Arginine-pyrimidine conjugates with therapeutic and prophylactic activity in lethal bacterial infections

Article abstract of DOI:10.1016/j.bmcl.2009.09.090

Arginine-pyrimidine conjugates represent a novel class of compounds that exhibits therapeutic and prophylactic activity in lethal infections by Gram-positive and Gram-negative bacteria without showing antibacterial activity in vitro.

Full text of DOI:10.1016/j.bmcl.2009.09.090

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6317–6318
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Arginine–pyrimidine conjugates with therapeutic and prophylactic activity
in lethal bacterial infections
b
a
Dieter Haebich a, , Hein-Peter Kroll , Hans-Georg Lerchen
*
a Medicinal Chemistry, Bayer Schering Pharma AG, D-42096 Wuppertal, Germany
b Antiinfective Research, Bayer Schering Pharma AG, D-42096 Wuppertal, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Arginine–pyrimidine conjugates represent a novel class of compounds that exhibits therapeutic and pro-
phylactic activity in lethal infections by Gram-positive and Gram-negative bacteria without showing
antibacterial activity in vitro.
Received 2 September 2009
Revised 22 September 2009
Accepted 23 September 2009
Available online 27 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterials
Host–pathogen interaction
Immunostimulant
Signal transduction
The discovery and development of the classical antibacterial
agents has been one of medicine’s major achievements.1 But at
present, the efficacy of once powerful drugs is continually lessen-
ing as more and more antibiotic-resistant bacteria emerge.2 Inno-
vative non-traditional approaches—alone or in combination with
classical antibiotics—could help to complement and secure our fu-
ture therapeutic options.3 To better understand the complex inter-
play of bacterial pathogens and the innate immune defense in
infective disease states, it seems wise to also explore host–patho-
gen interactions4 and the quorum sensing cross-talk between mi-
crobes.5 Efficient host defense against bacterial pathogens relies
on immune recognition and coordination of various signaling path-
ways. Bacteria respond to adverse environmental conditions by
adaptive responses that activate stress-dependent regulatory net-
works. The biosynthesis of bacterial virulence factors, special pro-
teins that disturb essential processes of immune defense, is
triggered by such environmental signals. For example, uropatho-
genic Escherichia coli depends on S fimbrial adhesins to bind to
eukaryotic receptors containing sialic acid and to colonize tissues.6
The associated signal transduction mechanism is a trans-acting
process that utilizes a membrane bound signal sensor (transactiva-
tor gene product) and a cytoplasmic regulator which after activa-
tion binds to regulatory regions of the bacterial DNA. In vitro test
systems which use this pathway of bacterial signal transduction
to measure adaptive responses have been developed and used to
discover novel lead structures.7
Here, we report the synthesis and biological profile of novel
arginine–pyrimidine conjugates 1 that exhibit therapeutic and pro-
phylactic activity in lethal infections by Gram-positive and Gram-
negative bacteria without showing antibacterial activity in vitro
(Fig. 1). The conjugates 1 bear some vague structural resemblance
to the natural polyamines spermidine and spermine, which due to
their polycationic structure interact with negatively charged mac-
romolecules and cellular components interfering with cell prolifer-
ation.8 In addition, polycations such as poly-
used as immunostimulants.9
L
-arginine have been
O
NH
N
N
R
N
H2N
N
Me
N
H
NH2
1a: R=H
1b: R=Me
O
NH
NH
N
H
H2N
N
HN
N
H
N
1c
O
* Corresponding author. Tel.: +49 202 368610; fax: +49 202 368149.
E-mail address: dieter.haebich@bayerhealthcare.com (D. Haebich).
Figure 1. New arginine–pyrimidine conjugates. The initial hit 1a was discovered in
an E. coli transactivator assay.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.090

6318
D. Haebich et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6317–6318
Initially, conjugate 1a attracted our attention by reducing the
N
N
N
production of intracellular b-galactosidase in an E. coli transactiva-
tor assay.6,7 Prototype 1a showed no antibacterial activity in vitro
but exhibited efficacy in vivo in lethal murine sepsis models (infec-
tious dose: LD50–LD90) with Gram-negative and Gram-positive
bacteria (Table 1).
Incorporation of an additional N-methyl-group provided conju-
gate 1b which was more stable in mouse serum and showed a pro-
longed half-live in vivo.10 Conjugate 1b exhibited no antibacterial
a
HN
Me
+
Cl
NH
Me
HN
Me
N
Me
N
N
72%
N
2
3
4
5
O
NH
b
Cbz
N
N
Cbz
N
N
Me
H
95 %
NH Me
activity in vitro as well (MIC >250 lg/mL against E. coli and Staph-
Cbz
ylococcus aureus). Intraperitoneal (ip) treatment of lethal infections
by E. coli in mice with 30 mg/kg 0.5 h and 4 h post infection re-
sulted in 80% survival (Table 1). Notably, the observed efficacy de-
pended on the infective dose (inoculum).
c
1b
80%
In an E. coli sepsis, a 1.5 log reduction (7.8 ꢀ 104 to 2.7 ꢀ
103 CFU11/mL) of bacteria from blood was observed 5 h after ip
administration of a single dose of 50 mg/kg of 1b 0.5 h post infec-
tion (pi). Repeated administration at 0.5 h and 4 h pi resulted in a
more pronounced reduction of 2.5 log units (7.8 ꢀ 104 to
3.7 ꢀ 102 CFU/mL). No active metabolites were produced after
incubation of 1b in mouse serum and no antibacterial activity
could be detected in the serum of mice which were treated with
1b. In vivo efficacy in various sepsis models12 strongly depended
on the route of application. While intraperitoneal (ip) and subcuta-
neous (sc) application of 1b showed good efficacy, peroral (po) and
intravenous (iv) application produced only negligible effects. An
active support by the innate immune defense seemed mandatory,
since in immunocompromised mice 1b showed no activity at
all.13 Prophylactic efficacy in a lethal E. coli sepsis model after ip
treatment with 50 mg/kg of 1b could be demonstrated up to 3 h
before infection (Table 2).
Scheme 1. Exemplary synthesis of arginine–pyrimidine conjugate 1b. Reagents and
conditions: (a) 1,4-dioxane, 48 h, rt; (b) (Cbz)3Arg-OH, EDC, HOBt, CH2Cl2, 16 h, rt; (c)
HBr, HOAc, 4 h, rt; basic ion exchange resin M 600. Cbz: benzyloxycarbonyl, EDC: 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide, HOBt: 1-hydroxybenzotriazole.
In summary we have described the synthesis and the prelimin-
ary biological profile of novel arginine–pyrimidine conjugates 1
that exhibit therapeutic and prophylactic activity in lethal infec-
tions by Gram-positive and Gram-negative bacteria without show-
ing antibacterial activity in vitro. In the presence of conjugates 1
bacteria seemed no longer able to adapt to the host environment
and were eradicated by the host immune system. An interference
with the bacterial signal transduction pathway could be a possible
reason. Additional studies are required to fully understand the role
of conjugates 1 in host–pathogen interaction and to elucidate their
mode of action.
Up to 450 mg/kg (ip) no acute and no subchronic toxic effects
were observed in mice over 5 days. Neither an Ames-test nor broad
in vitro and in vivo screening of 1b provided hints for potential
pharmacological or toxicological side effects.
Acknowledgments
We thank K.-D. Bremm, W. Springer, and R. Endermann for
stimulating discussions, and A. DiBetta, M. Torner, and D. Wolter
for technical assistance.
An exemplary synthesis of 1b is shown in Scheme 1. Thus,
nucleophilic substitution of 2-chloropyrimidine 3 by N,N0-dimeth-
ylpropane-1,3-diamine (2) in dioxane gave diamine 4 that was
coupled with tris-benzyloxycarbonyl-protected L-arginine in the
References and notes
presence of EDC and HOBt to afford protected conjugate 5. Cleav-
age of the benzyloxycarbonyl groups by HBr in acetic acid and re-
moval of excessive acid by addition of basic ion exchange resin M
600 provided 1b in three steps and 55% overall yield (Scheme 1).
Structural variations of the guanidine, the pyrimidine and the
diamine spacer afforded less active congeners. However, reduction
of rotational bonds and integration of conformational constraints
in diketopiperazine 1c were tolerated.14
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Table 1
Therapeutic efficacy in lethal murine sepsis models after ip treatment with 30 mg/kg
0.5 h and 4 h post infectiona
6. Schmoll, T.; Ott, M.; Oudega, B.; Hacker, J. J. Bacteriol. 1990, 172, 5103.
7. Danese, P. N.; Snyder, W. B.; Cosma, C. L.; Davis, L. J. B.; Silhavy, T. J. Genes Dev.
1995, 9, 387.
Compound
Survivors (%)
S. aureus
E. coli
8. Keinänen, T. A.; Järvinen, A.; Uimari, A.; Vepsäläinen, J.; Khomutov, A. R.;
Grigorenko, N. A.; Hyvönen, M. T.; Cerrada-Gimenez, M.; Alhonen, L.; Jänne, J.
Mini Rev. Med. Chem. 2007, 7, 813.
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1a
1b
40
60
40
20
40
80
60
40
Ciprofloxacin
Control
10. Conjugate 1b was completely stable in mouse plasma after 3 h at 37 °C.
a
Five mice per group.
Pharmacokinetic parameters, mouse: t1/2 = 1 h, cmax = 56
ip application of 100 mg/kg 1b).
lg/mL (30 min after
11. CFU = colony forming units.
12. Handbook of Animal Models of Infection; Zak, O., Sande, M. A., Eds.; Academic
Press: San Diego, USA, 1999.
13. Wound infection by Pseudomonas aeruginosa or muscle infection by
Streptococcus pyogenes in cyclophosphamide-treated (2 ꢀ 100 mg/kg ip on
day 4 and day 1 before infection) immunocompromised mice.
14. Compared to 1b, bacteria were more efficiently eliminated from the blood of
mice with diketopiperazine 1c. In preliminary studies a superior efficacy in
infection models (E. coli, S. aureus) was observed.
Table 2
Prophylactic efficacy in a lethal murine E. coli sepsis model after ip treatment with
50 mg/kg of 1b before infectiona
Time before infection (h)
Survivors (%)
3
2
1
0.5
80
Control
20
40
60
80
a
Five mice per group.