The synthesis of paleic acid, an antimicrobial agent effective against Mannheimia and Pasteurella, and its structurally related derivatives

Article abstract of DOI:10.1016/j.bmcl.2010.07.115

A synthetic route to paleic acid 1, antimicrobial agent effective against Mannheimia haemolytica and Pasteurella multocida, has been established. The absolute configuration of the secondary hydroxyl group was controlled by a catalytic asymmetric alkylation of an aldehyde using a chiral titanium sulfonamide complex and the cis double bond was installed using a Wittig reaction. This synthetic route was also applied to the preparation of structurally related analogs, which were used in structure-activity relationship studies for antibacterial activity.

Full text of DOI:10.1016/j.bmcl.2010.07.115

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5843–5846
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The synthesis of paleic acid, an antimicrobial agent effective against
Mannheimia and Pasteurella, and its structurally related derivatives
*
Takumi Watanabe , Ikuko Kurata, Chigusa Hayashi, Masayuki Igarashi, Ryuichi Sawa, Yoshikazu Takahashi,
Yuzuru Akamatsu
Institute of Microbial Chemistry, Tokyo, 3-14-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A synthetic route to paleic acid 1, antimicrobial agent effective against Mannheimia haemolytica and
Pasteurella multocida, has been established. The absolute configuration of the secondary hydroxyl group
was controlled by a catalytic asymmetric alkylation of an aldehyde using a chiral titanium sulfonamide
complex and the cis double bond was installed using a Wittig reaction. This synthetic route was also
applied to the preparation of structurally related analogs, which were used in structure–activity relation-
ship studies for antibacterial activity.
Received 20 July 2010
Revised 22 July 2010
Accepted 26 July 2010
Available online 3 August 2010
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Mannheimia haemolytica
Pasteurella multocida
Bovine respiratory disease
Antibacterial
Structure–activity relationship
Asymmetric synthesis
Mannheimia haemolytica1 and Pasteurella multocida2 are patho-
genic and cause a wide range of diseases in food-producing ani-
mals such as cattle, poultry, pigs and rabbits. Antimicrobial
agents are powerful tools for controlling infection. In fact, tilmico-
sin,3,4 a derivative of desmycosin, was found to be effective against
bovine respiratory disease (BRD) and was introduced into the US
market in 1990. Since that time, a variety of antibiotics including
b-lactams, macrolides, tetracyclines and sulfonamides have been
revealed to be effective against the infectious diseases.5,6 One of
the most successful veterinary drugs available is the triamilide
macrolide, tulathromycin7 (the active ingredient in Draxxin Inject-
able Solution launched by Pfizer Animal Health), which has been
approved for use in the treatment and prevention of BRD and the
treatment of swine respiratory disease in the EU and the USA.
The prevalence of antimicrobial resistance among pathogens8
urges us to search for new classes of antibiotics with unique struc-
ture and/or mode of action.
Figure 1. Structure of paleic acid.
action, the compound is expected to be a new lead in veterinary
drugs.
Herein we describe the synthesis of paleic acid 1 in detail. The
synthesis was easily applied to the preparation of a variety of pa-
leic acid analogs with structural modification at C16 and the car-
boxylate moiety. Once the synthesis of paleic acid analogs was
achieved, a study on the structure–activity relationship (SAR)
was conducted.
The structure of paleic acid is characterized as a (1) hydroxyl-
ated, (2) cis-unsaturated C18 fatty acid. We envisioned installing
the Z double bond through a Wittig reaction. An asymmetric alkyl-
ation of an aldehyde using diethylzinc and a titanium sulfonamide
ligand complex reported by Ohno and co-workers10–12 was chosen
to provide the chiral secondary alcohol at C16. The synthetic route
is displayed in Scheme 1.
In the previous research,9 the isolation and structural elucida-
tion of paleic acid 1 (Fig. 1) is described. Paleic acid is the first re-
ported fatty acid derivative to display antibacterial activity against
diverse strains of Mannheimia haemolytica and Pasteurella multo-
cida. Since paleic acid has a structure completely different from
those of other known antibiotics and may have a unique mode of
The 7-hydroxyheptylaldehyde, protected as a tert-butyldiphen-
ylsilyl (TBDPS) ether 2,13 was uneventfully alkylated in an enantio-
selective manner (62% yield, >99% ee; vide infra) according to the
reported procedure employing (1S,2S)-bis(trifluoromethanesulfo-
* Corresponding author. Tel.: +81 3 3441 4173; fax: +81 3 3441 7589.
E-mail address: twatanabe@bikaken.or.jp (T. Watanabe).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.115

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T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5843–5846
Scheme 1. Reagents and conditions: (a) (1S,2S)-1,2-N,N0-bis (trifuluoromethylsulfonylamino)cyclohexane 3, Et2Zn, Ti(Oi-Pr)4, toluene, ꢀ78 °C to ꢀ50 °C, 5 h, 62%, >99% ee; (b)
BzCl, pyridine, DCM, rt, 16 h, 67%, (c) TBAF, THF, rt, 16 h, quant.; (d) TPAP, NMO, MS4A, DCM, rt, 1 h, 53%; (e) BrꢀPh3P+(CH2)8COOH (8), KHMDS, THF, rt, 2 h, 72%; (f) NaOH, aq
MeOH, rt, 3 d, 82%.
nylamino)cyclohexane 3 as a chiral ligand. Although the enantiose-
lectivity of the reaction could not be determined by HPLC at this
stage, the absolute configuration of the newly formed stereocenter
was confirmed to be R by modified Mosher’s method. Based on the
analysis of the NMR spectra, the Mosher esters were not contami-
nated by ester derived from the S-isomer. The resulting alcohol 4
was protected as the benzoate 5 (67%), and the subsequent desily-
lation gave the primary alcohol 6 in quantitative yield, with which
the enantioselectivity of the first reaction was determined to be
>99% ee by chiral HPLC. Oxidation of the alcohol 6 with TPAP affor-
ded the desired aldehyde 7 in 53% yield, which is the substrate for
the subsequent Wittig reaction.
bond was constructed (14: 59%). After hydrolysis of the acetal
(15: 93%), the liberated carbonyl group was reacted with hydroxyl-
amine hydrochloride to give the oxime derivative of paleic acid 16.
The compound was produced as an inseparable equilibrating mix-
ture of E and Z-isomers (in approximately a 1:1 ratio in CDCl3).
To examine the significance of the carboxyl group of paleic acid
on antibacterial activity, a primary amide derivative 18 was pre-
pared (Scheme 4). Benzoyl paleic acid (9) was treated with trim-
ethylsilyldiazomethane to give methyl ester 17 (99%), which was
converted to 18 with methanolic ammonia and a catalytic amount
of sodium cyanide (NaCN)16 in 67% yield.
A preliminary experiment showed that the antibacterial activity
of paleic acid and the related compounds was ruined in the pres-
ence of serum albumin (unpublished results). Since the deleterious
effect may come from the high affinity of acidic carboxylate groups
albumin, paleic acid derivatives in which the carboxyl groups are
replaced with its bioisosters were synthesized,17 namely, hydroxa-
mate 19, sulfonimide 24, and tetrazole 27 (Scheme 5).
The hydroxamate analog 19 was obtained in a single step from
compound 17 (treated with hydroxylamine in methanol) in 34%
yield. Hydrolysis of the product by water gave rise to a reduction
in the yield of the reaction.
The sulfonamide 24 could be synthesized by changing the phos-
phonate 8 to 22 that was prepared from 9-bromononanoic acid 20
in three steps (formation of the sulfonamide 21, 46% over two steps
and substitution by triphenylphosphine, quantitative yield). The
Wittig reaction with phosphonium salt 22 produced the cis-olefin
23 in low yield (23% isolated yield). The poor yield was likely
due to the instability of the phosphonium ylide generated from
22. The final deprotection was performed to afford the sulfonimide
24 in 86% yield.
A phosphonium salt 814 was treated with potassium bis(tri-
methylsilyl)amide (KHMDS) in THF to give the corresponding
ylide, to which aldehyde 7 was added to afford the desired cis-ole-
fin 9 (72% yield, based on the aldehyde). Last, saponification affor-
ded paleic acid 1 in 82% yield. All the physicochemical properties
and antimicrobial activity of the synthetic sample were indistin-
guishable from those of the natural product.
In order to obtain insight into the structural features responsible
for the biological activity of paleic acid, a number of analogs were
synthesized utilizing a synthetic route similar to 1 (Scheme 2).
Epimeric paleic acid 12 was prepared to evaluate the effect of
the stereochemistry at C16 on antibacterial activity. The carboxyl-
ate of paleic acid 1 was esterified with trimethylsilyldiazomethane
to give compound 10 in quantitative yield. The subsequent Mitsun-
obu reaction using benzoic acid as a nucleophile resulted in benzo-
ate 11 (67%), with the stereochemistry at C16 position inverted.
Finally, hydrolysis of the both protecting groups in a basic condi-
tion afforded the requisite epimeric paleic acid 12 in 49% yield.
The oxime-derivative 16 could be obtained synthetically as
shown in Scheme 3. A C9 aldehyde 13,15 with the C16 carbonyl
protected as an acetal, was subjected to a Wittig reaction under
the same reaction conditions to that in the paleic acid synthesis,
where the whole 18-carbon framework containing a cis double
The tetrazole derivative 27 was prepared from benzoyl paleic
acid 9 in three steps according to Duncia’s procedure.18 The com-
pound 9 and 3-aminopropionitrile were condensed by a standard
coupling method employing N-ethyl-N0-(3-dimethylaminopro-
Scheme 2. Reagents and conditions: (a) TMSCHN2, MeOH, 0 °C, 20 min, quant.; (b) PhCOOH, DIAD, PPh3, Et2O, rt, 16 h, 67%; (c) NaOH, aq MeOH, rt, 5 h, 49%.
Scheme 3. Reagents and conditions: (a) BrꢀPh3P+(CH2)8COOH (8), KHMDS, THF, rt, 16 h, 59%; (b) AcOH, aq THF, 50 °C, 5 h, 93%; (c) NH2OHꢁHCl, AcONa, MeOH, rt, 2 h, 96%.

T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5843–5846
5845
has essentially the same antibacterial spectra as the parent com-
pound 1. This result shows that the stereochemistry of the hydro-
xyl group of paleic acid has almost no influence on its biological
activity. The C16 position allows structural modification with lim-
ited diversity. While the oxime 16 and ketone 15 analogs showed
almost the same antibacterial activity than that of paleic acid to-
ward many of the strains of M. haemolytica and P. multocida tested
here, the acetal derivative 14 was far less active. In addition, 1, 15,
and 16 have comparable antibacterial activity to that of the posi-
tive control, tilmicosin,3,4 against several strains examined in this
study.
The carboxyl group of paleic acid was revealed to be indispens-
able to its antibacterial activity, as the amide derivative 18 and the
bioisosteric analogs 19, 24, and 27 showed high MIC values com-
pared to the parent compound 1. Finally, no paleic acid-related
compounds displayed antibacterial activity against Histophilus
somni, another important pathogen that can cause BRD.
This study established a synthetic route toward paleic acid 1, a
novel naturally occurring anti-Mannheimia and anti-Pasteurella
substance. The synthesis could be applied to the preparation of a
variety of analogs, which demonstrated the structural require-
ments to display biological activity. Modifications of the oxygen
functionality at the C16 position were tolerated in limited degree
and the carboxylate group was found to be indispensable. This
study is expected to pave the way for further SAR studies to yield
Scheme 4. Reagents and conditions: (a) TMSCHN2, MeOH, 0 °C, 20 min, 99%; (b)
NH3, NaCN, MeOH, 45 °C, 16 h, 67%.
pyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotria-
zole (HOBt) to afford 25 in 70% yield. The amide was directly con-
verted to a tetrazole ring under Mitsunobu conditions in the
presence of trimethylsilylazide as the azide anion source to provide
26 in 86% yield. The final deprotection step completed the synthe-
sis of the tetrazole analog 27 in 76% yield.
Antibacterial activities of paleic acid and its derivatives synthe-
sized above were examined by subjection to a panel of bacteria
covering various strains of M. haemolytica and P. multocida, and
representative pathogens of human disease for comparison. To
evaluate the significance of oxygen functionality in the biological
activity of paleic acid, two intermediates in the oxime analog syn-
thesis, acetal 14 and ketone 15, were also tested.
Table 1 summarizes the results of the antibacterial assays. The
antibacterial activities of the synthetic and natural sample of paleic
acid 1 are identical. Interestingly, paleic acid epimeric at C16 12
Scheme 5. Reagents and conditions: (a) NH2OH, MeOH, rt, 16 h, 34%; (b) (i) (COCl)2, cat. DMF, 1 h, 70 °C; (ii) MeSO2NH2, pyridine, DCM, rt, 46% for two steps; (c) PPh3, MeCN,
95 °C, 4 d, quant.; (d) 22, KHMDS, THF, rt, 16 h, 23%; (e) NaOH, aq MeOH, rt, 5 h, 86%; (f) H2N(CH2)2CN, EDCI, HOBt, TEA, DCM, rt, 16 h, 70%; (g) TMSN3, DIAD, PPh3, THF, rt,
16 h, 86%; (h) NaOH, aq MeOH, rt, 4 d, 76%.
Table 1
Antibacterial activity of paleic acid and its derivatives
Test Organisms
MIC (lg/mL)
1a
1b
12
14
15
16
18
19
24
27
Timicosin
Mannheimia haemolytica N791
M. haemolytica N811
M. haemolytica S801
Pasteurella multocida No.6
P. multocida Kobe
P. multocida TS-8
P. multocida M-17
Acrinobacillus pleuroneumoniae NB001
Histophilus somni 23N2359
0.78
1.56
1.56
6.25
>50
3.13
>50
6.25
>50
0.78
0.78
0.78
12.5
>50
6.25
>50
6.25
>50
0.78
0.78
0.39
50
>50
6.25
>50
12.5
>50
25
25
25
>50
50
3.13
6.25
6.25
50
>50
50
>50
6.25
>50
1.56
1.56
1.56
3.13
>50
25
>50
6.25
>50
25
12.5
50
>50
>50
50
>50
>50
>50
25
25
50
12.5
>50
50
>50
50
50
50
25
50
>50
25
>50
>50
>50
50
50
50
50
50
50
50
50
50
1.56
0.39
3.13
1.56
1.56
1.56
1.56
12.5
3.13
25
>50
>100
>50
>50
a
Natural.
Synthetic.
b

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T. Watanabe et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5843–5846
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studies along these lines are currently underway.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.115.
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