One-pot syntheses of pseudopteroxazoles from pseudopterosins: A rapid route to non-natural congeners with improved antimicrobial activity

Article abstract of DOI:10.1021/np2006555

Rapid one-pot methodologies to prepare pseudopteroxazole (1) and novel congeners from abundant natural pseudopterosins have been devised. This is highlighted here with the first synthesis of the marine natural product homopseudopteroxazole (2) utilizing a novel, silver(I)-mediated catechol to benzoxazole transformation. Pseudopteroxazoles and isopseudopteroxazoles exhibit potent activity against a range of important Gram-positive pathogens including Mycobacterium spp. and vancomycin-resistant Enterococcus faecium. Several non-natural pseudopteroxazoles exhibited strong activity against methicillin-resistant Staphylococcus aureus, thereby displaying a broader spectrum of antibiotic activity compared to pseudopteroxazole.

Full text of DOI:10.1021/np2006555

ARTICLE
pubs.acs.org/jnp
One-Pot Syntheses of Pseudopteroxazoles from Pseudopterosins:
A Rapid Route to Non-natural Congeners with Improved
Antimicrobial Activity
Malcolm W. B. McCulloch,† Fabrice Berrue,† Brad Haltli,† and Russell G. Kerr*,†,‡
†Department of Chemistry and Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island,
Charlottetown, PEI C1A 4P3, Canada
‡Nautilus Biosciences Canada, Inc., Charlottetown, PEI, C1A 4P3, Canada
S Supporting Information
b
ABSTRACT: Rapid one-pot methodologies to prepare pseu-
dopteroxazole (1) and novel congeners from abundant natural
pseudopterosins have been devised. This is highlighted here
with the first synthesis of the marine natural product homo-
pseudopteroxazole (2) utilizing a novel, silver(I)-mediated
catechol to benzoxazole transformation. Pseudopteroxazoles
and isopseudopteroxazoles exhibit potent activity against
a range of important Gram-positive pathogens including
Mycobacterium spp. and vancomycin-resistant Enterococcus faecium. Several non-natural pseudopteroxazoles exhibited strong activity
against methicillin-resistant Staphylococcus aureus, thereby displaying a broader spectrum of antibiotic activity compared to
pseudopteroxazole.
atural product scaffolds (NPS) are of tremendous value
in drug discovery, playing a vital role in the genesis of a
we have prepared several hundred milligrams of 1 using a one-
pot synthesis.
N
significant proportion of drugs, particularly anti-infectives.1,2
The supply of promising natural product-based lead com-
pounds may be an issue when the natural source is limited
and total synthesis is inefficient or impractical.3 Lengthy
synthetic routes to NPS may also delay or hamper medicinal
chemistry efforts to generate analogues for SAR studies. An
alternative to total synthesis is to utilize abundant, naturally
occurring molecular architectures as starting materials for
natural product-based drug discovery. Herein, we describe
such methodology to rapidly supply a promising low-abun-
dance natural product, pseudopteroxazole (1),4 the related
natural product homopseudopteroxazole (2),5 and novel
non-natural congeners including more potent antibacterial
compounds.
Pseudopteroxazole is a promising antibiotic because it
exhibits strong activity against Mycobacterium tuberculosis,
the causative agent of tuberculosis, with commensurate low
toxicity.4 Interest in 1 by the synthetic community6À8 has
resulted in elegant multistep total syntheses described by the
groups of Corey9 and Harmata.10,11 These syntheses, how-
ever, have been reported only on a low-milligram scale, and
they have not led to the syntheses of congeners of 1 nor the
natural product 2. To date, medicinal chemistry investigations
around 1 have not been described. An efficient route to both
larger quantities and congeners of pseudopteroxazoles is
therefore desirable. Using the methodologies described here,
’ RESULTS AND DISCUSSION
Given that 1 possesses the same backbone as the pseudopter-
osin GÀJ aglycone (3),12 we rationalized that 1, 2, and analogues
could be attainable semisynthetically from 3. This approach
seemed logical because the various pseudopterosin glycosides13
are available in multigram quantities, constituting up to about 5%
of the dry weight of the gorgonian Pseudopterogorgia elisabethae.
In order to rapidly build a series of pseudopteroxazole congeners
Received: August 5, 2011
Published: October 06, 2011
r
Copyright
2011 American Chemical Society and
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Journal of Natural Products
ARTICLE
for biological evaluation, we sought chemistry that could be per-
formed efficiently in one pot. Axiomatically, this strategy required
methodology to convert the catechol in 3 into a benzoxazole;
furthermore, such catechol to benzoxazole transformations will be
valuable given benzoxazoles are important scaffolds in medicinal
chemistry and new routes to their synthesis are desirable.14,15
For the required catechol to benzoxazole transformation we
devised two general methods both of which proceed through
oxidation of the catechol in 3 to the ortho-quinone 416 and yield
different ratios of pseudopteroxazoles and their regioisomers,
“isopseudopteroxazoles” (methods A and B, Schemes 1 and 2).
The titular isopseudopteroxazole (5) represents a benzoxazole
regioisomer where the location of the N and O on the oxazole is
reversed from that in 1. In developing these procedures we
sought methodology applicable to the rapid synthesis of various
congeners for biological evaluation.
Method A (Table 1 and Scheme 1) was developed first and
proceeds by oxidation of 3 and subsequent condensation with an
ammonium salt and an aldehyde. Our second-generation proce-
dure, method B (Scheme 2), proceeds by Ag(I)-mediated
oxidation and condensation with an amino acid. In both cases
the resulting pseudopteroxazoles possess different C-21 substit-
uents depending on the nature of the aldehyde or amino acid.
These two complementary methods provide synthetic flexibility,
resulting in substantial product structural diversity.
Using method A, the aglycone (3) is heated with a mild
oxidant, an ammonium salt, and an aromatic aldehyde or para-
formaldehyde, yielding pseudopteroxazoles (e.g., 1, 6a, 7a) and
isopseudopteroxazoles (e.g., 5, 6b, 7b) in moderate yields
(Scheme 1 and Table 1). The oxidant can be air or another mild
oxidant, but the reaction does not proceed in the absence of an
oxidant (Table 1, entry 5). Method A is based on the known
reaction between an ortho-quinone, an ammonium salt, and an
aldehyde (recent examples are given in refs 17À19).
The ratio of pseudopteroxazoles to isopseudopteroxazoles re-
turned by method A is ∼3:1. The pseudopteroxazoles are readily
1
distinguished from the isopseudopteroxazoles by H NMR: the
chemical shifts of the C-20 methyl and the C-7 methine are relatively
deshielded in the natural and iso pseudopteroxazoles, respectively,
presumably due to diamagnetic anisotropy that is dependent on the
orientation of the oxazole. The NMR data for isopseudopteroxazole
(5) are given in Table 2. The C-7 methine and the C-20 methyl
signals are observed at 3.45 and 2.33 ppm, respectively, whereas in
pseudopteroxazole these signals are observed at 3.27 and 2.45 ppm.
As a further example distinguishing psesudopteroxazole regioi-
somers, Table 2 also provides a side by side comparison of the
NMR assignments for the pseudopteroxazole derivative 6a and the
isopseudopteroxazole 6b. The C-7 methines in 6a and 6b are
observed at 3.31 and 3.54 ppm, respectively, whereas the C-20
methyls are observed at 2.51 and 2.36 ppm, respectively (also see
Supporting Information Figure S10).
Scheme 1. Method A Pseudopteroxazole Syntheses and
Plausible Mechanism
A plausible mechanism for the reaction following method A is
shown in Scheme 1. Initially, the aglycone 3 is oxidized to ortho-
quinone 4. Subsequently, nucelophilic attack of the ammonia onto
one of the ortho-quinone carbonyls gives an imine: following the
preferred pathway, attack on the less sterically hindered carbonyl gives
an imine of general nature 8, which may redox cycle to the aniline 9.
Condensation with the aldehyde yields imine 10, which undergoes
cyclization and oxidation to the generic benzoxazole product.
While method A allowed us to rapidly access pseudopterox-
azole and aryl-substituted oxazole congeners, a limitation of this
method is that it does not work effectively with aliphatic aldehydes
(e.g., Table 1, entries 13À15). We thus developed method B as a
complementary route to provide different pseudopteroxazole
Scheme 2. Method B Pseudopteroxazole Syntheses and Plausible Mechanisma
a Isolated yields after column chromatography. b Determined by NMR.
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Table 1. Method A Syntheses of Pseudopteroxazoles and Selected Reaction Conditions Screeneda
products Ptx-R/
iso-Ptx-R (R)
entry
X
aldehyde
equiv aldehyde
[O] (equiv)
solv
temp (°C) time (h)
yield (%)b
1
2
3
4
5
6
7
8
9
OAc paraformaldehyde
OAc paraformaldehyde
OAc paraformaldehyde
OAc paraformaldehyde
OAc paraformaldehyde
OAc paraformaldehyde
OAc paraformaldehyde
HCO3 paraformaldehyde
HCO3 anisaldehyde
13
100
100
100
100
100
100
100
2.5
24
air
air
air
air
AcOH
PhMe
AcOH
AcOH
20
110
118
118
118
118
118
65
24
18
15
18
15
4
1/5 (H)
1/5 (H)
1/5 (H)
44
52
41
AcOH (degassed, N2 atm)
AcOH
Ag2O (1), air
Ce(NH4)2 (NO3)6 (1) AcOH
Ce(NH4)2 (NO3)6 (1) MeOH
24
24
1/5 (H)
48
air
air
air
air
air
air
air
AcOH
AcOH
AcOH
AcOH
AcOH
AcOH
AcOH
118
118
118
118
118
118
118
3.5
10 HCO3 anisaldehdye
11 OAc 4-fluorobenzaldehyde
12 OAc 2-naphthaldehyde
13 OAc crotonaldehyde
14 OAc hexanal
15
16
18
16
20
20
6a/6b (2-MeO-Ph)
7a/7b (4-Fl-Ph)
(R = naphtha)
52
49
47
50
5
36
2.5
100
trace quantities of 2
trace quantities of 2
15 OAc hexanal
a Ratio determined by NMR. b Isolated yield after column chromatography (of both regioisomers). Where no yield is given: reaction was monitored by
ELSD-LCMS and no significant conversion to the desired oxazole was detected.
congeners to address this synthetic limitation and to provide
additional analogues for biological evaluation given that prelimin-
ary antimicrobial analyses indicated the aryl-substituted pseudop-
teroxazole derivatives 6a and 7a displayed no notable biological
activity (Table 4).
To develop method B, we first screened reactions between the
pseudopterosin aglycone 3 and glycine under a variety of condi-
tions (Table 3). Pleasingly we found that Ag2O effected the
desired transformation to pseudopteroxazole (1). As far as we are
aware, this is the first example of a silver(I)-mediated reaction
between a catechol and an amino acid that yields a benzoxazole;
however, there are a small number of literature reports of reac-
tions between ortho-quinones and amino acids that yield ben-
zoxazoles.20À22
In method B the aglycone (3) is heated with Ag2O (or another
Ag(I) salt) and an excess of an amino acid, yielding predomi-
nantly pseudopteroxazoles (e.g., 1, 2, 12À19, Scheme 2). In the
absence of a Ag(I) salt the reaction returns only trace quantities
of pseudopteroxazoles (Table 3, entries 1À4). Similarly, preoxi-
dation of the aglycone followed by filtration to remove insoluble
Ag and subsequent amino acid addition gives only trace quan-
tities of pseudopteroxazoles. Given our aim to quickly prepare
and evaluate pseudopteroxazole congeners, the reaction for each
amino acid has not been fully optimized. However, in general,
higher yields are obtained with periodic addition of both the
amino acid and the Ag2O; presumably this attenuates Ag(I)-
mediated decarboxylation of the amino acid,23 hence the re-
quired excess of the amino acid. Silver-mediated decarboxylation
presumably explains why reactions with glutamic and aspartic
acid returned only trace quantities of the benzoxazole products.
Nonetheless, the product formally derived from glutamic acid
(i.e., 20), was prepared by hydrolysis of 19, which in turn was
synthesized from the aglycone 3 and 2-amino-5-methoxy-5-
oxopentanoic acid.
A plausible mechanism for the reaction following method B is
shown in Scheme 2. As before, oxidation of the catechol 3 gives
the ortho-quinone 4, and subsequently attack of the nitrogen on
the amino acid yields the α-carboxy imine 11. Decarboxylation
(probably Ag(I)-mediated) yields the general imine 10, which
undergoes cyclization and oxidation to the benzoxazole products.
A similar mechanism for an ortho-quinone to benzoxazole trans-
formation has been previously proposed.22 In both methods A
and B there is a preference for the nucleophile to attack the less
hindered C-10 carbonyl; however, this preference is relatively
greater in method B compared to method A, due to the greater
steric bulk surrounding the nitrogenous nucleophile. Hence, the
ratio of the normal (e.g., 1) to iso (e.g., 5) pseudopteroxazole
products differs between method A (∼3:1) and method B
(∼10:1). The structures of the new pseudopteroxazole deriva-
tives were confirmed by 1D and 2D NMR analysis.
The semisynthetic pseudopteroxazoles were tested against six
microorganisms (Table 4), and some of our analogues show
more potent activity than pseudopteroxazole. Pseudopteroxazole
(1), isopseudopteroxazole (5), and several analogues (e.g., 16,
18, 20) displayed activity at a similar potency to that exhibited by
the clinically used drug rifampicin against two model mycobac-
teria, M. smegmatis and M. diernhoferi. The compounds were also
tested against vancomycin-resistant Enterococcus faecium (VRE),
methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas
aeruginosa, and Candida albicans. While no activity was observed
against the latter two pathogens, 1, 5, 12, 16, 18, and 20 showed
strong activity against VRE, and 16, 18, and 20 showed strong
activity against MRSA. These results reveal a further spectrum
of activity possessed by pseudopteroxazoles. Effects of variation
about the C-21 oxazole moiety on the biological activity are also
revealed: generally, analogues with more lipophilic side chains
are less active compared to the natural product. It is notable that
pseudopteroxazole did not show activity against MRSA, yet
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Table 2. NMR Data for 5, 6a, and 6ba
5
6a
6b
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
3.99, m
δC, type
δH (J in Hz)
1
36.7, CH
3.93, app q (8.9)
2.10, m
36.4, CH
36.8, CH
3.95, m
2a
2b
3
40.0, CH2
40.1, CH2
2.16, m
1.31, m
1.31, m
2.29, m
2.19, m
1.12, m
2.21, m
1.46, m
3.31, m
40.1, CH2
2.11, m
1.30, m
1.28, m
2.25, m
2.16, m
1.08, m
2.23, m
1.42, m
3.54, m
1.29, m
34.5, CH
44.8, CH
28.1, CH2
1.26, m
34.6, CH
44.8, CH
28.1, CH2
34.5, CH
44.9, CH
28.2, CH2
4
2.23, m
5a
5b
6a
6b
7
2.16, m
1.04, m
32.2, CH2
2.23, m
32.4, CH2
32.3, CH2
1.41, m
30.8, CH
131.0, C
3.45, m
30.4, CH
121.7, C
30.9, CH
130.7, C
8
9
136.1,b C
147.6, C
138.1, C
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
148.5, C
140.0, C
149.2, C
117.3, C
136.2,b C
126.2, C
116.9, C
134.3, C
135.4,b C
134.9,b C
130.8, CH
128.6, C
135.3, C
135.9, C
130.5, CH
128.9, C
4.99, d (9.3)
131.0, CH
128.5, C
5.02, d (9.6)
5.02, d (9.0)
25.4, CH3
17.6, CH3
19.8, CH3
23.7, CH3
12.3, CH3
150.6, CH
1.68, s
25.4, CH3
17.6, CH3
19.8, CH3
22.0, CH3
13.6, CH3
160.2, C
1.69, s
25.4, CH3
17.6, CH3
19.9, CH3
23.8, CH3
12.4, CH3
159.7, C
1.69, s
1.78, s
1.80, s
1.78, s
1.06, d (6.0)
1.51, d (6.8)
2.33, s
1.07, d (6.0)
1.55, d (6.6)
2.51, s
1.06, d (6.0)
1.58, d (6.6)
2.36, s
7.96, s
117.3, C
117.6, C
158.2, C
158.3, C
112.1, CH
132.0, CH
120.6, CH
131.2, CH
56.0, CH3
7.05, d (8.4)
7.46, m
112.3, CH
131.9, CH
120.7, CH
131.3, CH
56.2, CH3
7.03, d (8.4)
7.45, m
7.08, m
7.06, m
8.08, dd (6.6, 1.2)
3.97, s
8.03, dd (7.2, 1.5)
3.95, s
a CDCl3, 600 MHz (13C: 150 MHz); assigned by COSY, HSQC, and HMBC experiments. b Interchangeable assignments.
three semisynthetic derivatives with relatively polar C-21
substituents showed strong activity, with IC50's in the low
μg/mL range.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a Rudolph Autopol III polarimeter. Infrared spectra were
recorded using attenuated total reflectance, with samples deposited as a
thin film on a Thermo Nicolet 6700 FT-IR spectrometer (Smart iTR).
NMR spectra were obtained on a Bruker Avance III 600 MHz NMR
spectrometer operating at 600 and 150 MHz for 1H and 13C, respectively.
Chemical shifts (δ) are reported in ppm and were referenced to residual
solvent signals: CDCl3 (δH 7.26; δC 77.0). Coupling constants (J) are
reported in Hz with the abbreviations (s) singlet, (d) doublet, (t) triplet,
(q) quartet, (m) multiplet, and (app) apparent. Low-resolution (nominal
mass) mass spectra were obtained using a Finnigan LXQ ion trap mass
spectrometer fitted with either an APCI or ESI source: samples were
typically analyzed by LCMS using either an analytical HPLC or a UPLC
column, with hyphenated MS-ELSD-UV detection. High-resolution
mass spectra were measured by Xiao Feng at Dalhousie Univeristy, on
a Bruker microTof Focus orthogonal ESI-TOF mass spectrometer. Flash
chromatography was conducted utilizing a Teledyne Combiflash Rf, with
UV detection triggered fraction collection using RediSep columns.
Pseudopterosin starting materials were isolated from samples of
P. elisabethae obtained from Victory Reef, Bahamas. The pseudopterosins
’ CONCLUSION
The catechol to benzoxazole syntheses described in this report
are significant, as they provide a simple, versatile route to
pseudopteroxazole and congeners for biological evaluation.
The methodology makes use of a relatively abundant material
(pseudopterosins GÀJ), and the same process should be applic-
able to other pseudopterosin skeletons that possess alternative
backbone configurations (e.g., pseudopterosin A). Biological
evaluations suggest modification of the C-21 oxazole in 1 can
lead to analogues with more potent antibacterial activity.
Furthermore, this work has unambiguously confirmed the struc-
ture of homopseudopteroxazole (2) and its skeletal relationship
to the pseudopterosin GÀJ aglycone. Our results also tentatively
support a biosynthetic route to 1, which may proceed through
condensation of 3 (or a similar pseudopterosin derivative) with
glycine.
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were purified by a combination of solvent extraction, liquidÀliquid
partitioning, and chromatography. The major pseudopterosin in the
sample possesses the pseudopterosin GÀJ aglycone skeleton. All other
starting materials, reagents, and solvents were commercially available
and were generally obtained from one of the following three suppliers:
VWR, Sigma-Aldrich, or Fisher Scientific.
Pseudopterosin GÀJ Aglycone (3). The typical procedure to prepare
the aglycone is based on the literature method for the hydrolysis of
pseudopterosin C.24 A sample of pseudopterosins GÀJ (3) is refluxed,
under an atmosphere of N2, in MeOH + 1 N HCl for 2 h. After cooling,
the sample is neutralized with aqueous NaHCO3, and then the aglycone
isolated by partitioning the sample between H2O and EtOAc. Concentra-
tion of the organic phase yields the aglycone 3, which may be used without
further purification. Immobile brown oil; 1H NMR (CDCl3, 600 MHz)
consistent with literature values;12 13C NMR (CDCl3, 150 MHz) δ 140.0,
139.9, 131.9, 131.4, 130.1, 128.3, 125.3, 120.4, 44.4, 40.1, 36.9, 34.0, 32.0,
28.4, 27.8, 25.4, 23.1, 19.8, 17.5, 11.9; APCIMS m/z 301 [M + H]+.
Method A: Aldehyde Route to Pseudopteroxazole Derivatives.
Synthesis of Pseudopteroxazole (1) and Isopseudopteroxazole (5)
via Method A. Example 1: Utilizing Air As the Oxidant. A sample of
the aglycone (3, 179 mg, 0.6 mmol) was dissolved in AcOH (5 mL), and
air was bubbled into the solution for 10 min. Paraformaldehyde (100
mg) and NH4OAc (1 g) were then added, and the reaction was then
refluxed for 7 h. The reaction products were then partitioned between
EtOAc and H2O, and the EtOAc-soluble material was then subjected to
reversed-phase flash chromatography (43 g C-18 column) using a
MeOH/H2O gradient (40:60 to 100:0). After analysis by LCMS the
fractions were recombined and concentrated in vacuo. One fraction was a
mixture of pseudopteroxazole and isopseudopteroxazole (1/5 (3:1
ratio), 95.5 mg, 0.31 mmol, 52%). The pseudopteroxazole regioisomers
were separated by normal-phase flash chromatography (40 g silica
column) using a MTBE/hexane gradient (0:100 to 25:75) to give pure
pseudopteroxazole (1, 52 mg, 0.17 mmol, 29%) and isopseudopterox-
azole (5, 12.5 mg, 0.04 mmol, 7%).
Table 3. Method B Syntheses of Pseudopteroxazole and
Selected Reaction Conditions Screened
Example 2: Utilizing Ceric Ammonium Nitrate As the Oxidant. To
a stirred solution of the aglycone (3, 214 mg, 0.71 mmol) in MeOH
(10 mL) under an N2 atmosphere was added ceric ammonium nitrate
(409 mg, 0.74 mmol). The reaction was refluxed for 1 h and cooled to
room temperature (rt), and then excess NH4HCO3 (2.4 g, 31 mmol)
was added. The reaction was returned to reflux, and then paraformalde-
hyde was added in two batches (4 h: 500 mg; 18 h: 200 mg). After a total
of 24 h at reflux the reaction products were partitioned between EtOAc
and H2O. The EtOAc-soluble material (214 mg) was purified by normal-
phase flash chromatography (24 g silica column) using a MTBE/hexane
gradient (0:100 for 2 min, then to 5:95 over 2 to 17 min, then to 10:90
over 17 to 22 min, 35 mL/min) to yield isopseudopteroxazole (5, 25 mg,
0.08 mmol, 11%) and pseudopteroxazole (1, 78 mg, 36%).
entry equivs glycine [O] (equiv)
solvent
atm time (h) yield (%)a
1
2
3
4
5
6
7
8
11.4
5.0
MeOH
H2Ob
air
air
24
0.5
3
trace
trace
trace
trace
24
3.2
NaIO4 (0.3) H2O/MeOH air
NaIO4 (1.1) H2O/MeOH air
3.3
2
2.7
Ag2O (0.1) MeOH
Ag2Oc (1.0) MeOH
Ag2Oc (1.4) MeOH
Ag2Oc (2.1) MeOHd
air
air
air
N2
18
3
10.1
11.4
12.2
22
32
Pseudopteroxazole (1): oil; [α]25D +100 (c 0.2, CHCl3); 1H NMR
and 13C NMR consistent with literature values;4,9,10 APCIMS m/z 310
[M + H]+; HRESIMS m/z [M + H]+ 310.2154 (calcd for C21H28NO,
310.2165).
24
minor
a Isolated yield after column chromatography. b Autoclave 121 °C,
18 PSIG. c Amino acid and Ag2O were added periodically in two or
more batches. d Degassed MeOH.
Table 4. Antimicrobial Evaluation of Semisynthetic Pseudopteroxazole Congeners
compounda
M. smegmatis,b MIC μg/mL
M. diernhoferi,c MIC μg/mL
VRE,d IC50 μg/mL
MRSA,e IC50 μg/mL
1 (Ptx-H)
2
4
13
>128
>128
>128
>128
NT
2 (Ptx-(CH2)4CH3)
5 (iso-Ptx-H)
>64
8
>64
8
>128
13
6a (Ptx-(2-CH3O-Ph))
7a (Ptx-(4-F-Ph))
12 (Ptx-CH3)
>64
>64
8
>64
>64
4
>128
NTf
2.5
>128
>128
>128
>128
3
13 (Ptx-CH(CH3)CH2CH3)
14 (Ptx-(CH2)2SCH3)
15 (Ptx-CH2Ph)
>64
16
>64
4
>64
16
4
>128
>128
>128
4
16 (Ptx-CH2CONH2)
17 (Ptx-CHOHCH3)
18 (Ptx-(CH2)2CONH2)
19 (Ptx-(CH2)2CO2CH3)
20 (Ptx-(CH2)2CO2H)
vancomycing
2
16
4
8
>128
7
>128
7
4
>128
8
>128
4
>128
12
>128
12
NT
1
NT
0.25
4
NT
NT
8.75
12.25
NT
isonazidg
rifampicing
4
NT
a No activity was observed against Pseudomonas aeruginosa or Candida albicans. Compounds 1 and 16 showed no activity against Proteus vulgaris.
b ATCC 12051. c ATCC 19340. d Vancomycin-resistant Enterococcus faecium (VREF Ef 379). e Methicillin-resistant Staphylococcus aureus (ATCC
33591). f NT = not tested. g Antibiotic controls.
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ARTICLE
Isopseudopteroxazole (5): oil; [α]25D +114 (c 1.29, CHCl3); IR νmax
General Procedure for Method B. A mixture of the pseudopter-
osin aglycone 3 and Ag2O (0.7 to 1 equiv) is refluxed in MeOH or EtOH
for 1 h. A given amino acid (>1 equiv) is then added, and the reaction
further refluxed until the starting aglycone has been consumed
(monitored by TLC, LCMS, etc.). Additional batches of the amino acid
and Ag2O (0.7 to 1.4 equiv) are added periodically to drive the reaction
to completion. The crude products are typically isolated by filtering the
cooled crude reaction products through Celite. Purification is achieved
chromatographically.
1
2946, 2921, 2855, 1445, 1088 cmÀ1; H and 13C NMR, see Table 2;
APCIMS m/z 310 [M + H]+; HRESIMS m/z [M + H]+ 310.2150 (calcd
for C21H28NO, 310.2165).
o-Anisaldehyde Derivatives (6a and 6b). A sample of pseudopter-
osins GÀJ (38 mg, 0.078 mmol) was refluxed in methanolic HCl (1.5 N,
10 mL) under N2 for 2.5 h. The solvent was removed under a stream of
nitrogen, and then NH4HCO3 (1 g, excess) and AcOH(4 mL) were added
to the residue. After the subsequent effervescence ceased, air was bubbled
into the vessel for 10 min, and then ortho-anisaldehyde (45 mg, 0.3 mmol)
was added. The reaction was then heated at 110 °C overnight. The cooled
reaction contents were partitioned between CHCl3 and H2O, and the
CHCl3 layer was concentrated invacuoto give a residue that was purified by
flash chromatography (silica, hexane/EtOAc) to give the title compounds
(6a/6b (3:1 ratio), 16.7 mg, 0.040 mmol, 52%). A portion of this material
was further purified by RP-HPLC (Phenomenex, phenylhexyl, 5 μm,
250 Â 10 mm, 4.0 mL/min) eluted with MeOH/H2O (isocratic 95:1) to
give 6b (eluted 14.9 to 15.6 min) and 6a (eluted 15.6 to 16.8 min).
Homopseudopteroxazole (2). Homopseudopteroxazole (2) was synthe-
sized from the pseudopterosin GÀJ aglycone (3, 14.2 mg, 0.047
mmol), Ag2O (2.3 equiv), and 2-aminoheptanoic acid (3.1 equiv)
following the general procedure. Purification by flash chromatography
on a 4 g silica column, eluted with a gradient of MTBE/hexane (from
100% hexane to 95% hexane over 15 min), yielded homopseudopter-
1
oxazole (2, 6.1 mg, 0.016 mmol, 34%). Immobile oil; H NMR, 13C
NMR, and other spectroscopic data consistent with literature values;5
APCIMS m/z 380 [M + H]+; HRESIMS m/z [M + H]+ 380.2944
(calcd for C26H38NO, 380.2948).
Compound 6a: amorphous solid; [α]25 +76 (c 0.25, CHCl3); IR
D
νmax 2920, 2853, 1464, 1257, 1025 cmÀ1; 1H and 13C NMR see Table 2;
APCIMS m/z 416 [M + H]+; HRESIMS m/z [M + H]+ 416.2568 (calcd
for C28H34NO2, 416.2584).
Alanine Product (12). The pseudopteroxazole C-21 methyl deriva-
tive (12) was synthesized from the pseudopterosin aglycone (3, 9.1 mg,
0.03 mmol), Ag2O (2.4 equiv), and alanine (2.9 equiv) following the
general procedure. Purification by flash chromatography on a 4 g C18
column, eluted with a gradient of MeOH/H2O (from 4:6 to 1:0),
yielded 12 (3.2 mg, 0.01 mmol, 33%). Immobile oil; [α]25D +93 (c 0.38,
CHCl3); IR νmax 2922, 2855, 1609, 1583, 1445, 1376, 1063 cmÀ1; 1H
and 13C NMR, see Tables S1 and S2; APCIMS m/z 324 [M + H]+;
HRESIMS m/z [M + H]+ 324.2318 (calcd for C22H30NO, 324.2322).
Asparagine Product (16). The pseudopteroxazole C-21 acetamide
derivative (16) was synthesized from the pseudopterosin aglycone
(3, 174 mg, 0.58 mmol), Ag2O (1.3 equiv), and asparagine (7.8 equiv)
following the general procedure. Purification by flash chromatography
on a 30 g diol column, eluted with a gradient of hexane/MTBE (from 0:1
to 1:0), yielded 16 (42.1 mg, 0.11 mmol, 20%). A portion of this material
was further purified by RP-HPLC (Phenomenex, Gemini C18, 5 μm,
250 Â 10 mm, 3.0 mL/min) eluted with MeOH/H2O/HCO2H (isocratic
76:24:0.1) to remove the minor regioisomer. Light orange immobile oil;
[α]25D +107.3 (c 0.32, CHCl3); IR νmax 3342 (br), 3191, 2948, 2922, 2855,
1679 (CO), 1388, 1062 cmÀ1; 1H and 13C NMR see Tables S1 and S2;
APCIMS m/z 367 [M + H]+; HRESIMS m/z [M + Na]+ 389.2190 (calcd
for C23H30N2O2Na, 389.2199).
Compound 6b: amorphous solid; [α]25 +72 (c 0.07, CHCl3); IR
D
νmax 2923, 2854, 1484, 1257, 1025 cmÀ1; 1H and 13C NMR see Table 2;
APCIMS m/z 416 [M + H]+; HRESIMS m/z [M + H]+ 416.2569 (calcd
for C28H34NO2, 416.2584).
p-Fluoroaldehyde Derivative (7a). A solution of the aglycone
(3, 41 mg, 0.14 mmol), NH4OAc (1 g, excess), and 4-fluorobenzaldehyde
(800 μL, excess) was refluxed in AcOH (4 mL) for 16 h. The cooled
reaction mixture was subsequently partitioned between EtOAc and
aqueous HCl (1 N). The organic-soluble material was purified by silica
flash chromatography (hexane/EtOAc, 10:0 to 9:1 gradient) to give the
mixed oxazole regioisomers (7a/7b (3:1 ratio), 27.6 mg, 0.068 mmol,
49%). A portion of the major regioisomer (7a) was further purified by
RP-HPLC (Phenomenex, phenylhexyl, 5 μm, 250 Â 10 mm, 4.0 mL/
min) using isocratic MeOH (eluted across 10.0 to 10.4 min). Immobile
oil; [α]25D +99 (c 0.14, CHCl3); IR νmax 2922, 2855, 1501, 841 cmÀ1; 1H
NMR (CDCl3, 600 MHz) δ 8.23 (m, 2H, H-23/H-27), 7.19 (app t, 2H,
J = 8.6 Hz, H-24/H-26), 5.00 (d, 1H, J = 9.4 Hz, H-14), 3.96 (m, 1H,
H-1), 3.31 (m, 1H, H-7), 2.48 (s, 3H, H-20), 2.31À2.10 (m, 4H), 1.79 (s,
3H, H-17), 1.69 (s, 3H, H-16), 1.54 (d, 3H, J = 6.8 Hz, H-19), 1.48À1.42
(m, 1H, H-6b), 1.32À1.26 (m, 2H), 1.11 (m, 1H, H-5b), 1.06 (d, 3H, J =
5.7 Hz, H-18); 13C NMR (CDCl3, 150 MHz) δ 164.4 (d, J = 251.4 Hz),
160.6, 147.9, 140.1, 136.3, 134.7, 130.8, 129.54 (d, J = 8.6 Hz), 128.7,
126.4, 124.3, 121.9, 115.9 (d, J = 22.1 Hz), 44.8, 40.1, 36.5, 34.5, 32.3,
30.4, 28.1, 25.4, 22.3, 19.8, 17.6, 13.5; APCIMS m/z 404 [M + H]+;
HRESIMS m/z [M + H]+ 404.2384 (calcd for C27H31FNO, 404.2384).
Method B: Amino Acid Route to Pseudopteroxazole Derivatives.
Specific Example: Method B Synthesis of Pseudopteroxazole (1) and
Isopseudopteroxazole (5). A sample of 3 (33.8 mg, 0.11 mmol) and
Ag2O (18.7 mg, 0.08 mmol) was refluxed in MeOH (30 mL) for 1 h. The
solution was then cooled to rt, and glycine (85 mg, 1.3 mmol) was added.
The reaction mixture was refluxed for a further 19 h, with additional
Ag2O (0.08 mmol) being added 2 h into this time frame. The cooled
crude reaction mixture was then filtered through Celite and dried in
vacuo. The crude product (35 mg) was subjected to flash chromatog-
raphy on a 13 g C18 column eluted with a gradient of MeOH/H2O
(from 4:6 to 1:0) to yield a mixture of pseudopteroxazole regioisomers
(10:1 ratio 1: 5 by NMR). Subsequently, the individual regioisomers
were purified by flash chromatography on a 4 g silica column eluted with
a gradient of MTBE/hexane (from 100% hexane to 95% hexane over
15 min) to yield 1 (11.3 mg, 0.036 mmol, 32%) and 5 (1.4 mg,
0.004 mmol, 4%). The spectroscopic data were identical to that observed
for pseudopteroxazoles prepared by method A.
Glutamine Product (18). The pseudopteroxazole propanamide deri-
vative (18) was synthesized from the pseudopterosin aglycone (3, 51
mg, 0.17 mmol), Ag2O (1.4 equiv), and glutamine (8 equiv) following
the general procedure. Purification by flash chromatography on a 13 g
C18 column, eluted with a gradient of MeOH/H2O (from 4:6 to 1:0),
yielded 18 (10.3 mg, 0.027 mmol, 16%). Orange immobile oil;
[α]25 +73 (c 0.29, CHCl3); IR νmax 3346, 3198, 2923, 2855, 1672
D
(CO), 1444, 1374, 1064 cmÀ1; 1H and 13C NMR see Tables S1 and S2;
APCIMS m/z 381 [M + H]+; HRESIMS m/z [M + H]+ 381.2525 (calcd
for C24H33N2O2, 381.2537).
Carboxylic Acid 20. To a portion of the ester 19 (10 mg, 0.25 mmol)
in THF (3 mL) was added a LiOH solution (12 mL, 1 N, aqueous). The
mixture was stirred for 21 h at 35 °C. Dilute HCl (0.5 N) was then added
to adjust the pH to ∼1, and the mixture was then partitioned between
EtOAc and H2O. The combined EtOAc extracts were concentrated in
vacuo to yield the carboxylic acid 20 (7.2 mg, 0.19 mmol, 75%).
Amorphous, light yellow solid; [α]25D +73.0 (c 0.36, CHCl3); IR νmax
3000 (broad), 2921, 2856, 1714 (CO), 1576, 1443, 1167 cmÀ1; 1H and
13C NMR, see Tables S1 and S2; APCIMS m/z 382 [M + H]+;
HRESIMS m/z [M + H]+ 382.2360 (calcd for C24H32NO3, 382.2377).
Antimicrobial Evaluation. Activity against Mycobacterium smegmatis
ATCC 12051 and Mycobacterium diernhoferi ATCC 19340 was
conducted using a microbroth dilution antibiotic susceptibility assay.
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Journal of Natural Products
ARTICLE
Testing was conducted in accordance with Clinical Laboratory Stan-
dards Institute testing standards (susceptibility testing of Mycobacteria,
Nocardiae, and other aerobic actinomycetes; Approved Standard. M24-
A Volume 23, number 18. Microbroth dilution method for antimicrobial
testing of fast growing mycobacteria). Compounds were tested against
each organism in triplicate. Compounds were prepared in sterile 20%
DMSO and serially diluted to generate a range of eight concentrations.
Replicates were performed on separate 96-well plates. Each plate
contained four uninoculated contamination controls (media + 20%
DMSO), four untreated controls (media + 20% DMSO + organism),
and one column containing a concentration range of a control antibiotic.
Control antibiotics tested included rifampicin, ciprofloxacin, and doxy-
cycline. Growth relative to untreated control wells was assessed by visual
inspection after 5 days of incubation at 30 °C.
Activity against methicillin-resistant Staphylococcus aureus ATCC 33591
(MRSA), vancomycin-resistant Enterococcus faecium Ef 379 (VRE),
Pseudomonas aeruginosa ATCC 14210, Proteus vulgaris ATCC 12454, and
Candida albicans ATCC 14035 was conducted using the microbroth
dilution antibiotic susceptibility assay. Testing was conducted in accordance
with Clinical Laboratory Standards Institute testing standards (Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aero-
bically; Approved Standard, Sixth Ed, M7-A6 Volume 23, number 2).
Compounds were tested against each organism in triplicate. Compounds
were prepared in sterile 20% DMSO and serially diluted to generate a range
of eight concentrations. Each plate contained eight uninoculated contam-
ination controls (media + 20% DMSO), eight untreated controls (media +
20% DMSO + organism), and one column containing a concentration
range of a control antibiotic (vancomycin, rifampicin, gentamycin, cipro-
floxacin, or nystatin, for MRSA, VRE, P. aeruginosa, P. vulgaris, and
C. albicans, respectively). The optical density of the plate was recorded at
600 nm at time zero and then again after incubation of the plates for 22 h at
37 °C. After subtracting the time zero OD600 from the final reading the
percentages of microorganism survival relative to vehicle control wells were
calculated and the IC50 was determined.
’ REFERENCES
(1) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2007, 70, 461–477.
(2) Newman, D. J.; Cragg, G. M. Future Med. Chem. 2009, 1,
1415–1427.
(3) Butler, M. S. J. Nat. Prod. 2004, 67, 2141–2153.
(4) Rodriguez, A. D.; Ramirez, C.; Rodriguez, I. I.; Gonzalez, E. Org.
Lett. 1999, 1, 527–530.
(5) Rodriguez, I. I.; Rodriguez, A. D. J. Nat. Prod. 2003, 66, 855–857.
(6) Yadav, J. S.; Bhasker, E. V.; Srihari, P. Tetrahedron 2010, 66,
1997–2004.
(7) Johnson, T. W.; Corey, E. J. J. Am. Chem. Soc. 2001, 123,
4475–4479.
(8) Harmata, M.; Hong, X.; Barnes, C. L. Org. Lett. 2004, 6,
2201–2203.
(9) Davidson, J. P.; Corey, E. J. J. Am. Chem. Soc. 2003, 125,
13486–13489.
(10) Harmata, M.; Hong, X. Org. Lett. 2005, 7, 3581–3583.
(11) Harmata, M.; Cai, Z.; Chen, Y. J. Org. Chem. 2009, 74,
5559–5561.
(12) Lazerwith, S. E.; Johnson, T. W.; Corey, E. J. Org. Lett. 2000, 2,
2389–2392.
(13) Berrue, F.; McCulloch, M. W. B.; Kerr, R. G. Article in press,
accepted manuscript 2011. doi: 10.1016/j.bmc.2011.06.083.
(14) Carpenter, R. D.; Kurth, M. J. Nat. Protoc. 2010, 5, 1731–1736.
(15) Kumar, R. V. Asian J. Chem. 2004, 16, 1241–1260.
(16) The aglycone (prepared by acid hydrolysis of natural
pseudopterosins) exists in the catechol form as evidenced by 13C
NMR (effectively 100%); see Figure S11.
(17) Eseola, A. O.; Li, W.; Adeyemi, O. G.; Obi-Egbedi, N. O.;
Woods, J. A. O. Polyhedron 2010, 29, 1891–1901.
(18) Ooyama, Y.; Ito, G.; Kushimoto, K.; Komaguchi, K.; Imae, I.;
Harima, Y. Org. Biomol. Chem. 2010, 8, 2756–2770.
(19) Eseola, A. O.; Zhang, M.; Xiang, J.; Zuo, W.; Li, Y.; Woods,
J. A. O.; Sun, W. Inorg. Chim. Acta 2010, 363, 1970–1978.
(20) Itoh, S.; Mure, M.; Suzuki, A.; Murao, H.; Ohshiro, Y. J. Chem.
Soc., Perkin Trans. 2 1992, 1245–1251.
(21) Sleath, P. R.; Noar, J. B.; Eberlein, G. A.; Bruice, T. C. J. Am.
Chem. Soc. 1985, 107, 3328–3338.
(22) Vinsova, J.; Horak, V.; Buchta, V.; Kaustova, J. Molecules 2005,
10, 783–793.
(23) Clarke, T. G.; Hampson, N. A.; Lee, J. B.; Morley, J. R.; Scanlon,
B. F. J. Chem. Soc., C 1970, 815–817.
’ ASSOCIATED CONTENT
S
Supporting Information. Details of the syntheses of
b
compounds 13À15, 17, and 19, H and 13C NMR spectra of
1
new compounds (5, 6a, 6b, 7a, 12À20) and of semisynthetic
pseudopteroxazole (1) and homopseudopteroxazole (2), in
addition to the 13C NMR spectrum of the known pseudopterosin
GÀJ aglycone (3). This material is available free of charge via the
Internet at http://pubs.acs.org.
(24) Look, S. A.; Fenical, W.; Matsumoto, G. K.; Clardy, J. J. Org.
Chem. 1986, 51, 5140–5145.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +1-902-566-0565. Fax: +1-902-566-7445. E-mail: rkerr@
upei.ca.
’ ACKNOWLEDGMENT
The authors are grateful for experimental assistance from M.
Lanteigne (microbiology) and K. Ballem (synthesis) and for NMR
services provided by L. Kerry and C. Kirby (AAFC). The authors
gratefully acknowledge financial support from the Natural Sciences
and Engineering Council of Canada (NSERC), the Canada
Research Chair Program, the Atlantic Innovation Fund, and the
Jeanne and Jean-Louis Levesque Foundation to UPEI; and
Nautilus acknowledges funding from the National Research Coun-
cil’s IRAP program. We are indebted to the Government of The
Bahamas for providing a Marine Resource Collections Permit.
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