Structure-activity relationship of Kahalalide F synthetic analogues

Article abstract of DOI:10.1021/jm8000828

Kahalalide F (KF) is a natural product currently under phase II clinical trials. Here, we report the solid phase synthesis of 132 novel analogues of kahalalide F and their in vitro activity on a panel of up to 14 cancer cell lines. The structure-activity relationship of these analogues revealed that KF is highly sensitive to backbone stereotopical modification but not to side chain size modification. These observations suggest that this compound has a defined conformational structure and also that it interacts with chiral compounds through its backbone and not through its side chains. The N-terminal aliphatic acid appears to be a hydrophobic buoy in a membrane-like environment. Moreover, significant improvement of the in vitro activity was achieved.

Full text of DOI:10.1021/jm8000828

4920
J. Med. Chem. 2008, 51, 4920–4931
Structure-Activity Relationship of Kahalalide F Synthetic Analogues
José C. Jiménez,†,‡ Angel López-Macià,‡,§ Carol Gracia,‡ Sonia Varón,‡,| Marta Carrascal,‡ Josep M. Caba,† Miriam Royo,‡,|,
Andrés M. Francesch,§ Carmen Cuevas,§ Ernest Giralt,†,‡ and Fernando Albericio*,†,‡,
Institute for Research in Biomedicine, Barcelona Science Park, Josep Samitier 1, 08028 Barcelona, Spain, Department of Organic Chemistry,
UniVersity of Barcelona, Mart´ı i Franque´s 1, 08028 Barcelona, Spain, Pharma Mar, S.A., 28770 Colmenar Viejo, Madrid, Spain,
Combinatorial Chemistry Unit, Barcelona Science Park, 08028 Barcelona, Spain, and CIBER-BBN, Networking Centre on Bioengineering,
Biomaterials and Nanomedicine, Barcelona Science Park, Josep Samitier 1-5, 08028 Barcelona, Spain
ReceiVed January 26, 2008
Kahalalide F (KF) is a natural product currently under phase II clinical trials. Here, we report the solid
phase synthesis of 132 novel analogues of kahalalide F and their in vitro activity on a panel of up to 14
cancer cell lines. The structure-activity relationship of these analogues revealed that KF is highly sensitive
to backbone stereotopical modification but not to side chain size modification. These observations suggest
that this compound has a defined conformational structure and also that it interacts with chiral compounds
through its backbone and not through its side chains. The N-terminal aliphatic acid appears to be a hydrophobic
buoy in a membrane-like environment. Moreover, significant improvement of the in vitro activity was
achieved.
Introduction
every 3 weeks).5,6 KF phase II clinical trials in hepatocellular
carcinoma, non-small-cell lung cancer (NSCLC) and melanoma
finished in 2006. Moreover, KF is being tested in phase II
clinical trials in patients with severe psoriasis.
Kahalalide F (KF,a Figure 1) is a depsipetide that was first
isolated in 1993 by Hamman and Scheuer from a marine
mollusk, Elysia rufescens, and from the algae that the mollusk
feeds on, Bryopsis pennata.1 It has also been reported in another
mollusk, Elysia grandifolia.2,3 KF shows in vitro activity against
prostate and colon cancer cells and to a lesser extent, antiviral,
antifungal, and bactericide activity.4 Two clinical phase I trials
in adult patients with advanced prostate cancer and pretreated
solid tumors have established the recommended dose for two
distinct treatment regimes (once a week and 5 consecutive days
Phase I trials showed that the dose-limiting toxicity was a
reversible elevation of liver transaminases. The mechanism of
action of KF is mostly unknown. KF is a National Cancer
Institute COMPARE-negative compound, which indicates that
its cytotoxicity might be related to a unique mode of action. A
first study indicated that cells treated with KF became swollen
and developed large vacuoles, which appeared to be a conse-
quence of changes in lysosomal membranes.7 Recently, a further
study has revealed that KF induces oncosis8 and that it has a 5-
to 40-fold greater effect on cancer cells than healthy cells.6,9
The mechanism of action of KF does not affect the nuclear
structure of the cell, while the integrity of crucial organelles
such as mitochondria, endoplasmic reticulum, and lysosomes
is severely compromised. The alterations found indicate that
the osmotic balance of the cell may be altered, possibly as a
result of cellular membrane damage. Thus, it appears that KF
does not induce apoptosis, the main mechanism by which tumor
cells are killed by anticancer drugs or radiotherapy.10 Moreover,
the mechanism of action of KF is caspase-independent and not
affected by DNA expression.8
With the aim to identify new KF analogues with improved
pharmacological properties and examine the role of each residue
on the biological activity of this compound, our group developed
a structure-activity relationship (SAR) program. In order to
understand this study, it is noted that the KF structure is divided
into three domains: domain A includes the macrocycle; domain
B contains the peptide tail; domain C is the N-terminal aliphatic
acid (Figure 1). Here, we report the synthesis and in vitro
cytotoxicity of 132 KF analogues on several human cancer cell
lines.
* To whom correspondence should be addressed. Phone: 0034 934037088.
Fax: 0034 934037126. E-mail: albericio@pcb.ub.es.
†
Institute for Research in Biomedicine.
University of Barcelona.
Pharma Mar, S.A.
Combinatorial Chemistry Unit.
‡
§
|
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and
Nanomedicine.
a Abbreviations: Alloc, allyloxycarbonyl; Bip, 2-amino-3-biphenyl-4-
ylpropionic acid; Boc, tert-butyloxycarbonyl; Bza-OH, benzoic acid; Cha,
cyclohexylalanine or 2-amino-3-cyclohexylpropionic acid; p-CF3Bza-OH,
4-trifluoromethylbenzoic acid; p-TflPhAc-OH, 3-(4-trifluoromethylbenzy-
l)acetic acid; p-TflCinn-OH, 3-(4-trifluoromethylbenzyl)acrylic acid; Cl-
TrtCl-resin, 2-chlorotrityl chloride resin; Dha, 2-aminoacrylic acid; Z-Dhb,
R,ꢀ-didehydro-R-aminobutyric acid; DIPEA, N,N-diisopropylethylamine;
DMF, N,N-dimethylformamide; EDC ·HCl, 1-ethyl-3-(3′-dimethylamino-
propyl)carbodiimide hydrochloride; Fmoc, 9 fluorenylmethoxycarbonyl; 6,6-
dFHep-OH, 6,6-difluoroheptanoic acid; 3,5-dFPhAc-OH, (3,5-difluorophe-
nyl)acetic acid; 4-GuBut-OH, 4-guanidinobutyric acid; GI50, growth
inhibition at 50%; hCh, homocyclohexylalanine or 2-amino-4-cyclohexy-
lbutyric acid; Hep-OH, heptanoic acid; HOAc, acetic acid; HOAt, 1-hy-
droxy-7-azabenzotriazole (3-hydroxy-3H-1,2,3-triazolo[4,5-b]pyridine); HOBt,
1 hydroxybenzotriazole; Icos-OH, icosanoic acid; KF, kahalalide F; LC50,
lethal concentration at 50%; Lit-OH, litocholic acid; MeHex-OH, methy-
hexanoic acid; (c/t)-MecHex-OH, (cis/trans)-4-methylcyclohexanecarboxylic
acid; p-MeBza-OH, 4-methylbenzoic acid; MeOH, methanol; Mst-OH,
myristic acid or tetradecanoic acid; NaI, 2-amino-3-naphthalen-2-ylpropionic
acid; Oct-OH, octanoic acid; 6-Ohep-OH, 6-oxoheptanoic acid; Oic,
octahydroisoindole-1-carboxylic acid; Phf-OH, perfluoroheptanoic acid; Phg,
aminophenylacetic acid; (5R)-Ph-Pro, 5-(R)-phenylpirrolidine-2-carboxilic
acid; Pip, pipecolic acid; Pipe-OH, benzo[1,3]dioxole-5-carboxilic acid; SPS,
solid-phase synthesis; SRB, sulphorhodamine B; TCA, trichloroacetic acid;
Tfa, trifluoroacetyl; TFA, trifluoroacetic acid; TFAA, trifluoroacetic anhy-
dride; TGI, total growth inhibition; Thi, 2-amino-3-thiophen-2-ylpropionic
acid; Tic, 1,2,3,4-tetraisoquinoline-3-carboxilic acid; Und-OH, undecanoic
acid.
Results and Discussion
Synthesis of KF Analogues. The analogues were synthesized
following the scheme used for the first KF synthesis with minor
modifications (Scheme 1).11,24 These modifications were mainly
(i) the use of DIPCDI/HOBt as coupling reagents in the solid-
10.1021/jm8000828 CCC: $40.75
2008 American Chemical Society
Published on Web 08/01/2008

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4921
Figure 1. KF structure with its three domains highlighted.
Scheme 1. Synthetic Strategies for Solid-Phase Synthesis of KF Analoguesa
a Conditions: (a) Fmoc-D-Val-OH, DIEA, DMF; (b) piperidine-DMF (2:8); (c) Fmoc-aa-OH, DIPCDI, HOBt, DMF; (d) Alloc-Val-OH, DIPCDI, DMAP;
(e) 5-MeHex acid, DIPCDI, HOBt, DMF; (f) Pd(PPh3)4, PhSiH3, DCM; (g) Alloc-Phe-(Z)-Dhb-OH, DIPCDI, HOAt, DMF; (h) EDC ·HCl, CuCl, DMF-DCM
(1:9); (i) TFA-DCM (1:99); (j) DIPCDI, HOBt, DIEA, DCM; (k) TFA-DCM (19:1).
phase elongation (SPPS) instead of azabenzotriazole-based
reagents and (ii) the use of DIPCDI/HOBt/DIEA for the
cyclization step instead of PyBOP/DIEA. Azabenzotriazole
derivatives are expensive, and therefore, their substitution greatly
reduces the cost of the synthesis. Moreover, these changes imply
that the epimerization on the DVal(4) is prevented during
cyclization. For the synthesis of most of the analogues, one or
more amino acids, or the 5-MeHex acid, were either removed
or substituted by another properly protected amino acid or acid.
In Scheme 1, two alternative routes to incorporate the
nonproteinogenic amino acid Z-didehydroaminobutyric acid
[(Z)-Dhb] are shown. Route A was chosen for most of the
analogues. This procedure involves the incorporation of a
presynthesized dipeptide, Alloc-Phe-(Z)-Dhb-OH, to the peptide

4922 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 1. Mean Activity of KF in Assay A (Values in Molar (M))
Jiménez et al.
acyclic natural analogue of KF, kahalalide G, it is known that
the ring is essential for biological activity.4
GI50
TGI
LC50
The presence of D-amino acids, the non-natural amino acid
(Z)-Dhb, and the lack of a C-terminal amino acid as a result of
the lactamization confer a high degree of enzymatic stability to
this domain.15 These features also increase the rigidity of the
domain, which may be of relevance for activity.15
The most explored residue in this domain is Phe (3), with 20
analogues, the most active being those that introduce halogen
groups on the aromatic ring, with the greatest of the activity in
compounds 22 [Phe(2-Cl)], 23 [Phe(3-Cl)], 24 [Phe(4-Cl)], 25
[Phe(3,4-F2)], and 26 (NaI). Interestingly, 11 [Phe(3,4-Cl2)]
with two chlorines on the aromatic ring was not as active as
the monochlorinated compounds 22, 23, or 24, and the position
of the chlorine did not appear to be relevant. In contrast, 25
[Phe(3,4-F2)] with two fluorines was more active than the
analogue with a single fluorine (15) and the analogue with five
fluorines (12).
The analogues at Phe (3) suggest that a hydrophobic residue
is required in that position. Almost all halogen derivatives of
Phe were more active than KF. Compounds in which Phe was
replaced by Trp (9), hCh (10), and methylated Tyr (16) were
more active, while hydrophilic compounds such as Thi (17) and
Tyr (19) showed lower activity. Analogues that restricted the
rotation of the aromatic ring through N-alkylation either did
not improve activity (N-MePhe, 21) or blocked it (Oic 20, Tic
18). Finally, the Phg derivate (28), which can be considered a
Phe lacking the ꢀ-methylene atom, was slightly less active.
Given the ring nature of domain A, the L/D configuration of
the R-carbon of the backbone is crucial. Compound 29 explored
the possibility of Val(4) instead of D-Val.25 This analogue lost
all activity, thereby confirming the importance of the conforma-
tion of the cycle.
A presumably key amino acid in this domain is (Z)-Dhb.
Analogues at this residue included other didehydroamino acids
such as (Z)-Dhf (3) and Dha (4). These two compounds were
as active as KF, thereby indicating that its role revolves around
the rigidness and special configuration that didehydroamino
acids confer to the main chain,15 with no major relevance of
their side chains. This point could be reinforced by the
observation that analogues 1 and 2, which contain L and D
ethylglycine (Etg), the saturated versions of (Z)-Dhb, were less
active than KF. Analogues 5 and 6, which incorporate D-Thr
and D-allo-Thr, respectively, in that position showed almost no
activity. Compounds with aquiral amino acids such as Gly (7)
and Aib (8) did not show activity either.
Finally, the D-allo-Thr that closes the ring was the subject of
a set of analogues with D-Dapa (30), D-Thr (31), and D-Ser (32)
(30 is the aza version of 32). Analogue 32 (D-Ser) maintained
the activity of KF, which could indicate that the ꢀ-methyl group
of the D-allo-Thr is not relevant for the stabilization of the
structure. However, when this methyl group was misplaced in
analogue 31, activity was lost. When D-Ser (32) was substituted
by D-Dapa (30), activity was lost again. These observations
indicate that the pattern of hydrogen bond donors/acceptors or
the freedom around the heterodetic bond may be relevant to
the conformation.
So far, the term “activity” has been referred to the overall
activity of the cell lines compared to KF. On closer inspection,
we can rank activity depending on the tissue from which the
tumor derives. Cell lines were as follows, in order of sensitivity
from high to low: colon (HT-29, LOVO and LOVO-DOX),
ovary (IGROV and IGROV-ET), breast (SK-BR-3), prostate
(DU-145 and LN-caP) and melanoma, lung, bone marrow
prostate
ovary
DU-145
LN-caP
IGROV
IGROV-ET
SK-BR3
9.31 × 10-7 1.72 × 10-6 3.72 × 10-6
9.60 × 10-7 1.96 × 10-6 4.26 × 10-6
4.93 × 10-7 1.18 × 10-6 3.22 × 10-6
5.50 × 10-7 1.30 × 10-6 3.28 × 10-6
3.63 × 10-7 8.10 × 10-7 1.98 × 10-6
1.16 × 10-6 2.20 × 10-6 4.52 × 10-6
1.93 × 10-6 3.32 × 10-6 5.28 × 10-6
1.77 × 10-6 3.33 × 10-6 5.07 × 10-6
4.32 × 10-7 9.15 × 10-7 2.24 × 10-6
3.34 × 10-7 6.14 × 10-7 1.51 × 10-6
breast
melanoma
lung (NSLC) A549
leukemia
pancreas
colon
SK-MEL-28 1.19 × 10-6 2.26 × 10-6 4.50 × 10-6
K-562
PANC1
HT29
LOVO
LOVO-DOX 2.99 × 10-7 4.85 × 10-7 1.47 × 10-6
cervix
HELA
9.65 × 10-7 1.97 × 10-6 4.73 × 10-6
HELA-APL 9.75 × 10-7 2.02 × 10-6 4.29 × 10-6
sequence as a regular amino acid. Route B, which involves a
ꢀ-alcohol solid-phase dehydration, takes longer and gives a
lower yield12 and was therefore used only when the modification
was on the amino acids (Z)-Dhb or Phe.
Finally, another set of analogues was obtained by reaction
of KF or an intermediate with diverse reactants through the
amino group of Orn (acylation with common coupling reagents)
or the hydroxyl group of Thr (acylation with DIPCDI in the
presence of DMAP).13
Antitumoral Screening and Data Representation. The
cytotoxicity of each peptide was evaluated against a panel of
up to 14 human tumor cell lines. The cytotoxic assay (assay A
in methods section) gives an output of three parameters: GI50
(concentration at which the growth is inhibited by 50%), TGI
(concentration at which the growth is totally inhibited), and LC50
(lethal concentration that will kill 50% of the cells).14 Of these
parameters, only GI50 is shown in Tables 2–5 to facilitate
comparison. The mean activity for KF is shown in Table 1.
Moreover, the activity of several analogues was taken from
another assay (assay B in methods section), which was
performed at the very beginning of this study. The output data
of this assay is the IC50 (half-maximal inhibitory concentration).
Compounds that did not show relevant activity were not tested
again in assay A. For the sake of simplicity, the activity of these
compounds has been included in the table with the other
analogues and can be distinguished by an asterisk.
Activity data are shown in Tables 2–5 using a color palette
to describe GI50 concentration in a logarithmic scale.
SAR. From the 132 KF analogues synthesized and tested,
32 had modifications at domain A (analogues 1-32, Table 2),
59 at domain B (33-91, Table 3), and 34 at domain C
(analogues 92-125, Table 4). In addition, seven had modifica-
tions in more than one domain (analogues 126-132, Table 5).
The chemical structure of each substitution is given in Figures
24.
Several compounds in Tables 2 (domain A) and 3 (domain
B) had two modifications on their structure: one related to the
explored domain and the other corresponding to the N-terminal
moiety. The 5-MeHex in these compounds was substituted by
4(S)-MeHex in position 14. This switching did not affect the in
vitro activity, as demonstrated in parallel experiments that
showed that these two acids led to similar in vitro activity. In
contrast, the compound with 4(S)-MeHex (100, Table 4)
exhibited higher in vivo efficacy in breast and prostate xe-
nografts.19
Domain A Analogues. This domain contains the macro ring
formed by the six C-terminal amino acids closed by an ester
bond between the carboxylic acid of Val (1) and the ꢀ-hydroxyl
function of D-allo-Thr (6). Given the absence of activity of the

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4923
Table 2. Domain A KF Analoguesa
a The chemical structure of each modification can be found in Figure 2. “4” in 5-MeHex column indicates the presence of 4(S)-MeHex. Colored squares
represent the GI50 (or IC50 on marked (/) compounds) of each compound on the cells lines tested. Colored values at the bottom are expressed in mol/L.
(leukemia), and pancreas (MEL-28, A-545, K-562, and PANC-
1). Depending on the analogue, this selectivity was altered; for
example, for 9 (Trp3), selectivity for ovary cell lines was greater
than for colon. For 11 [Phe(3,4-Cl2)] the selectivity for lung
cell line was increased. For 27 (Bip3) the activity in lung was
higher than in any other cell line.
Domain B Analogues. This domain is composed of seven
amino acids. It is rich in aliphatic residues including a D-allo-
Ile, three Val (two D and one L), and one D-Pro. The domain is
completed with one Thr and one Orn, which is the only charged
amino acid in the peptide at physiological pH. It is also
noticeable that four of the residues have configuration D.
All analogues in which the chirality of any residue was
switched lost their activity [Table 3, analogues 54 (Pro), 66 (D-
Val11), and 76 (D-Thr)] except for 86 (Val13), which is the
closest residue to the N-terminal aliphatic acid. The importance
of the configuration of the R carbons of the amino acids suggests
that this domain is not merely a link without structure between
the other two domains but rather adopts or induces some sort
of folding and/or interactions with another molecule.
To confirm that the loss of activity was not induced by the loss
of the total chain length, compound 130 (Table 5), which
included the C14 myristic acid at the N-terminus, was also
explored simulating the approximate length of the total chain.
This also resulted in no activity. These observations corroborate
the need for chiral information on that part of the peptide.
Moreover, when a residue was substituted by a distinct amino
acid, the activity was conserved or even increased when
nonpolar amino acids with the same chirality were used
(analogues 59, 63, 65, 73, 75, 81, 83, 85, and 87). The exception
to this observation was, again, at position 13, where D-Val could
not be replaced by the more hindered amino acid D-Cha
(analogues 60 and 61).
It is also of note that the hydroxyl group of the Thr was not
required for antitumoral activity. Furthermore, the activity was
increased with aliphatic substitutions (analogues 63 and 65).
The substitution of D-Pro by L-Pro (54) and (R)-5Ph-L-Pro (57)
led to no activity, pointing again to the existence of some sort of
folding or recognition. Moreover, neither did the substitution by
six-member ring D-Pro analogues D-Pip (55) and D-Tic (56) produce
activity, which introduces another twist to the peptide conforma-
tional sensitivity in that region, since increasing from five- to six-
member ring had less impact on the orientation of the groups than
changing the chirality of the amino acid.
One set of compounds we synthesized explored the elimina-
tion of residues starting from D-allo-Ile 7 (analogues 46-52).
None of these analogues showed activity. In all these analogues
the positive charge of Orn was missing, which may explain the
loss of activity. To confirm this assumption, we synthesized
compound 53 in which the Orn was kept but the last three
residues were removed. This synthesis resulted in no activity.
Compound 58 has historical relevance in the determination
of KF structure. The first report on KF stereochemistry assigned
the configurations D and L to Val (10) and Val (11), respec-

4924 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Table 3. Domain B KF Analoguesa
Jiménez et al.
a The chemical structure of each modification is shown in Figure 3. “4” in 5-MeHex column indicates the presence of 4(S)-MeHex. “no” indicates the
absence of that residue in the compound. Colored squares represent the GI50 (or IC50 on marked (/) compounds) of each compound on the cell lines tested.
Colored values at the bottom are expressed in mol/L.
tively.16 A review of this work originated a second assignment
where the stereochemistry in these two residues was switched.17
The two compounds were synthesized and compared to natural
KF by NMR and by activity assay, solving the correct
assignation without any doubt.11 NMR spectra of these two
compounds showed a single set of signals for KF, while
analogue 58 presented two sets corresponding to cis/trans Pro
isomerization. This was the first example of the sensitivity of
the structure of domain B to the stereochemistry of its residues
and how these affected its activity.
Furthermore, several analogues were prepared by direct
reaction on the amino group of Orn in KF with diverse reagents,
taking advantage of the regioselectivity of this group over the
rest of the molecule as previously done on KF by other groups
(analogues 33-39 and 42-44).18 We completed this set with
the introduction of distinct amino acids instead of Orn (ana-
logues 40 and 41).
Here, we found the most active compounds, analogues 35
and 37. In general, compounds with aliphatic groups coupled
to the amino group of Orn were the most active (34-37). In
analogue 37 the activity fell when the Fmoc group was cleaved,
thereby generating a positive charge (analogue 38). This is
surprising, since this is the only charged amino acid in the whole
sequence, and it seems to be not only unnecessary but
undesirable. Moreover, these compounds open the door to
conjugates with aliphatic drugs at that point that cannot only
act in synergy with KF but can also increase its inner activity
in the same way as analogues 35 and 37 do.
Another important datum is that Lys (40) is as active as Orn.
This may seem strange because Lys is a coded amino acid and
therefore more likely to be used in a natural product. Recently,
it has been reported that this compound is also present (called
as kahalalide S) in small amounts in Elysia grandifolia and the
algae it feeds on, Bryopsis plumose.3

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4925
Table 4. Domain C KF Analoguesa
a The chemical structure of each modification is shown in Figure 4. “no” indicates the absence of that residue in the compound. Colored squares represent
the GI50 of each compound on the cell lines tested. Colored values at the bottom are expressed in mol/L.
Table 5. KF Analogues in More Than One Domaina
a The chemical structure of each modification is shown in Figures 2–4. “no” indicates the absence of that residue in the compound. Colored squares
represent the GI50 of each compound on the cell lines tested. Color values at the bottom are expressed in mol/L.
The broad range of active compounds modified on Orn
suggests that this residue is not involved in any recognition
interface.
When we examined the changes in cell line selectivity, we
observed that the analogues with hydrophobic molecules capping
the δ-amino group of Orn not only were the most active but
also showed the most changes in selectivity. Thus, analogue
37 [Orn(Fmoc-PEG)] was as active or even more active on
prostate, lung, bone marrow, and pancreas cell lines than on
colon, breast, and ovary cell lines. This effect was also observed

4926 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Jiménez et al.
Figure 2. Building blocks used for the substitution of residues at domain A.
Figure 3. Building blocks used for the substitution of residues at domain B.
in analogue 35 [Orn(cHP)]. Analogues 33, 36, and 43 increased
on the rest of cell lines. A remarkable case was compound 69
their activity in breast and colon cell lines with no major effect
(Orn11 instead of Val). This compound not only broke the rule

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4927
Figure 4. Building blocks used for the substitution of residues at domain C.
that aliphatic changes are preferred over polar substitutions but
also affected mainly leukemia and showed very low activity
against the rest of the panel cell lines.
found in the organism Elysia grandifolia.2 In KR the 5-meth-
ylhexanoic acid in position 14 is substituted by 7-methyloctanoic
acid, while in KS it is substituted by 5-hydroxy-7-methyloc-
tanoic acid. KR is as active as KF, while KS is 20-fold less
active than KF. It is noted that references 2 and 3 both use
letters R and S to describe four new kahalalide compounds.
Domain C Analogues. The analogues generated at this
domain required an aliphatic group in this position. Any
inclusion of polar groups able to generate hydrogen bonds
decreased the activity (analogues 117-123). Compounds with
no terminal acid (92) or a short aliphatic group (analogues
93-98) had lower activity than KF. Finally, the activity was
maintained or increased when the N-terminal acid was substi-
tuted by a more aliphatic group (99-116). An exception to this
rule was analogue 108 (icosanoic acid, C20), where the low
activity can be explained by the poor solubility of the compound.
In compound 125 [Lit(OTfa)] the ester group generated
hydrogen bonds but the relative size of the molecule must be
considered.
Compounds 100 and 101 showed an interesting effect. These
two compounds incorporated the two enantiomers of 4-metyl-
hexanoic acid, which is the compound closest to 5-methylhex-
anoic acid with a chiral center. The activity of the compound
that incorporated the R enantiomer was lower than that which
incorporated the S enantiomer, the activity of the latter being
similar to the parent peptide. Further in vivo studies on these
compounds showed that compound 100 has enhanced efficacy
against breast and prostate xenografts.19 On the basis of this
observation, we chose to incorporate 4(S)-MeHex instead of
5-MeHex into other analogues.
A practical example of this is the finding of two new natural
kahalalides called kahalalide R (KR) and kahalalide S (KS)

4928 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Jiménez et al.
Several analogues clearly showed higher in vitro activity than
the parent peptide. Here, we highlight compounds 102,
104-107, 111, 112, 116, and 125, the most active analogues
being 106 (undecanoic acid, Und), 107 (palmitic acid), 112 (p-
TflBza), 116 (p-TflCinn), and 125 [Lit(OTfa)]. Compounds 111
and 112 were very similar, the only difference being that 112
had a trifluoromethyl group instead of a methyl group attached
to the aromatic ring. These two groups exerted an opposite
inductive effect on the aromatic ring, with enhanced electronic
density on compound 111 and lower electronic density on
compound 112, the latter being more active.
The wide array of molecules that maintained or increased
the activity in domain C and the common characteristic of their
high aliphatic behavior suggest that this domain does not interact
selectively with any other molecule and that its main function
is as an aliphatic buoy.
The activity can be increased by enhancing the hydrophobicity
at any position on the molecule, solubility in water being a
limiting factor. Domain C is the most sensitive to this rule, since
it does not allow the inclusion of electronic-donor atoms such
as oxygen or nitrogen. Domain C accepts only highly hydro-
phobic groups with a low heteroatom load. This observation
suggests that this domain interacts with a highly hydrophobic
and unspecific environment such as a membrane, possibly with
the lysosomal membrane since KF affects this organule.7
Analogues with a hydrophobic group attached to the ε-amino
group of Orn open the door to conjugated drugs (i.e., 33, 34,
35, and 37). These compounds are more active than KF and
compounds with polar groups. This finding implies that KF
activity can be enhanced while simultaneously using KF as a
carrier of a hydrophobic drug with solubility or transport
limitations, thereby obtaining a synergic effect.
As an extension of the above discussion, we consider this
set of analogues to be useful also as control peptides in research
into the biological mechanism of KF. By examination of the
distinct behavior displayed by some analogues compared to KF,
it is possible to identify the role played by each part of the
peptide in the overall activity. An example of this approach
would be the enantiomer analogue 126. Studying its capacity
to internalize the cell would provide information as to whether
KF is actively internalized by a carrier or unspecifically
internalized.
Also relevant, this set of compounds opens up the possibility
of modulating KF selectivity when used in distinct cell lines. It
would therefore be feasible to develop analogues with different
tissue specificity. This would allow the development of KF-
related compounds targeted to a particular kind of tumor, thus
obtaining a wider therapeutic window.
As seen in other domains, a number of compounds showed
a change in their selectivity against the cell lines assayed.
Analogues 102, 106, 107, 112, 116, and 125 had predominant
activity against ovary cell lines. Compound 107 also significantly
increased activity against prostate cell lines.
Multidomain Analogues. Table 5 shows the analogues
modified in more than one domain. The first entry (126)
corresponds to the KF enantiomer, which showed no activity.
Analogues 127 and 128 showed once again that more
hydrophobic residues (D-Cha) than those of Val or Ile
increased the activity, and when the number of methylenes
was reduced (e.g., substituting D-allo-Ile by Val), the activity
diminished. This behavior can also be explained by confor-
mational changes induced by the volume of the side chains,
since Val is less voluminous than D-allo-Ile, thereby allowing
more conformations.
Finally, now that the impact of backbone stereochemistry on
KF activity is known, analogues that affect this stereochemistry
can be used to perform structural studies of KF by means of
molecular modeling and NMR. These analogues are 1, 2, 3, 7,
8, 30-32 in domain A and 54-58, 62, 66, 76, and 86 in domain
B.
Analogue 129, which is the combination of analogues 11 and
116, showed increased activity compared to KF but not
significantly higher than that of compound 11.
Finally, in compound 132, the amino group of D-Val13 was
alkylated with two heptane chains. This substitution led to no
activity, which can be explained by the generation of a very
stable positive charge at this nitrogen.
In this panel we observed changes in the cell line selectivity
of compounds 127, 129, and 131, where again the compounds
were most active on ovary cell lines. Also worth mentioning is
the increased activity against prostate cell lines in compound
127. In compound 129 the activity against lung cell line (A549)
was very high because of the presence of Phe(3,4-Cl2) at the
third position, as with compound 11.
Experimental Section
Cl-TrtCl-resin, protected Fmoc-amino acid derivatives, HOBt,
and HOAt were from ABI (Framingham, MA), Bachem (Buben-
dorf, Switzerland), and NovaBiochem (La¨ufelfingen, Switzerland).
4-MeHex derivatives were from Narchem. HATU was from ABI
(Framingham, MA). DIPEA, DIPCDI, EDC ·HCl, piperidine, and
TFA were from Aldrich (Milwaukee, WI). DMF and CH2Cl2 were
from SDS (Peypin, France). Acetonitrile (HPLC grade) was from
Scharlau (Barcelona, Spain). All commercial reagents and solvents
were used as received with the exception of CH2Cl2, which was
passed through an alumina column to remove acidic contaminants.
Alloc-amino acids were prepared essentially as described,20 and
alloc-Z-Dhb-Phe-OH compounds were prepared as described11 and
also in Supporting Information.
HRMS was performed in LTQ-FT Ultra from ThermoScientific
in nanospray mode by infusion and H2O/CH3CN (1:1) + 0.1%
formic acid as a solvent. MALDI-TOF and ES-MS analyses of
peptide samples were performed in a PerSeptive Biosystems
Voyager DE RP, using DHB matrix, on a Waters Micromass ZQ
spectrometer and on an Agilent ion trap 1100 series LC/MSDTrap.
Peptide-resinsampleswerehydrolyzedin12NaqueousHCl-propionic
acid (1:1) at 155 °C for 1-3 h, and peptide-free samples were
hydrolyzed in 6 N aqueous HCl at 155 °C for 1 h. Subsequent
amino acid analyses were performed on a Beckman System 6300
autoanalyzer. 1H NMR spectroscopy [1H, NOESY, TOCSY at 278
K] was performed on a Varian Unity Plus (500 MHz). Chemical
shifts (δ) are expressed in parts per million downfield from TMS.
Coupling constants are expressed in hertz. Analytical HPLC was
Conclusions
Kahalalide F has a more defined structure than expected. This
is not surprising in cyclic domain A, but astonishingly it also
occurs in domain B, which a priori is much more flexible. KF
is highly sensitive to stereotopical changes, namely, those
changes that affect chirality at the R-carbon of the residue. KF
is not sensitive to side chain substitutions in almost every
residue, the electronic density of the substituting chain being
more relevant than its volume. For almost every side chain, it
was possible to find a distinct side chain that could preserve or
even improve the activity.
Our results on enantiomer 126 suggest that KF interacts with
at least one chiral compound to develop cytotoxicity. In this
regard, we found a more hindered replacement in each side chain
able to keep or improve the activity; the interactive region should
be pointed at the backbone of the molecule, the cycle being the
more appealing target.

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4929
carried out on a Waters instrument comprising two solvent delivery
pumps (Waters 1525), automatic injector (Waters 717 autosampler),
dual wavelength detector (Waters 2487), and system controller
(Breeze, version 3.20) and on an Agilent 1100 instrument compris-
ing two solvent delivery pumps (G1311A), automatic injector
(G1329A), and DAD (G1315B). UV detection was at 215 or 220
nm, and linear gradients consisted of CH3CN (+0.036% TFA) into
H2O (+0.045% TFA).
and 3 equiv; 310 µL, for 2 mmol and 4 equiv; 388 µL, for 2.5
mmol and 5 equiv) and HOBt (230 mg, for 1.5 mmol and 3 equiv;
307 mg, for 2 mmol and 4 equiv; 395 mg, 2.5 mmol and 5 equiv)
for 90 min. In all cases, after 90 min of coupling, the ninhydrin
test was negative. Removal of the Fmoc group and washings were
carried out as described in the general procedures section.
Step 5 of 7. (4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-
Orn(Boc)-D-allo-Ile-D-allo-Thr(Val-Z-Dhb-Phe-Alloc)-D-allo-Ile-
D-Val-O-TrtCl-resin. The Alloc group was removed with Pd(P-
Ph3)4 (58 mg, 0.05 mmol, 0.1 equiv) in the presence of PhSiH3
(617 µL, 5 mmol, 10 equiv) under Ar atmosphere, and Alloc-Phe-
Z-Dhb-OH (666 mg, 2 mmol, 4 equiv) and HOAt (273 mg, 2 mmol,
4 equiv) were dissolved in DMF (1.25 mL) and added to the
peptidyl-resin. DIPCDI (310 µL, 2 mmol, 4 equiv) was then added,
and the mixture was stirred for 5 h, after which the ninhydrin test
was negative. After washings with DMF and CH2Cl2, an aliquot
of the peptidyl-resin was treated with TFA-H2O (1:99) for 1 min
and the product was characterized by MALDI-TOF-MS, calcd for
C88H146N14O21, 1735.08. Found: m/z 1758.67 [M + Na]+, 1,774.62
[M + K]+.
Here, we describe the synthesis of compound 100 as a general
example. Detailed synthesis and characterization of the rest of the
compounds can be found in Supporting Information.
(4S)-MeHex-D-Val-ThrVal-D-Val-D-Pro-Orn-D-allo-Ile-ciclo[D-
allo-Thr-D-allo-Ile-D-Val-Phe-(Z)-Dhb-Val], [(4S)-MeHex14]KF,
Compound 100. Step 1 of 7. H-D-Val-O-TrtCl-Resin. Cl-TrtCl-
resin (1 g, 1.64 mmol/g) was placed in a 20 mL polypropylene
syringe fitted with a polyethylene filter disk. The resin was then
washed with CH2Cl2 (5 × 0.5 min), and a solution of Fmoc-D-
Val-OH (238 mg, 0.7 mmol) and DIPEA (0.41 mL, 2.41 mmol) in
CH2Cl2 (2.5 mL) was added. The mixture was then stirred for 15
min. Extra DIPEA was then added (0.81 mL, 4.75 mmol), and the
mixture was stirred for 45 min. The reaction was completed by
addition of MeOH (800 µL, 19.78 mmol), after stirring for 10 min.
The Fmoc-D-Val-O-TrtCl-resin was subjected to the following
washings/treatments with CH2Cl2 (3 × 0.5 min), DMF (3 × 0.5
min), piperidine as indicated in the general procedures section, and
DMF (5 × 0.5 min). The loading was 0.50 mmol/g calculated by
Fmoc determination.
Step 2 of 7. Fmoc-D-allo-Ile-D-allo-Thr(Val-Alloc)-D-allo-Ile-
D-Val-O-TrtCl-Resin. Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4
equiv), Fmoc-D-allo-Thr-OH (free hydroxy group) (683 mg, 2
mmol, 4 equiv), and Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv)
were added sequentially to the H-D-Val-O-TrtCl-resin described
above using DIPCDI (310 µL, 2 mmol, 4 equiv) and HOBt (307
mg, 2 mmol, 4 equiv) in DMF (2.5 mL). In all cases, after 90 min
of coupling, the ninhydrin test was negative. Removal of the Fmoc
group and washings were carried out as described in the Supporting
Information. Alloc-Val-OH (502 mg, 2.5 mmol, 5 equiv) was
coupled using DIPCDI (387 mg, 2.5 mmol, 5 equiv) in the presence
of DMAP (30.6 mg, 0.25 mmol, 0.5 equiv) and DIPEA (88 µL,
0.5 mmol, 1 equiv) for 45 min. This coupling was repeated under
the same conditions twice. An aliquot of the peptidyl-resin was
treated with TFA, and the HPLC results (tR ) 7.8 min, conditions
S, column D) of the crude product obtained after evaporation
showed a purity of >98%. ESMS, calcd for C45H63N5O11, 849.45.
Found: m/z 850.1 [M + H]+.
Step 6 of 7. (4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-
Orn(Boc)-D-allo-Ile-D-allo-Thr(Val-Z-Dhb-Phe-H)-D-allo-Ile-D-
Val-OH. After washings with DMF and CH2Cl2, the Alloc group
was removed with Pd(PPh3)4 (58 mg, 0.05 mmol, 0.1 equiv) in the
presence of PhSiH3 (617 µL, 5 mmol, 10 equiv) under Ar
atmosphere. The protected peptide was cleaved from the resin by
TFA-CH2Cl2 (1:99) (5 × 30 s). Filtrate was collected on H2O (4
mL), and the H2O was partially removed under reduced pressure,
appearing as a light-yellow precipitation in the remaining water.
Acetonitrile was then added to dissolve the solid that appeared
during H2O removal until the solution was clear. Then lyophilization
gave 639 mg (387 µmol, 77% yield) of the title compound with a
purity of >95% as shown by HPLC (condition R, column C, tR )
10.5 min).
Step 7 of 7. (4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D-
allo-Ile-cyclo(D-allo-Thr-D-allo-Ile-D-Val-Phe-Z-Dhb-Val). The
protected peptide from step 6 (639 mg, 387 µmol) was dissolved
in CH2Cl2 (390 mL, 1 mM). Then we added compounds in the
following order: HOBt (237 mg, 1.55 mmol) dissolved in the
minimum volume of DMF, DIPEA (203 µL, 1.16 mmol, 2.3 equiv),
and DIPCDI (240 µL, 1.55 mmol, 4 equiv). The mixture was stirred
for 1 h, and the course of the cyclization step was monitored by
HPLC. The solvent was removed by evaporation under reduced
pressure. The protected cyclic peptide was dissolved in TFA-H2O
(19:1, 85 mL), and the mixture was stirred for 1 h. The solvent
was removed by evaporation under reduced pressure, dioxane was
added (30 mL), and the solvent was removed by evaporation under
reduced pressure (the process was repeated three times). H2O (40
mL) was then added, and the solution was lyophilized. The crude
product was purified by HPLC (Kromasil C8 5 µm, 205 mm × 50
mm), isocratic 44% acetonitrile (+0.05% TFA) in water (+0.05%
TFA), 55 mL/min, detection at 220 nm, to give the title product
(192 mg, 0.13 mmol, 26% yield, 92.3%). HRMS m/z: found (M +
H)+ 1477.937 91, calcd for C75H125N14O16 (M + H)+ 1477.939 25.
The 1H NMR (2.5 mM, 500 MHz, H2O-D2O (9:1) data of the
compound are shown in Table 6.
Bioassays for Antitumoral Screening. Since activity data had
been collected over several months, the activity value of each
compound was normalized using the activity of KF on the assay
day and the mean KF activity.
Assay A. The objective of this assay is to interrupt the growth
of an in vitro tumor cell culture by means of continued exposure
of the cells to the sample to be tested. Cancer cell lines used are
listed in Table 7.
Inhibition of Cell Growth by Colorimetric Assay. A colori-
metric type of assay using the sulforhodamine B reaction was
adapted for quantitative measurement of cell growth and viability.21
This assay employed 96-well cell culture microplates of 9 mm
diameter.22,23 Most of the cell lines were obtained from American
Type Culture Collection (ATCC) derived from a range of types of
human cancer. Cells were maintained in RPMI 1640 10% FBS,
Step 3 of 7. Fmoc-D-Val-D-Pro-Orn(Boc)-D-allo-Ile-D-allo-
Thr(Val-Alloc)-D-allo-Ile-D-Val-O-TrtCl-Resin. The Fmoc group
was removed from the peptidyl-resin obtained in step 2 using
piperidine/DMF 20:80 for 1 × 1 min, 2 × 5 min, 1 × 10 min, and
Fmoc-Orn(Boc)-OH (912 mg, 2 mmol, 4 equiv), Fmoc-D-Pro-OH
(843 mg, 2.5 mmol, 5 equiv), and Fmoc-D-Val-OH (255 mg, 2.5
mmol, 5 equiv) were sequentially incorporated using DIPCDI (310
µL, for 2.0 mmol and 4 equiv; 388 µL, for 2.5 mmol and 5 equiv)
and HOBt (307 mg, for 2.0 mmol and 4 equiv; 395 mg, for 2.5
mmol and 5 equiv) for 90 min. The ninhydrin test after incorporation
of Orn and D-Pro was negative. The chloranil test after incorporation
of D-Val was slightly positive, and this residue was recoupled with
Fmoc-D-Val-OH (678 mg, 2.0 mmol, 4 equiv), DIPCDI (310 µL,
2.0 mmol, 4 equiv) and HOBt (307 mg, 2.0 mmol, 4 equiv) for 90
min. An aliquot of the peptidyl-resin was treated with TFA, and
the HPLC results (tR ) 10.1 min, conditions S, column D) of the
crude product obtained after evaporation showed a purity of >98%.
MALDI-TOF-MS, calcd for C65H97N9O16, 1,259.71. Found: m/z
1282.16 [M + Na]+.
Step 4 of 7. (4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-
Orn(Boc)-D-allo-Ile-D-allo-Thr(Val-Alloc)-D-allo-Ile-D-Val-O-
TrtCl-resin. The Fmoc group was removed, and Fmoc-Val-OH
(678 mg, 2 mmol, 4 equiv), Fmoc-Thr(tBu)-OH (992 mg, 2.5 mmol,
5 equiv), Fmoc-D-Val-OH (678 mg, 2 mmol, 4 equiv), and (4S)-
MeHex-OH (195 mg, 1.5 mmol, 3 equiv) were added sequentially
to the peptidyl-resin (step 3) using DIPCDI (233 µL, for 1.5 mmol

4930 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Jiménez et al.
Table 6. [4(S)-MeHex14]KF (Compound 100) 1H NMR Chemical Shifts
residue
N-H
9.59 (s)
8.82 (d, J ) 9.0 Hz)
8.75 (d, J )5.5 Hz)
8.67(d, J ) 9.0 Hz)
8.13 (d, J ) 7.5 Hz)
8.29 (d, J ) 7.5 Hz)
7.92 (d)
HR
Hꢀ
other
(Z)-Dhb
D-allo-Ile 1
L-Phe
D-allo-Thr
D-Val 3
L-Orn
D-allo-Ile 2
D-Val 5
L-Thr
D-Val 2
L-Val 4
L-Val 1
D-Pro
6.63 (q, J )7.5 Hz)
1.87
1.19 (d, γ-CH3)
4.42
4.63
4.64
4.33
4.31
4.18
4.08
4.29
4.32
4.10
4.02
4.36
1.25, 1.09, 0.82 (γ-CH2, γ-CH3, δ-CH3)
7.31 (2H Ar, t), 7.25 (3H Ar, d)
1.21 (γ-CH3)
3.08 (m)
5.05 (m)
2.01
1.66 (2H)
1.80
2.07
4.14 (m)
2.11
0.90 (2 γ -CH3)
+
1.88 (γ-CH2), 2.96 (bs, δ-CH2), 7.56 (ε-NH3
1.25, 1.09, 0.81 (γ-CH2, γ-CH3, δ-CH3)
0.87 (2 γ-CH3)
)
8.01 (d)
8.19 (d, J ) 7.5 Hz)
7.89 (d, J ) 7.5 Hz)
8.04 (d)
1.13 (γ-CH3)
0.78 (γ-CH3)
2.07
1.52
0.90 (2 γ-CH3)
7.19 (d, J ) 9.0 Hz)
0.75 (γ-CH3), 0.65 (d, γ-CH3)
2.23, 1.99 (m, ꢀ-CH2), 1.85 (m, γ-CH2), 3.83 (1H, m, δ-CH2), 3.64 (1H, m, δ-CH2)
1.57 (ꢀ-CH2), 1.26, 1.10, 1.33, 0.79 (δ-CH2, δ-CH3, γ-CH, ε-CH3)
4(S)-MeHex
2.26 (2H)
Table 7. Cellular Lines Used in Assay A
name
no. ATCC
species
tissue
prostate
prostate
ovary
ovary
breast
melanoma
lung
bone marrow;
pancreas
colon
description
DU-145
LN-caP
IGROV
IGROV-ET
SK-BR-3
MEL-28
A-549
K-562
PANC-1
HT-29
HTB-81
CRL-1740
human
human
human
human
human
human
human
human
human
human
human
human
human
human
prostate carcinoma, not androgen receptors
prostate adenocarcinoma, with androgen receptors
ovary adenocarcinoma
ovary adenocarcinoma, characterized as ET-743 resistant cells
breast adenocarcinoma, Her2/neu+, (pleural effusion)
malignant melanoma
HTB-30
HTB-72
CCL-185
CCL-243
CRL-1469
HTB-38
CCL-229
lung carcinoma ”NSCL”
chronic myelogenous leukemia
pancreatic epitheloid carcinoma
colon adenocarcinoma
colon adenocarcinoma
LoVo
LoVo-Dox
HELA
colon
colon
cervix
cervix
colon adenocarcinoma (MDR)
CCL-2
CCL-3
cervix epitheloid carcinoma
HELA-APL
cervix epitheloid carcinoma, characterized as aplidine resistant cells
supplemented with 0.1 g/L penicillin and 0.1 g/L streptomycin
sulfate and then incubated at 37 °C, 5% CO2, and 98% humidity.
For the experiments, cells were harvested from subconfluent cultures
using trypsin and resuspended in fresh medium before plating. Cells
were seeded in 96-well microtiter plates, at 5 × 103 cells per well
in aliquots of 195 µL medium, and allowed to attach to the plate
surface by growing in drug-free medium for 18 h. Afterward,
samples were added in aliquots of 5 µL in a range between 10 and
8 µg/mL, and dissolved in DMSO/EtOH/PBS (0.5:0.5:99). After
48 h of exposure, the antitumor effect was measured by the
sulforhodamine B (SRB) methodology: cells were fixed by adding
50 µL of cold 50% (wt/vol) trichloroacetic acid and then incubated
for 60 min at 4 °C. Plates were washed with deionized water and
dried. An amount of 100 µL of SRB solution (0.4 wt %/vol in 1%
acetic acid) was added to each microtiter well and incubated for
10 min at room temperature. Unbound SRB was removed by
washing with 1% acetic acid. Plates were air-dried, and the bound
stain was solubilized with Tris buffer. Optical densities were read
on an automated spectrophotometric plate reader at a single
wavelength of 490 nm. The values for mean ( SD of data from
triplicate wells were calculated. Some parameters for cellular
responses were calculated: GI ) growth inhibition, TGI ) total
growth inhibition (cytostatic effect), and LC ) cell killing (cytotoxic
effect).
Assay B. This assay used 24-well multidishes of 16 mm in
diameter. The tumor cell lines employed were A-549 (ATCC CCL
185) (monolayer culture of a human lung carcinoma), HT-29
(ATCC HTB-38) (monolayer culture of a human colon carcinoma),
MEL-28 (ATCC HTB-72) (monolayer culture of a human mela-
noma), and DU-145 (ATCC HBTB-81) (monolayer culture of a
human prostate carcinoma).
Cells were maintained in the logarithmic phase of growth in
Eagle’s minimum essential medium, with Earle’s balanced salts,
with nonessential amino acids, with 2.0 mM L-glutamine, without
sodium bicarbonate (EMEM/neaa), supplemented with 10% fetal
calf serum (FSC), 10-2 M sodium bicarbonate, and 0.1 U/L
penicillin G + 0.1 g/L streptomycin sulfate. For the experiments,
cells were harvested from subconfluent cultures using trypsin and
resuspended in fresh medium before plating.
A-549, HT-29, MEL-28, and DU-145 cells were seeded into 16-
mm diameter wells at 1 × 104 cells per well in 1 mL aliquots of
EMEM 5% FCS containing a range of concentrations of the sample
to be tested. A separate set of cultures without drug was seeded as
control of growth to ensure that cells remained in an exponential
phase of growth. All determinations were carried out in duplicate.
After 3 days of incubation at 37 °C, 5% CO2 in a 98% humid
atmosphere, cells were stained with 0.1% crystal violet. An
approximate IC50 was determined by comparing the growth in wells
containing the drug to the growth in control wells.
For quantification of the activity, after the incubation time cells
were trypsinized and counted in a Coulter counter ZM. All counts
(net cells per well) represented the average of duplicate wells, % G,
percent of growth relative to cultures without drug. The results of
these assays were used to generate dose-response curves from
which more precise IC50 values were determined (sample concen-
tration that produces 50% cell growth inhibition).
The results obtained may predict the usefulness of a certain drug
as a potential cancer treatment. For this technique, compounds that
showed IC50 values smaller than 1 µM were selected for further
studies. IC50 data allowed us to predict that a drug not only may
be cystostatic but may also provide potential benefit in terms of
tumor reduction.
Acknowledgment. This study was partially supported by
PharmaMar, S.A., CICYT (Grant CTQ2006-03794/BQU, PET-
RI), the Generalitat de Catalunya (Grant 2005SGR 00662), the
ISCIII (CIBER, nanomedicine), the Institute for Research in
Biomedicine, and the Barcelona Science Park.
Supporting Information Available: Synthesis procedures and
characterization by HPLC and MS, as well as in vitro assay data.
This material is available free of charge via the Internet at http://
pubs.acs.org.

Synthetic analogues of Kahalalide F
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4931
(14) Monks, A.; Scudiero, D.; Scudiero, D; Skehan, P; Shoemaker, R; Paull,
K; Vistica, D; Hose, C; Langley, J; Cronise, P; Vaigro-Wolff, A.
Feasibility of a high-flux anticancer drug screen using a diverse panel
of cultured human tumor cell lines. J. Natl. Cancer Inst. 1991, 83,
757–766.
(15) Joshi, R. M.; Chauan, V. S. Synthesis of Peptides Based on
R,ꢀ-Didehydro-R-amino Acids. In Methods of Organic Chemistry,
E22c; Goodman, M., Felix, A., Moroder, L, Toniolo, C., Eds.; Houben-
Weyl: New York, 2003; Vol. 63, pp 6-662.
(16) Goetz, G.; Yoshida, W. Y.; Scheuer, P. J. The absolute stereochemistry
of kahalalide F. Tetrahedron 1999, 55, 7739–7746.
References
(1) Hamann, M. T.; Scheuer, P. J. Kahalalide F: a bioactive depsipeptide
from the sacoglossan mollusk Elysia rufescens and the green alga
Bryopsis sp. J. Am. Chem. Soc. 1993, 115, 5825–5826.
(2) Ashour, M.; Edrada, R.; Ebel, R.; Wray, V.; Waetjen, W.; Padmaku-
mar, K.; Mueller, W. E. G.; Lin, W. H.; Proksch, P. Kahalalide
derivatives from the Indian sacoglossan mollusk Elysia grandifolia.
J. Nat. Prod. 2006, 69, 1547–1553.
(3) Tilvi, S.; Naik, C. G. Tandem mass spectrometry of kahalalides:
identification of two new cyclic depsipeptides, kahalalide R and S
from Elysia grandifolia. J. Mass. Spectrom. 2007, 42, 70–80.
(4) Hamann, M. T.; Otto, C. S.; Scheuer, P. J.; Dunbar, D. C. Kahalalides:
bioactive peptides from a marine mollusk Elysia rufescens and its algal
diet Bryopsis sp. J. Org. Chem. 1996, 61, 6594–6600.
(5) Ciruelos, C.; Trigo, T.; Pardo, J.; Paz-Ares, L.; Estaun, N.; Cuadra,
C.; Dominguez, M.; Marin, A.; Jimeno, J.; Izquierdo, M. A phase I
clinical and pharmacokinetic (PK) study with kahalalide F (KF) in
patients (pts) with advanced solid tumors (AST) with a continuous
weekly (W) 1-h iv infusion schedule. Eur. J. Cancer 2002, 38 (Suppl.
7), S33.
(6) Rademaker-Lakhai, J. M.; Horenblas, S.; Meinhardt, W.; Stokvis, E.;
de Reijke, T. M.; Jimeno, J. M.; Lopez-Lazaro, L.; Lopez Martin,
J. A.; Beijnen, J. H.; Schellens, J. H. M. Phase I clinical and
pharmacokinetic study of kahalalide F in patients with advanced
androgen refractory prostate cancer. Clin. Cancer Res. 2005, 11, 1854–
1862.
(7) Garcia-Rocha, M.; Bonay, P.; Avila, J. The antitumoral compound
kahalalide F acts on cell lysosomes. Cancer Lett. 1996, 99, 43–50.
(8) Janmaat, M. L.; Rodriguez, J. A.; Jimeno, J.; Kruyt, F. A. E.; Giaccone,
G. Kahalalide F induces necrosis-like cell death that involves depletion
of ErbB3 and inhibition of Akt signaling. Mol. Pharmacol. 2005, 68,
502–510.
(17) Bonnard, I.; Manzanares, I.; Rinehart, K. L. Stereochemistry of
kahalalide F. J. Nat. Prod. 2003, 66, 1466–1470.
(18) Shilabin, A. G.; Kasanah, N.; Wedge, D. E.; Hamann, M. T. Lysosome
and HER3 (ErbB3) selective anticancer agent kahalalide F: semisyn-
thetic modifications and antifungal lead-exploration studies. J. Med.
Chem. 2007, 50, 4340–4350.
(19) Faircloth, G. T.; Elices, M.; Sasak, H.; Aviles, P. M.; Cuevas, C.
Preparation of Kahalalide Antitumoral Compounds. PCT Int. Appl.
WO 2004035613 A2 20040429, CAN 140:357671, 2004.
(20) Dangles, O.; Guibe, F.; Balavoine, G.; Lavielle, S.; Marquet, A.
Selective cleavage of the allyl and (allyloxy)carbonyl groups through
palladium-catalyzed hydrostannolysis with tributyltin hydride. Ap-
plication to the selective protection-deprotection of amino acid
derivatives and in peptide synthesis. J. Org. Chem. 1987, 52, 4984–
4993.
(21) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New
colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl.
Cancer Inst. 1990, 82, 1107–1112.
(22) Mosmann, T. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol.
Methods 1983, 65, 55–63.
(9) Suarez, Y.; Gonzalez, L.; Cuadrado, A.; Berciano, M.; Lafarga, M.;
Munoz, A. Kahalalide F, a new marine-derived compound, induces
oncosis in human prostate and breast cancer cells. Mol. Cancer Ther.
2003, 2, 863–872.
(23) Faircloth, G. T.; Stewart, D. A simple screening procedure for the
quantitative measurement of cytotoxicity assay. J. Tissue Cult. Methods
1988, 11, 201–205.
(10) Finkel, E. Does cancer therapy trigger cell suicide? Science 1999, 286,
2256–2558.
(11) Lopez-Macia, A.; Jimenez, J. C.; Royo, M.; Giralt, E.; Albericio, F.
Synthesis and structure determination of kahalalide F. J. Am. Chem.
Soc. 2001, 123, 11398–11401.
(12) Jime´nez, J. C.; Bayo´, N.; Chavarria, B.; Lopez-Macia, A.; Royo, M.;
Nicolas, E.; Giralt, E.; Albericio, F. Synthesis of peptides containing
R,ꢀ-didehydroamino acids. Scope and limitations. Lett. Pept. Sci. 2002,
9, 135–141.
(13) Ho¨fle, G.; Steglich, W.; Vorbrueggen, H. New synthetic methods. 25.
4-Dialkylaminopyridines as acylation catalysts. 4. Puridine syntheses.
1. 4-Dialkylaminopuridines as highly active acylation catalysts. Angew.
Chem., Int. Ed. Engl. 1978, 17, 569–583.
(24) Recently, a convergent methodology for the rapid synthesis of KF
analogues has been developed in our group: Gracia, C.; Isidro-Llobet,
A.; Cruz, L. J.; Acosta, G. A.; Alvarez, M.; Cuevas, C.; Giralt, E.;
Albericio, F. Convergent approaches for the synthesis of the antitu-
moral peptide, kahalalide F. Study of orthogonal protecting groups.
J. Org. Chem. 2006, 71, 7196-7204. However, it was not used for
the generation of this set of compounds.
(25) This compound was obtained as side product during cyclization with
PyAOP/DIEA. See ref 11. This epimerization is totally avoided when
DIPCDI/HOBt is used as cyclization method.
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