Geminal dialkyl derivatives of N-substituted pantothenamides: Synthesis and antibacterial activity

Article abstract of DOI:10.1016/j.bmc.2011.02.053

As a key precursor of coenzyme A (CoA) biosynthesis, pantothenic acid has proven to be a useful backbone to elaborate probes of this biosynthetic pathway, study CoA-utilizing systems, and design molecules with antimicrobial activity. The increasing prevalence of bacterial strains resistant to one or more antibiotics has prompted a renewed interest for molecules with a novel mode of antibacterial action such as N-substituted pantothenamides. Although numerous derivatives have been reported, most are varied at the terminal N-substituent, and fewer at the β-alanine moiety. Modifications at the pantoyl portion are limited to the addition of an ω-methyl group. We report a synthetic route to N-substituted pantothenamides with various alkyl substituents replacing the geminal dimethyl groups. Our methodology is also applicable to the synthesis of pantothenic acid, pantetheine and CoA derivatives. Here a small library of new N-substituted pantothenamides was synthesized. Most of these compounds display antibacterial activity against sensitive and resistant Staphylococcus aureus. Interestingly, replacement of the ProR methyl with an allyl group yielded a new N-substituted pantothenamide which is amongst the most potent reported so far.

Full text of DOI:10.1016/j.bmc.2011.02.053

Bioorganic & Medicinal Chemistry 19 (2011) 2696–2706
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Geminal dialkyl derivatives of N-substituted pantothenamides: Synthesis
and antibacterial activity
⇑
T. Olukayode Akinnusi, Kenward Vong, Karine Auclair
Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montréal, Québec, Canada, H3A 2K6
a r t i c l e i n f o
a b s t r a c t
Article history:
As a key precursor of coenzyme A (CoA) biosynthesis, pantothenic acid has proven to be a useful back-
bone to elaborate probes of this biosynthetic pathway, study CoA-utilizing systems, and design molecules
with antimicrobial activity. The increasing prevalence of bacterial strains resistant to one or more anti-
biotics has prompted a renewed interest for molecules with a novel mode of antibacterial action such
as N-substituted pantothenamides. Although numerous derivatives have been reported, most are varied
at the terminal N-substituent, and fewer at the b-alanine moiety. Modifications at the pantoyl portion are
Received 5 January 2011
Revised 23 February 2011
Accepted 28 February 2011
Available online 4 March 2011
Keywords:
Antibiotic
limited to the addition of an
x-methyl group. We report a synthetic route to N-substituted pantothena-
mides with various alkyl substituents replacing the geminal dimethyl groups. Our methodology is also
applicable to the synthesis of pantothenic acid, pantetheine and CoA derivatives. Here a small library
of new N-substituted pantothenamides was synthesized. Most of these compounds display antibacterial
activity against sensitive and resistant Staphylococcus aureus. Interestingly, replacement of the ProR
methyl with an allyl group yielded a new N-substituted pantothenamide which is amongst the most
potent reported so far.
Pantothenamide
Diastereoselective synthesis
Resistance
Ó 2011 Elsevier Ltd. All rights reserved.
O
O
1. Introduction
pantoyl fragment
HS
OH
N
H
N
H
O
O
N
The discovery and clinical use of antibacterial agents in the
early 1900’s has dramatically improved our quality of life and lon-
gevity, yet infectious diseases remain a leading cause of morbidity
and mortality worldwide. The problem is exacerbated by the rapid
spread of bacterial resistance to clinically used antibacterial
agents.1–3 Since antibiotics have a finite lifetime before resistance
occurs, maintaining a regular pipeline of novel antibiotics is
increasingly recognized as highly important. Molecules with a no-
vel mode of action are of special interest due to the limited possi-
bility of cross-resistance. Although some steps in coenzyme A
(CoA) biosynthesis have been validated as targets for the develop-
ment of antibacterial agents, none of the currently approved anti-
bacterials affect this pathway. The discovery of pantothenic acid
(Fig. 1) as a key precursor in the biosynthesis of CoA at the turn
of the century4 led to significant efforts towards generating ana-
logues with antibacterial, antimalarial and antifungal activity.5
Modifications reported at the b-alanine fragment of pantothenic
acid mainly involved replacement of the carboxylic acid end with
other functional groups.6–12 Early on, limited variations in the pan-
toyl fragment of pantothenic acid were also exploited.7,13–16
N-Substituted pantothenamides are an important family of pan-
tothenic acid derivatives (Fig. 1). Their antibacterial activity was
HO
O
P
O
P
O
H
OH OH
CoA
O
O
OH
NH2
N
biosynthetic
enzymes
beta-alanine
fragment
N
N
OH
N
O
CoA
Pantothenic acid
O
P
OH
O
OH
OH
O
O
R
OH
N
H
N
H
O
O
R
N
H
N
H
O
P
O
P
O
CoA
OH OH
O
O
OH
OH
NH2
N
biosynthetic
enzymes
R = n-pentyl,
cyclopentyl,
benzylethyl, 2-propynyl,
cyclopropylmethyl, allyl,
or others
N
N
N
O
O
P
OH
OH
CoA
Analogues
O
Pantothenamides
OH
Figure 1. Structure of pantothenic acid and N-substituted pantothenamides.
first demonstrated by Skinner and coworkers17 against Escherichia
coli and subsequently against Staphylococcus aureus.18 This class of
⇑
Corresponding author. Tel.: +1 514 398 2822; fax: +1 514 398 3797.
E-mail address: karine.auclair@mcgill.ca (K. Auclair).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.02.053

T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
2697
compounds among which N-pentylpantothenamide (N5-pan, 9k)
is regarded as the benchmark, was originally assumed to exert its
activity by inhibition of CoA biosynthesis. A study performed by
Strauss and Begley however revealed that N5-pan (9k) is converted
in vitro to ethyldethia-CoA by the E. coli CoA biosynthetic en-
zymes.19 The authors postulated that the antibacterial activity of
N5-pan (9k) may result from the inhibition of CoA-utilizing en-
zymes. This hypothesis was later corroborated by Jackowski and
coworkers who demonstrated that pantothenamides were con-
verted in vivo to CoA derivatives which irreversibly inhibited the
acyl carrier protein (ACP) of the fatty acid biosynthetic machin-
ery.20 Studies with S. aureus indicate a similar mode of action.21
These studies and a recent one22 also suggest that the mechanism
of action of pantothenamides may be more complex than de-
scribed above.
been synthesized as diastereomeric mixtures, and a didemethyl
CoA analogue has also been reported,27 but variations at this posi-
tion have otherwise not been explored. We believe that this posi-
tion may provide an interesting diversification point, especially if
the configuration at the quaternary carbon can be controlled. We
report here a stereoselective synthetic route to N-substituted pant-
othenamide analogues with various geminal dialkyl substituents
replacing the geminal dimethyl groups. This route is easily adapted
to the synthesis of pantothenic acid, pantetheine, or CoA deriva-
tives. We also report on the biological activity of the newly gener-
ated N-substituted pantothenamides.
2. Results and discussion
2.1. Chemistry
With the rapid spread of drug-resistant pathogens, there has
been a renewed interest for N-substituted pantothenamides.5,23–
Synthesis of the target compounds was achieved via a sequen-
tial double alkylation of (R)-diethyl malate (2). Compound 2 was
generated from commercial (R)-malic acid (1) in 95% yield via thio-
nyl chloride mediated esterification in refluxing absolute ethanol.
Using a method reported by Seebach et al.28 the lithium dianion
generated by the addition of 2 equivalents of LDA to 2 was alkyl-
ated with methyl iodide, propyl iodide or allyl bromide, affording
6:1, 9:1 and 11:1 diastereomeric mixtures, respectively (3,
Scheme 1). Initial attempts to carry out the second alkylation on
26
Although numerous pantothenamides have been reported, the
pantoyl fragment is typically conserved amongst them all, except
for a few variants with an added
x
-methyl group.13 To the best
of our knowledge, there is no stereoselective methodology re-
ported to replace the geminal dimethyl groups of pantothenic acid,
pantothenamide, or pantetheine with other substituents. Panto-
thenic acid derivatives with one of the geminal dimethyl groups
lacking or replaced with ethyl,15 phenyl7 or cyclopentyl16 have
O
LDA, R1-I, THF
SOCl2, EtOH
LDA, R2-I, THF
-78oC - rt.
O
R1
-78oC - rt
OEt
reflux, 7h
OEt
EtO
(R)-Malic acid
EtO
95%
67-75%
dr 6:1 to 11:1
OH
O
32-70%
dr >99:1
OH
3
O
1
2
O
R1
O
R2
1) LiAlH4, THF
50oC, 16h
NaClO2, NaH2PO4
acetone:DCM 3:1
R1
O
R2
Dess-Martin
CH2Cl2, 1h
O
R1
OH
R2
H
HO
OEt
EtO
O
O
H2O, 15 min
2) PMPCH(OMe)2
CSA, DCE, 4h
35-72%
O
PMP
PMP
4
5
6
O
O
O
O
O
R1
R2
R1
O
R2
O
R1
O
R2
90%
EDC, HOBt, 12
R3
R3
N
H
N
H
HO
AcOH aq.,16h
DIPEA, THF, 16h
N
H
N
H
OH OH
O
45-96 %
41-79% from 5
PMP
PMP
7
9
8
R1
R2
R3
a: Me
b: Me
c: Me
d: Me
e: Me
f: Me
g: Me
h: Propyl
i: Allyl
j: Me
k: Me
l: Me
Ethyl
Propyl
iButyl
Pentyl
Pentyl
Pentyl
CH2cyButyl Pentyl
Hexyl
Allyl
CH2Ph
Propyl
Allyl
Allyl
Me
Me
Pentyl
Pentyl
Pentyl
Pentyl
Pentyl
Nonyl
Pentyl
Nonyl
R3NH2, EDC, HOBt
DIPEA, THF, 16h
O
O
R3
NHFmoc
HO
N
H
NHR4
85-93%
10
11
12
R
R
4 = Fmoc
4 = H
Piperidine
DMF
Scheme 1. Synthetic route to new N-substituted pantothenamides.

2698
T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
the lithium dianion of 3 (R1 = Me) with alkyl bromides (instead of
iodides) was only successful with benzyl and allyl bromides. The
reaction with other bromides (propyl-, cyclobutylmethyl-, and 2-
methylpropyl-) proceeded to <5% with 90% recovered starting
material. Even higher concentrations and longer reaction times
did not help improve the yields. Alkylation of 3 (R1 = Me) with allyl
bromide produced a single adduct 4f, whereas reaction with benzyl
bromide resulted in a 16:1 diastereomeric ratio of 4g favoring the S
configuration at the quaternary center. The S configuration (based
on diethyl malate) is favored at the quaternary center due to the
expected chelation of the lithium alkoxide generated in the reac-
tion to the carbonyl group, thereby promoting addition from the
opposite face relative to the alkoxide.29,30 A switch to the more
reactive alkyl iodides yielded exclusively single adducts of 4a–4e
(>99% diastereoselectivity based on 1H NMR of the crude reaction
mixture). Similarly, a second alkylation of the lithium dianion of
3 (R1 = propyl or allyl) with propyl iodide and allyl iodide respec-
tively afforded the corresponding 4h and 4i with both geminal di-
methyl groups replaced.
In an attempt to reverse the configuration at the quaternary
center of compounds 4a–4g, the order of alkylation was reversed,
with the larger electrophile added first, followed by the smaller
one. Unfortunately, this methodology did not proceed with good
diastereoselectivity and produced inseparable mixtures of
compounds.
Diesters 4a–4l were next extended to the target N-substituted
pantothenamides 9a–9l as follows. Global reduction to the corre-
sponding triols using LiAlH4, followed by selective protection of
the 1,3-diol using a method reported by Shiina and coworkers,30
afford the p-anisaldehyde acetals 5a–5l. Dess–Martin oxidation of
5a–5l31 followed by Pannick oxidation as reported by Burkart32
furnished acids 7a–7l which were used without further
purification.
were lower than for the other derivatives (<50%). This is presum-
ably the result of side reactions during the Pannick oxidation. Ace-
tic acid-mediated deprotection of the p-anisaldehyde acetals 8a–8l
in the presence of water furnished the desired N-substituted pant-
othenamides 9a–9l.
Compound 9m was prepared in 33% yield over three steps from
9f as described in Scheme 2. After TBS protection of 9f to 13, the
product was subjected to cyclopropanation according to a protocol
reported by Takemoto and co-workers.34 TBAF mediated deprotec-
tion afforded the desired compound 9m.
2.2. Biology
Of the antibiotic resistant microbes, S. aureus is one of the most
notorious. In recent years this multi-drug resistant pathogen (often
termed MRSA) has emerged as the major cause of nosocomial
infection.35 The antibacterial activity of target compounds 9a–9m
was evaluated against the bacterial S. aureus strains ATCC 29213
(oxacillin susceptible) and ATCC 43300 (oxacillin resistant, abbre-
viated MRSA), according to a standard procedure.36 The MIC values
obtained are listed in Table 1. A preliminary screen of these pant-
othenamides against other strains (Acinetobacter baumannii ATCC
19606, Klebsiella penumoniae ATCC 13883, E. coli ATCC 25922 and
11775, and Bacillus subtilis ATCC 6051 and 6633) using the agar dif-
fusion method suggests no antimicrobial activity (inhibition zone
<1.1 cm for a 1 cm disc impregnated with the desired compound).
Varying the chain length of R2 provides important structure–
activity relationships (SARs) about the antibacterial activity of N-
substituted pantothenamides. In our hands, the well characterized
N5-pan (9k) shows a MIC activity of 7 lM, in agreement with pre-
vious reports.18 Replacement of the ProR methyl group (R2) with
small alkyl chains, such as ethyl (9a), appears to have little effect
on the MIC. As the alkyl group gets longer however, the antibacte-
rial activity decreases sharply, with MIC values ranging from
In parallel, Fmoc protected b-alanine (10) prepared according to
a literature protocol33 was coupled to the appropriate alkyl amines
in the presence of EDC to furnish 11 (R3 = pentyl or nonyl) in excel-
lent yields. Following deprotection with piperidine in DMF, the
resulting compounds 12 (R3 = pentyl or nonyl) were coupled to
acids 7a–7l using EDC or HBTU. Products 8a–8l were obtained in
41–79% yields over the four step sequence from 5a–5l. Not surpris-
ingly, the yields for the formation of allyl derivatives 8f, 8i and 8j
13 l
M for R2 = nPr (9b), to below detection for R2 = hexyl (9e). R2
substituents with more steric bulk are also detrimental. This trend
is observed with the methylcyclopropyl (9m), isobutyl (9c), meth-
ylcyclobutyl (9d), and benzyl (9g) derivatives, which show activi-
ties of 24, 101, 374 and >703 lM, respectively (MICs for 9c and
9d are within error). Interestingly the allyl substituted N-pentyl-
pantothenamide 9f makes exception to these rules. Compound 9f
is the most potent N5-pan derivative reported here (MIC of
3.2
l
M), and twice as potent as the parent compound N5-pan (9k).
Compounds 9h and 9i were designed to investigate the effect of
different substituents at R1. The diallyl substituted derivative (9i)
had an MIC of 376 M, which is much weaker than that of the sin-
gly substituted compound (9f, MIC of 3.2 M). Confirmation of this
trend was obtained with the singly (9b) and doubly (9h) substi-
tuted propyl derivatives, with respective MICs of 13 M and
>744 M. These results are consistent with a poor tolerance of
O
O
l
N
H
N
H
l
OR OR
TBSCl
9f
, R = H
l
imidazole,
DMF, 16h
65%
l
13
, R = TBS
the system for variations at R1, and hence no further attempts were
made to prepare other derivatives at R1.
A previous report18 has shown that when the R3 pentyl group is
replaced with either a heptyl or a nonyl group, the antibacterial
activity is increased. Our data for the known N9-pan (9l) corrobo-
rate the previous report by others.18 With this trend in mind, an R2-
allyl substituted pantothenamide was synthesized with a nonyl
Et2Zn
CH2l2
CH2Cl2
(9j) group at R3. The MIC of compound 9j (1
l
M), was significantly
M), consistent
O
O
improved over that of the parent compound 9f (3.2
l
OR
N
N
with the trend reported.18 In this case however, the methyl to allyl
modification (9l vs 9j) did not lead to an improved activity (MICs
are within error).
H
H
OR
14
, R =TBS
As mentioned above, the generally accepted mechanism of ac-
tion for N-substituted pantothenamides involves conversion to
the corresponding CoA analog by the bacterial CoA biosynthetic
enzymes pantothenate kinase (PanK or coaA), phosphopantetheine
TBAF, THF, 16h
51% from 13
9m
, R = H
Scheme 2. Synthesis of the methyl cyclopropyl pantothenamide analogue 9m.

T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
2699
O
O
O
O
Table 1
PanK
Antibacterial activity of N-substituted pantothenamides 9a–9m
HN
N
H
HN
N
H
ATP ADP
OH OH
OH
O
MIC (
S. aureusb
l
M)a
O
P
OH
OH
9f
MRSAc
16f
ATP
PPi
O
O
O
O
O
O
O
O
O
O
PPAT
O
N
H
N
H
7
6
7
3
OH OH
OH OH
OH OH
OH OH
OH OH
O
O
O
9a
HN
N
H
HN
N
H
DPCK
OH
O
O
P
O
P
O
OH
O
O
P
O
P
O
OH
OH
OH
ADP ATP
N
H
N
H
NH2
NH2
N
13
7
13 2
N
N
N
N
N
O
OH
O
9b
N
N
O
O
18f
17f
O
P
OH
OH
OH OH
N
H
N
H
101 45
374 187
>715
51 15
374 153
>715
O
OH
9c
Figure 2. Proposed cellular transformation of 9f into 16f, 17f, and 18f by the CoA
biosynthetic enzymes PanK, PPAT, and DPCK.
N
H
N
H
9d
fatty acid biosynthesis.19,20 To probe whether the N-substituted
pantothenamides reported here proceed via a similar mechanism,
compound 9f was treated with PanK, the rate-limiting enzyme of
the CoA biosynthetic pathway. The Km and kcat for this transforma-
N
H
N
H
9e
l
M and 0.87 0.04 sÀ1, respectively. Although
O
O
O
O
tion were 534 159
the Km is many folds higher than that of the natural substrate pan-
tothenic acid (17
M), the kcat is unaffected (1.07 0.04 sÀ1).19
N
H
N
H
3.2 0.8
>703
3.2 0.9
>703
OH OH
2
l
9f
Overall, the conversion of compound 9f by the CoA biosynthetic
enzymes PanK, PPAT, and DPCK is expected to yield compound
18f (Fig. 2). An in vitro assay was performed with compounds 9f
and all three enzymes. LC–MS analysis of the reaction mixture
(Fig. 3) confirms the capacity of this enzyme system to produce
18f.
Ph
N
H
N
H
OH OH
OH OH
OH OH
9g
O
O
N
H
N
H
>744
>744
3. Conclusions
9h
In summary, we have developed a stereoselective synthetic
approach to geminal dialkyl analogues of N-substituted pantoth-
enamides. This route should easily adapt to the preparation of
various derivatives of the biologically important pantothenic
acid, pantetheine, and CoA. Thus our synthetic methodology pro-
vides access to new probes for mechanistic and structural stud-
ies of the numerous pathways generating or utilizing these
O
O
O
N
H
N
H
376 217
376 109
9i
O
N
H
N
H
1
7
0.9
2
1
7
0.7
2
OH OH
9j
O
O
N
H
N
H
OH OH
9k (N5-pan)
O
O
N
N
H
0.4 0.2
24 14
0.4 0.2
24 14
H
OH OH
9l (N9-pan)
O
O
N
H
N
H
OH OH
9m
a
Minimum inhibitory concentrations (MICs) were determined using the fol-
lowing concentrations: 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 g/mL. The lowest
concentration where complete cell death was observed was determined to be the
MIC and then converted to M using the molecular weight of each compound.
l
l
b
Staphylococcus aureus strains ATCC 29213 (oxacillin susceptible).
ATCC 43300 (oxacillin resistant or MRSA).
c
Figure 3. LC–MS analysis of the transformation of compound 9f by the CoA
biosynthetic enzymes PanK, PPAT and DPCK. The enzymatic reaction mixtures were
allowed to react either with water as a control (A) or 9f (B). The control with natural
substrate is not shown. The mass observed for 17f and 18f were 723.98 and 803.86,
respectively.
adenylyltransferase (PPAT or coaD), and dephosphocoenzyme A ki-
nase (DPCK or coaE). The resulting CoA antimetabolites are be-
lieved to inhibit acyl carrier protein function, and thereby disrupt

2700
T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
cofactors. The antibacterial studies reported here for a small li-
brary of new pantothenamides reveal some interesting trends.
Because of the numerous enzymes and pathways involved in
the mode of action of pantothenamides,18–22 it is not possible
at this time to explain these SARs in terms of the targets struc-
tures. Although replacement of the ProS methyl group (R1 substi-
tuent) with larger alkyl groups was detrimental, substitution of
the ProR methyl group (R2 substituent) was successful at
improving the antibacterial activity of N5-pan (9k) against S.
aureus and MRSA. Interestingly, the size of the substituent nega-
tively correlated with activity, except for the allyl group which
was favored over methyl. Thus besides providing new SARs for
the design of pantothenamides with improved antibacterial
activity, this study has generated three compounds that are
equivalent (9a) or superior (9f and 9j) to N5-pan (9k), the model
pantothenamide. Investigations into the mode of action for com-
pound 9f strongly suggest that, as with other N-substituted
pantothenamides,19,20 9f is first converted to the corresponding
CoA analogue before exerting its antibacterial effect. This is sup-
ported by kinetic studies with purified PanK, and by evidence of
full conversion to the corresponding CoA derivative in the pres-
ence of PanK, PPAT, and DPCK. Prior to this study, very few mod-
ifications at the pantoyl moiety of pantothenamides had been
reported. We hope that this study will encourage a more sys-
tematic exploration of this part of the molecule.
4.3. PanK enzyme activity
E. coli pantothenate kinase (PanK or coaA enzyme) was ex-
pressed and purified as previously described.38 Enzyme activity
was measured as described in the literature.19 This assay couples
the production of ADP to the consumption of NADH through the
activity of pyruvate kinase and lactic dehydrogenase. NADH con-
sumption was monitored at 340 nm. Reactions were performed
at 25 °C in an Agilent 8453 UV–vis spectrophotometer coupled to
an Agilent 89090A Peltier temperature controller. Kinetic parame-
ters were determined by fitting the rate data into the Michaelis–
Menten equation using Prism 4.0 (GraphPad). Each reaction
mixture (500 lL) contained ATP (1.5 mM), NADH (0.3 mM), phos-
phor(enol)pyruvate (0.5 mM), MgCl2 (10 mM), KCl (20 mM),
pyruvate kinase (5 units), lactic dehydrogenase (5 units), and PanK
(5
was initiated with the addition of the desired substrate (10–
160 M).
lg, 278 nM) in Tris-HCl buffer (50 mM, pH 7.6). The reaction
l
4.4. Transformation of N-substituted pantothenamides by a
mixture of PanK, PPAT and DPCK
The E. coli enzymes pantothenate kinase (PanK or coaA en-
zyme), phosphopantetheine adenylyltransferase (PPAT or coaD en-
zyme), and dephosphocoenzyme A kinase (DPCK or coaE enzyme)
were expressed and purified as previously described.38 Each reac-
tion mixture (500
lL) contained ATP (5.0 mM), MgCl2 (10 mM),
4. Experimental section
PanK (5 g, 278 nM), PPAT (5
l
l
g, 500 nM), DPCK (5 g, 454 nM)
l
in Tris-Cl buffer (50 mM, pH 7.6). The reaction was initiated with
the addition of pantetheine (3.0 mM) for comparison, water as a
negative control, or compound 9f (3.0 mM). Reactions were
incubated for 5 hours at room temperature and then stopped by
heating the mixture to 95 °C for 5 min. The precipitated protein
was removed by centrifugation (13,000 rpm for 5 min) and the
supernatant was analyzed by LC/MS. Reversed-phase analytical
HPLC was performed with an analytical 4.60 Â 250 mm, SYNERGI
4.1. Antibacterial activity (broth dilution method)
Antimicrobial activity of compounds 9a–9m were tested
against S. aureus ATCC 29213 (oxacillin susceptible) and 43300
(oxacillin resistant), which were grown on Trypticase soy media
(BD, Mississauga, ON). Antibacterial activity was determined using
the microdilution broth method in 96-well microtiter plates
according to the National Committee for Clinical Laboratory Stan-
dards (NCCLS).36 For solubility purposes, compounds 9a–9m were
dissolved in 10% (v/v) acetone in water and then serially diluted to
4l Hydro-RP 80A (Phenomenex, Torrance, CA) column coupled to
an Agilent 6120 Quadrupole LC/MS system for ESI-MS analysis.
The HPLC conditions had the sample elute at a flow rate of
0.5 mL minÀ1 using a combination of mobile phase A (H2O) and
mobile phase B (acetonitrile). Elution conditions are as follows:
isocratic 1% phase B from 0 to 3 min; followed by linear gradients
of phase B: 1–10% from 3 to 5 min; 10–15% from 5 to 10 min; 15–
30% from 10 to 13 min; and finally isocratic phase B at 30% from 13
to 26 min. The detector was set to 260 nm.
concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 lg/mL. A
bacterial suspension of 106 cfu/mL was then used to inoculate each
well, which was incubated for 18 h at 37 °C. The MIC was taken as
the lowest concentration demonstrating no visible growth in the
broth. All data are the average of the results from at least four sep-
arate experiments.
4.2. Antibacterial activity (agar diffusion method)
4.5. Synthesis of compounds
4.5.1. (Iodomethyl) cyclobutane37
The antimicrobial activity of compounds 9a–9m was measured
against S. aureus ATCC 29213, A. baumannii ATCC 19606, K. pneumo-
niae ATCC 13883, E. coli ATCC 25922 and 11775, and B. subtilis ATCC
6051 and 6633. Antibacterial activity was determined using the agar
diffusion method according to the National Committee for Clinical
Laboratory Standards (NCCLS).36 Müeller-Hinton agar plates (Fluka)
were inoculated with bacteria by streaking with a cotton swab and
allowing to dry for 5 min before applying drug impregnated discs.
To a flask containing sodium iodide (5.33 g, 35.56 mmol) was
added (bromomethyl)cyclobutane (2.65 g, 17.78 mmol) and ace-
tone (10 mL). The mixture was refluxed at 60 °C for 18 h. The solid
was filtered off and the filtrate was concentrated. Diethyl ether
was added to the concentrate to trigger the precipitation of sodium
iodide and sodium bromide which were filtered off. This was re-
peated three times until no solid was seen in the oil. The filtrate
was then concentrated to afford a light yellowish oil that was
stored over a copper wire (2.14 g, 65% yield). 1H NMR (CDCl3,
300 MHz) d 3.23 (d, J = 7.8, 2H), 2.72–2.56 (m, 1H), 2.12–2.02 (m,
1H), 1.89–156 (m, 4H). 13C NMR (CDCl3, 75 MHz) d 38.4, 29.1,
15.8, 14.5.
Solutions of compounds 9a–9m (150
lg/mL) were prepared and
then aliquoted (40 L) onto sterile discs (1 cm diameter, Fluka).
l
Upon placement of drug impregnated discs, plates were incubated
at 37 °C (S. aureus, A. baumannii, K. pneumoniae, and E. coli) or 30 °C
(B. subtilis) for 24 h. Antibacterial activity is expressed as the diam-
eter of the inhibition zone (mm) produced by the compounds. Ampi-
cillin (Sigma) was used as a positive control (2.0 cm for E. coli 11775;
1.6 cm for E. coli 25922; 3.3 cm for B. subtilis 6051; 3.2 cm for B. sub-
tilis 6633; 1.6 cm for S. aureus 29213; 1.1 cm for K. pneumoniae
13883; and 1.1 cm for A. baumannii 19606).
4.5.2. General procedure for alkylations28
To freshly prepared LDA at À78 °C [2.2 equiv, prepared by add-
ing nBuLi (2.2 equiv) to diisopropylamine (2.4 equiv) in THF (0.05–
0.1 M total reaction concentration) at 0 °C and stirred for 15 min]

T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
2701
was added dropwise (R)-diethyl malate (1 equiv) dissolved in THF.
The reaction was allowed to warm up to À40 °C over 1 h, and re-
cooled to À78 °C before addition of the alkyl halide (2.7 equiv).
The mixture was then stirred for 16 h while warming up to room
temperature. The reaction was cooled down to À78 °C and
quenched with saturated ammonium chloride. The organic layer
was separated and the aqueous layer was extracted with ethyl ace-
tate (3 Â 30 mL). The combined organic layers were washed with
brine, dried over sodium sulphate and evaporated to give an oil
that was purified by flash chromatography on silica gel (1:1.5
diethyl ether/hexanes, v/v).
4.5.8. (2S,3R)-Diethyl-2-allyl-3-hydroxy-2-methylsuccinate (4f)
Prepared in 70% yield from 3a according to the general alkyl-
ation procedure. Rf 0.33 (Et2O/hexanes, 1:1, v/v). 1H NMR (CDCl3,
500 MHz) d 5.80–5.71 (m, 1H), 5.13–5.08 (m, 2H), 4.28–4.22 (m,
3H), 4.18 (q, J = 7.2, 2H), 3.36 (d, J = 6.9, 1H), 2.54 (dd, J = 7.0,
13.7, 1H), 2.34 (dd, J = 7.8, 13.7, 1H), 1.31 (t, J = 7.2, 3H), 1.28 (t,
J = 7.2, 3H), 1.18 (s, 3H). 13C NMR (CDCl3, 125 MHz) d 174.3,
172.8, 133.0, 118.9, 75.3, 61.8, 61.0, 49.9, 39.5, 17.9, 14.1, 14.1.
HRMS for C12H20O5 [M+Na]+ calcd 267.1203, found 267.1205.
4.5.9. (2S,3R)-Diethyl-2-benzyl-3-hydroxy-2-methylsuccinate
(4g)1,2
4.5.3. (2S,3R)-Diethyl-2-ethyl-3-hydroxy-2-methylsuccinate
(4a)28
Prepared from 3a according to the general alkylation procedure
in 60% yield with a 16:1 ratio of diastereoisomers. Rf 0.33 (Et2O/
hexanes, 1:1, v/v). 1H NMR (CDCl3, 500 MHz) major isomer d
7.29–7.19 (m, 5H), 4.28–4.21 (m, 2H), 4.17–4.31 (m, 3H), 3.70 (d,
J = 8.3, 1H), 3.22 (d, J = 13.3, 1H), 3.00 (d, J = 13.3, 1H), 1.32 (t,
J = 7.1, 3H), 1.23 (t, J = 7.1, 3H), 1.12 (s, 3H). 13C NMR (CDCl3,
125 MHz) d 174.6, 172.8, 136.3, 130.5, 130.5, 128.1, 128.1, 126.8,
75.3, 61.7, 61.1, 50.7, 41.2, 17.7, 14.2, 14.0. HRMS for C16H22O5
[M+Na]+ calcd 317.1359, found 317.1364.
Prepared in 40% yield from 3a according to the general proce-
dure. Rf 0.40 (Et2O/hexanes, 1:1). 1H NMR (CDCl3, 500 MHz) d
4.28–4.18 (m, 5H), 3.36 (d, J = 7.9, 1H), 1.88–1.81 (m, 1H), 1.63–
1.56 (m, 1H), 1.30 (t, J = 7.2, 3H), 1.28 (t, J = 7.1, 3H), 1.15 (s, 3H),
0.88 (t, J = 7.5, 3H). 13C NMR (CDCl3, 125 MHz) d 174.8, 172.9,
75.6, 61.7, 60.9, 50.4, 28.2, 16.8, 14.2, 14.2, 8.7. HRMS for
C
11H20O5 [M+Na]+ calcd 255.1203, found 255.1205.
4.5.4. (2S,3R)-Diethyl-3-hydroxy-2-methyl-2-propylsuccinate
(4b)1,2
4.5.10. (R)-Diethyl 3-hydroxy-2,2-dipropylsuccinate (4h) and
(R)-diethyl 2,2-diallyl-3-hydroxysuccinate (4i)
Prepared in 48% from 3a according to the general alkylation
procedure. Rf 0.39 (Et2O/hexanes, 1:1, v/v). 1H NMR (CDCl3,
500 MHz) d 4.81 (q, J = 7.1, 2H), 4.29–4.21 (m, 3H), 3.37 (d,
J = 5.9, 1H), 1.75 (ddd, J = 4.4, 12.5, 13.4, 1H), 1.52 (ddd, J = 4.6,
12.5, 13.4, 1H), 1.40–1.32 (m, 1H), 1.31 (t, J = 7.2, 3H), 1.28 (t,
J = 7.1, 3H), 1.24–1.17 (m, 1H), 1.15 (s, 3H), 0.91 (t, J = 7.3, 3H).
13C NMR (CDCl3, 125 MHz) d 174.9, 172.9, 75.8, 61.7, 60.9, 50.1,
37.7, 17.6, 17.4, 14.6, 14.2, 14.1. HRMS for C12H22O5 [M+Na]+ calcd
269.1359, found 269.1361.
They were difficult to purify from the reaction mixture because
their Rf was very close to that of the starting materials (3h and 3i,
respectively). A mixture containing some starting materials 3h and
3i was carried over to the next reaction (reduction and selective
protection) from where pure 5h and 5i were obtained.
4.5.11. General procedure for reduction and protection (4–5)
LiAlH4 (3 mol equivalent) was suspended in THF (0.07 M total
concentration) at 0 °C under an atmosphere of nitrogen. A solution
of the appropriate diester (4) in THF was added dropwise over
3 min and the ice bath was removed. The reaction was stirred at
50 °C for 16 h, cooled to room temperature and the excess reagent
was quenched with solid sodium sulphate decahydrate (Na2SO4Á10
H2O, added until evolution of hydrogen stops). H2SO4 (18 N) was
added until pH 2 and the reaction was neutralized with solid so-
dium bicarbonate (NaHCO3). The solids were filtered off through
CeliteÒ, through Na2SO4, and washed with excess THF. The filtrate
was concentrated to give an oil which was re-dissolved in dichlo-
roethane (0.02 M). Anisaldehyde dimethyl acetal (1.5 mol equiva-
lent) and camphor sulfonic acid (CSA, 0.1 mol equivalent) were
added and the mixture was stirred for 4 h at room temperature.
Triethyl amine (excess, 1–2 mL) was added, the mixture was con-
centrated and the residue was purified by flash chromatography
on silica gel [EtOAc/hexanes 1:4 (v/v) ? 3:7 (v/v)].
4.5.5. (2S,3R)-Diethyl-3-hydroxy-2-isobutyl-2-methylsuccinate
(4c)
Prepared in 52% from 3a according to the general alkylation
procedure. Rf 0.34 (Et2O/hexanes, 1:1, v/v). 1H NMR (CDCl3,
500 MHz) d 4.30–4.15 (m, 5H), 3.40 (d, J = 8.2, 1H), 1.83 (dd,
J = 7.1, 13.9, 1H), 1.73–1.65 (m, 1H), 1.50 (dd, J = 5.2, 13.9, 1H),
1.30 (t, J = 7.1, 3H), 1.29 (t, J = 7.1, 3H), 1.16 (s, 3H), 0.93 (d,
J = 6.6, 3H), 0.85 (t, J = 6.6, 3H). 13C NMR (CDCl3, 125 MHz) d
175.1, 172.6, 76.7, 61.7, 60.9, 49.9, 44.0, 24.8, 24.6, 23.29, 17.04,
14.2, 14.1. HRMS for C13H24O5 [M+Na]+ calcd 283.1516, found
283.1519.
4.5.6. (2S,3R)-Diethyl-2-(cyclobutylmethyl)-3-hydroxy-2-
methylsuccinate (4d)
Prepared in 58% from 3a according to the general alkylation
procedure. Rf 0.32 (Et2O/hexanes, 1:1, v/v). 1H NMR (CDCl3,
500 MHz) d 4.27–4.14 (m, 4H), 3.42 (d, J = 8.3, 1H), 2.40–2.30 (m,
1H), 2.07–2.10 (m, 1H), 1.99–1.93 (m, 2H), 1.88–1.59 (m, 4H),
1.30 (t, J = 7.2, 3H), 1.29 (t, J = 7.2, 3H), 1.11 (s, 3H). 13C NMR (CDCl3,
125 MHz) d 174.9, 172.8, 76.1, 61.7, 60.9, 49.7, 42.5, 32.5, 30.3,
29.4, 19.0, 17.2, 14.2, 14.1. HRMS for C14H24O5 [M+Na]+ calcd
295.1516, found 295.1518.
4.5.12. ((4R,5R)-5-Ethyl-2-(4-methoxyphenyl)-5-methyl-1,3-
dioxan-4-yl)methanol (5a)
Prepared in 58% yield from 4a according to the general proce-
dure. Rf 0.20 (EtOAc/hexanes, 3:7). 1H NMR (CDCl3, 500 MHz) d
7.44 (d, J = 8.5, 2H), 6.91 (d, J = 8.5, 2H), 5.44 (s, 1H), 3.81 (s, 3H),
3.80 (d, J = 11.0, 1H), 3.76–3.62 (m, 4H), 1.37–1.23 (m, 2H), 1.13
(s, 3H), 0.87 (t, J = 7.8, 3H). 13C NMR (CDCl3, 125 MHz) d 160.1,
130.9, 127.5, 127.5, 113.7, 113.7, 101.8, 85.1, 76.8, 61.4, 55.3,
34.0, 28.2, 16.8, 7.3. HRMS for C15H22O4 [M+H]+ calcd 267.1591,
found 267.1594.
4.5.7. (2S,3R)-Diethyl-2-hexyl-3-hydroxy-2-methylsuccinate
(4e)
Prepared in 60% from 3a according to the general alkylation
procedure. Rf 0.40 (Et2O/hexanes, 1:1, v/v). 1H NMR (CDCl3,
500 MHz) d 4.29–4.15 (m, 5H), 3.37 (d, J = 8.1, 1H), 1.80–1.74 (m,
1H), 1.52 (dd, J = 4.0, 13.2, 1H), 1.31–1.26 (m, 14H), 1.15 (s, 3H),
0.88 (t, J = 7.0, 3H). 13C NMR (CDCl3, 125 MHz) d 174.9, 172.9,
75.8, 61.7, 60.9, 50.0, 35.5, 31.6, 29.7, 24.2, 22.6, 17.3, 14.2, 14.2,
14.0. HRMS for C15H28O5 [M+Na]+ calcd 311.1829, found 311.1834.
4.5.13. ((4R,5R)-2-(4-Methoxyphenyl)-5-methyl-5-propyl-1,3-
dioxan-4-yl)methanol (5b)
Prepared in 72% yield from 4b according to the general proce-
dure. Rf 0.23 (EtOAc/hexanes, 3:7, v/v). 1H NMR (CDCl3, 500 MHz)
d 7.44 (d, J = 8.5, 2H), 6.91 (d, J = 8.5, 2H), 5.45 (s, 1H), 3.81 (s,
3H), 3.80 (d, J = 11.0, 1H), 3.75–3.63 (m, 4H), 1.35–1.16 (m, 4H),

2702
T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
1.14 (s, 3H), 0.91 (t, J = 6.9, 3H). 13C NMR (CDCl3, 125 MHz) d 160.1,
130.9, 127.5, 127.5, 113.7, 113.7, 101.8, 85.2, 77.1, 61.4, 55.3, 38.3,
34.2, 17.4, 16.1, 15.0. HRMS for C16H24O4 [M+H]+ calcd 281.1747,
found 281.1745.
4.5.19. ((4R)-2-(4-Methoxyphenyl)-5,5-dipropyl-1,3-dioxan-4-
yl)methanol (5h)
Prepared in 62% from 4h according to the general procedure
(90% purity). Rf 0.31 (EtOAc/hexanes, 3:7). 1H NMR (CDCl3,
500 MHz) d 7.43 (d, J = 8.8, 2H), 6.91 (d, J = 8.8, 2H), 5.45 (s, 1H),
3.98 (d, J = 11.4, 1H), 3.86 (dd, J = 3.2, 8.3, 1H), 3.82–3.80 (m, 1H),
3.81 (s, 3H), 3.72 (d, J = 12.8, 1H), 3.62 (d, J = 11.4, 1H), 1.94 (ddd,
J = 4.5, 13.0, 13.5, 1H), 1.47–1.12 (m, 8H), 0.94 (t, J = 7.2, 3H),
0.91 (t, J = 6.9, 3H). 13C NMR (CDCl3, 125 MHz) d 160.1, 130.9,
127.6, 127.6, 113.7, 113.7, 101.9, 85.7, 73.6, 61.4, 55.3, 36.6, 36.3,
33.6, 17.2, 16.8, 15.2, 15.1. HRMS for C18H28O4 [M+H]+ calcd
309.2060, found 309.2062.
4.5.14. ((4R,5R)-5-Isobutyl-2-(4-methoxyphenyl)-5-methyl-1,3-
dioxan-4-yl)methanol (5c)
Prepared in 84% from 4c according to the general procedure. Rf
0.24 (EtOAc/hexanes, 3:7, v/v). 1H NMR (CDCl3, 500 MHz) d 7.44 (d,
J = 8.5, 2H), 6.91 (d, J = 8.5, 2H), 5.45 (s, 1H), 3.90 (d, J = 11.1, 1H),
3.81 (s, 3H), 3.73–3.71 (m, 3H), 3.64 (dd, J = 9.8, 11.8, 1H), 1.73
(sep, J = 6.6, 1H), 1.20 (dd, J = 5.9, 14.4, 1H), 1.18 (s, 3H), 1.14 (dd,
J = 5.6, 14.4, 1H), 0.96 (d, J = 6.6, 3H), 0.93 (d, J = 6.6, 3H). 13C
NMR (CDCl3, 125 MHz) d 160.1, 130.9, 127.5, 127.5, 113.7, 113.7,
101.8, 85.4, 77.3, 61.4, 55.3, 44.8, 34.9, 25.51, 25.46, 23.3, 17.8.
HRMS for C17H26O4 [M+H]+ calcd 295.1904, found 295.1905.
4.5.20. ((4R)-5,5-Diallyl-2-(4-methoxyphenyl)-1,3-dioxan-4-
yl)methanol (5i)
Prepared in 46% from 4i with a minor modification to the gen-
eral procedure; the excess LiAlH4 from the reduction reaction was
quenched with NaOH (5 mL of 1 M) at 0 °C and stirred for 30 min at
room temperature. Rf 0.26 (EtOAc/hexanes, 3:7, v/v). 1H NMR
(CDCl3, 500 MHz) d 7.44 (d, J = 8.8, 2H), 6.91 (d, J = 8.8, 2H), 5.96–
5.80 (m, 2H), 5.46 (s, 1H), 5.19–5.10 (m, 4H), 3.96 (d, J = 11.5,
1H), 3.93 (dd, J = 2.8, 8.7, 1H), 3.81 (s, 3H), 3.81 (dd, J = 2.8, 5.2,
1H), 3.78 (d, J = 2.8, 1H), 3.74 (dd, J = 8.7, 11.7, 1H), 3.64, (dd,
J = 1, 11.5, 1H), 2.83 (dd, J = 7.5, 13.8, 1H), 2.10–2.00 (m, 3H). 13C
NMR (CDCl3, 125 MHz) d 160.2, 133.8, 132.6, 130.7, 127.6, 127.6,
118.9, 118.8, 113.7, 113.7, 102.0, 84.6, 72.9, 61.0, 55.3, 37.7, 37.3,
35.2. HRMS for C18H24O4 [M+Na]+ calcd 327.1567, found 327.1576.
4.5.15. ((4R,5R)-5-(Cyclobutylmethyl)-2-(4-methoxyphenyl)-5-
methyl-1,3-dioxan-4-yl)methanol (5d)
Prepared in 35% from 4d according to the general procedure. Rf
0.38 (EtOAc/hexanes, 1:1.5, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42
(d, J = 8.5, 2H), 6.90 (d, J = 8.5, 2H), 5.43 (s, 1H), 3.81 (s, 3H), 3.76 (d,
J = 11.6, 1H), 3.72–3.61 (m, 4H), 2.43–2.37 (m, 1H), 2.07–2.03 (m,
2H), 1.91–1.83 (m, 1H), 1.78–1.72 (m, 1H), 1.69–1.59 (m 2H),
1.40–1.25 (m 2H), 1.11 (s, 3H). 13C NMR (CDCl3, 125 MHz) d
160.1, 130.9, 127.5, 127.5, 113.7, 113.7, 101.8, 85.1, 61.5, 55.3,
43.3, 35.1, 31.6, 30.8, 30.5, 30.5, 18.9, 17.7. HRMS for C18H26O4
[M+Na]+ calcd 329.1723, found 329.1727.
4.5.21. General procedure for amide coupling (5–8)
4.5.16. (4R,5R)-5-Hexyl-2-(4-methoxyphenyl)-5-methyl-1,3-
dioxan-4-yl)methanol (5e)
Dess–Martin periodinane (2 mol equiv) was suspended in CH2Cl2
and the alcohol in wet CH2Cl2 (1 mol equiv) was added. The mixture
was stirred at room temperature for 1 h (monitored by TLC), diluted
with diethyl ether and washed with a solution of Na2S2O3 in sat.
NaHCO3 (1.5 g Na2S2O3 in 5 mL of aqueous saturated NaHCO3). The
organic layer was dried over anhydrous sodium sulfate and concen-
trated to give the product which was used in the next reaction with-
out purification. To a suspension of the crude aldehyde in a mixture
of acetone and CH2Cl2 (3:1, v/v) was added a freshly prepared solu-
tion of NaH2PO4 (10 mol equiv) and NaClO2 (5 mol equiv) in water
(1:3 v/v ratio of water to the total volume of acetone/CH2Cl2). The
mixture was stirred at rt for 15 min. After evaporation of the solvent
in vacuo, the residue was dissolved in a saturated solution of sodium
sulfite (1 mL per mol equivalent of NaClO2). The aqueous solution
was extracted with ethyl acetate. The combined organic layers were
dried over sodium sulfate and concentrated to give the acid which
was also used without purification. The appropriate Fmoc protected
amine (11, 1.5 mol equiv) was dissolved in DMF (10 mL per 200 mg)
and piperidine (1.5 mL per 200 mg sample) was added. The solvent
and excess piperidine were evaporated off under vacuum. To this
residue was added EDC (3 mol equiv), HOBt (3.6 mol equiv) and
the flask was purged with nitrogen. A solution of the above crude
acid in THF was added followed by DIPEA (10 equiv) and the mixture
was stirred overnight at room temperature (16 h). The reaction mix-
ture was poured into a separatory funnel containing an aqueous
solution of saturated ammonium chloride, before extraction with
ethyl acetate. The combined organic layers were dried over sodium
sulfate and evaporated to give a solid. Purification by flash chroma-
tography on silica gel (EtOAc/hexanes, 1:1 ? 5:1) afforded the de-
sired product.
Prepared in 57% from 4e according to the general procedure. Rf
0.30 (EtOAc/hexanes, 3:7, v/v). 1H NMR (CDCl3, 500 MHz) d 7.44 (d,
J = 8.8, 2H), 6.91 (d, J = 8.8, 2H), 5.45 (s, 1H), 3.81 (s, 3H), 3.79 (d,
J = 12.8, 1H), 3.75 (ddd, J = 2.5, 6.6, 9.0, 1H), 3.71–3.63 (m, 3H),
1.31–1.18 (m, 10H), 1.14 (s, 3H), 0.89 (t, J = 7.0, 3H). 13C NMR
(CDCl3, 125 MHz) d 160.1, 130.9, 127.5, 127.5, 113.7, 113.7,
101.8, 85.2, 77.1, 61.4, 55.3, 36.0, 34.1, 31.7, 30.2, 22.7, 22.6,
17.4, 14.1. HRMS for C19H30O4 [M+H]+ calcd 323.2217, found
323.2223.
4.5.17. (4R,5R)-5-Allyl-2-(4-methoxyphenyl)-5-methyl-1,3-
dioxan-4-yl)methanol (5f)
Prepared in 82% yield from 4f with a minor modification to the
general procedure; the excess LiAlH4 from the reduction reaction
was quenched with NaOH (5 mL of 1 M) at 0 °C and stirred for
30 min at room temperature. Rf 0.18 (EtOAc/hexanes, 3:7, v/v).
1H NMR (CDCl3, 500 MHz) d 7.43 (d, J = 8.5, 2H), 6.91 (d, J = 8.5,
2H), 5.81–5.68 (m, 1H), 5.44 (s, 1H), 5.13–5.07 (m, 2H), 3.82–
3.69 (m, 4H), 3.81 (s, 3H), 3.67 (dd, J = 9.0, 11.6, 1H), 2.06–1.96
(m, 2H), 1.16 (s, 3H). 13C NMR (CDCl3, 125 MHz) d 160.1, 132.2,
130.7, 127.5, 127.5, 118.8, 113.7, 113.7, 101.8, 84.6, 77.0, 61.3,
55.3, 40.3, 34.3, 17.5. HRMS for C16H22O4 [M+H]+ calcd 279.1591,
found 279.1589.
4.5.18. ((4R,5R)-5-Benzyl-2-(4-methoxyphenyl)-5-methyl-1,3-
dioxan-4-yl)methanol (5g)
Prepared in 62% from 4g according to the general procedure.
1H NMR (CDCl3, 500 MHz) d 7.41 (d, J = 8.6, 2H), 7.32–7.29 (m,
2H), 7.26–7.24 (m, 1H), 7.12 (d, J = 6.8, 2H), 6.89 (d, J = 8.6,
2H), 5.40 (s, 1H), 3.90–3.82 (m, 3H), 3.80 (s, 3H), 3.78–3.72
(m, 2H), 3.59 (d, J = 11.2, 1H), 2.59 (s, 2H), 1.17 (s, 3H). 13C
4.5.22. (4R,5R)-5-Ethyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8a)
Prepared in 58% yield from 5a according to the general proce-
dure. Rf 0.31 (EtOAc/hexanes 5:1, v/v). 1H NMR (CDCl3, 500 MHz)
d 7.42 (d, J = 8.6, 2H), 7.00 (t, J = 6.2, 1H), 6.92 (d, J = 8.6, 2H),
5.78 (br s, 1H), 5.43 (s, 1H), 4.17 (s, 1H), 3.85 (d, J = 11.4, 1H),
NMR (CDCl3, 125 MHz)
d 160.1, 135.9, 130.7, 130.2, 130.2,
128.2, 128.2, 127.5, 127.5, 126.7, 113.7, 113.7, 101.7, 85.6,
77.0, 61.5, 55.3, 42.1, 34.9, 17.6. HRMS for C20H24O4 [M+H]+
calcd 329.1747, found 329.1749.

T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
2703
3.82 (s, 3H), 3.77 (d, J = 11.4, 1H), 3.61–3.46 (m, 2H), 3.22–3.18 (m,
4.5.27. (4R,5R)-5-Allyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
2H), 2.41 (t, J = 6.2, 2H), 1.79–1.71 (m, 1H), 1.60–1.53 (m, 1H),
1.49–1.43 (m, 2H), 1.33–1.25 (m, 4H), 1.09 (s, 3H), 0.91 (t, J = 7.6,
3H), 0.87 (t, J = 7.2, 3H). 13C NMR (CDCl3, 125 MHz) d 170.6,
169.6, 160.2, 130.2, 127.5, 127.5, 113.8, 113.8, 101.1, 82.7, 76.2,
55.3, 39.6, 36.3, 35.5, 35.0, 29.2, 29.0, 28.1, 22.3, 17.2, 14.0, 7.4.
HRMS for C23H36N2O5 [M+H]+ calcd 421.2697, found 421.2712.
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8f)
Prepared in 48% from 5f according to the general procedure. Rf
0.31 (EtOAc/hexanes, 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42 (d,
J = 8.6, 2H), 7.05 (t, J = 5.2, 1H), 6.92 (d, J = 8.6, 2H), 5.90–5.82 (m,
1H), 5.78 (s, 1H), 5.42 (s, 1H), 5.15–5.11 (m, 2H), 4.19 (s, 1H),
3.82 (s, 3H), 3.81 (d, J = 11.2, 1H), 3.78 (d, J = 11.2, 1H), 3.62–3.48
(m, 2H), 3.23–3.19 (m, 2H), 2.43–2.34 (m, 4H), 1.49–1.43 (m,
2H), 1.35–1.23 (m, 4H), 1.11 (s, 3H), 0.88 (t, J = 6.9, 3H). 13C NMR
(CDCl3, 125 MHz) d 170.6, 169.5, 160.2, 132.8, 130.1, 127.5,
127.5, 118.8, 113.7, 113.7, 101.1, 82.1, 76.2, 55.3, 40.1, 39.6, 36.2,
35.5, 35.0, 29.2, 29.0, 22.3, 17.7, 14.0. HRMS for C24H36N2O5
[M+H]+ calcd 433.2697, found 433.2714.
4.5.23. (4R,5R)-5-Propyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8b)
Prepared in 67% from 5b according to the general procedure. Rf
0.41 (EtOAc/hexanes, 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42 (d,
J = 8.6, 2H), 7.01 (t, J = 6.1, 1H), 6.92 (d, J = 8.6, 2H), 5.78 (br s, 1H),
5.43 (s, 1H), 4.17 (s, 1H), 3.85 (d, J = 11.4, 1H), 3.82 (s, 3H), 3.78 (d,
J = 11.4, 1H), 3.75 (ddd, J = 6.4, 12.5, 13.9, 1H), 3.39 (ddd, J = 6.2,
12.5, 13.9, 1H), 3.22–3.18 (m, 2H), 2.41 (t, J = 6.2, 2H), 1.66 (ddd,
J = 5.0, 12.2, 14.0, 1H), 1.52–1.43 (m, 3H), 1.39–1.23 (m, 6H), 1.10
(s, 3H), 0.93 (t, J = 7.2, 3H), 0.88 (t, J = 7.2, 3H). 13C NMR (CDCl3,
125 MHz) d 170.6, 169.6, 160.2, 130.2, 127.5, 127.5, 113.7, 113.7,
101.2, 82.8, 76.6, 55.3, 39.6, 38.0, 36.3, 35.6, 35.0, 29.2, 29.0,
22.3, 17.7, 16.2, 14.9, 14.0. HRMS for C24H38N2O5 [M+H]+ calcd
435.2854, found 435.2860.
4.5.28. (4R,5R)-5-Benzyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8g)
Prepared in 79% from 5g according to the general procedure. Rf
0.19 (EtOAC/hexanes 3:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.38 (d,
J = 8.8, 2H), 7.30–7.27 (m, 4H), 7.25–7.22 (m, 1H), 7.14 (t, J = 6.0,
1H), 6.90 (d, J = 8.8, 2H), 5.84 (br s, 1H), 5.30 (s, 1H), 4.26 (s, 1H),
3.82 (d, J = 11.5, 1H), 3.81 (s, 3H), 3.68 (d, J = 11.5, 1H), 3.65–3.53
(m, 2H), 3.25–3.21 (m, 2H), 3.10 (d, J = 13.8, 1H), 2.88 (d, J = 13.8,
1H), 2.46 (t, J = 6.3, 2H), 1.51–1.45 (m, 2H), 1.35–1.24 (m, 4H),
1.15 (s, 3H), 0.88 (t, J = 7.1, 3H). 13C NMR (CDCl3, 125 MHz) d
170.6, 169.8, 160.2, 136.5, 130.9, 130.9, 130.0, 128.0, 128.0,
127.4, 127.4, 126.4, 113.7, 113.7, 100.9, 81.3, 76.1, 55.3, 41.6,
39.6, 36.2, 36.0, 35.0, 29.2, 29.0, 22.3, 18.3, 14.0. HRMS for
4.5.24. (4R,5R)-5-Isobutyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8c)
Prepared in 75% from 5c according to the general procedure. Rf
0.17 (EtOAC/hexanes, 3:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42
(d, J = 8.7, 2H), 7.02 (t, J = 6.1, 1H), 6.92 (d, J = 8.7, 2H), 5.84 (br s,
1H), 5.44 (s, 1H), 4.13 (s, 1H), 3.95 (dd, J = 11.4, 12.8, 1H), 3.82 (s,
3H), 3.81 (d, J = 12.1, 1H), 3.61–3.47 (m, 2H), 3.24–3.17 (m, 2H),
2.42 (t, J = 6.2, 2H), 1.79–1.71 (m, 2H), 1.50–1.40 (m, 3H), 1.35–
1.23 (m, 4H), 1.14 (s, 3H), 0.97 (d, J = 6.3, 3H), 0.96 (d, J = 6.3,
3H),0.89 (t, J = 6.9, 3H). 13C NMR (CDCl3, 125 MHz) d 170.7, 169.6,
160.2, 130.2, 127.5, 127.5, 113.8, 113.8, 101.1, 83.2, 76.8, 55.3,
44.2, 39.6, 36.4, 36.2, 35.0, 29.2, 29.0, 25.52, 25.48, 23.3, 22.3, 17.8,
14.0. HRMS for C25H40N2O5 [M+H]+ calcd 449.3010, found 449.3016.
C
28H38N2O5 [M+H]+ calcd 483.2853, found 483.2864.
4.5.29. (4R)-5,5-Dipropyl-2-(4-methoxyphenyl)-N-(3-oxo-3-
(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8h)
Prepared in 43% from 5h according to the general procedure. Rf
0.41 (EtOAc/hexanes, 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42 (d,
J = 8.8, 2H), 7.07 (t, J = 6.0, 1H), 6.92 (d, J = 8.8, 2H), 5.83 (s, 1H),
5.43 (s, 1H), 4.31 (s, 1H), 3.98 (d, J = 11.6, 1H), 3.82 (s, 3H), 3.76
(d, J = 11.6, 1H), 3.56–3.52 (m, 2H), 3.27–3.14 (m, 2H), 2.45–2.38
(m, 2H), 1.82 (ddd, J = 4.4, 12.5, 13.0, 1H), 1.64–1.20 (m, 13H),
0.93 (t, J = 7.0, 3H), 0.90 (t, J = 7.0, 3H), 0.88 (t, J = 7.0, 3H). 13C
NMR (CDCl3, 125 MHz) d 170.6, 169.8, 160.2, 130.2, 127.5, 127.5,
113.7, 113.7, 101.2, 81.7, 73.3, 55.3, 39.6, 38.2, 36.2, 35.7, 34.9,
34.0, 29.2, 29.0, 22.3, 16.72, 16.69, 15.1, 15.0, 14.0. HRMS for
4.5.25. (4R,5R)-5-(Cyclobutylmethyl)-2-(4-methoxyphenyl)-5-
methyl-N-(3-oxo-3-(pentylamino)propyl)-1,3-dioxane-4-
carboxamide (8d)
Prepared in 61% from 5d according to the general procedure. Rf
0.46 (EtOAc/hexanes, 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.41 (d,
J = 8.8, 2H), 7.00 (t, J = 6.2, 1H), 6.92 (d, J = 8.8, 2H), 5.76 (br s, 1H),
5.42 (s, 1H), 4.12 (s, 1H), 3.83 (d, J = 11.5, 1H), 3.82 (s, 3H), 3.73 (d,
J = 11.5, 1H), 3.61–3.54 (m, 1H), 3.52–3.46 (m, 1H), 3.22–3.18 (m,
2H), 2.48–2.39 (m, 3H), 2.08–1.99 (m, 2H), 1.90–1.82 (m, 2H),
1.77–1.66 (m, 3H), 1.60 (dd, J = 7.1, 14.7, 1H), 1.49–1.44 (m, 2H),
1.35–1.23 (m, 4H), 1.08 (s, 3H), 0.89 (t, J = 7.2, 3H). 13C NMR (CDCl3,
125 MHz) d 170.6, 169.6, 160.2, 130.2, 127.4, 127.4, 113.7, 113.7,
101.1, 83.0, 77.3, 55.3, 42.8, 39.6, 36.6, 36.3, 35.0, 31.7, 30.65,
30.58, 29.2, 29.0, 22.3, 19.0, 17.6, 14.0. HRMS for C26H40N2O5
[M+Na]+ calcd 483.2829, found 483.2836.
C
26H42N2O5 [M+H]+ calcd 463.3166, found 463.3176.
4.5.30. (4R)-5,5-Diallyl-2-(4-methoxyphenyl)-N-(3-oxo-3-
(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8i)
Prepared in 45% from 5i with modifications to the general
procedure. HBTU was used to couple 7i to 12a in DMF instead
of EDC in THF. Rf 0.43 (EtOAC/hexanes, 5:1, v/v). 1H NMR (CDCl3,
500 MHz) d 7.42 (d, J = 8.7, 2H), 7.09 (t, J = 5.8, 1H), 6.92 (d,
J = 8.7, 2H), 6.04–5.96 (m, 1H), 5.89–5.81 (m, 1H), 5.77 (br s,
1H), 5.42 (s, 1H), 5.18–5.10 (m, 4H), 4.35 (s, 1H), 3.92 (d,
J = 11.5, 1H), 3.82 (s, 3H), 3.77 (d, J = 11.5, 1H), 3.56–3.52 (m,
2H), 3.22–3.18 (m, 2H), 2.69 (dd, J = 7.7, 13.5, 1H), 2.58 (dd,
J = 7.0, 14.1, 1H), 2.41 (t, J = 6.3, 2H), 2.15 (dd, J = 8.3, 14.1, 1H),
2.03 (dd, J = 7.5, 13.5, 1H), 1.49–1.43 (m, 2H), 1.33–1.25 (m,
4H), 0.88 (t, J = 6.9, 3H). 13C NMR (CDCl3, 125 MHz) d 170.7,
169.4, 160.3, 133.4, 133.4, 130.1, 127.5, 127.5, 119.1, 118.8,
113.8, 113.8, 101.3, 80.7, 72.2, 55.3, 39.6, 38.4, 37.3, 36.1, 35.7,
35.0, 29.2, 29.0, 22.3, 13.9. HRMS for C26H38N2O5 [MÀH]À calcd
457.2708, found 457.2717.
4.5.26. (4R,5R)-5-Hexyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
oxo-3-(pentylamino)propyl)-1,3-dioxane-4-carboxamide (8e)
Prepared in 71% from 5e according to the general procedure. Rf
0.28 (EtOAc/hexanes, 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42 (d,
J = 8.8, 2H), 7.01 (t, J = 6.3, 1H), 6.92 (d, J = 8.8, 2H), 5.97 (s, 1H),
5.43 (s, 1H), 4.18 (br s, 1H), 3.84 (d, J = 11.5, 1H), 3.82 (s, 3H),
3.78 (d, J = 11.5, 1H), 3.62–3.46 (m, 2H), 3.22–3.18 (m, 2H), 2.42–
2.40 (m, 2H), 1.68–1.60 (m, 1H), 1.54–1.43 (m, 3H), 1.35–1.22
(m, 12H), 1.09 (s, 3H), 0.89 (t, J = 7.0, 3H), 0.88 (t, J = 7.0, 3H). 13C
NMR (CDCl3, 125 MHz) d 170.6, 169.6, 160.2, 130.2, 127.5, 127.5,
113.7, 113.7, 101.1, 82.8, 76.6, 55.3, 39.6, 36.3, 35.7, 35.6, 35.0,
31.8, 30.2, 29.2, 29.0, 22.9, 22.7, 22.3, 17.7, 14.1, 14.0. HRMS for
4.5.31. (4R,5R)-5-Allyl-N-(3-(nonylamino)-3-oxopropyl)-2-(4-
methoxyphenyl)-5-methyl-1,3-dioxane-4-carboxamide (8j)
Prepared in 41% from 5k according to the general procedure. Rf
0.46 (EtOAc/hexanes, 3:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42 (d,
J = 8.8, 2H), 7.04 (t, J = 6.2, 1H), 6.92 (d, J = 8.8, 2H), 5.90–5.82 (m,
C
27H44N2O5 [MÀH]À calcd 475.3177, found 475.3187.

2704
T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
1H), 5.77 (br s, 1H), 5.42 (s, 1H), 5.15–5.11 (m, 2H), 4.19 (s, 1H),
3.82 (s, 3H), 3.81–3.76 (m, 2H), 3.62–3.48 (m, 2H), 3.23–3.19 (m,
2H), 2.43–2.34 (m, 4H), 1.49–1.43 (m, 2H), 1.31–1.23 (m, 12H),
1.11 (s, 3H), 0.88 (t, J = 7.1, 3H), 13C NMR (CDCl3, 125 MHz) d
170.6, 169.5, 160.2, 132.8, 130.1, 127.5, 127.5, 118.8, 113.8,
113.8, 101.2, 82.1, 76.2, 55.3, 40.1, 39.7, 36.2, 35.5, 35.0, 31.8,
29.51, 29.50, 29.3, 29.2, 26.9, 22.7, 17.7, 14.1. HRMS for
0.89 (t, J = 7.2, 3H). 13C NMR (CDCl3, 125 MHz) d 173.8, 171.4,
77.7, 68.8, 41.8, 39.7, 36.0, 35.9, 35.3, 29.2, 29.0, 22.3, 19.0, 16.5,
15.0, 13.9. HRMS for C16H32N2O4 [M+Na]+ calcd 339.2254, found
339.2250. Purity, >95% (tR = 16.60 min).
4.5.37. (2R,3R)-2-Hydroxy-3-(hydroxymethyl)-3,5-dimethyl-N-
(3-oxo-3-(pentylamino)propyl)hexanamide (9c)
C
28H44N2O5 [M+Na]+ calcd 511.3142, found 511.3142.
Prepared in 83% yield from 8c according to the general proce-
dure. Rf 0.43 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.44 (m, 1H), 5.89 (t, J = 4.6, 1H), 3.99–3.97 (m, 2H), 3.65–3.48
(m, 5H), 3.24–3.20 (m, 2H), 2.43 (dt, J = 1.2, 6.5, 2H), 1.76–1.68
(m, 1H), 1.52–1.47 (m, 3H), 1.36–1.25 (m, 4H), 1.22 (dd, J = 5.7,
14.2, 1H), 1.01 (s, 3H), 0.94 (t, J = 6.5, 6H), 0.89 (t, J = 7.2, 3H). 13C
NMR (CDCl3, 125 MHz) d 173.7, 171.4, 78.0, 69.0, 42.5, 42.0, 39.7,
35.8, 35.3, 29.2, 29.0, 25.5, 25.3, 23.5, 22.3, 19.4, 14.0. HRMS for
4.5.32. (4R,5R)-5-Allyl-2-(4-methoxyphenyl)-5-methyl-N-(3-
(nonylamino)-3-oxopropyl)-1,3-dioxane-4-carboxamide (8k)
Prepared in 66% from 5l according to the general procedure. Rf
0.21 (EtOAC/hexanes 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.43
(d, J = 8.8, 2H), 7.01 (t, J = 5.8, 1H), 6.92 (d, J = 8.8, 2H), 5.78 (brs,
1H), 5.46 (s, 1H), 4.07 (s, 1H), 3.82 (s, 3H), 3.71(d, J = 11.4, 1H),
3.66 (d, J = 11.4, 1H), 3.62–3.55 (m, 2H), 3.54–3.47 (m, 2H), 3.23–
3.19 (m, 2H), 2.41 (t, J = 6.2, 2H), 1.49–1.44 (m, 2H), 1.35–1.23
(m, 2H), 1.10 (s, 3H), 0.88 (t, J = 7.0, 3H); 13C NMR (CDCl3,
125 MHz) d 170.6, 169.6, 160.2, 130.1, 127.5, 127.5, 113.7, 113.7,
101.3, 83.8, 78.5, 55.3, 39.7, 36.2, 35.0, 33.1, 29.2, 29.0, 22.3,
21.8, 19.1, 14.0. HRMS for C22H34N2O5 [M+H]+ calcd 407.2540,
found 407.2547.
C
17H34N2O4 [M+Na]+ calcd 353.2411, found 353.2405. Purity
>95% (tR = 17.66 min).
4.5.38. (2R,3R)-4-Cyclobutyl-2-hydroxy-3-(hydroxymethyl)-3-
methyl-N-(3-oxo-3-(pentylamino)propyl)butanamide (9d)
Prepared in 60% yield from 8d according to the general proce-
dure. Rf 0.44 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.83 (br s, 1H), 5.80 (br s, 1H), 3.96 (s, 1H), 3.66–3.59 (m, 1H),
3.55–3.47 (m, 3H), 3.25–3.21 (m, 2H), 2.46–2.38 (m, 3H), 2.04–
2.00 (m, 2H), 1.88–1.79 (m, 1H), 1.76–1.62 (m, 4H), 1.52–1.47
(m, 2H), 1.41 (dd, J = 6.9, 14.0, 1H), 1.35–1.25 (m, 4H), 0.95 (s,
3H), 0.90 (t, J = 6.8, 3H). 13C NMR (CDCl3, 125 MHz) d 173.5,
171.3, 77.8, 68.8, 42.4, 40.6, 39.7, 35.8, 35.4, 32.0, 30.52, 30.49,
4.5.33. (4R)-2-(4-Methoxyphenyl)-5,5-dimethyl-N-(3-
(nonylamino)-3-oxopropyl)-1,3-dioxane-4-carboxamide (8l)
Prepared in 75% from 5l according to the general procedure. Rf
0.29 (EtOAc/hexanes 5:1, v/v). 1H NMR (CDCl3, 500 MHz) d 7.42
(d, J = 8.8, 2H), 7.01 (t, J = 5.7, 1H), 6.92 (d, J = 8.8, 2H), 5.85 (br s,
1H), 5.45 (s, 1H), 4.06 (s, 1H), 3.81 (s, 3H), 3.70 (d, J = 11.4, 1H),
3.65 (d, J = 11.4, 1H), 3.61–3.46 (m, 2H), 3.21–3.17 (m, 2H), 2.40
(t, J = 6.3, 2H), 1.48–1.43 (m, 2H), 1.31–1.20 (m, 12H), 1.10 (s,
3H), 1.09 (s, 3H), 0.87 (t, J = 7.1, 3H), 13C NMR (CDCl3, 125 MHz) d
170.6, 169.5, 160.2, 130.1, 127.5, 127.5, 113.7, 113.7, 101.3, 83.8,
78.5, 55.3, 39.6, 36.2, 35.0, 33.1, 31.7, 29.5, 29.5, 28.9, 28.9, 26.9,
22.6, 21.8, 19.1, 14.0.
29.2, 29.0, 22.3, 19.13, 19.06, 14.0. HRMS for
C18H34N2O4
[M+Na]+ calcd 365.2411, found 365.2412. Purity >95%
(tR = 18.11 min).
4.5.39. (2R,3R)-2-Hydroxy-3-(hydroxymethyl)-3-methyl-N-(3-
oxo-3-(pentylamino)propyl)nonanamide (9e)
Prepared in 79% yield from 8e according to the general proce-
dure. Rf 0.42 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.43 (m, 1H), 5.90 (t, J = 4.6, 1H), 4.00–3.98 (m, 2H), 3.66–3.59
(m, 2H), 3.56–3.48 (m, 3H), 3.25–3.18 (m, 2H), 2.47–2.38 (m,
2H), 1.55–1.46 (m, 3H), 1.35–1.26 (m, 13H), 0.96 (s, 3H), 0.89 (t,
J = 7.0, 3H), 0.87 (t, J = 7.0, 3H). 13C NMR (CDCl3, 125 MHz) d
173.8, 171.0, 77.7, 68.8, 41.7, 39.7, 35.8, 35.3, 33.8, 31.9, 30.2,
29.2, 29.0, 23.3, 22.7, 22.3, 19.0, 14.1, 13.9. HRMS for C19H38N2O4
[M+Na]+ calcd 381.2724, found 381.2720. Purity >95%
(tR = 20.27 min).
4.5.34. General procedure for deprotection (8–9)
Acetic acid 90% in water was added to compounds 8a–8l (0.03–
0.05 M) at room temperature and the mixture stirred overnight
(16 h). The solvent was evaporated and the residue purified by
flash chromatography on silica gel (100% EtOAc ? 10% MeOH in
EtOAc) to give the titled compounds. Samples for biological testing
were further purified by prep TLC (10% MeOH in EtOAc) to give
>95% pure compounds by reverse phase HPLC (gradient elution
with H2O/CH3CN, 99:1 ? 1:99 in 30 min on analytical Phenome-
nex Synergi 4
l
Hydro-RP 80A C18 column)
4.5.40. (2R,3R)-2-Hydroxy-3-(hydroxymethyl)-3-methyl-N-(3-
oxo-(pentylamino)propyl)hex-5-enamide (9f)
4.5.35. (2R,3R)-2-Hydroxy-3-(hydroxymethyl)-3-methyl-N-(3-
oxo-3-(pentylamino)propyl)pentanamide (9a)
Prepared in 96% yield from 8f according to the general proce-
dure. Rf 0.34 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.45 (m, 1H), 5.91 (t, J = 5.3, 1H), 5.88–5.80 (m, 1H), 5.12–5.08
(m, 2H), 4.01 (br s, 2H), 3.65–3.49 (m, 5H), 3.22 (q, J = 7.0, 2H),
2.43 (m, 2H), 2.29 (dd, J = 7.5, 13.7, 1H), 2.12 (dd, J = 7.5, 13.7,
1H), 1.52–1.46 (m, 2H), 1.36–1.25 (m, 4H), 0.97 (s, 3H), 0.89 (t,
J = 7.2, 3H). 13C NMR (CDCl3, 125 MHz) d 173.6, 171.4, 134.0,
118.3, 77.2, 68.6, 42.0, 39.7, 38.3, 35.7, 35.3, 29.2, 29.0, 22.3,
18.8, 13.9. HRMS for C16H30N2O4 [M+Na]+ calcd 337.2098, found
337.2093. Purity, >95% (tR = 16.19 min).
Prepared in 77% yield from 8a according to the general proce-
dure. Rf 0.36 (EtOAc/MeOH 9:1). 1H NMR (CDCl3, 500 MHz) d 7.43
(m, 1H), 5.91 (br s, 1H), 4.00 (s, 2H), 3.66–3.60 (m, 2H), 3.54–
3.47 (m, 3H), 3.24–3.20 (m, 2H), 2.44–2.41 (m, 2H), 1.63–1.56
(m, 1H), 1.52–1.46 (m, 2H), 1.40–1.25 (m, 5H), 0.94 (s, 3H), 0.89
(t, J = 6.9, 3H), 0.87 (t, J = 7.6, 3H). 13C NMR (CDCl3, 125 MHz) d
173.9, 171.4, 77.6, 68.4, 41.4, 39.7, 35.8, 35.4, 29.2, 29.0, 26.0,
22.3, 18.4, 14.0, 7.8. HRMS for
301.2133, found 301.2131. Purity, >95% (tR = 15.46 min).
C
15H30N2O4 [MÀH]À calcd
4.5.41. (2R,3R)-3-Benzyl-2,4-dihydroxy-3-methyl-N-(3-oxo-3-
(pentylamino)propyl)butanamide (9g)
4.5.36. (2R,3R)-2-Hydroxy-3(hydroxymethyl)-3-methyl-N-(3-
oxo-3-(pentylamino)propyl)hexanamide (9b)
Prepared in 52% yield from 8g according to the general proce-
dure. Rf 0.52 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.51 (m, 1H), 7.28–7.18 (m, 5H), 5.92 (t, J = 4.9, 1H), 4.28 (br s,
1H), 4.00 (s, 1H), 3.82 (br s, 1H), 3.67–3.61 (m, 1H), 3.56–3.48
(m, 2H), 3.51 (s, 1H), 3.20 (q, J = 6.7, 2H), 2.84 (d, J = 13.2, 1H),
2.75 (d, J = 13.2, 1H), 2.46–2.42 (m, 2H), 1.49–1.43 (m, 2H), 1.33–
Prepared in 81% yield from 8b according to the general proce-
dure. Rf 0.38 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.43 (m, 1H), 5.90 (t, J = 4.7, 1H), 4.00 (s, 2H), 3.66–3.59 (m, 2H),
3.56–3.48 (m, 3H), 3.25–3.18 (m, 2H), 2.44–2.41 (m, 2H), 1.53–
1.46(m, 3H), 1.35–1.23 (m, 7H), 0.96 (s, 3H), 0.92 (t, J = 6.6, 3H),

T. O. Akinnusi et al. / Bioorg. Med. Chem. 19 (2011) 2696–2706
2705
1.22 (m, 4H), 0.87 (t, J = 6.9, 3H), 0.86 (s, 3H). 13C NMR (CDCl3,
125 MHz) d 173.9, 171.4, 137.4, 130.9, 130.9, 127.9, 127.9, 126.2,
77.3, 67.8, 42.8, 39.7, 39.0, 35.7, 35.4, 29.1, 29.0, 22.3, 18.9, 13.9.
HRMS for C20H32N2O4 [M+Na]+ calcd 387.2254, found 387.2253.
Purity >95% (tR = 16.19 min).
1.53–1.47 (m, 2H), 1.44 (dd, J = 7.1, 14.1, 1H), 1.35–1.26 (m, 5H),
1.07 (s, 3H), 0.90 (t, J = 7.2, 3H), 0.71–0.64 (m, 1H), 0.48–0.44 (m,
2H), 0.09–0.07 (m, 2H). 13C NMR (CDCl3, 125 MHz) d 173.6,
171.3, 77.7, 68.9, 42.9, 39.7, 38.5, 35.8, 35.4, 29.2, 29.0, 22.3,
19.3, 14.0, 5.7, 4.9, 4.7. HRMS for C17H32N2O4 [M+Na]+ calcd
351.2254, found 351.2259. Purity >95% (tR = 18.03 min).
4.5.42. (R)-2-Hydroxy-3-(hydroxymethyl)-N-(3-oxo-3-
(pentylamino)propyl)-3-propylhexanamide (9h)
4.5.46. (9H-Fluoren-9-yl)methyl-3-oxo-3-(pentylamino)propyl-
carbamate (11a)
Prepared in 45% yield from 8h according to the general proce-
dure. Rf 0.35 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.43 (m, 1H), 5.78 (br s, 1H), 4.08 (s, 1H), 3.68–3.47 (m, 4H),
3.25–3.20 (m, 2H), 2.47–2.38 (m, 2H), 1.52–1.24 (m, 16H), 0.92–
0.88 (m, 9H). 13C NMR (CDCl3, 125 MHz) d 174.0, 171.4, 76.7,
67.4, 44.1, 39.7, 35.7, 35.4, 34.6, 33.6, 29.2, 29.0, 22.3, 16.6, 16.3,
15.0, 15.0, 14.0. HRMS for C18H36N2O4 [M+Na]+ calcd 367.2567,
found 367.2575. Purity >95% (tR = 19.05 min)
Fmoc protected b-alanine (2.00 g, 6.42 mmol), EDC (1.60 g,
8.36 mmol) and HOBt (1.59 g, 10.40 mmol) were weighed into a
flask which was purged with nitrogen. THF (30 mL), pentylamine
(0.88 mL, 7.62 mmol) and diisopropylamine (6.04 mL, 34.68 mmol)
were added sequentially and the mixture was stirred overnight un-
der nitrogen at room temperature. The reaction mixture was
poured into a separatory funnel containing saturated aqueous
ammonium chloride (30 mL), the organic layer was separated
and the aqueous layer was extracted with ethyl acetate
(3 Â 30 mL). The combined organic layer was washed with brine,
dried over sodium sulfate and concentrated to yield a yellow solid.
The solid was suspended in diethyl ether/hexanes (50 mL, 1.5/10 v/
v), filtered and the residue was washed with the same solvent sys-
tem (3 Â 25 mL) to afford a white solid that is homogeneous by TLC
and NMR (2.04 g, 83% yield). 1H NMR (CDCl3, 500 MHz) d 7.76 (d,
J = 7.5, 2H), 7.59 (d, J = 7.4, 2H), 7.40 (d, J = 7.5, 2H), 7.31 (d,
J = 7.5, 2H), 5.53 (br s, 1H), 5.49 (br s, 1H), 4.37 (d, J = 6.9, 2H),
4.20 (t, J = 6.9, 1H), 3.50 (br s, 2H), 3.27–3.23 (m, 2H), 2.41 (t,
J = 5.2, 2H), 1.53–1.47 (m, 2H), 1.35–1.23 (m, 4H), 0.90 (t, J = 6.69,
3H). 13C NMR (CDCl3, 125 MHz) d 171.2, 156.6, 143.9, 143.9,
141.3, 141.3, 127.7, 127.7, 127.0, 127.0, 125.1, 125.1, 119.9,
119.9, 66.7, 47.2, 39.6, 37.1, 36.0, 29.3, 29.0, 22.3, 14.0. HRMS for
4.5.43. (R)-3-Allyl-2-hydroxy-3-(hydroxymethyl)-N-(3-oxo-3-
(pentylamino)propyl)hex-5-enamide (9i)
Prepared in 52% yield from 8i according to the general procedure.
Rf 0.16 (EtOAc/hexanes, 9:1). 1H NMR (CDCl3, 500 MHz) d 7.25 (br s,
1H), 5.98–5.82(m, 3H), 5.17–5.09(m, 4H), 4.15 (s, 1H), 3.65–3.59(m,
4H), 3.25–3.21 (m, 2H), 2.44 (t, J = 6.7, 2H), 2.34 (dd, J = 7.6, 14.1, 1H),
2.24–2.16 (m, 4H), 1.52–1.46 (m, 2H), 1.36–1.25 (m, 5H), 0.89 (t,
J = 7.2, 3H). 13C NMR (CDCl3, 125 MHz) d 173.6, 171.2, 134.6, 133.9,
118.5, 118.4, 75.7, 67.1, 45.1, 39.7, 37.7, 35.7, 35.6, 35.4, 29.2, 29.0,
22.3, 13.9. HRMS for C18H32N2O4 [M+Na]+ calcd 363.2254, found
363.2255. Purity >95% (tR = 18.03 min)
4.5.44. (2R,3R)-2-Hydroxy-3-(hydroxymethyl)-3-methyl-N-
(3(nonylamino)-3-oxopropyl)hex-5-enamide (9j)
Prepared in 48% yield from 8j according to the general proce-
dure. Rf 0.45 (EtOAc/MeOH, 9:1). 1H NMR (CDCl3, 500 MHz) d
7.43 (br s, 1H), 5.92 (br s, 1H), 5.89–5.81 (m, 1H), 5.13–5.09 (m,
2H), 4.02 (s, 1H), 3.66–3.52 (m, 4H), 3.25–3.21 (m, 2H), 2.47–
2.44 (m, 2H), 2.30 (dd, J = 7.7, 14.0, 1H), 2.12 (dd, J = 7.3, 14.0,
1H) 1.52–1.46 (m, 2H), 1.29–1.26 (m, 14H), 0.98 (s, 3H), 0.88 (t,
J = 7.1, 3H). 13C NMR (CDCl3, 125 MHz) d 173.4, 171.3, 134.0,
118.4, 77.2, 68.6, 42.1, 39.7, 38.4, 35.7, 35.4, 31.8, 29.50, 29.48,
29.3, 29.2, 26.9, 22.6, 18.9, 14.1. HRMS for C20H38N2O4 [M+Na]+
calcd 393.2724, found 393.2724. Purity, >95% (tR = 21.41 min).
C
23H28N2O3 [M+Na]+ calcd 403.1992, found 403.1994.
4.5.47. (9H-Fluoren-9-yl)methyl 3-(nonylamino)-3-
oxopropylcarbamate (11j)
Compound 11j was synthesized in 84% yield from 10 and nonyl-
amine in a manner similar to 11a. 1H NMR (CDCl3, 500 MHz) d 7.76
(d, J = 7.5, 2H), 7.59 (d, J = 7.5, 2H), 7.40 (d, J = 7.4, 2H), 7.32 (d,
J = 7.4, 2H), 5.49 (br s, 2H), 4.37 (d, J = 7.2, 2H), 4.20 (t, J = 6.8,
1H), 3.51–3.48 (m, 2H), 3.27–3.23 (m, 2H), 2.41 (t, J = 5.7, 2H),
1.50–1.47 (m, 2H), 1.29–1.26 (m, 12H), 0.87 (t, J = 7.1, 3H). 13C
NMR (CDCl3, 125 MHz) d 171.1, 156.6, 143.9, 143.9, 141.3, 141.3,
127.7, 127.7, 127.0, 127.0, 125.1, 125.1, 119.9, 119.9, 66.7, 47.2,
39.6, 37.1, 36.0, 31.8, 29.6, 29.5, 29.3, 29.2, 26.9, 22.7, 14.1. HRMS
for C27H36N2O3 [M+Na]+ calcd 459.2618, found 459.2624.
4.5.45. (2R,3R)-4-Cyclopropyl-2-hydroxy-3-(hydroxymethyl)-3-
methyl-N-(3-oxo-3-(pentylamino)propyl)butanamide (9m)
To a solution of compound 13 (84 mg, 0.155 mmol) in CH2Cl2
(10 mL) at 0 °C was added drop wise a solution of Et2Zn in hexanes
(1.55 mL, 1.55 mmol, 1 M solution in hexanes) under nitrogen. The
mixture was stirred for 15 min followed by the addition of CH2I2
(0.25 mL, 3.09 mmol). The reaction mixture was brought to room
temperature (the formation of a white precipitate was observed)
and stirred for 24 h. Saturated aqueous NH4Cl (20 mL) was added,
the mixture extracted with ethyl acetate (3 Â 15 mL) and the com-
bined organic layer was dried (Na2SO4) and evaporated to afford a
light yellow oil that was used directly in the next reaction. To the
oil dissolved in THF (5 mL) at room temperature was added a solu-
tion of TBAF in THF (0.62 mL, 0.62 mmol, 1 M in THF). The reaction
mixture was stirred overnight, diluted with saturated aqueous
NaHCO3 (5 mL) and the mixture was extracted with ethyl acetate
(3 Â 10 mL). The combined organic layer was dried over Na2SO4
and evaporated to yield an oil that was purified by flash chroma-
tography on silica gel (gradient elution, 9:1 EtOAC/hexanes v/
v ? 19:1 EtOAc/methanol, v/v) to give the desired product
(25.8 mg, 51%) over the 2 steps. Rf 0.36 (EtOAc/MeOH, 9:1, v/v).
1H NMR (CDCl3, 500 MHz) d 7.45 (br s, 1H), 5.99 (br s, 1H), 4.07
(s, 1H), 3.68 (d, J = 11.3, 1H), 3.65–3.60 (m, 1H), 3.61 (d, J = 11.3,
1H), 3.56–3.50 (m, 1H), 3.26–3.21 (m, 2H), 2.47–2.44 (m, 2H),
Acknowledgments
The authors are grateful to G.D. Wright at McMaster University
for sharing his PanK, PPAT and DPCK expression plasmids. This
work was supported by the Canadian Institute of Health Research
(CIHR) and the National Science and Engineering Research Council
of Canada (NSERC). KV was supported through CIHR Chemical Biol-
ogy Training and Fonds québécois de la recherche sur la nature et
les technologies (FQRNT) scholarships.
Supplementary data
Supplementary data (NMR spectra and HPLC traces for key
intermediates and final targets) associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2011.02.053.
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