U.S. patent application number 10/357759 was filed with the patent office on 2003-08-07 for derivatives of dehydrodidemnin b.
This patent application is currently assigned to Pharma Mar S.A.. Invention is credited to Lithgow-Bertelloni, Anna M., Rinehart, Kenneth L..
Application Number | 20030148933 10/357759 |
Document ID | / |
Family ID | 27671034 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148933 |
Kind Code |
A1 |
Rinehart, Kenneth L. ; et
al. |
August 7, 2003 |
Derivatives of dehydrodidemnin B
Abstract
Dehydrodidemnin with useful biological activity has formula (1).
It can be isolated from natural sources or synthesized, and it
forms active derivatives. 1
Inventors: |
Rinehart, Kenneth L.;
(Urbana, IL) ; Lithgow-Bertelloni, Anna M.;
(Salamanca, ES) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
28 STATE STREET
28th FLOOR
BOSTON
MA
02109-9601
US
|
Assignee: |
Pharma Mar S.A.
Madrid
ES
|
Family ID: |
27671034 |
Appl. No.: |
10/357759 |
Filed: |
February 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10357759 |
Feb 4, 2003 |
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09619354 |
Jul 19, 2000 |
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09619354 |
Jul 19, 2000 |
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09182688 |
Oct 30, 1998 |
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09182688 |
Oct 30, 1998 |
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08280110 |
Jul 25, 1994 |
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5834586 |
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08280110 |
Jul 25, 1994 |
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07844567 |
Apr 24, 1992 |
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Current U.S.
Class: |
435/6.16 ;
514/21.1; 514/3.4; 530/317 |
Current CPC
Class: |
C07K 5/0205 20130101;
A61K 38/12 20130101; C07K 11/02 20130101 |
Class at
Publication: |
514/9 ;
530/317 |
International
Class: |
A61K 038/12; C07K
007/50 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 1995 |
WO |
PCT/GB90/01495 |
Claims
What is claimed is:
1. The substantially pure compound, Dehydrodidemnin B, essentially
free of the cellular debris of the Mediterranean tunicate, Aplidium
albicans, said compound having the formula: 3
2. An acylated, alkylated or arylated derivative of dehydrodidemnin
B wherein the acyl, alkyl, or aryl groups are selected from C.sub.1
to C.sub.6 alkyl, C.sub.1 to C.sub.6 acyl, phenyl, benzyl, or
C.sub.1 to C.sub.6 alkylphenyl.
3. A pharmaceutical composition which comprises dehydrodidemnin B
or an active acylated, alkylated or arylated derivative thereof,
together with a pharmaceutically acceptable carrier, wherein the
acyl, alkyl, or aryl groups are selected from either C.sub.1 to
C.sub.6 alkyl, C.sub.1 to C.sub.6 acyl, phenyl, benzyl, or C.sub.1
to C.sub.6 alkylphenyl.
4. A semisynthetic process for the preparation of dehydrodidemnin
B, which comprises the coupling of pyruvyl-proline to didemnin
A.
5. A method for isolation of dehydrodidemnin B which comprises
extraction from a dehydrodidemnin B-containing tunicate.
6. The monoacetyl derivative of dehydrodidemnin B.
Description
FIELD OF THE INVENTION
[0001] This invention relates to dehydrodidemnin B and, in
particular, to the isolation of dehydrodidemnin B, a cyclic
depsipeptide, from a tunicate of the Ascidiacea class. This novel
compound has been shown to have antiviral, antitumoral and
cytotoxic activities.
BACKGROUND OF THE INVENTION
[0002] The didemnins form a class of cyclic depsipeptides which
have been isolated from various species of the Trididemnum genus.
They have been shown to have potent activity against viruses and
tumor cells (Rinehart, Jr., et al., J. Am. Chem. Soc., 103, 1857-59
(1981). Didemnin B, up to now the most active compound of this
class, has been shown to have potent immunosuppressive activity
(Montgomery et al., Transplantation, 40, 49-56 (1985) and a more
potent inhibition of binding of prolactin to human lymphocytes than
other didemnin compounds (Montgomery et al., Fed. Prac., 44, 634
(1987).
SUMMARY OF THE INVENTION
[0003] This invention provides a novel and more active compound of
this class, unexpectedly isolated from the Mediterranean tunicate
Alpidium albicans, namely dehydrodidemnin B (or "DDB"), having the
formula: 2
[0004] where R is hydrogen; and derivatives thereof with the same
class of biological activity, i.e., where R is Acyl, Alkyl or
Aryl.
BRIEF DESCRIPTION OF THE FIGURES
[0005] FIGS. 1A and 1B each illustrate a scheme by which DDB can be
isolated from tunicates of the Ascidiacea class, subphylum
Urochordata;
[0006] FIGS. 2-5 are mass spectra of compounds disclosed
herein;
[0007] FIGS. 6 and 7-9 are .sup.1H NMR spectra of compounds
disclosed herein; and
[0008] FIGS. 10 and 11-14 are .sup.13C NMR spectrum of compounds
disclosed herein;
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0009] The compound of the present invention, DDB, is characterized
by the following properties, considering also that, in solution,
two conformers (at least) are possible:
[0010] TLC R.sub.f=0.4; 0.35 (Silica gel, 2:3,
CH.sub.2Cl.sub.2/EtOAc); 0.5; 0.44 (Silica gel; 9:1,
CHCl.sub.3/MeOH);
[0011] RP-HPLC t.sub.R=10.7; 11.9 min (Spherisorb C.sub.18 column,
250 mm.times.10 mm, 10 .mu.m particle size, 9.1, MeOH/H.sub.2); 2
mL/min);
[0012] [.alpha.].sub.D.sup.25.degree.=-86.degree. (c 1, MeOH);
[0013] HR FABMS (M+M) C.sub.57H.sub.88N.sub.7O.sub.15 m/z calcd.
1110.6366; (M-side chain+H): C.sub.42H.sub.66N.sub.5O.sub.11 m/z
calcd. 816.4781 (found 816.4755); (M-chain):
C.sub.15H.sub.23N.sub.2O.sub.4 m/z calcd. 295.1657 (found
295.1657);
[0014] IR (CHCl.sub.3) vmax cm.sup.-1 3680, 3600, 2970, 2940, 2880,
1740, 1650, 1605, 1540, 1510;
[0015] .sup.1H NMR (CDCl.sub.3, .delta., ppm): 7.82 (d, J=9 Hz,
1H); 7.79 (d, J=9 Hz, 1H); 7.62 (d, J=6 Hz, 1H); 7.21 (d, J=9 Hz,
1H); 7.19 (d, J-9 Hz, 1H); 7.08 (d, J=8.5 Hz, 2H); 6.85 (d, J=8.5
Hz, 2H); 3.77 (s, 3H); 3.13 (s, 3H); 3.08 (s, 3H); 2.54 (s, 3H);
2.50 (s, 3H); 2.1 (s, 3H); 2.02 (s, 3H); 0.82-0.88 (overlapped d
and t, 3OH); and
[0016] .sup.13C NMR (CDCl.sub.3, .delta., ppm): 204.93 (s); 204.77
(s); 201.23 (s); 197.55 (s); 173.05 (s); 173.05 (s); 172.36 (s);
171.84 (s); 171.21 (s); 171.16 (s); 170.59 (s); 169.58 (s); 169.51
(s); 169.35 (s); 168.36 (s); 168.28 (s); 161.31 (s); 161.06 (s);
158.64 (s); 158.62 (s), 130.31 (d); 114.12 (d); 114.01 (d); 81.47
(d), 81.43 (d); 70.68 (d); 70.33 (d); 67.97 (d); 67.76 (d); 66.38
(d); 66.22 (d); 60.39 (t); 50.88 (d); 57.80 (d); 66.38 (d); 66.22
(d); 60.39 (t); 50.88 (d); 57.80 (d); 57.45 (d); 57.26 (d); 57.18
(d); 57.12 (d); 55.61 (d); 55.57 (d); 55.26 (q); 54.65 (d); 49.55
(d); 49.49 (d); 48.85 (t); 48.41 (t); 46.98 (t); 41.29 (t); 41.24
(t); 38.78 (q); 38.74 (q); 38.68 (q); 36.42 (t); 36.22 (t); 34.06
(d); 33.99 (d); 33.96 (t); 31.57 (d); 31.38 (q); 31.34 (q); 31.30
(q); 30.69 (d); 29.68 (t); 29.64 (d); 27.28 (t); 27.94 (t); 27.30
(t); 27.17 (t); 27.08 (t); 25.91 (t); 25.87 (t); 25.73 (d); 25.68
(d); 25.63 (d); 25.52 (d); 25.48 (d); 24.80 (q); 24.70 (q); 24.44
(q); 24.31 (q); 22.21 (q); 22.12 (q); 21.92 (q); 21.79 (q); 21.76
(q); 19.46 (q); 17.76 (q); 17.72 (q); 17.18 (q); 16.87 (q); 16.08
(q); 15.62 (q); 15.48 (q); 15.05 (q); 12.55 (q); 12.50 (q).
[0017] The structure determination of DDB was accomplished by
comparison of mass spectrometry low and high resolution FABMS
(Rinehart, Jr. et al., Pure and Appl. Chem., 54, 2409-2424 (1982))
and NMR data with other didemnin data, and confirmed by synthesis
of DDB involving coupling of natural didemnin A with the
appropriate side chain. The low resolution FAB mass spectra showed
peaks at m/z 1110 (M+H), 816 (M+2H-side chain) and 295 (side
chain). The lack of two mass units in the molecular ion and side
chain peaks, in addition to the same m/z ratio for the ring,
suggested that the difference between dehydrodidemnin B and
didemnin B was represented by one more degree of unsaturation in
the side chain. The molecular formula deduced from high resolution
FABMS was C.sub.57H.sub.88N.sub.7O.sub.15 (M+H, .DELTA.2.8 mmu);
and for the fragment ions corresponding to the ring and the side
chain; C.sub.42H.sub.66N.sub.5O.sub.11 (.DELTA.0.4 mmu) and
C.sub.15H.sub.23N.sub.2O.sub.4 (.DELTA.2.6 mmu), respectively.
Tandem mass spectrometry on these peaks showed the typical cleavage
pattern of didemnins.
[0018] From the NMR data, the presence of peptide linkages was
indicated by peaks near .delta.8 ppm and the methyl signals
corresponding to the amino acid residues. Even though some of these
peaks are doubled or tripled due to the presence of, at least two
main conformers in solution at room temperature, these peaks are
very similar to those of didemnins. The main difference observed
between DDB and didemnin B is the methyl singlet peak at 2.04 ppm
which could be assigned to a methyl ketone and the absence of the
signal corresponding to the .alpha.-proton of the hydroxyl group in
the lactyl moiety at 4.3 ppm.
[0019] Biological Activities
[0020] The compound of this invention has been shown to inhibit in
vitro L1210 and P-388 mouse leukemia cells; L-929, mouse areolar
and adipose tissue, B-16, mouse melanoma cells; A-549, human lung
carcinoma cells; HeLa, human cervix epithelioid carcinoma cells and
KB, human cervix epithelioid carcinoma cells and KB, human oral
epidermoid carcinoma cells, and in vivo, P-338, mouse leukemia
cells, Lewis lung carcinoma cells and B-16 melanoma cells. Thus,
DDB is useful as an antitumor agent and therefore is useful
inhibiting the growth of tumor cells in mammals exhibiting such
tumor cells.
[0021] The following table summarizes the IC.sub.50 values for each
line cells in vitro:
1 TABLE 1 Cell Line IC.sub.50 (ng/mL) L-1210 0.3 P-388 0.175 L-929
1.9 B-16 0.225 A-549 0.5 HeLa 0.5 KB 5.6
[0022] The following table shows the % T/C in vivo after
administration of DDB:
2TABLE 2 Dehydrodidemnin B in vivo Activity Compound Control DDB
DDB Dose (.mu.g/kg/injn) -- 160 80 Schedule and Route QD 1-9, IF QD
1-9, IP QD 1-9, IP P-388, Median 10.0 21.0 19.5 Survival Time, Days
100 210 195 P-388 % T/C.sup.a Lewis Lung, Mean 1512 0 189 Tumor
Volume mm.sup.3 1.00 0.00 0.13 Lewis Lung % T/C.sup.b 3-16 Melanoma
17.0 >27.0 >27.0 Median Survival Time Days B-16 Melanoma 100
>158 >158 % T/C.sup.a .sup.a.Significant activity T/C
.gtoreq. 125. .sup.b.Significant activity T/C .gtoreq. 0.40.
[0023] Dehydrodidemnin B, like didemnin B, (Montgomery et al.,
Transplantation, 40, 49-56 (1985)), is a powerful
immunomodulator.
[0024] Dehydrodidemnin B has also shown activity against Herpes
simplex virus, type 1, in CV-1 cells (monkey kidney cells); thus it
is also useful as an antiviral agent. The IC.sub.50 determined was
60 ng/mL (e.g., 10 fold greater than for L-1210 cells) and 1
.mu.g/mL, respectively.
[0025] The compound of present invention is preferably presented
for administration to humans and animals in unit dosage form in
pharmaceutically appropriate carriers containing the active
ingredient in the appropriate quantity.
[0026] Illustratively, dosage levels of the administered active
ingredient can be intravenous 0.05 to about 50 mg/Kg,
intraperitoneal, subcutaneous and intramuscular 1 to 100 mg/Kg;
oral 1 to 150 mg/Kg of animal (body) weight.
[0027] The administration of DDB is useful to inhibit the growth of
cancer cells in animals or humans bearing a neoplastic disease, for
example, acute myelocytic leukemia, acute lymphocytic leukemia,
malignant melanoma, adenocarcinoma of the lung, small cell
carcinoma of the lung, and the like.
[0028] The compound can be isolated from tunicates of the genus
Aplidium, and more especially from the species Aplidium albicans.
The species is found in the Iberian Mediterranean Coast as well as
in the Balearic Islands. The species has been also found in Great
Britain, English Channel as well as in the Africa Coast and
Portugal. It seems to prefer detritic, coralligenic and sciafilae
algae communities. They also can be found in more photophilic
habitats.
[0029] Colonies of the tunicate are generally flat and lobed (2.5
cm diameter). It is jelly like, totally encrusted with sand which
confers a sandy color to the colony. Zooides are of a whitish color
10 mm long; the oral siphon has 6 lobes, and the cloacal languet is
trifid, which is a species characteristic. Generally there are
10-11 rows of stigmas. The stomach has 6 marked folds. Gonads are
of the family type with one or several ovocites below the digestive
track and numerous testicular follicles forming one or double row
in the post abdomen. Larvae are incubated in the number of 1 to 9
in the atrial cavity; they have 3 cupping-glasses and several
vesicular formations in the anterior part.
[0030] Thus in a typical procedure in accordance with the present
invention, isolation method generally comprises alcoholic
extraction of the homogenized tunicate and selective purification
of the desired DDB.
[0031] As shown in FIG. 1, the tunicate was extracted with MeOH,
filtered and dissolved in MeOH: Toluene 3:1 and partitioned with
10% NaNO.sub.3. The aqueous layer was successively extracted with
CH.sub.2Cl.sub.2, EtOAc and n-BuOH. The organic fractions were
combined after monitoring by normal-phase TLC developed
CHCl.sub.3:MeOH 9:1, affording a 2:1 (v/v) and the activity was
concentrated in the methanolic layer. The polar fraction is passed
through Silica gel Step-gradient Chromatography. The last fraction
is further purified by reverse phase HPLC at a flow rate of 2
ml/min. Two mean peaks were collected and readily interconverted to
a mixture of I and II, to establish an approximately 1:1 ratio.
[0032] The DDB can also be prepared by total synthesis, or
semisynthesis from natural Didemnin A, following in both cases
standard procedures of protection and activation in peptide
chemistry.
Pyruvic acid+L-Pro.fwdarw.Side Chain
Side Chain+Didemnin A.fwdarw.Dihydrodidemnin B
[0033] Thus for example, Pro-OBzl, in DMF is mixed with pyruvic
acid and HOBt, and DCC in CH.sub.2Cl.sub.2 added. The reaction
product can be purified and shows the chemical and physical
properties corresponding to Pyruvyl-Pro-OBzl.
[0034] To a solution of this last product in CH.sub.2Cl.sub.2, EDC
and then Didemnin A was added. The evaporated residue is purified
yielding DDB having chemical, physical, spectroscopical and
biological characteristics in accord with natural Dehydrodidemnin
B.
[0035] Apart from DDB itself, the present invention extends to
derivatives of DDB, comprising acylated, alkylated or arylated
derivatives of DDB, where R could be a group COR' or R', where R'
represents the following substituents:
[0036] CH.sub.3, CH.sub.2R.sub.1, CH.sub.2R.sub.1,
CHR.sub.1R.sub.2R.sub.3 or C.sub.6H.sub.5-- where R.sub.1 R.sub.2,
R.sub.3, could be alkyl (either linear or branched), aryl or
alkylaryl, the aryl groups, bearing or not the substituents
described under R'. The residues R.sub.1, R.sub.2, R.sub.3 could be
either the same or different.
[0037] The derivatives can be more preferably alkyl, aryl or
acyl-derivatives, where R' is an aliphatic or aromatic group, more
preferably a 1-6 carbon atom residue.
[0038] In general, such derivatives from DDB of this kind, are
expected to show similar biological activity to that of DDB itself,
including specifically antitumoral, antiviral, cytotoxic and
immunosuppressive activity.
[0039] The acyl derivatives can be obtained by treatment of the
parent compound with the corresponding carboxylic anhydride in the
presence of pyridine or other nitrogenated organic base; by
reaction of DDB with the respective acylchloride; or by dehydration
with DCC from DDB and the corresponding carboxylic acid.
[0040] In the case of the alkyl or aryl derivatives (R/R'), they
can be obtained by reaction of DDB with the corresponding halide,
in the presence of an alkaline-organic weak base or by dehydration
between DDB and alkyl or aryl hydroxy derivative by an organic
dehydrating agent.
[0041] Instrumentation, Material and Methods
[0042] NMR spectra were obtained with a General Electric QE-300
(300 MHz, .sup.1H), a Nicolet NT-360 (360 MHz, .sup.1H) or a
General Electric GN 500 (500 MHz, .sup.1H) at the University of
Illinois or a Varian Unity 300 (300 MHz, .sup.1H and 75 MHz,
.sup.13C at PharmaMar, S. A. (Madrid, Spain) Chemical shifts are
reported in ppm referenced to the chloroform peak at .delta.7.26
ppm for .sup.1H. FABMS were measured on a VG Analytical ZAB at the
Mass Spectrometry laboratory of the University of Illinois. GC
analyses were carried out using a Varian GC (Model 3700) equipped
with an Alltech Associates, Inc., Chirasil-Val II capillary column
(25 m.times.0.32 mm) with Helium gas carrier at a flow rate of 1.2
ml/min with programmed oven temperature (90.degree. C., 4.degree.
C./min, 180.degree. C.). Reversed-phrase HPLC was performed on a
system equipped with an Altex pump (Model 110 A) and a Waters
Associates differential refractometer (Model R-401) and an Alltech
Spherisorb C18 column (25 cm.times.1 cm, particle size 10 .mu.m)
with MeOH: H.sub.2O 0:1 as the solvent system.
[0043] The following examples illustrate the invention.
EXAMPLE 1
[0044] 1. Structure Determination
[0045] The structure of DDB has been determined by physical and
spectroscopic methods.
[0046] 1.1 Spectroscopic Data
[0047] TLC R.sub.f=0.4; 0.35 (Silica gel, 2:3,
CH.sub.2Cl.sub.2/EtOAc); 0,5; 0.44 (Silica gel; 9:1,
CHCl.sub.3/MeOH);
[0048] RP-HPLC t.sub.r=10, 7; 11.9 min (Spherisorb C.sub.18 column,
250 mm.times.10 mm, 10 .mu.m particle size, 9:1, MeOH/H.sub.2O; 2
mL/min);
[0049] [.alpha.].sub.D.sup.25=86.degree. (C 1, MeOH);
[0050] HR FABMS (M+H) C.sub.57H.sub.88N.sub.7O.sub.15 m/z calcd.
1110.6382 (found 1110.6366); (M-side chain+H):
C.sub.42H.sub.66N.sub.5O.sub.11 m/z calcd. 816.4781 (found
816.4755): (M-side chain): C.sub.15H.sub.23N.sub.2- O.sub.4 m/z
calcd. 295.1657 (found 295.1657);
[0051] IR (CHCl.sub.3) v.sub.max cm.sup.-1: 3680, 3600, 2970, 2940,
2880, 1740, 1650, 1605, 1540, 1510;
[0052] .sup.1H NMR (CDCl.sub.3, .delta., ppm): 7.82 (d, J=9 Hz, 1
H); 7.79 (d, J=9 Hz, 1H); 7.62 (d, J=6 Hz, 1H); 7.21 (d, J=9 Hz,
1H); 7.19 (d, J-9 Hz, 1H); 7.08 (d, J=8.5 H, 2H; 6.85 (d, J=8.5 Hz,
2H); 3.77 (s, 3H); 3.13 (s, 3H); 3.08 (s, 3H); 2.54 (s, 3H); 2.50
(s, 3H); 2.1 (s, 3H); 2.02 (s, 3H); 0.82-0.88 (overlapped d and t,
3OH);
[0053] .sup.13C NMR (CDCl.sub.3, .delta., ppm): 204.93 (s); 204.77
(s); 201.23 (s); 197.55 (s); 173.05 (s); 173.05 (s); 172.36 (s);
171.16 (s); 170.59 (s); 169.58 (s); 169.35 (s); 168.36 (s); 168.28
(s); 161.31 (s); 161.06 (s); 158.64 (s); 158.62 (s), 130.31 (d);
114.12 (d); 114.10 (d); 81.47 (d), 81.43 (d); 70.68 (d); 70.33 (d);
67.97 (d); 67.76 (d); 66.38 (d); 66.22 (d); 60.39 (t); 50.88 (d);
57.80 (d); 66.38 (d); 66.22 (d); 60.39 (t); 50.88 (d); 57.80 (d);
57.45 (d); 57.26 (d); 57.18 (d); 57.12 (d); 55.61 (d); 55.57 (d);
55.26 (q); 54.65 (d); 49.55 (d); 49.49 (d); 48.85 (t); 48.41 (t);
46.98 (t); 41.29 (t); 41.24 (t); 38.78 (q); 38.74 (q); 38.68 (q);
36.42 (t); 36.22 (t); 34.06 (d); 33.99 (d); 31.57 (d); 31.38 (q);
31.34 (q); 31.30 (q); 30.69 (d); 29.68 (t); 29.64 (d); 27.28 (t);
27.94 (t); 27.30 (t); 27.17 (t); 27.08 (t); 25.91 (t); 25.87 (t);
25.87 (t); 25.73 (d); 25.68 (d); 25.63 (d); 25.52 (d); 25.48 (d);
24.80 (q); 24.70 (q); 24.44 (q); 24.31 (q); 24.44 (q); 22.21 (q);
22.12 (q); 21.92 (q); 21.79 (q); 21.76 (q); 19.46 (q); 17.76 (q);
17.72 (q); 17.18 (q); 16.87 (q); 16.08 (q); 15.62 (q); 15.48 (q);
15.05 (q); 12.55 (q); 12.50 (q).
[0054] 1.2 Acetylation of DDB
[0055] The structure of dehydrodidemnin B can be confirmed also by
comparison of the acetylation product with the acetyl derivative of
didemnin B.
[0056] Acetylation of DDB with acetic anhydride and pyridine gave a
monoacetyl derivative.
[0057] Low resolution mass spectrum showed peaks at ml z 1153.5
(M+H), 859.0 (M+2H-side chain) and 295.4 (side chain), indicating
the loss of one of the two possible sites of acetylation with
respect to didemnin B, and that the missing site is the hydroxyl
group of the lactyl moiety in the side chain.
[0058] 1.3 N-Trifluoroacetyl methyl esters of amino acid
Residues
[0059] The structure of DDB can also be determined by
identification of the individual subunits by total hydrolysis and
conversion of the amino acids to their N-trifluoroacetyl methyl
esters and analysis by GC.
[0060] The amino acids were identified by their retention times and
comparison of authentic samples obtained from the conversion of
didemnin B to the N-trifluoroacetyl methyl esters of the amino
acids.
[0061] t.sub.R (min): L-Threonine (1.23); D-N-Me-Leucine (1.70);
L-Leucine (2.05); L-Proline (2.38); (3S,4R,5S)-Isostatine (3.15,
4.13, 4.77); L-N, O-Me.sub.2-Tyrosine (6.75).
[0062] A mixture of DDB and glass-distilled HCl was heated during
18 hours at 110.degree. C. in a sealed Teflon-lined screw-capped
vial. The solvent was removed under a stream of N.sub.2 gas.
[0063] The hydrolysate was treated with MeOH/Acetyl chloride during
1 hour at 110.degree. C. The solution was cooled to room
temperature, the solvent was removed under a stream of N.sub.2 gas.
The solid was treated with a mixture of TFAA/TFA during 15 min at
100.degree. C. The solution, was cooled and the solvent evaporated.
The residue was dissolved in 2-propanol for GC analysis.
EXAMPLE 2
[0064] Biological Activity Assays
[0065] 2.1 Assay Against L-1210 Cells (Ascetic Fluid from
DBA/2mouse)
[0066] L-1210 cells were seeded into 16 mm wells at
1.times.10.sup.4 cells per well in 1 mL aliquots of MEN 10C
containing the indicated concentrations of drug. All determinations
were carried out in triplicate. Cells were counted after three
drugs was counted daily to ensure that the cells remained in
exponential growth over the period of observation.
3 Growth Inhibition of L-1210 cells by DDB net increase in cell
ng/mL DDB number % Inhibition 0 2.9 .times. 10.sup.5 0 0.05 2.7
.times. 10.sup.5 7 0.1 2.7 .times. 10.sup.5 7 0.2 2.1 .times.
10.sup.5 28 0.5 1.0 .times. 10.sup.5 66 1 2.5 .times. 10.sup.4 91 2
6.3 .times. 10.sup.3 98
[0067] 2.2 Assay Against P-388 Cells (Lymphoid Neoplasm from
DBA/2mouse)
[0068] P-388 cells were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 mL aliquots of MEM 10C containing the indicated
concentrations of drug. All determinations were carried out in
triplicate. Cells were counted after three days of incubation. A
separate set of cultures without drug was counted daily to ensure
that the cells remained in exponential growth over the period of
observation.
4 Growth Inhibition of P-388 cells by DDB net increase in cell
ng/mL DDB number % Inhibition 0 5.63 .times. 10.sup.5 0 0.12 3.97
.times. 10.sup.5 29 0.25 1.27 .times. 10.sup.5 77 0.5 4.47 .times.
10.sup.5 92
[0069] 2.3 Assay Against L-929 Cells (Mouse Areolar and Adipose
Tissue)
[0070] L-929 cells were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 mL aliquots of MEM 10C. The following day,
medium was replaced with 0.5 mL aliquots of MEM 10C. The following
day, the medium was replaced with 0.5 mL aliquots of MEM 10C
containing the indicated concentrations of drug. All determinations
were carried out in triplicate. A separate set of cultures without
drug was counted daily to ensure that the cells remained in
exponential growth over the period of observation. Cells were
trypsinized and counted 4 days after seeding.
5 Growth Inhibition of L-929 cells by DDB net increase in cell
ng/mL DDB number % Inhibition 0 3.17 .times. 10.sup.5 0 1 2.31
.times. 10.sup.5 27 2.5 1.13 .times. 10.sup.5 64 5 5 .times.
10.sup.5 84
[0071] 2.4 Assay Against B-16 Cells (Mouse Melanoma)
[0072] B-16 cells were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 mL aliquots of MEM 10C determinations were
carried out in triplicate. A separate set of cultures without drug
was counted daily to ensure that the cells remained in exponential
growth over the period for observation. Cells were trypsinized and
counted 4 days after seeding.
6 Growth Inhibition of B-16 cells by DDB net increase in cell ng/mL
DDB number % Inhibition 0 1.71 .times. 10.sup.5 0 0.16 1.71 .times.
10.sup.5 0 0.12 1.27 .times. 10.sup.5 25 0.25 8.25 .times. 10.sup.4
54 0.5 4.50 .times. 10.sup.4 74 1.0 2.88 .times. 10.sup.4 83
[0073] 2.5 Assay Against A-549 Cells (Human Lung Carcinoma)
[0074] A-549 cells were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 mL aliquots of MEM 10C. The following day, the
medium was replaced with 0.5 mL aliquots of MEM 10C containing the
indicated concentrations of drug. All determinations were carried
out in triplicate. A separate set of cultures without drug was
counted daily to ensure that the cells remained in exponential
growth over the period of observation. Cells were trypsinated and
counted 4 days after seeding.
7 Growth Inhibition of A-549 cells by DDB net increase in cell
ng/mL DDB number % Inhibition 0 8.16 .times. 10.sup.4 0 0.25 4.80
.times. 10.sup.4 41 0.50 4.00 .times. 10.sup.4 50 1.0 2.50 .times.
10.sup.4 68 2.5 1.30 .times. 10.sup.4 84
[0075] 2.6 Assay Against HeLa Cells (Human Cervix Epitheliod
Carcinoma)
[0076] HeLa cells were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 mL aliquots of MEM 10C. The following day, the
medium was replaced with 0.5 mL aliquots of MEM 10C containing the
indicated concentrations of drug. All determinations were carried
out in triplicate. A separate set of cultures without drug was
counted daily to ensure that the cells remained in exponential
growth over the period of observation. Cells were trypsinized and
counted 4 days after seeding.
8 Growth Inhibition of KB cells by DDB net increase in cell ng/mL
DDB number % Inhibition 0 4.50 .times. 10.sup.4 0 2.5 4.57 .times.
10.sup.4 0 5 2.40 .times. 10.sup.4 46 10 1.02 .times. 10.sup.4
77
[0077] 2.8 Assay Against HSV-1 (Herpes Simplex Virus Type-1)
[0078] 16 mm diameter wells were seeded each with 2.times.10.sup.5
CV-1 cells in 1 mL aliquots of MEM 10C. Four days after, cells were
infected with HSV-1 at 10C PFU per well. After adsorption for 1.5
hours, the inoculum was replaced in pairs of wells with 0.5 mL
aliquots of MEM 5C containing the indicated concentrations of drug.
Cells from two wells without drug were scraped into the medium and
frozen 4 hours after infection to provide a baseline for
calculating new virus production. The average of these samples was
2.5.times.10.sup.5+1.2.times.10.sup.6 PFU per mL. The remaining
samples were collected 24 hours after infection.
9 Inhibition of HSV-1 replication by DDB net virus produced ng/mL
DDB (PFU/mL) % Inhibition 0 4.5 .times. 10.sup.8 0 0.03 3.8 .times.
10.sup.8 16 0.1 1.5 .times. 10.sup.8 67 0.3 1.9 .times. 10.sup.8 96
1 0 100
[0079] 2.9 Immunosuppressive Activity
[0080] Dehydrodidemnin B is active as an immunosuppressive agent.
In the mixed lymphocyte reaction it suppresses the immune reaction
of murine cells. It also inhibits the growth of murine T-cells and
B-cells.
EXAMPLE 3
[0081] Extraction and Isolation
[0082] A white solitary tunicate was collected near Ibiza in the
Balearic Islands (Spain) and was identified by Dr. Xavier Turon of
the Universitat de Barcelona, Barcelona (Spain) as Aplidium
albicans. A sample is preserved at Centre d'Etudes Avancats, Blanes
(Germona, Spain). Preliminary tests on shipboard indicated
antiviral activity against VSV-1 (Vesicular stomatitis virus).
Further studies in the laboratory confirmed the antiviral activity
against Herpes simplex virus, type 1 (HSV-1) in monkey kidney cells
(CV-1) and also showed cytotoxicity against mouse lymphoid leukemia
in vitro (L1210 line cells).
[0083] The frozen tunicate was extracted with methanol. Solvent
partitioning of the residue afforded three active fractions which
were combined according to their similarity in TLC (Thin Layer
Chromatography). The crude active fraction was portioned and the
activity concentrated in the methanolic layer. The methanol layer
was chromatographed by silica gel gravity column (chloroform and
chloroform-methanol mixtures), affording one active fraction which
was further purified by Reversed-Phase High-Performance Liquid
Chromatography (RPC.sub.18HPLC), affording two peaks (I and II).
Analysis by TLC revealed two identical spots in each HPLC fraction.
Re-injection of each individual fraction led to two peaks with the
same retention times as I and II. Co-injection of I and II
confirmed the presence of two identical peaks (possible conformers)
in each fraction suggesting a rapid interconversion of I to II and
vice versa.
EXAMPLE 4
[0084] Semisynthesis of DDB from Didemnin A
[0085] Dehydrodidemnin B can also be obtained and its structure
confirmed by comparison with a semisynthetic sample prepared by
coupling of the appropriate side chain to natural didemnin A. The
data obtained for the semisynthetic sample totally agreed with data
for natural DDB.
[0086] 4.1 Synthesis of Pyruvyl-Pro-)Bzl
[0087] The hydrochloride salt of Pro-OBzl (10.2 g, 42 mmol) was
dissolved in dry DMF (30 ml), neutralized with NMM
(N-methylmorpholine, 4.7 mL, 42 mmol) at 0.degree. C., and the
solution was mixed with pyruvic acid (8.8 g, 100 mmol) and HOBt
(1-hydroxybenzotriazole, 16.8 g, 110 mmol) in CH.sub.2Cl.sub.2-DMF
(90 mL, 8:1). DCC (dicyclohexylcarbodiimide, 22.6 g, 110 mmol) in
CH.sub.2Cl.sub.2 (35 mL) was added to the above mixture at
0.degree. C. with stirring. The reaction mixture was stirred for 2
hours at 0.degree. C. and left overnight at room temperature. DCCl
was filtered off and washed with CH.sub.2Cl.sub.2 (20 mL). The
filtrate was evaporated to dryness, the residue taken up in EtOAc
and washed successively with 5% citric acid, water, 5% NaHCO.sub.3
and finally with water to neutral pH. The organic layer was dried
(Na.sub.2SO) and concentrated. The residue was chromatographed on
SiO.sub.2 with hexane-EtOAc (2:1) to give the title compound (11 g,
95%).
[0088] [.alpha.].sub.D.sup.25=-78.57 (c 0.14, CHCl.sub.3);
[0089] R.sub.f+0.63 (19:1, CHCl.sub.3/MeOH);
[0090] Anal. Calcd. for C.sub.15H.sub.18NO.sub.4 (M+H):
276.1235;
[0091] Found: 276.1235 (M+H, HRFABMS).
[0092] 4.2 Synthesis of Pyruvyl-Proline
[0093] The protected dipeptide from the previous synthesis (11.0 g,
40 mmol) was dissolved in EtOAc (75 mL) and stirred under hydrogen
over Pd/C for 2 h. The catalyst was then filtered off and the
filtrate was evaporated to dryness. The residue was crystallized
from EtoAc-hexane to give the unprotected peptide (6.9 g, 93):
[0094] [.alpha.].sub.D.sup.25=-103.99 (c 0.124, CHCl.sub.3);
[0095] R.sub.f=0.4163 (19:1:0.5, CHCl.sub.3/MeOH/AcOH;
[0096] Anal. Calcd. for C.sub.8H.sub.12NO.sub.4 (M+H):
186.0766;
[0097] Found: 186.0765 (M+H, HRFABMS).
[0098] 4.3 Synthesis of Dehydrodidemnin B
[0099] EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 4.27 g,
22.3 mmol) was added to a solution of Pyrvu-Pro (8.2 g, 44.5 mmol)
in dry CH.sub.2Cl.sub.2 (40 mL) at 10.degree. C. with stirring. The
mixture was stirred for 2 h at 10.degree. C. and then cooled to
0.degree. C. Didemnin A (1.4 g, 1.48 mmol) in CH.sub.2Cl.sub.2-DMF
(10 mL, 4:1) was added, and the clear solution was stirred at
0.degree. C. for 2 h and then left in the refrigerator
overnight.
[0100] DMAP (4-dimethylaminopyridine, 25 mg) was added to the
reaction mixture, and it was again left in the refrigerator for 48
h. The solvent was evaporated to dryness, and the residue was taken
up in EtOAc and washed with 5% NaHCO.sub.3 and water to neutral pH.
The organic layer was dried (Na.sub.2SO.sub.4) and concentrated.
The residue so obtained was chromatographed on silica gel using
CHCl.sub.3-MeOH (19:1) to give dehydrodidemnin B (1.4 g, 84%, 2
spots on TLC):
[0101] [.alpha.].sub.D.sup.25=-95.384 (c 0.06, MeOH).sub.3);
[0102] R.sub.f=0.51 and 0.44 (19:1, CHCl.sub.3/MeOH);
[0103] Anal. Calcd. for C.sub.57H.sub.88N.sub.7O.sub.15 (M+H):
1110.6338;
[0104] Found: 1110.6355 (M+H, HRFABMS).
[0105] The same series of reactions can be carried out with slight
modifications, in particular EDC can be replaced by DDC with
slightly lower yield.
[0106] The present invention has been described in detail,
including the preferred embodiments thereof. However, it will be
appreciated that those skilled in the art, upon consideration of
the present disclosure, may make modifications and/or improvements
on this invention and still be within the scope and spirit of this
invention as set forth in the following claims.
* * * * *