Synthesis and activity of a novel class of tribasic macrocyclic antibiotics: The triamilides

Article abstract of DOI:10.1016/S0960-894X(02)00526-7

The stereoselective synthesis of two novel series of tribasic macrocyclic antibiotics with potent in vitro activity against Pasteurella multocida and Escherichia coli strains of bacteria is described. The in vitro activity can be significantly influenced

Full text of DOI:10.1016/S0960-894X(02)00526-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2771–2774
Synthesis and Activity of a Novel Class of Tribasic Macrocyclic
Antibiotics: The Triamilides
Michael A. Letavic,* Brian S. Bronk,* Camilla D. Bertsche, Jeffrey M. Casavant,
Hengmiao Cheng, Kirsten L. Daniel, David M. George, Shigeru F. Hayashi,
Barbara J. Kamicker, Nicole L. Kolosko, Laura J. L. Norcia, Vanessa D. Oberton,
Margaret A. Rushing and Sheryl L. Santoro
Pfizer Global Research and Development, Groton Laboratories, Eastern Point Road, Groton, CT 06340, USA
Received 27 March 2002; accepted 6 June 2002
Abstract—The stereoselective synthesis of two novel series of tribasic macrocyclic antibiotics with potent in vitro activity against
Pasteurella multocida and Escherichia coli strains of bacteria is described. The in vitro activity can be significantly influenced by the
nature of the substituents on the C-400 aminoalcohol, with the stereochemistry of the C-400 alcohol playing a less critical role. The
effect of substitution and stereochemistry on the in vivo activity in a murine model of respiratory infection is also described.
# 2002 Elsevier Science Ltd. All rights reserved.
Respiratory disease is the leading cause of morbidity
and mortality in North American beef cattle, with out-
breaks typically associated with animals that have been
recently transported to a feedlot. It has been estimated
that bovine respiratory disease (BRD) afflicts up to 30%
of cattle in domestic feedlots, with an average mortality
rate of 5%.1 Swine production is also significantly
impacted by respiratory disease.2
the treatment of human respiratory infections caused by
a variety of pathogens.3 We were particularly interested
in investigating the antimicrobial properties of novel
antibiotics related to the 15-membered azalides due to
their favorable tissue distribution and half-life as well as
their broad-spectrum antimicrobial activity.4
Antibacterial agents continue to play a pivotal role in
the responsible management of livestock respiratory
disease. Difficulties in handling animals in a safe and
stress-free way present an ongoing challenge to provid-
ing the best antimicrobial therapy for livestock. Anti-
microbial agents that can provide adequate duration of
therapy from a single application possess considerable
advantage for both the animal and the livestock produ-
cer. Although a wide variety of agents are approved for
treating BRD, the 16-membered semisynthetic macro-
lide tilmicosin (Micotil1, 1) has emerged as one of the
most popular therapeutic agents1 based in part on an
extended duration of action.
Macrocyclic antibiotics such as clarithromycin (2) and
azithromycin (3) have been used for quite some time for
*Corresponding authors. Tel.: +1-860-441-1803; fax: +1-860-715-2467;
e-mail: michael_a_letavic@groton.pfizer.com (M.A. Letavic); Tel.: +1-
860-441-1712; fax: +1-860-715-8157; e-mail: brian_s_bronk@groton.
pfizer.com (B.S. Bronk).
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00526-7

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M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2771–2774
Scheme 2. (a) Me3S(O)I, NaH, DMSO, tetrahydrofuran, 0–23 ꢀC,
100%; (b) MeOH, KI, 1ꢀ or 2ꢀ amine, 50 ꢀC.
Scheme 1. (a) CBZ-Cl, CH2Cl2, 0 ꢀC, 100%; (b) 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride, pyridinium trifluoro-
acetate, 0–23 ꢀC, CH2Cl2 88%.
Our efforts in this area included modification of the
C-400 alcohol of the cladinose ring on the 15-membered
macrocyclic template via installation of a third basic
amine or other heteroatom functionality. The goals of
this research were to improve the tissue distribution and
to extend elimination half-life of the drug, while main-
taining or enhancing potency against Pasteurella multo-
cida and Escherichia coli, two common pathogens of
livestock. We now report the preparation and screening
of a novel series of tribasic 15-membered macrocyclic
antibiotics that have potent in vitro and in vivo anti-
microbial activity. We have named this new class of
antibiotics the ‘triamilides’.
Scheme 3. (a) Me3SBF4, KHMDS, toluene, tetrahydrofuran, ꢁ78 ꢀC,
58%; (b) MeOH, KI, 1ꢀ or 2ꢀ amine, 50 ꢀC.
Aminoalcohols 8 and 10 are readily prepared in four
steps from the previously described5 C-400 alcohol 4.
Treatment of 4 (Scheme 1) with one equivalent of
benzyl chloroformate in dichloromethane afforded the
C-20 protected alcohol 5 in high yield. The initial
strategy to protect the reactive functionality on 4 relied
on utilization of excess benzyl chloroformate to afford
the doubly protected macrolide resulting from reaction
at both the C-20 alcohol and the 9a N-H. While this
intermediate could be used to prepare 8 and 10, the
sequence required a difficult hydrogenolysis step in
order to deprotect the 9a N-H. Following the obser-
vation that it was possible to efficiently protect only the
C-20 alcohol, we discovered that protection of the
nitrogen was not required for the later steps in
the sequence. Thus, the reaction of 4 with one equiva-
lent of benzyl chloroformate was found to proceed with
excellent selectivity and provide the C-20 protected
alcohol 5 as the sole product that could be used in
subsequent steps without the need for further purifica-
tion. Selective oxidation of the C-400 alcohol of 5 with
EDC/PTFA proceeded smoothly to give ketone 6 in
88% yield.
removing the benzyl carbonate at C-20 to provide the
desired macrocycle in modest to good yield.
The preparation of the epimeric aminoalcohol 10 is
shown in Scheme 3. In this sequence ketone 6 was trea-
ted with the more reactive ylide produced via the reac-
tion of trimethylsulfonium tetrafluoroborate and
potassium bis(trimethylsilyl)amide at low temperature
to afford epoxide 9 in moderate yield and excellent dia-
stereomeric purity (ca. 20:1). This epoxide was then
converted to aminoalcohol 10 upon reaction with the
appropriate amine in methanol as described for the
preparation of 8.
Unequivocal proof of the stereochemical outcome of the
epoxidation reaction to form 9 was obtained from a
single crystal X-ray structure of 10c. The conformation
of crystalline 10c is shown in Figure 1. The crystal
structure reveals several interesting facts concerning the
orientation of the macrocyclic ring and the stereo-
chemistry of the modified cladinose ring structure. One
of the most notable features is that the oxygen bearing
the macrocyclic ring system is in an axial orientation on
the cladinose ring. In addition, the aminoalcohol
formed from the addition of propyl amine to the epox-
ide is that derived from axial addition of the sulfur ylide
to the ketone. This is the expected epoxide based on lit-
erature precedence for unstabilized ylides to form the
epoxide via an axial attack on the ketone.
Scheme 2 depicts the preparation of aminoalcohols 8.
Treatment of ketone 6 with the stabilized ylide formed
from the reaction of trimethylsulfoxonium iodide and
sodium hydride provided the epoxide 7 in quantitative
yield as the major diastereomer (ca. 10:1).6 Epoxide 7
was converted to the aminoalcohol 8 upon reaction with
the appropriate amine in methanol. In all cases, use of
excess amine smoothly converted the epoxide to the
corresponding aminoalcohol while simultaneously
The above procedures efficiently provided the alkyl and
aryl aminoalcohols 8a–m, 8p–q, and 10a–j. We were also
interested, however, in products resulting from opening

M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2771–2774
2773
Table 1. MIC values against P. multocida and E. coli, and ED50
values in a mouse P. multocida infection model for compounds 8
Compd
R2
P. multocida
MIC
(Mg/mL)
E. coli
MIC
(mg/mL)
Mouse
ED50
(mg/kg)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H2N—
n-BuNH—
n-HexylNH—
CyclopropylNH—
t-BuNH—
F3CCH2NH—
MeO(CH2)2NH—
Me2N—
0.39
<0.1
0.2
0.05
0.1
<0.1
<0.1
<0.1
<0.1
0.1
0.05
<0.1
0.2
6.25
0.39
0.78
0.2
0.39
1.56
0.78
0.39
0.78
0.78
0.2
0.39
3.13
3.13
0.78
1.56
>50
1.56
30
23
>80
20
14
46
9.5
9.9
7.2
17
7.3
40
49
20
23
12
n.d.
51
Et(Me)N—
Cyclopropyl(Me)N—
Pyrrolidine
Piperidine
Morpholine
Imidazole
1,2,3-Triazole
HO(CH2)2(Me)N—
(HO(CH2)2)2N—
HO(CH2)2S—
0.2
0.05
0.1
3.13
0.2
Figure 1. The configuration of 10c in the solid state. Carbon atoms are
in green, oxygen atoms in red and nitrogen atoms in blue.
Table 2. MIC values against P. multocida and E. coli, and ED50
values in a mouse P. multocida infection model for compounds 10
Compd
R2
P. multocida
MIC
(mg/mL)
E. coli
MIC
(mg/mL)
Mouse
ED50
(mg/kg)
10a
10b
10c
10d
10e
10f
10g
10h
10i
H2N—
n-BuNH—
n-PropylNH—
CyclopropylNH—
t-BuNH—
Me2N—
Et(Me)N—
CylcopropylCH2NH—
Pyrrolidine
Piperidine
0.39
0.1
0.1
0.1
0.05
0.05
0.1
6.25
0.78
0.39
0.78
0.39
0.39
0.39
0.39
0.2
n.d.
17
12
15
9.6
5.9
<5
5.7
<10
n.d.
>40
Scheme 4. (a) Pyridinium-HCl, methanol, 55 ꢀC; imidazole (8n), 1,2,3-
triazole (8o).
0.1
0.05
0.05
0.39
10j
10k
0.39
25
epoxides 7 and 9 with other nucleophiles. Although
preliminary efforts did not afford conditions to effi-
ciently open the epoxides with oxygen nucleophiles, we
were able to produce the alkyl thiols 8r and 10k by the
reaction of 2-mercaptoethenol and the epoxide in
refluxing benzene in the presence of potassium carbon-
ate. In addition, the heteroaryl substituted alcohols 8n
and 8o were obtained by reaction of 7 with the hetero-
cycle in methanol in the presence of pyridinium-HCl as
shown in Scheme 4.
HO(CH2)2S—
0.39 mg/mL while more lipophilic substituents display
slightly improved activity. More compact, lipophilic
side chains such as trifluoroethyl (8f), cyclopropyl (8d
and 10d), t-butyl (8e and 10e), and pyrrolidine (8k and
10i) were the most active against this pathogen. The
polarity of the side chain has a more significant effect on
the in vitro activity against E. coli. Again, all of the
lipophilic substituents improved the E. coli activity over
the unsubstituted analogues 8a and 10a. Introduction of
polar substituents at C400 generally reduced the E. coli
activity as exemplified with 8q and 10k.
The aminoalcohols 8 and 10 were tested in vitro for
their ability to inhibit the growth of P. multocida and E.
coli. The minimum inhibitory concentration (MIC) was
determined by the microdilution method described in
the NCCLS guidelines.7 Most of the representative
amino-alcohols in Tables 1 and 2 effectively inhibited
the growth of P. multocida (MIC<0.39 mg/mL). The
least active is the bishydroxyethylamine 8q (MIC=3.13
mg/mL). Presumably the increased polarity resulting
from introduction of the second hydroxyethyl sub-
stituent causes this diminished activity, as both the bis
methoxyethylamine 8g and hydroxyethyl(methyl)amine
8p retain potency against P. multocida (MICꢂ0.1 mg/
mL). Modest differences in the activity against P. mul-
tocida are observed within the lipophilic alkyl sub-
stituents in both series of aminoalcohols. Thus, the
unsubstituted aminoalcohols 8a and 10a have MIC’s of
Tables 1 and 2 also show the in vivo activity of many of
the amino-alcohols as measured utilizing a murine
model of respiratory disease.8 In this model, mice are
infected intranasally with P. multocida. The drug is
subsequently administered subcutaneously. The in vivo
activity of aminoalcohols 8 reveals that larger alkyl
groups are disfavored (i.e., n-hexyl, piperidine and
morpholine; 8c, 8l, and 8m) while smaller groups gen-
erally show improved activity (i.e., cyclopropyl, di-
methyl and ethylmethyl; 8d, 8h, and 8i) although the
effects are not dramatic. The isomeric aminoalcohols 10
also show good in vivo activity in this model. As was

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M. A. Letavic et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2771–2774
observed with the aminoalcohols 8, the smaller alkyl
substituted analogues are the most potent of this group.
Dibrino, J. N.; Nowakowski, J.; Vincent, L.; Watrous, R. M.;
Sciavolino, F. C.; English, A. R.; Retsema, J. A.; Anderson,
M. R.; Brennan, L. A.; Borovoy, R. J.; Cimochowski, C. R.;
Faiella, J. A.; Girard, A. E.; Girard, D.; Herbert, C.; Manou-
sos, M.; Mason, R. J. Antibiot. 1988, 41, 1029.
5. Djokic, S.; Kobrehel, G.; Lazaravski, G.; Lopotar, N.;
Tamburasev, Z. J. Chem. Soc., Perkin Trans. 1 1986, 1882.
6. The stereochemical outcome of this reaction was verified by
reaction of the epoxide of 7 with lithium aluminum hydride to
form the methyl carbinol and comparison to the product
resulting from addition of methyl magnesium bromide to the
ketone 6. The stereochemical outcome of the latter reaction
was demonstrated through a single crystal X-ray structure, the
details of which will be published in due course (manuscript in
preparation).
In conclusion, we have discovered a new class of anti-
biotics with very promising in vitro activity against two
common veterinary pathogens. In addition, many
members of this new series of antimicrobial agents
demonstrate excellent in vivo activity against P. multo-
cida in a murine infection model indicating that these
compounds are promising candidates for the treatment
of livestock respiratory disease. Several of the more
interesting members of this series of compounds war-
rant further investigation of pharmacokinetic char-
acteristics and antimicrobial efficacy.
7. The E. coli strain 51A0150 (poultry lung origin) and P.
multocida strain 59A0067 (turkey origin) were used in this
assay to test the antibacterial activity. Both strains were grown
on Brain Heart Infusion (BHI) plates overnight. Several colo-
nies were suspended into saline and adjusted to
OD625nm=0.09 (0.5 McFarland unit). The inoculum solution
was made by preparing a 1:100 dilution of 0.5 McFarland sal-
ine suspension using BHI broth and 100 mL of this suspension
was added to 100 mL of BHI broth containing various con-
centrations of test antibiotics. The test antibiotic solution was
serially diluted 2-fold by automatic pipette in a 96-well micro-
titer format. After inoculation with both strains (final density
was approximately 5ꢃ105 cfu/mL), the microtiter plates were
incubated at 37 ꢀC for 18 h. MIC was determined as the lowest
concentration of the test compound in which the absorbency
at 600 nm is less than or to equal 0.025. For reference, tilmi-
cosin gave an MIC of 0.78 mg/mL against P. multocida and 50
mg/mL against E. coli in these assays.
8. The experimental parameters for the murine model are as
follows; twenty gram female CF-1 mice were infected intrana-
sally with 50 mL of a suspension of 2ꢃ104 P. multocida ser-
otype 5A. Compounds were administered subcutaneously 0.5
h post-infection at doses of 5–80 mg/kg. The ED50 was calcu-
lated based on the number of surviving mice 4 days after
infection. For reference, Oxytetracycline has an ED50 of 17
mg/kg in this assay.
Acknowledgements
The authors would like to acknowledge the contribu-
tions of Bingwei Yang (chemistry discussions) and Nick
Vamvakides (biology technical assistance). The authors
would also like to thank Jon Bordner for solving the
crystal structure of 10c and Frank DiCapua for provid-
ing Figure 1.
References and Notes
1. USDA. 2000. Part I-III: Beef Feedlot Study, 1999. USDA:
APHIS:VS, CEAH, National Animal Health Monitoring Sys-
tem (NAHMS). Fort Collins, CO. #N327.0500, #N335.1000
and #N336.1200.
2. USDA. 2001. Part 1: Reference of Swine Health and Man-
agement in the United States, 2000, National Animal Health
Monitoring System (NAHMS). Fort Collins, CO.
#N338.0801.
3. Wu, Y.-J.; Su, W.-G. Curr. Med. Chem. 2001, 8, 1727.
4. Bright, G. M.; Nagel, A. A.; Bordner, J.; Desai, K. A.;