Studies on the novel anti-staphyloccal compound nematophin

Article abstract of DOI:10.1016/S0960-894X(00)00331-0

A number of analogues of the recently described compound nematophin were prepared and studied for antibacterial activity. The 2-phenyl derivative was found to exhibit exceptional activity against methicillin resistant Staphylococcus aureus (MRSA) whereas the isosteric benzimidazole analogue was much less active. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00331-0

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1751±1754
Studies on the Novel Anti-staphyloccal Compound Nematophin
Gordon Kennedy,* Monica Viziano, John Alexander Winders,,
Palmina Cavallini, Monica Gevi, Fabrizio Micheli, Pamela Rodegher,
Pierfausto Seneci and Aurelia Zumerle
Medicines Research Centre, GlaxoWellcome SpA, Via Fleming 4, Verona 37100, Italy
Received 31 March 2000; accepted 6 June 2000
AbstractÐA number of analogues of the recently described compound nematophin were prepared and studied for antibacterial
activity. The 2-phenyl derivative was found to exhibit exceptional activity against methicillin resistant Staphylococcus aureus
(MRSA) whereas the isosteric benzimidazole analogue was much less active. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
The work of the Bayer group has established that sub-
stitution of the indole by a smaller heterocycle such as
pyridine or imidazole leads to inactive compounds, how-
ever, no data is available for sca€olds which are known to
be (bio)isosteric with indoles. They have also demon-
strated the requirement for the a-ketoamide moiety within
the molecule and that the indole hydrogen can be replaced
by either an aryl, alkyl or benzyl group (e.g., 2) with con-
comitant increase in in vitro anti-staphylococcal activity
in the absence of serum.
The alarming rise in the incidence of nosocomial
acquired infections caused by antibacterial resistant
organisms has become a cause for increasing concern
over the last few years. It has been estimated that at
least half of the approximately two million patients who
acquire infections within the hospital environment are
infected with bacteria resistant to most current antibiotic
treatments.1 Bacteraemia caused by the Gram positive
organism Staphylococcus aureus is a signi®cant cause of
morbidity in intensive care units and there is a real need
for the identi®cation of both novel essential targets within
bacteria, as well as new agents capable of combatting
these pathogens.2,3
Results and Discussion
The parent nematophin and the N-benzyl derivative 2
were prepared as standards according to the Bayer refer-
ence. The Suzuki cross-coupling reaction has recently
been applied to N-phthalimide protected 2-bromotrypt-
amine for the synthesis of biologically active molecules
and was used here to introduce aryl groups as shown in
Scheme 1.7 Bromination of the indole nucleus with pyr-
idinium tribromide required careful monitoring by
HPLC to ensure a good yield of the monobromo com-
pound and to avoid over-halogenation. The 2-(3-pyridyl)
derivative 5b was quaternized with both iodomethane
and iodoacetamide to give the compounds 6a and 6b
containing charged substituents.
Nematophin, 1, is a novel antibacterial compound isolated
from Xenorhabdus nematophilus, a bacterial symbiont of
a nematode (Fig. 1). The compound has only recently been
described by a Canadian group4,5 and some preliminary
SAR work has been published by them and a group of
researchers at the animal health division of Bayer AG.6
The available published data indicate that this compound
has potential as an anti-MRSA agent as it is very active
against both wild type and methicillin resistant strains of
S. aureus (MRSA), although the compound apparently
loses antibacterial activity in the presence of serum in
vitro. The mechanism of action of nematophin is not
known.
The benzimidazole nucleus represents a readily acces-
sible heterocyclic system which is isosteric with the
indole ring. The direct alkylation of either benzimida-
zole or 2-phenylbenzimidazole with N-(2-bromoethyl)
phthalimide using standard alkylation conditions (e.g.,
NaH/DMF or K2CO3/DMF) was not successful therefore
,This paper is dedicated to the memory of John Alexander Winders
9.12.59±12.4.99
*Corresponding author. Fax: +39-45-921-8191; e-mail: gk27184@
glaxowellcome.co.uk
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00331-0

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G. Kennedy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1751±1754
Figure 1. Nematophin (1) and its N-benzyl analogue (2).
Scheme 2. Reagents: (a) N-Boc ethylenediamine/Et3N/toluene/re¯ux/8
h; (b) TFA/CH2Cl2/ re¯ux (c) N-carboethoxyphthalimide/Et3N/THF/
re¯ux/24 h; (d) SnCl22H2O/DMF/60 ꢀC/12 h; (e) 8a: triethylortho-
formate/H+/toluene/100 ꢀC/1 h; for 8b triethyl orthobenzoate/H+/tol-
uene/100 ꢀC/1 h; (f) Me2NH 2.0 M in MeOH; (g) (rac)-3-methyl-2-
oxovaleric acid/HOBT/EDCI/DMF/ overnight; (h) MeI/CH2Cl2.
Table 1. Miniminum inhibitory concentrations (mg ml 1) against
Gram positive bacteria for compounds described in the texta
Compoundb
Staphylococcus
aureus 853
Staphylococcus
aureus COL
Bacillus
subtilis
Scheme 1. Reagents: (a) pyridinium tribromide/THF/0 ꢀC; (b)
Pd(PPh3)4/LiCl/2 M Na2CO3/phenylboronic acid or 3-pyridylboronic
acid 1,3-propanediol cyclic ester/EtOH/toluene; (c) 2 M Me2NH in
MeOH; (d) (rac)-3-methyl-2-oxovaleric acid/HOBT/EDCI/DMF/
overnight; (e) 6a: MeI/CH2Cl2/rt; 6b: ICH2CONH2/CH2Cl2/rt.
1
2
2
2
16
>32
0.06
4
>32
>32
>32
16
>32
>32
8
>32
>32
>32
>32
>32
>32
>32
5a
5b
6a
6b
9a
9b
10a
10b
<0.03
0.5
>32
>32
>32
8
these analogues were prepared by the less direct route
shown in Scheme 2.
>32
>32
>32
>32
The reaction of mono-Boc-protected ethylenediamine
with 2-¯uoronitrobenzene is known8 and was followed
by switching of the protecting group to phthalimide.
The nitro compound was easily reduced using tin (II)
chloride in DMF, although the reaction was somewhat
sluggish.9 The intermediate diamine was cyclised
through reaction with trimethylorthoformate or triethyl-
orthobenzoate in the presence of acid to form the benzi-
midazoles 8a and 8b, respectively.10 In the latter case, the
product recrystallised directly from the reaction medium.
The phthalimide protecting group was removed and the
amines coupled with 3-methyl-2-oxovaleric acid to give
the benzimidazole analogues 9a and 9b. Methylation of
these gave the charged benzimidazolium species 10a and
10b, respectively.
aMeasured according to the standard method described in ref 13.
bSpectroscopic data for compounds 2±10b is provided in note17
.
those against the sensitive strain and no activity was
observed for either compound against B. subtilis. This
observation contrasts with the reported literature values
and deserves comment. First of all, the previously
described values were measured in clinical isolates of
MRSA which exhibit di€erent phenotypes to the well-
characterised strain used routinely in this work and sec-
ondly, the MIC measurements were taken using the
serial broth dilution method, whereas previous workers
report MICs obtained using the agar method.
The antibacterial activities of the compounds prepared
along with those of the two standards 1 and 2 are collected
in Table 1. The minimum inhibitory concentrations
(MIC) of the compounds were measured against the
methicillin sensitive organism S. aureus 853 and the
methicillin-resistant organism S. aureus COL,11 as well
as Bacillus subtilis to determine if the activity observed
extended to non-staphylococcal bacteria. It is note-
worthy that the MIC values of 1 and 2 against the
MRSA organism S. aureus COL are much higher than
Introduction of a phenyl substituent in the 2-position of
the indole (5a) a€orded considerable gains in activity
with respect to the parent nematophin. This compound
displayed an MIC at the lower limit of detection against
S. aureus 853 and 8-dilutions lower than the parent
against S. aureus COL. This gain in activity was re¯ected
in the 3-pyridyl compound 5b although not to quite the
same levels. Interestingly, the 2-phenyl analogue was the
only compound prepared that also displayed activity,

G. Kennedy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1751±1754
1753
albeit modest, against the Gram positive organism B.
Acknowledgements
subtilis. The incompatibility of charge with maintenance
of activity is most clearly demonstrated in this series: the
methylpyridinium compound 6a being devoid of activity
against both S. aureus and B. subtilis. Neither 5a nor 5b
The authors would like to thank the sta€ of the spec-
troscopy departments and A. Felici and E. Domenici
for discussions and support given during this work.
1
showed signs of cytotoxicity at 10 mg ml (data not
shown).12
References and Notes
The benzimidazole analogue of nematophin 9a proved to
be completely inactive showing that the benzimidazole
ring cannot be considered to be bio-isosteric with an
indole in this system and that factors more subtle than
simple shape are at play here. Interestingly, the 2-phenyl
analogue 9b displayed some activity against both S.
aureus 853 and S. aureus COL, although the MIC values
are not comparable with those obtained for the corre-
sponding 2-phenyl indole derivative 5a. This shows that
the presence of an aryl ring in this position is sucient
to overcome some of the negative characteristics of the
benzimidazole nucleus. As with the pyridinium species
6a described above, the presence of charge in the com-
pounds 10a and 10b was not compatible with anti-
bacterial activity.
1. Roth, V. R.; Jarvis, W. R. Emerg. Ther. Targets 1999, 3,
73.
2. Ehlert, K. Curr. Pharm. Des. 1999, 5, 45.
3. Setti, E. L.; Quattrocchio, L.; Micetich, R. G. Drugs Future
1997, 22, 271.
4. Li, J.; Chen, G.; Webster, J. M. Can. J. Microbiol. 1997, 43,
770.
5. Li, J.; Chen, G.; Webster, J. M. Bioorg. Med. Chem. Lett.
1997, 7, 1349.
6. Himmler, T.; Pirro, F.; Schmeer, N. Bioorg. Med. Chem.
Lett. 1998, 8, 2045.
7. Lin, C.; Fisher, M. H.; Goulet, M. T.; Wyatt, M. Tetra-
hedron Lett. 1997, 38, 3871.
8. Boga, C.; Forlani, L.; Guardia, P. Gazz. Chim. Ital. 1997,
127, 259.
9. Karrer, F.; Benz, F.. Helv. Chim. Acta, 1029.
10. Katrizky, A.; Rachwal, S.; Ollmann, R. J. Heterocyclic
Chem. 1994, 31, 775.
11. S. aureus COL is a highly homogeneous MRSA carrying a
PBP 2 (Penicillin Binding Protein 2) mutation.
12. Mosmann, T. J. Immunol. Methods 1983, 65, 55.
13. National Committee for Clinical Laboratory Standards,
1993; Vol. 10, pp M7±A3.
14. Hecker, S. J. J. Antibiot. 1998, 51, 722.
15. Arnould, J. C.; Illingworth, R. N.; Nichols, W. W.; Wil-
son, R. G. Bioorg. Med. Chem. Lett. 1996, 6, 2449.
16. Shinagawa, H.; Yamaga, H.; Houchigai, H.; Sumita, Y.;
Sunagawa, M. Bioorg. Med. Chem. 1997, 5, 601.
17. Selected compound data. Note: all compounds containing
chiral centres were prepared as racemates.
The parent compound is known to lose activity in the
presence of serum and therefore the MIC values of the
compounds were also measured in the presence of 5%
bovine serum (data not shown). None of the compounds
prepared, including the charged pyridinium and the
benzimidazolium derivatives were active in the presence
of serum, despite several well-documented examples of
the use of charged groups as modi®ers of excessive
serum protein binding.14 16 This result indicates that the
loss of activity in the presence of serum is a general
property of the class.
1
Compound 2: waxy solid; IR (®lm) cm 3410, 1714, 1685,
1
1517, 1464, 1451; H NMR (400 MHz, CDCl3) d 0.89 (3H, t),
Conclusions
1.09 (3H, d), 1.39 (1H, m), 1.79 (1H, m), 3.03 (2H, t), 3.49
(1H, m), 3.64 (2H, m), 5.29 (2H, s), 6.97, (1H, s), 7.06 (1H, s),
7.13 (3H, m), 7.21, (1H, m), 7.26±7.34 (2H, m), 7.62 (1H, bd)
ES±MS m/z 363 (MH)+ 234 (M-C6H10NO2)+.
The biological data obtained show that this class is
probably not suited to systemic administration but that
such agents could be considered for topical use. The
restricted spectrum of activity of these compounds
which are apparently only active against some strains of
S. aureus (with some weak sign of activity against other
Gram positive bacteria) is a limiting factor for the class,
even though such narrow spectrum agents may become
more important in the medium-term with the advent of
rapid and accurate diagnostics.
Compound 5a (Æ)-N-[2-(2-phenyl-1H-indol-3-yl)ethyl]-3-methyl-
1
2-oxo-pentanamide: colourless oil; IR (®lm) cm 3393±3326,
1
1714±1671, 1457; H NMR (400 MHz, CDCl3) d 0.88 (3H, t),
1.05 (3H, d), 1.37 (1H, m), 1.69 (1H, m), 3.18 (2H, t), 3.44 (1H,
m), 3.64 (2H, t), 7.03 (1H, bt), 7.18±7.26 (2H, m), 7.40 (1H, t),
7.42 (1H, d), 7.49 (2H, t), 7.57 (2H, d), 7.67 (1H, d), 8.13 (1H,
bs), ES±MS m/z 220 (M NHCOCOCH(CH3)C2H5)+.
The surprising level of activity seen with the 2-phenyl
derivative 5a, as well as the apparent ability of the
phenyl group in the 2-position to allow a recovery of
activity in the otherwise inactive benzimidazole series,
points to quite speci®c substituent e€ects possibly related
to enhanced interaction with the target. This could
warrant further exploration. It would appear that these
compounds are optimally active against a target (or
targets) which are present in Staphylococcus sp.,
although there are considerable di€erences between
strains which could re¯ect structural di€erences at the
level of the target or strain dependent expression of the
target.
Compound 5b (Æ)-N-[2-(2-(3-pyridyl)-1H-indol-3-yl) ethyl]-3-
1
methyl-2-oxo-pentanamide; pale yellow gum; IR (nujol) cm
1720, 1660; 1H NMR (400 MHz, CDCl3) d 0.86 (3H, t), 1.05 (3H,
d), 1.38 (1H, m), 1.69 (1H, m), 3.14 (2H, t), 3.44 (1H, m), 3.64
(2H, t), 7.19 (1H, m), 7.26 (1H, t) (+solvent), 7.44 (2H, m), 7.7
(1H, m), 7.94 (1H, m), 8.44 (1H, bs), 8.59 (1H, dd), 8.87 (1H, d);
ES±MS m/z 220 (M NHCOCOCH(CH3)C2H5)+ 100%.
Compound 6a (Æ)-N-[2-(2-(3-N-methylpyridinium)-1H-indol-3-
yl)ethyl]-3-methyl-2-oxo-pentanamide iodide. Yellow solid (mp
103±104 ꢀC softens); IR (nujol) cm 3383, 3178, 1668; 1H
1
NMR (400 MHz, CDCl3) d 0.89 (3H, t), 1.09 (3H, d), 1.37
(1H, m), 1.69 (1H, m), 3.13 (2H, t), 3.44 (1H, m), 3.54 (2H, t),
4.60 (3H, s), 7.15 (1H, bt), 7.27 (2H, m), 7.57 (1H, d), 7.69 (1H,

1754
G. Kennedy et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1751±1754
1
1
d), 7.91 (1H, m), 8.32 (1H, d), 8.97 (1H, d), 9.87 (1H, s), 11.18
(1H, s); ES±MS m/z 364 (M I)+ 100%.
(nujol) cm 3411, 1716, 1690; H NMR (400MHz, CDCl3) d
0.45 (3H, t), 1.01 (3H, d), 1.31 (1H, m), 1.62 (1H, m), 3.31 (1H,
m), 3.62 (2H, q), 4.48 (2H, t), 6.95 (1H, bt), 7.34 (2H, m), 7.51
(4H, m), 7.69 (2H, m), 7.84 (1H, m); ES±MS m/z 350 (MH)+
100%.
Compound 6b (Æ)-N-[2-(2-(3-N-(2-acetamido) pyridinium)-1H-
indol-3-yl)ethyl]-3-methyl-2-oxo pentanamide iodide. Yellow solid
(mp 210±211 ꢀC); IR (nujol) cm 3320, 3120, 1690, 1670; H
NMR (400 MHz, DMSO) d 0.79 (3H, t), 0.96 (3H, d), 1.29 (1H,
m), 1.57 (1H, m), 3.10 (t, 2H,), 3.30 (m, 1H,), 3.39 (2H, t), 5.52
(2H, s), 7.11 (1H, t), 7.24 (1H, d), 7.45 (1H, d), 7.68 (1H, d), 7.74
(1H, s), 8.07 (1H, s), 8.30 (1H, d), 8.83 (1H, d), 8.85 (1H, t), 8.92
(1H, d), 9.26 (1H, s), 11.71 (1H, s); ES±MS m/z 407 (M I)+ 100%.
1
1
Compound 10a (Æ)-N-[2-(3-methyl-1H-benzimidazolium)ethyl]-3-
methyl-2-oxo-pentanamide iodide. Pale yellow powder (mp 148±
149 ꢀC, softens); IR (nujol) cm 3330, 1708, 1666; H NMR
(400 MHz, DMSO) d) 0.70 (3H, t), 0.85 (3H, d), 1.17 (1H, m),
1.43 (1H, m), 3.11 (1H, m), 3.64 (2H, q), 4.07 (3H, s), 4.60 (2H,
t), 7.67 (2H, m), 8.00 (1H, m), 8.82 (1H, t), 9.96 (1H, s); ES±MS
m/z 288 (M I)+ 100%.
1
1
Compound 9a (Æ)-N-[2-(1-benzimidazolyl)ethyl]-3-methyl-2-oxo-
pentanamide. White solid (mp 101±102 ꢀC); IR (nujol) cm
1
1
3417, 1717, 1689; H NMR (400 MHz, CDCl3) d 0.88 (3H, t),
Compound 10b (Æ)-N-[2-(3-methyl-2-phenyl-1H-benzimidazo-
lium)ethyl]-3-methyl-2-oxo-pentanamide iodide. O€-white pow-
1.08 (3H, d), 1.39 (1H, m), 1.69 (1H, m), 3.44 (1H, m), 3.75
(2H, t), 4.41 (2H, t), 7.16 (1H, m), 7.32 (2H, m), 7.44 (1H, dd),
7.81 (1H, dd), 7.92 (1H, s); ES±MS m/z 274 (MH)+.
der (softens ca. 105ꢀC); IR (nujol) cm 3415, 1717, 1667; H
NMR (400 MHz, DMSO) d 0.70 (3H, t), 0.82 (3H, d), 1.14 (1H,
m), 1.40 (1H, m), 3.04 (1H, m), 3.42 (2H, q), 3.84 (3H, s), 4.45
(2H, t), 7.70±7.83 (7H, m), 8.06 (1H, m), 8.13 (1H, m), 8.71 (1H,
t); ES±MS m/z 364 (M I)+ 100%.
1
1
Compound 9b (Æ)-N-[2-(2-phenyl-1-benzimidazolyl) ethyl]-3-
methyl-2-oxo-pentanamide. White solid (mp 115±116ꢀC); IR