Synthesis of cortistatins A, J, K and L

Article abstract of DOI:10.1038/nchem.794

The cortistatins are a recently identified class of marine natural products characterized by an unusual steroidal skeleton, which have been found to inhibit differentially the proliferation of various mammalian cells in culture by an unknown mechanism. We describe a comprehensive route for the synthesis of cortistatins from a common precursor, which in turn is assembled from two fragments of similar structural complexity. Cortistatins A and J, and for the first time K and L, have been synthesized in parallel processes from like intermediates prepared from a single compound. With the identification of facile laboratory transformations linking intermediates in the cortistatin L synthetic series with corresponding intermediates to cortistatins A and J, we have been led to speculate that somewhat related paths might occur in nature, offering potential sequencing and chemical detail for cortistatin biosynthetic pathways.

Full text of DOI:10.1038/nchem.794

ARTICLES
PUBLISHED ONLINE: 1 AUGUST 2010 | DOI: 10.1038/NCHEM.794
Synthesis of cortistatins A, J, K and L
Alec N. Flyer, Chong Si and Andrew G. Myers
*
The cortistatins are a recently identified class of marine natural products characterized by an unusual steroidal skeleton,
which have been found to inhibit differentially the proliferation of various mammalian cells in culture by an unknown
mechanism. We describe a comprehensive route for the synthesis of cortistatins from a common precursor, which in turn
is assembled from two fragments of similar structural complexity. Cortistatins A and J, and for the first time K and L, have
been synthesized in parallel processes from like intermediates prepared from a single compound. With the identification of
facile laboratory transformations linking intermediates in the cortistatin L synthetic series with corresponding
intermediates to cortistatins A and J, we have been led to speculate that somewhat related paths might occur in nature,
offering potential sequencing and chemical detail for cortistatin biosynthetic pathways.
ince the structure of cortistatin A was elucidated by Kobayashi
and colleagues in 2006, more than ten natural cortistatins have The o-vinyl benzylzinc reagent 8, the A-ring precursor, was derived
Results
S
been described, which have a common modified steroidal skel-
from the benzyl bromide 14, which was prepared in amounts up to
eton with varying substitution of the A- and D-rings1–3. Many of 47 g by the four-step sequence shown in Fig. 3 (via intermediates
these have profound effects on cultured mammalian cells, and on 10–13). Direct insertion of zinc25 into the carbon–bromine bond
human umbilical vein endothelial cells (HUVECs) in particular1–4
.
of the benzyl bromide 14 was attempted only once, without
In 2008, Baran and colleagues reported the first laboratory synthetic success; however, a two-stage process for the preparation of organo-
route to cortistatin A (ref. 5), from prednisone, and subsequent to zinc reagent 8 proved to be both highly reliable and readily scalable.
this a number of different approaches to the synthesis of cortistatins Addition of benzyl bromide 14 to activated magnesium turnings26
have been pursued, focused largely on the most highly functiona- in ether at 23 8C led to rapid (,30 min) formation of a deep
lized (and potent) member of the family, cortistatin A (1). No green solution of the corresponding Grignard reagent, contami-
fewer than three independent routes to cortistatin A have been nated with ,5% of the product of Wurtz coupling. In contrast, for-
reported to date5–7, as well as a number of synthetic studies8–18 mation of the Grignard reagent in tetrahydrofuran as solvent was
and formal routes19,20. In addition, the Nicolaou–Chen group has accompanied by as much as 65% of the Wurtz coupling by-
described a synthesis of cortistatin J (ref. 21).
product. Addition of the freshly prepared ethereal Grignard
Structure–activity relationships among natural cortistatins and product to a solution of anhydrous zinc chloride in tetrahydrofuran
synthetic analogues have suggested that the 7-substituted isoquinoline at 23 8C led to the formation of a cloudy white suspension of the
appendage isa key determinant of the phenotypic effects ofcortistatins presumed o-vinyl benzylzinc reagent 8, which was used directly
upon cells grown in culture4,21–23. The Nicolaou–Chen group recently for coupling with the enol triflate 9 (see below).
reported that cortistatin A inhibits the function of a number of differ-
ent kinases in vitro and proposed that the natural product may occupy
the ATP-binding site of at least one of the enzymes they identified24.
OH
CH3
CH3
HO
N
N
C
Before this, Kobayashi and colleagues had shown that the addition
of cortistatin A to cultured HUVECs led to a marked decrease in
levels of an unidentified 110 kDa phosphoprotein4.
O
O
B
D
A
H
H
(CH3)2N
(CH3)2N
Cortistatin J (2)
Cortistatin A (1)
We envisioned that the naturally occurring cortistatins A, J, K
and L (1–4, respectively, in Fig. 1) and a diverse array of cortistatin
analogues, including compounds suitable for use as biological
probes in target identification experiments, could be prepared
from the single azido alcohol intermediate 5 (Fig. 2). We considered
it likely that one or more pathways linking the azido alcohol 5 to the
cyclohexadienone 6 could be developed. The oxabicyclic core of the
latter precursor could then be disconnected by an oxidative cycliza-
tion transform, a strategy independently conceived and previously
1.8 nM
1 nM
2.1 0.4 nM
Ref. 1
Ref. 21
(this work)
Ref. 3
Ref. 21
(this work)
8 nM
70 13 nM
46 36 nM
2
CH3
CH3
HO
(CH3)2N
N
N
O
O
H
H
(CH3)2N
Cortistatin K (3)
Cortistatin L (4)
40 nM
112 45 nM
Ref. 3
(this work)
23 nM
32 11 nM
Ref. 3
(this work)
demonstrated by Sarpong and colleagues, as well as others8,15,16,18
.
To allow for a convergent assembly process, we imagined an
earlier disconnection of the seven-membered B-ring by an olefin
metathesis reaction, giving rise to the triene precursor 7, which
we envisioned could be synthesized by coupling of the o-vinyl ben-
zylzinc reagent 8 and the enol triflate intermediate 9, structures of
similar complexity, but less than certain viability as stable
chemical entities.
Figure 1 | Cortistatins A, J, K and L and their IC50 values measured in
cultured HUVECs. Members of the cortistatin natural product family have a
common modified steroidal skeleton with varying substitutions, primarily
within the A, B and C rings. These substitutional variations are found to
modulate the potencies of natural cortistatins as growth inhibitors of
HUVECs in culture.
Harvard Department of Chemistry and Chemical Biology, 12 Oxford Street, Box 408, Cambridge, Massachussetts 02138, USA.
e-mail: myers@chemistry.harvard.edu
*
886
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
CH3
OTBS
CH3
OTBS
sequence, albeit using a substrate with different A-ring substituents
(1-methoxyl and 2-p-methoxybenzyl groups)8.
HO
N3
O
O
O
H
H
We found that we could achieve the desired transformation
(18 ꢁ 20) by reversing the order of the two operations and in
so doing we were able to avoid an isolation step. Thus, direct addition
of a solution of dimethyldioxirane (1.5 equiv.) in acetone34 to the
unpurified metathesis product solution at 0 8C led to stereoselective
epoxidation of the tetrasubstituted alkene, affording the acid-sensi-
tive tetracyclic diene monoepoxide 19. Without purification, the
epoxide 19 was hydrogenated in benzene under 500 psi hydrogen
in the presence of a mixture of Wilkinson’s catalyst (0.15 equiv.)
and suspended solid sodium bicarbonate (1.5 equiv.), the latter
being added as an acid scavenger. The saturated product, epoxide
20, then underwent selective eliminative opening with lithium
diethylamide (2 equiv.) in tetrahydrofuran at 215 8C (ref. 35), fur-
nishing the conjugated allylic alcohol 21, a sufficiently polar inter-
mediate to allow for facile chromatographic isolation (.95%
5
6
OTBS
OTIPS
CH3
CH3
OTBS
TIPSO
+
ZnCl
TfO
H
H
8
9
7
Figure 2 | A key precursor to cortistatins and its retrosynthetic
disconnection. The azido alcohol intermediate 5 serves as a common
intermediate for the synthesis of cortistatins A, J, K and L, as well as
cortistatin analogues. This intermediate arises in turn from the
cyclohexadienone 6 and the triene 7. The latter intermediate is formed by
coupling of the o-vinyl benzylzinc reagent 8 and the enol triflate
intermediate 9. TBS, tert-butyldimethylsilyl; TIPS, triisopropylsilyl; Tf,
trifluoromethanesulfonyl.
1
purity, H NMR (nuclear magnetic resonance) analysis, 50% yield
over the four-step sequence). Cleavage of the triisopropylsilyl protec-
tive group with tetra-n-butylammonium fluoride (1.1 equiv.) in
tetrahydrofuran and oxidative cyclization of the resulting phenol
with [bis(trifluoroacetoxy)iodo]benzene (1.8 equiv.) in a mixture
of dichloromethane and hexafluoroisopropanol provided the cyclo-
The enol triflate coupling partner 9, the CD-ring precursor, was
synthesized in a three-step sequence from the known a-methylene
ketone 15 (Fig. 4). The latter substance was first prepared in its
antipodal form by Danishefsky and colleagues27 (from the
Hajos–Parrish enedione)28 in the context of their synthesis of
taxol, and was also selected as a starting material by the
Nicolaou–Chen6 and Sorensen18 groups in their research directed
toward the synthesis of cortistatins. As illustrated in Fig. 4, phospho-
niosilylation29,30 of a-methylene ketone 15 with triphenylphosphine
(1.02 equiv.) and triethylsilyl trifluoromethanesulfonate (1.02 equiv.)
at 240 8C in chloroform, uniquely effective as a solvent in this
particular transformation, afforded the phosphonium salt 16
(containing a tetrasubstituted triethylsilyl enol ether), which was
observed to be stable towards aqueous workup and extractive
isolation. Previously, phosphoniosilylation–Wittig sequences
have been conducted without isolation of the intermediate
phosphonium salts30; this was not an option in the present case,
because chloroform was not compatible with formation of the
Wittig reagent. The oily phosphonium salt obtained upon extractive
isolation and concentration was dissolved in tetrahydrofuran,
and after cooling to 278 8C, the resulting solution was treated
sequentially with n-butyllithium (1.1 equiv.) and paraformaldehyde
(5 equiv.), affording the a-vinyl triethylsilyl enol ether 17 in 75%
yield over the two steps from the a-methylene ketone 15.
Methodology for triflation developed by Corey and colleagues was
used to transform the triethylsilyl enol ether 17 into the corres-
ponding enol triflate derivative 9 (92% yield after purification by
flash-column chromatography)31. This product, a colourless oil,
was stored frozen as a solution in benzene for no longer than a
few days; decomposition was observed to occur over longer
periods of time.
hexadienone 6 in 50% yield8,18,36
.
The synthesis of the key azido alcohol intermediate 5 was
achieved in three steps from the cyclohexadienone 6, a sequence
initiated by hydrosilylation with triethylsilane (2 equiv.) in the pres-
ence of Wilkinson’s catalyst (0.05 equiv.)37. The intermediate
triethylsilyl enol ether was not isolated; rather, pyridine was added
(14% by volume) followed by N-bromosuccinimide (2 equiv.),
forming the (3R)-bromo ketone 22 as a single diastereomer in
70% yield. In the absence of pyridine, or with lesser quantities of
pyridine, the bromination reaction was much less stereoselective,
suggesting the lower selectivity was related to the presence of
Wilkinson’s catalyst during the bromination (bromination of the
triethylsilyl enol ether generated in the absence of Wilkinson’s cat-
alyst was highly diastereoselective). Bromide displacement with tet-
ramethylguanidinium azide (2 equiv.) proceeded with clean
inversion of C3-stereochemistry and this was followed by direct
reduction of the resulting (3S)-a-azido ketone with catecholborane
(2 equiv.) in the presence of the (R)-Corey–Bakshi–Shibata (CBS)
catalyst38 (0.2 equiv., 240 8C) and tetramethylguanidine (1 equiv.),
producing a 15:1 mixture of the (2S)- and (2R)-configured alcohols,
respectively, in 85% yield after chromatographic isolation. Similar
stereocontrol of the C3-centre could not be achieved with other
reductants examined (lithium tri-tert-butoxyaluminum hydride
OH
OTIPS
OTIPS
PdCl2(PPh3)2
CH2 CHZnBr
TIPSOTf
2,6-lutidine
THF
93%
THF
100%
CO2CH3
CO2CH3
CO2CH3
I
10
I
Coupling of the enol triflate 9 (1 equiv.) with the benzylzinc
reagent 8 (ꢀ1.2 equiv., a solution in ether–tetrahydrofuran) in the
presence of tris(dibenzylideneacetone)dipalladium (0.045 equiv.)
and the ligand 2-dicyclohexylphosphino-2′,6′-dimethoxy-biphenyl
(S-Phos, 0.18 equiv.)32 in N-methylpyrrolidone at 70 8C (20 h)
afforded the metathesis substrate 7 in 70% yield on a 16 g scale.
Warming of a 0.2 M solution of the latter product with the
second-generation Grubbs catalyst (0.025 equiv.)33 in dichloro-
methane at 45 8C for 5 h led to highly efficient ring-closing meta-
11
12
DIBAL-H
95%
OTIPS
OTIPS
OTIPS
Br2, PPh3
imidazole
Mg, Et2O;
ZnCl
Br
OH
CH2Cl2
88%
ZnCl2, THF
8
14
13
thesis, forming the tetracyclic diene 18 (.95% yield, 11 g scale). In Figure 3 | Preparation of the o-vinyl benzylzinc reagent 8. The
our initial route design we had planned to hydrogenate selectively functionalized benzylzinc reagent 8 is prepared from the corresponding
the disubstituted alkene within this product and then epoxidize Grignard reagent and anhydrous zinc chloride. The Grignard reagent is
the remaining (tetra-substituted) alkene; however, we were not readily formed from the benzyl bromide 14, which is prepared in multigram
able to achieve selective 1,2-reduction of the diene. Sarpong and col- amounts by a four-step sequence. THF, tetrahydrofuran; Ph, phenyl;
leagues have reported the successful execution of this exact two-step DIBAL-H, diisobutylaluminium hydride.
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
887

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
OTIPS
ZnCl
OTBS
CH3
CH3 OTBS
OTBS
OTBS
CH3
H
CH3
H
n-BuLi
(CH2O)n
CH3
OTBS
TESOTf
PPh3
CsF
PhNTf2
8
TIPSO
THF
75%
DME
92%
O
TESO
TESO
Pd2dba3, S-Phos
70%
CHCl3
TfO
H
H
H
PPh3+ TfO−
15
16
17
9
7
Grubbs II
catalyst
CH3
OTBS
CH3
OTBS
CH3
OTBS
CH3
OTBS
(PPh3)3RhCl
500 psi H2
TIPSO
TIPSO
TIPSO
TIPSO
LiNEt2
DMDO
O
O
HO
THF
50%
PhH
H
H
H
H
21
20
19
18
CH3
OTBS
CH3
OTBS
Et3SiH
(PPh3)3RhCl
CH3
OTBS
O
O
Br
HO
N3
2
TBAF; PhI(OCOCF3)2
1. TMG azide
O
O
O
CH2Cl2, (CF3)2CHOH
50%
then Py, NBS
2. (R)-CBS catalyst
catecholborane
85%, 15:1 d.r.
H
H
H
70%, >20:1 d.r.
Azido alcohol 5
6
22
Figure 4 | Synthesis of azido alcohol 5 from a-methylene ketone 15. The a-methylene ketone 15 is transformed in three steps to the enol triflate
intermediate 9. Coupling with the functionalized benzylzinc reagent 8 affords the triene 7, which undergoes efficient ring-closing metathesis and oxidation
(in situ, with dimethyldioxirane) to form the tetracyclic diene monoepoxide 19. A sequence of hydrogenation, eliminative epoxide opening, silyl ether cleavage
and oxidative cyclization provides cyclohexadienone 6, which is converted stereoselectively in three steps to the key azido alcohol intermediate 5. TES,
triethylsilyl; DME, 1,2-dimethoxyethane; Pd2dba3, tris(dibenzylideneacetone)dipalladium(0); S-Phos, 2-dicyclohexylphosphino-2’,6’-dimethoxybiphenyl; DMDO,
dimethyl dioxirane; TBAF, tetra-n-butylammonium fluoride; Py, pyridine; NBS, N-bromosuccinimide; TMG azide, tetramethylguanidinium azide; CBS, Corey–
Bakshi–Shibata. d.r., diastereometric ratio.
was the next most selective reductant, affording a 5:1 mixture of pro- and diol protection with chlorotriethylsilane then furnished the pro-
ducts favouring the (2S)-isomer).
tected 17-keto cortistatin A precursor (29) in 65% yield for the two
Shown in parallel in Fig. 5 are the sequential pathways by which steps (75 mg scale).
the key intermediate 5 was transformed into the corresponding 17-
Syntheses of the 17-keto precursors to cortistatins J, K and L were
keto precursors to cortistatins A, J, K and L. The synthetic route to substantially more direct (Fig. 5). The 17-keto cortistatin J precursor
the 17-keto precursor to cortistatin A (29) is depicted first, and was (32) was synthesized in just three steps from the azido alcohol 5.
initiated by protection of the 2-hydroxyl substituent of intermediate Following reductive (di)methylation to form the dimethylamino
5 with excess chloroacetyl chloride in pyridine. Cleavage of the tert- alcohol 30 (85% yield), 1,6-elimination of water and deprotection
butyldimethylsilyl ether within the resulting product occurred upon of the tert-butyldimethylsilyl ether occurred simultaneously upon
exposure to aqueous hydrofluoric acid in a 1:1 mixture of aceto- stirring the dimethylamino alcohol 30 with a biphasic mixture of
nitrile and tetrahydrofuran at 0 8C, affording the alcohol 23 in chloroform and concentrated hydrochloric acid (20 min, 23 8C).
80% yield. Significantly, we observed that if during the latter trans- Without purification, the product of the latter transformation (31)
formation the reaction mixture was allowed to warm from 0 to was subjected to oxidation with the Dess–Martin periodinane,
23 8C, 1,6-elimination of the chloroacetate ester accompanied providing the 17-keto cortistatin J precursor (32) as a white solid
deprotection of the tert-butyldimethylsilyl ether, an observation (77% yield following chromatographic purification, two steps).
later used to advantage in the synthesis of the 17-keto precursor
The synthetic route to the 17-keto precursor to cortistatin K (35)
to cortistatin (32). Oxidation of alcohol 23 with the also proceeded through the dimethylamino alcohol 30 (derived
J
Dess–Martin periodinane (2.4 equiv.) in dichloromethane at 0 8C from the azido alcohol 5, as described in the paragraph above), in
followed by addition of methanol and solid potassium carbonate a four-step sequence that was initiated by acetylation at 0 8C with
gave in a single operation the keto alcohol 24, in 85% yield. acetic anhydride (a large excess) and scandium triflate as catalyst
Addition of N-bromosuccinimide (1.02 equiv.) to a solution of (0.05 equiv.) in a 1:1 mixture of acetonitrile and dichloromethane
keto alcohol 24 in a 4:1 mixture of acetonitrile and methanol, (93% yield)41. Regioselective reductive cleavage of the allylic C–O
respectively, at 23 8C led to stereoselective (trans-diaxial) 1,4-bro- bond of the resulting acetate occurred in the presence of lithium
moetherification of the conjugated diene, forming the bromide 25, borohydride (excess) and tetrakis(triphenylphosphine)-palladium
which, without purification, was directly subjected to nucleophilic (0.2 equiv.)42. This afforded the dimethylamino diene 34 in
displacement at 0 8C with potassium superoxide (2.1 equiv.) in a complex with borane; decomplexation was achieved by treatment
3:1 mixture of toluene and dimethyl sulfoxide, respectively39,40
.
of this intermediate with Raney nickel in methanol43, providing
After extractive isolation and chromatography, the trans-diol the free amine as a pale yellow solid (90% yield after chromato-
methyl ether 26 was obtained in diastereomerically pure form graphic purification over Davisil silica gel). Silyl ether cleavage
(41% yield, 100 mg scale, two steps). When combined with scan- (tetra-n-butylammonium fluoride, 87% yield) and oxidation
dium triflate (0.03 equiv.) and trifluoroacetic acid (9.4 equiv.) in (Dess–Martin periodinane, 90%) then completed the route to the
dioxane at 23 8C, this substance was found to undergo efficient 17-keto cortistatin K precursor (35), also obtained as a pale
1,2-elimination of methanol, affording the azido diol 27 (94% yellow solid.
yield). The methoxyl substituent of substrate 26 is axially oriented
Finally, the 17-keto precursor to cortistatin L (38) was syn-
and therefore stereoelectronically disposed towards elimination; thesized from the azido alcohol 5 by a straightforward four-step
none of the other allylic C–O bonds of substrate 26 (or product sequence involving silyl ether deprotection (conducted at 0 8C, as
27) is so oriented. Reductive (di)methylation of the azido group in the cortistatin A series, to avoid 1,6-elimination), selective
888
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
a
Cortistatin A series
Cl
O
Br CH3O
CH3
CH3
OTBS
CH3
OH
DMP
CH2Cl2;
CH3
O
HO
N3
O
HO
N3
O
HO
N3
1. ClCH2COCl, Py
NBS
O
H
O
O
O
2. HF, 0°C
80%
K2CO3,
CH3OH
85%
CH3CN, CH3OH
H
H
H
N3
5
23
24
25
KO2, 18-Cr-6
41%
HO
OTES
OH
OH
CH3O
O
TESCl
DMAP
CH3
CH3
O
CH3
O
CH3
O
Sc(OTf)3, TFA
HO
N3
TESO
(CH3)2N
HO
(CH3)2N
P(CH3)3;
HO
N3
O
O
O
O
CH2Cl2
Formalin
NaBH3CN
Dioxane
94%
H
H
H
H
65%
29
28
27
26
b
Cortistatin J series
CH3
OH
CH3
O
CH3
OTBS
CH3
OTBS
HO
HO
P(CH3)3;
DMP
HCl
O
O
O
O
CH2Cl2
77%
Formalin
NaBH3CN
85%
CHCl3
H
H
H
(CH3)2N
(CH3)2N
H
(CH3)2N
N3
5
30
31
32
c
Cortistatin K series
1. P(CH3)3;
formalin
NaBH3CN
Pd(PPh3)4
LiBH4;
CH3
O
CH3
OTBS
CH3
OTBS
CH3
OTBS
HO
N3
1. TBAF
AcO
O
O
O
O
2. Ac2O
Sc(OTf)3
79%
Raney-Ni
CH3OH
90%
2. DMP
78%
H
H
(CH3)2N
H
H
(CH3)2N
(CH3)2N
5
33
34
35
Cortistatin L series
d
CH3
O
CH3
OTBS
CH3
OH
CH3
OH
TBSO
HO
N3
TBSO
N3
TBSO
P(CH3)3;
DMP
1. HF, 0 °C
O
O
O
O
CH2Cl2
90%
Formalin
NaBH3CN
2. TBSCl
DBU
74%
H
H
(CH3)2N
H
H
(CH3)2N
5
36
37
38
90%
Figure 5 | Synthetic pathways to 17-keto precursors to cortistatins A, J, K and L from azido alcohol 5. a, Synthesis of the 17-keto precursor to cortistatin A
(29) by an eight-step sequence. b, Three-step sequence to the 17-keto precursor to cortistatin J (32). c, Synthesis of the 17-keto precursor to cortistatin K
(35) by a five-step sequence. d, Four-step sequence to the 17-keto precursor to cortistatin L (38). DMP, Dess–Martin periodinane;18-Cr-6, 18-crown-6; TFA,
trifluoroacetic acid; DMAP, 4-dimethylaminopyridine; Ac, acetyl; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene.
OTES
OTES
OH
CH3
O
CH3
CH3
TESO
TESO
HO
N
N
O
O
O
OH
H
H
H
(CH3)2N
(CH3)2N
(CH3)2N
1. TFAA, Py
DMAP
2. Bu3SnH, AIBN
PhH, 100°C
3. TBAF
40
62%
Cortistatin A precursor (29)
Cortistatin A (1)
61%
CH3
O
CH3
CH3
CH3
CH3
TBSO
HO
TBSO
N
N
N
N
N
O
O
O
O
O
OH
OH
OH
N
H
41
52%
H
H
(CH3)2N
(CH3)2N
(CH3)2N
Li
39
Cortistatin L precursor (38)
Cortistatin L (4)
TMEDA
72%
THF, −78°C
CH3
CH3
O
O
O
H
H
H
(CH3)2N
(CH3)2N
(CH3)2N
1. TFAA, Py
DMAP
2. Bu3SnH, AIBN
PhH, 100°C
42
60%
Cortistatin J precursor (32)
Cortistatin J (2)
65%
CH3
O
CH3
N
O
O
H
H
(CH3)2N
(CH3)2N
H
(CH3)2N
43
55%
Cortistatin K precursor (35)
Cortistatin K (3)
65%
Figure 6 | Syntheses of cortistatins A, L, J and K. Parallel paths of 17-keto addition of 7-lithioisoquinoline followed by reductive deoxygenation transform
each 17-keto cortistatin precursor into the corresponding cortistatin. TMEDA, N,N,N′,N′-tetramethylethylenediamine; TFAA, trifluoroacetic anhydride; AIBN,
2,2′-azobis(2-methylpropionitrile).
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.
889

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
CH3
OTBS
CH3
OTBS
HO
MsCl, Et3N
O
O
H
H
CH2Cl2, 0°C
75%
(CH3)2N
(CH3)2N
30
44
OH
CH3
OTBS
CH3
OTBS
CH3
DMDO
Na2SO4, NaHCO3
O
TESO
Br
TESO
Br
C6H6
TESO
Br
OTBS
O
O
O
CH2Cl2, 0°C
23°C, 12h
28% (2 steps)
H
H
H
45
46
47
OH
CH3
CH3
O
HO
HO
(CH3)2N
R
R
Epoxidation
Activation
1,4-Elimination
1,6-Elimination
O
O
O
H
H
H
(CH3)2N
CH3
CH3
HO
(CH3)2N
R
R
Oxidation
Cortistatin A system
O
O
H
H
(CH3)2N
CH3
CH3
NaO3SO
(CH3)2N
R
R
Cortistatin K system
Cortistatin L system
O
H
(CH3)2N
R = isoquinoline appendage or its biosynthetic precursor
Cortistatin J system
Figure 7 | Chemical interconversions among intermediates of cortistatin series A, J and L and a hypothesized parallel biosynthetic sequence.
Intermediates of the cortistatin L series are found to undergo facile laboratory transformations to form corresponding intermediates of the A and J series,
suggesting a plausible sequencing for the latter stages of cortistatin biosyntheses, which are unknown. Ms, methanesulfonyl.
protection44, reductive (di)methylation and oxidation (Fig. 5, 60% each tertiary alcohol intermediate was readily transformed into
yield overall).
the corresponding trifluoroacetate ester with trifluoroacetic anhy-
Depicted in Fig. 6 are the parallel paths by which each advanced dride and pyridine-N,N-dimethylaminopyridine. When heated
17-keto cortistatin precursor was transformed into the correspond- with tri-n-butyltin hydride (excess) and azobisisobutyronitrile in
ing cortistatin. The method we used to introduce stereoselectively benzene in a sealed tube at 100 8C for 1–2 h, each trifluoroacetate
the 7-isoquinolyl substituent differs from earlier reported syntheses ester underwent efficient deoxygenation to provide the (17S)-con-
of cortistatin A, where in each case a variation of the sequence 17- figured reduction product. Attempted deoxygenation under the
keto ꢁ vinyl iodide5,7 or vinyl triflate6 followed by cross-coupling conditions originally reported by Jang and colleagues (Ph2SiH2,
with a 7-metalloisoquinoline derivative, then stereoselective hydro- (t-BuO)2, 130 8C)46,47 led primarily to elimination of the trifluoro-
genation, was pursued. The hydrogenation step in particular has acetate ester, a problem that was almost completely circumvented
been established to be challenging to execute with intermediates by the use of tri-n-butyltin hydride as reductant at 100 8C.
containing the conjugated diene function of cortistatin A5,7, and
In the cortistatin J and K series, deoxygenation led to these sub-
we believed it likely that this would be even more problematic stances directly; spectroscopic data for the synthetic materials
with precursors to cortistatins J, K and L. As a general alternative, matched values reported for the natural products. In the cortistatin
we developed the three-step 17-keto addition–reductive deoxygena- A and L series, cleavage of the silyl ether protective group(s) with
tion sequence shown in Fig. 6. While our work was in progress, tetra-n-butylammonium fluoride at 23 8C following deoxygenation
Hirama and colleagues reported a conceptually similar but differ- provided these targets; spectroscopic data obtained from the syn-
ently executed approach to introduce the isoquinolyl substituent thetic substances were found to be the same as those reported for
in their formal synthesis of cortistatin A (ref. 19). We observed the natural products. In all four series (A, J, K and L), the yield
that lithiation of 7-iodoisoquinoline (5 equiv.) with an equimolar for the two- or three-step deoxygenation (deprotection) procedure
amount of n-butyllithium in tetrahydrofuran at 278 8C followed was 61–72% (1–20 mg amounts of final product).
by sequential addition of N,N,N′,N′-tetramethylethylenediamine
In addition to spectroscopic characterization, synthetic cortista-
(TMEDA, 15 equiv.) and a 17-keto cortistatin precursor (1 equiv.) tins A, J, K and L were evaluated for their ability to inhibit the
reproducibly afforded the corresponding (17S)-1,2-addition growth of HUVECs in culture (96-h incubation). The GI50 values
product in 52–62% yield in all cases45. In the absence of TMEDA, we measured are summarized in Fig. 1 together with literature
1,2-addition did not occur, undoubtedly a consequence of compet- values. For synthetic cortistatin A, we observed growth inhibition
ing enolization of the 17-keto substituent. Enolization likely occurs, of HUVECs consistent with reported values, whereas measurements
but to a much lesser extent, in our optimized conditions as well (in for synthetic cortistatin J more closely matched reports from the
the presence of TMEDA) for we typically recover between 34–40% Nicolaou–Chen group than initial reports, and GI50 values for syn-
of each 17-keto precursor. The Hirama group developed a very thetic cortistatins K and L fell within the range of those originally
different protocol for metalation (using n-butyllithium and reported or were slightly higher1,3,21
cerium (III) chloride with 1-chloro-7-iodoisoquinoline as substrate)
.
and with this organometallic reagent observed highly efficient Discussion
addition to the carbonyl group of the Nicolaou–Chen 17-keto cor- In the course of our studies we observed that several compounds
tistatin A precursor. They then transformed the resulting tertiary closely related to cortistatin L undergo facile 1,6-elimination to
alcohol into a thionocarbamate derivative and the latter was deoxy- form the conjugated triene function of cortistatin J (see 30 ꢁ 31,
genated using tri-n-butyltin hydride19.
Fig. 5, discussion of intermediate 23 above, and the additional
We briefly explored a similar method for hydroxyl activation in example of the transformation of dienyl alcohol 30 to triene 44,
our deoxygenation studies, but our attempts to form a thionocarbo- Fig. 7). In an interesting and somewhat related transformation, we
nyl ester intermediate were unsuccessful. Instead, we adapted meth- observed that reaction of diene 45 (cortistatin L oxidation state)
odology by Jang and colleagues for free-radical deoxygenation of with dimethyldioxirane afforded the highly sensitive epoxide 46 as
alcohols via their trifluoroacetate esters46,47. As shown in Fig. 6, the primary product, which upon standing in benzene solution
890
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
18. Frie, J. L., Jeffrey, C. S. & Sorensen, E. J. A hypervalent iodine-induced double
annulation enables a concise synthesis of the pentacyclic core structure of the
cortistatins. Org. Lett. 11, 5394–5397 (2009).
19. Yamashita, S., Kitajima, K., Iso, K. & Hirama, M. Efficient and stereoselective
installation of isoquinoline: formal total synthesis of cortistatin A. Tetrahedron
Lett. 50, 3277–3279 (2009).
20. Simmons, E. M., Hardin-Narayan, A. R., Guo, X. & Sarpong, R. Formal total
synthesis of (+)-cortistatin A. Tetrahedron 66, 4696–4700 (2010).
21. Nicolaou, K. C. et al. Total synthesis and biological evaluation of
cortistatins A and J and analogues thereof. J. Am. Chem. Soc. 131,
10587–10597 (2009).
22. Shi, J. et al. Stereodivergent synthesis of 17-a and 17-b-aryl steroids: application
and biological evaluation of D-ring cortistatin analogues. Angew. Chem. Int. Ed.
48, 4328–4331 (2009).
23. Czako, B., Kurti, L., Mammoto, A., Ingber, D. E. & Corey, E. J. Discovery of
potent and practical antiangiogenic agents inspired by cortistatin A. J. Am.
Chem. Soc. 131, 9014–9019 (2009).
24. Cee, V. J., Chen, D. Y. K., Lee, M. R. & Nicolaou, K. C. Cortistatin A is a high-
affinity ligand of protein kinases ROCK, CDK8 and CDK11. Angew. Chem. Int.
Ed. 48, 8952–8957 (2009).
25. Berk, S. C., Knochel, P. & Yeh, M. C. P. General approach to highly
functionalized benzylic organometallics of zinc and copper. J. Org. Chem. 53,
5789–5791 (1988).
26. Pearson, D. E., Cowan, D. & Beckler, J. D. A study of the entrainment method for
making Grignard reagents. J. Org. Chem. 24, 504–509 (1959).
27. Isaacs, R. C. A., Digrandi, M. J. & Danishefsky, S. J. Synthesis of an
enantiomerically pure intermediate containing the CD substructure of taxol.
J. Org. Chem. 58, 3938–3941 (1993).
underwent spontaneous 1,4-elimination to form a product (dienyl
alcohol 47) with functionality characteristic of cortistatin A.
Consideration of these findings has led us to speculate that cortista-
tins A and J may derive from cortistatin L or a precursor of the same
oxidation state, as opposed to an alternative sequencing such as J ꢁ
A (although this cannot be ruled out)2. Cortistatin L might in turn
be derived by C2 oxidation of cortistatin K. We note that many
sterols bearing 2b-hydroxy and 2b-sulfato groups have been iso-
lated from marine sponges, but detailed biosynthetic routes to
these substances have not been determined48,49
.
We have shown that the protected azido alcohol intermediate 5,
synthesized in a nine-step sequence beginning with the coupling of
the benzylzinc reagent 8 and the enol triflate 9, is readily trans-
formed into advanced 17-keto precursors to cortistatins A, J, K
and L. Each of these intermediates is in turn converted into the cor-
responding cortistatin final product by a three- or four-step
sequence involving addition of a 7-isoquinolyl organometallic inter-
mediate followed by deoxygenation (deprotection). The latter
sequence appears to be a general route to cortistatins with divergent
substitutions of the A, B and C rings and it is anticipated that it will
allow for late-stage introduction of diversely substituted isoquino-
line groups and other heterocycles at position C17.
Received 26 May 2010; accepted 21 June 2010;
published online 1 August 2010
28. Hajos, Z. G. & Parrish, D. R. (þ)-(7aS)-7a-methyl-2,3,7,7a-tetrahydro-1H-
indene-1,5-6H-dione. In Organic Syntheses, Vol. 63, 26–31
(Wiley & Sons, 1985).
References
29. Evans, D. A., Hurst, K. M. & Takacs, J. M. New silicon-phosphorus reagents in
organic synthesis: carbonyl and conjugate addition reactions of silicon phosphite
esters and related systems. J. Am. Chem. Soc. 100, 3467–3477 (1978).
30. Kozikowski, A. P. & Jung, S. H. Phosphoniosilylation: an efficient and practical
method for the b-functionalization of enones. J. Org. Chem. 51,
3400–3402 (1986).
31. Mi, Y., Schreiber, J. V. & Corey, E. J. Total synthesis of (þ)-a-onocerin in four
steps via four-component coupling and tetracyclization steps. J. Am. Chem. Soc.
124, 11290–11291 (2002).
1. Aoki, S. et al. Cortistatins A, B, C and D, anti-angiogenic steroidal alkaloids,
from the marine sponge Corticium simplex. J. Am. Chem. Soc. 128,
3148–3149 (2006).
2. Watanabe, Y., Aoki, S., Tanabe, D., Setiawan, A. & Kobayashi, M. Cortistatins E,
F, G and H, four novel steroidal alkaloids from marine sponge Corticium
simplex. Tetrahedron 63, 4074–4079 (2007).
3. Aoki, S. et al. Cortistatins J, K, L, novel abeo-9(10-19)-androstane-type steroidal
alkaloids with isoquinoline unit, from marine sponge Corticium simplex.
Tetrahedron Lett. 48, 4485–4488 (2007).
4. Aoki, S. et al. Structure–activity relationship and biological property of
cortistatins, anti-angiogenic spongean steroidal alkaloids. Bioorg. Med. Chem.
15, 6758–6762 (2007).
32. Walker, S. D., Barder, T. E., Martinelli, J. R. & Buchwald, S. L. A rationally
designed universal catalyst for Suzuki–Miyaura coupling processes. Angew.
Chem. Int. Ed. 43, 1871–1876 (2004).
33. Scholl, M., Ding, S., Lee, C. W. & Grubbs, R. H. Synthesis and activity of a new
generation of ruthenium-based olefin metathesis catalysts coordinated with 1,3-
dimesityl-4,5-dihydroimidazol-2-ylidene ligands. Org. Lett. 1, 953–956 (1999).
34. Murray, R. W. & Singh, M. Synthesis of epoxides using dimethyldioxirane: trans-
stilbene oxide. In Organic Syntheses, Vol. 74, 91–100 (Wiley & Sons, 1997).
35. Thummel, R. P. & Rickborn, B. Base-induced rearrangement of
epoxides. V. Phenyl-substituted epoxides. J. Org. Chem. 37, 3919–3923 (1972).
36. Kita, Y., Tohma, H., Kikuchi, K., Inagaki, M. & Yakura, T. Hypervalent iodine
oxidation of N-acyltyramines: synthesis of quinol ethers, spirohexadienones and
hexahydroindol-6-ones. J. Org. Chem. 56, 435–438 (1991).
37. Kogure, T. & Ojima, I. Reduction of carbonyl compounds via hydrosilylation. 4.
Highly regioselective reductions of a,b-unsaturated carbonyl compounds.
Organometallics 1, 1390–1399 (1982).
38. Corey, E. J. & Helal, C. J. Reduction of carbonyl compounds with chiral
oxazaborolidine catalysts: a new paradigm for enantioselective catalysis and a
powerful new synthetic method. Angew. Chem. Int. Ed. 37, 1986–2012 (1998).
39. San Filippo, J., Chern, C. I. & Valentine, J. S. Reaction of superoxide with alkyl
halides and tosylates. J. Org. Chem. 40, 1678–1680 (1975).
40. Corey, E. J., Nicolaou, K. C., Shibasaki, M., Machida, Y. & Shiner, C. S.
Superoxide ion as a synthetically useful oxygen nucleophile . Tetrahedron Lett.
37, 3183–3186 (1975).
41. Ishihara, K., Kubota, M., Kurihara, H. & Yamamoto, H. Scandium
trifluoromethanesulfonate as an extremely active acylation catalyst. J. Am. Chem.
Soc. 117, 4413–4414 (1995).
42. Hutchins, R. O., Learn, K. & Fulton, R. P. Reductive displacement of allylic
acetates by hydride transfer via catalytic activation by palladium(0) complexes.
Tetrahedron Lett. 21, 27–30 (1980).
43. Couturier, M., Tucker, J. L., Andresen, B. M., Dube, P. & Negri, J. T. Palladium
and Raney nickel catalyzed methanolic cleavage of stable borane–amine
complexes. Org. Lett. 3, 465–467 (2001).
5. Shenvi, R. A., Guerrero, C. A., Shi, J., Li, C.-C. & Baran, P. S. Synthesis of (þ)-
cortistatin A. J. Am. Chem. Soc. 130, 7241–7243 (2008).
6. Nicolaou, K. C., Sun, Y. P., Peng, X. S., Polet, D. & Chen, D. Y. K. Total synthesis
of (þ)-cortistatin A. Angew. Chem. Int. Ed. 47, 7310–7313 (2008).
7. Lee, H. M., Nieto-Oberhuber, C. & Shair, M. D. Enantioselective synthesis of
(þ)-cortistatin A, a potent and selective inhibitor of endothelial cell
proliferation. J. Am. Chem. Soc. 130, 16864–16866 (2008).
8. Simmons, E. M., Hardin, A. R., Guo, X. & Sarpong, R. Rapid
construction of the cortistatin pentacyclic core. Angew. Chem. Int. Ed. 47,
6650–6653 (2008).
9. Yamashita, S., Iso, K. & Hirama, M. A concise synthesis of the pentacyclic
framework of cortistatins. Org. Lett. 10, 3413–3415 (2008).
10. Craft, D. T. & Gung, B. W. The first transannular [4 þ 3] cycloaddition reaction:
synthesis of the ABCD ring structure of cortistatins. Tetrahedron Lett. 49,
5931–5934 (2008).
11. Dai, M. J. & Danishefsky, S. J. A concise synthesis of the cortistatin core.
Tetrahedron Lett. 49, 6610–6612 (2008).
12. Dai, M. J., Wang, Z. & Danishefsky, S. J. A novel a,b–unsaturated nitrone-aryne
[3 þ 2] cycloaddition and its application in the synthesis of the cortistatin core.
Tetrahedron Lett. 49, 6613–6616 (2008).
13. Kotoku, N., Sumii, Y., Hayashi, T. & Kobayashi, M. Synthesis of CD-ring
structure of cortistatin A, an anti-angiogenic steroidal alkaloid from marine
sponge. Tetrahedron Lett. 49, 7078–7081 (2008).
14. Kurti, L., Czako, B. & Corey, E. J. A short, scalable synthesis of the carbocyclic
core of the anti-angiogenic cortistatins from (þ)-estrone by B-ring expansion.
Org. Lett. 10, 5247–5250 (2008).
15. Dai, M. J. & Danishefsky, S. J. An oxidative dearomatization cyclization model
for cortistatin A. Heterocycles 77, 157–161 (2009).
16. Liu, L. Z. et al. A model study for the concise construction of the
oxapentacyclic core of cortistatins through intramolecular Diels–Alder
and oxidative dearomatization–cyclization reactions. Chem. Commun.
662–664 (2009).
17. Magnus, P. & Littich, R. Intramolecular cyclopropene-furan [2 þ 4]
cycloaddition followed by a cyclopropylcarbinyl rearrangement to synthesize the
BCD rings of cortistatin A. Org. Lett. 11, 3938–3941 (2009).
44. Moon, S. S., Stuhmiller, L. M. & McMorris, T. C. Synthesis of oogoniol. J. Org.
Chem. 54, 26–28 (1989).
45. Foy, N. et al. Synthesis, receptor binding, molecular modeling and proliferative
assays of a series of 17a-arylestradiols. Chembiochem. 4, 494–503 (2003).
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
891
© 2010 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.794
ARTICLES
measurements as well as a gift of the HUVEC. We acknowledge helpful discussions with
C. T. Walsh and E. Balskus.
46. Jang, D. O., Kim, J. G., Cho, D. H. & Chung, C. M. Radical deoxygenation of
alcohols via their trifluoroacetate derivatives with diphenylsilane. Tetrahedron
Lett. 42, 1073–1075 (2001).
47. Kim, J. G., Cho, D. H. & Jang, D. O. Radical deoxygenation of tertiary alcohols
via trifluoroacetates. Tetrahedron Lett. 45, 3031–3033 (2004).
48. D’ Auria, M. V., Minale, L. & Riccio, R. Polyoxygenated steroids of marine
origin. Chem. Rev. 93, 1839–1895 (1993).
49. Sarma, N. S., Krishna, M. S. R. & Rao, S. R. Sterol ring system oxidation pattern
in marine sponges. Mar. Drugs 3, 84–111 (2005).
Author contributions
A.N.F., C.S. and A.G.M conceived the synthetic route. A.N.F and C.S. conducted
all experimental work and analysed the results. A.N.F., C.S. and A.G.M. wrote
the manuscript.
Additional information
Acknowledgements
The authors declare no competing financial interests. Supplementary information and
chemical compound information accompany this paper at www.nature.com/
naturechemistry. Reprints and permission information is available online at http://npg.nature.
com/reprintsandpermissions/. Correspondence and requests for materials should be
addressed to A.G.M.
Financial support from the National Institutes of Health (Stimulus grant no. CA047148-
22S1) is gratefully acknowledged. A.N.F. acknowledges a scholarship from Eli Lilly and
Company. We gratefully acknowledge G. Zou for measuring the GI50 values of cortistatins
A, J, K and L and A.W.G. Burgett and M.D. Shair for their assistance with these
892
NATURE CHEMISTRY | VOL 2 | OCTOBER 2010 | www.nature.com/naturechemistry
© 2010 Macmillan Publishers Limited. All rights reserved.