Isolation and first total synthesis of PM050489 and PM060184, two new marine anticancer compounds

Article abstract of DOI:10.1021/ja404578u

Microtubules continue to be one of the most successful anticancer drug targets and a favorite hit for many naturally occurring molecules. While two of the most successful representative agents in clinical use, the taxanes and the vinca alkaloids, come fro

Full text of DOI:10.1021/ja404578u

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Article
Isolation and First Total Synthesis of PM050489 and
PM060184, Two New Marine Anticancer Compounds
Maria Jesús Martín, Laura Coello, Rogelio Fernández, Fernando Reyes, Alberto
Rodríguez, Carmen Murcia, María Garranzo, Cristina Mateo, Francisco Sánchez, Santiago
Bueno, Carlos de Eguilior, Andres M. Francesch, Simon Munt, and Carmen Cuevas
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja404578u • Publication Date (Web): 10 Jun 2013
Downloaded from http://pubs.acs.org on June 11, 2013
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Journal of the American Chemical Society
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Isolation and First Total Synthesis of PM050489 and PM060184, Two
New Marine Anticancer Compounds
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María Jesús Martín, Laura Coello, Rogelio Fernández, Fernando Reyes,$ Alberto Rodríguez, Car-
men Murcia, María Garranzo, Cristina Mateo, Francisco Sánchez-Sancho,† Santiago Bueno, Car-
los de Eguilior, Andrés Francesch, Simon Munt, and Carmen Cuevas*
R&D, PharmaMar, S. A., 28770 - Colmenar Viejo, Madrid, Spain
ABSTRACT: Microtubules continue to be one of the most successful anticancer drug targets and a favourite hit for many
naturally occurring molecules. Whilst two of the most successful representative agents in clinical use, the taxanes and the
vinca alkaloids, come from terrestrial sources, the sea has also proven to be a rich source of new tubulin-binding mole-
cules. We describe herein the first isolation, structural elucidation and total synthesis of two totally new polyketides iso-
lated from the Madagascan sponge Lithoplocamia lithistoides. Both PM050489 and PM060184 show antimitotic properties
in human tumor cells lines at sub-nanomolar concentrations and a display distinct inhibition mechanism on microtu-
bules. The development of an efficient synthetic procedure has solved the supply problem and, following pharmaceutical
development, has allowed PM060184 to start clinical studies as a promising new drug for cancer treatment.
antimitotic activity, a new biochemical mechanism of inter-
INTRODUCTION
action with tubulin3 and potent in vivo activity in different
The discovery and development of new drugs that can be
animal models, thereby demonstrating that tubulin contin-
used in the treatment of cancer remains one of the biggest
ues to be a valid target for cancer treatment. Based on its
challenges for the scientific community and the pharmaceu-
activity, distinct mechanism of action and good safety pro-
tical industry. In recent decades there has been a boom in so-
file, PM060184 has entered clinical development and Phase I
called targeted therapies and one of the most successful
trials are currently underway in France, Spain and the United
anticancer drug targets, tubulin, continues to be the focus of
States of America
intense research efforts. Interestingly, nature has proven to
be one of the best sources of new molecules that bind to
tubulin and two of the most successful microtubule-targeting
drugs, paclitaxel and the natural vinca alkaloids, were origi-
nally isolated from terrestrial sources.
The sea has also proven to be a highly productive source of
compounds that bind to tubulin and some of them, such as
discodermolide,1 dolastatins and hemiasterlin, have entered
clinical trials but were later discontinued due to toxicity
issues. More successfully, plinabulin, a synthetic analog of
halimide, continues in clinical development (Figure 1) and
eribulin (Halaven™), an analog of the marine natural macro-
lide halichondrin B synthesized by Kishi,2 was approved by
the FDA and European authorities for breast cancer in 2010
and 2011, respectively.
As part of our continuing research programme to discover
new anticancer compounds from marine sources, we de-
scribe herein the first isolation, full stereo-chemical structur-
al determination, total synthesis and biological activity of
Figure 1. Chemical structures of tubulin binders. Marine
natural products: (a) Discordemolide, (b) Hemiasterlin, (c)
Dolastatin 10. Synthetic compounds inspired by marine natu-
ral compounds: (d) Plinabulin, (e) Eribulin mesylate.
two new marine natural products, PM050489 and PM060184
(1 and 2, Figure 2a), belonging to a new class of polyketides
isolated from the sponge Lithoplocamia lithistoides collected
in Madagascar and, to the best of our knowledge, hitherto
uninvestigated. PM050489 and PM060184 have shown sub-
nanomolar in vitro activity in human cancer cell lines, potent
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HMBC spectrum. The two NH2 protons of this latter group
RESULTS AND DISCUSSION
1
1
2
3
4
5
6
7
8
appeared in the H NMR spectrum as a broad signal that
changed intensity and chemical shift depending on the tem-
perature and concentration of the sample. Finally, to com-
plete the planar structure of 1, the chlorine atom present in
the molecule was placed at C24 in agreement with the substi-
tution pattern observed for the C23-C24 double bond and the
chemical shifts measured for these two olefinic carbons.4
Isolation and structural elucidation. PM050489 (1) and
PM060184 (2) were isolated from extracts (CH2Cl2:MeOH,
50:50) of the sponge Lithoplocamia lithistoides and purified
by reverse-phase RP-18 chromatography and semipreparative
HPLC. Initially, 1.6 mg (0.003%) of PM050489 (1) was ob-
tained from a 61 g sample of frozen L. lithistoides. Isolation of
further 1 from a second larger group of sponge samples yield-
ed 160.8 mg of 1 (0.002%) and 2.6 mg of 2 (0.00003%) from
7.66 Kg. of frozen animal material. The positive ion high-
resolution (ESI) mass spectrum of 1 displayed a pseudomo-
lecular ion at m/z 606.2940 [M+H]+ with an isotopic distribu-
tion consistent with the presence of a chlorine atom in the
molecule. These MS data and the presence of 31 signals in its
13C NMR spectrum (Table 1) gave a molecular formula of
C31H44ClN3O7 for the compound (calcd. for C31H4535ClN3O7
606.2946).
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The analysis of the HSQC spectrum revealed the presence
in the molecule of six aliphatic and one oxygenated methyl
groups, three aliphatic methylene units, and 13 methines,
including one aliphatic, one nitrogenated, two oxygenated,
and nine olefinic. In addition, the 13C spectrum displayed
signals for eight quaternary carbons, attributed to the pres-
ence in the molecule of three carbonyl groups, one carba-
mate, three quaternary olefinic, and one quaternary aliphatic
1
carbon. Two signals in the H spectrum at δH 8.78 and 6.51
ppm did not display any correlations in the HSQC spectrum
and were therefore assigned to two NH amide groups based
on COSY and HMBC correlations.
Careful study of the COSY and HMBC spectra of 1 led to
the identification of four discrete spin systems (A-D) repre-
sented in Figure 2b (H3-H28, H9-H12, NH14-H15 and NH17-
H25). Connectivity between these different systems was
1
established by H-13C long range correlations obtained from
HMBC experiments. Indeed, cross peaks between Me28 and
C7, C8 and C9, H9 and C7, and from H7 and H10 to C8 linked
segments A and B. Likewise, correlations from H11, H12,
NH14 and H15 to C13 coupled segment B to C through an
amide carbonyl group (C13). Finally, segments C and D were
linked in a similar manner based on the observation of
HMBC correlations from H15 and NH17 to C16.
Figure 2. (a) Chemical structures of PM050489 (1) and
PM060184 (2). (b) Key 2D NMR correlations observed for
PM050489 (1). (c) ꢀδ(R,S) values for the MTPA esters 5a and
5b.
The geometry of the double bonds in 1 was determined on
The presence of a tert-butyl group in the molecule was in-
ferred from the presence of a single signal at δH 1.04 ppm in
3
the basis of NOESY correlations (Figure 2b) and JH-H cou-
pling constant values. Values of 3JH9-H10 = 11.6 Hz, 3JH11-H12 = 11.6
1
the H NMR spectrum integrating for nine protons, which
3
Hz, and JH18-H19 = 9.7 Hz, and NOESY correlations between
correlated in both the HSQC and HMBC spectra with an
intense 13C signal at δC 26.7 ppm accounting for three of the
carbons in the molecule. Other HMBC correlations observed
between the methyl groups Me30, Me31, and Me32 and car-
bons C15 and C29, and from H15 to carbons C29 to C32
showed the presence of a tert-leucine (tert-butylglycine) in
the molecule and confirmed the location of this amino acid
residue in the structure of 1. Additionally, the presence of a
6-substituted 3-methoxy-5,6-dihydro-2H-pyran-2-one in
PM050489 (1) was shown by cross-peaks in the HMBC spec-
trum from H3, both H4 protons, and the oxygenated methyl
group Me26 to C2, and from H3 and H5 (weak) to the car-
bonyl group C1.
the pairs H9/H10, H11/H12, and H18/H19 gave a Z geometry
for the double bonds at C9-C10, C11-C12, and C18-C19. On the
other hand, the E geometry of the olefins at C2-C3, C7-C8,
and C23-C24 was established through NOESY cross peaks
observed between Me26/H3, H6/Me28, and H22/Me25, re-
spectively. Additionally, the E geometry of the C7-C8 olefin
was also supported by a NOESY correlation between H7 and
H9.
To determine the absolute configuration at C15, the
Marfey´s method for chiral amino acid analysis was used.5
Compound 1 was dissolved in 6 N HCl and heated at 110 ºC
for 16 hours in a sealed vial. After evaporation of the solvent
under a N2 stream and reaction of the residue with L-FDAA
(Nα-(2,4-dinitro-5-fluorophenyl)-L-alanamide), the mixture
was subjected to reversed phase HPLC-MS analysis. Compar-
ison of the retention time of the derivatized tert-leucine with
that of the derivatized R and S standards of the amino acid
A signal in the 13C NMR spectrum at δC 157.2 ppm ac-
counted for the presence in the molecule of a carbamate
1
group that was placed at C21 on the basis of a H-13C long
range correlation observed between H21 and C33 in the
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established the S configuration for the chiral center at C15 in
PM050489 (1).
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2
3
4
Table 1. 1H and 13C NMR (CDCl3, 500 and 125 MHz) assignments for PM050489 (1) and PM060184 (2).
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PM050489 (1)
δH (m, J in Hz)
PM060184 (2)
δH (m, J in Hz)
Position
δC, mult.
δC, mult.
1
2
3
161.6 C
145.2 C
-
-
161.6 C
145.2 C
-
-
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10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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108.2 CH
5.63 (dd, 6.5, 2.6)
2.45 (ddd, 17.3, 11.5, 2.6)
2.37 (ddd, 17.3, 6.5, 4.1)
4.24 (ddd, 11.5, 7.1, 4.1)
2.85 (ddq, 9.8, 7.1, 6.7)
5.29 (d, 9.8)
-
108.2 CH
5.63 (dd, 6.6, 2.7)
2.44 (m)
4
26.1 CH2
26.1 CH2
2.39 (m)
5
81.9 CH
37.1 CH
134.1 CH
133.7 C
140.2 CH
124.6 CH
137.6 CH
120.7 CH
166.3 C
-
81.9 CH
37.1 CH
134.1 CH
133.8 C
140.2 CH
124.4 CH
137.5 CH
120.8 CH
166.3 C
-
4.25 (ddd, 11.3, 7.0, 4.0)
2.85 (ddq, 9.9, 7.0, 6.7)
5.29 (d, 9.9)
-
6
7
8
9
6.17 (d, 11.6)
7.30 (dd, 11.6, 11.6)
6.91 (dd, 11.6, 11.6)
5.70 (d, 11.6)
-
6.15 (d, 11.6)
7.31 (dd, 11.6, 11.6)
6.90 (dd, 11.6, 11.6)
5.72 (br d, 11.6)
-
10
11
12
13
14
15
16
17
18
19
6.51 (d, 9.5)
4.41 (d, 9.5)
-
6.53 (d, 9.6)
4.44 (d, 9.6)
-
60.8 CH
168.2 C
-
60.7 CH
168.2 C
-
8.78 (d, 10.8)
6.84 (br dd, 10.8, 9.7)
4.80 (m)
8.69 (d, 10.4)
6.82 (ddd, 10.4, 9.1, 0.9)
4.82 (m)
124.5 CH
105.0 CH
124.2 CH
105.8 CH
2.46 (m)
2.46 (m)
20
30.7 CH2
30.9 CH2
2.09 (ddd, 14.1, 8.4, 8.1)
4.41 (m)
2.12 (ddd, 14.1, 8.0, 8.0)
4.45 (m)
21
22
23
24
25
26
27
28
29
30
31
32
33
74.9 CH
33.0 CH2
122.4 CH
132.0 C
75.6 CH
31.4 CH2
124.9 CH
127.1 CH
13.0 CH3
55.4 CH3
16.4 CH3
17.1 CH3
34.8 C
2.33 (m), 2H
5.61 (br t, 6.8)
-
2.35 (m), 2H
5.40 (m)
5.60 (m)
21.0 CH3
55.4 CH3
16.3 CH3
17.1 CH3
34.7 C
2.06 (s), 3H
3.66 (s), 3H
1.15 (d, 6.7), 3H
1.82 (s), 3H
-
1.63 (dd, 6.8, 1.0), 3H
3.66 (s), 3H
1.15 (d, 6.7), 3H
1.82 (s), 3H
-
26.7 CH3
26.7 CH3
26.7 CH3
157.2 C
1.04 (s), 3H
1.04 (s), 3H
1.04 (s), 3H
-
26.7 CH3
26.7 CH3
26.7 CH3
157.6 C
1.04 (s), 3H
1.04 (s), 3H
1.04 (s), 3H
-
The S absolute stereochemistry at C5 was elucidated by
Mosher analysis of the corresponding hydroxyl group,6
obtained after reduction of the lactone and protection of
the resulting primary hydroxyl group at C1 as its TBS (tert-
butyldimethylsilyl) ether (Scheme 1). Treatment of 1 with
NaBH4 (Sodium borohydride) yielded 1,5-diol 3 that was
regioselectively converted into its 1-TBS derivative 4 by
reaction with TBSCl (tert-butyldimethylsilyl chloride).
Esterification of this compound with S- and R-MTPA-Cl (α-
methoxy-α-trifluoromethylphenylacetyl chloride) yielded
compounds 5a and 5b in which elimination of the C26
methyl group and conversion of the resulting enol to the
ketone had taken place in addition to the formation of the
corresponding R- or S-MTPA ester at C5. Analysis of the
ꢀδ(R,S) values obtained for each derivatives (Figure 2c)
indicated the S absolute stereochemistry for the C5 chiral
center according to Riguera’s predictive models for MTPA
esters.7
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and 2, respectively. A metal-promoted coupling8 between
the vinylic fragment A and the organometallic fragment B
Scheme 1. Synthesis of the MTPA esters at C5 (5a
and 5b).a
1
2
was expected to afford the main skeleton of each molecule.
3
4
5
6
7
8
9
Many cross-coupling type reactions could be employed
to form the C10-C11 bond, such as the Stille, Suzuki or
Hiyama reactions, with the halide (or triflate) and the or-
ganometallic moiety being on either fragment A or B. The
Sonogashira coupling reaction between a terminal alkyne
and a vinyl halide could also be a good option. Retrosyn-
thetic analysis of fragment A (Figure 3) includes double
bond formation at C2-C3 through Horner-Wadsworth-
Emmons olefination, double bond formation at both C7-C8
and C9-C10 via Wittig olefination, and Evans oxazoli-
dinone-mediated aldol condensation between aldehyde C
and (R)-4-Benzyl-3-propionyl-2-oxazolidinone to control
the C5 and C6 carbon centers. For fragment B, the C21
stereocenter is introduced through resolution of racemic
epoxide D, addition of propyne, and regioselective hy-
drozirconation followed by Cl substitution with N-
chlorosuccinimide or by catalytic hydrogenation.
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In the case of PM060184 (2), the positive ion high-
resolution (ESI) mass spectrum displayed a pseudomolecu-
lar ion at m/z 594.3152 [M+Na]+ with an isotopic distribu-
tion consistent with the absence of a chlorine atom in the
molecule. These MS data and the presence of 31 signals in
its 13C NMR spectrum (Table 1) gave a molecular formula of
C31H45N3O7 for the compound (calcd. for C31H45N3O7Na
1
594.3150). The H NMR spectra of 2 were identical to the
corresponding spectra for 1 except for the presence of an
extra olefinic proton (at 5.60 ppm), which was assigned to
the C24 position by COSY and HMBC. The geometry of the
C23-C24 olefin was assigned as Z by the ROESY cross-peak
between H22/Me25. The configuration at C5, C6, C15 and
C21, and the geometry of the double bonds C2-C3, C7-C8,
C9-C10, C11-C12 and C18-C19 was seen to be identical to
those of PM050489 (1) by comparing the NMR data.
Figure 3. Retrosynthetic analysis of PM050489 (1) and
PM060184 (2).
At this point, the small amounts of each molecule isolat-
ed from the natural source had served not only to show
their promise as potent anticancer agents but also for initial
structural elucidation as described above. Nevertheless, the
absolute configuration of the stereocenters at C6 and C21
remained unknown and we turned our attention to the
design of a synthetic process that would not only allow the
configuration of the remaining two stereocenters to be
determined but that would also solve the supply problem
and open the way to the possible future clinical develop-
ment of these compounds. The supply-issue is often one of
the biggest barriers to the successful development of ma-
rine natural products.
Synthesis of fragment A. The starting point for the im-
plementation of this strategy was the alcohol 8 (Scheme 2)
obtained in six steps from 1,3-propanodiol and (R)-4-
benzyl-3-propionyl-2-oxazolidinone.9 Alcohol 8 was oxi-
dized to aldehyde 9 in 90% yield using sulfur trioxide pyri-
dine complex. Wittig reaction of aldehyde 9 with the stabi-
lized
ylide
(1-
Ethoxycarbonylethylidene)triphenylphosphorane afforded
stereoselectively (E:Z >95:5) the corresponding ester 10,
which was reduced with DIBAL (diisobutylaluminum hy-
dride) to allylic alcohol 11 in 77% yield. To introduce the
diene moiety, alcohol 11 was oxidized with manganese(IV)
oxide to aldehyde 12, which was then converted to vinyl
Total synthesis. PM050489 (1) and PM060184 (2) are
formed by an α,β-unsaturated δ-lactone, a conjugated
triene system and a small natural amino acid (L-tert-
Leucine) that are linked via a (Z)-enamide to a diene sys-
tem containing a carbamate subunit. Of the different pos-
sible synthetic approaches, we chose to adopt a convergent
strategy, disconnecting the C10-C11 bond to give two subu-
nits, fragments A and B (Figure 3). Whilst fragment A is
common to both compounds and crucial for elucidating the
absolute stereochemistry at C6, fragment B needed to be
prepared both with and without chlorine at C24 to obtain 1
iodide
13
by
olefination
with
(iodome-
thyl)triphenylphosphonium iodide10 in 84% yield with a
95:5 ratio of Z:E isomers.11 Homologation of 13 began with
regioselective deprotection of the primary TBS ether using
PPTS (pyridinium p-toluenesulfonate) in EtOH to give
alcohol 14 in excellent yield (93%) before oxidation to alde-
hyde 15 with sulfur trioxide pyridine complex in 80% yield.
Horner-Wadsworth-Emmons olefination of aldehyde 15
with
diethyl
(meth-
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oxy(methoxycarbonyl)methyl)phosphonate12 provided ester
16 as a 90:10 mixture of (E:Z) isomers. On treatment of the
1
2
3
4
5
isomers with HCl in MeOH, only the major E-isomer was
able to cyclise, giving the desired lactone 17 (5S,6S, relative
anti configuration) in a yield of 94%. Lactone 17 serves as
fragment A for the synthesis of both 1 and 2.
6
7
8
Scheme 2. Synthesis of Fragment A.a
9
I
(Ph3PCH2I)+I-
e
O
TBSO
OH
TBSO
TBSO
a
Ph3P
CO2Et
TBSO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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42
43
44
45
46
47
48
49
50
51
52
53
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60
R
OTBS
OTBS
OTBS
b
OTBS
8
9
10 R= CO2Et
11 R= CH2OH
12 R= CHO
c
13
d
f
CO2Me
MeO
CO2Me
I
I
I
O
i
g
MeO
PO(OEt)2
h
HO
MeO
O
O
OTBS
OTBS
I
OTBS
14
17, 5S
24, 5R
16, 5S
23, 5R
15, 5S
22, 5R
j
n
I
I
PivO
PivO
OH
OTBS
21
18
m
k
I
I
PivO
PivO
l
18
+
OH
O
20
19
.
aConditions: (a) SO3 Py, Et3N, CH2Cl2:DMSO (2.2:1), 0 ºC, 2 h, 90%; (b) carboethoxyethylidene-triphenylphosphorane, toluene, 60 ºC, 17 h, 96%; (c) DIBAL,
THF, -78 ºC, 4 h, 77%; (d) MnO2, Et2O, 23 ºC, 2 h, 94%; (e) iodomethyl triphenylphosphonium iodide, NaHMDS, DMPU, THF, -78 ºC, 2 h, 84%; (f) PPTS, EtOH,
.
23 ºC, 25 h, 93%; (g) SO3 Py, Et3N, CH2Cl2:DMSO (2.2:1), 0 ºC, 2 h, 80%; (h) diethyl(methoxy[methoxycarbonyl]methyl)phosphonate, KHMDS, 18-crown-6, THF,
-78 ºC, 1.5 h, 59% (16) and 85% (diastereomer 23); (i) HCl 37%, MeOH, 23 ºC, 6 h, 94% (17) and 57% (diastereomer 24); (j) (i) pivaloyl chloride, Et3N, THF, 23
ºC, 17 h; (ii) HCl 37%, MeOH, 23 ºC, 3 h, 33%; (k) Dess-Martin periodinane, CH2Cl2, 23 ºC, 45 min, 95%; (l) NaBH4, MeOH, -78 ºC, 40 min, 80%; (m) TBSOTf,
2,6-lutidine, CH2Cl2, 23 ºC, 30 min, 95%; (n) (i) NaOH, MeOH, 40 ºC, 4 h, 91%, (ii) Dess-Martin periodinane, CH2Cl2, 23 ºC, 1 h, 51%.
In order to complete the full stereochemical elucidation
of 1 and 2, we also synthesized the (5R,6S)-lactone (relative
syn configuration), from intermediate 14 following the
strategy of oxidation/reduction of the alcohol at C5. Or-
thogonal protection of alcohol 14 with pivaloyl chloride and
removal of the TBS ether gave intermediate 18. Oxidation of
18 with DMP (Dess-Martin periodinane)13 afforded ketone
19 which was nonselectively reduced with NaBH4 to obtain
a mixture of diastereoisomers (R,S)-20 and (S,S)-18 which
were separated by column chromatography. Protection of
the secondary alcohol of 20 as a TBS ether with TBSOTf
(tert-butyldimethylsilyl trifluoromethanesulfonate) afford-
ed compound 21. Finally, deprotection of the pivaloyl ether
with NaOH and oxidation of the resulting primary alcohol
with DMP yielded aldehyde 22 which was transformed into
lactone 24 (5R,6S, relative syn configuration) via ester 23 as
described previously. Instead of using J-based configura-
tional14 analysis to determine the configuration at C6, we
compared the NMR spectroscopic data of lactones 17
(5S,6S) and 24 (5R,6S) and observed multiplicity patterns
for protons H5 and H6 of the anti lactone 17 that were very
similar to those of 1, whilst great differences were observed
in the case of the syn lactone 24 (Figure 4). As such, the syn
configurations (5R,6S) and (5S,6R) could be eliminated
leaving only the two anti configurations, (5S,6S) and
(5R,6R), as remaining possibilities. Then, since the absolute
configuration at C5 had already been unambiguously as-
signed as S (see above), the C6 stereochemistry could also
now be unambiguously assigned as S.
Figure 4. Lactones 17 and 24.
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amines 42 and 43 with longer reaction times giving degra-
dation.
The reagents HOAt (1-hydroxy-7-azabenzotriazole) and
HATU (N,N,N',N'-Tetramethyl-O-(7-azabenzotriazol-1-
yl)uronium hexafluorophosphate) are widely used in pep-
tide synthesis to give rapid couplings with minimal loss of
chiral integrity.22 As such, these reagents were used to
couple amines 42 and 43 to the carboxylic acid 44 affording
stannanes 45 and 46 in 66-77% yield. Carboxylic acid 44
was prepared in two steps by hydrostannation of ethyl
propiolate with tributyltin hydride followed by hydrolysis
with aqueous LiOH in THF.
1
2
3
4
5
6
7
8
Synthesis of fragment B. Having established efficient
access to the common lactone-diene framework of 1 and 2,
the next challenge consisted in the synthesis of fragment B.
Our starting material was but-3-en-1-yloxy-tert-butyl-
dimethylsilane (Scheme 3). The required chiral center at
C21 was obtained from racemic butene oxide (±)-25 by way
of Jacobsen’s hydrolytic kinetic resolution.15 Addition of a
lithium anion derived from propyne to (R)-26 in THF then
yielded alcohol 27, and subsequent conversion to the PMB
(p-methoxybenzyl) ether provided 28. Gratifyingly, when
chiral alkyne 28 was reacted with three equivalents of
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12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
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60
Schwartz´s
reagent
(Cp2Zr.HCl,
bis(cyclopentadienyl)zirconium(IV) chloride hydride) in
toluene at 50 ºC, followed by addition of N-
chlorosuccinimide, 29 was obtained in high yield and with
excellent control of the olefin geometry (99% yield, 100%
regioselectivity). PMB proved to be a key group in the hy-
drozirconation of alkyne 2816 with the TBS or TBDPS (tert-
butyldiphenylsilyl) ether analogs of 28 being found to give
low levels of regioselectivity with Schwartz´s reagent and
mixtures of the (E)-2-choroalkene and (E)-3-chloroalkene
isomers. Cleavage of the TBS group of 29 with TBAF (tetra-
n-butylammonium fluoride) solution in THF, afforded
alcohol 30 which was converted to the vinyl iodide 32 using
similar methodology as employed in fragment A. The most
favourable oxidation process for alcohol 30 were Piancatelli
conditions,17 employing BAIB ((diacetoxyiodo)benzene)
and
a
catalytic
amount of TEMPO (2,2,6,6-
Tetramethylpiperidinin-1-yl)oxyl.
After several attempts to use the PMB ether throughout
the synthetic sequence, problems with the final deprotec-
tion of the PMB ether forced us to abandon this intermedi-
ate in favour of the TBS analogue 34. The PMB protecting
group of 32 was removed with DDQ (2,3-dichloro-5,6-
18
dicyano-1,4-benzoquinone) in aqueous CH2Cl2 and purifi-
cation of 33 simplified by treating the mixture with NaBH4
to reduce the p-methoxy-benzaldehyde by-product to the
alcohol. Protection of secondary alcohol 33 using TBSOTf
and 2,6-lutidine was straightforward and gave the key vinyl
iodide 34 required for the synthesis of 1.
For the synthesis of 2, we used alcohol 27 as starting ma-
terial to prepare vinyl iodide 39 which is an analog of 34 but
without chlorine. In this case, we decided to use TBDPS as
protecting group and reaction of 27 with TBDPSCl (tert-
butyldiphenylsilyl chloride) in the presence of DMAP (4-
dimethylaminopyridine) afforded intermediate 35 which
was subjected to Lindlar catalytic hydrogenation19 at at-
mospheric pressure to obtain stereoselectively Z alkene 36.
Finally, vinyl iodide 39 was obtained via intermediates 37
and 38 using the same synthetic strategy as used for 32.
With vinyl iodides 34 and 39 in hand, we now needed to
introduce the final chiral part of fragment B. Boc-tBu-Gly-
NH2 (N-(tert-butoxycarbonyl)-L-tert-leucine-amide), pre-
pared following Pozdnev procedure,20 was reacted with 34
and 39 under Buchwald conditions,21 affording enamides 40
and 41 in moderate yield (44-53%). The propensity of both
the TBS and TBDPS protecting groups to cleavage under
acidic conditions forced us to remove the Boc group by
pyrolysis. Dissolution of 40 and 41 in ethylenglycol and
heating at 200 ºC for not more than 20 minutes afforded
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Scheme 3. Synthesis of Fragment B.a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Final steps. At this point, the fragment A (lactone 17)
Scheme 4. Final steps of the synthesis of PM050489
(1) and PM060184 (2).a
and fragment B (vinyl stannnes 45 and 46) intermediates
required to make 1 and 2 were now all available. For the
final coupling, use of CuTc (copper(I) thiophene-2-
carboxylate) in NMP (1-methyl-2-pyrrolidinone) under
Liebeskind conditions23 provided trienes 47 and 48 in 66%
yield (Scheme 4). Cleavage of the TBS or TBDPS groups
with TBAF in THF generated compounds 49 and 50 in
adequate yields. Introduction of the C21 carbamate moiety
was then achieved by reaction of 49 and 50 with trichloroa-
cetyl isocyanate and subsequent treatment with neutral
alumina finally yielded the desired compounds 1 and 2 in
13
81-96% yield.24 All the spectral data (1H & C NMR, optical
rotation, IR, etc), HPLC retention times and biological
activities of the synthetic samples exactly matched those of
the isolated natural products.
Following completion of the total synthesis of 1 as de-
scribed above, the R absolute configuration could finally be
assigned to C21. This was achieved by using racemic epox-
ide (±)-25 to prepare intermediate 34 as a mixture of epi-
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mers and subsequent downstream conversion to obtain 49
1
2
3
4
5
6
7
8
as a mixture of the two alcohol epimers at C21 with
(5S,6S,15S,21R) and (5S,6S,15S,21S) stereochemistry. The two
epimers were separated by HPLC and the stereochemistry
of each assigned by preparing the Mosher derivatives,6
before the introduction in each derivative the carbamate
moiety. The NMR spectra of the natural product 1 clearly
matched those of the 21-R epimer whereas major differ-
ASSOCIATED CONTENT
Experimental procedures, spectral and other characteriza-
tion data. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
ences were observed for the H and 13C resonances in the
AUTHOR INFORMATION
region around the C21 chiral center in the 21-S stereoisomer
thereby confirming the R configuration for C21 in 1. In the
case of 2, the lack of chlorine at C22 means that although
there is no change in the spatial disposition of the different
groups around C21, the descriptor of the C21 configuration
is now S and that of the double link C23-C24 is now E (not
Z as in 1) due to the different assignment of priorities ac-
cording to the CIP (Cahn-Ingold-Prelog) priority rules.
9
Corresponding Author
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*ccuevas@pharmamar.com
Present Addresses
$Fundación Medina, 18016 – Granada, Spain. †Instituto de
Química Médica, CSIC, 28006 – Madrid, Spain.
In summary, the first total synthesis of PM050489 (1) and
PM060184 (2) was achieved in a total of 35 and 33 steps,
respectively. 18 steps from 1,3-propanediol is the longest
linear sequence required to synthesize these marine natural
products. The highly convergent synthesis we report herein
has been industrialized and has also been used for the
synthesis of new analogues.
ACKNOWLEDGMENT
We thank Dr. L. F. García-Fernández and Dr. A. Losada for
the cytotoxicity and antimitotic assays, J. L. Carballo for the
taxonomic identification of the sponge, and Dr. S. González
for recording the NMR spectra. We thank Profs. Federico
Gago, Claudio Palomo and Antonio M. Echavarren for
constructive and helpful comments during preparation of
the manuscript.
Biological evaluation. Two bioassays were conducted
in parallel during fractionation of the sponge extracts: (i)
cell killing ability, evaluated against a panel of three human
tumor cell lines, including colon (HT-29), lung (A-549),
and breast (MDA-MB-231), and (ii) antimitotic activity,
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(ELISA). The in vitro antiproliferative activity was assessed
using the colorimetric SRB (sulforhodamine B) method,
performed as described previously.25 The GI50 (nM), con-
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