Azalide 3,6-ketals: Antibacterial activity and structure-activity relationships of aryl and hetero aryl substituted analogues

Article abstract of DOI:10.1016/S0960-894X(03)00100-8

Aryl and hetero aryl substituted 3,6-ketals of 15-membered azalide analogues were synthesized and were found to have potent in vitro antibacterial activity against veterinary pathogens, including Staphylococcus aureus and Pasteurella multocida.

Full text of DOI:10.1016/S0960-894X(03)00100-8

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1373–1375
Azalide 3,6-Ketals: Antibacterial Activity and Structure–Activity
Relationships of Aryl and Hetero Aryl Substituted Analogues
Subas M. Sakya,* Peter Bertinato, Bryan Pratt, Melani Suarez-Contreras,
Kristin M. Lundy, Martha L. Minich, Hengmiao Cheng, Carl B. Ziegler,
Barbara J. Kamicker, Shigeru F. Hayashi, Sheryl L. Santoro, David M. George
and Camilla D. Bertsche
Veterinary Medicine Research and Development, Pfizer Inc, Eastern Point Road, Box 4063, Groton, CT 06340, USA
Received 17 September 2002; accepted 16 December 2002
Abstract—Aryl and hetero aryl substituted 3,6-ketals of 15-membered azalide analogues were synthesized and were found to have
potent in vitro antibacterial activity against veterinary pathogens, including Staphylococcus aureus and Pasteurella multocida.
# 2003 Elsevier Science Ltd. All rights reserved.
Mastitis is an inflammation of the mammary gland,
usually caused by a variety of bacteria. One of the most
prevalent pathogens for non-lactating cow (dry cow)
mastitis is Staphylococcus aureus. Mastitis is the most
important disease in the worldwide dairy industry and
causes considerable economic loss. The US National
Mastitis Council estimates that annual losses to the
dairy industry amount to US$1.8–2 billion, or US$185–
200 per cow. Yearly, between 30 and 50% of all dairy
cattle in the US are affected by mastitis.1,2
ella multocida and Mannheimia (Pasteurella) haemoly-
tica, responsible for BRD.3 These analogues,
exemplified by 1, also showed potent in vivo activity
against those pathogens.4 We initiated synthetic efforts
to identify compounds with improved activity against
Gram-positive mastitis pathogens using the 3,6-ketal
azalide as a template. Recently we reported the SAR of
the benzyl and aryl amide modifications on the 3,6-ketal
nitrogen (Fig. 1) in which we showed improved activity
by incorporation of heteroaryl moieties.4 In this paper,
we will describe the results of the modification of the
piperidine ketal nitrogen with aryl and heteroaryl
derivatives.
Antibiotic therapy is widely used to eliminate mastitis-
causing infections and return udders to a healthy con-
dition. These antibacterial agents include penicillins,
tetracyclines, macrolides and a number of cephalospor-
ins. Intramammary infusion (IMI) is commonly used to
deliver the antibiotics. However, none of the treatments
is significantly effective against mastitis caused by S.
aureus. Here, we report our attempts to discover anti-
bacterial agents with improved activity against S. aureus
when administered by subcutaneous injection.
In the search for active compounds against bovine
respiratory disease (BRD), Lundy et al. discovered that
3,6-ketals of the 9a-azalides showed excellent in vitro
activity against the Gram-negative pathogens, Pasteur-
*Corresponding author. Fax: +1-860-715-7312; e-mail: sakyas@
groton.pfizer.com
Figure 1. Lead 3,6-piperidine ketal azalides.
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00100-8

1374
S. M. Sakya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1373–1375
Compound 6 was used as precursor material for syn-
thesis of the 3,6-ketal analogues. The synthesis of 6 was
carried out using established procedures according to
Scheme 1.4
coli. The nitrophenyl derivatives (7d and 7e) showed
only moderate activity against all three pathogens.
Other functional groups containing aryl analogues did
not improve upon the activity against S. aureus.
The syntheses of aryl piperidine ketal derivatives were
accomplished by heating 3,6-ketal 6 with aryl fluorides
in the presence of base as shown in Scheme 2.
Among the pyridine analogues, nitro- and cyano-sub-
stituted analogues (8a–8c) showed good activity against
S. aureus and P. multocida, and reduced activity against
E. coli. Adding a more polar functionality to the mol-
ecule (8d) improved the activity against E. coli. The
hydroxy methyl substituted analogue (8e) had the best
activity against all three pathogens. Most analogues had
reasonable activity against all the pathogens with the
exception of 8k (Table 1).
Scheme 3 shows the preparation of the 2-pyridyl, 2-
pyrimidine, and 2-triazine derivatives. Generally mod-
erate to good yields of the products were obtained.
Similar reaction conditions were used for the prepara-
tion of other heterocyles (8t and 8v–8y). The pyrimidine
groups were further functionalized to make several ester
and amide derivatives.
The pyrimidine analogues had the weakest activity
against E. coli and P. multocida while having good
activity against S. aureus. The more lipophilic 8l and
8m had the best activity against S. aureus. Several of
the pyrimidine analogues (8n, 8o, 8r, and 8s) had
moderate activity against all three pathogens. Further
modifications of the pyrimidine ring did not improve
activity.
While broad spectrum agents are desirable, activity
against the Gram-positive bacteria S. aureus is essential.
Among the substituted aryl analogues, 2-fluoro or 3-
CF3-4-benzyloxycarbonyl substituted derivatives (7a
and 7c) with 9a N-H showed potent activity against S.
aureus (MIC: 0.39–0.78 mg/mL) but had less activity
against the Gram-negative Escherichia coli. The corre-
sponding 3-CF3-4-benzyloxycarbonyl aryl derivative
(7b) with 9a N-Me showed improved activity against E.
The heterocyclic analogues 8s–8x did not show any
enhanced activity over the other analogues. Overall,
very slight differences in the activity profile of the
various aryl and heteroaryl derivatives of the 3,6-
piperidine ketals were seen. A majority of the aryl
and heteroaryl analogues showed improved activity
against S. aureus over the unsubstituted 3,6-ketal 6
and reduced activity against E. coli. With the excep-
tion of the rather lipophilic pyrimidine analogues,
most analogues were active against P. multocida. Sev-
eral of the active aryl, pyridine and pyrimidine analo-
gues (7a, 7b, 7d, 7e, 8a, 8f, 8h, 8l, 8m, 8o, 8r, and 8u)
were tested in a S. aureus murine intraperitoneal infec-
tion model and showed poor in vivo activity (>20 mg/
kg ED50). Previous studies5 had shown that polarity was
essential for improved in vivo activity for macrolides.
Therefore, the lack of in vivo activity may be due to
the significantly increased lipophilicity of these new
analogues.
Scheme 1. (a) pTsOH, 4, CH2Cl2, 60%; (b) H2, Pd/C, 99%.
Aryl and heteroaryl linked piperidine 3,6-ketal azalides
with potent in vitro activity against both Gram-positive
and Gram-negative bacteria were discovered. There was
no significant difference in the in vitro activity against S.
aureus between 9a N-H and N-Me analogues. Unlike
the previously reported compounds4 these lipophilic
derivatives had minimal or no activity in a S. aureus in
vivo murine model.
Scheme 2. (a) CsCO3, aryl fluoride, base, 80–95 ꢀC, 60–85%.
In Vitro Assays
The microdilution method based on the NCCLS guide-
lines6 has been reported previously.4 Minimum inhibi-
tory concentration (MIC) was determined at the lowest
drug concentration that completely inhibits bacterial
growth. The following three strains were used for the
MIC determinations: 01A0758 (S. aureus), 51A0150 (E.
coli), and 59A0067 (P. multocida).
Scheme 3. (a) Et3N, 2-chloro pyridine, 2-chloro pyrimidine, or 2-
chloro triazine, CH3CN or iPrOH 80–95 ꢀC, 60–85%.

S. M. Sakya et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1373–1375
Table 1. Biological data for aryl and hetero aryl piperidine amine-3,6-ketals
1375
Compd
Ar
MIC (mg/mL)
E. coli
R (9a)
S. aureus
P. multocida
ClogP
6
N/A
2-Fluoro-4-benzyloxycarbonyl phenyl
3-CF3-4-Benzyloxycarbonyl phenyl
3-CF3-4-Benzyloxycarbonyl phenyl
2-Nitro phenyl
4-Nitro phenyl
2-Fluoro-4-pyridyl methyl amido phenyl
4-Cyano phenyl
Me
H
Me
H
6.25
0.39
0.78
0.78
1.56
1.56
3.13
3.13
25
0.39
>50
3.13
>50
<0.05
3.13
0.39
6.25
0.39
0.39
0.2
0.39
3.13
0.39
1.56
1.56
1.56
0.20
0.78
1.56
0.1
1.454
5.529
7.61
7a
7b
7c
7d
7e
7f
7g
7h
7i
6.757
4.698
4.698
3.754
4.419
2.554
6.638
5.573
5.097
3.362
2.695
2.898
3.114
2.974
2.49
2.399
1.929
2.695
3.805
3.548
3.426
4.675
5.528
2.849
2.923
2.408
3.776
3.72
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
H
H
H
H
H
H
H
Me
H
H
Me
Me
H
Me
Me
H
Me
Me
Me
Me
Me
Me
Me
3.13
6.25
1.56
25
12.5
3.13
>50
50
12.5
3-CF3-4-COOH-phenyl
4-Benzyloxycarbonyl phenyl
4-(4-F-Phenylsulfonyl) phenyl
4-Benzoylamino phenyl
4-Amino phenyl
3.13
3.13
12.5
6.35
0.78
0.78
0.78
1.56
1.56
1.56
1.56
1.56
1.56
3.13
6.25
0.78
1.17
1.56
1.56
3.52
6.25
1.56
1.56
1.56
1.56
3.13
3.13
3.13
6.25
7j
7k
7l
8a
8b
8c
8d
8e
8f
8g
8h
8i
3-Nitro-2-pyridyl
6.25
3-Cyano-6-Me-2-pyridyl
4-Methyl-5-nitro-2- pyridyl
5-Nitro-6-amino-2-pyridyl
4-Hydroxymethyl-6-chloro-2-pyridyl
5-Cyano-2-pyridyl
6.25
6.25
3.13
0.78
6.25
6.25
3.13
12.5
6.25
50
>50
>50
1.56
3.13
>50
12.5
0.2
0.20
0.20
0.39
0.78
0.39
3.13
>50
>50
0.10
0.78
>50
1.56
0.2
5-Amido-2-pyridyl
5-Nitro-2-pyridyl
5-CF3-2-pyridyl
5-Nitro-2-pyridyl
8j
8k
8l
5-Ethoxycarbonyl 2-pyridyl
4-CF3-5-Benyloxycarbonyl-2-pyrimidine
4-CF3-5-Benyloxycarbonyl-2-pyrimidine
2-Pyrimidine
4-CF3-2-Pyrimidine
5-(Pyrid-4-ylmethyl amido)-2-pyrimidine
4-CF3-2-Pyrimidine
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
8y
2,4-Methoxy-6-pyrimidine
2,4-Methoxy-6-pyrimidine
6-Chloro-2-pyrazine
3.13
6.25
6.25
0.39
0.39
>50
0.1
4.576
3.537
4.63
4.561
3.519
4.411
3.183
4-Chloro-6-phenyl-2-1,3,5-triazine
2-Benzimidazole
2-Thiazole
>50
1.56
3.13
6.25
3.13
0.39
0.78
0.20
2-Benzoxazole
6-Methyl-3-pyridazine
Murine Models
ger, P. S.; Todhunter, D. A.; Hueston, W. D.; Pritchard, D. E.;
Bowman, G. L.; Heider, L. E.; Brockett, B. L.; Conrad, H. R.
J. Dairy Sci. 1989, 72, 1547. (b) Clements, M. In Animal
Pharm Reports, Bovine Mastitis: Products and Markets; PJB,
Richmond, UK: June 30, 1998 p 9.
3. (a) Minich, M. L.; Lundy, K. M. Abstract of Papers,
Structural Elucidation of Descladinose Azithromycin-3,6-
(Azacyclohexyl) Ketal by One- and Two-D NMR Spec-
troscopy, 218th National Meeting of the American Che-
mical Society, New Orleans, LA, Aug 22–26, 1999. (b)
Minich, M. L.; Lundy, K. M.; Rafka, R. J.; Morton, B. J.
Abstract of Papers, Azalide 3,6-Ketals: Discovery of a Novel
Class of Azalide Antibiotics, 218th National Meeting of the
American Chemical Society, New Orleans, LA, Aug 22–26,
1999.
A S. aureus intraperitoneal infection model was descri-
bed in a previous report.4 The number of surviving mice
was counted after 4 days, and the effective dose for 50%
survival (ED50) was calculated using a regression
equation.
Acknowledgements
We are grateful to Dr. Robert Rafka’s group for pro-
viding us multi-gram quantities of 6 and Dr. John Dir-
lam for many helpful discussions.
4. Cheng, H.; Dirlam, J. P.; Ziegler, C. B.; Lundy, K. M.;
Hayashi, S. F.; Kamicker, B. J.; Dutra, J. K.; Daniel, K. L.;
Santoro, S. L.; George, D. M.; Bertsche, C. D.; Sakya, S. M.;
Suarez-Contreras, M. Bioorg. Med. Chem. Lett. 2002, 12,
2431.
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Approved standard, M31-A, Vol. 19, No. 11, June, 1999.
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