Synthesis and cytotoxicity of novel Sansalvamide A derivatives

Article abstract of DOI:10.1021/ol051161g

(Chemical Equation Presented) Described are the syntheses of 14 derivatives of the natural product Sansalvamide A, where two are more active against HCT 116 colon cancer cell lines than the natural product. These derivatives were synthesized using a combi

Full text of DOI:10.1021/ol051161g

ORGANIC
LETTERS
2005
Vol. 7, No. 16
3481-3484
Synthesis and Cytotoxicity of Novel
Sansalvamide A Derivatives
Chris L. Carroll,† Jennifer V. C. Johnston,† Ahmet Kekec,† Joseph D. Brown,†
Emily Parry,† Julia Cajica,† Irene Medina, Kristina M. Cook, Ricardo Corral,
Po-Shen Pan, 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 May 17, 2005
ABSTRACT
Described are the syntheses of 14 derivatives of the natural product Sansalvamide A, where two are more active against HCT 116 colon
cancer cell lines than the natural product. These derivatives were synthesized using a combinatorial-type strategy that permits elucidation of
the amino acid role in the cytotoxicity, and they lay the groundwork for development of new anticancer agents.
Sansalvamide A is a depsipeptide exhibiting antitumor
activity.1-3 It was discovered by Fenical and co-workers from
a marine fungus of the genus Fusarium3. It has been
characterized as a cytotoxin and antiviral agent. It demon-
strates a mean IC50 of 27.4 µg/mL in the NCI’s 60 cell line
panel3 and a potency of 3.5 µg/mL against colon cancer cell
line COLO205. It is more potent than a drug currently on
the market (mitomycin C), which exhibits an IC50 value of
5.3 µg/mL against this same cell line.3 It is also known to
inhibit topoisomerase activity or, more specifically, topoi-
somerase-catalyzed DNA relaxation.2 Topoisomerase activity
is important for biological processes such as DNA replica-
tion, repair, and transcription.
phase synthesis of Sansalvamide A4 and the Sansalvamide
A peptide5 has been completed by Silverman et al, and the
peptide demonstrates 10 times greater potency against the
human colon carcinoma than the natural product depsipeptide
(cell line HCT-116). However, little is known about the
mechanism of action of Sansalvamide A or the structure-
activity relationship (SAR) between active compounds and
the biological target. It is important to establish the SAR
and determine if further development of this class as
antitumor and antiviral agents would be useful. Additionally,
it is valuable to examine the biological activity of not only
derivatives relating to the natural product with the L-amino
acids but also the opposite enantiomers of these derivatives.
As a result of our experience working in solution phase and
the hydrophobicity of the residues, we developed a solution
phase route for the synthesis of Sansalvamide A derivatives.
Figure 1 depicts the two fragments involved in our synthesis
route.
Sansalvamide A is composed of four hydrophobic amino
acids and one hydrophobic hydroxy acid. An elegant solid-
† Contributed equally to the work reported.
(1) Cueto, M.; Jensen, P. R.; Fenical, W. Phytochemistry 2000, 55, 223.
(2) Hwang, Y.; Rowley, D.; Rhodes, D.; Gertsch, J.; Fenical, W.;
Bushman, F. Mol. Pharm. 1999, 55, 1049.
(3) Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron Lett. 1999,
40, 2913.
(4) Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743.
(5) Gu, W.; Liu, S.; Silverman, R. B. Org. Lett. 2002, 4, 4171.
10.1021/ol051161g CCC: $30.25
© 2005 American Chemical Society
Published on Web 07/08/2005

Scheme 1. Synthesis of Sansalvamide A Fragmentsa
Figure 1. Retrosynthetic approach.
Herein we describe the synthesis of 14 novel Sansalvamide
A derivatives using the amino acids shown (Figure 2). The
significant aspects of our work include the consecutive
a Conditions: (a) coupling agent [TBTU and/or HATU (1.25
equiv ea)], DIPEA (3 equiv), CH3CN; (b)TFA, anisole (2 equiv),
CH2Cl2; (c)LiOH (4 equiv), MeOH.
monomer 3a-d gave the desired tripeptide (Fragment 1) in
good yields (80-95%).6
The synthesis of Fragment 2 was completed by coupling
residue 4a,b to residue 5a-d to give the dipeptide 4-5-Boc
(90-95% yield) (Scheme 1). The amine was deprotected in
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,7 yielding 14 ex-
amples of linear pentapeptides (50-74% yield) (Scheme 2).6
The linear pentapeptides were amine deprotected using
HCl (pH < 2). Upon completion, the reaction was concen-
trated in vacuo, and the acid was deprotected by neutralizing
the reaction with lithium hydroxide and then adding an
additional 4 equiv of lithium hydroxide in methanol to give
pH ) ∼11.8 Following acid deprotection, the reaction was
concentrated in vacuo and subjected to HATU, TBTU,
PyAOP, PyBrop, and/or DEPBT coupling reagents (∼0.5
(6) All dipeptide and tripeptide structures were confirmed using 1H NMR.
All linear hexameric peptides were confirmed using LCMS and 1H NMR,
and cyclized peptides were all confirmed using LCMS. (Note: 1H NMR
was also used for cyclized peptides, but because of their complexity, they
were not seen as the primary confirmation for cyclized compounds).
(7) (a) Bolla, M. L.; Azevedo, E. V.; Smith, J. M.; Taylor, R. E.; Ranjit,
D. K.; Segall, A. M.; McAlpine, S. R. Org. Lett. 2003, 5, 109. (b) Robinson,
J. L.; Taylor, R. E.; Liotta, L. A.; Bolla, M. L.; Azevedo, E. V.; Medina,
I.; McAlpine, S. R. Tetrahedron Lett. 2004, 45, 2147 (c) Liotta, L. A.;
Medina, I.; Robinson, J. L.; Carroll, C. L.; Pan, P.-S.; Corral, R.; Johnston,
J. V. C.; Cook, K. M.; Curtis, F. A.; Sharples, G. J.; McAlpine, S. R.
Tetrahedron Lett. 2004, 45, 8447 (d) Ring-closing reactions are slow and
typically low yielding. Unpublished results from the Guy lab at UCSF and
recently our lab have found that the use of several coupling reagents
facilitates ring-closing reactions by providing a choice of reagents for the
specific substrate.
Figure 2. Monomers used in the synthesis of derivatives.
placement of N-methyl amino acids within the macrocycles
and comparison of their cytotoxicity to those containing NH
amino acids. This resulted in the discovery of two derivatives
that exhibited cytotoxicity against colon cancer cell lines
(HCT-116). In addition, we synthesized derivatives utilizing
all L-amino acids and all D-amino acids. Furthermore, the
synthesis of the peptide derivatives (as opposed to depsipep-
tide derivatives) provided macrocycles biologically more
stable than those containing a labile lactone. The peptide
derivatives demonstrate higher potency in the colon cancer
assays (presumably due to their greater stability in cells).5
Our synthesis utilized a convergent approach (Figure 1).
Using 2(1-H-benzotriazole-1-yl)-1,1,3-tetramethyl-uronium
tetrafluoroborate (TBTU) as a coupling reagent and diiso-
propylethylamine (DIPEA), acid-protected residue 1a,b and
N-Boc-protected residue 2a-f (Scheme 1) were coupled to
give the dipeptide 1-2-Boc (80-94% yield). Deprotection
of the amine on residue 2 using TFA gave the free amine
1-2 (∼quantitative yields). Coupling of this dipeptide to
(8) All linear protected pentamers were amine deprotected and then
confirmed via LCMS. Upon confirmation that the linear amine deprotections
were completed, the acid deprotections were undertaken, and confirmation
of the fully amine and acid deprotected pentamers were determined via
LCMS.
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Org. Lett., Vol. 7, No. 16, 2005

These 14 macrocyclic compounds were tested for their
cytotoxicity against HCT 116 colon cancer cells.10 Described
in Table 1 are the compounds and their IC50 against this cell
line.
Scheme 2. Cyclization of Sansalvamide Derivativesa
Table 1. Cytotoxicity Assay Results
compound
structure
IC50a (µg/mL)
San A
A
B
C
D
E
F
G
H
I
J
K
L
M
N
3.5
1.5
1a-2a-3a-4a-5a
1b-2b-3b-4b-5b
1a-2c-3a-4a-5a
1b-2d-3b-4b-5b
1a-2c-3a-4a-5c
1b-2d-3b-4b-5d
1a-2c-3c-4a-5a
1a-2a-3a-4a-5c
1b-2b-3b-4b-5d
1a-2e-3a-4b-5a
1a-2e-3a-4b-5a
1a-2e-3a-4b-5a
1a-2e-3a-4b-5a
1a-2e-3a-4b-5a
NSA
NSA
NSA
NSA
NSA
2.6
N/A
N/A
NSA
N/A
N/A
N/A
N/A
a Conditions: (a) coupling agent [for linear peptides, typically
HATU and TBTU (∼0.75 equiv ea); for cyclizations, HATU,
DEPBT, PyAOP, PyBROP, and/or TBTU (∼0.5 equiv ea)], DIPEA
(3 equiv), CH3CN; (b) HCl, anisole (2 equiv), CH2Cl2; (c) LiOH
(4 equiv), MeOH.
equiv each) and DIPEA (∼6 equiv until pH ) 8).7 The final
macrocyclizations took approximately 4 days because of the
low concentration (0.005-0.01 M) required to maximize the
yield. The one-pot ring-closing yields varied from 20% to
35%. The final compounds were then purified using reverse
phase HPLC and confirmed via LCMS.9 The final isolated
compounds are shown in Figure 3.
a NSA, g78 µg/mL; N/A, not available.
The two active compounds (Figure 4) include the nonm-
ethylated peptide derivative of Sansalvamide A (A), and the
Figure 4. Structures of two active compounds.
mono-N-methylated derivative on the three residue (G). The
error limits of the assay (0.1 µg/mL) are such that the mono-
N-methylated derivative (G) is clearly more active than
Sansalvamide A, and Sansalvamide A peptide (A) is twice
as active as the natural product. The 3-N-methyl derivative
(G) is a new compound, and interestingly enough, the 2-N-
methyl depsipeptide derivative (i.e., the natural product
termed N-methyl Sansalvamide) is not nearly as active as
compound G. Thus, our result suggests an interesting
structure-activity relationship (SAR) between the compound
and its biological target. This novel derivative and others
are being tested in additional assays, and new methylated
derivatives are currently being synthesized in order to explore
this SAR.
Figure 3. Structures of Sansalvamide derivatives
(9) The 14 macrocyclic peptides in Scheme 2 have LCMS data and yields
given in Supporting Information. One representative example of a macro-
cyclic pentapeptide is as follows (note: MS data is given as major peaks
with +23 [Na+] and +1 being those peaks):
(10) The protocol for the HCT 116 colon cancer assay is described in
the Supporting Information.
Org. Lett., Vol. 7, No. 16, 2005
3483

In summary, the synthesis of 14 Sansalvamide A deriva-
tives have provided two compounds that exhibited potency
greater than that of the natural product. Compounds A and
G demonstrated improved cytotoxicity against HCT-116
colon adeno carcinoma cancer cell lines over that seen with
Sansalvamide A. Compound G was a new structure, one that
had not previously been synthesized and tested for antitumor
activity. The two active derivatives are being assessed for
incorporation of photoaffinity tags so that the cellular target
can be isolated. Additional derivatives are currently being
synthesized. These derivatives, along with those described
in this paper, will be tested against other colon cancer cell
lines, as well as against four other types of cancer cell lines.
The results of these assays will be published in due course.
program for support of J.C. (spring 2005-current, NIH-
5R25GM58906) and the SDSU McNair program for support
to C.L.C. (summer 2003 and 2004) and I.M. (summer 2004).
We thank Merck and Co. for support of J.V.C.J. (summer
2004) and the Howell Foundation for support for C.L.C.
(spring 2004), K.C. (spring 2004), E.P. (spring 2005), and
J.V.C.J. (spring 2005). We thank the MIRT program for their
travel support of I.M. (2003-2004), C.L.C. (2004), and E.P.
(2005). We thank San Diego State University for financial
support. S.R.M. was supported by NIH (AI058241-01).
Note Added after ASAP Publication. There was an error
in the listing of contributing authors and a missing author
name in the version published ASAP July 8, 2005; the
corrected version was published ASAP July 15, 2005.
Acknowledgment. We thank Professor W. Fenical and
Lisa Zeigler for useful discussions and for running the HTC-
116 colon cancer assays. We thank Pfizer, La Jolla, for
equipment and financial donations, as well as their fellow-
ships to I.M. (2003-2005) and C.L.C. (SURF, summer
2004). We thank the U.S. Coast Guard for support of J.D.B.
(2004-2005). We thank the NIH/NIGMS-MBRS-IMSD
Supporting Information Available: General experimen-
tal procedures, cytotoxicity assay protocols, and mass spectral
data for final compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL051161G
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