. E.; Tidwell, R. R. J. Med.
Chem. 1998, 41, 124. (d) Tao, B.; Huang, T. L.; Zhang, Q.;
Jackson, L.; Queener, S. F.; Donkor, I. O. Eur. J. Med. Chem.
1999, 34, 531. (e) Francesconi, I.; Wilson, W. D.; Tanious,
F. A.; Hall, J. E.; Bender, B. C.; Tidwell, R. R.; McCurdy, D.;
Boykin, D. W. J. Med. Chem. 1999, 42, 2260.
.
.
2H), 9.14 (s, 2H). Anal. calcd C27H40N6 2HCl 0.6H2O: C,
60.91; H, 8.18; N, 15.79; found: C, 60.88; H, 7.89; N, 15.78.
N,N0-Bis[4-(N-tert-butylamidino)phenyl]homopiperazine (6):
HCl salt, 78% yield, mp 271–273 ꢂC; H NMR (DMSO-d6) d
1
1.26 (m, 18H), 1.98 (m, 2H), 3.43 (m, 4H), 3.67 (s, 4H), 6.76
(q, J=8.8 Hz, 2.8 Hz, 4H), 7.64 (d, J=8.8 Hz, 4H), 7.97 (s,
.
.
4H), 8.40 (bs, 2H). Anal. calcd C27H40N6 2HCl H2O: C, 62.18;
H, 8.12; N, 16.11; found: C, 62.15; H, 7.99; N, 16.24.
N,N0-Bis[4-(N-isopropylamidino)phenyl]homopiperazine (7):
HCl salt, 71% yield, mp >300 ꢂC; 1H NMR (DMSO-d6) d
1.22 (d, J=6.3 Hz, 12H), 1.93 (m, 2H), 3.49 (m, 4H), 3.73 (s,
4H), 4.02(m, 2H), 6.90 (d, J=8.7 Hz, 4H), 7.60 (d, J=8.7 Hz,
4H), 8.66 (s, 2H), 9.09 (m, 4H). Anal. calcd
2. (a) Simpson, I. J.; Lee, M.; Kumar, A.; Boykin, D. W.;
Neidle, S. Bioorg. Med. Chem. Lett. 2000, 10, 2593. (b) Ste-
phens, C. E.; Tanious, F.; Kim, S.; Wilson, W. D.; Schell,
W. A.; Perfect, J. R.; Franzblau, S. G.; Boykin, D. W. J. Med.
Chem. 2001, 44, 1741.
.
.
C25H36N6 2HCl H2O: C, 58.70; H, 7.88; N, 16.43; found: C,
58.92; H, 7.50; N, 16.63.
3. (a) Dykstra, C. C.; McClernon, D. R.; Elwell, L. P.; Tid-
well, R. R. Antimicrob. Agents Chemother. 1994, 38, 1890. (b)
Hildebrandt, E.; Boykin, D. W.; Kumar, A.; Tidwell, R. R.;
Dykstra, C. C. J. Euk. Microbiol. 1998, 45, 112. (c) Fitzgerald,
D. J.; Anderson, J. N. J. Biol. Chem. 1999, 274, 27128.
4. N,N0-Bis[4-(4,5,6-trihydro-1H-1,3-piperidin-2-yl)phenyl]ho-
mopiperazine (2): HCl salt, 78% yield, mp >300 ꢂC; 1H NMR
(DMSO-d6) d 1.91–1.92(m, 6H), 3.40–3.43 (m, 8H), 3.50 (m,
4H), 3.74 (s, 4H), 6.90 (d, J=9.6 Hz, 4H), 7.61 (d, J=9.6 Hz,
4H), 9.64 (bs, 4H).
N,N0 -Bis[4-(N-cyclopropylamidino)phenyl]homopiperazine
(8): HCl salt, 69% yield, mp >300 ꢂC; 1H NMR (DMSO-d6) d
0.76 (m, 4H), 0.89 (m, 4H), 1.91 (m, 2H), 2.71 (m, 2H), 3.50 (s,
4H), 3.73 (s, 4H), 6.87 (d, J=8.8 Hz, 4H), 7.67 (d, J=8.8 Hz,
4H), 8.72 (bs, 2H), 9.31 (bs, 2H), 9.56 (bs, 2H). Anal. calcd
.
.
C25H32N6 2HCl 0.8H2O: C, 59.59; H, 7.12; N, 16.68; found:
C, 59.93; H, 7.05; N, 16.89.
N,N0 -Bis[4-(N-cyclopentylamidino)phenyl]homopiperazine
(9): HCl salt, 73% yield, mp >300 ꢂC; 1H NMR (DMSO-d6) d
1.46–1.64 (m, 12H), 1.94 (m, 6H), 3.46 (s, 4H), 3.68 (s, 4H),
4.04 (m, 2H), 6.90 (d, J=8.7 Hz, 4H), 7.78 (d, J=8.7 Hz, 4H),
8.60 (s, 2H), 9.04 (s, 2H), 9.16 (s, 2H). Anal. calcd
.
.
Anal. calcd C25H32N6 2HCl 0.6H2O: C, 60.02; H, 7.09; N,
16.80; found: C, 60.42; H, 6.75; N, 16.40.
N,N0-Bis[4-(N-2-hydroxyethylamidino)phenyl]homopiperazine
C29H40N6 2HCl 0.6H2O: C, 62.60; H, 7.83; N, 15.10; found:
C, 62.41; H, 7.89; N, 15.43.
.
.
(3): HCl salt, 73% yield, mp 281–283 ꢂC; H NMR (DMSO-
1