Total synthesis and biological evaluation of transvalencin Z

Article abstract of DOI:10.1021/np200972s

The emerging global epidemic of drug-resistant tuberculosis has created an urgent need to identify novel therapeutic approaches for disease treatment. Transvalencin Z (1) is a natural product from Nocardia transvalensis with relatively potent and selective antimycobacterial activity against Mycobacterium smegmatis, making it an attractive target for structure-activity and mechanism of action studies. The total synthesis of the four possible diastereomers of transvalencin Z was completed (1a-d), and the absolute configurations were defined using chemical synthesis, HPLC retention times, and optical rotation measurements. Surprisingly, none of the transvalencin Z diastereomers exhibited any inhibitory activity against a panel of microbial pathogens, including several species of mycobacteria.

Full text of DOI:10.1021/np200972s

Article
pubs.acs.org/jnp
Total Synthesis and Biological Evaluation of Transvalencin Z
Kathryn M. Nelson,†,‡ Christine E. Salomon,† and Courtney C. Aldrich*,†
†Center for Drug Design, University of Minnesota, Minneapolis, Minnesota 55455, United States
‡Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: The emerging global epidemic of drug-resistant
tuberculosis has created an urgent need to identify novel
therapeutic approaches for disease treatment. Transvalencin Z
(1) is a natural product from Nocardia transvalensis with
relatively potent and selective antimycobacterial activity
against Mycobacterium smegmatis, making it an attractive target
for structure−activity and mechanism of action studies. The
total synthesis of the four possible diastereomers of trans-
valencin Z was completed (1a−d), and the absolute
configurations were defined using chemical synthesis, HPLC retention times, and optical rotation measurements. Surprisingly,
none of the transvalencin Z diastereomers exhibited any inhibitory activity against a panel of microbial pathogens, including
several species of mycobacteria.
uberculosis (TB), a contagious pulmonary infection from
the slow growing bacillus Mycobacterium tuberculosis
mammalian cell lines.6 Compound 1 also shows striking
structural similarity to the mycobactin siderophores produced
by Mtb (Figure 1).
T
(Mtb), is the leading cause of mortality globally due to
bacterial infectious disease.1 One-third of the world’s
population is estimated to be infected with latent Mtb, an
asymptomatic infection that may be triggered to an active
infection through stress on the immune system. A predicted 5−
10% of these individuals will develop the active disease in their
lifetime.2 The current therapy recommended by the World
Health Organization (WHO), termed DOTS (directly
observed treatment short-course), involves a minimum of 6−
9 months on a combination drug therapy of isoniazid, rifampin,
ethambutol, and pyrazinamide, resulting in a global average
cure rate of 85%.2 This therapy employs antitubercular agents
that were developed over 4 decades ago and has a limited ability
to treat latent Mtb. Co-infection with HIV is especially
concerning, as it can trigger latent TB into active infection.
The emergence of multidrug-resistant TB (MDR-TB) and
extensively drug resistant TB (XDR-TB) has continued to limit
the number of patients cured during DOTS treatment.2,3
Recently, new totally drug resistant TB (TDR-TB) strains with
resistance to all second-line therapeutics have been isolated in
small patient populations in both Iran and India.4,5 New, more
effective antitubercular agents that operate via novel mecha-
nisms of action are highly desirable to increase the TB cure rate,
decrease the duration of treatment, target latent infection, and
increase activity against drug-resistant strains.
Figure 1. (A) Reported structure of transvalencin Z (1).6 (B)
Structure of mycobactin-T.7
Siderophores are small molecule iron (Fe3+) chelators
produced by many bacterial species.8 Bacterial pathogens
synthesize, secrete, and reimport siderophores to scavenge
essential iron in limiting environments such as a human host.
The biosynthetic pathway for mycobactin synthesis in Mtb is
essential for growth and virulence in iron-limiting conditions,
Transvalencin Z (1) was isolated by Mukai and co-workers
from a clinical strain of Nocardia transvalensis (IFM 10065) and
was found to exhibit potent and specific inhibition of
Mycobacterium smegmatis (MIC, minimum inhibitory concen-
tration that results in >99% reduction of observable growth, of
0.125 μg/mL or 0.344 μM) and other acid-fast Nocardia spp.,
with no activity against Gram-negative bacilli, fungi, or
Received: December 15, 2011
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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making it an attractive drug target.9 The structural similarity of
1 to mycobactins suggests that it may interfere with the
mycobactin-mediated iron-scavenging process based on the
hypothesis proposed by Snow that mycobactin analogues may
antagonize mycobacterial growth.10,11 Indeed, Miller and co-
workers have shown that mycobactin S, produced by M.
smegmatis, which differs by only one stereocenter from
mycobactin T, inhibits Mtb growth, while a mycobactin
analogue wherein the beta-hydroxybutyrate was replaced with
a 2,3-diaminopropionate moiety exhibited exceptionally potent
antimycobacterial activity.12,13 More recently, Miller and co-
workers demonstrated that small organic molecules derived
from the phenyloxazoline headgroup of the mycobactins display
antimycobacterial activity, although the activity is likely
unrelated to antagonism of iron acquisition.12 Quadri and
colleagues discovered a series of small molecules, which
incorporate a 2-hydroxyphenyl moiety and thus bear some
resemblance to the mycobactins, that elicit iron-dependent
antimycobacterial activity.13,14
Scheme 1. Retrosynthetic Plan for 1a−d
Scheme 2. Synthesis of Building Block 2
Herein we report our complete synthesis and attempt to
define the stereochemistry of 1, as well as our investigation into
its antimycobacterial activity. We began by designing a total
synthesis of the four possible diastereomers (1a−d) since the
relative and absolute configurations of the chiral centers at C-2
and C-9 were not reported (Figure 2). All transvalencin Z
Figure 2. Diastereomers of transvalencin Z.
penultimate peptide coupling reaction, a phenomenon that
has been documented in multiple siderophore syntheses.13,17,19
Formylation of Nα-Fmoc-L- or -D-lysine 7 was performed
using the mixed anhydride method reported by Hughes and
Waters to give acid 8 (Scheme 3).20,21 Elaboration of 8 to the
fully protected lysine conjugate 9 was possible using a number
of common conditions for benzyl ester formation; however,
conditions involving direct alkylation (DIPEA, BnBr) were
ultimately chosen to prevent possible racemization.20,22 Fmoc
diastereomers were tested against a panel of microbial
pathogens to compare the spectrum of activity of the synthetic
products with that reported for the natural product.
RESULTS AND DISCUSSION
■
Chemistry. Retrosynthetically, we envisioned the diaster-
eomers of transvalencin Z could be accessed through two major
building blocks: a 2-hydroxyphenyl oxazoline acid 2 and a ε-
formyl lysine 3, as shown in Scheme 1. The acid 2 is a common
intermediate in previously published syntheses of mycobactins
and their analogues.12−14
Scheme 3. Synthesis of Building Block 3
The synthesis of building block 2 began with the benzylation
of salicylic acid 4,15 followed by peptide coupling with L- or D-
serine benzyl esters as previously described by Miller and co-
workers to yield the serine adducts 5 in 90% average yield
(Scheme 2).12 Oxazoline cyclization utilizing the recently
reported molybdenum catalyst, ammonium molybdenum
tetrahydrate, afforded the benzyl protected 6.16 The molybde-
num catalyst coordinates the amide carbonyl, thereby electro-
philically activating it for intramolecular attack by the serine
hydroxyl nucleophile. This biomimetic dehydrative cyclization
contrasts the previous methods to effect this cyclization
through electrophilic activation of the serine hydroxyl group
and nucleophilic displacement by the amide carbonyl.17,18
Global deprotection of 6 by hydrogenolysis provided acids 2.
Deprotection of the phenol ether was required for the
B
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deprotection of 9 using excess piperidine afforded the free
amine 3.
phase HPLC. We also observed nearly equal and opposite
optical rotations for each set of enantiomers, giving further
support to their purity. As shown in Table 1, most of the H
1
Peptide coupling of acid 2a and lysine 3a with 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC) resulted in epime-
rization at position 9 to afford 11a and 11c in a 4.2:1 ratio, as
determined by HPLC. Therefore, the penultimate step was
performed using 3-(diethoxyphosphoryloxy)-1,2,3-benzotria-
zin-4(3H)-one (DEPBT), 10, a reagent known for minimizing
racemization at sensitive stereocenters (Scheme 4).23 Hydro-
genolysis of 11a provided free acid 1a. The remaining
diastereomeric products 1b−d were prepared in an analogous
fashion to those described for 1a.
NMR chemical shifts for 1a−d match those reported for
transvalencin Z. The C-2, 2-NH, and 6-NH protons show
significant differences in chemical shift for 1a−d compared to
the natural product. While the shift at the C-2 chiral center is
significant and concerning, amide protons can be highly
variable due to pH, sample concentration, and temperature of
the solution, so the 2-NH and 6-NH shifts may not be
diagnostic.24 Additional NMR analysis utilizing homonuclear
2D J resolution spectroscopy on a 700 MHz instrument
clarified the coupling constants for the C-2 proton; however the
C-3 diastereotopic protons were still too complicated to resolve
clearly. The optical rotation values for 1a−d also do not match
the reported value (+15.3°) for transvalencin Z;6 the sets of
enantiomers 1a,b (+29.2°/−31.6°) and 1c,d (−2.0°/+6.0°) are
nearly equidistant from this value.
Scheme 4. Peptide Coupling to Complete Synthesis of 1a−d
Analysis of the literature describing natural products
produced by Nocardia sp. and other structurally related natural
products (e.g., siderophores such as mycobactins from M.
tuberculosis, acinetobactin from Acinetobacter baumannii, and
structurally related amistatins, brasilibactin, formobactin,
nocardichelins, and nocobactin NA from Nocardia sp.) does
not show any trend or preference for stereochemistry. Several
studies of salicyl and oxazoline/thiazoline motifs show this
stereocenter (C-9 in transvalencin Z) to be S, resulting from
naturally occurring L-serine.17,18,25,26 However, other natural
products, such as brasilibactin from Nocardia brasiliensis, show
an R stereocenter, suggesting the incorporation of an epimerase
in the biosynthetic machinery or the use of the less common D-
serine.27 Indeed similar natural products such as the
amamistatins, formobactin, and nocobactin NA contain the
fully saturated oxazole instead of the oxazoline ring system.28
Biosynthetic feeding studies of natural products containing
lysine (C-2 in transvalencin Z) motifs have demonstrated an S
configuration by incorporation of predominantly L-lysine.26
However, the only other transvalencin isolated to date is
transvalencin A, whose absolute stereochemistry has not yet
been identified.29 These conflicting literature data, along with
our lack of correlation between activity and spectral data with
the original report, make the stereochemical assignment of
transvalencin Z impossible without the authentic isolated
sample.
In attempting to further confirm the properties of trans-
valencin Z, 1a and its diastereomers were screened against a
panel of representative organisms (Table 2). We were
particularly interested in expanding the number of mycobac-
teria strains tested to give a more complete picture of the
compound’s selectivity displayed in the original screen.6 In
contrast to the potent M. smegmatis activity reported for
transvalencin Z, none of the four synthetic diastereomers
inhibited the growth of several species of mycobacteria,
including M. smegmatis MC24517 and ATCC607, M. bovis
BCG, and M. tuberculosis H37Rv. These results suggest that the
original natural product may have been contaminated with a
small amount of a highly active compound. Alternatively, the
slight discrepancies in the spectroscopic data between the four
synthetic diastereomers compared to the natural product
suggest that the reported structure of transvalencin Z may be
incorrect. While the synthesis of 1a−d has unequivocally
produced the above structures, small differences in bond
connections may produce this misalignment data, and chemical
Microbiology. Biological evaluation of 1a−d was initially
performed against M. smegmatis MC24517. The minimum
inhibitory concentration (MIC99) was greater than 100 μM for
all four diastereomers (Table 2, complete data Table S1). We
then tested the diastereomers against the identical M. smegmatis
strain (ATCC 607) employed by Mukai and co-workers, but
again observed no activity. The four compounds were also
tested against a panel of 10 additional pathogens (Enterococcus
faecalis, Staphylococcus aureus, Acinetobacter baumannii, Escher-
ichia coli, Klebsiella pneumoniae, Bacillus subtilis, Pseudomonas
aeruginosa, Candida albicans, Mycobacterium bovis BCG, and
Mycobacterium tuberculosis), and 1a−d were inactive (MIC99
≥
100 μM, ≥ 32 μg/mL) against all strains evaluated. Five of
these pathogens from our screen (M. smegmatis, S. aureus, B.
subtilis, E. coli, and C. albicans) were also screened by Mukai
and co-workers,6 who reported MIC values of 0.125, 1, 16, >64,
and >164 μg/mL respectively, which is inconsistent with our
data (Table 2).
Discussion. Transvalencins 1a−d were isolated as amor-
1
phous, white solids. Spectral data including H and 13C NMR,
UV−vis, and IR absorbance were similar to those reported by
Mukai and co-workers.6 Absolute configuration can be assigned
on the basis of our chiral starting materials and knowledge of
the chemistry used to synthesize 1a−d. The methods were
chosen to reduce racemization of stereocenters in all steps. As
discussed above, diastereomers of compounds 11a−d and 1a−
d could be identified by distinct retention times on reversed-
C
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6
1
Table 1. H NMR Data for Transvalencin Z and Compounds 1a−d in d6-DMSO at 600 MHz
δH and δC (ppm), mult. (J in Hz)
transvalencin Z6
δH
1a/b
1c/d
position
δC
δC
δH
δC
δH
11.81, br s
1
173.1
53.8
11.76, br s
3.85, q-like (6.0)
7.75, d (6.0)
1.54, m
173.3
52.1
11.80, br s
173.8
51.9
a
a
2
4.18, dt (10.5, 5.6)
4.17, dt (11.9, 4.9)
7.85, ovlp m
1.65, m
2-NH
3
7.87, ovlp m
1.61, m
31.7
30.4
30.8
3′
1.68, m
1.71, m
1.74, m
4
22.5
28.8
37.1
1.2, m
22.8
28.5
36.8
1.26, m
22.7
28.4
36.7
1.31, m
5
1.32, m
1.34, m
1.40, m
6
2.98, q-like (6.0)
7.94, br t (6.0)
7.92, s
3.05 m
3.05, t (6.0)
8.51, d (7.2)
7.98, s
6-NH
7
8.50, d (6.6)
7.91, s
160.7
168.6
67.4
160.9
169.7
67.1
160.8
169.5
67.0
8
a
a
9
4.99, dd (8.0, 10.0)
4.48, t (8.0)
4.98, dd (7.7, 10.5)
5.01, dd (7.7, 9.8)
10
10′
11
12
13
14
15
16
17
69.7
69.2
4.48, t (7.8)
4.60, t (7.8)
69.2
4.50, t (7.8)
4.63, t (8.4)
4.63, dd (8.0, 10.0)
165.9
159.1
109.8
116.5
119.1
134.0
128.0
165.8
159.1
109.8
116.6
119.1
134.0
128.0
165.7
158.9
109.8
116.4
118.9
133.9
127.9
7.0, br d (8.0)
6.95, d (8.4)
6.89, t (7.8)
7.41, t (7.8)
7.59, d (7.8)
7.0, d (8.4)
6.95, t (7.8)
7.46, t (7.2)
7.64, d (7.8)
6.94, br t (7.5)
7.46, ddd (1.5, 7.5, 8.0)
7.63, dd (1.5, 7.5)
a
Determined by homonuclear 2D J spectroscopy on a Bruker 700 MHz with a 5 mm TXI 700 MHz Z-gradient probe.
derivatization of the freshly isolated natural product may aide in
elucidating the true structure.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All commercial reagents
were used as provided unless otherwise indicated. An anhydrous
solvent dispensing system (J. C. Meyer) using two packed columns of
neutral alumina was used for drying THF and CH2Cl2, while two
packed columns of molecular sieves were used to dry DMF, and the
solvents were dispensed under argon. Anhydrous grade MeOH and
toluene were purchased from Aldrich. Flash chromatography was
performed using Combiflash Companion equipped with flash column
silica cartridges with the indicated solvent system. All reactions were
performed under an inert atmosphere of dry Ar or N2 in oven-dried
Table 2. MIC99 and MIC50 Activity Data for Reported
Transvalencin Z (1) and Synthetic 1a−d
reported (1)6
(μg/mL)
1a−d (μg/
strain
mL)
M. smegmatis (ATCC 607)
M. smegmatis (MC24517)
0.125
>32
>32
>32
>32
1
(150 °C) glassware. H and 13C NMR spectra were recorded on a
a
n.d.
Varian 600 MHz spectrometer or a Bruker 700 MHz with a 5 mm TXI
700 MHz Z-Gradient probe. Proton chemical shifts are reported in
ppm from an internal standard of residual chloroform (7.26),
methanol (3.31), dichloromethane (5.32), or dimethyl sulfoxide
(2.50), and carbon chemical shifts are reported using an internal
standard of residual chloroform (77.23), methanol (49.15), dichloro-
methane (54.00), or dimethyl sulfoxide (39.51). Proton chemical data
are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad, ovlp =
overlapping), coupling constant, integration. High-resolution mass
spectra were obtained on an Agilent TOF II TOF/MS instrument
equipped with either an ESI or APCI interface. Optical rotations were
measured on a Rudolph Autopol III polarimeter. Melting points were
measured on an electrothermal Mel-Temp manual melting apparatus
and are uncorrected. IR spectra were obtained on a Jasco FT/IR-4100.
Analytical HPLC was performed on a Phenomenex Gemini 5u C18
b
M. tuberculosis (H37Rv)
n.d.
n.d.
M. bovis BCG strain TMC 1011 Pasteur
(ATCC 35734)
E. coli (ATCC 25922)
>64
n.d.
n.d.
n.d.
16
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>64
A. baumannii (ATCC 19606)
K. pneumoniae (ATCC 13883)
P. aeruginosa (ATCC 27853)
B. subtilis (ATCC 6633)
E. faecalis (ATCC 51299)
n.d.
n.d.
1
S. aureus (MSSA, clinical IDRL 8545)
S. aureus (MRSA, ATCC 43300)
S. aureus (MRSA, clinical IDRL 6169)
C. albicans (ATCC 10231)
Vero (ATCC CCL-81)
n.d.
>64
n.d.
a
b
n.d.: not determined. Testing performed by Dr. Helena I. Boshoff.
D
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110A (250 × 4.60 mm) column operating at 0.9 mL/min with
detection at 254 nm.
dried (MgSO4), and concentrated to an off-white, amorphous solid.
Purification by flash chromatography (linear gradient 0−50% EtOAc−
hexanes) on silica gel afforded the title compound (1.00 g, 55%) as an
off-white solid: mp 68−71 °C; [α]2D3 +79.1 (c 1.0, CHCl3); Rf 0.33
Microbiology. M. smegmatis (MC24715) was a generous gift of
Dr. William R. Jacobs, Jr. of the Howard Hughes Medical Institute and
Department of Microbiology and Immunology, Albert Einstein
College of Medicine, Bronx, NY. S. aureus (MRSA) clinical IDRL
6169 and S. aureus (MSSA) IDRL 8545 were the kind gifts of Dr.
Robin Patel, Department of Laboratory Medicine and Pathology,
Mayo Clinic College of Medicine, Rochester, MN; other pathogens
were obtained from the ATCC (A. baumannii ATCC 19606, C.
albicans ATCC 10231, E. faecalis (VRE) ATCC 51299, E. coli ATCC
25922, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, S.
aureus (MRSA) ATCC 43300, M. smegmatis ATCC 607, M. bovis BCG
strain TMC1011 Pasteur ATCC 35734) and cultured as follows. All
bacterial strains were streaked from frozen glycerol stocks onto solid
agar plates before being subcultured in liquid medium for assay. Agar
plates consisted of the following media plus agar (Difco) to a 1.5%
concentration: E. coli, P. aeruginosa, and all S. aureus, trypticase soy; A.
baumannii, K. pneumoniae, M. bovis BCG, and all M. smegmatis nutrient
broth; E. faecalis, brain heart infusion; and C. albicans, YM broth. All
bacterial pathogens were grown at 37 °C, while C. albicans was grown
at 30 °C. After 24 h incubation, single colonies were picked and grown
in the respective broth above to an OD600 of 1.0, diluted to an OD600
of 0.003, and plated in 96-well sterile plates with 1% DMSO stock
solutions of varying concentrations of 1a−d. Plates were read at 24
and 48 h at 600 nm using a plate reader (Molecular Devices
Spectramax M5e); plate background (690 nm) was subtracted and
values were standardized to the DMSO controls. In initial micro-
biological testing against M. smegmatis, it was observed that the
bacteria would often clump together during growth, causing large
variations in optical density readings. Addition of the supplemental
detergent Tyloxapol prevented clumping of the mycobacterium
without the potentially toxic side effects observed when using
Tween detergents with mycobacteria.30 Mukai and co-workers did
not report the use of any detergents for M. smegmatis growth.
Cytotoxicity. Cytotoxicity of each compound was determined with
a standard tetrazolium assay using Vero green monkey kidney cells
(ATCC CCL-81).31,32 All tissue culture reagents were purchased from
Gibco, Invitrogen (Carlsbad, CA, USA). Cells were grown in MEM
media supplemented with 10% fetal bovine serum (FBS), 1% Penn-
Strep, and 1% Glutamax. Cells were seeded at 3.0 × 104 cells per well
in a 96-well microtiter plate (Corning) and allowed to adhere
overnight. Medium was carefully aspirated and replaced with 195 μL of
fresh medium, and compound solutions in DMSO (5 μL) were added
to give a final concentration ranging from 100 to 0.16 μM. All
concentrations were tested in triplicate. Plates were incubated for 72 h
at 37 °C and 4.5% CO2 in a humidified chamber. The solutions were
carefully removed, and RPMI without phenol red containing 1.0 mg/
mL 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide
(MTT) was added (200 μL). The mixture was incubated for 3 h,
after which the MTT medium was carefully removed. Isopropyl
alcohol (200 μL) was added to dissolve the precipitated purple
formazan crystals, and the plates were read at 570 nm using a plate
reader (Molecular Devices Spectramax M5e); plate background (690
nm) was subtracted and cell viability was estimated as the percentage
absorbance relative to the DMSO control. Dose−response curves were
generated using GraphPad Prism 5 software and used to determine the
MIC50 concentrations (minimal concentration that inhibits 50% of
growth).
1
(30% EtOAc−hexanes); H NMR, 13C NMR, and HRMS identical to
reported values.12
(4R)-Benzyl-2-[2-(benzyloxy)phenyl]-Δ2-1,3-oxazoline-4-carboxy-
late (6b). Reaction conditions were identical to those for 6a, except for
the use of N-[2-(benzyloxy)benzoyl]-D-serine benzyl ester (5b): [α]2D3
−81.7 (c 1.0, CHCl3).
(4S)-2-[2-Hydroxyphenyl]-Δ2-1,3-oxazoline-4-carboxylic Acid
(2a). (4S)-Benzyl-2-[2-(benzyloxy)phenyl]-Δ2-1,3-oxazoline-4-carbox-
ylate (6a) (0.385 g, 1.00 mmol) was dissolved in anhydrous methanol
(10 mL) and added to a Parr flask containing 10% by weight Pd/C
(0.039 g) under Ar. The reaction vessel was evacuated, then backfilled
with hydrogen gas to 3 atm, and the mixture was shaken at 25 °C for 1
h. The reaction vessel was opened, and the reaction mixture was
filtered through Celite. The filtrate was concentrated to a dark red-
orange oil (190 mg, 91%) and used directly in the next step without
further purification. However, for analytical characterization, purifica-
tion by flash chromatography (isocratic 50% EtOAc−hexanes with 1%
formic acid) afforded the title compound as a clear, colorless oil: [α]D23
+39.2 (c 1.0, MeOH); Rf 0.22 (1:1 hexanes−EtOAc with 1% formic
1
acid); H NMR (CD3OD, 600 MHz) δ 4.54−4.59 (m, 2H), 4.92
(apparent br t, J value not discernible due to broadening, 1H), 6.81 (t,
J = 7.8 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.57
(d, J = 7.8 Hz, 1H); 13C NMR (CD3OD, 150 MHz) δ 68.4, 70.7,
111.3, 117.7, 120.1, 129.5, 135.2, 161.1, 168.5, 174.1; HRMS (ESI−)
calculated for C10H8NO4 [M − H]− 206.0459, found 206.0435 (error
11.6 ppm) and C9H8NO2 [M − CO2]− 162.0561, found 162.0552
(error 5.6 ppm).
(4R)-2-[2-Hydroxyphenyl]-Δ2-1,3-oxazoline-4-carboxylic Acid
(2b). Reaction conditions were identical to those for 2a, except for
the use of (4R)-benzyl-2-[2-(benzyloxy)phenyl]-Δ2-1,3-oxazoline-4-
carboxylate (6b): [α]2D3 −27.5 (c 1.0, MeOH).
(2S)-({[(9H-Fluoren-9-yl)methyloxy]carbonyl}amino)-6-formami-
dohexanoic Acid (8a). Formic acid (1.5 mL, 39 mmol) was added
dropwise to acetic anhydride (4.5 mL, 48 mmol) at 0 °C followed by
heating to 60 °C for 2 h. The mixture was cooled to 0 °C, and a slurry
of 2-N-fluorenylmethyloxycarbonyl-L-lysine (7a) (2.5 g, 6.79 mmol) in
CH2Cl2 (25 mL) was added. The reaction was warmed to 25 °C and
stirred for 3 h. The reaction mixture was partitioned between EtOAc
(75 mL) and H2O (75 mL), and the organic layer was washed
successively with H2O (75 mL) and saturated aqueous NaCl (75 mL),
dried (MgSO4), and concentrated. Purification by flash chromatog-
raphy (linear gradient 0−7% MeOH−CH2Cl2 with 1% formic acid)
1
afforded the title compound (1.61 g, 60%) as a foamy, yellow oil: H
NMR, 13C NMR, and HRMS identical to reported values.20
(2R)-({[(9H-Fluoren-9-yl)methyloxy]carbonyl}amino)-6-formami-
dohexanoic Acid (8b). Reaction conditions were identical to those for
8a, except for the use of 2-N-fluorenylmethyloxycarbonyl-D-lysine
20
1
(7b): H NMR, 13C NMR, and HRMS identical to reported values.
(2S)-Benzyl-2-({[(9H-fluoren-9-yl)methyloxy]carbonyl}amino)-6-
formamidohexanoate (9a). To a stirring slurry of 2-(N-fluorenylme-
thyloxycarbonyl)-6-N-formyl-L-lysine (8a) (1.00 g, 2.52 mmol, 1.0
equiv) in CH2Cl2 (25 mL) was added diisopropylethylamine (0.66
mL, 3.78 mmol, 1.5 equiv) followed by benzyl bromide (0.45 mL, 3.78
mmol, 1.5 equiv) to afford a homogeneous clear solution. The reaction
was stirred for 20 h at 25 °C, then partitioned between EtOAc (100
mL) and H2O (100 mL), and the organic layer was washed
successively with saturated aqueous NaHCO3 (100 mL) and saturated
aqueous NaCl (100 mL), dried (MgSO4), and concentrated.
Purification by flash chromatography (linear gradient 10−100%
EtOAc−hexanes) on silica gel provided the title compound (740
mg, 60%) as a yellow oil: [α]2D3 −6.2 (c 1.0, CHCl3); Rf 0.38 (1:1
CH2Cl2−EtOAc with 1% formic acid); 1H NMR (CD2Cl2, 600 MHz)
δ 1.17−1.30 (m, 2H), 1.32−1.42 (m, 2H), 1.57−1.63 (m, 1H), 1.71−
1.78 (m, 1H), 3.05−3.12 (m, 2H), 4.12 (t, J = 6.6 Hz, 1H), 4.22−4.32
(m, 3H), 5.03 (d, J = 12.6 Hz, 1H), 5.08 (d, J = 12.6 Hz, 1H), 7.19−
7.27 (m, 7H), 7.30 (t, J = 7.8 Hz, 2H), 7.52 (t, J = 6.6 Hz, 2H), 7.67
Chemistry. Compounds 5a and 5b were prepared according to
literature procedures.12
(4S)-Benzyl-2-[2-(benzyloxy)phenyl]-Δ2-1,3-oxazoline-4-carboxy-
late (6a). To a stirring solution of N-[2-(benzyloxy)benzoyl]-L-serine
benzyl ester (5a) (2.0 g, 4.9 mmol, 1.0 equiv) in toluene (50 mL) was
a d d e d a m m o n i u m m o l y b d a t e ( V I ) t e t r a h y d r a t e
((NH4)6MoO24·4H2O) (2.4 g, 1.9 mmol, 0.39 equiv), and the
solution was refluxed for 18 h using a Dean−Stark trap to remove the
generated water. The reaction was concentrated to a dark, green oil
and partitioned between EtOAc and 5% aqueous NaHCO3. The
organic layer was washed with saturated aqueous NaCl (100 mL),
E
dx.doi.org/10.1021/np200972s | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(d, J = 7.2 Hz, 2H), 7.95 (s, 1H); 13C NMR (CD2Cl2, 150 MHz) δ
22.9, 29.4, 32.4, 37.8, 47.8, 54.4, 67.3, 67.5, 120.5, 125.6, 127.6, 128.2,
128.6, 128.8, 129.1, 136.2, 141.8, 144.4, 156.5, 161.6, 172.8; HRMS
(APCI+) calcd for C29H31N2O5+ [M + H]+ 487.2227, found 487.2228
(error 0.2 ppm).
(2R)-Benzyl-2-({[(9H-fluoren-9-yl)methyloxy]carbonyl}amino)-6-
formamidohexanoate (9b). Reaction conditions were identical to
those for 9a, except for the use of 2-(N-fluorenylmethyloxycarbonyl)-
6-N-formyl-D-lysine (8b): [α]D23 +7.9 (c 1.0, CHCl3).
Hz, 2H), 4.85 (dd, J = 9.0, 5.4 Hz, 1H), 4.56−4.64 (m, 2H), 5.01 (dd,
J = 10.6, 7.8 Hz, 1H), 5.09 (d, J = 12 Hz, H), 5.17 (d, J = 12 Hz, H),
6.91 (t, J = 7.2 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H), 7.27−7.32 (m, 5H),
7.42 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 8.01 (s, 1H); 13C
NMR (CD3OD, 150 MHz) δ 24.2, 29.9, 32.0, 38.7, 54.1, 68.1, 69.3,
70.5, 111.7, 117.8, 120.1, 129.3, 129.4, 129.6, 129.7, 133.6, 135.1,
137.2, 161.0, 163.9, 168.6, 173.1, 173.2; HRMS (ESI+) calcd for
C24H28N3O6 [M + H]+ 454.1973, found 454.2004 (error 6.8 ppm).
(2R,9S)-Benzyl-6-formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazo-
line-4-carboxamido]hexanoate (11d). Reaction conditions were the
same as those for 11a except for the use of 3b: [α]2D3 +10.8 (c 1.0,
MeOH).
(2S)-Benzyl-2-amino-6-formamidohexanoate (3a). To a stirring
solution of (2S)-benzyl-2-[([(9H-fluoren-9-yl)methyloxy]carbonyl)-
amino]-6-formamidohexanoate (9a) (0.600 g, 1.23 mmol, 1.0 equiv)
in DMF (12 mL) was added piperidine (0.61 mL, 6.17 mmol, 5.0
equiv), and the mixture stirred for 1 h at 25 °C. The reaction was
concentrated by rotary evaporation under high vacuum to remove
DMF, and the crude residue was partitioned between H2O (40 mL)
and hexane (40 mL). The hexane layer was discarded, and the aqueous
layer was basified to a pH 10 with saturated aqueous NaHCO3, then
extracted with 6:1 EtOAc−MeOH (3 × 40 mL). The combined
organic extracts were dried (MgSO4) and concentrated. Purification by
flash chromatography (linear gradient 0−10% MeOH−EtOAc) over
basic alumina yielded the title compound (140 mg, 50%) as a yellow
oil: [α]2D3 +3.9 (c 1.0, CH3OH); Rf 0.13 (10% MeOH−EtOAc with 1%
(2S,9S)-6-Formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazoline-4-
carboxamido]hexanoic Acid (1a). Solid Pd/C (10% by weight, 1.7
mg) was added to a solution of (2S,9S)-benzyl-6-formamido-2-([2-
hydroxyphenyl]-Δ2-1,3-oxazoline-4-carboxamido)hexanoate (11a)
(16.6 mg, 0.037 mmol) in MeOH (10 mL) under Ar. The reaction
vessel was evacuated then backfilled with hydrogen gas to 3 atm, and
the mixture was shaken at 25 °C for 2 h. The reaction vessel was
opened, the reaction mixture was filtered through Celite, and the
filtrate was concentrated. Purification by flash chromatography (linear
gradient 0−10% EtOH−CH2Cl2 plus 1% formic acid) afforded the
title compound (13 mg, 100%) as an amorphous, colorless solid.
Purity was determined by analytical HPLC using a Phenomenex
Gemini 5u C18 110A (250 × 4.6 mm) column operating at 0.9 mL/
min and a gradient of 10−100% MeOH−0.05% aqueous formic acid
over 20 min followed by 100% MeOH for 5 min. The retention time
of the product was 19.4 min, purity >96%: [α]2D3 +29.2 (c 0.1, MeOH);
Rf 0.59 (10% MeOH−CH2Cl2 with 1% formic acid); 1H NMR
(DMSO-d6, 600 MHz) δ 1.26−1.35 (m, 2H), 1.35−1.44 (m, 2H),
1.64−1.67 (m, 1H), 1.75−1.79 (m, 1H), 3.00−3.10 (m, 2H), 4.16−
4.20 (m, 1H), 4.48 (t, J = 7.8 Hz, 1H), 4.60 (t, J = 7.8, 1H), 4.98 (t, J =
7.8 Hz, 1H), 6.89 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 7.41 (t,
J = 8.4 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.83−7.90 (ovlp m, 1H),
7.91 (ovlp s, 1H), 8.50 (d, J = 6.6 Hz, 1H), 11.80 (br s, 1H); 13C
NMR (DMSO-d6, 150 MHz) δ 22.8, 28.5, 30.4, 36.8, 52.1, 67.1, 69.2,
109.8, 116.6, 119.1, 128.0, 134.0, 159.1, 160.9, 165.8, 169.7, 173.3;
HRMS (ESI−) calcd for C17H20N3O6 [M − H]− 362.1358, found
362.1331 (7.5 ppm error).
1
Et3N); H NMR (CD3OD, 600 MHz) δ 1.31−1.40 (m 2H), 1.46−
1.51 (m, 2H), 1.65−1.67 (m, 1H), 1.72−1.77 (m, 1H), 3.17 (t, J = 7.2
Hz, 2H), 3.52 (t, J = 6.6 Hz, 1H), 5.15 (d, J = 12.6 Hz, 1H), 5.21 (d, J
= 12.6 Hz, 1H), 7.32−7.39 (m, 5H), 8.00 (s, 1H); 13C NMR
(CD3OD, 150 MHz) δ 23.8, 30.2, 35.0, 38.8, 55.1, 67.9, 129.5, 129.6,
129.7, 137.5, 163.9, 176.1; HRMS (ESI+) calcd for C14H21N2O3 [M +
H]+ 265.1547, found 265.1546 (error 0.4 ppm).
(2R)-Benzyl-2-amino-6-formamidohexanoate (3b). Reaction con-
ditions were identical to those for 3a, except for the use of 9b: [α]D23
−1.2 (c 1.0, CH3OH).
(2S,9S)-Benzyl-6-formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazo-
line-4-carboxamido]hexanoate (11a). To a stirring solution of (4S)-
2-[2-hydroxyphenyl]-Δ2-1,3-oxazoline-4-carboxylic acid (2a) (31 mg,
0.15 mmol, 1.0 equiv), (2S)-benzyl-2-amino-6-formamidohexanoate
(3a) (40 mg, 0.15 mmol, 1.0 equiv), and DEPBT (49 mg, 0.16 mmol,
1.06 equiv) in THF (2.5 mL) was added Et3N (40 μL, 0.30 mmol, 2.0
equiv) at 25 °C. After 48 h, the reaction mixture was concentrated, and
the residue was partitioned between EtOAc (50 mL) and H2O (50
mL). The organic layer was washed with saturated aqueous NaCl (50
mL), dried (MgSO4), and concentrated to a yellow oil. Purification by
flash chromatography (linear gradient 0−10% MeOH−CH2Cl2)
afforded the title compound (16.6 mg, 24%) as an amorphous,
colorless solid: [α]2D3 +6.0 (c 1.0, CH3OH); Rf 0.65 (10% MeOH−
CH2Cl2); 1H NMR (CD3OD, 600 MHz) δ 1.37−1.42 (m, 2H), 1.47−
1.54 (m, 2H), 1.75−1.82 (m, 1H), 1.87−1.93 (m, 1H), 3.16 (t, J = 7.2
Hz, 2H), 4.48 (dd, J = 9.0, 4.8 Hz, 1H), 4.58 (d, J = 9.0 Hz, 2H), 5.00
(t, J = 9.0 Hz, 1H), 5.15 (d, J = 12.6 Hz, 2H), 5.21 (d, J = 12.6 Hz,
1H), 6.90 (t, J = 7.8 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 7.30−7.33 (m,
1H), 7.34−7.38 (m, 4H), 7.41 (td, J = 7.2, 1.2 Hz, 1H), 7.67 (d, J =
7.8, 1.2 Hz, 1H), 7.97 (s, 1H); 13C NMR (CD3OD, 150 MHz) δ 24.2,
29.9, 32.0, 38.7, 54.1, 68.2, 69.3, 70.5, 111.6, 117.8, 120.1, 129.46,
129.52, 129.6, 129.7, 135.2, 137.3, 161.1, 163.9, 168.6, 173.1, 173.3;
HRMS (ESI+) calcd for C24H28N3O6 [M + H]+ 454.1973, found
454.1994 (4.6 ppm error).
(2R,9R)-6-Formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazoline-4-
carboxamido]hexanoic Acid (1b). Reaction conditions were identical
to those for 1a, except for the use of 11b: [α]2D3 −31.6 (c 0.1, MeOH).
(2S,9R)-6-Formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazoline-4-
carboxamido]hexanoic Acid (1c). Reaction conditions were identical
to those for 1a, except for the use of 11c (15.5 mg, 0.03 mmol).
Purification by flash chromatography (linear gradient 0−10% EtOH−
CH2Cl2 with 1% formic acid) afforded the title compound (5.8 mg,
53%) as an amorphous solid. Purity was determined by analytical
HPLC using a Phenomenex Gemini 5u C18 110A (250 × 4.6 mm)
column operating at 0.9 mL/min and a gradient of 10−100% MeOH−
0.05% aqueous formic acid over 20 min followed by 100% MeOH for
5 min. The retention time of the product was 18.8 min, purity >97%:
[α]2D3 −2.0 (c 0.1, MeOH); Rf 0.59 (10% MeOH−CH2Cl2 with 1%
1
formic acid); H NMR (DMSO-d6, 600 MHz) δ 1.31−1.34 (m, 2H),
(2R,9R)-Benzyl-6-formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxa-
zoline-4-carboxamido]hexanoate (11b). Reaction conditions were
identical to those for 11a, except for the use of 2b and 3b: [α]2D3 −6.0
(c 1.0, CH3OH).
1.37−1.41 (m, 2H), 1.64−1.68 (m, 1H), 1.74−1.78 (m, 1H), 3.05 (t, J
= 6.0 Hz, 2H), 4.15−4.21 (m, 1H), 4.50 (t, J = 7.8, 1H), 4.63 (t, J =
8.4 Hz, 1H), 5.01 (t, J = 8.4 Hz, 1H), 6.95 (t, J = 7.8 Hz, 1H), 7.00 (d,
J = 8.4 Hz, 1H), 7.46 (t, J = 7.2 Hz, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.85
(ovlp m, 1H), 7.98 (ovlp s, 1H), 8.51 (d, J = 7.2 Hz, 1H), 11.81 (br s,
1H); 13C NMR (DMSO-d6, 150 MHz) δ 23.0, 28.9, 31.0, 37.2, 53.0,
67.5, 69.6, 110.2, 116.8, 119.2, 128.3, 134.2, 159.4, 161.3, 166.3, 169.9,
173.8; HRMS (ESI−) calcd for C17H20N3O6 [M − H]− 362.1358,
found 362.1360 (0.55 ppm error).
(2S,9R)-Benzyl-6-formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazo-
line-4-carboxamido]hexanoate (11c). Reaction conditions were
identical to those for 11a, except for the use of (4R)-2-[2-
hydroxyphenyl]-Δ2-1,3-oxazoline-4-carboxylic acid (2b). Purification
by flash chromatography (linear gradient 0−10% MeOH−CH2Cl2)
afforded the title compound (49.9 mg, 58%) as an amorphous,
colorless solid: [α]2D3 −6.7 (c 1.0, CH3OH); Rf 0.61 (10% MeOH−
CH2Cl2); 1H NMR (CD3OD, 600 MHz) δ 1.38−1.42 (m, 2H), 1.50−
1.55 (m, 2H), 1.78−1.82 (m, 1H), 1.89−1.92 (m, 1H), 3.20 (t, J = 7.2
(2R,9S)-6-Formamido-2-[(2-hydroxyphenyl)-Δ2-1,3-oxazoline-4-
carboxamido]hexanoic acid (1d). Reaction conditions were identical
to those for 1a, except for the use of 11d: [α]2D3 +6.0 (c 0.1, MeOH).
F
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Article
(20) Hughes, R. M.; Waters, M. L. J. Am. Chem. Soc. 2006, 128,
13586−13591.
(21) The reported conditions require overnight heating and a large
excess (14 equivalents) of formic acid; however transformation was
achieved in 3 h at room temperature using only 7 equivalents of formic
acid.
(22) Wuts, P. G. M.; Greene, T. W. Greene’s Protecting Groups in
Organic Synthesis, 4th ed.; John Wiley & Sons, Inc.: Hoboken, NJ,
2007.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 1a−d, 2a,b, 3a,b, 6a,b,
8a,b, 9a,b, 11a−d. COSY and HMBC correlations in 1a−d.
Complete data table for bacterial and mammalian cell activity
screens. This material is available free of charge via the Internet
at http://pubs.acs.org.
(23) Li, H.; Jiang, X.; Ye, Y.-h.; Fan, C.; Romoff, T.; Goodman, M.
Org. Lett. 1999, 1, 91−93.
AUTHOR INFORMATION
■
(24) Field, L. D.; Sternhell, S.; Kalman, J. R. Organic Structures from
Spectra, 4th ed. ed.; John Wiley & Sons Ltd: West Sussex, England,
2008.
Corresponding Author
*Phone: 612-625-7956. Fax: 612-626-5173. E-mail: aldri015@
umn.edu.
(25) Schneider, K.; Rose, I.; Vikineswary, S.; Jones, A. L.;
Notes
Goodfellow, M.; Nicholson, G.; Beil, W.; Sussmuch, R. D.; Fiedler,
̈
The authors declare no competing financial interest.
H.-P. J. Nat. Prod. 2007, 70, 932−935.
(26) Young, D. C.; Kasmar, A.; Moraski, G.; Cheng, T. Y.; Walz, A. J.;
Hu, J.; Xu, Y.; Endres, G. W.; Uzieblo, A.; Zajonc, D.; Costello, C. E.;
Miller, M. J.; Moody, D. B. J. Biol. Chem. 2009, 284, 25087−25096.
(27) Mitchell, J. M.; Shaw, J. T. Org. Lett. 2007, 9, 1679−1681.
(28) Fennell, K. A.; Mollmann, U.; Miller, M. J. J. Org. Chem. 2008,
73, 1018−1024.
ACKNOWLEDGMENTS
■
This research was supported by a grant from the NIH
(R01AI070219 to C.C.A.). K.M.N. was supported in part by the
3M Science and Technology Fellowship of the University of
Minnesota. The authors would like to thank Dr. H. I. Boshoff
and Dr. C. E. Barry III for testing of compounds 1a−d against
M. tuberculosis H37Rv, and Mr. C. Gelbmann, Dr. B. Duckworth,
and Dr. A. Meissner for assistance with the microbiology and
cell culture experiments. Funding for the 700 MHz NMR
instrumentation was provided by the Office of the Vice
President for Research, the Medical School, the College of
Biological Science, NIH, NSF, and the Minnesota Medical
Foundation.
(29) Hoshino, Y.; Mukai, A.; Yazawa, K.; Uno, J.; Ando, A.; Mikami,
Y.; Fukai, T.; Ishikawa, J.; Yamaguchi, K. J. Antibiot. 2004, 57, 803−
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