Discovery of potent and practical antiangiogenic agents inspired by cortistatin A

Article abstract of DOI:10.1021/ja902601e

The discovery that cortistatins A and J show noteworthy antiangiogenic activity prompted an investigation of the possibility that simpler and much more easily made compounds based on a steroid core might have useful bioactivity. These studies have led to

Full text of DOI:10.1021/ja902601e

Published on Web 05/26/2009
Discovery of Potent and Practical Antiangiogenic Agents
Inspired by Cortistatin A
Barbara Czako´,† La´szlo´ Ku¨rti,† Akiko Mammoto,‡ Donald E. Ingber,‡ and
E. J. Corey*,†
Department of Chemistry and Chemical Biology, HarVard UniVersity, 12 Oxford Street,
Cambridge, Massachusetts 02138, and Department of Pathology, HarVard Medical School,
Vascular Biology Program, Childrens Hospital, 300 Longwood AVenue,
Boston, Massachusetts 02115
Received April 1, 2009; E-mail: corey@chemistry.harvard.edu
Abstract: The discovery that cortistatins A and J show noteworthy antiangiogenic activity prompted an
investigation of the possibility that simpler and much more easily made compounds based on a steroid
core might have useful bioactivity. These studies have led to the development of several potent, water-
soluble compounds that may be suitable for local application to treat ocular wet macular degeneration, an
important cause of blindness, as well as for treatment of various other angiogenesis-dependent diseases.
One of these substances was tested in a mouse retinal angiogenesis model and found to inhibit angiogenesis
at a locally administered dose of 500 pmol. Comparison of cell migration data for this and two other synthetic
compounds with published data on cortistatin A indicate that they inhibit vascular endothelial growth factor-
induced cell migration of human umbilical vein endothelial cells more strongly than cortistatin A.
Introduction
One of the most significant recent developments in cancer
therapy is the discovery of a high-affinity antibody that
neutralizes the action of the vascular endothelial growth factor
(VEGF).1 That antibody (Avastin, Genentech) now has sales
of several billion dollars per annum. A modification of this
recombinant DNA-derived antibody is also useful for the
treatment of wet macular degeneration. Marketed as Lucentis
by Genentech, it has annual sales of about a billion dollars. In
cancer therapy, the antibody functions to inhibit the formation
of new blood vessels that are required for solid tumor growth.1,2
In the case of wet macular degeneration, the antibody inhibits
the excessive proliferation of blood vessels that leads to the
destruction of healthy tissue and consequently compromises
vision.3
Our research was stimulated by the discovery of a potent
antiangiogenic natural product, cortistatin A (1, Figure 1),
isolated as a trace component from a marine sponge, Corticulum
simplex, by Kobayashi in 2006.4 This complex steroidal natural
product was shown to exhibit highly selective antiproliferative
activity against human umbilical vein endothelial cells (HUVEC)
at nanomolar concentrations. It also was found to inhibit VEGF-
induced migration and basic fibroblast growth factor (bFGF)-
induced tubular network formation of HUVECs at ca. 200 nM
Figure 1. Structures of cortistatin natural products.
concentrations.5 In addition to cortistatin A (1), 10 related natural
products have been isolated from the same sponge, one of which,
cortistatin J (10), also exhibited good antiangiogenic activity
(Figure 1).6,7 To date, the exact cellular target of the cortistatins
has not been determined, and in ViVo studies of 1 have not been
reported.
Initially we were intrigued by the challenge of devising a
synthesis of cortistatin A and carried out preliminary studies
† Harvard University.
‡ Harvard Medical School.
(1) Kim, K. J.; Li, B.; Winer, J.; Armanini, M.; Gillett, N.; Phillips, H. S.;
Ferrara, N. Nature 1993, 362, 841–844.
(5) Aoki, S.; Watanabe, Y.; Tanabe, D.; Arai, M.; Suna, H.; Miyamoto,
K.; Tsujibo, H.; Tsujikawa, K.; Yamamoto, H.; Kobayashi, M. Bioorg.
Med. Chem. 2007, 15, 6758–6762.
(2) Folkman, J. N. Engl. J. Med. 1971, 285, 1182–1186.
(3) Ferrara, N.; Damico, L.; Shams, N.; Lowman, H.; Kim, R. Retina
2006, 26, 859–870.
(6) Aoki, S.; Watanabe, Y.; Tanabe, D.; Setiawan, A.; Arai, M.;
Kobayashi, M. Tetrahedron Lett. 2007, 48, 4485–4488.
(7) Watanabe, Y.; Aoki, S.; Tanabe, D.; Setiawan, A.; Kobayashi, M.
Tetrahedron 2007, 63, 4074–4079.
(4) Aoki, S.; Watanabe, Y.; Sanagawa, M.; Setiawan, A.; Kotoku, N.;
Kobayashi, M. J. Am. Chem. Soc. 2006, 128, 3148–3149.
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Antiangiogenic Agents Inspired by Cortistatin A
A R T I C L E S
on a synthetic pathway to 1 from the readily available starting
material, estrone.8 As this work progressed, we realized that
instead of targeting the total synthesis of 1, it would be a more
important contribution to take the natural product as a lead
molecule and initiate a research program to devise and
synthesize analogues that have equal or greater biological
activity, but are structurally less complex and much more readily
available for further biological studies than the natural product.
To date, three total syntheses of cortistatin A have been reported.
However, only milligram quantities of synthetic 1 have been
prepared.9
Design and Synthesis of Analogues of Cortistatin A
In order to design and synthesize structurally simple but
biologically active analogues of the natural cortistatins, we first
identified the minimal structural elements present in the natural
products that are essential for antiangiogenic activity. Based
on the biological data reported for the various cortistatins, the
following conclusions can be drawn:5 [1] The most active
members of the coristatin family, coristatin A (1) and cortistatin
J (10), incorporate a dimethylamino group at the C3 position
and an isoquinoline appendage at C17, suggesting that these
subunits contribute significantly to biological activity. [2]
Cortistatin J (10) does not have hydroxyl groups at C1 and C2
positions in contrast to cortistatin A (1), implying that these
groups may not be essential. [3] Substitution at the C16 and
C17 positions is not tolerated and leads to a significant decrease
of growth inhibition of HUVECs [coristatins B (2) and D (4)].
[4] Replacement of the isoquinoline subunit with a 4-isopentyl-
1,3-dimethylpiperidine [cortistatins E (5) and F (9)], a 3-methyl-
4-(3-methylbut-1-enyl)pyridine [cortistatin G (6)], or a 4-isopentyl-
3-methylpyridine side chain [cortistatin H (7)] results in
decreased biological activity and selectivity.
Figure 2. Structurally simple analogue of cortistatin A.
Scheme 1. Synthesis of Analogues 12 and 13
On the basis of these data and the assumption that the
distance between the dimethylamino and isoquinoline sub-
stituents should be maintained, it seemed logical to evaluate
compounds having a steroidal core, such as 12 (Figure 2).
Such structures have the advantage of being synthetically
accessible and, hence, potentially practical for therapeutic
use. We were attracted to compounds containing a C16-C17
double bond because overlay studies suggested a better fit
for this compound than the corresponding saturated deriva-
tive,10 and for ease of synthesis. Our plan also encompassed
the study of diastereomeric 3R- and 3ꢀ-amino compounds
and 19-norsteroids, as well as 19-methyl-containing steroids.
Analogues 12 and 13 were prepared starting from the 17-
ketal of 3-O-methyl estrone 14 using the sequence shown in
Scheme 1: Birch reduction of 14, selective acidic hydrolysis
of the resulting enol ether, base-mediated isomerization of
the double bond,11 and Li/NH3 reduction of the R,ꢀ-enone
provided ketone 15. Although reductive amination of 15 using
(8) Kürti, L.; Czakó, B.; Corey, E. J. Org. Lett. 2008, 10, 5247–5250.
(9) For total synthesis of cortistatin A, see: (a) Shenvi, R. A.; Guerrero,
C. A.; Shi, J.; Li, C.-C.; Baran, P. S. J. Am. Chem. Soc. 2008, 130,
7241–7243. (b) Nicolaou, K. C.; Sun, Y.-P.; Peng, X.-S.; Polet, D.;
Chen, D. Y. K. Angew. Chem., Int. Ed. 2008, 47, 7310–7313. (c) Lee,
H. M.; Nieto-Oberhuber, C.; Shair, M. D. J. Am. Chem. Soc. 2008,
130, 16864–16866. For approaches toward the cortistatin core, see:
(d) Simmons, E. M.; Hardin, A. R.; Guo, X.; Sarpong, R. Angew.
Chem., Int. Ed. 2008, 47, 6650–6653. (e) Dai, M.; Danishefsky, S. J.
Tetrahedron Lett. 2008, 49, 6610–6612. (f) Craft, D. T.; Gung, B. W.
Tetrahedron Lett. 2008, 49, 5931–5934. (g) Kürti, L.; Czakó, B.;
Corey, E. J. Org. Lett. 2008, 10, 5247–5250.
dimethylamine afforded a 1.4:1 mixture of diastereomers, the
ꢀ-dimethylamino ketone 16 could be obtained diastereose-
lectively via the sequence: (1) reduction of the ketone 15
with K-Selectride, (2) Mitsunobu inversion to the azide, (3)
reduction, (4) methylation of the resulting amine, and (5)
removal of the ketal protecting group. Subsequently, ketone
16 was converted to the corresponding enol triflate, which
(10) Overlay of stereochemical models was done using ChemBio3D Ultra
11.0 based on the minimal energy conformations of 1 and 12.
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Scheme 2. Synthesis of 7-Tributylstannyl Isoquinoline 20
Scheme 4. Varying the Heterocycle at the C17 Position
Scheme 3. Synthesis of 21 and 22
Scheme 5. Reduction of 23
was coupled with 7-tributylstannyl isoquinoline (20) using
the Corey/Han/Stoltz-modified Stille reaction to provide the desired
analogue 12.12 7-Tributylstannyl isoquinoline (20) was prepared
by treatment of 7-bromoisoquinoline (19) with n-BuLi and tribu-
tyltin chloride (Scheme 2). 7-Bromoisoquinoline (19) was synthe-
sized following a procedure described for 7-chloroisoquinoline,13
affording a 1.5:1 mixture of 7-bromoisoquinoline and 5-bromoiso-
quinoline. Fortunately, the HBr salts of these isomers could be
separated readily by crystallization. To arrive at R-dimethylamino
ketone 13, enone 17 was reduced to the corresponding ꢀ-alcohol
using Li/NH3/EtOH and subsequently converted to analogue 13
following a sequence similar to that described for 12.
Scheme 6. Preparation of Pyrrolidine and Morpholine Analogues of
Compounds 12 and 23
Detailed biological evaluation of 12 and 13 indicated that
the 3ꢀ-dimethylamino diastereomer 12 is more active, and
consequently further studies concentrated on compounds in the
3ꢀ-series. Intermediate 12 was used to prepare the related 17ꢀ-
oriented isoquinoline 21 by diimide reduction of the double bond
(potassium azodicarboxylate, AcOH)14,15 and the tetrahydroiso-
quinoline derivative 22 by reductive methylation (CH2O and
NaBH3CN), as shown in Scheme 3.
Intermediate 16 allowed systematic variation of the appendage
at C17, as shown in Scheme 4. Specifically, compounds 23-28
were synthesized for biological studies from 16. Further, the
17ꢀ analogue (29) of 23 was produced by diimide reduction
(Scheme 5).
We also investigated a series of compounds in which the 3ꢀ-
dimethylamino group was replaced by a 3ꢀ-pyrrolidino or 3ꢀ-
morpholino group. These compounds were prepared as shown
in Scheme 6.
(11) Fedorova, O. I.; Pekarskaya, E. S.; Lukashina, I. V.; Grinenko, G. S.
Pharm. Chem. J. 1974, 8, 462.
Finally, we prepared the steroidal analogues 39-42 based
on the 19-norsteroids 12, 23, 25, and 27, as summarized in
Scheme 7, for biological evaluation.16
(12) Han, X.; Stoltz, B. M.; Corey, E. J. J. Am. Chem. Soc. 1999, 121,
7600–7605.
(13) We slightly modified Brown’s procedure describing the preparation
of 7-chloroisoquinoline: Brown, E. V. J. Org. Chem. 1977, 42, 3208–
3209.
Biological Evaluation of Cortistatin Analogues
(14) Pasto, D. J.; Taylor, R. T. Organic Reactions; Wiley: Hoboken, NJ,
1991; Vol. 40, pp 91-150.
The antiangiogenic activity of the new steroidal and 19-
norsteroidal analogues of cortistatin were evaluated in Vitro
using assays for VEGF-stimulated HUVEC growth, migration,
and tubular network formation. The data obtained indicates that
(15) Catalytic hydrogenation using Pd/C under a variety of conditions led
to partial reduction of the isoquinoline. Raney nickel hydrogenation
provided a very low yield of the isolated product.
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Scheme 7. Synthesis of Analogues 39-42
Figure 5. Inhibition of HUVEC growth by compounds 12 and 13.
the activity of 1 and 12 directly, comparison of the literature
data reported for 1 with the experimental results obtained
for 12 suggests that the inhibitory effect of 12 on cell
migration is similar to that of 1 and that compounds 23, 25,
and 27 exhibit stronger inhibitory activity on VEGF-induced
cell migration of HUVECs than 1.5 The IC50 of compound
23 for inhibition of VEGF-induced cell migration was 70.7
nM.
B. Studies of HUVEC Growth. The effect of the active
compounds on HUVEC growth was examined by measuring
nuclear incorporation of BrdU into DNA.18 Lead compounds
12 and 13 inhibited BrdU incorporation at doses greater than 1
µM, respectively, and 12 inhibited BrdU incorporation at 200
nM concentration (Figure 5).
Compound 23, which was the most active analogue based
on migration assays, also exhibited strong inhibition of cell
growth induced by VEGF, bFGF, and PDGF (platelet-derived
growth factor), with IC50’s of 16.7, 64.1, and 79.5 nM,
respectively, when tested individually against each of these
growth factors (see Figure 11, below) [IC50(1) ) 1.8 nM].
Compound 27 inhibited cell growth induced by VEGF at 50
nM concentration, but it inhibited growth induced by bFGF
and PDGF only at higher concentrations (Figure 6).
Compounds 39-42 did not significantly inhibit cell growth
induced by VEGF (20 ng/mL) at 200 nM, although they
inhibited growth by about half at 1000 nM (Figure 7).
C. Studies of Tubular Network Formation. The effect of
these compounds on in Vitro angiogenesis was investigated
using the Matrigel tube formation assay.19,20 It was found
that lead compounds 12 and 13 (2 µM) inhibited tubular
network formation when cultured with VEGF in the basal
EBM2 medium with a 0.5% serum, but 12 was more potent
(Figure 8).
the most active of the compounds described above were 23,
25, and 27. In the VEGF-stimulated migration of HUVECs, for
instance, these compounds showed a 50% or greater inhibition
vs controls at a concentration of 50 nM (Figure 3).
A. Cell Migration Studies. Cell migration is important for
angiogenesis; therefore, it was examined whether the above-
prepared compounds control HUVEC motility stimulated by
VEGF using a transwell migration assay.17 These cell
migration assays revealed that compounds 23, 25, and 27
exhibited strong inhibitory activity at a concentration of 50
nM, while 24 and 26 were moderately active at this
concentration and 28 was inactive (Figure 4). Lead com-
pounds 12 and 13 exhibited moderate inhibitory activity at
200 nM concentration. Steroidal analogues 39-42, based on
the structure of 12 and 23, were less active than the
corresponding 19-norsteroidal analogues at 50 nM concentra-
tion. Reduced derivates of 12 (i.e., 21 and 22) did not show
inhibition at 50 nM concentration, but 21 was active at 200
nM concentration. Compound 29, obtained by the reduction
of 23, was less active than the parent compound. Pyrrolidine
and morpholine derivatives 32, 33, 35, and 36 did not inhibit
cell migration at 50 nM concentration. Although authentic
samples of cortistatin A were not available for us to compare
The most active compound based on cell migration and
growth assays, 23, produced significant inhibition of tubular
network formation at 50 nM concentration when cultured with
VEGF in the basal EBM2 medium (Figure 9). Quantified data
Figure 3. Most active analogues.
Figure 4. Inhibition of VEGF-induced migration of HUVECs by synthetic amino steroids and 19-norsteroids.
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Figure 10. Effect of 12 on retinal vessel formation of P7 mice.
Figure 6. Inhibition of VEGF-, bFGF-, and PDGF-induced HUVEC growth
by compounds 23 and 27.
Figure 11. Effect of 23 on retinal vessel formation of P6 mice.
by intravitreous injection of these compounds into the eyes of
newborn mice.21 Compound 12 appeared to inhibit retinal angio-
genesis based on morphological analysis with 5-10 nmol in a
single injection (Figure 10).
Figure 7. Inhibition of VEGF-induced HUVEC growth by compounds
39-42.
Based on the very impressive in Vitro biological results
obtained for compound 23, inhibition of retinal vessel formation
by 23 in P6 (6 days post birth) mice was examined. These
experiments revealed that compound 23 produced significant
inhibition of retinal angiogenesis in P6 mice after a single
injection of 500 pmol of 23. Compound 23 was more effective
than lead compound 12, which required more than 10 times
higher doses (5-10 nmol) to produce similar effects (Figure
11). Similar in ViVo experiments have not been reported for 1.
In Vitro cell toxicity measurements of 12 and 23 with several
cell cultures revealed no observable toxicity at 50 µM.
Conclusion
Figure 8. Inhibition of VEGF-induced tubular network formation by 12
and 13.
Based on the structure of the highly complex sterodial
antiangiogenic natural products, the cortistatins, a series of
analogues were designed and synthesized that inhibit capillary
cell growth, migration, in Vitro tube formation, and in ViVo
angiogenesis in the living retina. These compounds are
structurally less complex and much more readily accessible
by synthesis than the natural product. The most active and
most promising of the new compounds reported herein were
the 3ꢀ-dimethylamino-19-norsteroids with ∆16,17 unsaturation.
The substituent at C17 could be varied somewhat, but a basic
(16) Reyes, M.; Rosado, A.; Alvarez, Y. M.; Ruiz, J. A.; Aguero, J.; Velez,
H. J. Chem. Res., Synop. 2003, 234–235.
(17) Shimizu, A.; Mammoto, A.; Italiano, J. E., Jr.; Pravda, E.; Dudley,
A. C.; Ingber, D. E.; Klagsbrun, M. J. Biol. Chem. 2008, 283, 27230–
27238.
(18) Huang, S.; Chen, C. S.; Ingber, D. E. Mol. Biol. Cell 1998, 9, 3179–
3193.
Figure 9. Inhibition of tubular network formation by 23.
(19) Grant, D. S.; Kinsella, J. L.; Kibbey, M. C.; LaFlamme, S.; Burbelo,
P. D.; Goldstein, A. L.; Kleinman, H. K. J. Cell Sci. 1995, 108, 3685–
3694.
on the inhibition of VEGF-induced tubular network formation
by cortistatin A (1) have not been reported.
D. In ViWo Studies with Mice Using Compounds 12 and
23. To begin exploring the clinical relevance of these compounds,
the effect of compound 12 on retinal angiogenesis was examined
(20) Mammoto, A.; Connor, K. M.; Mammoto, T.; Yung, C. W.; Huh, D.;
Aderman, C. M.; Mostoslavsky, G.; Smith, L. E. H.; Ingber, D. E.
Nature (London, U.K.) 2009, 457, 1103–1108.
(21) Chen, J.; Connor, K. M.; Aderman, C. M.; Smith, L. E. H. J. Clin.
InVest. 2008, 118, 526–533.
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A R T I C L E S
heterocycle seemed important for bioactivity. These com-
pounds inhibit the angiogenic effects of VEGF, and some
have the ability to inhibit cell sensitivity to multiple
angiogenic factors, the most active analogue being 23, which
exhibits highly potent antiangiogenic activity at low nano-
molar concentrations in in Vitro assays. Comparison of the
biological activity of the synthetic compounds 23, 25, and
27 with published data on cortistatin A indicates that the
synthetic compounds inhibit VEGF-induced cell migration
of HUVECs more strongly than 1. Most importantly, locally
administered picomole quantities of 23 were shown to inhibit
retinal vessel formation in P6 mice, a recognized animal
model for ocular wet macular degeneration. These data
indicate that these water-soluble, apparently nontoxic com-
pounds (at 50 µM) may be suitable for local application to
treat ocular wet macular degeneration, an important cause
of blindness, as well as for treatment of various other
angiogenesis-dependent diseases, including malignant and
inflammatory conditions.
Acknowledgment. L.K. is a Fellow of the Damon Runyon
Cancer Research Foundation (DRG: 1961-07). This work was
supported by NIH grants CA45548 and CA55833 (to D.E.I.). D.E.I.
is a recipient of a Department of Defense Breast Cancer Innovator
Award.
Supporting Information Available: Detailed experimental
procedures and characterization data for all new compounds.
This material is available free of charge via the Internet at http://
pubs.acs.org.
JA902601E
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