Synthesis of Sansalvamide A derivatives and their cytotoxicity in the MSS colon cancer cell line HT-29

Article abstract of DOI:10.1016/j.bmc.2006.04.031

We report the synthesis of thirty-six Sansalvamide A derivatives, and their biological activity against colon cancer HT-29 cell line, a microsatellite stable (MSS) colon cancer cell-line. The thirty-six compounds can be divided into three subsets, where t

Full text of DOI:10.1016/j.bmc.2006.04.031

Bioorganic & Medicinal Chemistry 14 (2006) 5625–5631
Synthesis of Sansalvamide A derivatives and their cytotoxicity
in the MSS colon cancer cell line HT-29
Thomas J. Styers,a Ahmet Kekec,a Rodrigo Rodriguez,a Joseph D. Brown,a
Julia Cajica,a Po-Shen Pan,a Emily Parry,a Chris L. Carroll,a Irene Medina,a
Ricardo Corral,a Stephanie Lapera,a Katerina Otrubova,a Chung-Mao Pan,a
Kathleen L. McGuireb,* and Shelli R. McAlpinea,*
aDepartment of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA
bDepartment of Biology, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-4630, USA
Received 5 March 2006; revised 1 April 2006; accepted 13 April 2006
Available online 11 May 2006
Abstract—We report the synthesis of thirty-six Sansalvamide A derivatives, and their biological activity against colon cancer HT-29
cell line, a microsatellite stable (MSS) colon cancer cell-line. The thirty-six compounds can be divided into three subsets, where the
first subset of compounds contains L-amino acids, the second subset contains D-amino acids, and the third subset contains both
D- and L-amino acids. Five compounds exhibited excellent inhibitory activity (>75% inhibition). The structure–activity relationship
(SAR) of the compounds established that a single D-amino acid in position 2 or 3 gave up to a 10-fold improved cytotoxicity over
Sansalvamide A peptide. This work highlights the importance of residues 2 and 3 and the role of D-amino acids in the extraordinary
SAR for this compound class.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Currently, only the MSS colon cancers are known to re-
spond to chemotherapeutic drugs. The drug of choice
for treatment, 5-fluorouracil (5-FU) [IC50 = 5 lM], has
severe side effects, making it desirable to develop a drug
with improved efficacy. Because MSI colon cancers do
not respond to 5-FU,1,2 or to current chemotherapeutic
drugs, finding new structures that target both cancer
pathways would be very valuable.
Carcinogenesis in the colonrectum is thought to occur
through two different pathways. The existing model sug-
gests that 80–85% of colon cancers involve chromosom-
al instability, where point mutations are found in loci
within RAS, p53, and other checkpoint proteins.1 The
remaining 15–20% of colon cancers involve a loss in
the DNA mismatch repair system, which leads to point
mutations in repetitive sequences. These repetitive
sequences are known as microsatellites and occur in sev-
eral important growth regulators. Mutations in these
microsatellites lead to instability within these areas,
which ultimately impacts the function of these growth
regulator proteins. The two pathways are usually re-
ferred to as having microsatellite stability (MSS) or
microsatellite instability (MSI), respectively.
It has been shown that Sansalvamide A (San A), which
is a depsipeptide isolated from a marine fungus (Fusar-
ium ssp.), exhibits anti-tumor activity.3–5 Syntheses and
evaluation of all-peptide analogs against HCT-116
(MSI) have revealed that several derivatives have greater
potency against HCT-116 than the natural depsipep-
tide.6–8 Previous work by our group9 has shown that
several derivatives are potent against HT-29 (MSS).
Determining the structure–activity relationship (SAR)
of this compound class will facilitate understanding of
San A’s mechanism of action in MSS cell lines. Further,
very few analogs have been made (to date ꢀ30 derivatives
have been reported by us and others). Here, we describe
the synthesis of 36 compounds, where 18 compounds
are new structures not previously reported. The evalua-
tion of these derivatives provides a clear structural motif
Keywords: Macrocycles; Peptides; Macrocyclic peptides; Cytotoxicity;
Colon cancer; MSS; Sansalvamide A.
*
Corresponding authors. Tel.: +1 619 993 8609; fax: +1 619 594
5580; e-mail: mcalpine@chemistry.sdsu.edu
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.031

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T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
important for cytotoxicity. Comparison of these to the
current drug on the market emphasizes the relevance of
San A as a new therapeutic class of compounds.
2. Results and discussion
2.1. Synthetic strategy
San A is composed of four hydrophobic amino acids
and one hydrophobic hydroxy-acid. We report here
the synthesis of thirty-six San A derivatives. All deriva-
tives are peptide analogs, which involve the exchange of
the hydroxy acid in position 4 to an amino acid (Fig. 1).
We used a solution phase synthesis route because of the
hydrophobic nature of the residues. Our convergent ap-
proach, involving two fragments (Fig. 1),8 is amenable
to inserting L- and D-amino acids systematically within
San A. This route was also designed to facilitate large-
scale synthesis in extensive biological studies.
Synthesis of thirty-six San A derivatives was completed
using amino acids shown (Fig. 2) via the synthetic route
outlined.
2.2. Synthesis
Using
2(1-H-benzotriazole-1-yl)-1,1,3-tetramethyl-
uronium tetrafluoroborate (TBTU), and diisopropyleth-
ylamine (DIPEA), acid protected residue 1(a,b) and
N-Boc protected residue 2(a–i) (Fig. 2) were coupled to
give the dipeptides 1-2-Boc (80–94% yield). Deprotection
of the amine on residue 2 using TFA gave the free amines
1 and 2 (ꢀquantitative yields). Coupling of this dipeptide
to monomer 3(a–h) gave the desired tripeptides
(Fragment 1) in good yields (80–95%).11 The synthesis
of Fragment 2 was completed by coupling residue
4(a–d) to residue 5(a–e) to give the dipeptide 4- and
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. Fragment 1 and
Fragment 2 were coupled using multiple coupling agents
8,10,12,13
yielding thirty-six examples of linear pentapep-
tides (66–90% yield).11
Cyclizing large macrocycles is usually very challenging,
and typically the yields are low. The recent discovery
of high-yielding conditions14 provided most of the final
macrocycles in good yields. Dissolving the linear penta-
Amino Acid 3
Hydroxy Acid 4
O
Amino Acid 4
O
N
O
RHN
O
OH
R
O
O
RN
NH
O
Figure 2. Synthesis of macrocycles. Reagents: (a) coupling agent
(TBTU (1.2 equiv), and/or HATU (0.75 equiv)10), DIPEA (3 equiv),
CH2Cl2 (0.1 M); (b) TFA (20%), anisole (2 equiv), CH2Cl2; (c) LiOH
(4 equiv), MeOH; (d) HCl in THF (0.05 M), anisole (2 equiv); (e)
HATU (0.7 equiv), DEPBT (0.7 equiv), TBTU (0.7 equiv), DIPEA
(6 equiv), THF/CH3CN/DCM (2:2:1) 0.007 M.
RN
HN
NH
H3CO
HN
NHBoc
O
O
Amino Acid 5
O
Amino
Acid 2
O
R = H or Me
Amino Acid 1
Sansalvamide A
peptide in THF (0.05 M), addition of 2 equiv of anisole,
and approximately eight drops concentrated HCl per
Figure 1. Retrosynthetic strategy.

T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
5627
0.3 mmol of linear pentapeptide led to partially depro-
tected amine within 24 h. Four drops of HCl per
0.3 mmol of peptide were added. The reaction mixture
was allowed to stir at room temperature and then
checked after 24 h by LC–MS. Typically deprotection
of the acid and amine was complete within four days.15
Upon completion, the reaction is concentrated in vacuo
and dried on the high-vac. The dried, crude, free-amine
free-acid linear pentapeptide was dissolved in 2:2:1 ratio
ofTrypanosoma THF/CH3CN/CH2Cl2 (0.007 M). Addi-
tion of DIPEA (6 equiv), and three coupling agents
(HATU, DEPBT, and TBTU 0.7 equiv ea) to reaction
gave a clear solution. Reactions were usually complete
in 4–6 h.16 Workup with methylene chloride and ammo-
nium chloride, concentration in vacuo, purification via
flash chromotagraphy and subsequently LC–MS, pro-
vided the final products (yields ranged from 23% to
90% depending on the substrate).17
is exchanged to the enantiomeric D-amino acid. The four
active compounds in subset 3 are compounds (26) and
(33), which contain D-amino acids in position 2, and
compounds (27) and (32), which contain D-amino acids
at position 3. Importantly, compounds (4) and (32) only
differ by the exchange of an L-amino acid to a D-amino
acid in position 3, yet (32) has a 40-fold increased poten-
cy over (4) [(4) IC50 = 302 lM and (32) jC50 = 7.5 lM].
Further, comparison of compound (1) to compounds
(2), (4), and (15) shows that the inclusion of N-methyl
groups on L-amino acids leads to a decrease in potency
when in positions 1, 2, or 3. It also appears the SAR is
related to a single D-amino acid in positions 2 or 3, but
not multiple D-amino acids within the San A structure.
For example, compounds (20) and (22), which contain
all D-amino acids including the same N-methyl D-amino
acids that enhance potency in compounds (33) and (32),
are significantly less active than (33) or (32).
2.3. Structures of macrocyclic derivatives
The only exception to our observed SAR is when a sin-
gle N-methyl group is in position 5. This compound
exhibited an unexpected and significant cytotoxicity.
The synthesis of a new generation of compounds and
testing on HT-29 will explore this trend. Interestingly,
compounds that contain N-methyl moieties in position
5 combined with other positions [compounds (3), (6),
and (7)] do not exhibit the same level of potency as com-
pound (5). Perhaps having a single N-methyl in position
5 promotes a conformational change that enhances
binding to the biological target.
We designed and synthesized three subsets of San A
derivatives. The first subset of compounds contains all
L-amino acids (compounds 1–18, Fig. 3).
The second subset contain all D-amino acids (com-
pounds 19–24, Fig. 4).
The third subset contain all both L- and D-amino acids
(compounds 25–36, Fig. 5).
2.4. Thymidine uptake assays
2.6. Significance
The SAR was established using San A peptide as a con-
trol [compound (1)]. [3H]thymidine uptake assays on
HT-29 revealed five compounds, (5), (26), (27), (32),
and (33), had greater than 75% inhibitory activity
(Chart 1). Comparison of the three subsets showed that
subset 3, which contains compounds with both D- and L-
amino acids, has the most active macrocycles. Signifi-
cantly, 63% (7/11) of the compounds in subset 3 exhibit
greater than 70% inhibition. By comparison, subset 1
has 17% (3/18) and subset 2 has 0% of compounds with
greater than 70% inhibition. Clearly, subset 3 contains
the most intriguing pharmacophores.
In summary, five San A derivatives [(5), (26), (27), (32)
and (33)] exhibit effective potency against an MSS colon
cancer cell line (HT-29). Compound (32) has potency
comparable to that of a current drug on the market
(5-FU). Interestingly, subset 3 contains four of the five
most active compounds. This is the first thorough
SAR exploration for an MSS colon cancer cell line,
and it highlights the importance of compounds contain-
ing a single D- and four L-amino acids. The SAR seen in
our data provides extraordinary promise in facilitating
the design of new, potentially potent San A analogs.
Evaluation of these derivatives provides a clear structur-
al motif important for cytotoxicity. Further investiga-
tion will refine the roles of D- and N-methyl amino
acids. Mechanistic and cytotoxicity assays of these com-
pounds against other colon cancer cell lines are under-
way, and synthesis of next generation derivatives
utilizing the information described here is also in pro-
gress. These results will be reported in due course.
Compounds (5), (26), (27), (32), and (33) have IC50 val-
ues of 32 lM, 34 lM, 19 lM, 7.5 lM, and 33 lM,
respectively (San A peptide IC50 = 77 lM, and 5-
FU = 5 lM) (Chart 2). Compound (32) not only exhib-
ited 89% growth inhibition and a 10-fold increased
potency compared to San A peptide [compound (1)], it
is almost as potent as 5-FU.
2.5. Summary of structure–activity relationships
2.7. Experimental procedures
It has been proposed by Silverman and co-workers7 that
the N-methyl moieties may be important for antitumor
activity of San A derivatives in the HCT 116 (MSI) cell
line. With the exception of compound (5), our data sug-
gest that in the MSS cancer cell line HT-29, the N-meth-
yl moiety does not play a significant role in potency.
Rather potency is enhanced when a single L-amino acid
2.7.1. General remarks. All coupling reactions were per-
formed under argon. All reagents were used as received.
Anhydrous methylene chloride Dri Solv (EM), and
anhydrous Acetonitrile Dri Solv (EM) were bought
from VWR, and were packed under nitrogen with a
septum cap. Diisopropylethylamine (DIPEA) and
anhydrous THF were purchased from Aldrich,

5628
T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
3a
O
3a
O
O
3a
4a
4a
4a
O
O
O
N
N
H
N
H
H
N
2c
NH
N
HN
NH
NH
2a
O
O
O
2c
O
NH
N
NH HN
O
NH HN
O
O
O
5a
5c
5a
O
1a
1a
1a
Compound 3
Compound 1
Compound 2
3a
O
O
3c
3c
4a
1a
4a
O
O
O
4a
N
N
O
N
H
HN
HN
NH
HN
HN
2a
NH
2a
2a
O
5c
HN
O
NH
O
5c
N
N
O
O
NH HN
O
O
5a
O
O
1a
1a
Compound 5
Compound 6
Compound 4
O
3c
O
3c
O
O
3a
O
4a
4a
1a
4a
O
N
N
N
H
N
N
NH
NH
HN
NH
2c
2c
O
5a
O
5c
O
2e
HN
NH
O
N
HN
NH HN
O
O
O
5a
O
O
1a
1a
Compound 9
Compound 7
Compound 8
O
O
3h
O
3g
O
4a
3a
4a
O
4a
N
N
O
H
H
N
H
HN
HN
NH
NH
2a
O
O
N
NH
O
2f
2a
NH HN
O
NH HN
O
O
O
NH HN
O
5a
5a
4a
5a
O
1a
1a
1a
Compound 10
Compound 11
3a
Compound 12
3a
O
O
3a
O
O
4a
4a
O
O
N
N
N
H
H
H
HN
N
NH
HN
NH
HN
NH
O
O
O
2i
2h
2a
NH
O
NH HN
O
NH HN
O
O
O
O
5a
5a
5a
1a
1d
1a
Compound 13
Compound 15
Compound 14
O
3c
3e
4a
1d
O
3a
O
4c
O
O
N
4a
5e
N
O
HN
N
H
NH
H
2a
HN
NH
O
5a
HN
NH
O
O
N
NH
O
O
NH HN
O
NH HN
O
2g
O
2a
O
5a
1a
1a
Compound 17
Compound 18
Compound 16
Figure 3. Subset 1: L-amino acid San A derivatives.
packaged under nitrogen in a sure seal bottle. The
coupling agent HATU and PyAOP were purchased
from Perspective:Applied Biosystems at 850 Lincoln
Center Dr. Foster City, CA 94404, Telephone: +1 800
327 3002. The coupling agents TBTU and PyBOP were
purchased from NovaBiochem. DEPBT [3(diethoxy-
phosphoryloxy)-1,2,3-benzotriazine-4(3H)] was pur-
1
chased from Aldrich (order number 49596-4). The H
NMR spectra were recorded on a Varian at 500 MHz.
LC–MS were obtained at San Diego State University

T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
5629
3b
3b
O
O
O
4b
3b
O
4b
4b
O
O
N
N
N
H
H
N
NH
N
2d
NH
H
2d
HN
NH
O
O
O
NH HN
O
N
HN
2b
O
O
NH HN
O
O
5d
4b
5b
5b
O
1b
1b
Compound 21
1b
Compound 19
Compound 20
O
3d
O
O
N
3d
O
3d
O
4b
N
4b
O
N
N
NH
HN
HN
NH
O
2b
O
HN
NH
O
2d
N
HN
2b
O
N
O
NH HN
O
5d
O
5d
O
O
5b
1b
1b
1b
Compound 22
Compound 23
Compound 24
Figure 4. Subset 2: D-amino acid San A derivatives.
Figure 5. Subset 3: L- and D-amino acid San A derivatives.
using HP1100 Finnnigan LCQ. Flash column chroma-
tography was performed on 230–400 mesh 32–74 lm
2.7.2. General peptide synthesis. All peptide coupling
reactions were carried out under argon with dry solvent,
using methylene chloride for dipeptide and tripeptide
˚
60 A silica gel from Bodman Industries.

5630
T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
Chart 1. Activity of 36 compounds in colon cancer (50 lM) detected in 3H-thymidine uptake assays: Subset 1 = compounds 1-18, Subset
2 = compounds 19-24, and Subset 3 = compounds 25-36. (Error bars are within approximately 2%).
2.7.4. General acid deprotection. Acids were deprotected
using ꢀ4 equiv of lithium hydroxide (or until pH ꢀ11)
in methanol (0.1 M). The peptide was placed in a flask,
along with lithium hydroxide and methanol, and stirred
overnight. Within 12 h the acid was usually deprotected.
Workup of reactions involved the acidification of reac-
tion solution using HCl to pH 1. The aqueous solution
was extracted three times with methylene chloride, and
the combined organic layer was dried, filtered, and con-
centrated in vacuo.
IC50Values
77.0
80
70
60
50
40
30
20
10
0
34.0
33.0
33
32.0
19.0
27
7.5
32
5.0
5-FU
1
5
26
Compound Number
2.7.5. Macrocyclization procedure (in situ). All pentapep-
tides were acid and amine deprotected using HCl
(8 drops per 0.3 mmol of linear pentapeptide) in THF
(0.05 M). Addition of anisole (2 equiv) was added to
the reaction and the reaction mixture was stirred at
room temperature. The reaction typically took 4 days,
but TLC and LC–MS were used to monitor the reaction
every 12 h. LC–MS data typically indicated the reaction
was ꢀ50% complete after the first day. Addition of four
drops of HCl per 0.3 mmol of pentapeptide, stirring at
RT overnight, and checking the reaction via LC–MS
usually showed ꢀ75% completion. On the fourth day
verification of the presence of the free amine and free
acid and disappearance of the starting linear protected
pentapeptide permitted workup. The reaction mixture
was concentrated in vacuo and the crude, dry, double
deprotected peptide (free acid and free amine) was dis-
solved in a minimum solution of THF: acetonitrile:
methylene chloride (2:2:1 ratio). Three coupling agents
(DEPBT, HATU, and TBTU) were used at ꢀ0.5 to
0.75 equiv each. These coupling agents were dissolved
in a calculated volume of dry 40% THF, 40% acetoni-
trile, and 20% methylene chloride that would give a
0.007 M overall solution when included in the volume
used for the deprotected peptide. The coupling agents
was then added to the deprotected peptide solution.
DIPEA (6 equiv or more in order to neutralize the
pH) were then added to the reaction. The coupling
agents are typically not very soluble in acetonitrile,
which is why a combination of solvents is used.
Chart 2. IC50 of five most active compounds detected in [3H]thymidine
uptake assays.
couplings and acetonitrile for pentapeptide couplings.
The amine (1.1 equiv) and acid (1 equiv) were weighed
into a dry flask along with 4 equiv of DIPEA and
1.1 equiv of TBTU. Anhydrous methylene chloride
was added to generate a 0.1 M solution. The solution
was stirred at room temperature and reactions were
monitored by TLC. Reactions were run for 1 h before
checking via TLC. If reaction were not complete addi-
tional 0.25 equiv was of HATU and TBTU were added.
If reaction were complete, then workup was done by
washing with saturated ammonium chloride. (Note: if
acetonitrile was used for the reaction, methylene chlo-
ride was added to reaction upon workup and the result-
ing solution was washed with ammonium chloride).
After back extraction of aqueous layers with methylene
chloride, organic layers were combined, dried over sodi-
um sulfate, filtered, and concentrated. Flash chromatog-
raphy using a gradient of ethyl acetate–hexane gave our
desired peptide.
2.7.3. General amine deprotection. Amines were depro-
tected using 20% TFA in methylene chloride (0.1 M)
with 2 equiv of anisole. The reactions were monitored
by TLC, where the TLC sample was first worked up
in a mini-workup using DI water and methylene chlo-
ride to remove TFA. Reactions were allowed to run
for 1–2 h and then concentrated in vacuo.
After 1 h, TLC and LC–MS (where the LC–MS sample
was worked up prior to injection) indicated that a
product spot was developing. The comparison Rf value
in the product spot on TLC was the protected linear
pentapeptide. The reactions were always complete after
2 h, and monitoring the starting material deprotected
Some coupling reactions would not go to completion using only
TBTU and therefore ꢀ0.25 equiv of HATU and/or DEPBT were
used. In a few cases up to 1.1 equiv of all three coupling reagents were
used.

T. J. Styers et al. / Bioorg. Med. Chem. 14 (2006) 5625–5631
5631
pentapeptide via LC–MS was the easiest method of
determining completion. Upon completion, the reaction
was worked up by washing with ammonium chloride.
After back extraction of aqueous layers with large quan-
tities of methylene chloride, the organic layers were
combined, dried, filtered, and concentrated. All macro-
cycles were purified by initially running a crude plug
of compound using an ethylacetate/Hexane gradient
on silica gel, then running a column on the isolated
product. Finally, if necessary reverse-phase HPLC was
used for additional purification using a gradient of ace-
tonitrile and DI water with 0.1% TFA.
(2005). We thank San Diego State University for
financial support. SRM was supported by NIH
(AI058241-01).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2006.04.031.
References and notes
2.8. Biological assay protocol
1. Boland, C. R.; Sinicrope, F. A.; Brenner, D. E.; Carethers,
J. M. Gastroenterology 2000, 118, S115–S128.
2. Carethers, J. M.; Chauhan, D. P.; Fink, J. L.; Nebel, S.;
Bresalier, R. S.; Howell, S. B.; Boland, C. R. Gastroen-
terology 1999, 117, 123–131.
3. Cueto, M.; Jensen, P. R.; Fenical, W. Phytochemistry
2000, 55, 223–226.
4. Hwang, Y.; Rowley, D.; Rhodes, D.; Gertsch, J.; Fenical,
W.; Bushman, F. Mol. Pharmacol. 1999, 55, 1049–1053.
5. Belofsky, G. N.; Jensen, P. R.; Fenical, W. Tetrahedron
Lett. 1999, 40, 2913–2916.
6. Lee, Y.; Silverman, R. B. Org. Lett. 2000, 2, 3743–3746.
7. 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.
8. 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.
9. Otrubova, K.; Styers, T. J.; Pan, P.-S.; Rodriguez, R.;
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2.8.1. Thymidine uptake assays. Proliferation of the
HT29 colon cancer cell line was tested in the presence
and absence of the compounds using [3H]thymidine up-
take assays. Cells treated with the compounds were
compared to DMSO-treated controls for their ability
to proliferate as indicated by the incorporation of
[3H]thymidine into their DNA. Cells were cultured in
96-well plates at a concentration of 50,000 cells/well.
The media was McCoy’s 5a with L-glutamine, 10% fetal
bovine serum, and antibiotics. After incubation for
approximately 6 h, the compounds were added. The
compounds were dissolved in DMSO for a final concen-
tration of 2 mM and tested at the concentrations indi-
cated in the manuscript. The DMSO concentration
was held constant in all wells at 2.5%. After the cells
had been incubated with the compounds for 24 h,
1 lCi [3H]thymidine per well was added and the cells
were cultured for an additional 16 h (for the cells to have
a total of 40 h with the drug), at which time the cells
were harvested using a PHD cell harvester from Cam-
bridge Technology Incorporated. The samples were then
counted in a scintillation counter for 2 min each using
ScintiVerse universal scintillation fluid from Fisher.
Decreases in [3H]thymidine incorporation, as compared
to controls, are an indication that the cells are no longer
progressing through the cell cycle or synthesizing DNA.
10. Unpublished results from the Guy lab at UCSF and
published results from our laboratory show that the use of
several coupling reagents facilitates formation of the
peptide bond in high-yields.
11. Dipeptide and tripeptide structures were confirmed using
1H NMR. All linear pentapeptides were confirmed using
1
1
LC–MS and H NMR. (Note: H NMR were taken for
cyclized peptides, but due to their complexity, they were
not seen as the primary confirmation for cyclized com-
pounds). See Supplementary material for spectra.
12. 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, 109–112.
Acknowledgments
We thank Pfizer, La Jolla, for equipment and financial
donations as well as their fellowships to I.M.
(2003–2005), RC (2003–2005), CLC (SURF, Summer
2004), and T.J.S. (SDSU-Pfizer Summer 2005). We
thank the Howell Foundation for support for C.L.C.
(Spring 2004), EP (Spring 2005), J.V.C.J. (Spring
2005), and T.J.S. (Spring 2006). We thank the US Coast
Guard for support of JDB (2004-current). We thank the
NIH/NIGMS-MBRS-IMSD program for support of JC
(Spring 2005-Spring 2006 NIH-5R25GM58906) and the
SDSU McNair program for support to CLC (Summer
2003 and 2004) and IM (Summer 2004). We thank the
NIH/MARC (5T34GM08303) program for support of
RR (2005/2006). We thank the MIRT program for their
travel support of IM (2003-2004), CLC (2004), and EP
13. 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, 8447–8450.
14. Styers, T. J.; Rodriguez, R.; Pan, P.-S.; McAlpine, S. R.
Tetrahedron Lett. 2006, 47, 515–517.
15. For details on the reaction conditions, see Supplementary
material.
16. It was straightforward to follow the reactions via LC–MS
as the starting material free-acid–free amine linear pre-
cursor would appear at 5.0–5.5 min and the cyclized
product would appear between 6.1 and 7.0 min.
17. The thirty-six macrocyclic peptides have LC–MS spectra
given in Supplementary material. In addition, data for
intermediates involved in the synthesis of active com-
pounds (5, 26, 27, 32, and 33) are also shown.