Improved antibacterial activities of coumarin antibiotics bearing 5',5'-dialkylnoviose: Biological activity of RU79115

Article abstract of DOI:10.1016/S0960-894X(00)00304-8

A new series of coumarin inhibitors of DNA gyrase B bearing a N-propargyloxycarbamate at C-3' of various 5',5'-dialkylnoviose, including RU79115, were synthesised and their antibacterial activities have been delineated. Introduction of dialkyl substituents at 5'5'-position of noviose leads to coumarin analogues with improved in vitro and in vivo antibacterial activity. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00304-8

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1695±1699
Improved Antibacterial Activities of Coumarin Antibiotics Bearing
50,50-Dialkylnoviose: Biological Activity of RU79115
Branislav Musicki,a,* Anne-Marie Periers,a Patrick Laurin,a Didier Ferroud,a
Yannick Benedetti,a Sylvette Lachaud,a Fabienne Chatreaux,a Jean-Luc Haesslein,a
Alain Iltis,a Christine Pierre,a Jean Khider,a Nicole Tessot,a Marlene Airault,a
Jacques Demassey,a Claudine Dupuis-Hamelin,b Patrice Lassaigne,b
Alain Bonnefoy,b Pascale Vicatc and Michel Klicha
aMedicinal Chemistry, Hoechst Marion Roussel/Aventis, 102 route de Noisy, 93235 Romainville Cedex, France
bInfectious Disease, Hoechst Marion Roussel/Aventis, 102 route de Noisy, 93235 Romainville Cedex, France
cCentral Research, Hoechst Marion Roussel/Aventis, 102 route de Noisy, 93235 Romainville Cedex, France
Received 2 February 2000; accepted 26 May 2000
AbstractÐA new series of coumarin inhibitors of DNA gyrase B bearing a N-propargyloxycarbamate at C-30 of various 50,50-di-
alkylnoviose, including RU79115, were synthesised and their antibacterial activities have been delineated. Introduction of dialkyl
substituents at 5050-position of noviose leads to coumarin analogues with improved in vitro and in vivo antibacterial activity.
# 2000 Elsevier Science Ltd. All rights reserved.
di€erent Gram-positive strains6 indicated that entero-
Introduction
coccal and streptococcal gyrase B activity could be more
In the past several years we have seen intensive e€orts
by several research groups, including ours,1 in ®nding
potential inhibitors of bacterial DNA gyrase, a DNA
topoisomerase that catalyses a variety of interconver-
sions between topological isomers of DNA.2 Speci®-
cally, the subunit of DNA gyrase, gyrase B, was
identi®ed as a target for two structurally di€erent classes
of natural products: coumarins (novobiocin 1, clorobiocin
2)3 and cyclothialidines,4 as well as synthetic triazines.5
Our work in this ®eld has been directed toward the
coumarin antibiotics and their structural modi®cation.
Recently, we disclosed the marked in¯uence of 50,50-
dimethyl group of noviose in determining the antibacterial
spectrum of coumarin antibiotics.1d Furthermore, com-
parison of gyrase B aminoacid sequences from twelve
in¯uenced by the 50,50-dialkyl group of noviose com-
pared to that of gyrase B of E. coli. (Fig. 1). Their
hydrophobic pockets, consisting of Val 94 and Phe 95,
are slightly larger compared to that of gyrase B of E.
coli.7,8 As we have developed ecient stereoselective
synthetic approaches to 50-monoalkyl and 50,50-di-
alkylnoviose derivatives,9 these provided us with a useful
tool to probe structure±activity relationships for this
part of the molecule.
In this report we disclose the synthesis, in vitro and in
vivo biological activities of a novel series of coumarin
congeners incorporating various 50,50-dialkylnoviose.
Also, the pharmacological pro®le of RU79115, the most
potent candidate within the series is reported.
*Corresponding author. Tel.: +33-1-49-91-40-07: fax: +33-1-49-91-
50-87; e-mail: branislav.musicki@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00304-8

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B. Musicki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1695±1699
Biological Results and Discussions
The in vitro pro®les of the inhibition of the supercoiling
and antibacterial activities of S. aureus DNA gyrase by
novobiocin, clorobiocin and the novel series of coumarin
inhibitors 3b±h possessing 30-N-propargyloxycarbamate
on the modi®ed sugar noviose are shown in Table 1.
Comparison of the inhibitory potencies between the
noviose analogue (3a)1f with those containing 50,50-dia-
lkylnoviose (3c±h), reveals that the introduction of bulkier
alkyl substituents at the C-50 of noviose does not produce
signi®cant change in their supercoiling inhibitory activi-
ties. The dialkyl derivative (3c) seems to display better
inhibition of the negative supercoiling of gyrase B than
50-monoalkyl counterpart (3b).
Figure 1. Comparison of the amino acid sequences of the hydrophobic
pocket surrounding 50,50-dimethyl group of noviose part in di€erent
gyrase B.
Chemistry
General synthetic route towards 50,50-dialkylnoviosyl
coumarin antibiotics is illustrated by the preparation of
50,50-spirocyclopentylnoviose analogue 3g (RU79115)
(Scheme 1). The noviose derivative9 4 was transformed
to the corresponding 20,30-noviose carbonate 5 with 1,10-
carbonyldiimidazole in dichloroethane at re¯ux in 67%
yield. The carbonate was then coupled with coumarin
building block 61e under Mitsunobu conditions to a€ord
the a-glycoside 7 as the major product (80% yield), easily
separable by chromatography from b-glycoside (7%
yield). The benzhydryl protecting group in 7 was readily
removed by hydrogenolysis and the 3-acetyl functionality
in 4-hydroxycoumarin was introduced by the base cata-
lysed C-3 acylation of coumarin part in dichloromethane
in the presence of DMAP to a€ord 8 in quantitative
yield.1c Opening of the carbonate 8 under thermodynamic
conditions with excess O-propargylhydroxylamine in
pyridine in the presence of lithium perchlorate or lithium
tri¯ate, provided a mixture of 30- 9a and 20-N-propargyl-
oxycarbamate 9b in the ratio 3:1. The regioisomers were
separated by chromatography using CH2Cl2:EtOAc:
AcOH 80:20:1 as eluent. The opening of the carbonate
was accompanied by formation of O-propargyloxime at
C-3 acetyl group of the coumarin. This oxime could be
easily exchanged for methoxy 3 by reaction with excess of
O-methylhydroxylamine in ethanol at room temperature.
Regarding the antibacterial properties, improved anti-
enterococcal activity was observed with the 50,50-di-
alkylnoviose series. Low antibacterial activity of analogue
3b (MIC from 0.3±40 mg/mL) lacking the C-50 axial
methyl group compared to 3c (MIC from ꢀ0.04±0.3 mg/
mL) con®rms our previous ®ndings observed within the
rhamnose series1e that the presence of a C-50 axial sub-
stituent is important in conferring good antibacterial
activity.
Further in vitro and in vivo pharmacological activities
of 3g (RU79115) are presented in Tables 2±5. Against
staphylococci (Table 2), whatever their phenotype of
resistance, RU79115 showed MIC50's 7.5 times lower
than eperezolid10 and far lower than vancomycin (0.08,
0.6 and 1.2 mg/mL, respectively).
Scheme 1. Reagents and conditions: (a) Im2CO, ClCH2CH2Cl re¯ux, 67%; (b) PPh3, EtO2CN=NCO2Et, DMF, rt, 80%; (c) H2, Pd-C/10%, THF,
rt, quant; (d) Ac2O, DMAP, CH2Cl2, 0 ꢁC, quant; (e) HCꢂCCH2ONH2-HCl, LiClO4, rt, quant; (f) MeONH2-HCl, KOAc, EtOH, rt, 90%.

B. Musicki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1695±1699
1697
Table 1. In vitro activity of coumarin inhibitors against S. aureus DNA gyrase supercoiling (IC50),a and selected in vitro antibacterial activity
(MIC)b
MIC (mg/mL)
Compound
Ratio
IC50nov
IC50comp
S.aureus
011HT3
S. aureus
011GO64
O¯oOxaEry-R
S. aureus
011HT1
Nov-R
S. epidermidis
012GO42
Oxa-R
S. pyogenes
02A1UC1
E. faecium
02D31P2
VanTeiEry-R
a
Novobiocin
Clorobiocin
3a
3b
3c
3d
3e
3f
3g (RU79115)
3h
1
ꢀ0.04
ꢀ0.04
0.16
ꢀ0.04
ND
0.16
10
0.6
5
ꢀ0.04
ND
0.08
0.3
ꢀ0.04
0.6
2.5
0.08
1.2
0.3
0.6
0.15
0.6
0.6
ꢀ0.04
2.5
40
0.15
1.2
0.6
0.6
0.15
0.15
1.7
12.5
2.6
11.1
2.6
2.6
4.0
2.6
2.6
0.3
0.6
ꢀ0.04
20
0.3
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
0.3
2.5
1.2
2.5
1.2
0.6
0.3
ꢀ0.04
0.08
0.15
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
ꢀ0.04
0.15
aIC50 was determined for gyrase B of S. aureus against novobiocin (0.5 mg/mL) as reference. For the details see ref 1f.
bMIC, Minimum Inhibitory Concentrations (mg/mL) were measured by using a 2-fold broth microdilution after 24 h incubation. Particular pheno-
type of resistance (-R) of the tested bacterial strains were mentioned: O¯o for o¯oxacin, Oxa for oxacillin, Ery for erythromycin, Nov for novo-
biocin, Tei for teicoplanin, Van for vancomycin. Otherwise, strains were fully susceptible.
Table 2. In vitro anti-staphylococcal activity of RU79115 (MIC mg/mL)
Table 3. In vitro anti-streptococcal activity of RU79115 (MIC mg/mL)
VAN
Eperezolid
RU79115
VAN
Eperezolid
RU79115
S. sppa
GMb
MIC90
[91]d
1.15
2.5
[91]
0.74
1.2
[91]
0.099
0.15
St. spp (groupable)a
GM
MIC90
[38]c
0.35
0.6
[38]
0.52
0.6
[38]
0.47
1.2
S. spp Oxa-R
GM
MIC90c
[66]
1.16
2.5
[66]
0.73
1.2
[66]
0.097
0.15
St. spp (viridans)
GM
MIC90
[25]
0.4
0.6
[25]
0.39
1.2
[25]
0.59
1.2
S. spp O¯o-R
GM
MIC90
[59]
1.2
2.5
[59]
0.71
1.2
[59]
0.082
0.15
P. Pen-Rb
GM
MIC90
[29]
0.24
0.3
[29]
0.25
0.3
[29]
0.12
0.3
S. spp Oxa-R O¯o-R
GM
MIC90
[47]
1.21
2.5
[47]
0.72
1.2
[47]
0.081
0.15
aSt.: streptococci.
bP.: pneumococci; Pen.: penicilin; R: resistant.
c[ ]: number of isolates.
aS.: staphylococcus; Oxa: oxacillin; O¯o: o¯oxacin; R: resistant.
bGM: geometric mean.
cMIC's were determined using a 2-fold agar-dilution (mg/mL).
d[ ]: number of isolates.
Table 4. In vitro anti-enterococcal activity of RU79115 (MIC mg/
mL)
VAN
[26]b
Eperezolid
RU79115
Against streptococci (Table 3), including penicillin-resis-
tant pneumococci, RU79115, vancomycin and eperezolid
displayed very similar activities with MIC50's ranging
from 0.15 to 0.6 mg/mL. In septicemic mice infected by
these species (Table 5), the ecacy of RU79115 was close
to those of the reference compounds, with PD50's10 from 1
to 37.6 mg/kg.
E. spp VAN-S
GM
MIC90
[26]
0.51
0.6
[26]
0.26
1.2
0.75
0.6
E. spp VAN-R
GM
MIC90
[65]
23.7
>20
[65]
0.72
1.2
[65]
0.26
0.6
E. spp VAN-R TEI-Ra
GM
MIC90
[17]
36.9
>20
[17]
0.43
1.2
[17]
0.16
0.3
Against enterococci (Table 4), RU79115 exhibited simi-
lar activities against vancomycin-susceptible and resis-
tant strains (MIC50: 0.15±0.3 mg/mL). In comparison,
eperezolid was twice less active. In vivo (Table 5), in 12
systemic infections induced in mice by various Gram+
cocci, including vancomycin resistant enterococci,
PD50's for RU79115 ranged from 10 to 77 mg/kg, i.e., at
a higher level than that of eperezolid and linezolid.
aVAN: vancomycin; TEI: teicoplanin; R: resistant; E.:enterococci.
b[ ] : number of isolates.
Pharmacokinetic properties of RU79115 (Table 6) were
in accordance with the pharmacological pro®le.
In summary, our current work demonstrates that the
novel series of coumarin inhibitors of gyrase B bearing
50,50-dialkylsubstituted noviose displays improved in vitro
and in vivo antibacterial properties compared to natural
series possessing noviose. Improvement of the anti-
In vitro, RU79115 bactericidal activity against E. fae-
cium (Fig. 2) and S. aureus (not shown) was time
dependent and similar to that of vancomycin in the case
of S. aureus.

1698
B. Musicki et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1695±1699
Table 5. In vivo anti-bacterial activity of RU79115
a
Strains Reference Pheno-
type
Antibiotic
MIC
PD50
(mg/mL) (mg/kg)
S.a.b
HT18
VANc
eperezolid (SC)d
RU79115
VAN
eperezolid (SC)
RU79115
linezolid (SC)
RU79115
linezolid (SC)
RU79115
linezolid (SC)
RU79115
VAN
eperezolid (SC) <=0.15
RU79115
VAN
eperezolid (SC)
RU79115
VAN
eperezolid (SC)
linezolid
RU79115
VAN
eperezolid (SC)
linezolid
RU79115
VAN
eperezolid (SC)
linezolid
RU79115
VAN
eperezolid (SC)
linezolid
1.2
0.6
<=0.04
1.2
1.2
7.8
20
5.6
4
5
1
5.8
2.8
8.7
7.1
3
GR56
OXA-Re
0.08
1.2
0.15
2.5
0.3
1.2
1.2
0.6
HT17
GO3
S.pn.b
PW3
8.5
<1.5
22.8
37.6
17.2
>50
27.7
16.7
6.4
4.2
10
>50
1.5
1.8
40
>50
27.2
8.6
77
>50
8.7
2.3
44
>50
2.8
2.1
32
8.7
3
2.4
112
S. py.b
A1UC1
0.15
0.3
0.6
Ð
2.5
0.6
S. ag.b
E. fs.b
B1HT3
D2HT6
Ð
0.3
>40
0.3
E. fm.b D3HT12 VAN-Re
Ð
Figure 2. In vitro bactericidal activity of RU 79115 against E. faecium
D3AP9. Time killing curves were determined using a micromethod in
broth.
0.15
>40
0.3
D3HM4 VAN-R
Ð
Acknowledgements
0.6
>40
1.2
Ð
0.6
>40
Ð
Ð
0.08
2.5
0.6
ÐÐ
0.15
D3AP9
VAN-R
We are grateful to Dr John Larkin and Dr John Weston
for the helpful discussions. We would like also to thank
the Analytical Department (HMR, Romainville) for
performing the spectral analysis.
RU79115
VAN
eperezolid (SC)
linezolid
RU79115
VAN
eperezolid (SC)
linezolid
RU79115
D3HM3 VAN-R
D3HT7
References and Notes
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bS.a.: S. aureus; S. pn.: S. pneumoniae; S. py.: S. pyogenes; S. ag.: S.
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Mouse
Dose
10 mg/kg
Tmax
Cmax
Half life
2.5 H
F%
IV
PO
0.5 H
5.4 mg/ml
62%
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sentative of the new family of coumarin antibiotics
and is currently undergoing further pharmacological
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