Structure-activity relationships of the didemnins

Article abstract of DOI:10.1021/jm960048g

Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.

Full text of DOI:10.1021/jm960048g

J . Med. Chem. 1996, 39, 2819-2834
2819
Str u ctu r e-Activity Rela tion sh ip s of th e Did em n in s1,2
Ryuichi Sakai,† Kenneth L. Rinehart,*,† Vimal Kishore,† Bijoy Kundu,† Glynn Faircloth,‡ J ames B. Gloer,†
J ohn R. Carney,† Michio Namikoshi,† Furong Sun,† Robert G. Hughes, J r.,§ Dolores Garc´ıa Gra´valos,‡
Teresa Garc´ıa de Quesada,‡ George R. Wilson,† and Richard M. Heid†
Roger Adams Laboratory, University of Illinois, Urbana, Illinois 61801, Roswell Park Cancer Institute,
Buffalo, New York 14263, PharmaMar, U.S.A., Inc., Cambridge, Massachusetts 02139, and PharmaMar,
S.A., 28760 Tres Cantos (Madrid), Spain
Received J anuary 16, 1996X
Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun
solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been
compared. The growth inhibition of various murine and human tumor cells and plaque
reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess
cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein,
DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA
polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the
mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was
determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the
native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites
of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain
portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular,
dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice,
as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in
murine systems, showed remarkable gains in their in vitro and in vivo activities compared to
didemnin B.
In tr od u ction
ribosomal A site, preventing translocation but not
peptide bond formation.11c Most recently, didemnin B
has been reported to induce apoptosis in human HL-60
cells at the most rapid rate yet recorded.11d
The didemnins, marine organism-derived cyclic depsi-
peptides, were isolated from the Caribbean tunicate
Trididemnum solidum by our group as antitumor and
antiviral agents3-5 and numerous biological studies on
didemnins have been conducted.6,7 In in vitro studies,
didemnin B (DB, Chart 1, 1), one of the most potent
components, was cytotoxic to L1210 murine leukemia
cells at very low concentrations and to CV-1 monkey
kidney cells at much higher concentrations.3 In in vivo
studies, 1 was effective in P388 and B16 murine tumor
models and in a Yoshida ascites model.3,8 Compound 1
was the first marine-derived compound to be evaluated
in phase I and phase II clinical trials by the National
Cancer Institute. As an antitumor agent9 it has shown
complete or partial response in previously treated non-
Hodgkins lymphomas.10 Inhibition of protein synthesis
and, to a lesser extent, DNA synthesis was earlier
proposed as the mode of action for the cytotoxicity of
the didemnins.11a Recently, Crews et al. reported
purification of a didemnin-binding protein from a bovine
brain homogenate which appeared to be identical to
human translational enlongation factor-1R (EF-1R) by
affinity chromatography using N-biotinylbis(ꢀ-amino-
caproyl)didemnin A as a ligand. Didemnin A binds to
EF-1R in a GTP-dependent manner, i.e., it binds to the
GTP-EF-1R complex, but does not inhibit EF-1R’s
GTPase activity.11b More recently, SirDeshpande and
Toogood have reported that didemnin B inhibits protein
synthesis by stabilizing aminoacyl-tRNA binding to the
Both didemnins A (DA, Chart 1, 2) and B (1) have
been shown to be strong inhibitors of various viruses
in vitro.3,12 Compounds 1 and 2 also showed activity
in vitro against strains of the lethal RNA viruses
Venezuelan equine encephalomyelitis, yellow fever,
sandfly fever, Rift Valley fever, and a Pichinde virus,
for all of which no effective chemotherapeutic agents
exist.13 In vivo, 1 protected 90% of Rift Valley fever
virus infected mice, although considerable toxicity to the
host animal was observed.5,13
In vivo testing of 1 and 2 against herpes simplex
virus-2 (HSV-2) in mice has shown some efficacy in
topical administration, but intracranial administration
against encephalitis HSV-1, subcutaneous administra-
tion against Semliki-Forest virus, and cutaneous ap-
plication against HSV-1 failed because of narrow thera-
peutic indexes.5,12 On the whole, the high toxicity of 1
and 2 precludes their use as antiviral agents.
Didemnin B (1) inhibited lymphocyte blastogenesis
and the mixed lymphocyte reaction (MLR) in vitro in
murine cells,14 requiring lower concentrations than
cyclosporin A tested in a human lymphocyte system.15
Some efficacy was observed in the graft vs host (GVH)
reaction in mice and allograft transplantation in a rat
with an auxiliary heart graft after treatment with 1.14,16
These results suggest the potential of the didemnins as
immunosuppressive agents.
† University of Illinois.
While 1 has shown a remarkable spectrum of biologi-
cal activities, each has been accompanied by consider-
able toxicity. Hence, modifications of the compound to
‡ PharmaMar U.S.A. and S.A.
§ Roswell Park Cancer Institute.
X Abstract published in Advance ACS Abstracts, May 1, 1996.
S0022-2623(96)00048-9 CCC: $12.00 © 1996 American Chemical Society

2820 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
Ch a r t 1
increase a specific bioactivity while attenuating its
general toxicity is a worthy endeavor.
Na tu r a l Did em n in s. A total of 15 didemnin ana-
logues isolated by us from extracts of the Caribbean
tunicate T. solidum have been characterized.2,3,20 Of
these, 11 congeners (1, 2, 12, 13, 17, 26, and 38-42)
were evaluated in the present study.
Additionally, [pyruvyl9]DB (dehydrodidemnin B, 30),
recently isolated from the Mediterranean tunicate A.
albicans, was also tested.21
We have previously reported some structural modi-
fications and structure-activity relationships (SAR’s)
of didemnins.7 These preliminary results revealed that
simple acylation of the N-terminus of didemnin A
enhanced activities dramatically.3c,7 Several limited
SAR studies of didemnins have been recently reported
by others. J ouin and co-workers reported a total
synthesis of nordidemnin B and four nordidemnin
congeners and their in vitro and in vivo activities:
mandelyl-Pro-, (p-hydroxyphenyl)propionyl-Pro-, and
palmityl-Pro-nordidemnin A and [L-MeLeu7]nordidemnin
B.17 Kessler and co-workers reported the preparation
and solution structure of [L-MeLeu7]didemnin B.18 Most
recently J oullie´ and co-workers reported synthesis and
bioactivity of didemnin B congeners: [dehydro-L-Pro8]-
and [trans-4-hydroxyPro8]DB, dehydro-L-Pro-DA and
trans-4-hydroxyPro-DA as well as L-Pro-DA.19 The
biological results can be summarized as follows: man-
delyl-Pro-norDA, (p-hydroxyphenyl)propionyl-Pro-nor
DA, and [dehydroPro8] analogues showed comparable
activity to 1, but trans-4-hydroxyPro8 analogues showed
cytotoxicity somewhat weaker than DB. Palmityl-Pro-
norDA and [L-MeLeu7] congeners had significantly
diminished activity in the inhibition of tumor cell
growth. The [dehydroPro8] analogues showed compa-
rable antiproliferative activity in an in vitro immuno-
suppressive assay.19 These studies demonstrated some
SAR’s of didemnins, but a more systematic study has
been needed to determine the structural features re-
sponsible for their antitumor, antiviral, and immuno-
suppressive activities, which is essential information in
studying the molecular level mechanism of action of the
didemnins and in developing more selective drugs.
Over 60 didemnin analogues have been obtained
during semisynthetic and synthetic studies of didemnins
and the isolation of bioactive components from extracts
of T. solidum and Aplidium albicans.2 We describe
herein preparation and bioactivities of 42 of these
analogues, representing a great part of the SAR of
didemnins, and discuss the structural requirements of
these remarkable peptides.
The two most abundant didemnins, B (1) and A (2),
are the lead compounds in this study. Didemnin A (2)
has the basic structure common to many other con-
geners, consisting of a cyclic depsihexapeptide with a
D-N-methylleucine (D-MeLeu) side chain attached to
Thr1. Compound 2 (DA) was used as the starting
material in the preparation of 20 congeners since its
N-terminus, a free secondary amino group, offers a site
to attach various acyl groups to the cyclic depsipeptide.
Didemnin B (1), which has a Lac-Pro-acyl unit attached
to the N-terminus of 2, was also used as a starting
molecule for some semisynthetic modifications.
Con gen er s w ith th e Cyclic Dep sip ep tid e Mod i-
fied . 1. Th e Isosta tin e2 Su bu n it. One of the most
unusual subunits, isostatine (Ist), is an intriguing target
site for modifications. During the total synthesis of the
didemnins, all eight possible stereoisomers of Ist were
synthesized,22 and selected stereoisomers have been
incorporated into the cyclic backbone of the peptide to
observe the effects of the stereochemistry of Ist on
bioactivities. Since Z-didemnin A (3) is known to
possess more potent bioactivities than 2 itself,1,6,7 bio-
activities of three compounds varying in a single
stereocentersZ-[3R,4R,5S-Ist2]-, Z-[3S,4S,5S-Ist2]-, and
Z-[3S,4R,5R-Ist2]didemnins A, compounds 4, 5, and 6,
respectivelyswere compared. These compounds were
prepared following the method used in the total syn-
thesis of 2, which contains [3S,4R,5S-Ist2] (Scheme 1).23
The hydroxyl group at the C-3 position of Ist2 was also
modified. Z-Didemnin A (3) was acetylated, and hydro-
genolysis of N-Z-O-acetyl[Ist2]didemnin A afforded
O-acetyl[Ist2]didemnin A (7).
Ch em istr y
Treatment of 1 with phthalimide, diethyl azodicar-
boxylate (DEAD), and PPh3 gave two products, 8 ([Phth-
Ala9]DB) and an anhydro byproduct, 9 ([Anhydro-
Ist2][Phthal-Ala9]DB), which has a trans-2,3-olefin in
the Ist2 unit.20,24
Sequences and some physicochemical properties of all
didemnin congeners described in this paper are listed
in Table 1. Structures of unusual subunits are shown
in Chart 2.

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2821

2822 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
Ch a r t 2
Sch em e 1
2. Th e Hip 3 Su bu n it. The Hip (hydroxyisovaleryl-
propionyl) subunit is also an important modification site
of the didemnins. It has been proposed that the keto
group of the Hip unit plays a major role in the bio-
activities of didemnins.4a Treatment of 2 with NaBH4
afforded the reduced Hip analogue, [(2S,3R,4S)-
H2Hip3]didemnin A ()[H2Hip3]DA, 10)4a,20 as a major
product.
Epididemnin A1 ()[4-epiHip3]DA, 12) from T. solidum
contains a (2S,4R)-hydroxyisovalerylpropionyl (Hip)
residue in place of (2S,4S)-Hip in 1.20
3. Me2Tyr 6 Su bu n it. Didemnin N ()[Tyr6]DB, 13),
isolated from T. solidum, has tyrosine (Tyr) in place of
the N,O-dimethyltyrosine (Me2Tyr) of 1.20 Several
chemical modifications of the Tyr unit were carried out
(see Chart 2 for structures). Treatment of 1 with I2/
AgI afforded the derivative 14 with iodine ortho to the
methoxyl (Chart 2). Catalytic hydrogenation of 1 (Pt/C
Treatment of 1 with hydroxylamine gave [Hip3 oxime]-
didemnin B (11).

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2823
Ta ble 2. Cytotoxicitya and Inhibition of Macromolecule
H2) reduced the aromatic ring of Me2Tyr to give
[H6Me2Tyr6]didemnin B (15) and [H6-N-MePhe6]-
didemnin B (16).
4. Acyclod id em n in A. Acyclodidemnin A (17),
isolated from T. solidum, is an acyclic derivative of 2 in
which the ester linkage between [Thr1] and [Me2Tyr6]
has been hydrolyzed.20
Synthesisb by Didemnin Congeners
inhibition of
macromolecule
cytotoxicity, IC50 (ng/mL)
synthesis IC50 (mg/mL)
compd
no.
P388 A549 HT-29 MEL-28 protein DNA RNA
30
31
32
33
19
20
21
14
16
18
22
26
27
28
35
37
40
1
0.2
0.2
0.2
0.2
0.5
0.5
0.5
0.5
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2.5
5
5
3
5
10
10
20
0.2
0.2
0.2
0.2
0.5
0.5
0.5
1
1
1
1
2
2
2
2
2
2
2
2
2
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
1
2
2
2
2
2
2
2
2
2
2
2.5
2.5
2.5
2.5
5
5
10
10
10
20
0.5
0.2
0.2
0.5
0.5
0.5
0.5
1
1
1
2.5
2
2
2
2
2
2
2
2
2
2
2
2
2.5
5
5
20
20
10
10
5
0.5
0.1
0.1
0.05
0.07
0.08
0.04
0.1
0.07
0.06
0.09
0.1
0.5
0.3
0.1
0.5
0.4
0.1
0.2
0.4
>1
>1
>1
1
0.1
0.2
1
0.3
0.4
0.5
0.5
0.6
0.4
1
0.1
0.2
0.1
0.1
0.1
0.1
0.1
0.4
0.1
0.1
0.1
0.3
0.1
1
>1
1
>1
1
1
1
1
>1
>1
1
Con gen er s w ith a Mod ified Lin ea r P ep tid e P or -
tion . 1. Acyl Der iva tives of DA a n d DB. In an
earlier study, N-acetyldidemnin A (18) was found to be
more active than 2 itself, showing cytotoxicity to L1210
cells comparable to that of DB (1).4a,5 The lipophilicity
added to the molecule, and/or endcapping of the N-
terminus to neutralize the peptide, presumably in-
creases its cell permeability, resulting in more potent
activity. In the present study various fatty acids were
condensed with the N-terminus of 2 to evaluate the
effects of lipophilicity and size of the acyl side chain on
the activities. Compounds semisynthetically prepared
include NR-propionyldidemnin A (19), NR-n-butyryl-
didemnin A (20), NR-pentanoyldidemnin A (21), NR-
hexanoyldidemnin A (22), NR-octanoyldidemnin A (23),
NR-dodecanoyldidemnin A (24), and NR-octadecanoyl-
didemnin A (25).2 Didemnin G ()NR-formyl DA, 26),
an N-formyl derivative of 2, was isolated from T.
solidum.4b
2. Am in o a cid d er iva tives of 2 were also prepared
to evaluate the effect of the eighth subunit on activities.
Condensation of Z-L-Leu, Z-L-Pro, and Z-D-Pro with 2
followed by catalytic hydrogenation gave NR-L-leucyl-
didemnin A (27),3c NR-L-prolyldidemnin A (28),3c and NR-
D-prolyldidemnin A (29), respectively.
3. Did em n in B-typ e a n a logu es which have two
acyl units after the N-terminus of the didemnin A core
were prepared to examine the structural factors con-
tributing to didemnin B’s potent bioactivities. The
diacyl compounds acetyl-Pro-OH, propionyl-Pro-OH,
isobutyryl-Pro-OH, isobutyryl-D-Pro-OH, O-benzyl-Lac-
D-Pro-OH, and O-benzyl-Lac-L-Ala-OH were prepared
and condensed with 2 by the mixed anhydride method.
Deprotection and purification afforded the correspond-
ing didemnin B-type analogues [acetyl9]didemnin B (31),
[propionyl9]didemnin B (32), [isobutyryl9]didemnin B
(33), [isobutyryl9-D-Pro8]didemnin B (34), [Ala8]didemnin
B (35), and [D-Pro8]didemnin B (36).
1
>1
>1
NAc
>1
NA
NA
>1
1
NA
NA
NA
NA
>1
1
NA
NA
NA
NA
>1
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
0.2
>1
NA
0.4
1
8
38
39
41
42
3
15
34
9
1
2
2
2
2.5
5
5
3
5
10
10
20
10
20
50
200
200
200
200
500
500
500
2000
NA
NA
>1
0.5
0.3
0.5
>1
1
1
1
1
>1
1
1
NA
NA
NA
>1
>1
NA
NA
>1
5
2
11
23
29
36
13
17
7
5
20
20
10
20
50
20
20
50
100
200
200
200
200
500
1000
1000
2000
200
200
200
200
500
500
500
2000
200
200
200
200
500
1000
1000
2000
NA
>1
NA
>1
NA
NA
NA
>1
4
10
24
25
6
12
a
P388 ) murine lymphoma. A549 ) human lung carcinoma.
HT-29 ) human colon carcinoma. MEL-28 ) human melanoma.
b
P388 cells were used. c NA ) not active.
(monolayer culture of human melanoma) cell lines.
Cytotoxicity data of all didemnin congeners are sum-
marized in Table 2. An obvious point is the similarity
in IC50 values of each congener vs the four cell lines,
i.e., a lack of selectivity to these cell lines.
All the above compounds have the fundamental
skeleton of didemnin A through D-MeLeu7, but differ
in the extended linear peptide portion of didemnin B.
4. Glu ta m in yld id em n in s. O-pGlu-didemnin B (37)
was prepared to evaluate the effect of the number of
glutaminyl groups on the bioactivities. Didemnins M
(38),20,25 E (39), and D (40),4a,7 which are naturally
occurring, have O-[pGlu-(glutaminyl)n-] peptide chains
(n ) 1-3) acylating the hydroxyl group of the Lac unit
of 1. Didemnins X (41) and Y (42), also isolated from
T. solidum, have an (R)-3-hydroxydecanoyl terminus
after the oligo-Gln peptide chains.7,20
Wide differences were observed, however, in the
relative cytotoxicities of the didemnins tested, over a
10000-fold difference. The two lead compounds were
taken to be DA and DB. We had earlier noted7 that
didemnin A, the only basic didemnin (with a free
secondary amine group), was considerably less cytotoxic
than didemnin B, which is now in clinical trials. This
is true, but neither stands as an extreme of the series.
The most cytotoxic of the didemnins were compounds
30-33, containing less polar short acyl groups (C2-C4)
instead of the lactic acid of DB. These compounds all
had IC50’s of 0.2 ng/mL. Only slightly less active was
the class consisting of 19-21, nonpolar short-chain
Biologica l Resu lts
Cytotoxicity. A simple screening procedure de-
scribed by Bergeron et al.26a was used for the cytotoxicity
assays using P388 (suspension culture of a lymphoid
neoplasm from the DBA/2 mouse), A549 (monolayer
culture of a human lung carcinoma), HT-29 (monolayer
culture of a human colon carcinoma), and MEL-29
N-acyl (C3-C5) derivatives of didemnin A, with IC50
0.5 ng/mL.
)
At the other extreme were didemnin A analogues
containing stereoisomers or reduced analogues of Ist2

2824 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
Ta ble 3. In Vivo Antitumor Activities of Dehydrodidemnin B ([Pyruvyl9]didemnin B, 30) in Micea
doseb
(mg/kg/day)
body wt
change (g)
median
survival (day)
day of death
T/Cc (%)
alive
P388d
(day 23)
320
160
80
-3.3
-3.2
-1.4
4, 5, 6, 6, 6, 9
10, 19, 21, 21, 22, 22
13, 19, 19, 20, 20, 20
6.0
21.0
19.5
60
210
195
0
0
0
B16e
(day 24)
160
80
40
-1.3
-0.5
0.1
15, 31, 32, 32, 36, 38, 38, 39, 41, 42
30, 31, 31, 32, 32, 32, 32, 33, 34, 35
29, 29, 29, 30, 30, 30, 31, 31, 32, 32
37
32
30
218
188
178
9
10
10
dose
(mg/kg/day)
body wt
change (g)
median tumor
NPg (day 14)
vol (mm3)
T/Ch (%)
Lewis Lungf
160
80
40
-3.7
-4.5
-2.6
5
2
0
0
163
473
0.00
0.13
0.37
a
b
d
Assays were carried out by Athur D. Little Inc. Schedule 1-9 days ip. c Treated/control; significant activity, >125%. P388 murine
lymphoma. Median survival of control mice, 10.0 days. e B16 melanoma. Median survival of control mice, 17.0 days. f Lewis lung carcinoma,
median tumor volume of control mice 1372 mm3. NP ) number of nonpalpable tumors on day 14. Significant activity: T/C < 0.40.
g
h
or Hip3 (4, 6, 10, 12), together with long-chain acyl (C12-
C18) derivatives of didemnin A (24, 25). It is of interest
that N-acyl derivatives of didemnin A are among the
most and the least cytotoxic didemnins, depending on
chain length. It is also of interest that some (but not
all) steric modifications in the Ist2-Hip3 portion of the
ring can have profoundly negative effects on the cyto-
toxicity. Thus, the epimers of Ist at C-3 or C-4 are much
less active, but the C-5 epimer, stereoisomeric in the
alkyl group, is like DA. The hydroxyl group of Ist2 may
be involved in H-bonding since the derivative (7)
containing an O-acetylisostatine2 is much less active.
Even more dramatic is the analogue containing the C-4
epimer of Hip in DA (12), which is nearly inactive. The
keto group in Hip3 is also important, since the DA
analogue with dihydro Hip3 (10) is much less active.
[Hip3 oxime]DB (11), however, is less cytotoxic by only
5-fold, a far less significant loss than for the reduced
analogue 10, suggesting that the sp2 carbon at C-3 of
Hip in 11 may maintain didemnin’s backbone conforma-
tion and is essential.
The results from Table 2 show that analogues which
are site-modified within the depsipeptide ring backbone,
the cyclic structure itself, tend to lose their cytotoxicity.
Acyclodidemnin A (17) showed much weaker cytotoxicity
than its cyclic counterpart 2, indicating the importance
of the ring. Generally, analogues which have modified
stereocenters or functional groups directly attached to
the cyclic peptide backbone showed significant losses of
their bioactivities. The N-methyl in Me2Tyr may be
important for the conformation of DA, since iododi-
demnin (14) and reduced didemnins 15 and 16, which
retain the N-methyl group, showed bioactivities com-
parable to those of the parent compounds, while the
Me2Tyr analogue didemnin N ([Tyr6]DB, 13), lacking
the methyls, was much less active. These data indicate
the inherent importance of the cyclic backbone structure
of the depsipeptide.
cytotoxicity of N-acetyldidemnin A (18) is comparable
to that of 1, compound 18 was inactive against P388
leukemia in vivo in the range 0.08-8 mg/kg.
L-Amino acid-substituted didemnins A, 27 and 28,
were approximately as active as 1, while D-amino acid-
substituted 29 was less cytotoxic.
Didemnin B-type analogues 30-33, which have a
ninth subunit more hydrophobic than that of 1 (Lac),
generally were more active than 1. Among these
analogues, naturally occurring dehydrodidemnin B (30)
was tested in vivo (Table 3). Compound 30 exhibited
potent in vivo antitumor activities in mice implanted
with B16 melanoma or P388 leukemia, at lower doses
than 1. Most remarkably, 30 showed effectiveness
against the Lewis lung carcinoma system, for which 1
failed to show any activity.21 On the other hand,
compound 33 was inactive against P388 leukemia in
vivo in the range 0.8-50 mg/kg.
[D-Pro8] analogues of didemnin B, 34 and 36, showed
substantially weaker cytotoxicity compared to the cor-
responding [L-Pro8] compounds, 33 and 1, respectively,
but [L-Ala8]didemnin B (35) was as active as 1.
Glutaminyl didemnins, 37-42, showed potent cyto-
toxicity, but a clear correlation of cytotoxicities with the
number of Gln’s in the side chain was not observed.
An tivir a l Activity. The plaque reduction method
was used for antiviral assays.26b The RNA virus ve-
sicular stomatitis virus (VSV) was grown on baby
hamster kidney cells (BHK) and the DNA virus Herpes
simplex virus type I (HSV-1) was grown on CV-1
monkey kidney cells. Antiviral activities for some
analogues are listed in Table 4. None of the didemnins
significantly inhibited proliferation of the human im-
munodeficiency virus (HIV) in CEM cells (data not
shown).
Cytotoxicity to the tumor cells and antiviral activities
of didemnins usually varied in a parallel fashion. It
should be noted that in the antiviral assays most
didemnins tested gave good virus inhibition accompa-
nied by toxicity to the host cells as marked by LS (light
staining) or PT (partial toxicity) in Table 4. Like the
cytotoxicity, the antiviral activity vs HSV (a DNA virus)
and VSV (an RNA virus) was strong for short-chain acyl
analogues of DB (30-34), and for N-Ac-DA (18, the most
On the other hand, modifications in the linear peptide
portion of the didemnins in some cases resulted in
increased cytotoxicities. Of the acyl derivatives of 2
(18-25), short-chain NR acyl groups generally enhanced
cytotoxicity, but longer chain acyl groups (>C8) de-
creased activities substantially. Although the in vitro

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2825
active of all). On the other extreme acycloDA (17) and
epi-HipDA (12) were inactive. Surprisingly, N-Pro-DA
(28), [Ala8]DB, and [D-Pro8]DB (35 and 36) were quite
active against VSV and glutaminyl didemnins (37, 39,
41) were quite active. Didemnins which showed notice-
able RNA virus (VSV) activity generally showed inhibi-
tion of RNA synthesis (Table 2). This explains the fact
that antiviral activity in the plaque reduction assay is
always accompanied by cytotoxicity to the host cells.
for 18 and 22, and NR-propionyl-, butyryl- and pen-
tanoyl-DA’s were all in the top half. Short-chain [acyl9]
analogues of DB were also quite active.
The native compounds didemnins A and B were in
the middle ranking of all compounds tested, whereas
most site-modified classifications (chiefly Ist2, Hip3) and
long-chain acyl derivatives of DA were in the lower
third, containing less potent compounds, with NR-
octadecanoyl-DA (25) having the largest IC50 at 5110
nM (6040 ng/mL).
In h ibition of Ma cr om olecu la r Syn th esis. Inhibi-
tions of protein, DNA, and RNA synthesis were evalu-
ated by measuring cellular incorporation of tritiated
precursors into P388 cells (Table 2).27 Didemnins B (1)
and A (2) have been previously shown to inhibit macro-
molecular synthesis.11,12 In the present study, most
congeners which showed potent cytotoxicities and anti-
viral activities were strong inhibitors of protein and, to
a lesser extent, DNA synthesis. The strongest inhibitors
of protein synthesis were short-chain acyl derivatives
of DA (18-22). Similar acyl analogues of DB (30-33)
also were among the most active inhibitors. Inhibition
of DNA synthesis was generally at slightly higher
concentrations than protein synthesis, while RNA syn-
thesis was not as routinely inhibited. Short-chain (up
to C6) N-acyl DA’s and some other analogues with
relatively hydrophobic side chains showed weak inhibi-
tion of RNA synthesis. These data suggest that cyto-
toxicity and antiviral activity of the didemnins may be
attributed to a combination of the inhibition of macro-
molecular syntheses, especially protein and DNA, though
the differential between macromolecular synthesis (µg/
mL) and cytotoxicity (ng/mL) is still great. The spread
of activity (SAR) within cytotoxicity is also much
greater.
2. Lym p h ocyte Via bility Assa y. All of the 42
compounds were evaluated for cytotoxicity to one of the
lymphocyte populations in the MLR (i.e. Balb/c spleno-
cytes). The purpose of the lymphocyte viability assay
(LcV) is to measure metabolic activity in lymphocytes
after they have been exposed to the compounds for the
duration of and under conditions equivalent to the MLR.
The inverse of the data yields cytotoxicity information
about the compounds on murine lymphocytes. The
rationale of using resting cells, in this case unstimulated
lymphocytes, to measure cytotoxicity has a strategic
importance. In the MLR the heterogeneous cell popula-
tions are predominantly resting cells when the assay
begins. Transformation (i.e. induction) is initiated upon
costimulation by differing lymphocyte populations, but
cellular proliferation (i.e. lymphoblasts, cells in mitosis)
occurs between days 2 and 4 in the murine MLR.33
Therefore, the first-stage cytotoxic effects of compounds
in the assay are most likely to occur early, before
lymphoblasts develop.
The results show that cytotoxicity of the compounds
to lymphocytes occurs over a narrower range of concen-
trations (3 logs vs 7 logs) as well as at much lower
potencies (0.074-11.0 µM) than their immune inhibitory
effects (Table 5), thus creating a large therapeutic index,
in most cases.
For unexplained reasons, the greatest cytotoxicity was
observed for [H6Me2Tyr6]DB (15) and NR-butyryl-DA
(20) with LC50 values of 0.074 µM (0.08 µg/mL) and
0.099 µM (0.10 µg/mL), respectively. The remaining
compounds have much lower cytotoxicities.
3. Lym p h ocyte Non cytotoxic Im m u n osu p p r es-
sion . A ratio of cytotoxicity-to-inhibitory effects of the
didemnins and their analogues (i.e., a therapeutic index)
is also shown in Table 5. This index identifies those
compounds which may inhibit an in vitro cell-mediated
immune response by noncytotoxic means.
Five compounds showed very large ratios (more than
105). These are the most active compounds (in the same
order) in the mixed lymphocyte reactionsthe pyro-
glutamyl-substituted didemnins B and the short-chain
acyl-substituted didemnins A. Similarly, at the bottom
of the list is NR-octadecanoyldidemnin A (25), with a
ratio of 2, which correlates well with its lowest ranking
in the MLR (IC50 5.110 nM) and moderate cytotoxicity
(>8.50 µM). O-pGlu-DB (37) is the only synthetic
analogue of didemnin B represented in this group, with
a noncytotoxic inhibition ratio of 1 500 000.
The parent compounds, didemnins A and B, showed
similar midrange ratios between 10 000 and 15 000,
with site-modified analogues of each distributed about
equally above and below each parent compound.
4. Lym p h obla st Via bility Assa y. The didemnins
were also evaluated for cytotoxicity to a blastogenic form
of the lymphocytes. Concanavalin A, the T-cell mitogen,
En zym e In h ibition Assa ys. The didemnins were
also assayed for inhibiton of the enzymes DNA and RNA
polymerases,28 topoisomerases I and II,29 dihydrofolate
reductase,30 thymidylate synthase,31 and adenosine
deaminase.32 None of the compounds tested signifi-
cantly inhibited these enzymes. (Data not shown.)
Im m u n osu p p r essive Act ivit ies. 1. Tw o-Wa y
Mixed Lym p h ocyte Resp on se (MLR) Assa y. The
MLR is a cell-mediated immune response induced by
co-culturing two sources of murine splenocytes. In the
present case the overall immunomodulatory properties
of the didemnins and didemnin analogues were evalu-
ated using a bidirectional MLR derived from murine
splenocytes of genetically dissimilar strains of mice.
Table 5 summarizes the variable biological activities of
42 didemnins in suppressing an immune response in
this in vitro assay system.
The results show that all of the compounds suppress
the immune reaction, but over a wide range of potencies.
Of these, didemin M (38) showed the strongest inhibi-
tion of the immune response, with an IC50 value of 0.76
pM (1.0 pg/mL). Other pyroglutamyl didemninssO-
pGlu-DB (37) and didemnin E (39)sare the second and
third most active (IC50 5.3 and 8.4 pM, respectively). In
fact, all four of the pyroglutamyl compounds (37-40)
show strong suppression of the MLR. Although the NR-
acyl-DA class of nine compounds was distributed over
the entire range of potencies, two of the short-chain
members, NR-Ac-DA (18) and NR-hexanoyl-DA (22),
were particularly effective, with IC50’s of 0.027 nM
(0.027 ng/mL) and 0.021 nM (0.022 ng/mL), respectively,

2826 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
Ta ble 4. Antiviral Activities of Some Analogues of Didemnins
VSV/BHK
HSV/CV-1
cytotoxicitya
compd
mg/mL
cytotoxicitya
antiviral activityb
compd
mg/mL
antiviral activityb
18
1
0, PT
0, PT
0, PT
0, PT
12, PT
12, PT
0
+++
++
++
++
+++
+
18
10
3
1
0.3
10
1
10, LS
10, LS
8, LS
0, LS
0, LS
0, LS
0
+++
+++
+++
++
+++
+++
-
0.5
0.2
0.1
1
0.1
0.01
0.001
1
0.1
0.01
0.001
1
0.1
0.01
0.001
1
1
32
31
33
37
39
41
34
0.1
0
-
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0
0, PT
0, PT
0, PT
0, PT
+++
+
10
1
0.1
0, LS
0, LS
0
+++
++
-
(
(
+++
+
10
1
0.1
0, LS
0, LS
0
+++
++
-
-
-
+++
+
10
3
1
0.3
0.1
10
3
0, LS
0, LS
0, LS
0
+++
++
+
0.1
0.01
0.001
-
-
(
-
0
28
34
39
41
37
2
1
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0
+++
++
+
36
28
30
1
0, LS
0
0
++
+
0.5
0.2
0.1
1
0.1
0.01
1
-
(
++
+
10
3
1
0.3
1
0.1
0.01
0.001
1
0.1
0.01
0.001
1
0.1
0.01
0.001
1
0.1
0.01
0.001
1
0.1
0.01
0.001
10
3
0.1
10
5
2
1
10
3
1
0.3
10
1
0.1
10
3
0, LS
0, LS
0
0
0, LS
0
0
0
0, LS
0
0
0
0, LS
0
0
0
0, LS
9
0
0
0, LS
0
0
0
0, LS
0
0
12
8
5
5
8, LS
8, LS
5
0, LS
0, LS
0
0
0, LS
5
0
0
0
0
0
11
8
5
+++
+
-
(
-
1
0.1
0.01
T
-
+
-
+++
(
10, PT
0
-
-
1
0.1
0.01
T
+++
(
10, PT
0
+
-
-
1
0.1
0.01
T
8, PT
6
31
32
33
+++
(
+
-
-
-
1
0, PT
0, PT
0
+
+++
(
0.5
0.2
0.1
1
0.1
0.01
0.001
1
0.1
0.01
1
0.1
0.01
+
(
-
-
0
-
30
0, PT
0, PT
0
+++
+++
(
-
-
-
-
0
35
36
0, PT
0, PT
0
0, PT
0, PT
0, PT
++
(
35
2
+++
(
0
-
++
+++
(
-
+
-
-
29
1
0.5
0.2
0.1
1
0.1
10
10
5
2
1
10
5
2
0, PT
0, PT
0, PT
0
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0, PT
0
27
+++
(
-
-
-
13
7
38
29
+++
-
-
-
-
++
(
-
-
1
(
0.3
10
1
0.1
10
5
-
12
27
17
7
(
-
-
-
-
-
-
-
0
-
1
0, PT
0, PT
0, PT
0, PT
-
0.5
0.2
0.1
-
1
1
-
5
-

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2827
Ta ble 4 (Continued)
VSV/BHK
HSV/CV-1
compd
mg/mL
cytotoxicitya
antiviral activityb
compd
mg/mL
cytotoxicitya
antiviral activityb
38
1
T
8, PT
0
5
0
0
-
-
-
-
-
-
12
10
5
2
1
0.1
0.01
0
5
2
0
0
0
-
-
-
-
-
-
0.1
0.01
10
1
0.1
17
13
a
b
0 (least toxic) to 16 (toxic); PT ) partial toxicity; LS ) light staining, indicating some toxicity. +++ ) complete inhibition, ++ )
strong inhibition, + ) moderate inhibition, ( ) marginal inhibition, - ) no inhibition.
Ta ble 5. In Vitro Immunosuppressive Activities of Didemninsa
two-way mixed
lymphocyte reaction (MLR)
cytotoxicity to lymphocytes
cytotoxicity to lymphoblasts
compd
no.
suppression MLR,
IC50 nM (ng/mL)
LcV,b
LC50 µM (µg/mL)
ratio LcV (LC50)/
MLR (IC50
LbV,c
LC50 µM
ratio LbV (LC50)/
compd name
didemnin M
)
MLR (IC50)
38
37
39
22
18
33
40
31
32
9
0.00076 (0.001)
0.0053 (0.007)
0.0084 (0.012)
0.021(0.022)
0.027 (0.027)
0.037 (0.041)
0.12 (0.20)
0.21 (0.22)
0.23 (0.26)
0.25 (0.30)
0.28 (0.30)
0.30 (0.30)
0.31 (0.33)
0.37 (0.37)
0.38 (0.43)
0.39 (0.49)
0.42 (0.46)
0.48 (0.48)
0.50 (0.83)
0.50 (0.89)
0.52 (0.57)
0.57 (0.54)
0.60 (0.67)
0.66 (0.81)
0.72 (0.81)
0.72 (0.70)
0.74 (0.78)
0.83 (0.92)
0.85 (0.96)
0.96 (1.00)
0.98 (0.93)
1.02 (1.10)
5.10 (5.50)
11.7 (12.2)
18.0 (19.0)
30.5 (29.0)
34.2 (37.0)
86.0 (81.0)
100 (100)
>7.41 (>10)
>8.20 (>10)
>6.77 (>10)
9.60 (>10)
>10000000
>1500000
>800000
>460000
>370000
60000
>50000
14000
8500
2.5E-4
7.9E-5
1.4E-3
1.1E-4
3.9E-4
2.04E-4
8.5E-4
1.6E-4
1.84E-4
1.9E-3
1.2E-2
7.4E-5
7.17E-4
6.8E-5
7.9E-5
8.1E-4
6.0E-4
5.9E-5
3.1E-3
1.1E-3
7.3E-4
0.24
4.81E-3
8.2E-4
1.6E-3
1.1E-3
3.8E-3
0.013
1.7E-3
5.1E-3
0.015
326
15
169
5
14
6
7
1
1
7
43
<1
2
<1
<1
2
1
<1
6
2
1
436
8
1
2
2
5
15
2
5
15
2
4
2
12
16
1
1
1
O-pGlu-DB
didemnin E
NR-hexanoyl-DA
NR-acetyl-DA
[isobutyryl9]DB
didemnin D
>10.0 (>10)
2.24 (2.40)
>6.22 (>10)
2.80 (3.10)
1.98 (2.20)
>8.2 (>10)
>9.3 (>10)
0.099 (0.1)
>9.20 (>10)
6.50 (6.50)
2.60 (2.90)
>8.1 (>10)
6.34 (7.00)
9.70 (>10)
6.01 (10.0)
>5.57 (>10)
>9.20 (>10)
>10.4 (>10)
5.60 (6.20)
>8.10 (>10)
0.074 (0.080)
>10.0 (>10)
>9.50 (>10)
>9.00 (>10)
>8.9 (>10)
>9.60 (>10)
>11.0 (>10)d
7.50 (8.10)
>9.35 (>10)
>9.60 (>10)
9.00 (9.70)
>10.6 (>10)
>9.20 (>10)
>10.6 (>10)
>10.0 (>10)
>8.90 (>10)
>9.3 (>10)
>8.50 (>10)
[acetyl9]DB
[propionyl9]DB
[anhydroist2][Phth-Ala9]DB
Z-[(3S,4R,5R)-Ist2]DA
NR-butyryl-DA
[Ala8]DB
33000
33000
300
>30000
18000
6800
21000
15000
20000
>2000
>11000
>18000
>18000
9000
5
20
35
19
30
8
NR-propionyl-DA
[pyruvyl9]DB (DDB)
[Phth-Ala9]DB
DB
1
21
41
42
16
17
34
14
15
26
27
36
11
28
2
NR-pentanoyl-DA
didemnin X
didemnin Y
[H6-NMePhe6]
acyclodidemnin A
[isobutyryl9-D-Pro8]DB
[iodoMe2Tyr6]DB
[H6-Me2Tyr6]DB
NR-formyl-DA
NR-leucyl-DA
>12000
100
>14000
>13000
11000
10000
>10000
11000
7400
[D-Pro8]DB
[Hip3 oxime]DB
NR-prolyl-DA
DA
Z-DA
3
1.9E-3
0.021
0.022
0.22
23
29
4
10
13
12
7
24
6
25
NR-octanoyl-DA
NR-D-prolyl-DA
Z-[(3R,4R,5S)-Ist2]DA
[H2-Hip3]DA
1800
>8000
500
350
>270
120
100
>84
34
0.46
didemnin N
2.1E-1
5.1E-2
7.7E-2
0.16
2.6
2.7
[epi-Hip3]DA
O-acetyl-DA
NR-dodecanoyl-DA
Z-[(3S,4S,5S)-Ist2]DA
NR-octadecanoyl-DA
106 (119)
270 (290)
5110 (6040)
2
9
1
>2
a
b
Arranged in order of IC50’s (most active to least active) in MLR. LcV ) Lymphocyte viability. c LbV ) Lymphoblast viability. E-4
is the same as 10-4
.
Highest concentration 10 µg/mL.
d
was used to induce cellular proliferation of the spleno-
cytes. Since cellular proliferation occurs between days
2 and 4 in the murine MLR, there may be secondary
cytotoxic effects of the compounds occurring later as
lymphoblasts develop. This form of the cell is more
fragile and therefore usually more susceptible to cyto-
toxicity.
concentrations (down to 7.9E-5 µM) were generally
observed compared to lymphocytes.
The order of cytotoxicity to lymphoblasts is generally,
but not exactly, like that seen for cytotoxicity to
lymphocytes. One exception seems to be for O-pGlu-
DB (37), which ranked higher in cytotoxicity to lym-
phoblasts than to lymphocytes. This change in rank
accounts for a marked difference in the ratios of non-
cytotoxic immunosuppression discussed below.
The results show that the cytotoxicity of the com-
pounds to lymphoblasts occurs over a much wider range
of concentrations than was observed toward lympho-
cytes (Table 5). Not unexpectedly, much lower cytotoxic
5. Lym p h obla st Non cytotoxic Im m u n osu p p r es-
sion . Some important differences between lymphocyte

2828 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
Ta ble 6. Immunosuppression of Didemnins on Graft vs Host Reaction (GVHR) Assaya
mean group spleen
wt normalized to
day 8 body wt
doseb
(mg/kg/day)
body wtc day 0
body wt day 5
(g ( SD)
body wt change
day 5 (g)
alive
day 8 (%)
compound
(g ( SD)
indexedd
didemnin B (1)
0.16
17.0 ( 0.9
17.2 ( 0.7
17.4 ( 0.8
16.6 ( 0.8
16.6 ( 0.5
15.8 ( 1.3
15.8 ( 1.2
16.4 ( 0.8
17.0 ( 1.5
16.8 ( 1.0
16.6 ( 0.5
16.3 ( 0.4
15.6 ( 1.6
18.2 ( 0.7
18.4 ( 0.8
16.4 ( 1.0
17.8 ( 0.7
17.2 ( 1.2
15.2 ( 1.2
17.2 ( 1.5
17.4 ( 1.7
18.0 ( 1.3
18.0 ( 1.3
13.8 ( 0.4
-1.4
1.0
1.0
1.0
-0.2
1.4
-0.6
0.8
0.4
1.2
1.4
-2.5
100
100
100
100
100
100
100
100
100
100
100
100
4.0
4.2
6.1
4.9
4.4
4.1
4.2
5.6
5.2
5.4
3.2
0.4
1.24
1.33
1.91
1.54
1.38
1.27
1.32
1.76
1.63
1.70
1.00
0.14
0.016
0.0016
0.16
0.016
0.0016
0.16
didemnin M (38)
O-pGlu-DB (37)
0.016
0.0016
positive controle
syngeneticf
cyclophosphamide
200
a
b
Balb/c-to-CB6F1 GVHR model. See the Experimental Section for detail. Schedule qd 1-7, 0.5 mL of solution/mouse. c Mice were
d
weighed on days 0, 5, 8. Index ) spleen wt treated/spleen wt syngenetic injection. >1.3 (50% suppression of positive control is considered
to be significant suppression). e Vehicle only. f Syngenetic (CB6F1-CB6F1) injection.
and lymphoblast assays are observed. First, the toxic-
ity/suppression ratios are smaller in the lymphoblast
assay, indicating that the inhibition of the immune
response at later stages in the reaction would be most
affected by cytotoxicity to proliferating immune cells.
However, the exception to this, consistent with a judg-
ment of noncytotoxic immunosuppression, occurs for
acycloDA (17), didemnin M (38), and didemnin E (39).
The ratios are larger as a group to distinguish them as
compounds that might rely on other means to inhibit
the immune reaction than via cytotoxicity involving
lymphoblasts. Didemnins M and E have this distinction
whether the cytotoxicity data are for lymphocytes or
lymphoblasts.
6. Gr a ft vs Host Rea ction (GVHR). Three repre-
sentative compoundssdidemnin M (38), O-pGlu-DB
(37), and didemnin B (1)sshowing high (37, 38) or
moderate (1) in vitro immunosuppressive activity were
subsequently evaluated in a multidose assay (0.16,
0.016, and 0.0016 mg/kg per injection; qd 1-7) for their
in vivo immunosuppressive effects on the GVHR
splenomegaly assay.
The results show didemnin M (38) optimally sup-
pressed the allogeneically induced splenomegaly re-
sponse in CB6F1 mice grafted with Balb/c splenocytes
at 1.6 µg/kg/inj by 61% compared to control (Table 6).
Higher doses (16 and 160 µg/kg) were less effective, but
not toxic. Didemnin B (2) and O-pGlu-DB (37) were
equally effective but at optimal dosages of 160 µg/kg per
injection, showing 66% and 54% suppression, respec-
tively. These were the highest doses for each and
neither was toxic to the animals. Lower doses were less
effective for O-pGlu-DB. Didemnin B showed a 53%
suppression at the next lower dose of 16 µg/kg per
injection but was not effective at the lowest dose (1.6
µg/kg per injection). The relationship of these in vivo
effects correlates well with their in vitro data.
the compounds may interfere with the assembly of viral
DNA or RNA in the infected cells.
Structurally, the original cyclic depsipeptide backbone
has been shown to be essential for all three bioactivities,
except acyclodidemnin A (17) for immunosuppression.
All other modifications within the cyclic peptide portion
resulted in diminished bioactivities. Stereochemical
and functional changes in the peptide backbone led to
significant losses in bioactivities, suggesting that the
peptide conformation maintained by the original ste-
reocenters and functional groups is indispensable. An-
hydroIst analogue 9 and Hip oxime analogue 11 were
the only analogues with bioactivity comparable to their
counterparts, 8 and 1, respectively. These compounds
are presumably capable of maintaining the original
backbone conformation.
The alkyl and aryl side chains of Ist and Me2Tyr,
respectively, could be modified without drastic loss of
cytotoxicity or antiviral activity. The latter case was
somewhat surprising, since the aryl group of didemnin
B was proposed by Hossain et al. to be one possible
binding site to the receptors.34 In the MLR assay,
however, the Me2Tyr unit demonstrated some impor-
tance as all analogues site modified at this subunit
showed lower potency in MLR than their counterparts.
In contrast to the cyclic peptide portion, modification
by acyl groups extending from the N-terminus of the
MeLeu unit led to gains in bioactivities. Short-chain
acyl (C2-C6) and L-amino acid derivatives of 2 were
comparably active to 1 in vitro. In vivo testing of 18,
however, showed that at least some of those modifica-
tions are not appropriate to give significant efficacy.3c
In the didemnin B-type compounds, terminal (unit 9)
acyl groups less polar than and similar in size to Lac
gained in vitro and in vivo antitumor activity. Interest-
ingly, amino acid derivatives of 2 and didemnin B-type
analogues which contained [D-Pro8] showed much lower
activities than their counterparts. NMR studies of these
[D-Pro8] analogues showed that they consist of several
conformers in solution (CDCl3, Figure 1). This suggests
that [Pro8] regulates the orientation of the peptide side
chain, by a â-turn structure according to the suggestions
of Kessler et al.,35 which may affect the entire peptide
shape.
Discu ssion
The present study has defined SAR’s for the di-
demnins regarding three important bioactivities: cyto-
toxicity, immunosuppression, and antiviral activity. The
data in Tables 2 and 4 suggest that the cytotoxicity and
antiviral activity of all congeners may be due to inhibi-
tion of macromolecular synthesis, i.e. protein, DNA, and
RNA. Inhibition of DNA and RNA synthesis appears
to be particularly important in antiviral activity, since
The immunology data suggest that the immunosup-
pressive activity of didemnins is mediated mostly by
cytotoxicity to lymphoblasts occurring at a later stage

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2829
F igu r e 1. 1H NMR spectra (500 MHz in CDCl3) of the amide proton regions for didemnin B (1, below) and [D-Pro8]DB (36,
above). In the latter compound, NH’s appeared as multiple signals, suggesting that [D-Pro8]DB exists as several stable conformers
in solution. It is noteworthy that the amide proton for the Thr subunit of 36 gave two distinct chemical shifts, group 1 and group
2. The assignments are based on COSY data.
penicillin G, and streptomycin sulfate. P388 cells (104 cells/
of activation in the cell-mediated immune response,
16-mm well) and A-549, HT-29, Mel-28 cells (2 × 104 cells/16-
except for some glutaminyl didemnins, 37-39, and
mm well) were seeded in 1 mL of the above medium with each
concentration of samples. All assays were carried out in
duplicate. After 3 days of incubation (37 °C, 98% humidity,
10% CO2) cells were visually counted against control wells to
acyclo-DA (17), as indicated by the relatively constant
therapeutic index utilizing cytotoxicity data to prolif-
erating lymphoblasts versus resting lymphocytes. These
results indicate that the immunosuppressive activity of
determine IC50
.
didemnins is, in most cases, a result of antiproliferation
of stimulated T-cells, as demonstrated for 1 by others.36
In the cases of 37-39 and 17, however, some unique or
specific mechanisms of action such as those seen in
cyclosporin A,37 FK 506, or rapamycin38 may be involved
in the immunosuppressive actions (e.g. noncytotoxic
immunosuppression during early stage events involving
T-cell activation).
More detailed biochemical study on the cellular mech-
anism for the antiproliferative activity of the above-
mentioned didemnin congeners should result in further
understanding of their interesting immunosuppressive
activity.
DNA, RNA, a n d P r otein Syn th esis. These assays were
carried out by following the method published previously, with
slight modification.27 Inhibitions of DNA, RNA, and protein
synthesis were determined by measuring cellular incorporation
of [methyl-3H]thymidine, [5-3H]uridine, and DL-[4,5-3H]leucine,
respectively, into P388 cells. Cells were cultured in MEM with
5% newborn calf serum (3 × 105 cells/mL). Compounds were
added with DMSO (1% final DMSO concentration) to testing
concentrations. These cells were added to the mixture of each
tritiated precursor (1-3 µCi/mL), the corresponding thymidine,
uridine, or leucine (4 mM) was incubated (37 °C, 5% CO2 in
air) and then collected on a Durapore filter (0.45 µm, Millipore)
by vacuum filtration, and the macromolecules were precipi-
tated by addition of 5% trichloroacetic acid (TCA). The filter
was washed by 5% TCA three times and then transferred to
scintillation vials. Scintillation cocktail Biogreen-2 was added
to the vial, and radioactivity was counted by a liquid scintil-
lation counter.
Exp er im en ta l Section
Gen er a l. Optical rotations were measured using a 5-cm
1
cell (1 mL). NMR spectra (200, 300, and 500 MHz, H) were
DNA P olym er a se Assa y. Each compound in DMSO (1%
final DMSO concentration) was added to a mixture (final
volume 50 µL) comprised of Escherichia coli DNA polymerase-1
in 50 mM Tris-HCl (pH 7.5), MgCl2 (10 mM), dithiothreitol (1
mM), bovine serum albumin (30 µg/mL), activated calf thymus
DNA (40 µg/mL), dGTP, dTTP, dCTP (35 µM, each), dATP (17
µM), and 1 µCi of [8-3H(N)]dATP (10-25 Ci/mmol). The
reaction was quenched by adding TCA (10%) and sodium
pyrophosphate (1.0 M). The macromolecule precipitate was
collected, and the radioactivity was counted as described above.
obtained using either deuteriochloroform (CDCl3), deuterio-
methanol (CD3OD), or a mixture of both as solvents and
internal standard [7.26 (1H) and 77.0 (13C) ppm for CDCl3, 3.30
(1H) and 49.0 (13C) ppm for CD3OD or a mixture of CD3OD-
CDCl3]. FABMS spectra and HRFABMS data were obtained
using magic bullet as a matrix. All solvents for reactions were
distilled over appropriate drying agents prior to use. RPHPLC
was perfomed by using a semipreparative C-18 silica gel
column with UV detection at 254 nm and a flow rate of 1 mL/
min. Solvent systems are indicated in each case.
RNA P olym er a se Assa y. Each compound in DMSO (1%
final DMSO concentration) was added to a mixture (final
volume 50 µL) comprised of E. coli RNA polymerase in 40 mM
Tris-HCl (pH 7.5), MgCl2 (10 mM), KCl (50 mM), dithiothreitol
(1 mM), bovine serum albumin (100 µg/mL), activated calf
thymus DNA (40 µg/mL), GTP, UTP, CTP (70 µM, each), ATP
(35 µM), and 1 µCi of [2,8-3H]ATP (25-40 Ci/mmol). The
Bioa ssa ys. Cytotoxicity Mea su r em en t. Cytotoxicities
were measured using the procedure described previously.25
Briefly, cells were maintained in logarithmic phase of growth
in a medium comprised of the following: Eagle’s minimum
essential medium (MEM) with Earle’s balanced salts [with
L-Gln (2.0 mM) and nonessential amino acids, without Na2CO3]
supplemented by 10% fetal calf serum, Na2CO3 (10-2 M),

2830 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
reaction was quenched, and the radioactivity of the macro-
molecule was counted as above.
phosphate-buffered saline) at one of three dose levels (0.16,
0.016, and 0.0016 mg/kg per injection) in a multidose assay,
cyclophosphamide (200 mg/kg per injection), or vehicle on days
1-7 (qd 1-7). On day 8 all groups were sacrificed, spleens
were excised, and a graft index was calculated for each group
by the following formula:
P r ep a r a tion of Sp len ocyte Cell Su sp en sion s for P r i-
m a r y Cu ltu r e. Separate splenocyte cell suspensions were
prepared by homogenization of spleens from 6-12 week old
female C57Bl/6 (H-2b) and BALB/c (H-2d) mice in cold (4 °C)
tissue culture medium [TCM: RPMI 1640 medium supple-
mented with 10% fetal calf serum, 4 mM HEPES, 1% anti-
mycotic solution (i.e. 100 units/mL penicillin, 0.25 µg/mL
amphotericin-B, 100 µg/mL streptomycin), 25 µg/mL gentami-
cin, and 2% L-glutamine]. The homogenate was centrifuged
at 500g for 5 min at 4 °C. The resulting pellet was resus-
pended in cold TCM at 10 mL/spleen and evaluated for
viability by hemacytometer using the trypan blue exclusion
method. The cell concentration was adjusted to 2.5 × 106 cells/
mL for each splenocyte population.
graft index )
[(spleen wt of test group)/(body wt of test group) × 100]
(spleen wt of syngeneic group)/(body wt of syngeneic group)
P r ep a r a t ion of Did em n in Con gen er s. Na t ive Di-
d em n in s. All native didemnins were isolated from the extract
of T. solidum, except for dehydrodidemnin B (30), which was
isolated from Aplidium albicans.21 Isolation and structure
determination of these peptides have been described else-
where.3,20,21
Mixed Lym p h ocyte Rea ction (MLR).39 Each compound
was dissolved in absolute ethanol (abs. EtOH) and diluted 16-
fold (from 10 µg/mL to 3.33 × 10-6 µg/mL). Duplicate volumes
(10 µL) were added into wells of a 96-well microtiter plate and
then evaporated to dryness at room temperature. Splenocytes
derived from Balb/c and C57Bl/6 mice were prepared as
described above, and 100 µL of each cell suspension was added
to each well. Wells containing 200 µL of media alone served
as nonspecific control wells.
Assay plates were incubated in a 5% CO2 humidified
incubator at 37 °C for 96 h and then pulsed overnight (about
15 h) with 1 µCi of [3H]thymidine (20 Ci/mmol) per well and
finally filtered to recover tritiated-thymidine incorporated into
newly synthesized DNA.
Boc-(3R,4R,5S)-Ist-OH. Boc-(3R,4R,5S)-Ist-OEt22 (130 mg,
0.43 mmol) was treated with KOH (1N, 0.5 mL) in dioxane
(1.0 mL) and H2O (0.5 mL) at room temperature for 1 h. An
oily product (98 mg, 83%) was afforded after the usual
workup: [R]25 +33° (c 0.13, CHCl3); 1H NMR (200 MHz,
D
CDCl3) δ 4.88 (1H, d, J ) 10.0 Hz), 4.22 (1H, m), 3.32 (1H, m),
2.6-2.5 (2H, m), 1.45 (9H, s); HRFABMS calcd for C13H26NO5
Mr 276.1811 (M + H), found 276.1816.
Boc-(3S,4R,5R)-Ist -OH . Boc-(3S,4R,5R)-Ist-OEt22 was
treated as above to give an oil (84%): [R]25 -1.2° (c 0.27,
D
CHCl3); 1H NMR (200 MHz, CDCl3) δ 4.50 (1H, d, J ) 9.8 Hz),
4.06 (1H, m), 3.60 (1H, m), 2.6-2.4 (2H, m), 1.45 (9H, s);
HRFABMS found 276.1804.
The MLR data were calculated as a percentage of immune
cell proliferative activity relative to control, and an IC50 value
was interpolated for each test compound.
Boc-(3S,4S,5S)-Ist-OH. Boc-(3S,4S,5S)-Ist-OEt was treated
as above to give an oil (84%): [R]25 -41.3° (c 0.29, CHCl3);
D
1H NMR22 (200 MHz, CDCl3) δ 4.90 (1H, d, J ) 10.0 Hz), 4.27
(1H, m), 3.22 (1H, m), 2.7-2.4 (2H, m), 1.45 (9H, s); HRFABMS
found 276.1804.
Lym p h ocyte Via bility (LcV) Assa y.40 Test compounds
were prepared in two sets as described above. Splenocytes
were prepared as described previously from one murine strain
(Balb/c), and a volume of 200 µL of the cell suspension was
added to one set of test compounds and control wells.
Assay plates were incubated as in the MLR and then pulsed
overnight with 75 µL per well MTT-thiazolyl blue solution
(150 µg). The plates were decanted, and the resulting insoluble
formazan crystals were dissolved in 200 µL of isopropyl alcohol.
Absorbance at 570 nm was measured.
Boc-(3R,4R,5S)-Ist(TBDMS)-OH. Boc-(3R,4R,5S)-Ist-OH
(97 mg, 0.35 mmol) was treated with tert-butyldimethylsilyl
(TBDMS) chloride (0.16 g, 0.45 mmol) in the presence of
imidazole (145 mg, 2.1 mmol) in DMF (1.1 mL) under N2 for
20 h. The product was chromatographed (SiO2, hexane-
EtOAc, 1:4) to give an oil (80 mg, 59%): [R]25 +21° (c 0.29,
D
CHCl3); 1H NMR (200 MHz, CDCl3) δ 4.72 (1H, d, J ) 9.0 Hz),
4.33 (1H, m), 3.37 (1H, m), 2.7-2.4 (2H, m), 1.46 (9H, s), 0.90
(9H, s), 0.09 and 0.07 (6H, 2:1 singlets); HRFABMS calcd for
C19H40NO5Si Mr 390.2676 (M + H), found 390.2662.
The LcV data were calculated as a percentage of basal
metabolic activity, or percent viability, relative to BALB/c
control, and an LC50 value was interpolated for each test
compound.
Boc-(3S,4R,5R)-Ist(TBDMS)-OH. The above treatment of
Boc-(3S,4R,5R)-Ist-OH gave an oil (63%): [R]25D -0.74° (c 0.41,
CHCl3); 1H NMR (200 MHz, CDCl3) δ 6.64 and 4.50 (0.5 H
each, d, J ) 9.0 Hz), 4.17 (1H, m), 3.70 and 3.30 (0.5H each,
m), 2.6-2.4 (2H, m), 1.46 and 1.43 (9H, s), 0.88 (9H, brs), 0.11
and 0.02 (6H, 4:1singlets); HRFABMS found 390.2680.
Boc-(3S,4S,5S)-Ist (TBDMS)-OH. The above treatment
Lym p h obla st Via bility (LbV) Assa y. The cytotoxicity of
didemnins and didemnin analogues on lymphoblasts was
evaluated using a modification of the above LcV procedure.
Mitogen-induced lymphoblastic proliferation was initiated by
preincubation of splenocytes, as prepared above, with 1.0 µg/
mL of concanavalin A (Con A) in a 5% CO2 humidified
incubator at 37 °C for 30 min.41 Test compounds were
prepared as described above. Preincubated Con A splenocytes
were added in a volume of 200 µL to each well. Incubation,
processing, and data calculation were the same as described
above for the LcV assay.
Gr a ft vs Host GVH Rea ction . Three didemnins (didem-
nin B, didemnin M, and pGlu-didemnin B) were evaluated in
a modified Simonsen splenomegaly assay.42 Briefly, an F1
hybrid host animal is grafted with immunocompetent spleen
cells from the parent strain. The index used to measure the
success of the GVHR is splenomegaly (increased spleen weight
due to cellular proliferation of grafted lymphocytes). An index
> 1.3 (graft index) is considered to be a successful graft
rejection of the recipient animal. Immunosuppression is
considered as a reduction of the graft index.
of Boc-(3S,4S,5S)-Ist-OH gave an oil (58%): [R]25 -30.3° (c
D
1
0.37, CHCl3); H NMR (200 MHz, CDCl3) δ 4.75 (1 H, d, J )
9.0 Hz), 4.37 (1H, m), 3.30 (1H, m), 2.6-2.4 (2H, m), 1.46 (9H,
s), 0.90 (9H, brs), 0.12 (6H, s); HRFABMS found 390.2676.
Boc-(3R,4R,5S)-Ist(TBDMS)-Hip -Leu -OTMSE. A solu-
tion of DCC (1,3-dicyclohexylcarbodiimide, 44 mg, 0.22 mmol)
in CH2Cl2 (1 mL) was added to a stirred solution of Hip-Leu-
OTMSE23 (74 mg, 0.19 mmol), Boc-(3R,4R,5S)-Ist(TBDMS)-
OH (74 mg, 0.19 mmol), and DMAP ((N,N-dimethylamino)-
pyridine, 23 mg, 0.19 mmol) in CH2Cl2 (3 mL) at 0 °C. The
reaction mixture was stirred at 0 °C for 1 h then at room
temperature for 12 h. The product was chromatographed
(SiO2, EtOAc-hexane 9:1) to give a viscous oil (54 mg, 37%):
[R]25D -7.5° (c 0.28, CHCl3); HRFABMS calcd for C30H75N2O9-
Si Mr 759.5011 (M + H), found 759.5035.
Boc-(3S,4R,5R)-Ist (TBDMS)-H ip -Leu -OTMSE (51%):
On day 0, CB6F1 female mice, 4 weeks of age, were grafted
by intraperitoneal (ip) injection of 50 × 106 splenocytes from
BALB/c female mice in high-glucose (4500 mg/mL) Dulbecco’s
modified Eagles medium. A syngeneic control group (CB6F1-
to-CB6F1) was similarly prepared and served as the negative
control. Grafted mice were divided into treatment groups
containing six mice each. Groups were injected ip with test
compound (dissolved in vehicle; 1% abs. EtOH in sterile
[R]25 -19° (c 0.34, CHCl3); HRFABMS found 759.4998.
D
Boc-(3S,4S,5S)-Ist (TBDMS)-H ip -Leu -OTMSE (40%):
[R]25 -27°(c 0.33, CHCl3); HRFABMS found 759.5011.
D
Boc-(3S,4S,5S)-Ist -H ip -Leu -OH . A solution of Boc-
(3R,4R,5S)-Ist(TBDMS)-Hip-Leu-OTMSE (65 mg, 0.086 mmol)
in THF (0.6 mL) was stirred with tetrabutylammonium
fluoride (TBAF) solution (1 M, 0.195 µL in THF) at room
temperature for 3 days. Water was added to the product after

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2831
removal of the organic solvent. Crude oily material was
obtained after the usual workup which was separated (SiO2,
silica gel column with EtOAc-2-propanol (4:1). The residue
was purified by HPLC (silica gel, EtOAc) to give 7 (6 mg,
CHCl3-MeOH, 9:1) to give an oil (45 mg, 96%): [R]24 -9.8°
17%): [R]24 -136° (c 0.38, CHCl3); white powder; IR (film)
D
D
(c 0.31, CHCl3); HRFABMS calcd for C27H49N2O9, Mr 545.3438
(M + H), found 545.3444.
3319, 1734, 1653, 1635 cm-1
;
1H NMR (300 MHz);1 FABMS
m/z 986 (M + H); HRFABMS calcd for C51H81N6O13 Mr
985.5861 (M + H), found 985.5871.
Boc-(3S,4R,5R)-Ist-Hip -Leu -OH (87%): [R]24 -11° (c
D
[(2S,3R,4S)-H2Hip3]Didem n in A (10). A solution of NaBH4
(3.50 mg, 0.095 mmol) in THF-H2O (1:1, 2 mL) was added
dropwise to a solution of 2 (79.4 mg, 0.084 mmol) in THF (2
mL) at 0 °C. The mixture was stirred at 0 °C for 50 min and
the temperature was elevated to room temperature over 2 h;
HCl (1 N, 90 µL) was added, and the product was extracted
with CH2Cl2. The organic layer was concentrated to give a
solid (79.2 mg) which was chromatographed (silica gel, CHCl3-
MeOH, 6:1) to give nearly pure 10 (53.4 mg, 67%). A portion
was purified by RPHPLC (MeOH-NaCl (0.4 M), 7:1): HR-
FABMS calcd for C49H81N6O12 Mr 945.5912 (M + H), found
945.5934.
0.25, CHCl3); HRFABMS found 545.3444.
Boc-(3S,4S,5S)-Ist-Hip -Leu -OH (88%): [R]24D -21° (c 0.25,
CHCl3); HRFABMS found 545.3432.
Z-D-MeLeu -Th r -{O-[Boc-(3R,4R,5S)Ist-O-Hip -Leu -P r o-
Me2Tyr ]}-OTMSe. A solution of Boc-(3S,4R,5R)-Ist-Hip-Leu-
OH (30 mg, 0.055 mmol) and N-hydroxybenzotriazole (HOBT,
13.5 mg, 0.1 mmol) in THF (0.4 mL) was added to a solution
of Z-D-MeLeu-Thr-[O-(Pro-Me2Tyr)]-OH‚HCl (40 mg, 0.050
mmol) in DMF (0.3 mL) in the presence of N-methylmorpholine
(NMM, 4 µL). A solution of EDC [1-[3-(dimethylamino)propyl]-
3-ethylcarbodiimide hydrochloride, 11.5 mg, 0.056 mmol] in
THF (0.3 mL) was added at 0 °C. The reaction mixture was
stirred at 0 °C for 1 h and then at room temperature for 12 h.
The products were purified by RPHPLC (MeOH-H2O, 9:1).
Two products (epimers at the R-position of Hip, identical
[Hip 3 oxim e]Did em n in B (11). To a solution of 1 (25.1
mg, 22.6 mmol) in CH3OH (1 mL) was added NH2OH-HCl
(57.3 mg, 825 mmol) followed by (C2H5)3N (115 mL, 825 mmol).
The solution was stirred at room temperature for 1 week and
concentrated (N2). The residue was chromatographed as above
to give 11 (11.9 mg, 47%): 1H NMR (CDCl3, 500 MHz)1 δ 7.70
(1H, d, J ) 9.7 Hz), 7.67 (1H, d, J ) 5.2 Hz), 7.40 (1H, d, J )
10.0 Hz), 6.09 (1H, d, J ) 5.4 Hz, Hip-R); HRFABMS calcd for
C57H91N8O15 Mr 1127.6604 (M + H), found 1127.6619.
Iod od id em n in B (14). To a solution of 1 (5.5 mg, 0.005
mmol in CH2Cl2 0.2 mL) was added CF3CO2Ag (8.8 mg, 0.04
mmol) followed, dropwise, by a solution of I2 (10 mg, 0.04 mmol
in CH2Cl2, 0.2 mL). The suspension was stirred overnight at
room temperature. Excess reagents were removed (filtration,
Na2SO3 wash). The solvent was removed, and the crude
product was purified by HPLC (C-18, MeOH-H2O, 7:1) to give
14 (5.0 mg, 82%): 1H NMR (CDCl3)1 δ 7.51 (1H, s), 7.15 (1H,
d, J ) 8.0 Hz), 6.77 (1H, d, J ) 8.0 Hz), 3.87 (3H, s), 2.59 (3H,
s, N-CH3); HRFABMS calcd for C57H89IN7O15 Mr 1238.5461 (M
+ H), found 1238.5458.
FABMS data) were combined (yield 41.5 mg, 58%): [R]25
D
-0.9° (c 0.89, CHCl3); HRFABMS calcd for C67H107N6O17Si Mr
1295.7462 (M + H), found 1295.7460.
Z-D-MeLeu -Th r -{O-[Boc-(3S,4R,5R)Ist-O-Hip -Leu -P r o-
Me2Tyr ]}-OTMSe (50%): [R]25 +1.9° (c 0.89, CHCl3); HR-
D
FABMS calcd for C67H107N6O17Si Mr 1295.7462 (M + H), found
1295.7471.
Z-D-MeLeu -Th r -{O-[Boc-(3S,4S,5S)Ist-O-Hip -Leu -P r o-
Me2Tyr ]}-OTMSe (50%): [R]25 -9° (c 0.19, CHCl3); HR-
D
FABMS calcd for C67H107N6O17Si Mr 1295.7462 (M + H), found
1295.7460.
Z-[(3R,4R,5S)Ist2]d id em n in A (4). A stirred solution of
Z-D-MeLeu-Thr-{O-[Boc-(3R,4R,5S)Ist-O-Hip-Leu-Pro-Me2Tyr]}-
OTMSe (40 mg, 0.031 mmol) in THF (0.2 mL) was treated with
tetrabutylammonium fluoride (1 M in THF, 0.12 mL) for 16
h. A viscous oil (35 mg, FABMS m/z 1195) was obtained after
the usual workup which showed one spot on TLC (SiO2). To
this product (in CH2Cl2, 0.2 mL) was added TFA (0.25 mL) at
0 °C. The reaction mixture was stirred at 0 °C for 1 h. Excess
TFA was removed in vacuo to give the ninhydrin-positive
product (36 mg, m/z 1096 by FABMS, TLC one spot). NMM
(4 µL) was added to a solution (CH2Cl2, 1 mL) of the above
product at 0 °C, followed by CH2Cl2 (15 mL). To a stirred
solution of HOBT (14.1 mg, 0.10 mmol) and EDC (14.2 mg,
0.070 mmol) in DMF (0.5 mL) and CH2Cl2 (60 mL) was slowly
added the above mixture over 6 h at 0 °C. The reaction
mixture was stirred at room temperature for 4 days and then
concentrated. The EtOAc soluble portion was washed, and the
product (27 mg) was separated by RPHPLC (MeOH-H2O, 85:
15) to give a solid (2.3 mg, 6%). This product, a mixture of
Z-didemnin A and its epimer at the R-position of the Hip
residue, was separated by HPLC (silica gel, EtOAc-hexane,
3:2). The first fraction and the second fraction gave
Z-[(3R,4R,5S)Ist2]didemnin A (4) and Z-[(3R,4R,5S)Ist2,R-epi-
Hip3]didemnin A, respectively.23 Compound 4: colorless solid;
[H 6Me2Tyr 6]Did em n in
B
(15) a n d [H 6-N-MeP h e6]-
Did em n in B (16). A mixture of 1 (16.3 mg, 0.015 mmol), Pt/C
(10%, 38.3 mg), and TFA (20 µL) in CH3OH (5 mL) was stirred
under H2 for 4 h at room temperature. The mixture was
filtered through a C-18 Sep-pak with MeOH and concentrated
to give a solid (33.1 mg), which was separated by HPLC (C-
18, CH3OH-0.4 M NaCl, 7:1) to give 15 (3.3 mg, 20%) as a
1
white powder: [R]23 -29° (c 0.41, CHCl3); H NMR (CDCl3,
D
500 MHz);1 HRFABMS calcd for C57H96N7O15 Mr 1118.6964
(M + H), found 1118.7001.
Fraction 2 yielded 16 (5.2 mg, 32%) as a white solid: [R]24
D
-29° (c 0.41, CHCl3); 1H NMR (CDCl3, 500 MHz);1 HRFABMS
calcd for C56H94N7O14 Mr 1088.6859 (M + H), found 1088.6902.
Nr-Acetyld id em n in A (18) was prepared from 2 as de-
scribed:3c HRFABMS calcd for C51H81N6O13 Mr 985.5862 (M +
H), found 985.5880.
Nr-P r op ion yld id em n in A (19) was prepared from 2 as
described:3c HRFABMS calcd for C52H83N6O13 Mr 999.6018 (M
+ H), found 999.5985.
[R]25 -14° (c 0.16, CHCl3); HRFABMS calcd for C57H85N6O14
D
Mr 1077.6124 (M + H), found 1077.6115.
Nr-n -Bu tyr yld id em n in A (20). To a solution of 2 (30 mg,
31 mmol) in dry CH2Cl2 was added n-butyric anhydride (10
mg, 0.63 mmol) at 0 °C followed by a catalytic amount (2 mg)
of DMAP. The mixture was left at 0 °C for 48 h. EtOAc and
aqueous NaHCO3 were added, and the organic layer was dried
(Na2SO4), concentrated, and separated to give 20 (27 mg,
86%): colorless solid, HRFABMS calcd for C53H84N6O13 Mr
1013.6185 (M + H), found 1013.6175.
Z-[(3S,4R,5R)-Ist2]d id em n in A (5): yield 19%; colorless
solid; [R]25D -102° (c 0.11, CHCl3); HRFABMS found 1077.6124.
Z-[(3S,4S,5S)-Ist2]d id em n in A (6): yield 14%; colorless
solid; [R]25D -28° (c 0.11, CHCl3); HRFABMS found 1077.6124.
O-Acetyld id em n in A (7). To a solution of 2 (55.2 mg,
0.059 mmol) in benzene (0.8 mL) was added benzyl chlorofor-
mate (50 mL, 6 equiv) and Et3N (10 µL). The mixture was
stirred at room temperature for 24 h, and then the solvents
were removed by N2. The resulting solid was separated (silica
gel, EtOAc) to give N-Z-didemnin A. The product was treated
with pyridine (0.5 mL) and acetic anhydride (0.5 mL) for 12 h
at room temperature to give N-Z-O-acetyldidemnin A (63 mg,
92%): 1H NMR (CDCl3, 300 MHz);1 HRFABMS calcd for
C59H87N6O15 Mr 1119.6229 (M + H), found 1119.6217.
A mixture of N-Z-O-acetyldidemnin A (40 mg 0.035 mol) and
Pt/C (10%, 40 mg) in 2-propanol (1 mL) and acetic acid (10
mL) was stirred in a H2 atmosphere for 2.5 h at room
temperature. The product was filtered through a short (4 g)
Nr-Acyl[p en ta n oyl (21), h exa n oyl (22), octa n oyl (23),
d od eca n oyl (24), h exa d eca n oyl (25)]d id em n in s. Gen er a l
Meth od . EDC (0.31 mmol) was added at 10 °C to a stirred
solution of the free acid (0.63 mmol) in CH2Cl2 (2 mL). The
mixture was allowed to react for 1.5 h at 10 °C, 2 (0.31 mmol)
was added to the solution, and the reaction mixture was stirred
for 2 h at 10 °C and then left at -20 °C for 20 h. The mixed
anhydride (prepared as above, 0.31 mmol) was added, and the
reaction mixture was allowed to stand at 0 °C for 24 h. Solvent
was evaporated, and the product after usual workup was

2832 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14
Sakai et al.
purified (SiO2, CHCl3-MeOH 3-5%) to give the correponding
NR-acyldidemnins.
MHz);1 FABMS m/z 1097 (M + H), 281; HRFABMS calcd for
C57H90N7O14 Mr 1096.6556 (M + H), found 1096.6572.
[Isobu tyr yl9]d id em n in B (33) a n d [Isobu tyr yl9,D-P r o8]-
d id em n in B (34). L-Pro (1.15 g, 0.01 mol) was treated with
isobutyric anhydride in a procedure like the N-propionylproline
synthesis to give N-isobutyrylproline (1.8 g, 96%): fine crys-
tals; mp 80-82 °C; [R]25D -8.7° (c 1.56, CHCl3); 1H NMR.1 Anal.
(C9H15NO3) C, H, N.
Nr-P en ta n oyld id em n in A (21): 90%; HRFABMS calcd for
C54H87N6O13 Mr 1027.6331 (M + H), found 1027.6306.
Nr-Hexa n oyld id em n in A (22): 90%; HRFABMS calcd for
C55H89N6O13 Mr 1041.6488 (M + H), found 1041.6477.
Nr-Octa n oyld id em n in A (23): 90%; HRFABMS calcd for
C57H93N6O13 Mr 1069.6791 (M + H), found 1069.6785.
Nr-Dod eca n oyld id em n in A (24): 89%; HRFABMS calcd
for C61H101N6O13 Mr 1125.7427 (M + H), found 1125.7401.
Nr-Octa d eca n oyld id em n in A (25): 89%; HRFABMS calcd
for C67H113N6O13 (M + H), found 1181.8040.
N-Isobutyrylproline was coupled with 2 (26.4 mg, 0.028
mmol) using the method described earlier for the preparation
of 31. The product was separated (silica gel, EtOAc) to give
33 as the first fraction (8.7 mg, 28%), a white powder: 1H NMR
(CDCl3, 500 MHz) δ 8.11 (1H, d, J ) 5.5 Hz), 7.89 (1H, d, J )
9.0 Hz), 7.21 (1H, d, J ) 10.0 Hz), 7.06 (2H, d, J ) 8.5 Hz),
6.84 (2H, d, J ) 8.5 Hz), 3.79 (3H, s), 3.12 (3H, s), 2.54 (3H,
s); FABMS m/z 1112 (M + H), 295; HRFABMS calcd for
C58H92N7O14 Mr 1110.6302 (M + H), found 1110.6737.
The second fraction gave 34 (13 mg, 42%): colorless
N-(D-P r o)d id em n in A (29). DCC (24.4 mg, 0.12 mmol)
was added to a solution of Z-D-proline (Pro) (59 mg, 0.24 mmol)
in CH2Cl2 (0.5 mL) at 5 °C. The mixture was stirred at 5 °C
for 2 h. Compound 2 (75.4 mg, 0.08 mmol) in CH2Cl2 (0.5 mL)
was added, and the mixture was allowed to stand for 8 h at
10 °C and then was concentrated. The residue was suspended
in cold EtOAc, filtered, and concentrated in vacuo to an oil
which was separated (silica gel, EtOAc-CH2Cl2, 7:3) to give
N-(Z-D-Pro)didemnin A as a white powder (83.9 mg, 0.071
mmol, 89%): 1H NMR (CDCl3, 300 MHz) δ 7.9* (1H, m), 7.2-
7.4 (5H, m), 7.05 (2H, d, J ) 8.4 Hz), 6.82 (2H, d, J ) 8.4 Hz),
3.76 (3H, s), 2.88*, 2.89*, 2.77* (s), 2.55*, 2.52* (s) (*peaks
were observed as pairs); HRFABMS calcd for C62H92N7O15 Mr
1174.6651 (M + H), found 1174.6663.
A mixture of N-(Z-D-prolyl)didemnin A (80 mg, 0.077 mmol)
and Pd/C (10%, 36 mg) in MeOH (2 mL) was stirred vigorously
in a hydrogen atmosphere for 2 h at room temperature, filtered
through a C-18 Sep-pak column with MeOH, and concentrated
to a white powder (65.6 mg). A portion (17.0 mg) of the powder
was separated by HPLC to give pure 29 (11.9 mg, 57%): white
powder; 1H NMR (CDCl3, 300 MHz) δ 7.97-7.3 (br NH’s) 7.07
(2H, d, J ) 8.4 Hz), 6.83 (2H, d, J ) 8.4 Hz), 3.79 (3H, s), 2.92
(3H, brs), 2.53 (3H, s); HRFABMS calcd for C54H86N7O13 Mr
1140.6284 (M + H), found 1140.6285.
1
needles: mp 162-164 °C; H NMR (CDCl3, 500 MHz) δ 9.15
(1H, d, J ) 6.0 Hz), 7.92*, 7.87* (1H, d, J ) 9.0 Hz), 7.26*,
7.11* (1H, d, J ) 10.0 Hz), 7.06 (1H, d, J ) 9.0 Hz), 6.94 (1/4H,
d, J ) 9.0 Hz), 6.84 (2H, d, J ) 8.5 Hz), 3.78 (3H, s), 2.86 (3H,
s), 2.54, 2.53 (3H, s) (*peaks observed as pairs); FABMS m/z
1110 (M + H), 295; HRFABMS calcd for C58H92N7O14 Mr
1110.6302 (M + H), found 1110.6726.
O-Ben zyl-L-la ctyl-L-Ala Meth yl Ester . A mixture of
O-benzyl-L-lactic acid23 (57.7 mg, 0.35 mmol), L-alanine methyl
ester hydrochloride (50.0 mg, 0.36 mmol), N-hydroxysuccin-
imide (82 mg, 0.70 mmol), and NMM (35 mg, 0.35 mmol) in
CH2Cl2-DMF (6:4, 2 mL) was stirred at -10 °C. A solution
of DCC (100 mg, 0.49 mmol) and DMAP (2 mg) in CH2Cl2-
DMF (6:4, 2 mL) was added to the mixture, which was allowed
to warm from -10 °C to 4 °C over 2 h and then stood at 4 °C
for 30 h. The reaction mixture was concentrated in vacuo, and
the resulting product was suspended in cold EtOAc, filtered,
and separated (silica gel, EtOAc) to give O-benzyl-L-lactyl-L-
Ala methyl ester as a light yellow oil (81.7 mg, 94%, HPLC
data): [R]20D -20° (c 1.2, CHCl3); 1H NMR (CDCl3, 300 MHz);1
13C NMR (CDCl3,75 MHz);1 HRFABMS calcd for C14H19NO4
Mr 266.1392 (M + H), found 266.1398.
O-Ben zyl-L-la ctyl-L-Ala . Aqueous KOH (1 N, 300 µL) was
added to a solution of O-benzyl-L-lactyl-L-Ala methyl ester (62
mg, 0.25 mmol) in dioxane. The mixture was stirred for 12 h
at room temperature, HCl (1 N, 300 µL) was added, and the
solvent was removed in vacuo. The residue was suspended
in CH2Cl2, filtered, and concentrated in vacuo to give O-benzyl-
L-lactyl-L-Ala as a light yellow oil (56.9 mg, 98%): [R]26D -18.8°
(c 2.11, CHCl3); 1H NMR (CDCl3, 300 MHz);1 HRFABMS calcd
for C13H17NO4 Mr 252.1236 (M + H), found 252.1238.
N-(L-P r o)d id em n in A (28).3c Z-L-Pro was coupled to 2 and
deprotected as above to give a white powder (27% overall): 1H
NMR (CDCl3, 300 MHz)1 δ 8.03 (1H, d, J ) 8.7 Hz), 7.73 (1H,
br d, J ) 9.6 Hz), 7.33 (1H, br s), 7.06 (2H, d, J ) 8.4 Hz),
6.84 (2H, d, J ) 8.4 Hz), 3.88 (3H, s), 3.02 (3H, s), 2.53 (3H,
s); HRFABMS found 1040.6277.
N-(L-Leu )d id em n in A (27). A sample of 27 prepared
previously3c was repurified by RPHPLC (MeOH-NaCl (0.4 M),
7:1).
[Acetyl9]d id em n in B (31). To a suspension of L-Pro (247
mg, 2.15 mmol) in pyridine (1 mL) was added acetic anhydride
(1 mL), and the mixture was stirred at room temperature for
5 min. The solvent was removed in vacuo, and the resulting
oil was partitioned between EtOAc and HCl (1 N). The organic
layer was dried over Na2SO4 and concentrated, and the
resulting solid was recrystallized from EtOAc to give N-Ac-L-
Pro (167 mg, 44%), a white powder: mp 108 °C [lit. 115 °C
O-Ben zyl[L-Ala 8]d id em n in B. A solution of O-benzyl-L-
lactyl-L-Ala (22.6 mg, 0.095 mmol) and N-hydroxysuccinimide
(13.1 mg, 1.2 mmol) in CH2Cl2 (1 mL) was added to a solution
of DCC (21.5 mg, 0.10 mmol) in CH2Cl2 (0.5 mL) at -10 °C.
The mixture, which became a heterogeneous emulsion, was
stirred for 1 h at -10 °C. Compound 2 (81.4 mg, 0.086 mmol)
and NMM (9.0 mg, 0.088 mmol) were added, and the reaction
mixture stood at -10 °C for 3 h and then at 4 °C for 24 h. A
catalytic amount of DMAP (1 mg) was added to the mixture,
and the reaction stood for 16 h at 4 °C and then was
concentrated. The residue was suspended in EtOAc, filtered,
and concentrated in vacuo to give an oil, which was chromato-
graphed on a silica gel column, eluting with CHCl3-MeOH
(15:1), to give O-benzyl[L-Ala8]didemnin B (62 mg, 62%) as a
white powder: 1H NMR (300 MHz, CDCl3);1 FABMS m/z 1176
(M + H), 361; HRFABMS calcd for C62H94N7O15 Mr 1176.6808
(M + H), found 1176.6814.
(from CHCl3)43]; [R]25 -171° (c 0.91, CHCl3). Anal. (C7H11
-
D
NO3) C, H, N.
N-Ac-L-Pro (31 mg, 0.197 mmol) was treated with DCC (20.3
mg, 0.099 mmol) in CH2Cl2 (0.1 mL) for 4 h at 10 °C.
A
solution of 2 (62 mg, 0.068 mmol) in CH2Cl2-DMF (6:4, 1 mL)
was added to the mixture at 5 °C. The mixture was allowed
to stand at 5 °C for 12 h, filtered, and then concentrated in
vacuo. The resulting solid was separated (silica gel, EtOAc-
2-propanol, 10:1) to give 31 (63 mg, 0.058 mmol, 88%) as a
white powder: 1H NMR (CDCl3, 500 MHz);1 HRFABMS calcd
for C56H88N7O14 Mr 1082.6389 (M + H), found 1082.6396.
[P r op ion yl9]d id em n in B (32). Propionic anhydride (2.60
g, 0.02 mol) was added to a suspension of L-Pro (1.15 g, 0.01
mol) in pyridine (2 mL). The mixture was stirred for 30 min
at room temperature, solvent was removed in vacuo, and the
product was recrystallized from EtOAc to give N-propionyl-L-
Pro (1.60 g, 96%): colorless needles; mp 98-99 °C; [R]25D -186°
(c 1.7, CHCl3); 1H NMR (360 MHz).1 Anal. (C8H13NO3) C, H,
N.
[L-Ala 8]Did em n in B (35). A mixture of O-benzyl[L-Ala8]-
didemnin B (53.2 mg, 0.045 mmol) and Pd/C (10%, 50 mg) in
MeOH (2 mL, containing 100 µL of acetic acid) was vigorously
stirred in an H2 atmosphere for 3 h at room temperature. To
the mixture was added 10 mg of NaHCO3, and the reaction
mixture was filtered through a C-18 Sep-pak column with
MeOH and concentrated to give 35 (TLC, one spot; 47.6 mg,
97%). A portion of 35 was further purified for bioassay by
HPLC (C-18, MeOH-H2O, 7:1): IR (film) 3330, 1734, 1637,
1248 cm-1; 1H NMR (CDCl3, 500 MHz);1 13C NMR (CDCl3, 300
N-Propionyl-L-Pro was coupled with 2 (26.4 mg, 0.028 mmol)
as in the synthesis of 31 to give [propionyl9]didemnin B (32)
(28.7 mg, 93%) as a white powder: 1H NMR (CDCl3, 500

Structure-Activity Relationships of the Didemnins
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 14 2833
MHz);1 FABMS m/z 1087 (M + H), 271; HRFABMS calcd for
Refer en ces
C
55H88N7O15 Mr 1086.6338 (M + H), found 1086.6359.
(1) Data in this paper are taken in part from the following: Sakai,
R. Biologically Active Compounds from Tunicates and a Sponge.
Ph.D. Thesis, University of Illinois, Urbana, 1991.
(2) Preparation and some bioactivity of these and some other
congeners were listed in the following: Rinehart, K. L., J r.
Pharmaceutical Compositions Containing Didemnins. U.S. Patent
5,294,603, March 15, 1994; Chem. Abstr. 1994, 121, 887. Either
the threonine or the isostatine unit was assigned as unit1 in our
previous publications. In this paper, we use [Thr1].
O-Ben zyl-L-la ctyl-D-P r o Meth yl Ester . A mixture of
O-benzyl-L-lactic acid (194.4 mg, 1.08 mmol) and DCC (111.2
mg, 0.54 mmol) in CH2Cl2 (1 mL) was stirred at 0 °C for 30
min. D-Pro-OMe‚HCl (53.1 mg, 0.32 mmol) and NMM (33.0
mg, 0.33 mmol) in DMF (∼1 mL) were added, and the mixture
stood at 4 °C for 9 h. The product was filtered, concentrated
in vacuo, suspended in cold EtOAc, filtered, and concentrated
in vacuo to an oil. The crude product was separated (silica
gel, EtOAc) to give O-benzyl-L-lactyl-D-Pro methyl ester (47.6
(3) (a) Rinehart, K. L., J r.; Gloer, J . B.; Cook, J . C., J r.; Mizsak, S.
A.; Scahill, T. A. Structures of the Didemnins, Antiviral and
Cytotoxic Depsipeptides from a Caribbean Tunicate. J . Am.
Chem. Soc. 1981, 103, 1857-1859. (b) Rinehart, K. L., J r.; Gloer,
J . B.; Hughes, R. G., J r.; Renis, H. E.; McGovren, J . P.;
Swynenberg, E. B.; Stringfellow, D. A.; Kuentzel, S. L.; Li, L.
H. Didemnins: Antiviral and Antitumor Depsipeptides from a
Caribbean Tunicate. Science 1981, 212, 933-935. (c) Rinehart,
K. L., J r. Didemnins A, B, C, and Derivatives Thereof as
Antiviral Agents. U.S. Patent 4,493,796, J an 15, 1985; Chem.
Abstr. 1985, 103, 76241v. (d) Rinehart, K. L., J r. Composition
of Matter and Process. U.S. Patent 4,548, 814, Oct 22, 1985.
(4) (a) Gloer, J . B. Structures of the Didemnins. Ph.D. Thesis,
University of Illinois, Urbana, 1983; Chem. Abstr. 1984, 101,
122692b; Diss. Abstr. Int. B 1984, 45, 188-189. (b) Gutowsky,
R. E. Isolation and Identification of Didemnins. M.S. Thesis,
University of Illinois, Urbana, 1984.
(5) Rinehart, K. L., J r.; Cook, J . C., J r.; Pandey, R. C.; Gaudioso, L.
A.; Meng, H.; Moore, M. L.; Gloer, J . B.; Wilson, G. R.; Gutowsky,
R. E.; Zierath, P. D.; Shield, L. S.; Li, L. H.; Renis, H. E.;
McGovren, J . P.; Canonico, P. G. Biologically Active Peptides
and Their Mass Spectra. Pure Appl. Chem. 1982, 54, 2409-2424.
(6) Rinehart, K. L. Didemnin and Its Biological Properties. In
Peptides, Chemistry and Biology; Proc. 10th Am. Peptide Sym-
posium; Marshall, G. R., Ed.; ESCOM: Leiden, 1988; pp 626-
631 and references therein.
mg, 44%) as a colorless oil: [R]26 +1.42° (c 1.83, CHCl3); IR
D
(film) 1736, 1639, 1450, 1200, 1170 cm-1; 1H NMR (CDCl3, 500
MHz);1 13C NMR (CDCl3, 300 MHz);1 HRFABMS calcd for
C
16H22NO4 Mr 292.1549 (M + H), found 292.1550.
O-Ben zyl-L-la ctyl-D-P r o. A solution of O-benzyl-L-lactyl-
D-Pro methyl ester (124.7 mg) in dioxane (1 mL) and aqueous
KOH (1 N, 0.5 mL) stood at room temperature for 20 h. The
mixture was concentrated to give an aqueous emulsion, which
was acidified to pH 2 (HCl), extracted with CH2Cl2, dried
(Na2SO3), and evaporated to give an oil (117.9 mg, 100%): IR
(film) 3400-2500 br, 1736, 1640 cm-1; HRFABMS calcd for
C
15H20NO4 Mr 278.1392 (M + H), found 278.1394.
O-Ben zyl[D-P r o8]d id em n in B. A mixture of O-benzyl-L-
lactyl-D-Pro (52.4 mg, 0.19 mmol) and DCC (19.6 mg, 0.095
mmol) in CH2Cl2 (1 mL) was stirred at 0 °C for 2 h. A solution
of 2 (59.7 mg, 0.063 mmol) in CH2Cl2 (1 mL) was added, and
the reaction mixture was allowed to stand at 0 °C for 12 h
and concentrated in vacuo. The residue was suspended in
EtOAc and filtered, and the product was separated (silica gel,
EtOAc) to give O-benzyl[D-Pro8]didemnin B (61.9 mg, 83%
based on unreacted 2) as a white solid: 1H NMR (CDCl3, 300
MHz) δ 7.94* (1H, d × 2, 6.9, 6.3), 7.22-7.42 (5H, m), 7.13
(1H, d, J ) 9.9 Hz), 7.07 (2H, d, J ) 8.7 Hz), 6.95 (1H, d, J )
9.0 Hz), 3.79 (3H, s), 2.95 and 2.88* (3H, s), 2.56 and 2.55*
(3H, s) (*appearing as pairs of signals due to conformers);
HRFABMS calcd for C64H96N7O15 Mr 1202.6664 (M + H), found
1202.6671.
[D-P r o8]Did em n in B (36). A mixture of O-benzyl[D-Pro8]-
didemnin B (43.1 mg, 0.036 mmol) and Pd/C (10%, 40 mg) in
MeOH (2 mL) and acetic acid (20 mL) was stirred under
hydrogen for 2.5 h at room temperature. To the mixture was
added 10 mg of NaHCO3, and the product was filtered and
concentrated in vacuo to a glass which was purified by HPLC
(C-18, MeOH-H2O, 7:1) to give pure 36 (39 mg, 97%) as a
white powder: IR (film) 3420, 3330, 1732, 1635, 1539, 1248,
1176 cm-1; 1H NMR (CDCl3, 500 MHz, mixture of conformers,
Figure 1) δ 8.62:8.57 (3:1, 1/3H, d’s, J ) 6.5 Hz), 7.93, 7.92,
7.86, 7.83 (5:1:4:2, 1H, d’s, J ) 9.5 Hz), 7.29:7.14 (1:1, 1H, d’s,
J ) 10.0 Hz), 7.07 (2H, d, J ) 8.5 Hz), 6.97:6.85 (1:4, 2/1H, J
) 9.5 Hz), 6.84 (2H, d, J ) 8.5 Hz), 5.19:5.16 (2:3, 1H, d’s, J )
3.5 Hz, Hip H-4), 3.79 (3H, s, Me2Tyr-OCH3), 2.94:2.93:2.89:
2.88 (5:2:4:1, 3H, singlets), 2.56:2.54 (2:3, 3H, singlets); HR-
FABMS calcd for C57H90N7O15 Mr 1112.6491 (M + H), found
1112.6493.
(7) Rinehart, K. L.; Kishore V.; Bible, K. C.; Sakai, R.; Sullins, D.
W.; Li, K.-M. Didemnins and Tunichlorin: Novel Natural
Products From the Marine Tunicate Trididemnum solidum. J .
Nat. Prod. 1988, 51, 1-21 and references therein.
(8) Fimiani, V. In vivo Effect of Didemnin B on Two Tumors of the
Rat. Oncology 1987, 44, 42-46.
(9) (a) National Cancer Institute Clinical Brochure, Didemnin B.
NSC 325319. IND. Division of Cancer Treatment, NCI, Be-
thesda, MD, J une 1984. (b) Chun, H. G.; Davies, B.; Hoth, D.;
Suffness, M.; Plowman, J .; Flora, K.; Grieshaber, C.; Leyland-
J ones, B. Didemnin B: The First Marine Compound Entering
Clinical Trials as an Antineoplastic Agent. Invest. New Drugs
1986, 4, 279-284. (c) Dorr, F. A.; Kuhn, J . G.; Phillips, J .; Von
Hoff, D. D. Phase I Clinical and Pharmacokinetic Investigation
of Didemnin B, a Cyclic Depsipeptide. Eur. J . Cancer Clin. Oncol.
1988, 24, 1699-1706. (d) J ones, D. V., J r.; Ajani, J . A.; Black-
burn, R.; Daugherty, K.; Levin, B.; Patt, Y. Z.; Abbruzzese, J . L.
Phase II Study of Didemnin B in Advanced Colorectal Cancer.
Invest. New Drugs 1992, 10, 211-213. (e) Queisser, W. New
Anti-cancer Agents in Phase I/II. Onkologie 1992, 15, 454-462.
(f) Malfetano, J . H.; Blessing, J . A.; J acobs, A. J . A Phase II Trial
of Didemnin B (NSC #325319) in Patients with Previously
Treated Epithelial Ovarian Cancer. A Gynecologic Oncology
Group Study. Am. J . Clin. Oncol. 1993, 16, 47-49.
(10) Annual Report to the Food and Drug Administration. Didemnin
B. NSC 325319. IND 24505. Division of Cancer Treatment, NCI,
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didemnin A4b) and didemnin M for the present compound,1,20
we shall retain our previous nomenclature.
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