Fine tuning of physico-chemical parameters to optimise a new series of novobiocin analogues

Article abstract of DOI:10.1016/S0960-894X(01)00257-8

A novel series of novobiocin analogues has been synthesised by removing the lipophilic aryl chain in novobiocin and introducing an amino substituent. The structural modifications have been dictated by the control of lipophilicity and the dissociation constant of the resulting compounds. Antibacterial activity of the new coumarin derivatives could be correlated with the amount of uncharged form in physiological conditions.

Full text of DOI:10.1016/S0960-894X(01)00257-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1461–1464
Fine Tuning of Physico-Chemical Parameters to Optimise a New
Series of Novobiocin Analogues
Laurent Schio,a,* Fabienne Chatreaux,a Veronique Loyau,a Michel Murer,a
Anne Ferreira,b Pascale Mauvais,c Alain Bonnefoyc and Michel Klicha
aMedicinal Chemistry, Aventis Pharma, 102 route de Noisy, F-93235 Romainville Cedex, France
bStructural Analysis, Aventis Pharma, 102 route de Noisy, F-93235 Romainville Cedex, France
cInfectious Diseases, Aventis Pharma, 102 route de Noisy, F-93235 Romainville Cedex, France
Received 17 January 2001; revised 6 April 2001; accepted 9 April 2001
Abstract—A novel series of novobiocin analogues has been synthesised by removing the lipophilic aryl chain in novobiocin and
introducing an amino substituent. The structural modifications have been dictated by the control of lipophilicity and the dissocia-
tion constant of the resulting compounds. Antibacterial activity of the new coumarin derivatives could be correlated with the
amount of uncharged form in physiological conditions. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
lishing structure–activity relationships and identifying
new candidates for pre-clinical and clinical
evaluations.6ꢀ10 We report here our rational approach
relying on the control of physico-chemical parameters in
the development of a new series of gyrase B coumarin
inhibitors 4.11
The naturally occurring antibiotics novobiocin 1 and
clorobiocin 2 have attracted renewed attention because
of their impressive activity against b-lactamase produ-
cing and methicillin-resistant Staphylococcus aureus
(MRSA) strains.1 These coumarin-containing anti-
bacterial agents block the negative supercoiling of
relaxed DNA (a process involved in gene expression) by
inhibiting ATP hydrolysis in the B sub-unit of DNA
gyrase.2ꢀ5 We described in recent papers our extensive
programme of chemical modifications aimed at estab-
In structure 4, the noviose derived moiety of clorobiocin
has been retained since it is implicated in high energy
hydrogen bonding with gyrase B amino-acid residues.4
On the other hand, the C3-isopentenylhydroxy-
benzocarboxamide group has been removed in 4. This
*Corresponding author. Tel.: +33-1-49-91-35-76; fax: +33-1-49-91-50-87; e-mail: laurent.schio@aventis.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00257-8

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L. Schio et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1461–1464
lipophilic group has a limited contribution to the inhi-
bition of DNA gyrase activity but facilitates transport
of drugs 1 and 2 across the bacterial cytoplasmic mem-
brane by passive diffusion12 (compound 3 has no anti-
bacterial activity while being a gyrase B inhibitor only
2-fold less potent than novobiocin). Consequently,
novobiocin and clorobiocin are highly lipophilic deri-
vatives exhibiting low water solubility and high binding
to serum proteins.13,14 Amine introduction in the cou-
marin moiety (C4 position) was expected to increase
water solubility, reduce the negative serum effect on
activity as well as develop additional interactions with
gyrase B. Piperazine was selected because it provides a
basic function that could potentially co-exist in charged
(NH+–R4) and uncharged forms (N–R4) at neutral pH.
The charged species (more hydrophilic) could give aqu-
eous solubility and electrostatic interactions at the
membrane interface.15 The uncharged species (more
lipophilic) could be able to cross phospholipid bilayers
providing that lipophilicity of the whole molecule 4 is
appropriate for passive diffusion.16 Several coumarin
compounds bearing a differently substituted piperazine
moiety (4a–g) were synthesised as well as closely related
analogues (4h–j, Scheme 1). Their corresponding dis-
sociation constants (pKa) and lipophilicity values (log P)
were measured and correlated with their respective
potency against DNA gyrase supercoiling (IC50)6 and
antibacterial activity (MIC) determined in the absence
and in the presence of serum proteins.
Consequently additional protection-cleavage steps were
required for the successful preparation of analogues
such as 4b, 4c and 4d. Chlorination of coumarin 7 at the
C3 position was successfully achieved by addition of a
chlorine solution in acetic acid at 0 ꢁC to lead to the
coumarin precursor of compound 4i in 69% yield. To
overcome the low yield observed in the glycosylation
reaction to obtain 4j, we had to develop an alternative
strategy based on the terminal amination of tosylate
intermediate 1118 (Scheme 3).
Results andDiscussion
Lipophilicity of coumarin antibiotics 4 was estimated on
reversed-phase thin layer chromatography (RP-TLC)
plates and RMw values were determined (see Table 1).19
The RMw parameter was shown to be consistent with
the octanol/water partition coefficient for novobiocin
(RMw=5.2 vs log Po/w=5.5).20 Compound 4a bearing
a pyridine moiety, uncharged at neutral pH, is highly
lipophilic and hence exhibited a marked reduced anti-
bacterial activity against S. aureus in the presence of
serum. Introduction of a secondary amine (4b–d)
reduced lipophilicity, improved DNA gyrase inhibition
but was detrimental to antibacterial activity when com-
pared to novobiocin. The negative contribution of the
NH function to permeability21 could not be compen-
sated by addition of two a-methyl groups (4d vs 4b). On
the other hand, the N-methyl piperazine derived com-
pound 4e displayed a noteworthy antibacterial activity
against S. aureus (as well as other Gram-positive
strains)22 which was retained in the presence of serum.
When compared to the secondary amines 4b–d, the ter-
tiary amine 4e showed similar gyrase inhibition and
lipophilicity but decreased basicity. Inactivity of 4g
(piperazinium derivative) and lower antibacterial activ-
ity of 4f (N-ethyl piperazine derivative) when compared
to 4e confirm that the development of a positive charge
in the molecule at physiological pH diminishes anti-
bacterial activity. Chlorination of 4e (4i) failed to
decrease the basicity of the N-methyl piperazine group
by remote electron-withdrawing induction. Moreover,
the positive contribution of the chlorine atom to lipo-
philicity led to a more pronounced serum effect than for
Synthesis
The 4-amino derived novobiocin analogues 4 that were
synthesised are listed in Scheme 1.
Our general synthetic approach from 57 relied on ami-
nation of triflate17 intermediate 6 followed by a glycosy-
lation reaction carried out under Mitsunobu conditions
between 4-amino-7-hydroxycoumarin substrates and the
noviose derivative 96 (e.g., synthesis of 4e and 4g from
8; Scheme 2). All amines were commercially available.
Limited yields were observed in the glycosylation of
coumarin substrates bearing unprotected NH groups.
Scheme 1.

L. Schio et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1461–1464
1463
Scheme 2. Reagents and conditions: (a) (CF3SO2)2O, Et3N, CH2Cl2, 1 h, 0 ꢁC, 87%; (b) 1-methylpiperazine, THF, 1 h, rt, 98%; (c) Bu4NF, THF,
1 h, 0 ꢁC, 69%; (d) PPh3, EtO2CN¼NCO2Et, CH2Cl2, rt, 6 h, 50%; (e) CH3I, CH3CN, 1 h, 60 ꢁC, 83%.
Scheme 3. Reagents and conditions: (a) PPh3, EtO2CN¼NCO2Et, CH2Cl2, rt, 2.5 h, 42%; (b) 4-dimethylaminopiperidine, THF, 1.5 h, rt, 19%.
Table 1. Physico-chemical properties and biological activities of 4-aminocoumarin analogues 4
MICd (mg/mL)
on S. aureuse
MIC (mg/mL) on
S. aureus+10% serum
a
b
Compound
RMw
pKa
IC50c (mg/mL)
Novobiocin
5.2
6.4
3.6
5.5
8.3
8.3
8.0
7.3
7.5
—
0.25
1.1
0.125
0.125
0.16
0.3
0.25
0.14
0.35
0.5
ꢂ 0.04
0.30
5.0
2.5
2.5
0.15
0.6
>40
5.0
2.5
10
4a
4b
4c
4d
4e
4f
4g
4h
4i
4.37
4.44
4.70
4.48
4.78
—
4.50
4.71
4.60
5.0
2.5
2.5
0.30
2.5
>40
5.0
20
8.2
7.4
9.0
1.2
2.5
4j
0.28
2.5
aDetermined on RP-18 TLC plates in an incubator adjusted to 30 ꢁC. Methanol–Tris buffer (pH 7.4) mixtures were used as eluent.
bMeasured with the Sirius GLpKa instrument coupled to the Sirius D-PAS system.
cSee ref 6.
dMIC, minimum inhibitory concentration, measured by using a 2-fold broth microdilution after a 24-h incubation.
eStrain 011HT3 with no particular phenotype of resistance.
4e. The very close analogues 4h and 4j of compound 4e
exhibited a limited antibacterial activity which can be
attributed to their significantly higher basicity (these
two tertiary amines are at least as basic as the secondary
amines 4b–d).
was critical for maintaining the antibacterial activity in
the presence of serum proteins. Among the derivatives
synthesised, compound 4e exhibited the best biological
profile derived from optimised physico-chemical prop-
erties. This coumarin analogue was further investigated
in vivo.
In conclusion, the novel series of novobiocin analogues
4 bearing different amino groups on the coumarin moi-
ety displayed excellent inhibitory potency against DNA
gyrase supercoiling. Antibacterial activity and hence
permeability across the bacterial membrane was shown
to be correlated predominantly with basicity over lipo-
philicity of the molecule. The control of lipophilicity
Acknowledgements
The authors are grateful to F. Rocchiccioli and B. For-
gerit for their contribution in the development of the
RMw technology in our laboratory.

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A.; Vicat, P.; Klich, M. Bioorg. Med. Chem. Lett. 2000, 10,
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5; S. aureus 011HT26 (oflo R, oxa R, ery R), 0.15; S. epi-
dermidis 012GO20, 0.08; S. epidermidis 012HI1 (teico R, oxa
R, vanco R), 0.30; S. pyogenes 02A1IP1 (teico R, vanco R),
2.50; S. faecalis 02D2UC6, 20; E. faecium 02D3IP2 (teico R,
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