β-keto-ester chemistry and ketolides. Synthesis and antibacterial activity of 2-halogeno, 2-methyl and 2,3 enol-ether ketolides

Article abstract of DOI:10.1016/S0960-894X(00)00392-9

The effect of 2,3 modifications on the antibacterial activity of ketolides was evaluated by introducing substituents in position 2 and converting the C-1, C-2, C-3 β-keto-ester into stable 2,3 enol-ether or 2,3 anhydro derivatives. Introduction of a fluorine in C-2 is beneficial with regard to the overall antibacterial spectrum whereas the enol-ether and 2,3 unsaturated compounds, as well as the bulky gem dimethyl or 2-chloro derivatives, are less active particularly against erythromycin resistant strains. A 2-fluoro ketolide derivative demonstrates good antibacterial activity and in vivo efficacy against multi-resistant Streptococcus pneumoniae. Compared to azithromycin against Haemophilus influenzae, this compound is equivalent in vitro and slightly more active in vivo. These results demonstrate that within the ketolide class, to retain good antibacterial activity, position 2 needs to remain tetrahedral and tolerates only very small substituents such as fluorine. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00392-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2019±2022
ꢀ-Keto-Ester Chemistry and Ketolides. Synthesis and
Antibacterial Activity of 2-Halogeno, 2-Methyl and 2,3
Enol-Ether Ketolides
Alexis Denis,a,* Francois Bretin,a Claude Fromentin,a A. Bonnet,b
G. Piltan, Alain Bonnefoyd and Constantin Agouridasc
aMedicinal Chemistry, Aventis Pharma, 102 route de Noisy, 93235 Romainville Cedex, France
bChemistry, Aventis Pharma, 102 route de Noisy, 93235 Romainville Cedex, France
cCore Research Functions, Aventis Pharma, 102 route de Noisy, 93235 Romainville Cedex, France
dAnti-infectives Disease Group, Aventis Pharma, 102 route de Noisy, 93235 Romainville Cedex, France
Received 11 April 2000; revised 16 June 2000; accepted 5 July 2000
AbstractÐThe e€ect of 2,3 modi®cations on the antibacterial activity of ketolides was evaluated by introducing substituents in
position 2 and converting the C-1, C-2, C-3 b-keto-ester into stable 2,3 enol-ether or 2,3 anhydro derivatives. Introduction of a
¯uorine in C-2 is bene®cial with regard to the overall antibacterial spectrum whereas the enol-ether and 2,3 unsaturated com-
pounds, as well as the bulky gem dimethyl or 2-chloro derivatives, are less active particularly against erythromycin resistant strains.
A 2-¯uoro ketolide derivative demonstrates good antibacterial activity and in vivo ecacy against multi-resistant Streptococcus
pneumoniae. Compared to azithromycin against Haemophilus in¯uenzae, this compound is equivalent in vitro and slightly more
active in vivo. These results demonstrate that within the ketolide class, to retain good antibacterial activity, position 2 needs to
remain tetrahedral and tolerates only very small substituents such as ¯uorine. # 2000 Elsevier Science Ltd. All rights reserved.
The ketolides,1a,b exempli®ed by telithromycin,1b are a
major new class of semisynthetic macrolide derivatives
exhibiting antibacterial activity against erythromycin-
resistant Streptococcus pneumoniae and Haemophilus
in¯uenzae. The fact that the ketolides do not induce
MLSB resistance2 bestows therapeutic utility against
erythromycin-resistant S. pneumoniae and other Gram
positive pathogens. The ketolides, are characterized by a
3-keto function that replaces the l-cladinose, and thus
de®ne a b-keto-ester at the C-1, C-2, C-3 positions of
the macrolactone ring. In addition to the 3-keto func-
tion, the most important features for in vitro and in vivo
activities of ketolides are: a 11,12-cyclic carbamate
moiety and a hetero-aryl side chain, generally linked to
the ketolide backbone by the carbamate nitrogen. All
these groups are also present in di€erent series synthe-
sized by Abbott3 (e.g., 6-O-substituted ketolides and aza-
imino tricyclic ketolides). However, apart from the
recent report of 2,3 anhydro4 (Anhydrolides) derivatives,
very little is known about the relative importance of
position 2 for the overall activity of ketolides (Fig. 1).
To address this question, we have exploited the chemical
reactivity of the 1,3 b-keto-ester function of ketolides to
modify the C-2 and C-3 positions. This approach has
allowed us to introduce various electrophiles (halogen
atoms or a methyl group) in position 2 and to convert
the enolate intermediate into new stable 2,3 enol-ether
derivatives. At the same time we have synthesized the
2,3 anhydrolide homologue of telithromycin to compare
the overall e€ect of 2,3 modi®cations on antibacterial
activity of ketolides.
Chemistry
Fluorination at C-2 was achieved in 3 steps from teli-
thromycin (Scheme 1). First quantitative silylation of
the 20 alcohol with (TMS)2NH/imidazole gave a pro-
tected intermediate that was reacted with tBuOK and
N-¯uorosulfonimide (NSFI) to give stereospeci®cally,
after desilylation with Bu4N+F , the desired ¯uoro
ketolide I in 83% yield. The absolute stereochemistry of
this compound was later demonstrated by synthesizing I
from the ¯uoro-enone VII. The starting enone1a was
*Corresponding author. Tel.: +33-0-1-49-91-46-67 fax: +33-0-149-91-
34-08; e-mail: alexis.denis@aventis.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00392-9

2020
A. Denis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2019±2022
Figure 1.
Scheme 1. (a) (TMS)2 NH/imidazole/THF; (b) tBuOK/NSFI/THF/ 10ꢀC; (c) N+Bu4F /THF; (d) Ac2O/ CH2Cl2; (e) carbonyldiimidazole /DBU/
RNH2/THF/rt; (f) MeOH.
®rst ¯uorinated similarly to I to give VII in 69% yield.
This compound was then crystallized and the absolute
stereochemistry of C-2 determined (Fig. 2). Finally,
after acetylation in 20, VII was reacted with carbonyl-
diimidazole and DBU in THF and the corresponding 4-
[4-(3-pyridyl)-imidazolyl]lbutyl amine added to generate
I in 67% yield. As the two di€erent synthetic pathways
yielded the same compound (1H NMR, especially 2-Me d
(ppm) 1.79 (d, JH,F=21.5 Hz, 3H), and melting point=
118 ꢀC),5 the absolute S con®guration was attributed to
I . It should be mentioned that this ¯uorination reaction
was also carried out stereospeci®cally in the aza-imino
tricyclic series.6 The chlorine atom was easily introduced
by radical chlorination using N-chlorosuccinimide with
AIBN at 40 ꢀC. II was obtained in 38% yield as a single
isomer of unknown stereochemistry (Scheme 2).
Methylation at C-2 was carried out by treatment of the
20-OAc protected derivative of telithromycin VIII with
methyl iodide and sodium hydroxid0e in the presence of
Bu4N+HSO4 . Removing of the 2 -OAc in methanol
gave III in 13% yield (Scheme 2). The 2,3 enol-ether
derivatives were synthesized by O-alkylation of VIII
with known chloromethylethers. IV was obtained in
21% yield by treatment with Br(CH2)OCH2Cl and NaH
in DMF followed by amination using dimethylamine in
re¯uxing ethanol. Similarly V was obtained in 40% yield
by alkylation with C6H5(CH2)3OCH2Cl (Scheme 2).
Finally, the 2,3 anhydro compound VI was synthesized
in 37% yield from the known acyl-imidazole inter-
mediate4 by reaction with the corresponding amine in
acetonitrile and deprotection of the 20-OAc (Scheme 3);
in agreement with the observation made by Elliott4 for
the anhydrolide series, 17% of the corresponding C-10
epimer were also isolated.
Results and Discussion
All the ketolides and analogues were tested in vitro by
standard agar dilution method against both erythromycin-
susceptible and erythromycin-resistant staphylococci,
streptococci and pneumococci including constitutive
(EryRc) and inducible (EryRi) phenotype. In addition,
one strain of H. in¯uenzae was also tested. Without
Figure 2. X-ray structure of VII.

A. Denis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2019±2022
2021
Scheme 2. Synthesis of 2,3-enol-ether, 2-dimethyl and 2-chloro ketolides; (a) MeI/NaOH/ Bu4N+HSO4 /CH2Cl2; (b) MeOH; (c) N-chloro-
succinimide/AIBN/CCl4/40 ꢀC; (d) NaH/DMF/ Br(CH2)2OCH2Cl; (e) EtOH/(CH3)2NH/re¯ux; (f) NaH/DMF/C6H5(CH2)3OCH2Cl; (g) MeOH.
Scheme 3. Synthesis of 2,3-anhydro telithromycin; (a) NaH/ carbonyldiimidazole/CH3CN/rt; (b) RNH2/DMF; (c) MeOH.
exception, the reference macrolides clarithromycin and
azithromycin were inactive (MICs>40 mg/mL) against
erythromycin resistant strains whatever the phenotype.
e€ective against inducibly resistant S. aureus and S.
pneumoniae as well as constitutively resistant S. pneumo-
niae. I was equal to azithromycin and telithromycin
against H. in¯uenzae (Table 1). The 2-chloro II and 2-
methyl compounds III were both less active than the par-
ent compound; particularly they were almost ine€ective
against EryRi S. aureus and ®ve times less active against
EryR S. pneumoniae.
All the compounds were inactive against a constitutively
erythromycin-resistant strain of S. aureus (MIC>40 mg/
mL). The replacement of a C-2-hydrogen atom for a
¯uorine in I (HMR3562) gave a compound that demon-
strated good activities against strains susceptible to
erythromycin (at least one order of magnitude higher
than those of clarithromycin). Furthermore I was very
The two planar derivatives IV and V were generally
weakly active against most of the strains tested with a
Table 1. In vitro activity of 2,3 modi®ed ketolidesa
MIC (mg/mL)
S. a.
S. a.
S. a.
S. pyo.
S. p.
S. p.
S. p.
S. p.
H. i.
EryS
011UC4
EryRi
011GO25i
EryRc
011CB20
EryS
02A1UC1
EryS
032UC1
EryRc
030PW23c
EryRc
030SJ1
EryRi
030SJ5i
351HT3
(b lactamase +)
AZI
CLA
TEL
I
II
III
IV
0.3
0.3
0.04
0.02
0.6
0.6
2.5
5
>40
>40
0.08
0.08
>40
1.2
20
>40
0.3
>40
>40
>40
>40
>40
>40
>40
>40
>40
0.6
0.08
40.02
40.02
0.04
0.02
0.3
0.15
0.02
0.15
0.04
40.02
40.02
0.04
0.02
0.15
0.8
0.02
>40
>40
0.04
40.02
1.2
2.5
2.5
20
>40
>40
40.02
40.02
5
2.5
10
20
>40
>40
40.02
40.02
0.6
0.6
5
20
1.2
5
1.2
1.2
2.5
2.5
20
V
VI
>40
2.5
0.08
0.3
10
2.5
aAZI=azithromycin; CLA: clarithromycin; TEL: telithromycin; EryS=susceptible; EryRc=constitutive MLS resistance; EryRi=inducible MLS
resistance. S. a.=Staphylococcus aureus; S. pyo.=Streptococcus pyogenes; S. p.=Streptococcus pneumoniae; H. i.=Haemophilus in¯uenzae.

2022
A. Denis et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2019±2022
Table 2. In vivo ecacy of 2-¯uoro ketolide I
pathogen. These results demonstrate that within the
ketolide class, to retain good antibacterial activity,
position 2 needs to remain tetrahedral and tolerates
only very small substituents such as ¯uorine.
ED50 (mg/kg)a
S. a.b
S. p.
S. p.
S. p.
H. i.
EryS
EryS
EryRc
EryRi
AmpR 351RD7
011HT17 032UC1 030MV2 030SJ5i (b lactamase +)
Acknowledgements
CLA
AZI
I
12
72
9
2
4.5
1.5
>50
>50
2
>50
>50
4.3
>150
94
56
The authors wish to thank C. Lang (Structural Analysis
Department) for her support.
aE€ective dosage that protect 50% of mice from lethal infection after
oral administration; AmpR=ampicillin resistant.
bAbbreviations as footnote in Table 1.
References and Notes
complete loss of activity against the Ery R strains
(MICs>2.5±40 mg/mL). In contrast, the anhydrolide VI
retained activity against susceptible and EryRi S. aureus
strains. However it was poorly active (4 to 8 times less
active than I) against erythromycin resistant S. pneumo-
niae whatever the phenotype.
1. (a) Agouridas, C.; Denis, A.; Auger, J.-M.; Benedetti, Y.;
Bonnefoy, A.; Bretin, F.; Chantot, J.-F.; Dussarat, A.; Fro-
mentin, C.; Gouin D'Ambrieres, S.; Lachaud, S.; Laurin, P.;
Le Martret, O.; Loyau, V.; Tessot, N. J. Med. Chem. 1998, 41,
4080. (b) Denis, A.; Agouridas, C.; Auger, J.-M.; Benedetti,
Y.; Bonnefoy, A.; Bretin, F.; Chantot, J.-F.; Dussarat, A.;
Fromentin, C.; Gouin D'Ambrieres, S.; Lachaud, S.; Laurin,
P.; Le Martret, O.; Loyau, V.; Tessot, N.; Pejac, J.-M.; Per-
ron, S. Bioorg. Med. Chem. Lett. 1999, 9, 3075.
In vivo evaluation
2. (a) Inoue, M.; Sato, Y.; Kuga, A.; Okamoto, R. 38th
Interscience Conference on Antimicrobial Agents and Che-
motherapy, San Francisco, CL, 1998, Abstract E-138. (b)
Bonnefoy, A.; Girard, A.-M.; Agouridas, C.; Chantot, J.-F. J.
Antimicrob. Chemother. 1997, 40, 85.
3. (a) Ma, Z.; Clark, R. F.; Nilius, A. M.; Flamm, R. K.; Or, Y.
S. 39th Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Francisco, CL, 1999, Abstract F-2133. (b)
Or, Y. S.; Ma, Z.; Clark, R. F.; Chu, D. T.; Plattner, J. J. WO 98/
09978 A1, 1998. (c) Phan, L. T.; Or, Y. S.; Spina, K. P.; Chen, Y.;
Tufano, M.; Chu, D. T.; Nilius, A. M.; Bui, M.-H.; Plattner, J. J.
37th Interscience Conference on Antimicrobial Agents and
Chemotherapy, Miami, FL, 1997, Abstract F-263.
The in vivo ecacies of compound I to VI and reference
compounds clarithromycin and azithromycin were
assessed by acute lethal murine infection models caused
by susceptible and erythromycin-resistant Gram positive
cocci and H. in¯uenzae (Table 2). Against infections
caused by erythromycin susceptible strains, I exhibited in
vivo ecacies close to clarithromycin and substantially
better than azithromycin. Unlike classical macrolides
(CLA, AZI) which show complete inactivity with ED50
up to 100 mg/kg, I demonstrated excellent anti-pneu-
mococcal ecacy in infections caused by EryRi and
EryRc S. pneumoniae, the corresponding e€ective doses
for I ranging between 2 to 4 mg/kg. Against H. in¯uen-
zae, I exhibited a 2-fold improvement in ecacy over
azithromycin while clarithromycin was inactive under
150 mg/kg.
4. Elliott, R. L.; Pireh, D.; Griesgraber, G.; Nilius, A. M.;
Ewing, P. J.; Ha Bui, M. ; Raney, P. M.; Flamm, R. K.; Kim,
K.; Henry, R. F.; Chu, D. T. W.; Plattner, J. J.; Sun Or, Y. J.
Med. Chem. 1998, 41, 1651.
5. Spectral data for I (HMR 3562): mp: 118 ꢀC; FAB±
MS=830+ (M+H+); 1H NMR (400 MHz, CDCl3): d 0.87 (t,
3H) CH3CH2, 1.01 (d, 3H) 10-CH3, 1.19 (d, 3H) 8-CH3, 1.21±
0
1.68 (m, 1H) H4 , 1.24 (d, 3H) 50-Me, 1.31 (d, 3H) 4-CH3, 1.34
(s, 3H) 6-CH3, 1.79 (d, J=21.5 Hz, 3H) 2-CH3, 1.50 (s, 3H)
12-CH3, 1.60±1.83 (m, 1H) H7, 1.68±1.86 (m, 4H) CH2±CH2,
1.50±1.97 (m, 2H) H14, 2.27 (s, 6H) N(CH3)2, 2.46 (m, 1H)
Conclusions
The b-keto-ester function of ketolides can be chemically
exploited to generate new 2-modi®ed ketolides. Introduc-
tion of a ¯uorine at C-2 by electrophilic ¯uorination results
in good antibacterial activities whereas introduction of
larger substituents or 2,3 anhydro or enol-ether mod-
i®cations result in loss of activity and higher MIC. The
2-¯uoro ketolide I (HMR3562) displays, with the excep-
tion of constitutively MLSB resistant S. aureus, good in
vitro and in vivo activities against all erythromycin
resistant Gram positive cocci, including multi-resistant
S. pneumoniae. In addition, I is active in vitro against H.
in¯uenzae to a similar extent as azithromycin and
demonstrates good in vivo activities against this
0
H3 , 2.59 (m, 1H) H8, 2.55 (s, 3H) 6-OCH3, 3.11 (q, 1H) H10,
0
0
3.18 (dd, J=7.5 and 10 Hz, 1H) H2 , 3.53 (m, 2H) H4 and H5 ,
3.42 (s, 1H) H11, 3.63±3.75(m, 2H) CH2NCO, 4.01 (t, 2H)
0
CH2N, 4.07 (d, J=10.5 Hz, 1H) H5, 4.31 (d, 1H) H1 , 4.86 (dd,
J=2 and 10.5 Hz, 1H) H13, [7.55 (d, 1H) H2, 7.33 (d, 1H) H5]
imidazole, [8.98 (s, 1H) H2, 8.09 (dt, 1H) H4, 7.29 (ddd,1H)
H5, 8.46 (dd, 1H) H6] pyridine. Anal. Calc. (%) for
C43H64N5O10F: C 62.33, H 7.77, N 8.44, F 2.29. Found : C
61.9, H 8.1, N 8.7, F 2.2.
6. Phan, L. T.; Or, Y. S.; Chu, D. T.; Ewing, P.; Nilius, A. M.;
Bui, M.-H.; Raney, P.; Hensey-Rudlo€, D.; Henry, R. F.; Mit-
ten, M. 38th Interscience Conference on Antimicrobial Agents
and Chemotherapy, San Diego, CL, 1998, Abstract F-127.