Synthesis and SAR of azalide 3,6-ketal aromatic derivatives as potent gram-positive and gram-negative antibacterial agents

Article abstract of DOI:10.1016/S0960-894X(02)00434-1

3,6-Ketals of 15-membered azalide pseudoaglycones are a novel series of macrolide antibiotics. The aromatic derivatives of the azalide 3,6-ketals demonstrated potent antibacterial activities against both Gram-positive and Gram-negative bacteria.

Full text of DOI:10.1016/S0960-894X(02)00434-1

Bioorganic& Medicinal Chemistry Letters 12 (2002) 2431–2434
Synthesis and SAR of Azalide 3,6-Ketal Aromatic Derivatives as
Potent Gram-Positive and Gram-Negative Antibacterial Agents
Hengmiao Cheng,* John P. Dirlam, Carl B. Ziegler, Kristin M. Lundy,
Shigeru F. Hayashi, Barbara J. Kamicker, Jason K. Dutra, Kirsten L. Daniel,
Sheryl L. Santoro, David M. George, Camilla D. Bertsche, Subas M. Sakya
and Melani Suarez-Contreras
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
Received 14 February 2002; accepted 15 May 2002
Abstract—3,6-Ketals of 15-membered azalide pseudoaglycones are a novel series of macrolide antibiotics. The aromatic derivatives
of the azalide 3,6-ketals demonstrated potent antibacterial activities against both Gram-positive and Gram-negative bacteria.
# 2002 Elsevier Science Ltd. All rights reserved.
Mastitis is an inflammation of the mammary gland
caused by a variety of bacterial pathogens. The most
commonly isolated pathogens in dairy cows are Sta-
phylococcus aureus, Escherichia coli, and several species
of Streptococcus.1 Mastitis has a significant economic
impact on the world dairy industry due to costs asso-
ciated with poor milk quality from infected cows, loss of
milk from cows on treatment, and the cost of treatment.
Between 30 and 50% of all dairy cows in the US are
estimated to be affected by mastitis on a yearly basis.1a,2
Azalide antibiotics such as azithromycin 1 (Fig. 1) are
well known for their broad-spectrum antibacterial
activity and long tissue half-life.3 In the search for novel
azalide derivatives to treat bovine respiratory disease
(BRD), Lundy et al. discovered that 3,6-ketal azalides
are potent antibacterial agents against Gram-negative
bacteria.4 Subsequent cattle studies also demonstrated
that 3,6-ketal azalides such as CP-456280 2 (Fig. 1) are
effective agents for treating BRD, which is caused typi-
cally by Pasteurella multocida and Mannheimia (Pas-
teurella) haemolytica.5 In our search for a single-shot
antibacterial agent using 3,6-ketal azalides as a template
to treat dry cow mastitis, we discovered that introduc-
tion of an aromaticmoiety to the piperidine nitrogen of
the 3,6-ketal azalides increases their activity against S.
aureus.
Antibiotictherapy is commonly used to eliminate sus-
ceptible mastitis-causing pathogens from the udder. The
most widely used antibacterial agents include penicillins,
cephalosporins, pirlimycin, novobiocin, and dihydro-
streptomycin. Intramammary infusion is widely used to
deliver antibiotics into the infected udder. However, this
treatment method may increase the chance of introdu-
cing environmental pathogens into the udder along with
the drug. Moreover, none of the treatments are
significantly effective against persistent S. aureus infec-
tions. Thus, an effective antibacterial agent administered
as a single subcutaneous treatment at the beginning of
the dry period would be preferred as a safe and con-
venient method of delivering an antimicrobial therapy.
*Corresponding author. Fax: +1-860-441-6952; e-mail: henry_cheng@
groton.pfizer.com
Figure 1. 15-Membered azalide antibiotics.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00434-1

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H. Cheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2431–2434
9a N–H and 9a N–Me 3,6-ketal templates were pre-
pared as shown in Scheme 1.6 9a N–H or 9a N–Me
descladinose azalide 3 was reacted with the methyl
enol ether of N-CBZ piperidone in the presence of
p-toluenesulfonicaicd monohydrate to afford the 3,6-
ketal derivative 4 in 60% yield. The CBZ protecting
group was then removed by hydrogenolysis to generate
the desired 3,6-ketal template in 99% yield.
NaB(OAc)3H as the reducing agent. In method B, the
piperidine nitrogen was alkylated following SN2 reac-
tion conditions with benzyl halides. Method A or B was
chosen based on the availability of the aldehydes or the
halides.
3,6-Ketal derivatives were tested against P. multocida
(59A0067), E. coli (51A0150), and S. aureus (01A0785).
Susceptibility testing was carried out by the microdilu-
tion method described in the NCCLS guidelines.8 MIC
(minimum inhibitory concentration) was determined at
the lowest drug concentration that prohibits bacterial
growth completely.
Scheme 2 illustrates the general synthetic method for
preparing piperidine-amide-3,6-ketal azalide derivatives.
Under standard HOBt/EDC coupling conditions, the
amide bond was formed only with piperidine nitrogen.
The macrolide ring nitrogen was not affected when R0 is
H at the 9a-position probably due to the sterichinder-
ance from the macrolide ring.
S. aureus intraperitoneal infection model. Twenty-gram
female CF-1 mice were infected intraperitoneally (IP)
with 1.6ꢀ105 CFU of S. aureus in 5% hog gastric
mucin. Compounds were administered subcutaneously
0.5 h post-infection at doses of 10–40 mg/kg. The num-
ber of surviving mice was counted for 4 days, and the
effective dose for 50% survival (ED50) was calculated
using a regression equation.
To make piperidine-amine-3,6-ketal azalide derivatives,
two methods were employed as shown in Scheme 3. In
method A, reductive amination was carried out using
S. aureus intramammary infection model. Forty-gram
female CD-1 lactating mice were infected in one mam-
mary gland with ꢁ38 CFU of S. aureus diluted in Dul-
becco’s phosphate buffered saline. Pups were removed
at the time of infection. Compounds were given sub-
cutaneously 0.5 h post-infection at doses of 5–30 mg/kg.
The number of bacteria in the infected mammary gland
was quantitated five days post-infection. Infected, non-
medicated mice have 5ꢀ109 CFU/gland at the time of
necropsy.
All the compounds in the piperidine-amide-3,6-ketal
derivative series demonstrated antibacterial activity
against Gram-positive and Gram-negative bacteria as
shown in Table 1. Regardless of the size of the aromatic
moiety, the ketal derivatives were very potent against P.
multocida with MIC values ranging from 0.05 to 0.3 mg/
mL. Compounds with an unsubstituted five-membered
or six-membered aromaticmoiety demonstrated good in
vitro activity against E. coli. However, compounds with
substituted or fused aromaticsystems demonstrated
decreased activity against E. coli. The MIC of the
piperidine-amide-3,6-ketal derivatives against S. aureus
varied from 0.2 to 3.13 mg/mL. There was no significant
difference for in vitro activity between 9a N–H and 9a
N–Me derivatives.
Scheme 1. Synthesis of the N–H and N–Me azalide piperidine-3,6-
ketal templates.
Scheme 2. General syntheticmethod for preparing piperidine-amide-
3,6-ketal azalide derivatives.
The in vivo activity of these ketal derivatives is also lis-
ted in Table 1. In general, more polar compounds were
more potent in the S. aureus intraperitoneal infection
model, and compounds with pyridine or quinoline moi-
eties were the most active. The more polar 9a N–H
derivatives were usually more active than 9a N–Me
analogues. There is a trend that compounds with lower
clogP values demonstrated better in vivo activity.
Our second in vivo model was a murine S. aureus
intramammary infection (IMI) model that was designed
to mimicbovine mastitis. In this model, potenyc is
measured by decreasing bacterial count, as indicated by
Scheme 3. General syntheticmethod for preparing piperidine-amine-
3,6-ketal derivatives. Reagents and reaction conditions: (a) ArCHO,
NaB(OAc)3H, AcOH, molecular sieves (3A), CH2Cl2; (b) ArCH2X,
Et3N, CH2Cl2.

H. Cheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2431–2434
Table 1. Biological data for piperidine-amide-3,6-ketals
2433
Compd
Ar
R (9a)
In vitro MIC (mg/mL)
S. aureus IP model
ED50 (mg/kg)
S. aureus IMI model
Log10 CFU at 15 mg/kg
clogP
P. multocida
E. coli
S. aureus
1
5
8
9
N/A
N/A
Phenyl
Phenyl
Me
Me
H
Me
H
H
Me
H
Me
H
H
H
H
H
H
Me
H
Me
H
H
H
H
H
0.1
0.05
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.3
0.1
0.1
0.05
0.05
0.05
0.1
0.05
0.05
0.1
0.1
0.1
0.05
0.1
0.1
0.1
0.1
0.1
0.2
1.56
0.2
0.2
4.1
—
20
—
20
7.6
12
6.4
10
6.1
19
9.4
>20
11
2.8
—
2.9
—
1.826
1.454
1.847
2.699
2.066
0.746
0.746
0.746
1.598
ꢂ0.232
1.088
1.441
2.043
1.023
1.023
1.875
1.522
2.476
0.861
1.102
2.245
2.245
2.13
6.25
0.78
—
1.56
3.13
3.13
1.56
1.56
3.13
3.13
1.56
6.25
3.13
3.13
0.78
1.56
0.78
1.56
1.56
0.78
1.56
3.13
6.25
1.56
6.25
3.13
6.25
6.25
6.25
6.25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
m-Methoxyphenyl
2-Pyridinyl
2-Pyridinyl
0.78
0.78
1.56
1.56
1.56
1.56
3.13
1.56
3.13
0.78
0.78
—
5.6
3.5
2.9
3.2
2.6
1.3
9.7
4.4
—
2.6
0.4
3.3
—
3-Pyridinyl
3-Pyridinyl
Pyrazine
6-Hydroxy-2-pyridinyl
2-Chloro-3-pyridinyl
4,5-Dichloro-3-pyridinyl
3-Furanyl
2-Furanyl
2-Furanyl
3-Methyl-2-furanyl
2-Thienyl
2-Pyrrole
12
>20
>20
>20
19
19
>20
11
14
>20
13
>20
7.6
20
0.78
—
—
0.78
0.78
0.78
0.39
0.2
0.39
0.39
0.39
0.39
0.39
0.78
4.7
2.4
—
N-Methyl-2-pyrrole
2-Indole
3-Indole
—
2-Quinoline
2-Quinoline
3-Quinoline
3-Quinoline
4-Quinoline
4-Quinoline
Biphenyl-4-yl
1.9
9.7
3.3
—
1.9
2.5
—
Me
H
Me
H
Me
H
2.982
1.362
2.215
2.13
2.982
3.735
—
Table 2. Biological data for piperidine-amine-3,6-ketals
Compd
Ar
R (9a)
In vitro MIC (mg/mL)
S. aureus IP model
ED50 (mg/kg)
S. aureus IMI model
Log10 CFU at 15 mg/kg
clogP
P. multocida
E. coli
S. aureus
1
N/A
Benzyl
Benzyl
Me
H
Me
H
Me
H
Me
H
Me
H
Me
H
Me
Me
Me
Me
H
Me
H
Me
H
H
Me
H
0.1
1.56
0.1
0.075
0.1
0.1
0.3
0.2
0.2
0.39
0.2
0.2
3.13
50
0.39
0.39
0.2
0.2
0.2
0.78
0.39
0.78
0.78
0.78
1.56
1.56
1.56
0.2
0.39
—
0.39
0.39
0.39
0.6
0.2
0.39
—
0.39
0.39
0.2
0.78
—
4.1
>20
>20
—
>20
—
>20
—
>20
—
>20
—
—
22
9.6
—
2.8
—
7.3
—
—
—
—
—
9.7
—
9.7
—
—
—
9.7
—
1.1
1.9
1.3
—
—
5.2
—
—
—
—
1.826
3.436
4.288
3.355
4.207
3.355
3.471
3.355
4.207
4.149
5.001
4.742
5.594
3.721
3.307
3.464
1.939
2.791
1.939
2.791
1.939
3.323
4.175
3.323
4.175
6.176
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
0.03
0.02
0.03
0.03
0.03
0.03
0.01
0.03
0.01
0.03
0.05
0.05
0.03
0.05
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.05
0.05
0.1
o-Methoxybenzyl
o-Methoxybenzyl
m-Methoxybenzyl
m-Methoxybenzyl
p-Methoxybenzyl
p-Methoxybenzyl
p-Chlorobenzyl
p-Chlorobenzyl
3,4-Dichlorobenzyl
3,4-Dichlorobenzyl
p-Cyanobenzyl
4-Acetaminobenzyl
3-Furanmethyl
2-Pyridyl
—
0.6
<5
17
<5
>20
6.4
2-Pyridyl
3-Pyridyl
3-Pyridyl
4-Pyridyl
0.78
0.39
0.78
1.56
0.2
0.39
0.2
0.39
0.2
2-Quinolinemethyl
2-Quinolinemethyl
4-Quinolinemethyl
4-Quinolinemethyl
Biphenyl-4-yl
22
>20
>20
>20
>20
Me
Me

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H. Cheng et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2431–2434
a low log10 CFU value. A few compounds demonstrated
very good efficacy. For example, when mice were trea-
ted with ketal derivative 20, the observed log10 CFU
was 0.4. This indicated that, among 10 mice treated with
20, most of the murine mammary glands were free of S.
aureus at day five.
demonstrated very good activity in the S. aureus intra-
mammary infection model. They are promising candi-
dates for further efficacy evaluations.
References and Notes
Compounds in the piperidine-amine-3,6-ketal series
have at least three basicnitrogens. The presenec of a
third basicnitrogen made these ketal derivatives more
potent against both Gram-negative and Gram-positive
bacteria as shown in Table 2. Most compounds demon-
strated very potent activity against P. multocida. In
general, the piperidine-amine-3,6-ketal derivatives were
also very potent against E. coli. When the size of the
aromatic moiety increased, the activity of the piper-
idine-amine-3,6-ketal against E. coli decreased. For
example, the MIC for compound 36 against E. coli was
0.075 mg/mL. When the phenyl ring in compound 36
was substituted by another phenyl ring, the MIC of the
resultant analogue 59 was increased to 1.56 mg/mL.
Compared to the piperidine-amide derivatives, the
piperidine-amine ketals demonstrated significantly more
potent in vitro activity against S. aureus. This is con-
sistent with the general observation that the more lipo-
philicthe macrolide derivative is, the more potent its in
vitro activity against S. aureus.7 In general, the piper-
idine-amine derivatives have much higher clogP values
than their amide analogues. For in vivo activity of
macrolides, the general trend is that the more polar
analogues usually demonstrate more potent activity.7
This was also the case with this series of compounds.
The piperidine-amine-3,6-ketals, which are more lipo-
philicthan the piperidine-amide-3,6-ketal derivatives,
were less active in the S. aureus intraperitoneal infection
model. The exceptions are pyridyl derivatives in the 9a
N–H series. For example, both compounds 50 and 52
demonstrated ED50s less than 5 mg/kg. The same is true
for their activity in the murine intramammary infection
model; the log10 CFU for mice treated with 50 and 52
were 1.1 and 1.3, respectively.
1. (a) Bramley, A. J.; Cullor, J. S.; Erskine, R. J.; Fox, L. K.;
Harmon, R. J.; Hogan, J. S.; Nickerson, S. C.; Oliver, S. P.;
Smith, L. K.; Sordillo, L. M. In Current Concepts of Bovine
Mastitis, 4th ed.; National Mastitis Council, 1996; p 11. (b)
Barkema, H. W.; Schukken, Y. H.; Lam, T. J. M.; Beiboer, M.
L.; Wilmink, H.; Benedictus, G.; Brand, A. J. Dairy Sci. 1998,
81, 411.
2. (a) Hogan, J. S.; Smith, K. L.; Hoblet, K. H.; Schoenberger,
P. S.; Todhunter, D. A.; Hueston, W. D.; Pritc hard, D. E.; Bow-
man, 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: 1998, p 9.
3. Bright, G. M.; Nagel, A. A.; Bordner, J.; Desai, K. A.;
Dibrino, J. N.; Nowakowski, J.; Vincent, Lawrence; Watrous,
R. M.; Sciavolino, F. C.; English, A. R.; Retsema, J. A.;
Anderson, M. R.; Brennan, L. A.; Borovoy, R. J.; Cimo-
chowski, C. R.; Faiella, J. A.; Girard, A. E.; Girard, D.; Her-
bert, C.; Manousos, M.; Mason, R. J. Antibiot. 1988, 41, 1029.
4. (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 Spectro-
scopy, 218th National Meeting of the American Chemical
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.
5. Lundy, K. M.; Minich, M.; Jaynes, B.; Hayashi, S.;
Kamicker, B.; Bertsche, C.; Cheng, H.; George, D.; Morton,
B.; Pratt, B.; Rafka, R.; Santoro, S.; Silvia, A. Abstract of
Papers, Azalide 3,6-Ketals: Synthesis and SAR of a Novel
Class of Macrolide Antibiotics, 2000 Pacifichem Meeting,
Honolulu, Hawaii, December 14–19, 2000; Poster number 264.
6. Cheng, H.; Bertinato, P. A.; Bertsche, C. D.; Daniel, K.;
Dutra, J. K.; George, D.; Hayashi, S. F.; Kamicker, B. J.;
Lundy, K. M.; Minich, M. L.; Morton, B. J.; Pratt, B.; Rafka,
R. J.; Sakya, S.; Santoro, S. L.; Suarez-Contreras, M.; Vam-
vakides, N.; Ziegler, C. B. Azalide 3,6-Ketals: SAR in An
Aromatic Analogue Series with Potent Gram-positive and
Gram-negative Activity, 2000 Pacifichem meeting, Honolulu,
HI, Dec14–19, 2000; Posternumber 265.
In conclusion, 3,6-ketal azalides with potent in vitro
activity against both Gram-negative and Gram-positive
bacteria were discovered by derivatization of the piper-
idine ketal nitrogen with lipophilicaromaticmoieties.
There was no significant difference in the in vitro activ-
ity between 9a N–H and 9a N–Me analogues. However,
compounds in the 9a N–H series usually demonstrated
more potent activity in the in vivo models. Compounds
20, 50, and 52, which have heterocyclic aromatic rings,
7. McFarland, J. W.; Berger, C. M.; Froshauer, S. A.; Haya-
shi, A. F.; Hecker, S. J.; Jaynes, B. H.; Jefson, M. R.;
Kamicker, B. J.; Lipinski, C. A.; Lundy, K. M.; Reese, C. P.;
Vu, C. B. J. Med. Chem. 1997, 40, 1340.
8. Performance standards for antimicrobial disk and dilution
susceptibility tests for bacteria isolated from animals;
approved standard, M31-A, Vol. 19, No. 11, June, 1999.