Synthesis and cytotoxicity of a new class of potent decapeptide macrocycles

Article abstract of DOI:10.1021/ol702403r

(Chemical Equation Presented) Described are the syntheses of five decapeptides that are C-2-symmetrical derivatives of the natural product pentapeptide sansalvamide A. Derivatives were made using a succinct convergent synthesis. These analogues share no s

Full text of DOI:10.1021/ol702403r

ORGANIC
LETTERS
2008
Vol. 10, No. 2
177-180
Synthesis and Cytotoxicity of a New
Class of Potent Decapeptide
Macrocycles
Melinda R. Davis, Thomas J. Styers, Rodrigo A. Rodriguez, Po-Shen Pan,
Robert C. Vasko, and Shelli R. McAlpine*
Department of Chemistry, Molecular Biology Institute, and Center for Applied and
Experimental Genomics, 5500 Campanile Road, 208 CSL, San Diego State UniVersity,
San Diego, California 92182-1030
mcalpine@chemistry.sdsu.edu
Received October 2, 2007
ABSTRACT
Described are the syntheses of five decapeptides that are C-2-symmetrical derivatives of the natural product pentapeptide sansalvamide A.
Derivatives were made using a succinct convergent synthesis. These analogues share no structural homology to current cancer drugs, are
cytotoxic at levels on par with existing drugs treating cancers, and demonstrate selectivity for drug-resistant pancreatic cancer cell lines over
noncancerous cell lines. These molecules are excellent chemotherapeutic leads in the search for new anticancer agents.
Pancreatic cancer is the fifth most deadly cancer in the U.S.
Only 10% of patients are eligible for surgery,1 and less than
20% of pancreatic cancers respond to the drug of choice,
gemcitabine.2 The 5-year survival rate for patients with
pancreatic cancers is less than 5%.2,3 With such a low
response rate to current chemotherapeutic treatments, there
is an immediate need for new drugs that provide options to
pancreatic cancer patients. Recent work describing the
synthesis and biological activity of pentapeptide derivatives
based on the natural product sansalvamide A (San A) as
potential therapies for pancreatic cancers has brought atten-
tion to this new compound class.4 San A is a penta-
depsipeptide that exhibits antitumor activity. It was discov-
ered by Fenical and co-workers from a marine fungus of
the genus Fusarium.15 San A derivatives have shown potent
(4) (a) Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743-3746. (b)
Gu, W.; Liu, S.; Silverman, R. B. Org. Lett. 2002, 4, 4171-4174. (c) Carroll,
C. L.; Johnston, J. V. C.; Kekec, A.; Brown, J. D.; Parry, E.; Cajica, J.;
Medina, I.; Cook, K. M.; Corral, R.; Pan, P.-S.; McAlpine, S. R. Org. Lett.
2005, 7, 3481-3484. (d) Liu, S.; Gu, W.; D., L.; Ding, X.-Z.; Ujiki, M.;
Adrian, T. E.; Soff, G. A.; Silverman, R. B. J. Med. Chem. 2005, 48, 3630-
3638. (e) Otrubova, K.; Styers, T. J.; Pan, P.-S.; Rodriguez, R.; McGuire,
K. L.; McAlpine, S. R. Chem. Commun. 2006, 1033-1034. (f) Styers, T.
J.; Kekec, A.; Rodriguez, R.; Brown, J. D.; Cajica, J.; Pan, P.-S.; Parry, E.;
Carroll, C. L.; Medina, I.; Corral, R.; Lapera, S.; Otrubova, K.; Pan, C.-
M.; McGuire, K. L.; McAlpine, S. R. Bioorg. Med. Chem. 2006, 14, 5625-
5631. (g) Rodriguez, R.; Pan, P.-S.; Pan, C.-M.; Ravula, S.; Lapera, S.;
Singh, E.; Styers, T. J.; Brown, J. D.; Cajica, J.; Parry, E.; Otrubova, K.;
McAlpine, S. R. J. Org. Chem. 2007, 72, 1980-2002.
(1) Murr, M. M.; Sarr, M. G.; Oishi, A. J.; Heerden, J. A. CA Cancer J.
Clin. 1994, 44, 304-318.
(2) (a) Burris, H. A.; Moore, M. J.; Andersen, J.; Greem, M. R.;
Rothenberg, M. I.; Modiano, M. R.; Cripps, M. C.; Portenoy, R. K.;
Sotorniolo, A. M.; Tarassaoff, P.; Nelson, R.; Dorr, F. A.; Stephens, C. D.;
vonHoff, D. J. Clin. Oncol. 1997, 15, 2403-2413. (b) Fennelly, D.; Kelsen,
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Am. Coll. Surg. 1999, 189, 1-7. (b) Niederhuber, J. E.; Brennan, M. F.;
Menck, H. R. The national cancer data base report on pancreactic cancer.
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10.1021/ol702403r CCC: $40.75
© 2008 American Chemical Society
Published on Web 12/21/2007

cytotoxicity against pancreatic,4b,5 colon,4c,e,f,6 breast, prostate,
and melanoma cancers,4d clearly indicating the potential of
this compound class as a platform useful in targeting multiple
cancers.
Synthesis of macrocyclic decapeptides is difficult due to
cyclizations that typically generate compounds in low yields.
There have been a number of recent examples in the synthesis
of large macrocyclic peptides that have utilized either
solution-phase, solid-phase, chemoenzymatic, or template-
directed synthesis.7 Further, there are a significant number
of large macrocycles, ranging from 4 to 12 amino acids that
have been successfully used as antitumor, antibiotic, and
immunosupression agents.7,8 In this paper, we describe a
succinct and convergent approach of the synthesis of five
San A-based decapeptides. One of these five compounds
displays extraordinary potency against pancreatic cancers and
has sub-nanomolar IC50 values for two pancreatic cancer cell
lines. In addition, this compound demonstrates a 33-fold
differential selectivity for cancer cells over normal cells and
is 43-fold more potent against pancreatic cancer cell lines
than the current drug of choice, gemcitabine (Gem). Thus,
this is the first of its structural class to be synthesized, and
this new class demonstrates extraordinary potency against
drug-resistant pancreatic cancer cell lines. Indeed, it shows
greater than 1000-fold more potency than its structural
“monomer”: macrocyclic pentapeptide San A. Further, these
decapeptides molecules share no homology to known
pancreatic cancer drugs.
Our succinct and convergent synthesis utilizes the amino
acids shown (Figure 1) and the synthetic strategy described
in Scheme 1. Our solution-phase approach, involving a single
linear pentapeptide, is amenable to inserting L- and D-amino
acids systematically within the di-San A derivative. This
route was also designed to facilitate large-scale synthesis for
extensive biological studies. Syntheses of five di-San A
derivatives were completed using amino acids shown (Figure
1) via the synthetic route outlined (Scheme 1). Using 2(1H-
benzotriazole-1-yl)-1,1,3-tetramethyluronium tetrafluorobo-
rate (TBTU) and diisopropylethylamine (DIPEA), acid-
protected residue 1(a-b) and N-Boc protected residue 2(a-
Figure 1. Retrosynthetic approach for di-SanA.
Scheme 1. Synthesis of Decapeptides Derivatives
(5) (a) Pan, P.-S.; McGuire, K. L.; McAlpine, S. R. Bioorg. Med. Chem.
Lett. 2007, 17, 5072-5077. (b) Ujiki, M.; Milam, B.; Ding, X.-Z.; Roginsky,
A. B.; Salabat, M. R.; Talamonti, M. S.; Bell, R. H.; Gu, W.; Silverman,
R. B.; Adrian, T. E. Biochem. Biophys. Res. Commun. 2006, 340, 1224-
1228.
(6) Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999,
40, 2913-2916.
(7) (a) Qin, C.; Bu, X.; Zhong, X.; Ng, N. L. J.; Guo, Z. J. Comb. Chem.
2004, 6, 398-406. (b) Qin, C.; Bu, X.; Wu, X.; Guo, Z. J. Comb. Chem.
2003, 5, 353-355. (c) Wadhwani, P.; Afonin, S.; Ieronimo, M.; Buerck,
J.; Ulrich, A. J. Org. Chem. 2005, 71, 55-61. (d) Tsuchida, K.; Chaki, H.;
Takakura, T.; Kotsubo, H.; Tanaka, T.; Aikawa, Y.; Shiozawa, S.; Hirono,
S. J. Med. Chem. 2006, 49, 80-91. (e) Wagner, B.; Schumann, D.; Linne,
U.; Koert, U.; Marahiel, M. A. J. Am. Chem. Soc. 2006, 128, 10513-10520.
(f) Malkinson, J. P.; Anim, M. K.; Zloh, M.; Searcey, M. J. Org. Chem.
2005, 70, 7654-7661. (g) Adrio, J.; Cuevas, C.; Manzanares, I.; Joullie´,
M. M. J. Org. Chem. 2007, 72, 5129-5138. (h) Gru¨newald, J.; Marahiel,
M. A. Microbiol. Mol. Biol. ReV. 2006, 70, 121-146. (i) van Maarseveen,
J. H.; Horne, W. S.; Ghadiri, M. R. Org. Lett. 2005, 7, 4503-4506.
Decapeptides, peptides, and macrocyclic peptides: (j) Dutton, F. E.; Lee,
B. H.; Johnson, S. S.; Coscarelli, E. M.; Lee, P. H. J. Med. Chem. 2003,
46, 2057-2073.
*TBTU (1.2 equiv) and/or HATU (0.75 equiv).19
(8) (a) Hruby, V. J. Nature ReV. Drug DiscoVery 2002, 1, 847-858. (b)
Loffet, A. Eur. Peptide Soc. 2002, 8, 1-7.
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Org. Lett., Vol. 10, No. 2, 2008

b) (Figure 1) were coupled to give the dipeptides 1-2-Boc
(90-95% yield). Deprotection of the amine on residue 2
using TFA gave the free amines 1-2 (∼quantitative yields).
Coupling of this dipeptide to monomer 3(a-b) gave the
desired tripeptides (fragment 1) in good yields (80%-95%).9
The synthesis of fragment 2 was completed by coupling
residue 4(a-c) to residue 5 (a-b) to give the dipeptide 4-5-
Boc (90-95% yield). The amine was deprotected on
fragment 1 using TFA and the acid was deprotected in
fragment 2 using lithium hydroxide. Fragments 1 and 2 were
coupled using multiple coupling agents,4c,e,g,10 yielding 31
examples of linear pentapeptides (70-90% yield).
Cyclizing large macrocycles is usually very challenging,
and typically, the yields are low. The recent discovery of
high-yielding conditions11 provided the final decapeptide
macrocycles in good yields. Dissolving the linear pentapep-
tide in THF (0.05 M), addition of 2 equiv of anisole, and
approximately 8 drops of concentrated HCl per 0.3 mmol
of linear pentapeptide led to partially deprotected amine
within 24 h. Four drops of HCl per 0.3 mmol of peptide
were added. The reaction was allowed to stir at room
temperature and then checked after 24 h by LCMS. Typi-
cally, deprotection of the acid and amine were complete
within 2 days.12 Upon completion, the reaction is concen-
trated in vacuo and dried on the high vac. The dried, crude,
free-amine free-acid linear pentapeptide was dissolved in a
1:1 ratio of CH3CN/CH2Cl2 (0.1 M). Addition of DIPEA (6
equiv) and three coupling agents (HATU, DEPBT, and
TBTU 0.5 equiv each) to reaction gave a clear solution.
Reactions were usually complete in 2-4 h.13 Workup with
methylene chloride and ammonium chloride, concentration
in vacuo, and purification via flash chromotagraphy and
subsequently LCMS provided the final products (yields
ranged from 30% to 90% depending on the substrate) (Figure
2).
Figure 2. Structures of five decapeptide derivatives.
Testing the chemotherapeutic activity of these derivatives
against two pancreatic cancer cell lines, PL-45 and BxPC3,
showed that compound 5 is extremely cytotoxic against both
pancreatic cancer cell lines (Figure 3). We have also
determined the IC50 values for compound 5 and found that
they are sub-nanomolar for both cancer cell lines (Figure
4)! Further, 5 is 33-fold less potent against normal skin
fibroblasts than against cancer cell lines, thus demonstrating
differential selectivity. In addition, 5 displayed up to 43-
fold improved cytotoxicity against PL-45 than the drug of
1
(9) Dipeptide and tripeptide structures were confirmed using H NMR.
All linear pentapeptides were confirmed using LCMS and 1H NMR.
(Note: 1H NMR were taken for cyclized peptides, but due to their
complexity, they were not seen as the primary confirmation for cyclized
compounds). See the Supporting Information for spectra.
(10) Unpublished results from the Guy laboratory at the Department of
Chemical Biology and Therapeutics, St Jude Children’s Research Hospital,
Memphis, TN 38103, and published results from our laboratory show that
the use of several coupling reagents facilitates formation of the peptide
bond in high yields.
Figure 3. Cytotoxicity assays of five decapaptides. Each data point
is an average of four wells from three assays at 5 µM. Error )
(5%; DMSO was used as a control () 100% growth).
choice, gemcitabine. Finally, compound 5 demonstrates
potency against drug-resistant colon cancer cell lines (HCT-
116), exhibiting an IC50 of 14-fold differential selectivity for
colon cancer cells over normal cells and 400-fold greater
potency against HCT-116 than gemcitabine. This suggests
that 5 is acting via a mechanism of action that involves a
target common to drug-resistant cell lines from multiple
tumorgenic tissues.
(11) Styers, T. J.; Rodriguez, R.; Pan, P.-S.; McAlpine, S. R. Tetrahedron
Lett. 2006, 47, 515-517.
(12) For details on the reaction conditions, see the Supporting Informa-
tion.
(13) It was straightforward to follow the reactions via LCMS as the
starting material double-deprotected linear precursor would appear at 5.0-
5.5 min and the cyclized product would appear between 6.1 and 7.0 min.
Org. Lett., Vol. 10, No. 2, 2008
179

two D-amino acids in position 5, compound 3 has four
D-amino acids at positions 1 and 5, and compound 4 has
four D-amino acids at positions 4 and 5. Based on the fact
that compound 5 is so potent (i.e., greater than 1000-fold
more cytotoxic than the other four compounds), while the
other compounds exhibit modest cytotoxicity, we believe that
5 is reaching a key biological target inside the cell or on the
cell surface. Presumably, the specific conformation of 5 plays
a significant role in binding to this biological target, which
is why only 5 is active, while the other decapeptides
derivatives show very limited potency.
In summary, we have outlined the synthesis of five
compounds that are related to a potent class of cytotoxic
agents, sansalvamide A. One compound, 5, demonstrated
extraordinary potency against the drug-resistant pancreatic
cancer cell lines PL-45 and BxPC3. Future derivatives that
incorporate additional D-amino acids at positions 2 and 3
will be synthesized and tested. Their potency will be reported
in due course.
Figure 4. IC50 assay of most potent decapeptide run in HCT-116
(colon cancer cell lines), PL-45, BxPC3, and WS-1 (WS-1 )
normal cells, skin fibroblasts). Data represents results from a
concentration curve taken from four concentrations, where each
concentration data point is from four separate experiments per-
formed in quadruplicate. Margin of error ) (5%. Concentrations
are nM.
Acknowledgment. We thank San Diego State University
for funding, as well as the Howell foundation for their
fellowships to T.J.S. and R.A.R. R.A.R was also supported
by NIH PREP program and NIH SDSU MARC 5T34-
GM08303.
Supporting Information Available: General experimen-
tal procedures, cytotoxicity assay protocol, and NMR and
mass spectral data for compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
The structural differences between compounds 1-4 and
compound 5 are subtle. Compound 5 has four D-amino acids
in positions 2 and 3, respectively. By comparison, compound
1 has two D-amino acids in position 3, compound 2 also has
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