Synthesis and biological activity of chimeric structures derived from the cytotoxic natural compounds dolastatin 10 and dolastatin 15

Article abstract of DOI:10.1021/jm970800t

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide

Full text of DOI:10.1021/jm970800t

1524
J . Med. Chem. 1998, 41, 1524-1530
Syn th esis a n d Biologica l Activity of Ch im er ic Str u ctu r es Der ived fr om th e
Cytotoxic Na tu r a l Com p ou n d s Dola sta tin 10 a n d Dola sta tin 15†
J oe¨l Poncet,*,‡ Magali Busquet,‡ Florence Roux,‡,§ Alain Pierre´,| Ghanem Atassi,| and Patrick J ouin‡
Laboratoire des Me´canismes Mole´culaires des Communications Cellulaires, UPR 9023 CNRS, Centre CNRS-INSERM de
Pharmacologie Endocrinologie, 141, rue de la Cardonille, 34094 Montpellier Cedex 5, France, and Institut de Recherches
Servier, 11, rue des Moulineaux, 92150 Suresnes, France
Received November 24, 1997
The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure
and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging
the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were
synthesized in order to evaluate the possible equivalence of these units. These compounds
can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the
former and the C-terminal part of the latter and vice versa. Both analogues exhibited a marked
decrease in their cytotoxic activity but showed similar differential cytotoxicity with regard to
the cell lines assayed compared with the parent compounds. HT-29 cell line was the least
sensitive one. However, this activity was in the nanomolar level and close to that of vincristine.
The differences in their effect on tubulin polymerization were less pronounced. We confirmed
the already known crucial role of the Dil residue in this assay. The nonequivalence of the Dil
unit and the MeVal-Pro dipeptide probably reflects modification in the relative positions of
the N-dimethylamino and the phenyl moieties.
In tr od u ction
Dolastatins are a family of antineoplastic pseudopep-
tides isolated from the sea hare Dolabella auricularia.
Most of them were originally identified by the group of
Pettit,1-7 and more recently the number of active
metabolites present in this mollusk was extended by the
groups of Yamada8-14 and Pettit.15 Among them, do-
lastatins 10 and 15 show promising antitumoral capac-
ity. They are both highly potent inhibitors of the prolif-
eration of murine and human leukemia cell lines16-19
and also inhibitors of hematopoietic progenitor cells.20
These compounds typically behave as antimitotic
agents.21,22 Dolastatin 10 inhibits in vitro microtubule
assembly by binding to the tubulin â subunit near the
vinca domain in a similar fashion as the noncytotoxic
pseudopeptide phomopsin A.23 Like vinblastin, the
binding of dolastatin 10 to tubulin induces aggregation
in vitro, but at higher concentration than that which
inhibits cell proliferation.24 A similar study has not
been reported for dolastatin 15. However, both com-
pounds22 as well as several analogues25,26 induce disrup-
tive effects on the cytoplasmic microtubule network.
The similarity between the cellular effects of dola-
statin 10 and dolastatin 15 is probably a reflection of
their structural analogies.22 A formal comparison of
both structures shows that, first, the N-termini are
identical from N-25a to C-19 for dolastatin 10 and from
N-29 to C-22 for dolastatin 15; second, the C-termini
are constituted of phenylalanine-derived residues where
the carboxylic acid group is replaced by a heterocycle;
and third, a pyrrolidine occupies the center of both
molecules. These considerations have led us to the
assumption that the Dil-Dap or Pro-Pro bond might
act as a hinge in the middle of the molecules. To
evaluate this hypothesis, we envisaged the synthesis of
compounds 1 and 2 formed by replacing the MeVal-Pro
† The different residues of dolastatins 10 and 15 were named
according to refs 2 and 3. Abbreviations follow IUPAC-IUB J oint
Commission on Biochemical Nomenclature for amino acids and pep-
tides (Eur. J . Biochem. 1984, 138, 9-37). Additional abbreviations used
herein are as follows: BroP, tris(dimethylamino)bromophosphonium
hexafluorophosphate; COMMOD, 2,2′-carbonylbis(3,5-dioxo-4-methyl-
1,2,4-oxadiazolidine); DIEA, diisopropylethylamine; DMAP, 4-(dim-
ethylamino)pyridine; IPCC, isopropenyl chlorocarbonate; PyBOP, (1H-
benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate;
PyBroP, tripyrrolidinobromophosphonium hexafluorophosphate; Py-
CloP, tripyrrolidinochlorophosphonium hexafluorophosphate; TEA,
triethylamine; TFA, trifluoroacetic acid.
* To whom correspondence should be addressed.
‡ Centre CNRS-INSERM.
§ Present address: Synt:em, Parc Scientifique Georges Besse, 30000
Nimes, France.
| Institut de Recherches Servier.
S0022-2623(97)00800-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/28/1998

Chimeric Structures Derived from Dolastatin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1525
dipeptide in dolastatin 15 by the central Dil residue of
dolastatin 10 and vice versa. These molecules could be
considered as chimeric structures associating the dola-
statin 10 N-terminus and the dolastatin 15 C-terminus
in the case of 1 and vice versa in the case of 2. We
report here our results concerning the synthesis of these
compounds and the evaluation of their biological activ-
ity.
ment under various conditions. We tried to circumvent
this difficulty by coupling Dap-Doe before elaborating
the tertiary amine function. Thus, the ester function
of 9 was hydrolyzed by TFA treatment to furnish 12
(Scheme 3). Coupling of this compound with Dap-Doe
yielded 14. Hydrogenolysis of the Z group by using Pd
catalysts was unsatisfactory, whereas deprotection un-
der acidic conditions (HBr/AcOH) led surprisingly to
extensive breaking of the MeVal-Pro amide bond to give
the tetrapeptide 14. These latter results can be ex-
plained in the light of Anteunis’s studies concerning the
sensitivity of imino acids to acid-catalyzed peptoly-
sis.29,30
Finally, the compound 2 was synthesized according
to a stepwise Fmoc strategy as depicted in Scheme 4.
Eva lu a tion of Biologica l Activity
Cytotoxic activities of analogues 1 and 2 were tested
on either human (HT-29, MCF7) or murine (L1210) cell
lines and compared with those of the parent compounds
and vincristine, another natural antimitotic agent taken
as reference. Drug effects on tubulin polymerization
were also evaluated. The results are given in the Table
1.
Ch em istr y
Both analogues, just like dolastatins 10 and 15 and
vincristine, showed similar differential cytotoxicity with
regard to the cell lines assayed, HT-29 being the least
sensitive one, particularly in the case of 1. Both
analogues exhibited a loss of activity by comparison with
the parent compounds. This loss was uniformly within
the range of about 2 orders of magnitude except for 1
against the HT-29 cell line where the loss of activity
was even greater. Nevertheless, all the IC50 values were
at the nanomolar level and in the same range as that
of vincristine.
On the other hand, the inhibition of tubulin polym-
erization by these analogues could not be correlated with
their effects on cell growth. Interestingly, the IC50 value
of dolastatin 10 was similar to that of compound 1,
which possesses the N-terminal part of dolastatin 10
(2.2 vs 3.6), and the IC50 value of dolastatin 15 was
similar to that of compound 2, which possesses the
N-terminal part of dolastatin 15 (17 vs 13).
Compound 1 was obtained by following a stepwise
peptide synthesis starting from the previously described
compound Hiva-Dpy (Scheme 1).27 The unusual amino
acids were all synthesized according to previously
described procedures.27,28
To prepare compound 2 we decided to proceed by
fragment coupling between protected peptides Val-Val-
MeVal-Pro and Dap-Doe. Thus, the tetrapeptide 10 was
elaborated as depicted in Scheme 2.
Hydrogenolysis of 9 in the presence of formaldehyde
followed by treatment with TFA yielded the free tet-
rapeptide 11 (Scheme 3). Unfortunately, we were
unable to couple this compound to the Dap-Doe frag-
Sch em e 1a
Discu ssion
The development of synthetic routes to dolastatin
1028,31-33 and dolastatin 1527,34 has permitted prelimi-
nary studies of structure-activity relationships.35-39
However, most of the studies reported so far have dealt
with modifications of the side chains. In this context,
it has been shown that there is only a limited correlation
between inhibition of tubulin polymerization and in
vitro cell growth.35,36 Moreover, various modifications
of the Dil or Dap units lead to opposite effects whether
assayed in vitro36 or in vivo.37 These discrepancies could
suggest the involvement of complex interaction mech-
anisms and also differential transport into cells or
intracellular metabolism. Nevertheless, these studies
have led to the elucidation of some key features of the
structure-activity relationship: in the case of dolastatin
10, Dil unit with an erythro configuration seems to be
crucial for both antitumoral and antitubulin activities;
a phenyl moiety at the C-terminus and at least one
a
Reagents: (a) IPCC, TEA, DMAP, 5 °C; (b) TFA; (c) PyBOP,
DIEA; (d) PyCloP, DIEA; (e) HCHO, H2, Pd/C.
Sch em e 2a
a
Reagents: (a) PyBroP, DIEA; (b) H2, Pd/C; (c) BOP, DIEA.

1526 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Poncet et al.
Sch em e 3a
a
Reagents: (a) HCHO, H2, Pd/C; (b) TFA; (c) Dap-Doe, PyBroP, DIEA; (d) HBr/AcOH.
Sch em e 4a
IC50 ratios of 1 to dolastatin 15 and 2 to dolastatin 10
shows that the replacement of MeVal-Pro by Dil in
dolastatin 15 and Dil by MeVal-Pro in dolastatin 10 led
to more or less identical decreases. From this point of
view, these two units seem to induce an almost equiva-
lent perturbation relative to the parent compounds.
The difference between the two compounds was less
pronounced by considering the effects on in vitro tubulin
polymerization. As already observed by other authors,
this activity did not correlate with the cytotoxic potency
as both compounds exhibited an inhibitory effect mid-
way between that of dolastatins 10 and 15. Substitution
of the MeVal-Pro dipeptide by the Dil residue into
dolastatin 15 (compound 1) led to a lower inhibition
constant (3.6 instead of 17 µM), while the introduction
of the MeVal-Pro dipeptide into dolastatin 10 (compound
2) decreased this inhibition (13 instead of 2.2 µM). Our
results are in agreement with the findings of Bai et al.
who showed that the Dil residue is critical in the tubulin
assays and that the N-terminal tripeptide of dolastatin
10 is responsible for nearly the entire antitubulin
activity of the molecule.36
a
Reagents: (a) PyBroP, DIEA; (b) DEA; (c) BroP, DIEA; (d)
COMMOD.
Ta ble 1. Inhibition of Cell Growth (IC50, nM) and in Vitro
Tubulin Polymerization (IC50, µM)
cell line
tubulin
compound
HT-29
MCF7
0.03
0.04
0.84
L1210 polymerization
0.03 2.2
17
dolastatin 10
dolastatin 15
vincristine
0.06
0.29
2.06
0.13
0.73
1.8
3.6
13
1
2
396 (1365)a 0.19 (5)a 1.58 (12)a
11.4 (190)b 2.48 (83)b 2.72 (90)b
In the absence of a well-defined biological mechanism,
the understanding of the structure-activity relation-
ships of dolastatins is not easy. The ability of dolastatin
10 to bind to tubulin is now well-documented.24 How-
ever, the molecular mechanisms involved are unknown.
The assumption that dolastatin 15 binds to tubulin in
the same site is based on its structural analogy to
dolastatin 10 and on its inhibitory effect on GTP
hydrolysis.22 Within this context, one could assume that
conformational modifications are at the origin of the
differences between both analogues and their parent
compounds. Several approaches to the conformational
analysis of dolastatin 10 have been tried based on X-ray
diffraction analysis41 or theoretical molecular design.42,43
NMR studies41,44 showed that, in solution, dolastatin 10
exists in two different conformations corresponding to
a cis-trans isomerization of the Dil-Dap amide bond,
conferring an important bend shape around the Dil-Dap
junction. Adversely, in solution all amide bonds are
trans in dolastatin 15 thus inducing an extended form
of the molecule.45 The difference between 1 and 2 and
their parent compounds could result from perturbation
of such an equilibrium. Similarly, if one considers the
distal N-dimethylamino and phenyl moieties, which
were shown to be crucial for the activity of both natural
a
Ratio IC50(1)/IC50(dolastatin 15). b Ratio IC50(2)/IC50(dolastatin
10).
N-methyl group at the N-terminus are necessary to
maintain the activity in both compounds. In the case
of dolastatin 15, almost no data have been published
concerning modifications of the backbone although
several modifications have been revendicated in a
patent.40 Surprisingly, whereas residue deletion is
detrimental to cytotoxicity in the case of dolastatin 10,36
the shortened LU103793 analogue retains the ability
of dolastatin 15 to inhibit not only cell proliferation in
vitro and in vivo but also tubulin polymerization.26
Thus, backbone modifications introduced into com-
pounds 1 and 2 could provide another insight into how
the Dil unit or the MeVal-Pro dipeptide acts upon the
biological activity of both dolastatins 10 and 15.
Coupling the N-terminal part of dolastatin 10 to the
C-terminal part of dolastatin 15 (compound 1) and vice
versa (compound 2), i.e., a Dil versus MeVal-Pro per-
mutation, resulted in a marked decrease in cytotoxic
activity (Table 1) depending on the cell line. However,
all IC50 values (except for the activity of 1 on the HT-
29 cell line) remained at the nanomolar level and in the
same range as that of vincristine. Comparison of the

Chimeric Structures Derived from Dolastatin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1527
mg, 70%) as a white powder after preparative HPLC (gradient
from 20% to 80% B in 90 min) and lyophilization: mp 190 °C
compounds, they are 19 bonds apart in dolastatin 10
and 21 bonds apart in dolastatin 15. Permutation of
the MeVal-Pro and Dil units led to both analogues 1
and 2 with a 20-bond backbone. Thus, our biological
results could be interpreted in terms of a structural
model where both pharmacophores (the phenyl and the
dimethylamino groups) are maintained at an optimum
distance. Such a model was already discussed by Bai
et al.21 and could also explain the activity of the
analogue LU103793.26
In conclusion, although dolastatin 10 and dolastatin
15 exhibit some analogies, our study shows that the Dil
unit and the MeVal-Pro dipeptide are not equivalent on
the basis of their effects on cell growth nor on the basis
of their effects on tubulin polymerization. It is conceiv-
able that they play a determinant role in both phenom-
ena through the induction of an optimal conformation
of the drugs. Alternatively, the 4- versus 5-bond back-
bone could position differently the N- and C-termini.
These hypotheses are currently being addressed by
structural studies on compounds 1 and 2 and backbone
modifications of dolastatins 10 and 15 leading to blocked
structures.
dec; [R]20 -60° (c 0.1, MeOH); tR 7.9 min (gradient from 20%
D
to 80% in 15 min); FABMS m/e (relative intensity) 810 (M +
H, 10), 471 (H-Pro-Dap-Doe + H, 5), 437 (Dov-Val-MeVal-Pro+,
3), 340 (Dov-Val-MeVal+, 33), 138 (H-Dap+ - MeOH, 53), 100
(Me2N+dCHiPr, 100). Anal. (C43H67N7O6S) C, H, N.
Boc-P r o-Hiva -Dp y (3). Following the procedure described
by Zeggaf et al.,46 to a cooled (-5 °C) solution of Boc-Pro (0.90
g, 4.12 mmol), Hiva-Dpy (1.25 g, 4.12 mmol) prepared accord-
ing to Patino et al.,27 TEA (0.58 mL, 4.12 mmol), and DMAP
(0.15 g) in ethanol-free CH2Cl2 (8 mL) was dropped a solution
of IPCC (0.45 mL, 4.12 mmol) in ethanol-free CH2Cl2 (2 mL).
After stirring for 30 min at -5 °C and for 4 h at room
temperature, the reaction mixture was worked up as usual to
yield 3 which was crystallized in AcOEt/hexane (2.00 g, 97%):
mp 168-169 °C (lit.34 158-159 °C); [R]20 135° (c 1, MeOH);
D
Rf 0.60 (AcOEt/hexane/AcOH, 40:60:1); 1H NMR (DMSO-d6) δ
0.88 (d, J ) 6.8 Hz, 3 H), 0.99 (d, J ) 6.8 Hz, 3 H), 1.38 (s, 9
H), 1.78-1.98 (m, 2 H), 2.05-2.20 (m, 2 H), 2.21-2.31 (m, 1
H), 2.98 (dd, J 1 ) 13.7 Hz, J 2 ) 3.0 Hz, 1 H), 3.32 (dd, J 1
)
13.7 Hz, J 2 ) 8.6 Hz, 1 H), 3.39-3.46 (m, 2 H), 3.80 (s, 3 H),
4.34 (dd, J 1 ) 3.0 Hz, J 2 ) 8.6 Hz, 1 H), 4.91-4.93 (m, 1 H),
5.10 (s, 1 H), 5.78 (d, J ) 2.8 Hz, 1 H), 7.01-7.04 (m, 2 H),
7.17-7.24 (m, 3 H). Anal. (C27H36N2O7) C, H, N.
Boc-Dil-P r o-Hiva -Dp y (4). Compound 3 (2.00 g, 4.00
mmol) was treated with TFA for 30 min. The solvent was
evaporated under reduced pressure to yield the amino-free
compound as an oil (2.03 g). This compound was coupled to
Boc-Dil (1.43 g, 4.95 mmol) by using PyBOP. After purification
by column chromatography (AcOEt/hexane, 30:70), 4 was
crystallized in AcOEt/hexane (2.42 g, 88%): mp 155-156 °C;
[R]20D 94° (c 1, MeOH); Rf 0.47 (AcOEt/hexane, 40:60); FABMS
m/e (relative intensity) 686 (M + H, 9), 586 (Dil-Pro-Hiva-Dpy
+ H, 7), 401 (Pro-Hiva-Dpy + H, 7), 286 (Boc-Dil, 6), 91
(tropylium, 22), 57 (tBu+, 40). Anal. (C37H55N3O9) C, H, N.
Exp er im en ta l P a r t
HPLC analyses were performed using an Ultrabase C8 (5
µm, 4.6 × 150 mm; Shandon) column and mixtures of 0.1%
TFA in water (solvent A) and 0.1% TFA in CH3CN (solvent B)
at a flow rate of 1.5 mL/min. Preparative HPLC was per-
formed using a Hypersil C18 (10 µm, 25 × 500 mm) column
and mixtures of water (solvent A) and CH3CN (solvent B) at
a
flow rate of 9 mL/min. Column chromatography was
Boc-Va l-Dil-P r o-Hiva -Dp y (5). Compound 4 (0.60 g, 0.90
mmol) was treated with 3 N HCl in dioxane solution (3 mL)
for 1 h at room temperature. The mixture was lyophilized to
yield the HCl salt of the amino-free compound as a white
powder (0.55 g). This compound was coupled to Boc-Val (0.33
g, 1.35 mmol) by using PyCloP. After the mixture stirred for
12 h, 1 equiv of Boc-Val, 1 equiv of PyCloP, and 2 equiv of
DIEA were added. The reaction was complete within 24 h.
After purification by column chromatography (AcOEt/hexane,
70:30), 5 was crystallized in AcOEt/hexane (0.51 g, 88%): mp
performed on 70-200-µm silica gel. 1H NMR spectra were
recorded at 360 MHz.
Cou p lin g Gen er a l P r oced u r e. To a solution of P-Aaa-
OH (1 equiv) and H-Bbb-P′ (1 equiv) in ethanol-free CH2Cl2
(2 mL/mmol) were added successively DIEA (3.5 equiv) and
the coupling reagent (1.5 equiv). After stirring for 1 h (unless
specified otherwise) at room temperature, the reaction mixture
was concentrated to dryness under reduced pressure. The
residue was dissolved in AcOEt (10 mL/mmol), washed twice
with 5% KHSO4 (equal to one-tenth the volume of the organic
phase), water (ibid.), 5% NaHCO3 (ibid.), and saturated brine
(ibid.), dried over anhydrous Na2SO4, filtered, and concentrated
under reduced pressure. Further purification was performed
by column chromatography on silica gel and crystallization.
Samples for elemental analysis were dried at 78 °C on KOH
for 12 h.
125-130 °C; [R]20 50° (c 1, MeOH); Rf 0.42 (AcOEt/hexane/
D
AcOH, 70:30:1); FABMS m/e (relative intensity) 785 (M + H,
10), 685 (Val-Dil-Pro-Hiva-Dpy + H, 20), 586 (Dil-Pro-Hiva-
Dpy + H, 3), 401 (Pro-Hiva-Dpy + H, 5), 186 (Dil + H, 24),
154 (Dil - MeOH + H, 25), 100 (Val + H, 55), 72 (H2NdCH+-
iPr, 60), 70 (pyrrolinium, 70), 57 (tBu+, base). Anal.
(C42H63N4O10) C, H, N.
Z-Va l-Va l-Dil-P r o-Hiva -Dp y (6). Compound 5 (0.50 g,
0.64 mmol) was treated with TFA for 30 min. The solvent was
evaporated under reduced pressure to yield the amino-free
compound as an oil (0.50 g). This compound was coupled to
Z-Val by using PyBOP. After purification by column chroma-
tography (AcOEt/hexane, 70:30), 6 was crystallized in AcOEt/
hexane (0.42 g, 72%): mp 115-118 °C; [R]20D 29° (c 1, MeOH);
Rf 0.39 (AcOEt/hexane/AcOH, 70:30:1); FABMS m/e (relative
intensity) 918 (M + H, 5), 886 (M - MeOH + H, 3), 685 (Val-
Dil-Pro-Hiva-Dpy + H, 1), 586 (Dil-Pro-Hiva-Dpy + H, 2), 100
(Val + H, 25), 91 (tropylium, base), 72 (H2NdCH+iPr, 60), 70
(pyrrolinium, 35). Anal. (C50H71N5O11‚2H2O) C, H, N.
Dov-Va l-Dil-P r o-Hiva -Dp y (1). Compound 6 (0.30 g, 0.33
mmol) was solubilized in a mixture of MeOH (4 mL) and water
(3 mL) and hydrogenolyzed over 10% Pd on charcoal (60 mg)
in the presence of 30% aqueous formaldehyde (2.1 mL) for 48
h at room temperature and atmospheric pressure. The reac-
tion mixture was filtered and concentrated under reduced
pressure to furnish an oily residue (0.25 g) which was chro-
matographed (CH2Cl2/MeOH, 90:10). 1 was obtained as a
white solid (0.122 g, 46%): mp 98-100 °C; [R]20 25° (c 1,
D
MeOH); Rf 0.17 (AcOEt/hexane/AcOH, 70:30:1); FABMS m/e
(relative intensity) 812 (M + H, 37), 780 (M - MeOH + H, 7),
685 (Val-Dil-Pro-Hiva-Dpy + H, 2), 586 (Dil-Pro-Hiva-Dpy +
H, 4), 401 (Pro-Hiva-Dpy + H, 6), 186 (Dil + H, 20), 154 (Dil
- MeOH + H, 20), 128 (Dov, 15), 100 (Val + H, base), 72
(H2NdCH+iPr, 95), 70 (pyrrolinium, base). Anal. (C44H69N5O9)
C, H, N.
Dov-Va l-MeVa l-P r o-Da p -Doe (2). The Fmoc group of 17
(81 mg, 89 µmol) was removed as described for 16 to yield the
free peptide (62 mg). Dov (22 mg, 140 µmol) was activated
using COMODD according to a previously described proce-
dure28 and coupled to the preceding free peptide to yield 2 (39
Z-MeVa l-P r o-OtBu (7). Z-MeVal (1.00 g, 3.80 mmol) was
coupled to Pro-OtBu (0.78 g, 4.5 mmol) by using PyBroP to
yield 7 (1.21 g, 76%) as a white solid: mp (Et2O/hexane); [R]20
D
-129° (c 1, MeOH); Rf 0.49 (AcOEt/hexane, 50:50); FABMS
m/e (relative intensity) 419 (M + H, 35), 285 (MeVal-Pro-OtBu
+ H, 11), 91 (tropylium, base); 1H NMR (DMSO-d6) (two
conformers in a ratio of 2:1) major conformer δ 0.79 (d, J )
6.6 Hz, 3 H), 1.38 (s, 9 H), 1.71-1.92 (m, 3 H), 2.12-2.20 (m,
2 H), 2.81 (s, 3 H), 3.54-3.66 (m, 2 H), 4.11-4.17 (m, 1 H),

1528 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9
Poncet et al.
4.50 (d, J ) 10.9 Hz, 1 H), 5.05 and 5.18 (AB, J ) 12.1 Hz, 2
H), 7.30-7.40 (m, 5 H); minor conformer (distinguishable
signals) δ 0.92 (d, J ) 6.6 Hz, 3 H), 1.36 (s, 9H), 2.78 (s, 3 H),
4.33 (d, J ) 10.8 Hz, 1 H), 5.08 and 5.14 (AB, J ) 12.9 Hz, 2
H). Anal. (C23H34N2O5) C, H, N.
(relative intensity) 455 (M + H, 60), 340 (Dov-Val-MeVal+, 45),
100 (Me2N+dCHiPr, 100).
Z-Va l-Va l-MeVa l-P r o-OH (12). Compound 9 (55 mg, 0.090
mmol) was treated with 3 N HCl in dioxane (1.5 mL). After 5
h, the mixture was lyophilized to yield 12 as a white powder.
Z-Va l-Va l-MeVa l-P r o-Da p -Doe (13). Compound 12 (27
mg, 0.080 mmol) was coupled to Dap-Doe prepared according
to Roux et al.28 by using PyBroP to yield 13 as a white gum
(49 mg, 82%) after preparative HPLC (gradient from 40% to
90% B in 60 min) and lyophilization: Rf 0.25 (CH2Cl2/MeOH,
70:30); tR 8.1 min (gradient from 40% to 90% in 15 min);
FABMS m/e (relative intensity) 916 (M + H, 10), 543 (Z-Val-
Val-MeVal-Pro+, 10), 447 (H-Val-MeVal-Pro-Dap+ - MeOH,
90), 446 (Z-Val-Val-MeVal+, 100), 356 (HO2C-Val-Val-MeVal+,
15), 333 (Z-Val-Val+, 10), 234 (Z-Val+, 40), 188 (Doe - NH,
45), 138 (H-Dap+ - MeOH, 20), 114 (H-MeVal+, 15), 91
(tropylium, 100), 86 (MeHN+dCHiPr, 100), 72, (H2N+dCHiPr,
100), 70 (pyrrolinium, 70). Anal. (C49H69N7O8S) N. C: calcd,
64.24; found, 63.85. H: calcd, 7.59; found, 7.10.
H-P r o-Da p -Doe (14). To a cooled solution of 13 (30 mg,
0.032 mmol) in CH2Cl2 (0.5 mL) was added 30% HBr/AcOH
(0.5 mL). After the mixture stirred for 1 h, the solvent was
evaporated under reduced pressure and the residue purified
by preparative HPLC (gradient from 10% to 80% B in 60 min)
to yield 14 as a brown gum after lyophilization (10 mg, 64%):
tR 5.1 min (gradient from 20% to 80% in 15 min); FABMS m/e
(relative intensity) 471 (M + H, 83), 373 (H-Dap-Doe + H, 19).
Anal. (C25H34N4O3S) C, H, N.
Z-Va l-MeVa l-P r o-OtBu (8). Compound 7 (1.10 g, 2.63
mmol) was solubilized in MeOH (30 mL) and hydrogenolyzed
over 10% Pd on charcoal (0.22 g) in the presence of 12 N HCl
(90 µL) during 16 h at room temperature and atmospheric
pressure. The reaction mixture was filtered and concentrated
under reduced pressure to furnish the ammonium chlorhy-
drate as a white solid (0.88 g, 99%) which was coupled to Z-Val
by using PyBroP. After purification by chromatography
(AcOEt/hexane, 35:65), 8 was obtained as a pale-yellow oil
(1.28 g, 93%): [R]20 -162° (c 1, MeOH); Rf 0.53 (AcOEt/
D
pentane/AcOH, 30:70:1); FABMS, m/e (relative intensity) 518
(M + H, 12), 384 (M - Z + 2 H, 5), 347 (Z-Val-MeVal+, 100),
285 (H-MeVal-Pro-OtBu + H, 10), 257 (HO2C-Val-MeVal+, 10),
213 (H-Val-MeVal+, 10), 172 (H-Pro-OtBu + H, 5), 91 (tropyl-
ium, 100), 86 (MeHN+dCHiPr, 95), 70 (pyrrolinium, 25), 57
1
(tBu+, 25); H NMR (DMSO-d6) δ 0.70 (d, J ) 6.7 Hz, 3 H),
0.79 (d, J ) 6.7 Hz, 3 H), 0.88 (d, J ) 6.7 Hz, 6 H), 1.38 (s, 9
H), 1.70-1.80 (m, 2 H), 1.89-1.99 (m, 2 H), 2.08-2.20 (m, 2
H), 3.29 (s, 3 H), 3.47-3.54 (m, 1 H), 3.64-3.70 (m, 1 H), 4.10
(dd, J 1 ) 8.7 Hz, J 2 ) 3.3 Hz, 1 H), 4.22 (t, J ) 8.6 Hz, 1 H),
4.94 (d, J ) 10.9 Hz, 1 H), 4.98 and 5.07 (AB, J ) 12.7 Hz, 2
H), 7.29-7.36 (m, 5 H), 7.52 (d, J ) 8.6 Hz, 1 H). Anal.
(C28H43N3O6) C, H, N.
F m oc-P r o-Da p -Doe (15). Fmoc-Pro-OH (0.210 g, 0.60
mmol) was coupled to HCl-Dap-Doe prepared according to
Roux et al.28 by using PyBroP to furnish 15 as a white gum
(0.185 g, 90%): Rf 0.36 (CH2Cl2/MeOH, 95:5); tR 13.5 min
(gradient from 20% to 80% in 20 min). Anal. (C40H44N4O5S)
C, H, N.
F m oc-MeVa l-P r o-Da p -Doe (16). A solution of compound
15 (0.185 g, 0.27 mmol) in DMF (1 mL) was treated with
diethylamine (0.3 mL) at room temperature for 30 min. After
evaporation of the solvent, the resulting amino-free peptide
was obtained as a pale-yellow oil (0.124 g) and coupled to
Fmoc-MeVal-OH (0.180 g, 0.52 mmol) prepared according to
Freidinger by using BroP reagent to furnish 16 as a white gum
(0.188 g, 92%): Rf 0.43 (AcOEt); tR 14.5 min (gradient from
20% to 80% in 20 min); FABMS m/e (relative intensity) 806
(M + H, 20), 433 (Fmoc-MeVal-Pro+, 10), 188 (Doe - NH, 9),
138 (H-Dap+ - MeOH, 10), 179 (fluorenylmethylenium, 100).
Anal. (C46H55N5O6S) C, H, N.
Z-Va l-Va l-MeVa l-P r o-OtBu (9). Compound 8 (1.20 g, 2.32
mmol) was solubilized in MeOH (30 mL) and hydrogenolyzed
over 10% Pd on charcoal (0.24 g) in the presence of 12 N HCl
(80 µL) during 16 h at room temperature and atmospheric
pressure. The reaction mixture was filtered and concentrated
under reduced pressure to furnish the ammonium chlorhy-
drate as a white solid (0.96 g, 99%) which was coupled to Z-Val
by using BOP to lead to 9 as a white foam (1.10 g, 84%): [R]20
D
-155° (c 1, MeOH); Rf 0.51 (AcOEt/pentane/AcOH, 50:50:1);
FABMS m/e (relative intensity) 617 (M + H, 10), 446 (Z-Val-
Val-MeVal+, 65), 356 (HO2C-Val-Val-MeVal+, 15), 285 (H-
MeVal-Pro-OtBu + H, 20), 172 (H-Pro-OtBu + H, 10), 91
(tropylium, 100), 86 (MeHN+dCHiPr, 75), 72 (H2N+dCHiPr,
55), 70 (pyrrolinium, 25), 57 (tBu+, 50); 1H NMR (DMSO-d6) δ
0.67 (d, J ) 6.7 Hz, 3 H), 0.80 (d, J ) 6.7 Hz, 6 H), 0.82 (d, J
) 6.7 Hz, 3 H), 0.87 (d, J ) 6.7 Hz, 6 H), 1.38 (s, 9 H), 1.71-
1.80 (m, 2 H), 1.87-2.00 (m, 3 H), 2.08-2.20 (m, 2 H), 2.99 (s,
3 H), 3.47-3.54 (m, 1 H), 3.64-3.70 (m, 1 H), 3.94 (dd, J 1
)
F m oc-Va l-MeVa l-P r o-Da p -Doe (17). By following the
procedure described for the preparation of 16, 16 (0.188 g, 0.24
mmol) led to 17 (0.160 g, 80%): Rf 0.30 (AcOEt); tR 14.4 min
(gradient from 20% to 80% in 20 min); FABMS m/e (relative
intensity) 905 (M + H, 8), 471 (H-Pro-Dap-Doe + H, 8), 435
(Fmoc-Val-MeVal+, 50), 138 (H-Dap+ - MeOH, 60), 179
(fluorenylmethylenium, 100). Anal. (C51H64N6O7S) C, H, N.
P u r ifica t ion of Micr ot u b u le P r ot ein a n d Assem b ly
Assa y. Microtubule proteins were purified from bovine brain
by cycles of assembly at 37 °C and disassembly at 0 °C
essentially according to the method of Shelanski.47 Fresh
bovine brains were cooled on ice, minced, and homogenized in
buffer A (100 mM MES, 0.5 mM MgCl2, 1 mM EGTA, pH 6.6).
After centrifugation at 90000g for 45 min at 4 °C, the
supernatant was mixed with an equal volume of buffer B
(buffer A, 8 M glycerol, 1 mM GTP) and warmed at 37 °C for
30 min. The polymerized tubulin was collected as a pellet by
centrifugation at 98000g for 1 h at 30 °C. The pellet was
resuspended in buffer A, homogenized, and chilled at 4 °C for
30 min. Centrifugation of the suspension at 90000g for 30 min
at 4 °C led to a supernatant which was mixed with buffer B
and then warmed at 37 °C for 30 min. After centrifugation
for 60 min at 30 °C, the supernatant was suspended in buffer
A and 0.5 M GTP and stored at -80 °C until required.
Immediately before use, the protein solution was mixed with
buffer A and 1 mM GTP to give a concentration of 2 mg of
protein/mL, and the solution was kept on ice. Assembly was
evaluated by measuring turbidity at 350 nm and 37 °C.48
6.9 Hz, J 2 ) 8.9 Hz, 1 H), 4.10 (dd, J 1 ) 5.2 Hz, J 2 ) 8.6 Hz,
1 H), 4.48 (t, J ) 8.6 Hz, 1 H), 4.92 (d, J ) 10.9 Hz, 1 H), 5.01
(s, 2 H), 7.21 (d, J ) 8.6 Hz, 1 H), 7.22-7.35 (m, 5 H), 8.00 (d,
J ) 8.6 Hz, 1 H). Anal. (C33H52N4O7‚H2O) C, H, N.
Dov-Va l-MeVa l-P r o-OtBu (10). Compound 9 (1.00 g, 1.60
mmol) was solubilized in MeOH (30 mL) and hydrogenolyzed
over 10% Pd on charcoal (0.24 g) in the presence of 30%
aqueous formaldehyde (10.3 mL) during 48 h at room tem-
perature and atmospheric pressure. The reaction mixture was
filtered and concentrated under reduced pressure to furnish
10 as a white solid (0.98 g, 98%): mp 84-86 °C (AcOEt/
pentane); [R]20 -149° (c 1, MeOH); Rf 0.32 (AcOEt/hexane,
D
70:30); FABMS m/e (relative intensity) 511 (M + H, 50), 340
(Dov-Val-MeVal+, 25), 100 (Me2N+dCHiPr, 100); 1H NMR
(DMSO-d6) δ 0.69 (d, J ) 6.7 Hz, 3 H), 0.70 (d, J ) 6.7 Hz, 3
H), 0.81 (d, J ) 6.7 Hz, 3 H), 0.86 (d, J ) 6.7 Hz, 3 H), 0.88 (d,
J ) 6.7 Hz, 3 H), 0.92 (d, J ) 6.7 Hz, 3 H), 1.38 (s, 9 H), 1.70-
1.80 (m, 2 H), 1.86-2.08 (m, 3 H), 2.11-2.20 (m, 2 H), 2.20 (s,
6 H), 3.03 (s, 3 H), 3.47-3.54 (m, 1 H), 3.65-3.71 (m, 1 H),
4.09 (dd, J 1 ) 5.5 Hz, J 2 ) 8.5 Hz, 1 H), 4.51 (t, J ) 8.6 Hz, 1
H), 4.65 (d, J ) 6.2 Hz, 1 H), 4.95 (d, J ) 10.9 Hz, 1 H), 8.01
(d, J ) 8.6 Hz, 1 H). Anal. (C27H50N4O5) C, H, N.
Dov-Va l-MeVa l-P r o-OH (11). Ester 10 (51 mg, 0.10
mmol) was treated with TFA during 30 min, and the solvent
was evaporated under reduced pressure. The residue was
solubilized in 2 N HCl and lyophilized to yield the hydrochlo-
ride salt of 11 as a white powder (50 mg, 100%): FABMS m/e

Chimeric Structures Derived from Dolastatin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 9 1529
(15) Pettit, G. R.; Xu, J .-P.; Williams, F. H.; Schmidt, J . M.; Cerny,
R. L. Antineoplastic agents 370. Isolation and structure of
dolastatin 18. Bioorg. Med. Chem. Lett. 1997, 7, 827-832.
(16) Quentmeier, H.; Brauer, S.; Pettit, G. R.; Drexler, H. G.
Cytostatic effects of dolastatin 10 and dolastatin 15 on human
leukemia cell lines. Leukemia Lymphoma 1992, 6, 245-250.
(17) Steube, K. G.; Grunicke, D.; Pietsch, T.; Gignac, S. M.; Pettit,
G. R.; Drexler, H. G. Dolastatin 10 and dolastatin 15: effects of
two natural peptides on growth and differentiation of leukemia
cells. Leukemia 1992, 6, 1048-1053.
(18) Beckwith, M.; Urba, W. J .; Longo, D. L. Growth inhibition of
human lymphoma cell lines by the marine products, dolastatins
10 and 15. J . Natl. Cancer Inst. 1993, 85, 483-488.
(19) Hu, Z. B.; Gignac, S. M.; Quentmeier, H.; Pettit, G. R.; Drexler,
H. G. Effects of dolastatins on human B-Lymphocytic leukemia
cell lines. Leukemia Res. 1993, 17, 333-339.
(20) J acobsen, S. E. W.; Ruscett, F. W.; Longo, D. L.; Keller, J . R.
Antineoplastic dolastatins: potent inhibitors of hematopoietic
progenitor cells. J . Natl. Cancer Inst. 1991, 83, 1672-1677.
(21) Bai, R.; Pettit, G. R.; Hamel, E. Binding of dolastatin 10 to
tubulin at a distinct site for peptide antimitotic agents near the
exchangeable nucleotide and vinca alkaloid sites. J . Biol. Chem.
1990, 265, 17141-17149.
(22) Bai, R.; Friedman, S. J .; Pettit, G. R. Dolastatin 15, a potent
antimitotic depsipeptide derived from Dolabella auricularia.
Interaction with tubulin and effects on cellular microtubules.
Biochem. Pharmacol. 1992, 43, 2637-2645.
(23) Li, Y.; Kobayashi, H.; Hashimoto, Y.; Shirai, R.; Hirata, A.;
Hayashi, K.; Hamada, Y.; Shioiri, T.; Iwasaki, S. Interaction of
marine toxin dolastatin 10 with porcine brain tubulin: competi-
tive inhibition of rhizoxin and phomopsin A binding. Chem. Biol.
Interact. 1994, 175-183.
(24) Bai, R.; Taylor, G. F.; Schmidt, J . M.; Williams, M. D.; Kepler,
J . A.; Pettit, G. R.; Hamel, E. Interaction of dolastatin 10 with
tubulin: induction of aggregation and binding and dissociation
reactions. Mol. Pharmacol. 1995, 47, 965-976.
(25) Bai, R.; Roach, M. C.; J ayaram, S. K.; Barkoczy, J .; Pettit, G.
R.; Luduen˜a, R. F.; Hamel, E. Differential effects of active
isomers, segments, and analogues of dolastatin 10 on ligand
interactions with tubulin. Biochem. Pharmacol. 1993, 45, 1503-
1515.
Assembly was initiated by a temperature jump from 4 to 37
°C. Each drug was added as a solution in DMSO. The IC50 is
defined as the drug concentration inducing a 50% inhibition
of the rate of assembly.
In h ibition of Cell Gr ow th . L1210 cells were provided by
the National Cancer Institute and HT-29 and MCF7 cells by
the American Type Culture Collection (Frederick, MD). Cells
were cultivated in RPMI 1640 medium (Gibco) supplemented
with 10% fetal calf serum, 2 mM L-glutamine, 100 units/mL
penicillin, 100 µg/mL streptomycin, and 10 mM HEPES buffer
(pH 7.4). Cytotoxicity was measured by the microculture
tetrazolium assay as described.49 Cells were exposed to graded
concentrations of the compounds (nine serial dilutions in
triplicate) for about four doubling times, i.e., 48 h (L1210), 96
h (HT-29), or 168 h (MCF7). Results are expressed as IC50
,
the concentration which reduced by 50% the optical density
of treated cells with respect to untreated controls.
Ack n ow led gm en t. This research was supported by
funds from the Association pour la Recherche sur le
Cancer and the Ligue contre le Cancer. We are grateful
to Dr. F. Gueritte-Voegelein and Ms. S. Thoret for
tubulin assays and to Dr. S. L. Salhi for help in revising
the manuscript.
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