Stereochemical modification of geminal dialkyl substituents on pantothenamides alters antimicrobial activity

Article abstract of DOI:10.1016/j.bmcl.2014.06.013

Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial activity through interference with coenzyme A (CoA) biosynthesis or downstream CoA-utilizing proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark N- pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent antibacterial pantothenamides reported so far (MIC of 3.2 μM for both sensitive and resistant Staphylococcus aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a key diastereoselective reduction with Baker's yeast, and report on the scope of this reaction for modified systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the R-syn isomer proved to be significantly more potent against the malaria parasite (IC₅₀ of 2.4 ± 0.2 μM). Our research underlines the striking influence that stereochemistry has on the biological activity of pantothenamides, and may find utility in the study of various CoA-utilizing systems.

Full text of DOI:10.1016/j.bmcl.2014.06.013

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3274–3277
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Stereochemical modification of geminal dialkyl substituents
on pantothenamides alters antimicrobial activity
Annabelle Hoegl a, , Hamed Darabi a, , Elisa Tran a, Emelia Awuah a, Eleanor S. C. Kerdo b, Eric Habib a,
Kevin J. Saliba b,c, Karine Auclair a,
⇑
a Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec H3A 0B8, Canada
b Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australian Capital Territory 0200, Australia
c Medical School, College of Medicine, Biology and Environment, The Australian National University, Canberra, Australian Capital Territory 0200, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Pantothenamides are N-substituted pantothenate derivatives which are known to exert antimicrobial
Received 23 April 2014
Revised 2 June 2014
Accepted 5 June 2014
Available online 18 June 2014
activity through interference with coenzyme
proteins. A previous report has shown that replacement of the ProR methyl group of the benchmark
N-pentylpantothenamide with an allyl group (R-anti configuration) yielded one of the most potent anti-
A (CoA) biosynthesis or downstream CoA-utilizing
bacterial pantothenamides reported so far (MIC of 3.2 lM for both sensitive and resistant Staphylococcus
aureus). We describe herein a synthetic route for accessing the corresponding R-syn diastereomer using a
key diastereoselective reduction with Baker’s yeast, and report on the scope of this reaction for modified
systems. Interestingly, whilst the R-anti diastereomer is the only one to show antibacterial activity, the
Keywords:
Pantothenamides
Antibacterial
Antiplasmodial
Baker’s yeast
Coenzyme A
R-syn isomer proved to be significantly more potent against the malaria parasite (IC50 of 2.4 0.2 lM).
Our research underlines the striking influence that stereochemistry has on the biological activity of
pantothenamides, and may find utility in the study of various CoA-utilizing systems.
Ó 2014 Published by Elsevier Ltd.
Infectious diseases remain a major contributing factor to world-
wide mortality. Moreover, the development of antimicrobial resis-
tance is raising significant concerns about the increasingly limited
efficacy of currently available treatments.1 There have been con-
siderable efforts towards discovering and characterizing novel
therapeutic targets for antimicrobial drugs. One such target which
has emerged as a promising point-of-attack is coenzyme A (CoA)
biosynthesis and its associated cellular processes.2 This ubiquitous
cofactor is required for a diverse set of biological functions and is
essential to all organisms. Most rely on the exogenous uptake of
its natural precursor pantothenate (vitamin B5), and extend it into
CoA through a 5-step biotransformation.3,4 The inherent differ-
ences in the CoA biosynthetic machinery between humans and
microbial pathogens suggest that this pathway can be exploited
for therapeutic applications.1 In fact, a variety of pantothenate ana-
logues have been evaluated for antibacterial, antiplasmodial and
antifungal properties.2
have potent activity against Escherichia coli (in low pantothenate
media)5 and Staphylococcus aureus.6 It was later found that pant-
othenamides were, in fact, being extended by the CoA biosynthetic
enzymes into CoA analogues which affected downstream targets
such as the acyl carrier protein necessary for fatty acid synthesis.7,8
It has been suggested that pantothenamides may also act by inhib-
iting the CoA biosynthetic pathway.9,10 While much of the research
has focused on antibacterials, targeting the CoA pathway is also a
promising strategy for antiplasmodials. For example, the provita-
min pantothenol, as well as a range of other pantothenate ana-
logues, have been shown to repress the proliferation of the
malarial parasite Plasmodium falciparum.11–15
A synthetic route for accessing geminal dialkyl-substituted
pantothenamide derivatives was recently reported by some of
us.16 In this study, several compounds were synthesized and eval-
uated for antibacterial properties which revealed that larger sub-
stituents were not well tolerated at the gem-dimethyl position.
This led to the identification of a methyl-allyl derivative (1) with
potent antibacterial activity against both sensitive and resistant
S. aureus.16 It was envisaged that these structure–activity relation-
ships (SARs) could be extended through modification of the stereo-
chemistry of the alkyl-substituted pantoyl fragment. In designing a
target, we opted to focus on the 2-methyl-allyl derivative because
of its clear superiority in antibacterial activity assays,16 and to
An important class of such compounds are the N-substituted
pantothenamides, including the benchmark molecule in the field,
N-pentylpantothenamide (Fig. 1).5 This compound was shown to
⇑
Corresponding author. Tel.: +1 514 398 2822; fax: +1 514 398 3797.
E-mail address: karine.auclair@mcgill.ca (K. Auclair).
These authors contributed equally to the work.
http://dx.doi.org/10.1016/j.bmcl.2014.06.013
0960-894X/Ó 2014 Published by Elsevier Ltd.

A. Hoegl et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3274–3277
3275
O
O
O
O
to afford the prochiral ketone 6a in good yield, which was subse-
quently reduced to the R-alcohol 7a.
-O
N
H
N
H
N
H
OH OH
OH OH
Several commercially available chemical reducing agents were
tested to evaluate their ability to yield the syn alcohol product from
6a. As shown in Table S1, typical agents such as DIBAL-H, NaBH4
and Zn2(BH4)2, as well as chiral reducing agents such as (R)-CBS
and (S)-CBS showed poor diastereoselectivity and yielded predom-
inantly the anti-product. The potential of biocatalysts was thus
explored. To this end, we envisaged utilizing a whole cell mixture
of common Baker’s yeast (Saccharomyces cerevisiae) to reduce 6a to
the syn product 7a diastereoselectively. There is precedence for
Pantothenate
N-pentylpantothenamide
O
O
O
O
3
3
N
H
N
H
2
N
H
N
H
2
OH OH
OH OH
1
2
2R,3R-anti
2S,3R-syn
Figure 1. Structure of pantothenate and N-pentylpantothenamide analogues.
Baker’s yeast to reduce
a- and b-ketoesters to enantiopure alco-
hols.18 Baker’s yeast expresses several reducing enzymes which
can be selectively inhibited or favored by varying the reaction con-
ditions.19 Pre-treatment of the yeast with cross-linking agents such
as methyl-vinyl ketone (MVK), was found to favor syn-selectivity
and prevent over-reduction to the diol.20 Heat-denaturation
(50 °C, 30 min) also achieved the same goal and even worked syn-
ergistically with MVK.21,22
Indeed, reduction of 6a with Baker’s yeast yielded the desired
syn product (dr of >99:1) in 68% yield after purification. This high
selectivity was only possible when the yeast was pre-incubated
with MVK at 50 °C for 30 min before addition of the ketone. The
dr ratio was determined by integrating the characteristic NMR
peaks for the methyl group at the quaternary carbon, specifically
the signal at 1.16 ppm from the anti product,16 and the signal at
1.05 ppm from the syn product. The absolute stereochemistry of
the product was confirmed by derivatization using enantiomeric
auxiliary reagents and subsequent 1H NMR analysis as described
by Seco et al.23 (R)- and (S)-methoxyphenylacetic acid (MPA) were
thus coupled to 7a to generate the diastereoisomeric MPA deriva-
tives for NMR analysis. To evaluate the scope of the Baker’s yeast
maintain the R-configuration at C-3 based on previous studies sug-
gesting that this is the preferred stereochemistry.2 We report here
on a methodology for accessing the 2S,3R-syn allyl-substituted iso-
mer (2), and on the contrasting profiles of diastereomers 1 and 2
with regards to antibacterial and antiplasmodial activities.
Synthetically, the pantothenamide structure has been obtained
through a sequence of amide couplings on a modified pantoyl frag-
ment.16 In the synthesis of geminal dialkyl-substituted pantothena-
mide derivatives, the stereochemistry at the quaternary carbon is
determined by the initial configuration of the alcohol in the starting
material, and can be controlled via two successive alkylations anti
to the alcohol.16 This synthetic methodology, however, only pro-
vides access to 2R,3R-anti analogues diastereoselectively. Reversing
the configuration at the quaternary center by reversing the order of the
two alkylation reactions, for example adding the larger alkyl group
before methylation, proceeds with poor selectivity and produces
inseparable mixtures of 2R,3R-anti and 2R,3S-syn analogues.16,17 The
difficulty in obtaining the syn product by reversing the alkylation
sequence warrants an alternate synthetic route. In order to access
the novel 2S,3R diastereomer, we envisaged inverting the stereo-
chemistry of the fragment through oxidation, followed by diaste-
reoselective reduction. We expected this last step to pose the
greatest challenge, due to the unfavorable energetic barrier
reaction, a series of
a-ketosuccinates (6a–f) was synthesized and
reduced. The dr was measured by NMR on the crude product,
and the yield was calculated after purification (Table 1). Interest-
ingly, all compounds showed excellent diastereoselectivity
(dr P 98:1, with one exception at 80:20), although the yield of
the reactions generally decreased with increasing bulk of the alkyl
substituents, consistent with larger groups not being well tolerated
in the binding pocket of the reductase of interest. The low yields
observed in some of the examples are largely attributed to product
recovery issues resulting from inefficient extraction from the com-
plex matrix. Overall, Baker’s yeast shows excellent stereoselectivi-
ty for the reduction of these systems.
associated with forming the syn product. Thus,
(3S-alcohol) is used here to access the syn isomer (2) through inver-
L
-(À)-malic acid
sion of stereochemistry, while
D
-(À)-malic acid (3R-alcohol) was
previously used directly to synthesize the anti isomer (1).16
In order to generate the alkyl-substituted pantoyl fragment
with the desired stereochemistry,
L-(À)-malic acid was first ester-
ified under mild acidic conditions (Scheme 1). The Frater–Seebach
method of alkylating chiral b-hydroxy esters was used to install the
methyl and various alkyl groups onto 3 with excellent diastereose-
lectivity.17 The addition of two equivalents of strong base gener-
ates a di-anion which forms a six-membered ring chelate.17 The
stereoconfiguration of the secondary alcohol directs the electro-
philic addition from the less hindered face, thereby yielding the
anti product with a consistently high diastereomeric ratio (dr; as
measured by NMR of the crude sample). Swern oxidation was used
With the stereochemistry established, we were able to extend
the intermediates into full pantothenamides as shown in Scheme 2.
As mentioned above, the methyl-allyl derivative 7a was selected
for extension based on the reported antibacterial activity of 1.16
Table 1
Baker’s yeast reduction of di-alkyl substituted
a-ketomalonates
SOCl2,
LDA, CH3I,
THF, -78°C
73%
dr 11:1
Baker's Yeast
MVK,H2O
30°C, 24h
O
O
O
O
R
O
R
EtOH, rt, 6h
OH
OEt
OEt
OEt
OEt
HO
EtO
EtO
EtO
71%
EtO
EtO
OH
O
OH
O
OH O
O
O
OH
O
L-(-)-malic acid
3
4
6a-f
7a-f
LDA, R-X,
THF, -78°C
38-75%
dr 99:1
Compound
R
dra
Yieldb (%)
Baker's Yeast
MVK, H2O
30°C, 24h
21-68%
7a
7b
7c
7d
7e
7f
Allyl
>99:1
80:20
98:1
>99:1
>99:1
98:1
68
65
37
31
21
26
Swern Oxidation
DCM, -78°C
O
R
O
R
O
R
Propargyl
Ethyl
OEt
OEt
OEt
EtO
EtO
74-88%
OH
7a-f
O
O
O
OH
O
Propyl
Hexyl
_
dr >98:1*
6a-f
5a-f
Isobutyl
R= allyl, propargyl, propyl, ethyl, hexyl and isobutyl
*except R= propargyl (dr 80:20)
a
Diastereomeric ratio determined by NMR.
Isolated yield.
b
Scheme 1. Synthesis of compounds 7a–f.

3276
A. Hoegl et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3274–3277
2.4 0.2
(p ꢀ 0.001) when compared to its anti-isomer 1 (31
n = 3). Moreover, increasing the extracellular pantothenate concen-
tration (from 1 to 50 M) resulted in a higher IC50 for 2 (13 M,
n = 3, p ꢀ 0.001), consistent with 2 inhibiting parasite growth by
targeting CoA biosynthesis or utilization (Fig. 2). Compounds 1
and 2 were also tested in human cells and did not show significant
toxicity (see Figure S1).
l
M, n = 6) appeared to be considerably more potent
1. LiAlH4, THF
50°C, 16h
Dess-Martin
DCM, 1h
O
O
4
l
M,
OEt
EtO
HO
R
2. PMPCH(OMe)2
CSA, DCM, 4h
68%
74%
OH
O
O
O
O
O
l
1 l
PMP
PMP
R =H
7a
8
9
NaClO2, NaH2PO4
acetone:DCM 3:1
10 R =OH
13
,
EDC, HOBt,
A successful method was established for the synthesis of 2S,3R-
syn 2-methyl-alkyl pantothenamide derivatives. A key step of this
process involved the use of Baker’s yeast for the stereoselective
DIPEA, THF, 16h
45%
90% AcOH
(aq.) 4h
O
O
O
O
reduction of a-ketosuccinates. Stereochemical modification of the
N
H
N
H
N
H
N
H
57%
pantoyl fragment significantly influenced the biological activity
and antimicrobial selectivity of these compounds. This suggests
that such alterations may be applied to study other CoA-utilizing
systems. For example, N-substituted pantothenamides have found
use as chemical biology tools and as mechanistic probes of CoA-
dependent enzymes. Specific applications include both in vitro
and in-cell labeling of carrier proteins,24–26 as well as prodrug acti-
vation by the CoA biosynthetic pathway.27 Thus, we expect the
methodology developed herein to find utility in various areas.
OH OH
O
O
2
11
PMP
O
O
HO
NHBoc
NH2
N
NHR
EDC, HOBt,
H
DIPEA, THF, 16h
92%
TFA,
DCM 13 R = H
12 R = Boc
Scheme 2. Synthesis of compound 2.16
Acknowledgements
Thus, 7a was reduced to the corresponding triol using LiAlH4 and
selectively protected as the six-membered ring anisaldehyde acetal
8. Dess–Martin oxidation afforded the aldehyde 9, which was
further oxidized to the acid 10 via Pinnick oxidation. Due to its
instability, the crude acid (10) was used directly in the amide cou-
pling to deprotected amine 13. Finally, the acetal protecting group
of 11 was cleaved using 90% aqueous acetic acid to produce the
desired pantothenamide 2.
This work was supported by the Center in Green Chemistry and
Catalysis, as well as by research grants from the Canadian Institute
of Health Research (CIHR) and the Natural Sciences and Engineer-
ing Research Council of Canada (NSERC) to K.A. A.H. thanks CIHR
and the CIHR-McGill Drug Development Training Program (DDTP)
for financial support. We are grateful to the Canberra branch of the
Australian Red Cross Blood Service for the provision of erythrocytes
and to Dr. Giel van Dooren (ANU) for assistance with the HFF
assays.
The 2S,3R-syn pantothenamide 2 was tested for antibacterial
activity against S. aureus (sensitive and MRSA) but no appreciable
effect could be detected at the highest concentration tested
Supplementary data
(500
anti pantothenamide 1, which previously showed potent antibac-
terial activity (MIC of 3.2
M for both strains).9 Interestingly, this
lM; data not shown). This is in sharp contrast to the 2R,3R-
Supplementary data (experimental procedures, characteriza-
tion of all compounds, as well as Table S1, Figure S1, NMR spectra
and selected HPLC traces) associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.
013.
l
trend was reversed when 1 and 2 were evaluated for their antiplas-
modial activity. As shown in Figure 2, although both diastereomers
inhibited in vitro parasite growth, the syn derivative 2 (IC50 of
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