U.S. patent application number 10/257856 was filed with the patent office on 2004-01-01 for synthetic process for the manufacture of an ecteinaschidin compound.
Invention is credited to Chicharro, Jose Luis, Cuevas, Carmen, Fernandez, Carolina, Francesch, Andres, Gallego, Pilar, Manzanares, Ignacio, Martin, Maria Jesus, Munt, Simon, Perez, Marta, Zarzuelo, Maria.
Application Number | 20040002602 10/257856 |
Document ID | / |
Family ID | 42632476 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040002602 |
Kind Code |
A1 |
Francesch, Andres ; et
al. |
January 1, 2004 |
Synthetic process for the manufacture of an ecteinaschidin
compound
Abstract
Processes are provided for preparing compounds with a fused ring
structure of formula (XIV). Such products include ecteinascidins
and have a spiroamine-1,4-bridge. The process involving forming a
1,4 bridge using a 1-labile, 10-hydroxy, 18-protected hydroxy,
di-6,8-en-5-one fused ring compound. After formation of the 1,4
bridge, C-18 protection is removed before spiroamine introduction.
1
Inventors: |
Francesch, Andres; (Madrid,
ES) ; Fernandez, Carolina; (Madrid, ES) ;
Chicharro, Jose Luis; (Madrid, ES) ; Gallego,
Pilar; (Madrid, ES) ; Zarzuelo, Maria;
(Madrid, ES) ; Manzanares, Ignacio; (Madrid,
ES) ; Perez, Marta; (Madrid, ES) ; Cuevas,
Carmen; (Madrid, ES) ; Martin, Maria Jesus;
(Madrid, ES) ; Munt, Simon; (Madrid, ES) |
Correspondence
Address: |
Gottlieb Rackman & Reisman
270 Madison Avenue
New York
NY
10016-0601
US
|
Family ID: |
42632476 |
Appl. No.: |
10/257856 |
Filed: |
March 31, 2003 |
PCT Filed: |
May 15, 2001 |
PCT NO: |
PCT/GB01/02120 |
Current U.S.
Class: |
540/453 |
Current CPC
Class: |
C07D 491/22 20130101;
C07D 471/18 20130101; C07D 471/22 20130101; A61P 31/04 20180101;
A61P 35/00 20180101; C07D 515/22 20130101; Y02P 20/55 20151101 |
Class at
Publication: |
540/453 |
International
Class: |
C07D 225/04 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 2000 |
WO |
PCT/GB00/01852 |
Claims
1. A process for preparing an ecteinascidin product with a
spiroamine-1,4-bridge, the process involving forming a 1,4 bridge
using a 1-labile, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one
fused ring compound, wherein C-18 protection is removed before
spiroamine introduction.
2. A process according to claim 1, wherein the ecteinascidin
product has a 21-hydroxy group, the process including converting a
21-cyano group to the 21-hydroxy group.
3. A process according to claim 1 or 2, wherein the spiroamine is a
spiroquinoline.
4. A process according to any preceding claim, wherein the
18-protected group of the 1-labile, 10-hydroxy, 18-protected
hydroxy, di-6,8-en-5-one fused ring compound is protected with:
MOM, methoxymethyl; or MEM, methoxyethoxymethyl group.
5. A process according to any preceding claim, wherein the 1-labile
group is an N-protected cysteinyloxymethylene group of the formula
--CH.sub.2--O--CO--CNHProt.sup.1--CH.sub.2--S--H.
6. A process according to claim 5, where Prot.sup.1 is: Boc,
t-butyloxycarbonyl; Troc, 2,2,2-trichloroethyloxycarbonyl; Cbz,
benzyloxycarbonyl; or Alloc, allyloxycarbonyl.
7. A process according to claim 5 or 6, wherein Prot.sup.1 is
removed in the same step as C-18 protection.
8. A process according to claim 5, 6 or 7, wherein the 1-labile
group is generated from a 1-substituent of the formula:
--CH.sub.2--O--CO--CNHProt- .sup.1--CH.sub.2--S-Prot.sup.2.
9. A process according to claim 8, wherein Prot.sup.2 is Fm,
9-fluorenylmethyl.
10. A process according to claim 8 or 9, wherein the 1-substituent
of the formula:
--CH.sub.2--O--CO--CNHProt.sup.1--CH.sub.2--S-Prot.sup.2. is formed
by esterification of a --CH.sub.2--O--H substituent.
11. A process according to claim 10, wherein the esterification is
carried out before formation of the 10-hydroxy, di-6,8-en-5-one
structure.
12. A process according to claim 10, wherein the esterification is
carried out after introduction of the 10-hydroxy, di-6,8-en-5-one
structure.
13. A process according to any preceding claim, which starts from a
1-aminomethylene, 5-protected hydroxy, 7,8-dioxymethylene,
18-hydroxy, 21-cyano fused ring compound
14. A process according to claim 13, where the 1-aminomethylene
group is temporarily protected to allow protection at the
18-hydroxy group, and the temporary protection is removed.
15. A process according to claim 13, wherein the C-18 hydroxy group
is protected after formation of a 1-ester function.
16. A process according to claim 13, wherein the 1-aminomethylene
group is converted to a 1-hydroxymethylene group and the
1-hydroxymethylne group is temporarily protected, to allow
protection at the 18-hydroxy group, and the temporary protection is
removed.
17. A process according to claim 1, wherein the 1-labile,
10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused ring
compound is prepared by steps starting from a 21-Nuc compound with
a structure of formula (XIV): 243where at least one ring A or E is
quinolic, and where Nuc indicates the residue of a nucleophilic
agent.
18. A process according to claim 17, wherein the compound of
formula (XIV) is cyanosafracin B.
19. A process according to any preceding claim, wherein the product
is of formula (XXIIb): 244where: R.sup.1 and R.sup.4 together form
a group of formula (IV), (V), (VI) or (VII): 245R.sup.5 is --OH or
a protected or derivatised version of such a group; R.sup.14a and
R.sup.14b are both --H or one is --H and the other is --OH or a
protected or derivatised version of such a group, --OCH3 or
--OCH.sub.2CH.sub.3, or R.sup.14a and R.sup.14b together form a
keto group; R.sup.12 is --NCH.sub.3--; R.sup.15 is --OH or a
protected or derivatised version of such a group; and R.sup.18 is
--OH or a protected or derivatised version of such a group.
20. A process according to claim 19, wherein R.sup.5 is alkanoyloxy
of 1 to 5 carbon atoms.
21. A process according to claim 20, wherein R.sup.5 is
acetyloxy.
22. A process according to claim 19, 20 or 21, wherein R.sup.14a
and R.sup.14b are hydrogen.
23. A process according to any of claims 19 to 22, wherein R.sup.15
is hydrogen.
24. A process according to any of claims 19 to 23, wherein R.sup.21
is --OH or --CN.
25. A process according to claim 11, wherein R.sup.7 and R.sup.B
together form a group --O--CH.sub.2--O--.
26. A process according to any of claims 19 to 25, wherein R.sup.1
and R.sup.4 together form a group of formula (IV): 246
27. A process according to any preceding claim, wherein the
ecteinascidin product is ecteinascidin 743.
28. A process step in the manufacture of an ecteinascidin comopund,
the step comprising removing both protecting groups in a single
step, in accordance with the following scheme: 247where Prot.sup.NH
is amino protecting group, and Prot.sup.OH is a hydroxy protecting
group.
29. A process according to any of claims 1 to 27, which includes
the process step according to claim 28.
Description
[0001] The present invention relates to synthetic processes, and in
particular it relates to synthetic processes for producing
ecteinascidin compounds.
BACKGROUND OF THE INVENTION
[0002] European Patent 309,477 relates to ecteinascidins 729, 743,
745, 759A, 759B and 770. The ecteinascidin compounds are disclosed
to have antibacterial and other useful properties. Ecteinascidin
743 is now undergoing clinical trials as an antitumour agent.
[0003] Ecteinascidin 743 has a complex
tris(tetrahydroisoquinolinephenol) structure of the following
formula (I): 2
[0004] It is currently prepared by isolation from extracts of the
marine tunicate Ecteinascidin turbinata. The yield is low, and
alternative preparative processes have been sought.
[0005] A synthetic process for producing ecteinascidin compounds is
described in U.S. Pat. No. 5,721,362, see also WO 9812198 which is
incorporated herein by reference in full. The claimed method is
long and complicated, there being 38 Examples each describing one
or more steps in the synthetic sequence to arrive at ecteinascidin
743.
[0006] In the known synthetic process, a 1,4 bridge is formed using
a 1-labile, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused
ring compound. As shown in Example 33, a compound (13) is converted
to compound (14): 3
[0007] According to the known synthetic process, a spiroquinoline
is then formed in the 1,4 bridge by the steps of Examples 34 to 36,
and the 18-MOM protecting group is removed to give ecteinascidin
770 which can then be converted to ecteinascidin 743.
[0008] Claim 25 of U.S. Pat. No. 5,721,362 is directed at an
intermediate phenol compound of a given formula (11), which we
refer to also as Intermediate 11 or Int-11. It has the following
bis(tetrahydroisoquinolin- ephenol) structure (II): 4
[0009] where MOM is a methoxymethyl substituent and TBDPS is a
tert-butyldiphenylsilyl substituent.
[0010] From Intermediate 11 it is possible to synthesise another
interesting antitumour agent, phthalascidin, see Proc. Natl. Acad.
Sci. USA, 96, 3496-3501, 1999. Phthalascidin is a
bis(tetrahydroisoquinolineph- enol) derivative of formula (III):
5
[0011] In ecteinascidins 743 and 770, the 1,4 bridge has the
structure of formula (IV): 6
[0012] Other known ecteinascidins include compounds with a
different bridged cyclic ring system, such as occurs in
ecteinascidin 722 and 736, where the bridge has the structure of
formula (V): 7
[0013] ecteinascidins 583 and 597, where the bridge has the
structure of formula (VI): 8
[0014] and ecteinascidin 594 and 596, where the bridge has the
structure of formula (VII): 9
[0015] The complete structure for these and related compounds is
given in J. Am. Chem. Soc. (1996) 118, 9017-9023. This article is
incorporated by reference.
[0016] Other literature on the ecteinasdin compounds includes:
Corey, E. J., J. Am. Chem. Soc, 1996, 118 pp. 9202-9203; Rinehart,
et al., Journal of Natural Products, 1990, "Bioactive Compounds
from Aquatic and Terrestrial Sources", vol. 53, pp. 771-792;
Rinehart et al., Pure and Appl. Chem., 1990, "Biologically active
natural products", vol 62, pp. 1277-1280; Rinehart, et al., J. Org.
Chem., 1990, "Ecteinascidins 729, 743, 745, 759A, 759B, and 770:
potent Antitumour Agents from the Caribbean Tunicate Ecteinascidia
tuminatac, vol. 55, pp. 4512-4515; Wright et al., J. Org. Chem.,
1990, "Antitumour Tetrahydroisoquinoline Alkaloids from the
Colonial ascidian Ecteinascidia turbinata", vol. 55, pp. 4508-4512;
Sakai et al., Proc. Natl. Acad. Sci. USA 1992, Additional anitumor
ecteinascidins from a Caribbean tunicate: Crystal structures and
activities in vivo", vol. 89, 11456-11460; Science 1994, "Chemical
Prospectors Scour the Seas for Promising Drugs", vol. 266, pp.1324;
Koenig, K. E., "Asymmetric Synthesis", ed. Morrison, Academic
Press, Inc., Orlando, Fla., vol. 5, 1985, p. 71; Barton, et al., J.
Chem Soc. Perkin Trans., 1, 1982, "Synthesis and Properties of a
Series of Sterically Hindered Guanidine bases", pp. 2085; Fukuyama
et al., J. Am. Chem. Soc., 1982, "Stereocontrolled Total Synthesis
of (+)-Saframycin B", vol. 104, pp. 4957; Fukuyama et al., J. Am.
Chem. Soc., 1990, "Total Synthesis of (+)--Saframycin A", vol. 112,
p. 3712; Saito, et al., J. Org. Chem., 1989, "Synthesis of
Saframycins. Preparation of a Key tricyclic Lactam Intermediate to
Saframycin A", vol. 54, 5391; Still, et al., J. Org. Chem., 1978,
"Rapid Chromatographic Technique for Preparative Separations with
Moderate Resolution", vol. 43, p. 2923; Kofron, W. G.; Baclawski,
L. M., J. Org. Chem., 1976, vol. 41, 1879; Guan et al., J.
Biomolec. Struc. & Dynam., vol. 10, pp. 793-817 (1993); Shamma
et al., "Carbon-13 NMR Shift Assignments of Amines and Alkaloids",
p. 206 (1979); Lown et al., Biochemistry, 21, 419-428 (1982);
Zmijewski et al., Chem. Biol. Interactions, 52, 361-375 (1985);
Ito, CR.sup.C Crit. Rev. Anal. Chemn, 17, 65-143 (1986); Rinehart
et al., "Topics in Pharmaceutical Sciences 1989", pp. 613-626, D.
D. Breimer, D. J. A. Cromwelin, K. K. Midha, Eds., Amsterdam
Medical Press B. V., Noordwijk, The Netherlands (1989); Rinehart et
al., "Biological Mass Spectrometry", 233-258 eds. Burlingame et
al., Elsevier Amsterdam (1990); Guan et al., Jour. Biomolec.
Struct. & Dynam., vol. 10 pp. 793-817 (1993); Nakagawa et al.,
J. Am. Chem. Soc., 111: 2721-2722 (1989);; Lichter et al., "Food
and Drugs from the Sea Proceedings" (1972), Marine Technology
Society, Washington, D.C. 1973, 117-127; Sakai et al., J. Am. Chem.
Soc., 1996, 118, 9017; Garcia-Rocha et al., Brit. J. Cancer, 1996,
73: 875-883; and Pommier et al., Biochemistry, 1996, 35:
13303-13309.
[0017] Further compounds are known which lack a bridged cyclic ring
system. They include the bis(tetrahydroisoquinolinequinone)
antitumor-antimicrobial antibiotics safracins and saframycins, and
the marine natural products renieramicins and xestomycin isolated
from cultured microbes or sponges. They all have a common dimeric
tetrahydroisoquinoline carbon framework. These compounds can be
classified into four types, types I to IV, with respect to the
oxidation pattern of the aromatic rings.
[0018] Type I, dimeric isoquinolinequinones, is a system of formula
(VIII) most commonly occurring in this class of compounds, see the
following table I.
1TABLE I Structure of Type I Saframycin Antibiotics. 10
Substituents Compound R.sup.14a R.sup.14b R.sup.21 R.sup.25a
R.sup.25b R.sup.25c saframycin A H H CN O O CH.sub.3 saframycin B H
H H O O CH.sub.3 saframycin C H OCH.sub.3 H O O CH.sub.3 saframycin
G H OH CN O O CH.sub.3 saframycin H H H CN OH CH.sub.2COCH.sub.3
CH.sub.3 saframycin S H H OH O O CH.sub.3 saframycin Y.sub.3 H H CN
NH.sub.2 H CH.sub.3 saframycin Yd.sub.1 H H CN NH.sub.2 H
C.sub.2H.sub.5 saframycin Ad.sub.1 H H CN O O C.sub.2H.sub.5
saframycin Yd.sub.2 H H CN NH.sub.2 H H saframycin Y.sub.2b H
Q.sup.b CN NH.sub.2 H CH.sub.3 saframycin Y.sub.2b-d H Q.sup.b CN
NH.sub.2 H C.sub.2H.sub.5 saframycin AH.sub.2 H H CN H.sup.a
OH.sup.a CH.sub.3 saframycin AH.sub.2Ac H H CN H OAc CH.sub.3
saframycm AH.sub.1 H H CN OH.sup.a H.sup.a CH.sub.3 saframycin
AH.sub.1Ac H H CN OAc H CH.sub.3 saframycin AR.sub.3 H H H H OH
CH.sub.3 .sup.aassignments are interchangeable. .sup.bwhere the
group Q is of formula (IX): 11
[0019] Type I aromatic rings are seen in saframycins A, B and C; G
and H; and S isolated from Streptomyces lavendulae as minor
components. A cyano derivative of saframycin A, called
cyanoquinonamine, is known from Japanese Kokai JP-A2 59/225189 and
60/084,288. Saframycins Y.sub.3, Yd.sub.1, Ad.sub.1, and Yd.sub.2
were produced by S. lavendulae by directed biosynthesis, with
appropriate supplementation of the culture medium. Saframycins
Y.sub.2b and Y.sub.2b-d dimers formed by linking the nitrogen on
the C-25 of one unit to the C-14 of the other, have also been
produced in supplemented culture media of S. lavendulae.
Saframycins AR, (=AH.sub.2,), a microbial reduction product of
saframycin A at C-25 produced by Rhodococcus amidophilus, is also
prepared by nonstereoselective chemical reduction of saframycin A
by sodium borohydride as a 1:1 mixture of epimers followed by
chromatographic separation [the other isomer AH.sub.1 is less
polari. The further reduction product saframycin AR.sub.3,
21-decyano-25-dihydro-saframycin A, (=25-dihydrosaframycin B) was
produced by the same microbial conversion. Another type of
microbial conversion of saframycin A using a Nocardia species
produced saframycin B and further reduction by a Mycobacterium
species produced saframycin AH.sup.1Ac. The 25-O-acetates of
saframycin AH.sub.2 and AH.sub.1 have also been prepared chemically
for biological studies.
[0020] Type I compounds of formula (X) have also been isolated from
marines sponges, see Table II.
2TABLE II Structures of Type I Compounds from Marine Sponges. 12
Substituents R.sup.14a R.sup.14b R.sup.21 R renieramycin A OH H H
--C(CH.sub.3).dbd.CH--CH.sub.3 renieramycin B OC.sub.2H.sub.5 H H
--C(CH.sub.3).dbd.CH--CH.sub.3 renieramycin C OH O O
--C(CH.sub.3).dbd.CH--CH.sub.3 renieramycin D OC.sub.2H.sub.5 O O
--C(CH.sub.3).dbd.CH--CH.sub.3 renieramycin E H H OH
--C(CH.sub.3).dbd.CH--CH.sub.3 renieramycin F OCH.sub.3 H OH
--C(CH.sub.3).dbd.CH--CH.sub.3 xestomycin OCH.sub.3 H H
--CH.sub.3
[0021] Renieramycins A-D were isolated from the antimicrobial
extract of a sponge, a Reniera species collected in Mexico, along
with the biogenetically related monomeric isoquinolines renierone
and related compounds. The structure of renieramycin A was
initially assigned with inverted stereochemistry at C-3, C-11, and
C-13. However, careful examination of the .sup.1H NMR data for new,
related compounds renieramycins E and F, isolated from the same
sponge collected in Palau, revealed that the ring junction of
renieramycins was identical to that of saframycins. This result led
to the conclusion that the formerly assigned stereochemistry of
renieramycins A to D must be the same as that of saframycins.
[0022] Xestomycin was found in a sponge, a Xestospongia species
collected from Sri Lancan waters.
[0023] Type II compounds of formula (XI) with a reduced
hydroquinone ring include saframycins D and F, isolated from S.
lavendulae, and saframycins Mx-1 and Mx-2, isolated from Myxococcus
xanthus. See table III.
3TABLE III Type II Compounds 13 Substituents Compound R.sup.14a
R.sup.14b R.sup.21 R.sup.25a R.sup.25b R.sup.25c saframycin D O O H
O O CH.sub.3 saframycin F O O CN O O CH.sub.3 saframycin Mx-1 H
OCH.sub.3 OH H CH.sub.3 NH.sub.2 saframycin Mx-2 H OCH.sub.3 H H
CH.sub.3 NH.sub.2
[0024] The type III skeleton is found in the antibiotics safracins
A and B, isolated from cultured Pseudomonas fluorescens. These
antibiotics of formula (XII) consist of a
tetrahydroisoquinoline-quinone subunit and a
tetrahydroisoquninolinephenol subunit. 14
[0025] where R.sup.21 is --H in safracin A and is --OH in safracin
B.
[0026] Saframycin R, the only compound classified as the Type IV
skeleton, was also isolated from S. lavendulae. This compound of
formula (XIII), consisting of a hydroquinone ring with a glycolic
ester sidechain on one of the phenolic oxygens, is conceivably a
pro-drug of saframycin A because of its moderate toxicity. 15
[0027] All these known compounds have a fused system of five rings
(A) to (E) as shown in the following structure of formula (XIV):
16
[0028] The rings A and E are phenolic in the ecteinascidins and
some other compounds, while in other compounds, notably the
saframycins, the rings A and E are quinolic. In the known
compounds, the rings B and D are tetrahydro, while ring C is
perhydro.
OBJECT OF THE INVENTION
[0029] The need remains for alternative synthetic routes to the
ecteinascidin compounds and related compounds. Such synthetic
routes may provide more economic paths to the known antitumour
agents, as well as permitting preparation of new active
compounds.
SUMMARY OF THE INVENTION
[0030] This invention relates to synthetic processes for the
formation of intermediates, derivatives and related structures of
ecteinascidin or other tetrahydroisoquinolinephenol compounds.
[0031] In one aspect, the present invention provides a process for
preparing an ecteinascidin product with a spiroamine-1,4-bridge.
The process involving forming a 1,4 bridge using a 1-labile,
10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused ring
compound, where the fused ring is the formula (XIV). In the present
invention, the C-18 protection is removed before spiroamine
introduction.
[0032] Suitable starting materials for the new synthetic processes
include compounds related to the natural
bis(tetrahydroisoquinoline) alkaloids. Such starting materials may
be prepared either from the different classes of saframycin and
safracin antibiotics available from different culture broths as
detailed in WO 0069862 or by other synthetic or biochemical
processes. In this respect, WO 0069862 is incorporated herein in
full by reference. The present PCT application claims priority from
application PCT/GB 00/01852 which was published as WO 0069862. We
incorporate that text by reference to the extent that there is
disclosure therein which is not in the present specification.
PREFERRED EMBODIMENTS OF THE INVENTION
[0033] In one particular aspect, the present invention is directed
at the use of the compound Intermediate 21 in a number of new
synthetic processes for the preparation of ecteinascidin 743 and
related compounds, 17
[0034] The Intermediate 21 has a 5-allyloxy group, where the allyl
group serves to protect the 5-hydroxy group. It will be understood
that other protecting groups can easily be employed, and that the
present invention extends generally to the use of other such
5-protected hydroxy compounds.
[0035] Formation of Ecteinascidin 743 and Related Compounds
[0036] In general, the conversion of Intermediate 21, or a related
compound, to an ecteinascidin product involves the following key
transformations:
[0037] (a) Conversion of the NH.sub.2 to OH by reaction, for
example with sodium nitrite in acetic acid.
[0038] (b) E-ring phenol protection.
[0039] (c) Esterification by protecting the primary 1-hydroxy
function with a protected cysteine sidechain.
[0040] (d) Deprotection of allyl group and oxidation.
[0041] (e) Creation of the bridged ring by cyclization
reaction.
[0042] (f) Deprotections of E-ring phenol and the cysteine
moiety
[0043] (g) Quinoline Introduction by Trans-amination and Petter
Spengler reactions.
[0044] The high functionality of the intermediate compounds
necessitates the use of protecting groups for the E-ring phenol and
for the cysteine sidechain in order to prevent unwanted side
reactions.
[0045] As such, a number of alternative intermediates can be
generated dependent on the particular selection of protecting
groups.
[0046] Different possible sequences are possible for combining
these transformations dependent primarily on the protecting groups
selected for the phenol ring and for the amine of the cysteine
sidechain.
[0047] The total number of synthetic transformations is also a
function of the protecting groups selected.
[0048] By way of illustration, the use of different combinations of
protecting groups is described below for six typical routes for the
preparation of ET-743 from Intermediate 21, also referred to herein
as SF21.
4 Number Route Phenol Protection Cysteine Protection of steps 1 MOM
Boc 12 2 MEM Boc 10 3 MEM Cbz 11 4 MOM Alloc 13 5 MEM Alloc 13 6
MOM Cbz 15
[0049] As the skilled artisan will readily appreciate, the reaction
schemes described herein may be modified and/or combined in various
ways, and the alternative sequences of steps and the compounds
generated therefrom are part of this invention.
[0050] Additionally, the use of other protecting group strategies
not detailed is also part of this invention.
[0051] Process Details of Six Typical Synthetic Routes
[0052] Full reaction schemes for each route are in the following
Schemes 1 to 6. 181920 212223 242526 272829 303132 3334
[0053] In route 1, protection of the E-ring phenol is achieved in
three steps involving protection/deprotection of the amine of SF21
with Troc.
[0054] For routes 1 and 2, protection of the cysteine sidechain
with Boc allows the phenol and cysteine groups to be deprotected in
a single step rather than as two separate steps. For the rest of
the routes, an additional deprotection step is required.
[0055] For route 2, Intermediate 25 is avoided through the use of
the direct esterification methodology and the subsequent protection
of the phenol with the MEM group.
[0056] In routes 2 and 3 protection of the E-ring phenol is delayed
until after the diazotisation and esterification steps thereby
allowing the phenol to be protected in a single step rather than by
the three step sequence of route 1.
[0057] For routes 1, 2 and 3, direct esterification of the primary
alcohol with the cysteine derivative eliminates the unproductive
protection/deprotection steps of the primary alcohol with a silyl
group (routes 4 and 5) thereby shortening the sequence by two
steps.
[0058] Route 6 only contemplates herein the last steps from
intermediate 161, which can be easily obtained from intermediate
21.
[0059] In routes 4 and 5 the primary alcohol produced by the
initial diazotisation step is protected with silicon to allow
selective protection of the E-ring phenol and avoiding intermediate
25. Following modification of the A-ring (deprotection/oxidation),
the silicon group is removed and the primary alcohol esterified
with the cysteine derivative.
[0060] These changes are a direct consequence of the problems that
were found in the scale up of the route given in WO 0069862. As a
result of these changes the overall route 2 is three steps shorter
and potentially therefore more suitable and/or cheaper for routine
manufacture. Process Overview
[0061] Thus, in view of the routes 1 to 6, the present invention
extends to a process for preparing an ecteinascidin product with a
spiroamine-1,4-bridge, the process involving forming a 1,4 bridge
using a 1-labile, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one
fused ring compound, wherein C-18 protection is removed before
spiroamine introduction.
[0062] In one version of the process, the ecteinascidin product has
a 21-hydroxy group, and the process includes converting a 21-cyano
group to the 21-hydroxy group.
[0063] Typically the spiroamine is a spiroquinoline, especially the
spiroquinoline of ecteinascidin 743.
[0064] In a preferred process the 18-protected group of the
1-labile, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused
ring compound is protected with: MOM, methoxymethyl; or MEM,
methoxyethoxymethyl group.
[0065] Suitably the 1-labile group is an N-protected
cysteinyloxymethylene group of the formula
--CH.sub.2--O--CO--CNHProt.sup.1-CH.sub.2--S--H.
[0066] In this formula Prot.sup.1 is typically: Boc,
t-butyloxycarbonyl; Troc, 2,2,2-trichloroethyloxycarbonyl; Cbz,
benzyloxycarbonyl; or Alloc, allyloxycarbonyl.
[0067] With some embodiments of the process, Prot.sup.1 is removed
in the same step as C-18 protection.
[0068] The 1-labile group can be generated from a 1-substituent of
the formula:
--CH.sub.2--O--CO--CNHProt.sup.1-CH.sub.2--S-Prot.sup.2.
[0069] In this formula, Prot.sup.2 is typically Fm,
9-fluorenylmethyl.
[0070] A 1-substituent of the formula:
--CH.sub.2--O--CO--CNHProt.sup.1-CH.sub.2--S-Prot.sup.2
[0071] can be formed by esterification of a --CH.sub.2--O--H
substituent.
[0072] The esterification can be carried out before or after
formation of the 10-hydroxy, di-6,8-en-5-one structure.
[0073] In one version, the claimed process starts from a
1-aminomethylene, 5-protected hydroxy, 7,8-dioxymethylene,
18-hydroxy, 21-cyano fused ring compound
[0074] The 1-aminomethylene group can be temporarily protected to
allow protection at the 18-hydroxy group, and the temporary
protection removed.
[0075] Alternatively, the C-18 hydroxy group can be protected after
formation of a 1-ester function.
[0076] In another variation, the 1-aminomethylene group is
converted to a 1-hydroxymethylene group and the 1-hydroxymethylne
group is temporarily protected, to allow protection at the
18-hydroxy group, and the temporary protection is removed.
[0077] The fused ring structure is suitably of the formula: 35
[0078] especially where R.sup.15 is H. One or more or all of the
remaining subsitituents can be as in ecteinascidin 743.
[0079] Hemisythesis
[0080] The invention permits the use of a known compound, safracin
B, also referred to as quinonamine, in hemisynthetic synthesis.
[0081] More generally, the invention relates to a hemisynthetic
process for the formation of intermediates, derivatives and related
structures of ecteinascidin or other tetrahydroisoquinolinephenol
compounds starting from natural bis(tetrahydroisoquinoline)
alkaloids. Suitable starting materials for the hemi-synthetic
process include the classes of saframycin and safracin antibiotics
available from different culture broths, and also the classes of
reineramicin and xestomycin compounds available from marine
sponges.
[0082] A general formula (XV) for the starting compounds is as
follows: 36
[0083] where:
[0084] R.sup.1 is an amidomethylene group such as
--CH.sub.2--NH--CO--CR.s- up.25aR.sup.25bR.sup.25c where R.sup.25a
and R.sup.25b form a keto group or one is --OH, --NH.sub.2 or
--OCOCH.sub.3 and the other is --CH.sub.2COCH.sub.3, --H, --OH or
--OCOCH.sub.3, provided that when R.sup.25a is --OH or --NH.sub.2
then R.sup.25b is not --OH, and R.sup.25c is --H. --CH.sub.3 or
--CH.sub.2CH.sub.3, or R.sup.1 is an acyloxymethylene group such as
--CH.sub.2--O--CO--R, where R is --C(CH.sub.3).dbd.CH--CH.sub.3 or
--CH.sub.3;
[0085] R.sup.5 and R.sup.8 are independently chosen from --H, --OH
or --OCOCH.sub.2OH, or R.sup.5 and R.sup.8 are both keto and the
ring A is a p-benzoquinone ring;
[0086] R.sup.14a and R.sup.14b are both --H or one is --H and the
other is --OH, --OCH.sub.3 or --OCH.sub.2CH.sub.3, or R.sup.14a and
R.sup.14b together form a keto group;
[0087] R.sup.15 and R.sup.18 are independently chosen from --H or
--OH, or R.sup.5 and R.sup.8 are both keto and the ring A is a
p-benzoquinone ring; and
[0088] R.sup.21 is --OH or --CN.
[0089] A more general formula for these class of compounds is
provided below: 37
[0090] wherein the substituent groups defined by R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8,
[0091] R.sub.9, R.sub.10 are each independently selected from the
group consisting of H, OH, OCH.sub.3, CN, .dbd.O, CH.sub.3;
[0092] wherein X are the different amide or ester functionalities
contained in the mentioned natural products;
[0093] wherein each dotted circle represents one, two or three
optional double bonds.
[0094] Thus, according to the present invention, we now provide
hemisynthetic routes for the production of intermediates including
Intermediates 11 or 21 and thus for the production of the
ecteinascidin compounds as well as phthalascidin and additional
compounds. The hemisynthetic routes of the invention each comprise
a number of transformation steps to arrive at the desired product.
Each step in itself is a process in accordance with this invention.
The invention is not limited to the routes that are exemplified,
and alternative routes may be provided by, for example, changing
the order of the transformation steps, as appropriate or by a
change to the protecting groups used.
[0095] In particular, this invention involves the provision of a
21-cyano starting material of general formula (XVI): 38
[0096] where R.sup.1, R.sup.5, R.sup.8, R.sup.14a, R.sup.14b,
R.sup.15 and R.sup.18 are as defined.
[0097] Other compounds of formula (XVI) with different substituents
at the 21-position may also represent possible starting materials.
In general, any derivative capable of production by nucleophilic
displacement of the 21-hydroxy group of compounds of formula (XV)
wherein R.sup.21 is a hydroxy group cis a candidate. Examples of
suitable 21-substituents include but are not limited to:
[0098] a mercapto group;
[0099] an alkylthio group (the alkyl group having from 1 to 6
carbon atoms);
[0100] an arylthio group (the aryl group having from 6 to 10 carbon
atoms and being unsubstituted or substituted by from 1 to 5
substituents selected from, for example, alkyl group having from 1
to 6 carbon atoms, alkoxy groups having from 1 to 6 carbon atoms,
halogen atoms, mercapto groups and nitro groups);
[0101] an amino group;
[0102] a mono-or dialkylamino (the or each alkyl group having from
1 to 6 carbon atoms);
[0103] a mono-or diarylamino group (the or each aryl group being as
defined above in relation to arylthio groups);
[0104] an .alpha.-carbonylalkyl group of formula
--C(R.sup.a)(R.sup.b)--C(- .dbd.O)R.sup.c, where
[0105] R.sup.a and R.sup.b are selected from hydrogen atoms, alkl
groups having from 1 to 20 carbon atoms, aryl groups (as defined
above in relation to arylthio groups) and aralkyl groups (in which
an alkyl group having from 1 to 4 carbon atoms is substituted by an
aryl group a defined above in relation to arylthio groups), with
the proviso that one of R.sub.a and R.sub.b is a hydrogen atom;
[0106] R.sup.c is selected from a hydrogen atom, an alky group
having from 1 to 20 carbon atoms, aryl groups (as defined above in
relation to arylthio groups), an aralkyl group (in which an allyl
group having from 1 to 4 carbon atoms is substituted by an aryl
group a defined above in relation to aiylthio groups), an alkoxy
group having from 1 to 6 carbon atoms, an amino group or a mono- or
dialkylamino group as defined above.
[0107] Thus, in a more general aspect, the present invention
relates to processes where the first step is to form a
21-deriviative using a nucleophilic reagent. We refer to such
compounds as 21-Nuc compounds.
[0108] The presence of the 21-cyano group is required for some of
the end-products, notably ecteinascidin 770 and phthalascidin,
while for other end-products it acts as a protecting group which
can readily be converted to another substituent, such as the
21-hydroxy group of ecteinascidin 743 or of
21-hydroxyphthalascidin. The adoption of the 21-cyano compound as
the starting material effectively stabilises the molecule during
the ensuing synthetic steps, until it is optionally removed. Other
21-Nuc compounds can offer this and other advantages.
[0109] In one important aspect, the present invention consists in
the use of a 21-cyano compound of the general formula (XVI) in the
preparation of a bis- or tris-(tetrahydroisoquinolinephenol)
compounds. Products which may be prepared include intermediates
such as Intermediate 11 or 21, and the ecteinascidins, as well as
new and known compounds of related structure.
[0110] Preferred starting materials include those compounds of
formula (XV) or (XVI) where R.sup.14a and R.sup.14b are both
hydrogen. Preferred starting materials also include compounds of
formula (XV) or (XVI) where R.sup.15 is hydrogen. Furthermore, the
preferred starting materials include compounds of formula (XV) or
(XVI) where ring E is a phenolic ring. Preferred starting materials
further include compounds of formula (XV) or (XVI) where at least
one, better at least two or three of R.sup.5, R.sup.8, R.sup.15 and
R.sup.18 is not hydrogen.
[0111] Examples of suitable starting materials for this invention
include saframycin A, saframycin B, saframycin C, saframycin G,
saframycin H, saframycin S, saframycin Y.sub.3, saframycin
Yd.sub.1, saframycin Ad.sub.1, saframycin Yd.sub.2, saframycin
AH.sub.2, saframycin AH.sub.2Ac, saframycin AH.sub.1, saframycin
AH.sub.1Ac, saframycin AR.sub.3, renieramycin A, renieramycin B,
renieramycin C, renieramycin D, renieramycin E, renieramycin F,
xestomycin, saframycin D, saframycin F, saframycin Mx-1, saframycin
Mx-2, safracin A, safracin B and saframycin R. Preferred starting
materials have a cyano group in position 21, for the group
R.sup.21.
[0112] In a particularly preferred aspect, the invention involves a
hemisynthetic process wherein the transformation steps are applied
to safracin B: 39
[0113] Safracin B presents a ring system closely related to the
ecteinascidins. This compound has the same pentacycle structure and
the same substitution pattern in the right-hand aromatic ring, ring
E. Also, safracin B presents very close similarities to some of the
synthetic intermediates in the total synthesis of ET-743,
particularly to the intermediates 11 or 21. Such intermediate can
be transformed into Et-743 using a well established method.
Synthetic conversion of safracin B into intermediates 11 or 21 will
therefore provide an hemi-synthetic method to obtain ET-743.
[0114] Thus, we provide Intermediates 11 or 21 made from this
compound safracin B, and compounds derived from Intermediate 11 or
21, particularly ecteinascidin compounds. We further provide
phthalascidin made from safracin B. The invention also relates to
use of safracin B in the production of Intermediates 11 or 21,
ecteinascidin compounds and the other intermediates of the
invention. The invention also relates to compounds described herein
derived from the other suggested starting materials, and use of
those compounds in the production of such compounds.
[0115] The more preferred starting materials of this invention have
a 21-cyano group. The currently most preferred compound of the
present invention is the compound of Formula 2. This compound is
obtained directly from safracin B and is considered a key
intermediate in the hemisynthetic process. 40
[0116] In a related aspect, we provide cyanosafracin B by
fermentation of a safracin B-producing strain of Pseudomonas
fluorescens, and working up the cultured broth using cyanide ion.
The preferred strain of Pseudomonas fluorescens is strain A2-2,
FERM BP-14, which is employed in the procedure of EP 055,299. A
suitable source of cyanide ion is potassium cyanide. In a typical
work-up, the broth is filtered and excess cyanide ion is added.
After an appropriate interval of agitation, such as 1 hour, the pH
is rendered alkaline, say pH 9.5, and an organic extraction gives a
crude extract which can be further purified to give the
cyanosafracin B.
[0117] For the avoidance of doubt, the stereochemistries indicated
in this patent specification are based on our understanding of the
correct stereochemistry of the natural products. To the extent that
an error is discovered in the assigned stereochemistry, then the
appropriate correction needs to be made in the formulae given
throughout in this patent specification. Furthermore, to the extent
that the syntheses are capable of modification, this invention
extends to stereoisomers.
[0118] The products of this invention are typically of the formula
(XVIIb): 41
[0119] where
[0120] R.sup.1 and R.sup.4 together form a group of formula (IV),
(V) (VII) or (VII): 42
[0121] R.sup.5 is --H or --OH;
[0122] R.sup.7 and R.sup.8 together form a group
--O--CH.sub.2--O--;
[0123] R.sup.14a and R.sup.14b are both --H or one is --H and the
other is --OH, --OCH.sub.3 or --OCH.sub.2CH.sub.3, or R.sup.14a and
R.sup.14b together form a keto group; and
[0124] R.sup.15 is --H or --OH;
[0125] R.sup.21 is --H, --OH or --CN;
[0126] and derivatives including acyl derivatives thereof
especially where R.sup.5 is acetyloxy or other acyloxy group of up
to 4 carbon atoms.
[0127] In the formula (XVIIb), R.sup.1 typically with R.sup.4 forms
a group (IV) or (V). The group R.sup.18 is usually protected.
Usually R.sup.21 is cyano.
[0128] Preferably R.sup.14a and R.sup.14b are hydrogen. Preferably
R.sup.15 is hydrogen. The O-acyl derivatives are suitably aliphatic
O-acyl derivatives, especially acyl derivatives of 1 to 4 carbon
atoms, and typically an O-acetyl group, notably at the
5-position.
[0129] Suitable protecting groups for phenols and hydroxy groups
include ethers and esters, such as alkyl, alkoxyalkyl,
aryloxyalkyl, alkoxyalkoxyalkyl, alkylsilylalkoxyalkyl,
alkylthioalkyl, arylthioalkyl, azidoalkyl, cyanoalkyl, chloroalkyl,
heterocyclic, arylacyl, haloarylacyl, cycloalkylalkyl, alkenyl,
cycloalkyl, alyklarylalkyl, alkoxyarylalkyl, nitroarylalkyl,
haloarylalkyl, alkylaminocarbonylarylalk- yl,
alkylsulfinylarylalky, alkylsilyl and other ethers, and arylacyl,
aryl alkyl carbonate, aliphatic carbonate, alkylsulfinylarlyalkyl
carbonate, alkyl carbonate, aryl haloalkyl carbonate, aryl alkenyl
carbonate, aryl carbamate, alkyl phosphinyl, alkylphosphinothioyl,
aryl phosphinothioyl, aryl alkyl sulphonate and other esters. Such
groups may optionally be substituted with the previously mentioned
groups in R.sup.1.
[0130] Suitable protecting groups for amines include carbamates,
amides, and other protecting groups, such as alkyl, arylalkyl,
sulpho- or halo-arylalkyl, haloalkyl, alkylsilylalkyl, arylalkyl,
cycloalkylalkyl, alkylarylalkyl, heterocyclylalkyl, nitroarylalkyl,
acylaminoalkyl, nitroaryldithioarylalkyl,
dicycloalkylcarboxamidoalkyl, cycloalkyl, alkenyl, arylalkenyl,
nitroarylalkenyl, heterocyclylalkenyl, heterocyclyl,
hydroxyheterocyclyl, alkyldithio, alkoxy- or halo- or
alkylsulphinyl arylalkyl, hetercyclylacyl, and other carbamates,
and alkanoyl, haloalkanoyl, arylalkanoyl, alkenoyl,
heterocyclylacyl, aroyl, arylaroyl, haloaroyl, nitroaroyl, and
other amides, as well as alkyl, alkenyl, alkylsilylalkoxyalkyl,
alkoxyalkyl, cyanoalkyl, heterocyclyl, alkoxyarylalkyl, cycloalkyl,
nitroaryl, arylalkyl, alkoxy- or hydroxyarylalkyl, and many other
groups. Such groups may optionally be substituted with the
previously mentioned groups in R.sup.1.
[0131] Examples of such protecting groups are given in the
following tables.
5 protection for --OH group ethers abbreviation methyl
methoxymethyl MOM benzyloxymethyl BOM methoxyethoxymethyl MEM
2-(trimethylsilyl)ethoxymethy- l SEM methylthiomethyl MTM
phenylthiomethyl PTM azidomethyl cyanomethyl
2,2-dichloro-1,1-difluoroethyl 2-chloroethyl 2-bromoethyl
tetrahydropyranyl THP 1-ethoxyethyl EE phenacyl 4-bromophenacyl
cyclopropylmethyl allyl propargyl isopropyl cyclohexyl t-butyl
benzyl 2,6-dimethylbenzyl 4-methoxybenzyl MPM or PMB o-nitrobenzyl
2,6-dichlorobenzyl 3,4-dichlorobenzyl
4-(dimethylamino)carbonylbenzyl 4-methylsuflinylbenzyl Msib
9-anthrylmethyl 4-picolyl heptafluoro-p-tolyl tetrafluoro-4-pyridyl
trimethylsilyl TMS t-butyldimethylsilyl TBDMS t-butyldiphenylsilyl
TBDPS triisopropylsilyl TIPS esters aryl formate aryl acetate aryl
levulinate aryl pivaloate ArOPv aryl benzoate aryl
9-fluorocarboxylate aryl methyl carbonate 1-adamantyl carbonate
t-butyl carbonate BOC-OAr 4-methylsulfinylbenzyl carbonate Msz-Oar
2,4-dimethylpent-3-yl carbonate Doc-Oar aryl 2,2,2-trichloroethyl
carbonate aryl vinyl carbonate aryl benzyl carbonate aryl carbamate
dimethylphosphinyl Dmp-OAr dimethylphosphinothioyl Mpt-OAr
diphenylphosphinothioyl Dpt-Oar aryl methanesulfonate aryl
toluenesulfonate aryl 2-formylbenzenesulfonate
[0132]
6 protection for the --NH.sub.2 group carbamates abbreviation
methyl ethyl 9-fluorenylmethyl Fmoc 9-(2-sulfo)fluroenylmethyl
9-(2,7-dibromo)fluorenylmethyl 17-tetrabenzo[a,c,g,i]fluorenylmet-
hyl Tbfmoc 2-chloro-3-indenylmethyl Climoc benz[f]inden-3-ylmethyl
Bimoc 2,7-di-t-butyl[9-(10,10-dioxo-10,10- ,10,10-
tetrahydrothioxanthyl)]methyl DBD-Tmoc 2,2,2-trichloroethyl Troc
2-trimethylsilylethyl Teoc 2-phenylethyl hZ
1-(1-adamantyl)-1-methylethyl Adpoc 2-chlooethyl
1,1-dimethyl-2-chloroethyl 1,1-dimethyl-2-bromoethyl
1,1-dimethyl-2,2-dibromoethyl DB-t-BOC
1,1-dimethyl-2,2,2-trichloroethyl TCBOC
1-methyl-1-(4-biphenyl)ethyl Bpoc 1-(3,5-di-t-butylphenyl)-1-1-me-
thylethyl t-Burmeoc 2-(2'-and 4'-pyridyl)ethyl Pyoc
2,2-bis(4'-nitrophenyl)ethyl Bnpeoc n-(2-pivaloylamino)-1,1-dimet-
hylethyl 2-[(2-nitrophenyl)dithio]-1-phenylethyl NpSSPeoc
2-(n,n-dicyclohexylcarboxamido)ethyl t-butyl BOC 1-adamantyl 1-Adoc
2-adamantyl 2-Adoc vinyl Voc allyl Aloc or Alloc 1-isopropylallyl
Ipaoc cinnamyl Coc 4-nitrocinnamyl Noc 3-(3'-pyridyl)prop-2-enyl
Paloc 8-quinolyl n-hydroxypiperidinyl alkyldithio benzyl Cbz or Z
p-methoxybenzyl Moz p-nitrobenzyl PNZ p-bromobenzyl p-chlorobenzyl
2,4-dichlorobenzyl 4-methylsulfinylbenzyl Msz 9-anthrylmethyl
diphenylmethyl phenothiazinyl-(10)-carbonyl
n'-p-toluenesulfonylaminocarbonyl n'-phenylaminothiocarbonyl amides
formamide acetamide chloroacetamide trifluoroacetamide TFA
phenylacetamide 3-phenylpropanamide pent-4-enamide picolinamide
3-pyridylcarboxamide benzamide p-phenylbenzamide n-phthalimide
n-tetrachlorophthalimide TCP 4-nitro-n-phthalimide
n-dithiasuccinimide Dts n-2,3-diphenylmaleimide
n-2,5-dimethylpyrrole n-2,5-bis(triisopropylsioxyl)pyrrole BIPSOP
n-1,1,4,4- STABASE tetramethyldisiliazacyclopentante adduct
1,1,3,3-tetramethyl-1,3-disilaisoindoline BSB special --NH
protective groups n-methylamine n-t-butylamine n-allylamine
n-[2-trimethylsilyl)ethoxy]methylamine SEM n-3-acetoxypropylamine
n-cyanomethylamine n-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-
yl)amine n-2,4-dimethoxybenzylamine Dmb 2-azanorbornenes
n-2,4-dinitrophenylamine n-benzylamine Bn n-4-methoxybenzylamine
MPM n-2,4-dimethoxybenzylamine DMPM n-2-hydroxybenzylamine Hbn
n-(diphenylmethyl)amino DPM n-bis(4-methoxyphenyl)methylamine
n-5-dibenzosuberylamine DBS n-triphenylmethylamine Tr n-[(4- MMTr
methoxyphenyl)diphenylmethyl]amino n-9-phenylflurenylamine Pf
n-ferrocenylmethylamine Fcm n-2-picolylamine n'-oxide
n-1,1-dimethylthiomethyleneamine n-benzylideneamine
n-p-methoxybenzylideneamine n-diphenylmethyleneamine
n-(5,5-dimethyl-3-oxo-1- cyclohexenyl)amine n-nitroamine
n-nitrosoamine diphenylphosphinamide Dpp dimethylthiophosphinamide
Mpt diphenylthiophosphinamide Ppt dibenzyl phosphoramidate
2-nitrobenzenesulfenamide Nps n-1-(2,2,2-trifluoro-1,1- TDE
diphenyl)ethylsufenamide 3-nitro-2-pyridinesulfenamide Npys
p-toluenesulfonamide Ts benzenesulfonamide
[0133] Particular ecteinascidin products of this invention include
compounds of the formula (XVIII): 43
[0134] where R.sup.1 and R.sup.4 form a group of formula (IV), (V),
(VI) or (VII): 44
[0135] more particularly a group (IV) or (V);
[0136] R.sup.21 is --H, --OH or --CN, more particularly --OH or
--CN;
[0137] and acyl derivatives thereof, more particularly 5-acyl
derivatives including the 5-acetyl derivative.
[0138] In general, the conversion of the 21-cyano starting compound
to an ecteinascidin product of, for example, formula (XVIII)
involves:
[0139] a) conversion if necessary of a quinone system for the ring
E into the phenol system
[0140] b) conversion if necessary of a quinone system for the ring
A into the phenol system;
[0141] c) conversion of the phenol system for the ring A into the
methylenedioxyphenol ring;
[0142] d) formation of the bridged spiro ring system of formula
(IV), (VI) or (VII) across the 1-position and 4-position in ring B;
and
[0143] e) derivatisation as appropriate, such as acylation.
[0144] Step (a), conversion if necessary of a quinone system for
the ring E into the phenol system, can be effected by conventional
reduction procedures. A suitable reagent system is hydrogen with a
palladium-carbon catalyst, though other reducing systems can be
employed.
[0145] Step (b), conversion if necessary of a quinone system for
the ring A into the phenol system is analogous to step (a), and
more detail is not needed.
[0146] Step (c), conversion of the phenol system for the ring A
into the methylenedioxyphenol ring, can be effected in several
ways, possibly along with step (b). For example, a quinone ring A
can be demethylated in the methoxy substituent at the 7-position
and reduced to a dihydroquinone and trapped with a suitable
electrophilic reagent such as CH.sub.2Br.sub.2, BrCH.sub.2Cl, or a
similar divalent reagent directly yielding the methylenedioxy ring
system, or with a divalent reagent such as thiocarbonyldiimidazol
which yields a substituted methylenedioxy ring system which can be
converted to the desired ring.
[0147] Step (d) is typically effected by appropriate substitution
at the 1-position with a bridging reagent that can assist formation
of the desired bridge, forming an exendo quinone methide at the
4-position and allowing the methide to react with the 1-substituent
to bring about the bridged structure. Preferred bridging reagents
are of formula (XIX) 45
[0148] where Fu indicates a protected functional group, such as a
group --NHProt.sup.4a, Prot.sup.3 is a protecting group, and the
dotted line shows an optional double bond.
[0149] Suitably the methide is formed by first introducing a
hydroxy group at the 10-position at the junction of rings A and B
to give a partial structure of formula (XX): 46
[0150] or more preferably a partial structure of formula (XXI):
47
[0151] where the group R" is chosen for the desired group of
formula (IV), (V), (VI) or (VII). For the first two such groups,
the group R" typically takes the form --CHFu-CH.sub.2--SProt.sup.3.
The protecting groups can then be removed and modified as
appropriate to give the desired compound.
[0152] A typical procedure for step (d) is provided in U.S. Pat.
No. 5,721,362 incorporated by reference. Particular reference is
made to the passage at column 8, step (l) and Example 33 of the US
Patent, and related passages.
[0153] Derivatisation in step (e) can include acylation, for
instance with a group R.sup.a--CO--, where R.sup.a can be various
groups such as alkyl, alkoxy, alkylene, arylalkyl, arylalkylene,
amino acid acyl, or heterocyclyl, each optionally substituted with
halo, cyano, nitro, carboxyalkyl, alkoxy, aryl, aryloxy,
heterocyclyl, heterocyclyloxy, alkyl, amino or substituted amino.
Other acylating agents include isothiocyanates, such as aryl
isothiocyanates, notably phenyl isocyanate. The alkyl, alkoxy or
alkylene groups of R.sup.a suitably have 1 to 6 or 12 carbon atoms,
and can be linear, branched or cyclic. Aryl groups are typically
phenyl, biphenyl or naphthyl. Heterocyclyl groups can be aromatic
or partially or completely unsaturated and suitably have 4 to. 8
ring atoms, more preferably 5 or 6 ring atoms, with one or more
heteroatoms selected from nitrogen, sulphur and oxygen.
[0154] Without being exhaustive, typical R.sup.a groups include
alkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, arylalkylene,
haloalkylarylakylene, acyl, haloacyl, arlyalkyl, alkenyl and amino
acid. For example, R.sup.a--CO-- can be acetyl, trifluoroacetyl,
2,2,2-trichloroethoxycarbonyl, isovalerylcarbonyl,
trans-3-(trifluoromethyl)cinnamoylcarbonyl,
heptalluorobutyrylcarbonyl, decanoylcarbonyl,
trans-cinnamoylcarbonyl, butyrylcarbonyl,
3-chloropropyonylcarbonyl, cinnamoylcarbonyl,
4-methylcinnamoylcarbonyl, hydrocinnamoylcarbonyl, or
trans-hexenoylcarbonyl, or alanyl, arginyl, aspartyl, asparagyl,
cystyl, glutamyl, glutaminyl, glycyl, histidyl, hydroxyprolyl.,
isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, seryl,
threonyl, thyronyl, tryptophyl, tyrosyl, valyl, as well as other
less common amino acid acyl groups, as well as phthalimido and
other cyclic amides. Other examples may be found among the listed
protecting groups. Compounds wherein --CO--R.sup.a is derived from
an amino acid and include an amino group can themselves form acyl
derivatives. Suitable N-acyl commands include dipeptides which in
turn can form N-acyl derivatives.
[0155] By way of illustration, it is now feasible to transform
cyanosafracin B compound of formula 2 into ET-743 resulting in a
shorter and more straightforward way to make ET-743 than methods
previously described.
[0156] The retrosynthetic analysis to make ET-743 using compound 29
is depicted in scheme I. 48
[0157] Following the above scheme I it is possible to obtain ET-743
in 21 linear steps. This method transforms cyanosafracin B into
intermediate 25 through a sequence of reactions that involves
essentially (1) removal of methoxy group placed in ring A, (2)
reduction of ring A and formation of methylene-dioxy group in one
pot, (3) hydrolysis of amide function placed over carbon 1, (4)
transformation of the resulting amine group into hydroxyl group.
Furthermore the method avoids protection and de-protection of the
primary alcohol function at the position 1 in ring B of compound 25
using directly a cysteine residue 29 to form intermediate 27.
Cysteine derivative 29 is protected in the amino group with
.beta.-.beta.-.beta.-trichloroethoxycarbonyl protecting group in
order to have compatibility with the existing allyl and MOM groups.
Intermediate 27 is directly oxidized and cycled. These
circumstances, together with a different de-protecting strategy in
the later stages of the synthesis makes the route novel and more
amenable to industrial development than the process of U.S. Pat.
No. 5,721,362.
[0158] The conversion of the 2-cyano compound into Intermediate 25
usually involves the following steps (see scheme II):
[0159] formation of the protected compound of Formula 14 by
reacting 2 with tert-butoxycarbonyl anhydride;
[0160] converting of 14 into the di-protected compound of Formula
15 by reacting with bromomethylmethyl ether and
diisopropylethylamine in acetonitrile;
[0161] selectively elimination of the methoxy group of the quinone
system in 15 to obtain the compound of Formula 16 by reacting with
a methanolic solution of sodium hydroxide;
[0162] transforming of 16 into the methylene-dioxy compound of
Formula 18 by employing the next preferred sequence: (1) quinone
group of compound 16 is reduced with 10% Pd/C under hydrogen
atmosphere; (2) the hydroquinone intermediate is converted into the
methylenedioxy compound of Formula 17 by reacting with
bromochloromethane and caesium carbonate under hydrogen atmosphere;
(3) 17 is transformed into the compound of Formula 18 by protecting
the free hydroxyl group as a OCH.sub.2R group. This reaction is
carried out with BrCH.sub.2R and caesium carbonate, where R can be
aryl, CH.dbd.CH.sub.2, OR etc.
[0163] elimination of the tert-butoxycarbonyl and the
methyloxymethyl protecting groups of 18 to afford the compound of
Formula 19 by reacting with a solution of HCl in dioxane. Also this
reaction is achieved by mixing 18 with a solution of
trifluoroacetic acid in dichloromethane;
[0164] formation of the thiourea compound of Formula 20 by reacting
19 with phenylisothiocyanate;
[0165] converting compound of Formula 20 into the amine compound of
Formula 21 by reacting with a solution of hydrogen chloride in
dioxane;
[0166] transforming compound of Formula 21 into the N-Troc
derivative 22 by reacting with trichloroethyl chloroformate and
pyridine;
[0167] formation of the protected hydroxy compound of Formula 23 by
reacting 22 with bromomethylmethyl ether and
diisopropylethylamine;
[0168] transforming compound of Formula 23 into the N--H derivative
24 by reacting with acetic acid and zinc;
[0169] conversion of compound of Formula 24 into the hydroxy
compound of Formula 25 by reaction with sodium nitrite in acetic
acid. Alternatively, it can be used nitrogen tetroxide in a mixture
of acetic acid and acetonitrile followed by treatment with sodium
hydroxide. Also, it can be used sodium nitrite in a mixture of
acetic anhydride-acetic acid, followed by treatment with sodium
hydroxide. 49505152
[0170] The conversion of the Intermediate 25 compound into ET-743
using cysteine derivative 29 usually involves the following steps
(see scheme III): 535455
[0171] transforming compound of formula 24 into the derivative 30
by protecting the primary hydroxyl function with
(S)-N-2,2,2-tricloroethoxyc-
arbonyl-S-(9H-fluoren-9-ylmethyl)cysteine 29;
[0172] converting the protected compound of formula 30 into the
phenol derivative 31 by cleavage of the allyl group with
tributyltin hydride and dichloropalladium-bis
(triphenylphosphine);
[0173] transforming the phenol compound of Formula 31 into compound
of formula 32 by oxidation with benzeneseleninic anhydride at low
temperature;
[0174] transforming the hydroxy compound of formula 32 into the
lactone 33 by the following sequence: (1) Reacting compound of
formula 32 with 2 eq. of triflic anhydride and 5 eq. of DMSO. (2)
followed by reaction with 8 eq. of diisopropylethylamine. (3)
followed by reaction with 4 eq of t-butyl alcohol (4) followed by
reaction with 7 eq of 2-tert-Butyl-1,1,3,3,tetramethylguanidine (5)
followed by reaction with 10 eq of acetic anhydride;
[0175] transforming the lactone compound 33 into hydroxyl compound
34 by removal of MOM protecting group with TMSI;
[0176] cleaving the N-trichloroethoxycarbonyl group of the compound
of formula 34 into compound 35 by reaction with Zn/AcOH;
[0177] transforming the amino compound 35 into the corresponding
.alpha.-keto lactone compound 36 by reaction with
N-methylpyridinium carboxaldehyde chloride followed by DBU;
[0178] forming ET-770 by reacting compound of Formula 36 with
3-hydroxy-4-methoxyphenylethylamine;
[0179] transforming ET-770 into ET-743 by reaction with silver
nitrate in a mixture of AcN/H.sub.2O.
[0180] Formation of Intermediate 11 and Related Intermediates
[0181] The retrosynthetic analysis is described in the following
sequence. 56
[0182] In the present invention, a key class of intermediate
includes Intermediate 11 and has the general formula (XXI): 57
[0183] where Prot.sup.1 and Prot.sup.2 are hydroxy protecting
groups, preferably different. For Intermediate 11 itself, the group
Prot.sup.1 is a methoxymethyl group, and Prot.sup.2 is a
t-butyldiphenylsilyl group.
[0184] The conversion of the 21-cyano compound to Intermediate 11
or a related intermediate of formula (XXI) usually involves the
following steps:
[0185] a) conversion if necessary of a quinone system for the ring
E into the phenol system
[0186] b) formation of the --OProt.sup.1 group at the 18-position,
in ring E;
[0187] c) formation of the --CH.sub.2--OProt.sup.2 group at the
1-position, in ring B; and
[0188] d) conversion if necessary of a quinone system for the ring
A into the phenol system;
[0189] e) conversion of the phenol system for the ring A into the
methylenedioxyphenol ring.
[0190] Step (b), formation of the --OProt.sup.1 group at the
18-position in ring E, is a typical protection reaction for a
phenol group, and no special comments need to be made. Appropriate
conditions are chosen depending on the nature of the protecting
group. The other steps are similar to the other reactions.
[0191] Step (b), formation of the --CH.sub.2--OProt.sup.2 group at
the 1-position in ring B, is normally carried out by forming a
group --CH.sub.2NH.sub.2 at the 1-position and then converting the
amine function to a hydroxy function and protecting. Thus, where
the starting material has a group R.sup.1 which is
--CH.sub.2--NH--CO--CR.sup.25aR.sup- .25bR.sup.25c then it is
matter of removing the N-acyl group. Where the starting material
has a group R.sup.1 which is --CH.sub.2--O--CO--R then no change
may be needed for an ecteinascidin product where the substituent
R.sup.1 is the same. For other products, it is matter of removing
the O-acyl group. Various procedures are available for such
de-acylations. In one variation, the deacylation and conversion to
a hydroxy function are performed in one step. Thereafter, the
hydroxy group can be acylated or otherwise converted to give the
appropriate R.sup.1 group.
[0192] U.S. Pat. No. 5,721,362 describe synthetic methods to make
ET-743 through a long multistep synthesis. One of the Intermediates
of this synthesis is Intermediate 11. Using cyanosafracin B as
starting material it is possible to reach Intermediate 11 providing
a much shorter way to make such Intermediate and therefor improving
the method to make ET-743
[0193] Cyanosafracin B can be converted into Intermediate 25 by the
methods described above. From Intermediate 25 is possible to reach
Intermediate 11 using the following steps, see scheme VII.
[0194] formation of the protected hydroxy compound of Formula 26 by
reacting 25 with tert-butyldiphenylsilyl chloride in the presence
of a base;
[0195] final cleavage of the allyl group with tributyltin hydride
and dichloropalladium-bis (triphenylphosphine) in 26 that leads to
the formation of the intermediate 11. 58
[0196] One embodiment of the synthetic process of the present
invention to transform safracin B into intermediate 11 is a
modification and extension of Scheme VIII and comprises the
sequential steps of:
[0197] stereospecifically converting the compound Safracin B to the
compound
[0198] of Formula 2 by selective replacement of OH by CN by
reacting with KCN in acid media;
[0199] forming the thiourea compound of Formula 3 by reacting
compound of Formula 2 with phenyl isothiocyanate;
[0200] converting the thiourea compound of Formula 3 into the
acetamide of Formula 5 by an hydrolysis in acid media followed by
addition of acetic anhydride; The intermediate amine compound of
Formula 4 can be isolated by quenching the hydrolysis in acid media
with sodium bicarbonate, but this intermediate is highly unstable,
and is transformed quickly into a five member cyclic imine, named
compound 6;
[0201] forming the protected compound of Formula 7 by reacting with
bromomethylmethyl ether and diusopropylethylamine in
dichloromethane;
[0202] selectively de-methylating the methoxy group of the quinone
system of compound of Formula 7 into the compound of Formula 8 by
reacting with methanolic solution of sodium hydroxide;
[0203] transforming the compound of Formula 8 into
methylenedioxy-compound of Formula 9 by the preferred following
sequence: (1) quinone group of compound 8 is reduced with 10% Pd/C
under hydrogen atmosphere; (2) the hydroquinone intermediate is
converted into the methylene-dioxy compound of Formula 9 by
reacting with bromochloromethane and cesium carbonate under
hydrogen atmosphere; (3) compound of Formula 9 is transformed into
compound of Formula 10 by protecting the free hydroxyl group as a
OCH.sub.2R group, by reacting with BrCH.sub.2R and cesium
carbonate, where R can be aryl, CH.dbd.CH.sub.2, OR' etc.;
[0204] converting the acetamide group of compound of Formula 10
into the corresponding hydroxyl group of Formula 25 by reaction
with nitrogen tetroxide in a mixture of acetic acid and acetic
acetate followed by treatment with sodium hydroxide; alternatively
can be used sodium nitrite in a mixture of acetic anhydride acetic
acid, followed by treatment with sodium hydroxide; alternatively
the acetamide group of compound of Formula 10 can be converted into
the primary amine group by reacting with hydrazine or with
Boc.sub.2O, DMAP followed by hydrazine; such primary amine can be
converted into the corresponding hydroxyl group (compound of
Formula 25) by an oxidative conversion of the primary amine into
the corresponding aldehyde with 4-formyl-1-methylpyridiniurm
benzenesulphonate or other pyridinium ion, followed by DBU or other
base treatment and further hydrolization, and followed by the
reduction of the aldehyde to the corresponding hydroxyl group with
lithium aluminium hydride or other reducing agent;
[0205] forming the protected compound of Formula 26 by reacting
with t-butyldiphenylsilyl chloride and dimethylaminopyridine in
dichloromethane (Scheme VII);
[0206] transforming the silylated compound of Formula 26 into the
intermediate 11 by deprotection of the OCH.sub.2R protecting group,
by reacting under reductive conditions or acid conditions. Typical
procedures are with palladium black under hydrogen atmosphere, or
aqueous TFA, or tributyltin hydride and dichlorobis
(triphenylphosphine palladium). 5960
[0207] In yet another preferred modification, the cyano compound of
Formula 2 can be transformed into Intermediate 11 using an
extension of the scheme II, involving the further steps of.
[0208] formation of the protected hydroxy compound of Formula 26 by
reacting 25 with tert-butyldiphenylsilyl chloride in the presence
of a base;
[0209] final cleavage of the allyl group with tributyltin hydride
and dichloropalladium-bis (triphenylphosphine) in 26 that leads to
the formation of the intermediate 11.
[0210] Thus, by these and other routes, it is possible to transform
cyanosafracin B into a number of intermediates and derivatives with
potential antitumor therapeutic activity. These intermediates can
be made starting from already described compounds, or using
alternative routes.
[0211] Novel Intermediate Compounds
[0212] In the light of the preceding explanations, it can be seen
that the present invention provides novel intermediate compounds.
Depending on ring A, the intermediates are of formula (XXIIa):
61
[0213] or of formula (XXIIb): 62
[0214] where:
[0215] R.sup.1 is --CH.sub.2NH.sub.2 or --CH.sub.2OH, or a
protected or derivatised version of such a group and R.sup.4 is
--H; or
[0216] R.sup.1a and R.sup.4 together form a group of formula (IV),
(V), (VI) or (VII): 63
[0217] R.sup.5 is --OH or a protected or derivatised version of
such a group;
[0218] R.sup.14a and R.sup.14b are both --H or one is --H and the
other is --OH or a protected or derivatised version of such a
group, --OCH.sub.3 or --OCH.sub.2CH.sub.3,
[0219] or R.sup.14a and R.sup.14b together form a keto group;
[0220] R.sup.12 is --H--, --CH.sub.3-- or --CH.sub.2CH.sub.3--;
[0221] R.sup.15 is --H, --OH or a protected or derivatised version
of such a group; and
[0222] R.sup.18 is --OH or a protected or derivatised version of
such a group.
[0223] In one embodiment, preferably at least of R.sup.1, R.sup.5,
R.sup.14a, R.sup.14b, R.sup.15 or R.sup.18 is a protected or
derivatised group.
[0224] In one variation of this invention, the group R.sup.1 is not
a tert-butyldiphenylsilyl substituent and/or the group R.sup.18 is
not a methoxymethyl group.
[0225] Preferably R.sup.1 is --CH.sub.2NH.sub.2 or --CH.sub.2OH, or
a protected or derivatised version of such a group and R.sup.4 is
--H; or
[0226] R.sup.1a and R.sup.4 together form a group: 64
[0227] Preferably R.sup.14a and R.sup.14b are both --H.
[0228] One preferred class of intermediates includes the compound
which we identify as compound 25, of formula: 65
[0229] The preferred class is thus of the general formula where the
group MOM is replaced by any other protecting group.
[0230] Other preferred intermediates includes the compounds which
we identify as compound 45 and 43 (Scheme IX). 66
[0231] Other N-acyl derivatives may readily be made from compound
45 and are an important part of this invention. Suitable acyl
groups include those previously mentioned. The corresponding
21-hydroxy compounds are also useful and are among the active
compounds which we have found.
[0232] Novel Active Compounds
[0233] We have additionally found that certain of the compounds of
the invention which we initially prepared as intermediates have
exceptional activity in the treatment of cancers, such as
leukaemias, lung cancer, colon cancer, kidney cancer and
melanoma.
[0234] Thus, the present invention provides a method of treating
any mammal, notably a human, affected by cancer which comprises
administering to the affected individual a therapeutically
effective amount of a compound of the invention, or a
pharmaceutical composition thereof.
[0235] The present invention also relates to pharmaceutical
preparations, which contain as active ingredient a compound or
compounds of the invention, as well as the processes for their
preparation.
[0236] Examples of pharmaceutical compositions include any solid
(tablets, pills, capsules, granules, etc.) or liquid (solutions,
suspensions or emulsions) with suitable composition or oral,
topical or parenteral administration, and they may contain the pure
compound or in combination with any carrier or other
pharmacologically active compounds. These compositions may need to
be sterile when administered parenterally.
[0237] Administration of the compounds or compositions of the
present invention may be by any suitable method, such as
intravenous infusion, oral preparations, intraperitoneal and
intravenous administration. We prefer that infusion times of up to
24 hours are used, more preferably 2-12 hours, with 2-6 hours most
preferred. Short infusion times which allow treatment to be carried
out without an overnight stay in hospital are especially desirable.
However, infusion may be 12 to 24 hours or even longer if required.
Infusion may be carried out at suitable intervals of say 2 to 4
weeks. Pharmaceutical compositions containing compounds of the
invention may be delivered by liposome or nanosphere encapsulation,
in sustained release formulations or by other standard delivery
means.
[0238] The correct dosage of the compounds will vary according to
the particular formulation, the mode of application, and the
particular situs, host and tumour being treated. Other factors like
age, body weight, sex, diet, time of administration, rate of
excretion, condition of the host, drug combinations, reaction
sensitivities and severity of the disease shall be taken into
account. Administration can be carried out continuously or
periodically within the maximum tolerated dose.
[0239] The compounds and compositions of this invention may be used
with other drugs to provide a combination therapy. The other drugs
may form part of the same composition, or be provided as a separate
composition for administration at the same time or a different
time. The identity of the other drug is not particularly limited,
and suitable candidates include:
[0240] a) drugs with antimitotic effects, especially those which
target cytoskeletal elements, including microtubule modulators such
as taxane drugs (such as taxol, paclitaxel, taxotere, docetaxel),
podophylotoxins or vinca alkaloids (vincristine, vinblastine);
[0241] b) antimetabolite drugs such as 5-fluorouracil, cytarabine,
gemcitabine, purine analogues such as pentostatin,
methotrexate);
[0242] c) alkylating agents such as nitrogen mustards (such as
cyclophosphamide or ifosphamide);
[0243] d) drugs which target DNA such as the antracycline drugs
adriamycin, doxorubicin, pharmorubicin or epirubicin;
[0244] e) drugs which target topoisomerases such as etoposide;
[0245] f) hormones and hormone agonists or antagonists such as
estrogens, antiestrogens (tamoxifen and related compounds) and
androgens, flutamide, leuprorelin, goserelin, cyprotrone or
octreotide;
[0246] g) drugs which target signal transduction in tumour cells
including antibody derivatives such as herceptin;
[0247] h) alkylating drugs such as platinum drugs (cis-platin,
carbonplatin, oxaliplatin, paraplatin) or nitrosoureas;
[0248] i) drugs potentially affecting metastasis of tumours such as
matrix metalloproteinase inhibitors;
[0249] j) gene therapy and antisense agents;
[0250] k) antibody therapeutics;
[0251] l) other bioactive compounds of marine origin, notably the
didemnins such as aplidine;
[0252] m) steroid analogues, in particular dexamethasone;
[0253] n) anti-inflammatory drugs, in particular dexamethasone;
and
[0254] o) anti-emetic drugs, in particular dexamethasone.
[0255] The present invention also extends to the compounds of the
invention for use in a method of treatment, and to the use of the
compounds in the preparation of a composition for treatment of
cancer.
[0256] Cytotoxic Activity
[0257] Cell Cultures. Cells were maintained in logarithmic phase of
growth in Eagle's Minimum Essential Medium, with Earle's Balanced
Salts, with 2.0 mM L-glutamine, with non-essential amino acids,
without sodium bicarbonate (EMEM/neaa); supplemented with 10% Fetal
Calf Serum (FCS), 10.sup.-2 M sodium bicarbonate and 0.1 g/l
pencillin-G+streptomycin sulfate.
[0258] A simple screening procedure has been carried out to
determine and compare the antitumour activity of these compounds,
using an adapted form of the method described by Bergeron et al
(1984). The tumour cell line employed have been P-388 (suspension
culture of a lymphoid neoplasm from DBA/2 mouse), A-549 (monolayer
culture of a human lung carcinoma), HT-29 (monolayer culture of a
human colon carcinoma) and MEL-28 (monolayer culture of a human
melanoma).
[0259] P-388 cell were seeded into 16 mm wells at 1.times.10.sup.4
cells per well in 1 ml aliquots of MEM 5FCS containing the
indicated concentration of drug. A separate set of cultures without
drug was seeded as control growth to ensure that cells remained in
exponential phase of growth. All determinations were carried out in
duplicate. After three days of incubation at 37.degree. C., 10%
CO.sub.2 in a 98% humid atmosphere, an approximately IC.sub.50 was
determined by comparing the growth in wells with drug to the growth
in wells control.
[0260] A-549, HT-29 and MEL-28 were seeded into 16 mm wells at
2.times.10.sup.4 cells per well in 1 ml aliquots of MEM 10FCS
containing the indicated concentration of drug. A separate set of
cultures without drug was seeded as control growth to ensure that
cells remained in exponential phase of growth. All determinations
were carried out in duplicate. After three days of incubation at
37.degree. C., 10% CO.sub.2 in a 98% humid atmosphere, the wells
were stained with 0.1% Crystal Violet. An approximately IC.sub.50
was determined by comparing the growth in wells with drug to the
growth in wells control.
[0261] 1. Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J.
Kline. Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter.
Antineoplastic and antiherpetic activity of spermidine
catecholamide iron chelators. Biochem. Bioph Res. Comm. 1984,
121(3), 848-854.
[0262] 2. Alan C. Schroeder, Robert G. Hughes, Jr. and Alexander
Bloch. Effects of Acyclic Pyrimidine Nucleoside Analoges. J. Med.
Chem. 1981, 24 1078-1083.
7 Cytotoxic activity IC.sub.50 (.mu.M) Compound P-388 A-549 HT-29
MEL-28 CV-1 DU-145 67 0.009 0.018 0.018 0.018 0.023 68 0.15
>0.15 0.15 >0.15 69 1.44 1.44 1.44 1.44 70 >1.5 >1.5
>1.5 >1.5 71 1.4 1.4 1.4 1.4 72 0.01 0.01 0.01 0.01 73 0.08
0.16 0.01 0.16 74 0.01 0.01 0.01 0.01 75 0.019 0.019 0.019 0.019 76
0.014 0.014 0.014 0.014 0.014 0.014 77 0.13 0.13 0.13 0.13 0.13
0.13 78 0.18 1.8 1.8 1.18 1.8 1.8 79 0.2 0.2 0.2 0.2 0.2 80 0.008
0.008 0.008 0.008 81 0.01 0.01 0.01 0.01 82 0.001 0.001 0.001 0.001
0.001 0.001 83 0.13 0.13 0.13 0.13 0.13 84 0.008 0.016 0.008 0.008
0.016 85 0.001 0.001 0.001 0.001 0.001 86 0.01 0.01 0.01 0.01 0.01
87 0.015 0.015 0.015 0.015 0.018 88 2.171 2.171 2.171 2.171 2.171
89 0.005 0.005 0.005 0.005 90 0.22 0.22 0.22 0.22 0.22 91 >9
>18.1 >18.1 >18.1 >18.1 92 >1.77 >1.77 >1.77
>1.77 >1.77 93 >1.65 >1.65 >1.65 >1.65 >1.65
94 0.016 0.016 0.016 0.016 0.016 95 0.001 0.001 0.001 0.001 0.001
96 0.0008 0.001 0.0008 0.0008 0.001 97 0.007 0.007 0.007 0.007
0.007 98 0.0001 0.0001 0.0001 0.0001 0.0001 99 0.0001 0.0001 0.0001
0.0001 0.0001 100 0.001 0.001 0.001 0.001 0.001 101 0.0001 0.0001
0.0001 0.0001 0.0001 102 0.001 0.001 0.001 0.001 0.001 103 0.01
0.01 0.01 0.01 0.01 104 0.18 0.9 0.18 0.8 0.9 105 0.14 0.14 0.14
0.14 0.14 106 0.001 0.001 0.001 0.001 0.001 107 0.001 0.001 0.0005
0.001 0.0005 108 0.001 0.001 0.001 0.001 0.001 109 0.001 0.001
0.0005 0.0005 0.001 110 0.0001 0.0001 0.0001 0.0001 0.0001 111
0.001 0.001 0.001 0.001 0.001 112 0.0001 0.0005 0.0001 0.0001
0.0005 113 0.0001 0.0001 0.0001 0.0001 0.0001 114 0.0001 0.0001
0.0001 0.0001 0.0001 115 >1 >1 116 >1 >1 117 0.19 0.19
0.19 0.19 118 0.0055 0.0055 119 >1 >1 120 0.01 0.01 121 0.051
0.051 122 0.012 0.012 123 0.11 0.11 124 >1 >1 125 >1 >1
126 0.59 0.59 127 0.0013 0.0013 128 0.00015 0.00015 129 >1 >1
130 >1 >1 131 >1 >1 132 >1 >1 133 >1 >1 134
>1 >1 135 0.012 0.012 136 >1 >1 137 0.062 0.062
[0263] The active compounds of this invention thus include
compounds with the 10-hydroxy group and the 1-labile group.
[0264] An important method of this invention includes the reaction:
138
[0265] Another important method of this invention includes the
reaction: 139
[0266] Another important method of this invention includes the
reaction includes the reaction where a group R.sup.1 is
aminomethylene is converted to a hydroxymethylene group.
[0267] Another important method of this invention includes the
reaction wherein a compound with a group R.sup.1 which is
hydroxymethylene is reacted with a reagent of the formula (XIX)
140
[0268] where Fu indicates a protected functional group, Prot.sup.3
is a protecting group, and the dotted line shows an optional double
bond.
[0269] Another important method of this invention includes the
reaction for preparing a 21-cyano compound of formula (XVI) which
comprises reacting a compound of formula (XV): 141
[0270] where R.sup.1, R.sup.5, R.sup.8, R.sup.14a, R.sup.14b
R.sup.15 and R.sup.18 are as defined and R.sup.21 is a hydroxy
group, with a source of cyanide ion, to give the desired 21-cyano
compound.
[0271] In addition, processes using other nucleophile-containing
compounds, to produce similar compounds of formula (XVI) wherein
the 21-position is protected by another nucleophilic group, a
21-Nuc group, are also envisaged. For example, a 2 l-Nuc compound
of formula (XVI) with an alkylamino substituent at the 21-position
can be produced by reacting the compound of formula (XV) wherein
R.sup.21 is a hydroxy group with a suitable alkylamine. A 21-Nuc
compound of formula (XVI) with an alkylthio substituent at the
21-position can also be produced by reacting the compound of
formula (XV) wherein R.sup.21 is a hydroxy group with a suitable
alkanethiol. Alternatively, a 21-Nuc compound of formula (XVI) with
an .quadrature.-carbonylalkyl substituent at the 21-position can be
produced by reacting the compound of formula (XV) wherein R.sup.21
is a hydroxy group with a suitable carbonyl compound, typically in
the presence of a base. Other synthetic routes are available for
other 21-Nuc compounds.
[0272] Another important reaction of this invention involves
treatment of a 21-cyano product of this invention to form a
21-hydroxy compound. Such compounds have interesting in vivo
properties.
EXAMPLES
[0273] The present invention is illustrated by the following
examples.
Example 1
[0274] 142
[0275] To a solution of 2 (21.53 g, 39.17 mmol) in ethanol (200
ml), tert-butoxycarbonyl anhydride (7.7 g, 35.25 mmol) was added
and the mixture was stirred for 7 h at 23.degree. C. Then, the
reaction was concentrated in vacuo and the residue was purified by
flash column chromatography (SiO.sub.2, hexane:ethyl acetate 6:4)
to give 14 (20.6 g, 81%) as a yellow solid.
[0276] Rf: 0.52 (ethyl acetate:CHCl.sub.3 5:2).
[0277] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.49 (s, 1H),
6.32 (bs, 1H), 5.26 (bs, 1H), 4.60 (bs, 1H), 4.14 (d, J=2.4 Hz,
1H), 4.05 (d, J=2.4 Hz, 1H), 3.94 (s, 3H), 3.81 (d, J=4.8 Hz, 1H),
3.7 (s, 3H), 3.34 (br d, J=7.2 Hz, 1H), 3.18-3.00 (m, 5H), 2.44 (d,
J=18.3 Hz, 1H), 2.29 (s, 3H), 2.24 (s, 3H), 1.82 (s, 3H), 1.80-1.65
(m, 1H), 1.48 (s, 9H), 0.86 (d, J=5.7 Hz, 3H)
[0278] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 185.5, 180.8,
172.7, 155.9, 154.5, 147.3, 143.3, 141.5, 135.3, 130.4, 129.2,
127.5, 120.2, 117.4, 116.9, 80.2, 60.7, 60.3, 58.5, 55.9, 55.8,
54.9, 54.4, 50.0, 41.6, 40.3, 28.0, 25.3, 24.0, 18.1, 15.6,
8.5.
[0279] ESI-MS m/z: Calcd. for C.sub.34H.sub.43N.sub.5O.sub.8:
649.7. Found (M+H).sup.+: 650.3.
Example 2
[0280] 143
[0281] To a stirred solution of 14 (20.6 g, 31.75 mmol) in
CH.sub.3CN (159 ml), diisopropylethylamine (82.96 ml, 476.2 mmol),
methoxymethylene bromide (25.9 ml, 317.5 mmol) and
dimethylaminopyridine (155 mg, 1.27 mmol) were added at 0.degree.
C. The mixture was stirred at 23.degree. C. for 24 h. The reaction
was quenched at 0.degree. C. with aqueous 0.1N HCl (750 ml) (pH=5),
and extracted with CH.sub.2Cl.sub.2 (2.times.400 ml). The organic
phase was dried (sodium sulphate) and concentrated in vacuo. The
residue was purified by flash column chromatography (SiO.sub.2,
gradient hexane:ethyl acetate 4:1 to hexane:ethyl acetate 3:2) to
give 15 (17.6 g, 83%) as a yellow solid.
[0282] Rf: 0.38 (hexane:ethyl acetate 3:7).
[0283] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.73 (s, 1H),
5.35 (bs, 1H), 5.13 (s, 2H), 4.50 (bs, 1H), 4.25 (d, J=2.7 Hz, 1H),
4.03 (d, J=2.7 Hz, 1H), 3.97 (s, 3H), 3.84 (bs, 1H), 3.82-3.65 (m,
1H), 3.69 (s, 3H), 3.56 (s, 3H), 3.39-3.37 (m, 1H), 3.20-3.00 (m,
5H), 2.46 (d, J=18 Hz, 1H), 2.33 (s, 3H), 2.23 (s, 3H), 1.85 (s,
3H), 1.73-1.63 (m, 1H), 1.29 (s, 9H), 0.93 (d, J=5.1 Hz, 3H)
[0284] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 185.4, 180.9,
172.4, 155.9, 154.5, 149.0, 148.4, 141.6, 135.1, 131.0, 129.9,
127.6, 124.4, 123.7, 117.3, 99.1, 79.3, 60.7, 59.7, 58.4, 57.5,
56.2, 55.9, 55.0, 54.2, 50.0, 41.5, 39.9, 28.0, 25.2, 24.0, 18.1,
15.6, 8.5.
[0285] ESI-MS m/z: Calcd. for C.sub.36H.sub.47N.sub.5O.sub.9:
693.8. Found (M+H).sup.+: 694.3.
Example 3
[0286] 144
[0287] To a flask containing 15 (8 g, 1.5 ml) in methanol (1.6 l)
an aqueous solution of 1M sodium hydroxide (3.2 l) was added at
0.degree. C. The reaction was stirred for 2 h at this temperature
and then, quenched with 6M HCl to pH=5. The mixture was extracted
with ethyl acetate (3.times.11) and the combined organic layers
were dried over sodium sulphate and concentrated in vacuo. The
residue was purified by flash column chromatography (SiO.sub.2,
gradient CHCl.sub.3 to CHCl.sub.3:ethyl acetate 2:1) to afford 16
(5.3 mg, 68%).
[0288] Rf: 0.48 (CH.sub.3CN:H.sub.2O 7:3, RP-C18)
[0289] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.73 (s, 1H),
5.43 (bs, 1H), 5.16 (s, 2H), 4.54 (bs, 1H), 4.26 (d, J=1.8 Hz, 1H),
4.04 (d, J=2.7 Hz 1H), 3.84 (bs, 1H), 3.80-3.64 (m, 1H), 3.58 (s,
3H), 3.41-3.39 (m, 1H), 3.22-3.06 (m, 5H), 2.49 (d, J=18.6 Hz 1H),
2.35 (s, 3H), 2.30-2.25 (m, 1H), 2.24 (s, 3H), 1.87 (s, 3H),
1.45-1.33 (m, 1H), 1.19 (s, 9H), 1.00 (br d, J=6.6 Hz 3H)
[0290] .sup.13C NMR (75 MHz, .quadrature.CDCl.sub.3):.delta. 184.9,
180.9, 172.6, 154.7, 151.3, 149.1, 148.6, 144.7, 132.9, 131.3,
129.8, 124.5, 123.7, 117.3, 116.8, 99.1, 79.4, 59.8, 58.6, 57.7,
56.2, 55.6, 54.9, 54.5, 50.1, 41.6, 40.1, 28.0, 25.3, 24.4, 18.1,
15.7, 8.0.
[0291] ESI-MS m/z: Calcd. for C.sub.35H.sub.45N.sub.5O.sub.9:
679.7. Found (M+H).sup.+: 680.3.
Example 4
[0292] 145
[0293] To a degassed solution of compound 16 (1.8 g, 2.64 mmol) in
DMF (221 ml) 10% Pd/C (360 mg) was added and stirred under H.sub.2
(atmospheric pressure) for 45 min. The reaction was filtered
through celite under argon, to a flask containing anhydrous
CS.sub.2CO.sub.3 (2.58 g, 7.92 mmol). Then, bromochloromethane
(3.40 ml 52.8 mmol), was added and the tube was sealed and stirred
at 100.degree. C. for 2 h. The reaction was cooled, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2. The
organic layer was concentrated and dried (sodium sulphate) to
afford 17 as a brown oil that was used in the next step with no
further purification.
[0294] Rf: 0.36 (hexane:ethyl acetate 1:5, SiO.sub.2).
[0295] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.68 (s, 1H),
6.05 (bs, 1H), 5.90 (s, 1H), 5.79 (s, 1H), 5.40 (bs, 1H), 5.31-5.24
(m, 2H), 4.67 (d, J=8.1 Hz, 1H), 4.19 (d, J=2.7 Hz, 1H), 4.07 (bs,
1H), 4.01 (bs, 1H), 3.70 (s, 3H), 3.67 (s, 3H), 3.64-2.96 (m, 5H),
2.65 (d, J=18.3 Hz, 1H), 2.33 (s, 3H), 2.21 (s, 3H), 2.04 (s, 3H),
2.01-1.95 (m, 1H), 1.28 (s, 9H), 0.87 (d, J=6.3 Hz, 3H)
[0296] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 172.1, 162.6,
154.9, 149.1, 145.7, 135.9, 130.8, 130.7, 125.1, 123.1, 117.8,
100.8, 99.8, 76.6, 59.8, 59.2, 57.7, 57.0, 56.7, 55.8, 55.2, 49.5,
41.6, 40.1, 36.5, 31.9, 31.6, 29.7, 28.2, 26.3, 25.0, 22.6, 18.2,
15.8, 14.1, 8.8.
[0297] ESI-MS m/z: Calcd. for C.sub.36H.sub.47N.sub.5O.sub.9:
693.34. Found (M+H).sup.+: 694.3.
Example 5
[0298] 146
[0299] To a flask containing a solution of 17 (1.83 g, 2.65 mmol)
in DMF (13 ml), Cs.sub.2CO.sub.3 (2.6 g, 7.97 mmol), and allyl
bromide (1.15 ml, 13.28 mmol) were added at 0.degree. C. The
resulting mixture was stirred at 23.degree. C. for 1 h. The
reaction was filtered through a pad of celite and washed with
CH.sub.2Cl.sub.2. The organic layer was dried and concentrated
(sodium sulphate). The residue was purified by flash column
chromatography (SiO.sub.2, CHCl.sub.3:ethyl acetate 1:4) to afford
18 (1.08 mg, 56%) as a white solid.
[0300] Rf: 0.36 (CHCl.sub.3:ethyl acetate 1:3).
[0301] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.70 (s, 1H),
6.27-6.02 (m, 1H), 5.94 (s, 1H), 5.83 (s, 1H), 5.37 (dd,
J.sub.1=1.01 Hz, J.sub.2=16.8 Hz, 1H), 5.40 (bs, 1H), 5.25 (dd,
J.sub.1=1.0 Hz, J.sub.2=10.5 Hz, 1H), 5.10 (s, 2H), 4.91 (bs, 1H),
4.25-4.22 (m, 1H), 4.21 (d, J=2.4 Hz, 1H), 4.14-4.10 (m, 1H), 4.08
(d, J=2.4 Hz, 1H), 4.00 (bs, 1H), 3.70 (s, 3H), 3.59 (s, 3H),
3.56-3.35 (m, 2H), 3.26-3.20 (m, 2H), 3.05-2.96 (dd, J.sub.1=8.1
Hz, J.sub.2=18 Hz, 1H), 2.63 (d, J=18 Hz, 1H), 2.30 (s, 3H), 2.21
(s, 3H), 2.09 (s, 3H), 1.91-1.80 (m, 1H), 1.24 (s, 9H), 0.94 (d,
J=6.6 Hz, 3H)
[0302] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 172.0, 154.8,
148.8, 148.6, 148.4, 144.4, 138.8, 133.7, 130.9, 130.3, 125.1,
124.0, 120.9, 117.8, 117.4, 112.8, 112.6, 101.1, 99.2, 73.9, 59.7,
59.3, 57.7, 56.9, 56.8, 56.2, 55.2, 40.1, 34.6, 31.5, 28.1, 26.4,
25.1, 22.6, 18.5, 15.7, 14.0, 9.2.
[0303] ESI-MS m/z: Calcd. for C.sub.39H.sub.51N.sub.5O.sub.9:
733.4. Found (M+H).sup.+: 734.4.
Example 6
[0304] 147
[0305] To a solution of 18 (0.1 g, 0.137 mmol) in dioxane (2 ml),
4.2M HCl/dioxane (1.46 ml) was added and the mixture was stirred
for 1.2 h at 23.degree. C. The reaction was quenched at 0.degree.
C. with sat. Aqueous sodium bicarbonate (60 ml) and extracted with
ethyl acetate (2.times.70 ml). The organic layers were dried
(sodium sulphate) and concentrated in vacuo to afford 19 (267 mg,
95%) as a white solid that was used in subsequent reactions with no
further purification.
[0306] Rf: 0.17 (ethyl acetate:meihanol 10:1, SiO.sub.2)
[0307] .sup.1H NMR (300 MHz, CDCl.sub.3): S 6.49 (s, 1H),
6.12-6.00.(m, 1H), 5.94 (s, 1H), 5.86 (s, 1H), 5.34 (dd, J=1.0 Hz,
J=17.4 Hz, 1H), 5.25 (dd, J=1.0 Hz, J=10.2 Hz, 1H), 4.18-3.76 (m,
5H), 3.74 (s, 3H), 3.71-3.59 (m, 1H), 3.36-3.20 (m, 4H), 3.01-2.90
(m, 1H), 2.60 (d, J=18.0 Hz, 1H), 2.29 (s, 3H), 2.24 (s, 3H), 2.11
(s, 3H), 1.97-1.86 (m, 1H), 0.93 (d, J=8.7 Hz, 3H)
[0308] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 175.5, 148.4,
146.7, 144.4, 142.4, 138.9, 133.7, 131.3, 128.3, 120.8, 117.9,
117.4, 113.8, 112.4, 101.1, 74.2, 60.5, 59.1, 56.5, 56.1, 56.3,
56.0, 55.0, 50.5, 41.6, 39.5, 29.5, 26.4, 24.9, 21.1, 15.5,
9.33.
[0309] ESI-MS m/z: Calcd. for C.sub.32H.sub.39N.sub.5O.sub.6: 589.
Found (M+H).sup.+: 590.
Example 7
[0310] 148
[0311] To a solution of 19 (250 mg, 0.42 mmol) in CH.sub.2Cl.sub.2
(1.5 mmol), phenyl isothiocyanate (0.3 ml, 2.51 mmol) was added and
the mixture was stirred at 23.degree. C. for 1 h. The reaction was
concentrated in vacuo and the residue was purified by flash column
chromatography (SiO.sub.2, gradient Hexane to 5:1 hexane:ethyl
acetate) to afford 20 (270 mg, 87%) as a white solid.
[0312] Rf: 0.56 (CHCl.sub.3:ethyl acetate 1:4).
[0313] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 8.00 (bs, 1H),
7.45-6.97 (m, 4H), 6.10 (s, 1H), 6.08-6.00 (m, 1H), 5.92 (s, 1H),
5.89 (s, 1H), 5.82 (s, 1H), 5.40 (dd, J=1.5 Hz, J=17.1 Hz, 1H),
3.38 (bs, 1H), 5.23 (dd, J=1.5 Hz, J=10.5 Hz, 1H), 4.42-4.36 (m,
1H), 4.19-4.03 (m, 5H), 3.71 (s, 3H), 3.68-3.17 (m, 4H), 2.90 (dd,
J=7.8 Hz, J=18.3 Hz, 1H), 2.57 (d, J=18.3 Hz, 1H), 2.25 (s, 3H),
2.12 (s, 3H), 2.10 (s, 3H), 1.90 (dd, J=12.3 Hz, J=16.5 Hz, 1H),
0.81 (d, J=6.9 Hz, 3H).
[0314] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 178.4, 171.6,
148.6, 146.8, 144.3, 142.7, 138.7, 136.2, 133.6, 130.7, 129.8,
126.6, 124.2, 124.1, 120.9, 120.5, 117.7, 117.4, 116.7, 112.6,
112.5, 101.0, 74.0, 60.6, 59.0, 57.0, 56.2, 56.1, 55.0, 53.3, 41.4,
39.7, 26.3, 24.8, 18.3, 15.5, 9.2.
[0315] ESI-MS m/z: Calcd. for C.sub.39H.sub.44N.sub.6O.sub.6S:
724.8 Found (M+H).sup.+: 725.3.
Example 8
[0316] 149
[0317] To a solution of 20 (270 mg, 0.37 mmol) in dioxane (1 ml),
4.2N HCl/dioxane (3.5 ml) was added and the reaction was stirred at
23.degree. C. for 30 min. Then, ethyl acetate (20 ml) and H.sub.2O
(20 ml) were added and the organic layer was decanted. The aqueous
phase was basified with saturated aqueous sodium bicarbonate (60
ml) (pH=8) at 0.degree. C. and then, extracted with
CH.sub.2Cl.sub.2 (2.times.50 ml). The combined organic extracts
were dried (sodium sulphate), and concentrated in vacuo. The
residue was purified by flash column chromatography (SiO.sub.2,
ethyl acetate:methanol 5:1) to afford compound 21 (158 mg, 82%) as
a white solid.
[0318] Rf: 0.3 (ethyl acetate:methanol 1:1).
[0319] .sup.1H NMR (300 MHz, CDCl.sub.3): S 6.45 (s, 1H), 6.12-6.03
(m, 1H), 5.91 (s, 1H), 5.85 (s, 1H), 5.38 (dd, J.sub.1=1.2 Hz,
J.sub.2=17.1 Hz, 1H), 5.24 (dd, J.sub.1=1.2 Hz, J.sub.2=10.5 Hz,
1H), 4.23-4.09 (m, 4H), 3.98 (d, J=2.1 Hz, 1H), 3.90 (bs, 1H), 3.72
(s, 3H), 3.36-3.02 (m, 5H), 2.72-2.71 (m, 2H), 2.48 (d, J=18.0 Hz,
1H), 2.33 (s, 3H), 2.22 (s, 3H), 2.11 (s, 3H), 1.85 (dd,
J.sub.1=11.7 Hz, J.sub.2=15.6 Hz, 1H)).
[0320] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 148.4, 146.7,
144.4, 142.8, 138.8, 133.8, 130.5, 128.8, 121.5, 120.8, 118.0,
117.5, 116.9, 113.6, 112.2, 101.1, 74.3, 60.7, 59.9, 58.8, 56.6,
56.5, 55.3, 44.2, 41.8, 29.7, 26.5, 25.7, 15.7, 9.4.
[0321] ESI-MS m/z: Calcd. for C.sub.29H.sub.34N.sub.4O.sub.5:
518.3. Found (M+H).sup.+: 519.2.
Example 9
[0322] 150
[0323] To a solution of 21 (0.64 g, 1.22 mmol) in CH.sub.2Cl.sub.2
(6.13 ml), pyridine (0.104 ml, 1.28 mmol) and 2,2,2-trichloroethyl
chloroformate (0.177 ml, 1.28 mmol) were added at -10.degree. C.
The mixture was stirred at this temperature for 1 h and then, the
reaction was quenched by addition of 0.1N HCl (10 ml) and extracted
with CH.sub.2Cl.sub.2 (2.times.10 ml). The organic layer was dried
over sodium sulphate and concentrated in vacuo. The residue was
purified by flash column chromatography (SiO.sub.2, (hexane:ethyl
acetate 1:2) to afford 22 (0.84 g, 98%) as a white foam solid.
[0324] Rf: 0.57 (ethyl acetate:methanol 5:1).
[0325] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.50 (s, 1H),
6.10-6.00 (m, 1H), 6.94 (d, J=1.5 Hz, 1H), 5.87 (d, J=1.5 Hz, 1H),
5.73 (bs, 1H), 5.37 (dq, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.26
(dq, J.sub.1=1.8 Hz, J.sub.2=10.2 Hz, 1H), 4.60 (d, J=12 Hz, 1H),
4.22-4.10 (m, 4H), 4.19 (d, J=12 Hz, 1H), 4.02 (m, 2H), 3.75 (s,
3H), 3.37-3.18 (m, 5H), 3.04 (dd, J.sub.1=8.1 Hz, J.sub.2=18 Hz,
1H), 2.63 (d, J=18 Hz, 1H), 2.31 (s, 3H), 2.26 (s, 3H), 2.11 (s,
3H), 1.85 (dd, J.sub.1=12.3 Hz, J.sub.2=15.9 Hz, 1H).
[0326] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 154.3, 148.5,
146.7, 144.5, 142.8, 139.0, 133.8, 130.7, 128.7, 121.3, 120.8,
117.8, 117.7, 116.8, 112.7, 101.2, 77.2, 74.3, 60.7, 59.9, 57.0,
56.4, 55.3, 43.3, 41.7, 31.6, 26.4, 25.3, 22.6, 15.9, 14.1,
9.4.
[0327] ESI-MS m/z: Calcd. for
C.sub.32H.sub.35Cl.sub.3N.sub.4O.sub.7: 694.17. Found (M+H).sup.+:
695.2.
Example 10
[0328] 151
[0329] To a solution of 22 (0.32 g, 0.46 mmol) in CH.sub.3CN (2.33
ml), diisopropylethylamine (1.62 ml, 9.34 mmol), bromomethyl methyl
ether (0.57 ml, 7.0 mmol) and dimethylaminopyridine (6 mg, 0.046
mmol) were added at 0.degree. C. The mixture was heated at
30.degree. C. for 10 h. Then, the reaction was diluted with
dichloromethane (30 ml) and poured in an aqueous solution of HCl at
pH=5 (10 ml). The organic layer was dried over sodium sulphate and
the solvent was eliminated under reduced pressure to give a residue
which was purified by flash column chromatography (SiO.sub.2,
hexane:ethyl acetate 2:1) to afford 23 (0.304 g, 88%) as a white
foam solid.
[0330] Rf: 0.62 (hexane:ethyl acetate 1:3).
[0331] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.73 (s, 1H),
6.10 (m, 1H), 5.94 (d, J=1.5 Hz, 1H), 5.88 (d, J=1.5 Hz, 1H), 5.39
(dq, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.26 (dq, J.sub.1=1.8
Hz, J.sub.2=10.2 Hz, 1H), 5.12 (s, 2H), 4.61 (d, J=12 Hz, 1H), 4.55
(t, J=6.6 Hz, 1H), 4.25 (d, J=12 Hz, 1H), 4.22-4.11 (m, 4H), 4.03
(m, 2H), 3.72 (s, 3H), 3.58 (s, 3H), 3.38-3.21 (m, 5H), 3.05 (dd,
J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H), 2.65 (d, J=18 Hz, 1H), 2.32 (s,
3H), 2.23 (s, 3H), 2.12 (s, 3H), 1.79 (dd, J.=12.3 Hz, J.sub.2=15.9
Hz, 1H);
[0332] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 154.3, 148.6,
148.4, 144.5, 139.0, 133.6, 130.6, 130.1, 125.07, 124.7, 124.0,
121.1, 117.7, 112.6, 101.2, 99.2, 77.2, 74.4, 74.1, 59.8, 59.8,
57.7, 57.0, 56.8, 56.68, 55.3, 43.2, 41.5, 26.4, 25.2, 15.9,
9.3.
[0333] ESI-MS m/z: Calcd. for
C.sub.34H.sub.39Cl.sub.3N.sub.4O.sub.8: 738.20. Found (M+H).sup.+:
739.0.
Example 11
[0334] 152
[0335] To a suspension of 23 (0.304 g, 0.41 mmol) in 90% aqueous
acetic acid (4 ml), powder zinc (0.2 g, 6.17 mmol) was added and
the reaction was stirred for 7 hour at 23.degree. C. The mixture
was filtered through a pad of celite which was washed with
CH.sub.2Cl.sub.2. The organic layer was washed with an aqueous sat.
solution of sodium bicarbonate (pH=9) (15 ml) and dried over sodium
sulphate. The solvent was eliminated under reduced pressure to give
24 (0.191 g, 83%) as a white solid.
[0336] Rf: 0.3 (ethyl acetate:methanol 5:1).
[0337] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.68 (s, 1H),
6.09 (m, 1H), 5.90 (d, J=1.5 Hz, 1H), 5.83 (d, J=1.5 Hz, 1H), 5.39
(dq, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.25 (dq, J.sub.1=1.5
Hz, J.sub.2=10.2 Hz, 1H), 5.10 (s, 2H), 4.22-4.09 (m, 3H), 3.98 (d,
J=2.4 Hz, 1H), 3.89 (m, 1H), 3.69 (s, 3H), 3.57 (s, 3H), 3.37-3.17
(m, 3H), 3.07 (dd, J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H), 2.71 (m,
2H), 2.48 (d, J=18 Hz, 1H), 2.33 (s, 3H), 2.19 (s, 3H), 2.17 (s,
3H), 1.80 (dd, J.=12.3 Hz, J.sub.2=15.9 Hz, 1H)
[0338] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 148.5, 148.2,
144.3, 138.7, 133.7, 130.7, 129.9, 125.0, 123.9, 121.3, 117.9,
117.5, 113.6, 112.0, 101.0, 99.2, 74.0, 59.8, 59.7, 58.8, 57.6,
57.0, 56.2, 55.2, 44.2, 41.5, 31.5, 26.4, 25.6, 22.5, 16.7, 14.0,
9.2.
[0339] ESI-MS m/z: Calcd. for C.sub.31H.sub.38N.sub.4O.sub.6:
562.66. Found (M+H).sup.+: 563.1.
Example 12
[0340] 153
[0341] To a solution of 24 (20 mg, 0.035 mmol), in H.sub.2O (0.7
mmol) and THF (0.7 mmol), NaNO.sub.2 (12 mg, 0.17 mmol) and 90%
aqueous AcOH (0.06 ml) were added at 0.degree. C. and the mixture
was stirred at 0.degree. C. for 3 h. After dilution with
CH.sub.2Cl.sub.2 (5 ml), the organic layer was washed with water (1
ml), dried over sodium sulphate and concentrated in vacuo. The
residue was purified by flash column chromatography (SiO.sub.2,
hexane:ethyl acetate 2:1) to afford 25 (9.8 mg, 50%) as a white
solid.
[0342] Rf: 0.34 (hexane:ethyl acetate 1:1).
[0343] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.71 (s, 1H),
6.11 (m, 1H), 5.92](d, J=1.5 Hz, 1H), 5.87 (d, J=1.5 Hz, 1H), 5.42
(dq, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.28 (dq, J.=1.5 Hz,
J.sub.2=10.2 Hz, 1H), 5.12 (s, 2H), 4.26-4.09 (m, 3H), 4.05 (d,
J=2.4 Hz, 1H), 3.97 (t, J=3.0 Hz, 1H), 3.70 (s, 3H), 3.67-3.32 (m,
4H), 3.58 (s, 3H), 3.24 (dd, J.sub.1=2.7 Hz, J.sub.2=15.9 Hz, 1H),
3.12 (dd, J.sub.1=8.1 Hz, J.sub.2=18.0 Hz, 1H), 2.51 (d, J=18 Hz,
1H), 2.36 (s, 3H), 2.21 (s, 3H), 2.12 (s, 3H), 1.83 (dd,
J.sub.1=12.3 Hz, J.sub.2=15.9 Hz, 1H)
[0344] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.7, 148.4,
138.9, 133.7, 131.1, 129.4, 125.1, 123.9, 120.7, 117.6, 117.5,
113.2, 112.3, 101.1, 99.2, 74.0, 63.2, 59.8, 59.7, 57.9, 57.7,
57.0, 56.5, 55.2, 41.6, 29.6, 26.1, 25.6, 22.6, 15.7, 9.2.
[0345] ESI-MS m/z: Calcd. for C.sub.31H.sub.37N.sub.3O.sub.7:
563.64. Found (M+H).sup.+: 564.1.
Example 13
[0346] 154
[0347] The starting material (2.0 g, 5.90 mmol) was added to a
suspension of sodium hydride (354 mg, 8.86 mmol) in THF (40 ml) at
23.degree. C., following the suspension was treated with allyl
chloroformate (1.135 ml, 8.25 mmol) at 23.degree. C. and then
refluxed for 3 hours. The suspension was cooled, filtered off, the
solid washed with ethyl acetate (100 ml), and the filtrate was
concentrated. The oil crude was ground with hexane (100 ml) and
kept at 4.degree. C. overnight. After, the solvent was decanted and
the light yellow slury was treated with CH.sub.2Cl.sub.2 (20 ml),
and precipitated with hexane (100 ml). After 10 minutes, the
solvent was decanted again. The operation was repeated until
appearing a white solid. The white solid was filtered off and dried
to afford compound 29 (1.80 g, 65%) as a white solid.
[0348] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 7.74 (d, J=7.5
Hz, 2H), 7.62 (d, J=6.9 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 7.30 (t,
J=6.3 Hz, 2H), 5.71 (d, J=7.8 Hz, 1H), 4.73 (d, J=7.8 Hz, 2H), 4.59
(m, 1H), 4.11 (t, J=6.0 Hz, 1H), 3.17 (dd, J=6.0 Hz, J=2.7 Hz, 2H),
3.20 (dd, J=5.4 Hz, J=2.1 Hz, 2H).
[0349] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 173.6, 152.7,
144.0, 139.7, 137.8, 126.0, 125.6, 123.4, 118.3, 73.4, 52.4, 45.5,
35.8, 33.7.
[0350] ESI-MS m/z: Calcd. for C.sub.20H.sub.18Cl.sub.3NO.sub.4S:
474.8. Found (M+Na).sup.+: 497.8
Example 14
[0351] 155
[0352] A mixture of compound 25 (585 mg, 1.03 mmol) and compound 29
(1.47 mg, 3.11 mmol) were azeotroped with anhydrous toluene
(3.times.10 ml). To a solution of 25 and 29 in anhydrous
CH.sub.2Cl.sub.2 (40 ml) was added DMAP (633 mg, 5.18 mmol) and
EDCHCl (994 mg, 5.18 mmol) at 23.degree. C. The reaction mixture
was stirred at 23.degree. C. for 3 hours. The miture was
partitioned with saturated aqueous solution of sodium bicarbonate
(50 ml) and the layers were separated. The aqueous layer was washed
with CH.sub.2Cl.sub.2 (50 ml). The combined organic layers were
dried over sodium sulphate, filtered and concentrated. The crude
was purified by flash column chromatography (ethyl acetate/hexane
1:3) to obtain 30 (1.00 g, 95%) as a pale cream yellow solid.
[0353] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 7.72 (m, 2H),
7.52 (m, 2H), 7.38 (m, 2H), 7.28 (m, 2H), 6.65 (s, 1H), 6.03 (m,
1H), 5.92 (d, J=1.5 Hz, 1H), 5.79 (d, J=1.5 Hz, 1H), 5.39 (m, 1H),
5.29 (dq, J=10.3 Hz, J=1.5 Hz, 1H), 5.10 (s, 2H), 4.73 (d, J=11.9
Hz, 1H), 4.66 (d, J=11.9 Hz, 1H), 4.53 (m, 1H), 4.36-3.96 (m, 9H),
3.89 (t, J=6.4 Hz, 1H), 3.71 (s, 3H), 3.55 (s, 3H), 3.33 (m, 1H),
3.20 (m, 2H), 2.94 (m, 3H), 2.59 (m, 1H), 2.29 (s, 3H), 2.23 (s,
3H), 2.02 (s, 3H), 1.83 (dd, J=16.0 Hz, J=11.9 Hz, 1H).
[0354] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 169.7, 154.0,
148.8, 148.4, 145.7, 144.5, 140.9, 139.0, 133.7, 130.9, 130.6,
127.6, 127.0, 124.8, 124.6, 124.1, 120.8, 119.9, 118.2, 117.7,
117.3, 112.7, 112.1, 101.3, 99.2, 74.7, 73.9, 64.4, 59.8, 57.7,
57.0, 56.8, 55.4, 53.3, 46.7, 41.4, 36.5, 34.7, 31.5, 26.4, 24.9,
22.6, 15.7, 14.0, 9.1.
[0355] ESI-MS m/z: Calcd. for
C.sub.51H.sub.53Cl.sub.3N.sub.4O.sub.10S: 1020.4. Found
(M+H).sup.+: 1021.2
Example 15
[0356] 156
[0357] To a solution of 30 (845 mg, 0.82 mmol), acetic acid (500
mg, 8.28 mmol) and (PPh.sub.3).sub.2PdCl.sub.2 (29 mg, 0.04 mmol)
in anhydrous CH.sub.2Cl.sub.2 20 ml at 23.degree. C. was added,
dropwise, Bu.sub.3SnH (650 mg, 2.23 mmol). The reaction mixture was
stirred at this temperature for 15 min., bubbling was. The crude
was quenched with water (50 ml) and extracted with CH.sub.2Cl.sub.2
(3.times.50 ml). The organic layers were dried over sodium
sulphate, filtered and concentrated. The crude was purified by
flash column chromatography (ethyl acetate/hexane in gradient from
1:5 to 1:3) to obtain compound 31 (730 mg, 90%) as a pale cream
yellow solid.
[0358] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 7.72 (m, 2H),
7.56 (m, 2H), 7.37 (m, 2H), 7.30 (m, 2H), 6.65 (s, 1H), 5.89 (s,
1H), 5.77 (s, 1H), 5.74 (s, 1H), 5.36 (d, J=5.9 Hz, 1H), 5.32 (d,
J=5.9 Hz, 1H), 5.20 (d, J=9.0, 1H), 4.75 (d, J=12.0 Hz, 1H), 4.73
(m, 1H), 4.48 (d, J=11.9 Hz, 1H), 4.08 (m, 4H), 3.89 (m, 1H), 3.86,
(t, J=6.2 Hz, 1H), 3.70 (s, 3H), 3.69 (s, 3H), 3.38 (m, 1H), 3.25
(m, 1H), 3.02-2.89 (m, 4H), 2.67 (s, 1H), 2.61 (s, 1H), 2.51 (dd,
J=14.3 Hz, J=4.5 Hz, 1H), 2.29 (s, 3H), 2.23 (s, 3H), 1.95 (s, 3H),
1.83 (m, 1H).
[0359] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 168.2, 152.5,
148.1, 146.2, 144.4, 144.3, 143.3, 139.6, 134.6, 129.7, 129.6,
126.2, 125.6, 123.4, 123.3, 121.6, 118.5, 116.3, 110.7, 110.2,
105.1, 99.4, 98.5, 75.2, 73.3, 61.7, 58.4, 57.9, 56.3, 56.1, 55.1,
54.7, 53.9, 51.9, 45.2, 40.1, 35.6, 33.3, 24.8, 23.3., 14.5,
7.3.
[0360] ESI-MS m/z: Calcd. for
C.sub.48H.sub.49Cl.sub.3N.sub.4O.sub.10S: 980.3. Found (M+H).sup.+:
981.2
Example 16
[0361] 157
[0362] To a solution of 31 (310 mg, 0.32 mmol), in anhydrous
CH.sub.2Cl.sub.2 (15 ml) at -10.degree. C. was added a solution of
benzeneseleninic anhydride 70% (165 mg, 0.32 mmol), in anhydrous
CH.sub.2Cl.sub.2 (7 ml), via cannula, keeping the temperature at
-10.degree. C. The reaction mixture was stirred at -10.degree. C.
for 5 min. A saturated solution of sodium bicarbonate (30 ml) was
added at this temperature. The aqueous layer was washed with more
CH.sub.2Cl.sub.2 (40 ml). The organic layers were dried over sodium
sulphate, filtered and concentrated. The crude was purified by
flash column chromatography (ethyl acetate/hexane in gradient from
1:5 to 1: 1) to obtain 32 (287 mg, 91%, HPLC: 91.3%) as a pale
cream yellow solid and as a mixture of two isomers (65:35) which
were used in the next step.
[0363] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. (Mixture of
isomers) 7.76 (m, 4H), 7.65 (m, 4H), 7.39 (m, 4H), 7.29 (m, 4H),
6.62 (s, 1H), 6.55 (s, 1H), 5.79-5.63 (m, 6H), 5.09 (s, 1H), 5.02
(d, J=6.0 Hz, 1H), 4.99 (d, J=6.0 Hz, 1H), 4.80-4.63 (m, 6H), 4.60
(m, 1H), 4.50 (m, 1H), 4.38 (d, J=12.8 Hz, J=7.5 Hz, 1H), 4.27 (dd,
J=12.8 Hz, J=7.5 Hz, 1H), 4.16-3.90 (m, 10H), 3.84 (s, 3H), 3.62
(s, 3H), 3.50 (s, 3H), 3.49 (s, 3H), 3.33-2.83 (m, 14H), 2.45-2.18
(m, 2H), 2.21 (s, 6H), 2.17 (s, 6H), 1.77 (s, 6H), 1.67 (m,
2H).
[0364] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. (Mixture of
isomers) 168.6, 168.4, 158.6, 154.8, 152.8, 152.5, 147.3, 147.2,
146.8, 144.1, 144.0, 140.8, 139.7, 137.1, 129.8, 129.3, 128.4,
128.7, 126.5, 125.5, 123.7, 123.6, 123.5, 123.4, 122.2, 121.3,
118.3, 115.8, 115.5, 110.2, 106.9, 103.5, 103.2, 100.1, 99.6, 97.9,
97.7, 93.8, 73.4, 70.9, 69.2, 64.9, 62.5, 59.3, 58.9, 58.4, 56.7,
56.3, 56.2, 55.4, 55.2, 55.1, 54.9, 54.7, 54.3, 54.1, 53.8, 52.8,
45.5, 40.5, 40.0, 39.8, 35.8, 35.5, 33.9, 33.7, 30.1, 28.8, 24.2,
24.1, 21.2, 14.5, 14.4, 12.7, 6.0, 5.7.
[0365] ESI-MS m/z: Calcd. for
C.sub.48H.sub.49Cl.sub.3N.sub.4O.sub.11S: 996.3. Found (M+H).sup.+:
997.2
Example 17
[0366] 158
[0367] The reaction flask was flamed twice, purged vacuum/Argon
several times and kept under Argon atmosphere for the reaction. To
a solution of DMSO (39.1 ml, 0.55 mmol, 5 equivalents.) in
anhydrous CH.sub.2Cl.sub.2 (4.5 ml) was dropwise added triflic
anhydride (37.3 ml, 0.22 mmol, 2 equivalents.) at -78.degree. C.
The reaction mixture was stirred at -78.degree. C. for 20 minutes,
then a solution of 32 (110 mg, 0.11 mmol, HPLC: 91.3%) in anhydrous
CH.sub.2Cl.sub.2 (1 ml, for the main addition and 0.5 ml for wash)
at -78.degree. C. was added, via cannula. During the addition the
temperature was kept at -78.degree. C. in both flasks and the
colour changed from yellow to brown. The reaction mixture was
stirred at -40.degree. C. for 35 minutes. During this period of
time the solution was turned from yellow to dark green. After this
time, .sup.iPr.sub.2NEt (153 ml, 0.88 mmol, 8 equivalents.) was
dropwise added and the reaction mixture was kept at 0.degree. C.
for 45 minutes, the colour of the solution turned to brown during
this time. Then t-butanol (41.6 ml, 0.44 mmol, 4 equivalents.) and
2-.sup.tButyl-1,1,3,3-tetramethylguanidine (132.8 ml, 0.77 mmol, 7
equivalents.) were dropwise added and the reaction mixture was
stirred at 23.degree. C. for 40 minutes. After this time, acetic
anhydride (104.3 ml, 1.10 mmol, 10 equivalents.) was dropwise added
and the reaction mixture was kept at 23.degree. C. for 1 hour more.
Then the reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 ml)
and washed with aqueous saturated solution of NH.sub.4Cl (50 ml),
sodium bicarbonate (50 ml), and sodium chloride (50 ml). The
combined organic layers were dried over sodium sulphate, filtered
and concentrated. The residue was purified by flash column
chromatography (eluent: ethyl acetate/hexane gradient from 1:3 to
1:2) to afford compound 33 (54 mg, 58%) as a pale yellow solid.
[0368] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 6.85 (s, 1H),
6.09 (s, 1H), 5.99 (s, 1H), 5.20 (d, J=5.8 Hz, 1H), 5.14 (d, J=5.3
Hz, 1H), 5.03 (m, 1H), 4.82 (d, J=12.2, 1H), 4.63 (d, J=12.0 Hz,
1H), 4.52 (m, 1H), 4.35-4.17 (m, 4H), 3.76 (s, 3H), 3.56 (s, 3H),
3.45 (m, 2H), 2.91 (m, 2H), 2.32 (s, 3H), 2.28 (s, 3H), 2.21 (s,
3H), 2.12 (m, 2H), 2.03 (s, 3H).
[0369] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 168.5, 167.2,
152.7, 148.1, 147.1, 144.5, 139.6, 139.1, 130.5, 129.0, 123.7,
123.5, 123.3, 118.8, 116.5, 112.1, 100.6, 97.8, 73.3, 60.5, 59.4,
59.2, 58.3, 57.6, 57.4, 56.1, 53.3, 53.1, 40.6, 40.0, 31.0, 22.2,
18.9, 14.4, 8.1.
[0370] ESI-MS m/z: Calcd. for
C.sub.36H.sub.39Cl.sub.3N.sub.4O.sub.11S: 842.1. Found (M+H).sup.+:
843.1
Example 18
[0371] 159
[0372] To a solution of 33 (12 mg, 0.014 mmol)in dry
dichloromethane (1.2 ml) and HPLC grade acetonitrile (1.2 ml) was
added at 23.degree. C. sodium iodide (21 mg, 0.14 mmol) and freshly
distilled (over calcium hydride at atmospheric pressure)
trimethylsilyl chloride (15.4 mg, 0.14 mmol). The reaction mixture
turned to orange colour. After 15 min the solution was diluted with
dichloromethane (10 ml) and was washed with a freshly aqueous
saturated solution of Na.sub.2S.sub.2O.sub.4 (3.times.10 ml). The
organic layer was dried over sodium sulphate, filtered and
concentrated. It was obtained compound 34 (13 mg, quantitative) as
pale yellow solid which was used without further purification.
[0373] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 6.85 (s, 1H),
6.09 (s, 1H), 5.99 (s, -1H), 5.27 (d, J=5.8 Hz, 1H), 5.14 (d, J=5.3
Hz, 1H), 5.03 (d, J=11.9 Hz, 1H), 4.82 (d, J=12.2, 1H), 4.63 (d,
J=13.0 Hz, 1H), 4.52 (m, 1H), 4.34 (m, 1H), 4.27 (bs, 1H), 4.18 (m,
2H), 3.76 (s, 3H), 3.56 (s, 3H), 3.44 (m, 1H), 3.42 (m, 1H), 2.91
(m, 2H), 2.32 (s, 3H), 2.28 (s, 3H), 2.21 (s, 3H), 2.03 (s,
3H).
[0374] ESI-MS m/z: Calcd. for C.sub.34H.sub.35N.sub.4O.sub.10S:
798.1. Found (M+H).sup.+: 799.1
Example 19
[0375] 160
[0376] To a solution of 34 (13 mg, 0.016 mmol) in a mixture of
acetic acid/H.sub.2O (90:10, 1 ml) was added powder Zinc (5.3 mg,
0.081 mmol) at 23.degree. C. The reaction mixture was heated at
70.degree. C. for 6 h. After this time, was cooled to 23.degree.
C., diluted with CH.sub.2Cl.sub.2 (20 ml) and washed with aqueous
saturated solution of sodium bicarbonate (15 ml) and aqueous
solution of Et.sub.3N (15 ml). The organic layer was dried over
sodium sulphate, filtered and concentrated. The residue was
purified by flash column chromatography with Silica-NH.sub.2
(eluent: ethyl acetate/hexane gradient from 0:100 to 50:50) to
afford compound 35 (6.8 mg, 77% for two steps) as a pale yellow
solid.
[0377] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 6.51 (s, 1H),
6.03 (dd, J=1.3 Hz, J=26.5 Hz, 2H), 5.75 (bs, 1H), 5.02 (d, J=11.6
Hz, 1H), 4.52 (m, 1H), 4.25 (m, 2H), 4.18 (d, J=2.5 Hz, 1H), 4.12
(dd, J=1.9 Hz, J=11.5 Hz, 1H), 3.77 (s, 3H), 3.40 (m, 2H), 3.26 (t,
Jo 6.4 Hz, 1H), 2.88 (m, 2H), 2.30-2.10 (m, 2H), 2.30 (s, 3H), 2.28
(s, 3H), 2.18 (s, 3H), 2.02 (s, 3H).
[0378] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 174.1, 168.4,
147.8, 145.4, 142.9, 140.8, 140.1, 131.7, 130.2, 129.1, 128.3,
120.4, 118.3, 117.9, 113.8, 111.7, 101.7, 61.2, 59.8, 59.2, 58.9,
54.4, 53.8, 54.4, 41.3, 41.5, 34.1, 23.6, 20.3, 15.5, 9.4.
[0379] ESI-MS m/z: Calcd. for C.sub.31H.sub.34N.sub.4O.sub.8S:
622.7. Found (M+H).sup.+: 623.2.
Example 20
[0380] 161
[0381] A solution of N-methylpyridine-4-carboxaldehyde iodide (378
mg, 1.5 mmol) in anhydrous DMF (5.8 mL) was treated with anhydrous
toluene (2.times.10 mL) to eliminate the amount of water by
azeotropic removal of the toluene. A solution of 35 (134 mg, 0.21
mmol), previously treated with anhydrous toluene (2.times.10 mL),
in anhydrous CH.sub.2Cl.sub.2 (distilled over CaH.sub.2, 7.2 mL)
was added, via cannula, at 23.degree. C. to this orange solution.
The reaction mixture was stirred at 23.degree. C. for 4 hours.
After this time DBU (32.2 .quadrature.L, 0.21 mmol) was dropwise
added at 23.degree. C. and it was stirred for 15 minutes at
23.degree. C. A freshly aqueous saturated solution of oxalic acid
(5.8 mL) was added to the reaction mixture and was stirred for 30
minutes at 23.degree. C. Then the reaction mixture was cooled to
0.degree. C. and NaHCO.sub.3 was portionwise added followed by
addittion of aqueous saturated solution of NaHCO.sub.3. The mixture
was extracted with Et.sub.2O. K.sub.2CO.sub.3 was added to the
aqueous layer and it was extrated with Et.sub.2O. The combined
organic layers were dried over MgSO.sub.4 and the solvent was
removed under reduced pressure. The crude was purified by flash
column chromatography (AcOEt/hexane from 1/3 to 1/1) to afford
compound 36 (77 mg, 57%) as pale yellow solid.
[0382] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 6.48 (s, 1H),
6.11 (d, J=1.3 Hz, 1H), 6.02 (d, J=1.3 Hz, 1H), 5.70 (bs, 1H), 5.09
(d, J=11.3 Hz, 1H), 4.66 (bs, 1H), 4.39 (m, 1H), 4.27 (d, J=5.6 Hz,
1H), 4.21 (d, V 10.5 Hz, 1H), 4.16 (d, J=2.6 Hz, 1H), 3.76 (s, 3H),
3.54 (d, J=5.1 Hz, 1H), 3.42 (d, J=8.5 Hz, 1H), 2.88-2.54 (m, 3H),
2.32 (s, 3H), 2.24 (s, 3H), 2.14 (s, 3H), 2.04 (s, 3H).
.sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 186.7, 168.5, 160.5,
147.1, 146.4, 142.9, 141.6, 140.7, 130.4, 129.8, 121.7 (2C), 120.0,
117.8, 117.1, 113.5, 102.2, 61.7, 61.4, 60.3, 59.8, 58.9, 54.6,
41.6, 36.9, 29.7, 24.1, 20.3, 15.8, 14.1, 9.6.
[0383] ESI-MS m/z: Calcd. for C.sub.31H.sub.31N.sub.3O.sub.9S:
621.7. Found (M+H).sup.+: 622.2
Example 21
[0384] 162
[0385] To a solution of 36 (49 mg, 0.08 mmol) and
2-[3-hydroxy-4-methoxyph- enyl]ethylamine (46.2 mg, 0.27 mmol) in
ethanol (2.5 ml) was added silica gel (105 mg) at 23.degree. C. The
reaction mixture was stirred at 23.degree. C. for 14 h. It was
diluted with hexane and poured into a column of chromatography
(ethyl acetate/hexane from 1/3 to 1/1) to afford Et-770 (55 mg,
90%) as a pale yellow solid.
[0386] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 6.60 (s, 1H),
6.47 (s, 1H), 6.45 (s, 1H), 6.05 (s, 1H), 5.98 (s, 1H), 5.02 (d,
J=11.4 Hz, 1H), 4.57 (bs, 1H), 4.32 (bs, 1H), 4.28 (d, J=5.3 Hz,
1H), 4.18 (d, J=2.5 Hz, 1H), 4.12 (dd, J=2.1 Hz, J=11.5 Hz, 1H),
3.78 (s, 3H), 3.62 (s, 3H), 3.50 (d, J=5.0 Hz, 1H), 3.42 (m, 1H),
3.10 (ddd, J.sub.1=4.0 Hz, J.sub.2=10.0 Hz, J.sub.3=11.0 Hz, 1H),
2.94 (m, 2H), 2.79 (m, 1H), 2.61 (m, 1H), 2.47 (m, 1H), 2.35 (m,
1H), 2.32 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.09 (m, 1H), 2.04
(s, 3H).
[0387] ESI-MS m/z: Calcd. for C.sub.40H.sub.42N.sub.40O.sub.10S:
770.7. Found (M+H).sup.+: 771.2
Example 22
[0388] 163
[0389] To a solution of 21 (22 mg, 0.042 mmol) in CH.sub.2Cl.sub.2
(0.8 ml) was added phthalic anhydride (6.44 mg, 0.042 mmol) and the
reaction mixture was stirred for 2 h at 23.degree. C. Then,
carbonyldiimidazole (1 mg, 0.006 mmol) was added and the mixture
was stirred at 23.degree. C. for 7 h. Then, carbonyldiimidazole
(5.86 mg, 0.035 ml) was added and the reaction was stirred at
23.degree. C. for an additional 17 h. The solution was diluted with
CH.sub.2Cl.sub.2 (15 ml) and washed with 0.1 N HCl (15 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, hexane:ethyl
acetate 2:1) to afford 27 (26.4 mg, 96%) as a white solid.
[0390] Rf: 0.58 (ethyl acetate).
[0391] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.73-7.64 (m,
4H), 6.40 (s, 1H), 6.12-6.01 (m, 1H), 5.63 (s, 1H), 5.58 (d, J=1.5
Hz, 1H), 5.37 (dd, J.sub.1=1.8 Hz, J.sub.2=17.4 Hz), 5.23 (dd,
J.sub.1=1.8 Hz, J.sub.2=10.5 Hz, 1H), 5.12 (d, J=1.5 Hz, 1H),
4.22-4.15 (m, 3H), 4.08 (d, J=1.8 Hz, 1H), 3.68 (s, 3H), 3.59-3.55
(m 2H), 3.35 (d, J=8.1 Hz, 1H), 3.27-3.16 (m, 2H), 3.05 (dd,
J.sub.1=8.1 Hz, J.sub.2=18.3 Hz, 1H), 2.64 (d, J=18.0 Hz, 1H), 2.30
(s, 3H), 2.24 (s, 3H), 2.09 (s, 3H), 1.80 (dd, J.sub.1=11.4 Hz,
J.sub.2=15 Hz, 1H);
[0392] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 167.7, 148.9,
146.4, 144.2, 142.6, 139.5, 134.0, 133.5, 132.0, 131.0, 128.3,
123.0, 121.3, 120.9, 118.1, 117.5, 116.8, 113.6, 112.4, 100.8,
74.5, 60.6, 60.5, 57.7, 56.6, 55.6, 55.5, 42.3, 41.7, 26.6, 25.5,
15.9, 9.46.
[0393] ESI-MS m/z: Calcd. for C.sub.37H.sub.35N.sub.4O.sub.7:
648.79. Found (M+H).sup.+: 649.3.
Example 23
[0394] 164
[0395] To a solution of 27 (26 mg, 0.041 mmol) in CH.sub.2Cl.sub.2
(11 ml), acetic acid (11 ml), (PPh.sub.3).sub.2PdCl.sub.2 (2.36 mg)
and Bu.sub.3SnH (28 ml, 0.10 mmol) were added at 23.degree. C.
After stirring at that temperature for 2 h the reaction was poured
into a pad of flash column (SiO.sub.2, gradient Hex to hexane:ethyl
acetate 2:1) to afford 28 (24.7 mg, 99%) as a white solid.
[0396] Rf: 0.33 (hexane:ethyl acetate 2:1).
[0397] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.75-7.70 (m,
2H), 7.69-7.65 (m, 2H), 6.39 (s, 1H), 5.82 (bs, 1H), 5.50 (d, J=1.5
Hz, 1H), 5.0 (d, J=1.5 Hz, 1H), 4.45 (bs, 1H), 4.23-4.19 (m, 2H),
4.10-4.09 (m, 1H), 3.73 (s, 3H), 3.60-3.48 (m, 2H), 3.36-3.33 (m,
1H), 3.26-3.20 (m, 1H), 3.14-3.08 (m, 1H), 3.98 (d, J=14.4 Hz, 1H),
2.61 (d, J=18.3 Hz, 1H), 2.30 (s, 3H), 2.23 (s, 3H), 2.06 (s, 3H),
1.85 (dd, J.sub.1=12 Hz, J.sub.2=15.3 Hz);
[0398] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 167.8, 146.4,
145.1, 143.9, 142.7, 137.1, 133.5, 131.9, 130.8, 128.4, 122.9,
120.8, 118.0, 116.8, 114.0, 113.4, 106.4, 100.4, 60.6, 60.5, 57.8,
56.6, 55.5, 55.2, 42.6, 41.5, 25.6, 25.5, 15.8, 8.9.
[0399] ESI-MS m/z: Calcd. for C.sub.34H.sub.32N.sub.4O.sub.7:
608.6. Found (M+H).sup.+: 609.2.
Example 24
[0400] 165
[0401] To a solution of 28 (357 mg, 0.058 mmol) in CH.sub.2Cl.sub.2
(3 ml), acetyl chloride (41.58 ml, 0.58 mmol) and pyridine (47.3
ml, 0.58 ml) were added at 0.degree. C. The reaction mixture was
stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (15 ml) and washed with 0.1 N HCl (15 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (RP-18, CH.sub.3CN:H.sub.2O
60:40) to afford phthalascidin (354 mg, 94%) as a white solid.
[0402] Rf: 0.37 (CH.sub.3CN:H.sub.2O 7:3, RP-18).
[0403] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.72-7.68 (m,
2H), 7.67-7.63 (m, 2H), 6.38 (s, 1H), 5.69 (d, J=1.2 Hz, 1H), 5.64
(d, J=1.2 Hz, 1H), 5.30 (bs, 1H), 4.25-4.21 (m, 2H), 4.02 (d, J=2.1
Hz, 1H), 3.64-3.62 (m, 5H), 3.33 (d, J=8.4 Hz, 1H), 3.21-3.16 (m,
1H), 3.02 (dd, J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H), 2.76 (dd,
J.sub.1=1.8 Hz, J.sub.2=15.6 Hz, 1H), 2.63 (d, J=17.7 Hz, 1H), 2.29
(s, 3H), 2.28 (s,3H), 2.21.(s, 3H), 2.0 (s, 3H), 1.73 (dd,
J.sub.1=12.0 Hz, J.sub.2=15.3 Hz, 1H))
[0404] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 168.5, 167.6,
146.2, 144.2, 142.5, 141.0, 140.5, 133.4, 131.8, 130.7, 128.2,
120.9, 120.8, 117.9, 116.4, 113.6, 101.1, 60.4, 60.0, 57.0, 56.3,
55.6, 55.4, 41.6, 41.5, 26.5, 25.2, 20.2, 15.7, 9.4.
[0405] ESI-MS m/z: Calcd. for C.sub.36H.sub.34N.sub.4O.sub.8: 650.
Found (M+H).sup.+: 651.2.
Example 25
[0406] 166
[0407] To a solution of 17 (300 mg, 0.432 mmol) in CH.sub.2Cl.sub.2
(2 ml), acetyl chloride (30.7 ml, 0.432 mmol) and pyridine (34.9
ml, 0.432 mmol) were added at 0.degree. C. The reaction mixture was
stirred for 2 h at that temperature and then, the solution was
diluted with CH.sub.2Cl.sub.2 (15 ml) and washed with 0.1 N HCl (15
ml). The organic layer was dried over sodium sulphate, filtered,
and the solvent was eliminated under reduced pressure to afford 42
(318 mg, 100%) as a white solid that was used in subsequent
reactions with no further purification.
[0408] Rf: 0.5 (ethyl acetate:methanol 5:1).
[0409] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 6.66 (s, 1H),
5.93 (d, J=1.2 Hz, 1H), 5.83 (d, J=1.2 Hz, 1H), 5.42 (t, J=6.6 Hz,
1H), 5.07 (d, J=5.7 Hz, 1H), 4.98 (d, J=5.7 Hz, 1H), 4.16 (d, J=1.8
Hz, 1H), 4.11 (d, J=2.7 Hz, 1H), 3.98 (bs, 1H), 3.73-3.61 (m, 2H),
3.64 (s, 3H), 3.52-3.48 (m, 1H), 3.50 (s, 3H), 3.33 (d, J=9.6 Hz,
1H), 3.17-3.14 (m, 1H), 2.97-2.87 (m, 1H), 2.75-2.70 (d, J=16.8 Hz,
1H), 2.26 (s, 6H), 2.16 (s, 3H), 1.96 (s, 3H), 1.70 (dd,
J.sub.1=11.7 Hz, J.sub.2=15.6 Hz, 1H), 1.33 (s, 9H), 0.59 (d, J=6.0
Hz, 3H).
[0410] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 172.0, 168.3,
162.3, 148.2, 144.4, 140.4, 140.2, 130.9, 130.5, 125.3, 123.4,
120.8, 117.6, 112.7, 111.7, 101.4, 99.1, 79.2, 59.5, 58.8, 57.5,
57.4, 56.4, 55.5, 55.0, 41.3, 39.0, 28.2, 26.4, 24.6, 19.9, 18.4,
15.4, 9.1.
[0411] ESI-MS m/z: Calcd. for C.sub.38H.sub.49N.sub.50.sub.10:
735.82. Found (M+H).sup.+: 736.3.
Example 26
[0412] 167
[0413] To a solution of 42 (318 mg, 0.432 mmol) in CH.sub.2Cl.sub.2
(2.16 mmol), trifluoroacetic acid (1.33 ml, 17.30 mmol) was added
and the reaction mixture was stirred for 3.5 h at 23.degree. C. The
reaction was quenched at 0.degree. C. with saturated aqueous sodium
bicarbonate (60 ml) and extracted with CH.sub.2Cl.sub.2 (2.times.70
ml). The combined organic layers were dried (sodium sulphate) and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, ethyl acetate:methanol 20:1) to afford
43 (154 mg, 60%) as a white solid.
[0414] Rf: 0.22 (ethyl acetate:methanol 5:1).
[0415] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 6.47 (s, 1H),
6.22 (bs, 1H), 5.95 (d, J=1.2 Hz, 1H), 5.88 (d, J=1.2 Hz, 1H),
4.08-4.06 (m, 2H), 4.01 (bs, 1H), 3.69 (s, 3H), 3.49 (d, J=3.6 Hz,
1H), 3.33 (d, J=8.1 Hz, 1H), 3.26-3.22 (m, 1H), 2.95 (dd,
J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H), 2.80-2.76 (m, 2H), 2.58 (d,
J=18 Hz, 1H), 2.29 (s, 3H), 2.27 (s, 3H), 2.21 (s, 3H), 1.96 (s,
3H), 1.77 (dd, J.sub.1=12.3 Hz, J.sub.2=15.6 Hz, 1H), 0.90 (d,
J=6.9 Hz, 3H).
[0416] .sup.13C NMR (75 MHz, CDCb)): .delta. 174.8, 169.0, 146.8,
144.4, 142.8, 140.5, 140.2, 131.1, 128.8, 120.8, 120.5, 117.1,
112.9, 111.6, 101.5, 60.3, 59.0, 56.5, 56.3, 55.6, 55.1, 50.2,
41.6, 39.5, 26.8, 26.3, 24.9, 20.2, 15.4, 9.2.
[0417] ESI-MS m/z: Calcd. for C.sub.31H.sub.37N.sub.5O.sub.7:
591.65. Found (M+H).sup.+: 592.3.
Example 27
[0418] 168
[0419] To a solution of 43 (154 mg, 0.26 mmol) in CH.sub.2Cl.sub.2
(1.3 ml), phenyl isothiocyanate (186 ml, 1.56 mmol) was added and
the mixture was stirred at 23.degree. C. for 2 h. The reaction was
concentrated in vacuo and the residue was purified by flash column
chromatography (SiO.sub.2, gradient Hexane to hexane:ethyl acetate
1:1) to afford 44 (120 mg, 63%) as a white solid.
[0420] Rf: 0.41 (ethyl acetate:methanol 5:1).
[0421] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 8.17 (s, 1H),
7.49-7.44 (m, 3H), 7.31-7.24 (m, 3H), 7.05 (d, J=6.9 Hz, 1H), 5.98
(d, J=1.2 Hz, 1H), 5.87 (d, J=1.2 Hz, 1H), 5.52 (bs, 1H), 4.54 (t,
J=6.6 Hz, 1H), 4.15 (d, J=2.1 Hz, 1H), 4.03 (d, J=2.7 Hz, 2H), 3.80
(bs, 1H), 3.66 (s, 3H), 3.40 (bs, 1H), 3.32 (d, J=7.8 Hz, 1H), 3.16
(d, J=11.7 Hz, 1H), 2.82-2.61 (m, 3H), 2.29 (s, 3H), 2.20 (s, 3H),
2.01 (s, 3H), 1.99 (s, 3H), 1.80 (dd, J.sub.1=12.0 Hz, J.sub.2=15.9
Hz, 1H), 0.62 (d, J=6.0 Hz, 3H).
[0422] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 178.5, 171.9,
168.7, 146.7, 144.5, 142.6, 140.6, 140.3, 136.3, 131.0, 129.9,
128.9, 126.7, 124.4, 120.9, 120.6, 117.7, 116.6, 112.7, 111.9,
101.4, 60.4, 58.7, 57.5, 56.1, 55.7, 55.1, 53.3, 41.4, 38.8, 26.3,
24.4, 20.2, 18.1, 15.3, 9.2.
[0423] ESI-MS m/z: Calcd. for C.sub.38H.sub.42N.sub.6O.sub.7S:
726.3. Found (M+H).sup.+: 727.3.
Example 28
[0424] 169
[0425] To a solution of 44 (120 mg, 0.165 mmol) in dioxane (0.9
ml), 5.3N HCl/dioxane (1.8 ml) was added and the reaction was
stirred at 23.degree. C. for 2.5 h. Then, CH.sub.2Cl.sub.2 (10 ml)
and H.sub.2O (5 ml) were added to this reaction and the organic
layer was decanted. The aqueous phase was basified with saturated
aq sodium bicarbonate (20 ml) (pH=8) at 0.degree. C. and then,
extracted with CH.sub.2Cl.sub.2 (2.times.15 ml). The combined
organic extracts were dried (sodium sulphate), and concentrated in
vacuo to afford 45 (75 mg, 87%) as a white solid that was used in
subsequent reactions with no further purification.
[0426] Rf: 0.23 (ethyl acetate:methanol 5:1).
[0427] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.43 (s, 1H),
5.94 (d, J=1.2 Hz, 1H), 5.87 (d, J=1.2 Hz, 1H), 4.10 (d, J=2.1 Hz,
1H), 3.98 (d, J=2.4 Hz, 1H), 3.91 (bs, 1H), 3.69 (s, 3H), 3.34-3.25
(m, 2H), 3.05 (dd, J.sub.1=1.8 Hz, J.sub.2=8.1 Hz, 1H), 2.80-2.73
(m, 3H), 2.46 (d, J=18 Hz, 1H), 2.30 (s, 3H), 2.28 (s,3H), 2.20 (s,
3H), 1.98 (s, 3H), 1.79 (dd, J.sub.1=12.6 Hz, J.sub.2=16.2 Hz,
1H);
[0428] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 168.7, 146.7,
144.4, 142.9, 140.4, 130.4, 128.9, 121.1, 120.8, 117.8, 116.8,
113.6, 111.5, 101.4, 67.6, 60.5, 59.8, 58.4, 56.6, 55.8, 55.3,
43.6, 41.8, 31.3, 25.6, 20.2, 15.6, 9.2.
[0429] ESI-MS m/z: Calcd. for C.sub.28H.sub.32N.sub.4O.sub.6:
520.58. Found (M+H).sup.+: 521.3.
Example 29
[0430] 170
[0431] To a solution of 45 (10 mg, 0.02 mmol) in CH.sub.2Cl.sub.2
(0.4 ml) was added phthalic anhydride (2.84 mg, 0.02 mmol) and the
reaction mixture was stirred for 2 h at 23.degree. C. Then,
carbonyldiimidazole (0.5 mg, 0.003 mmol) was added and t he mixture
was stirred at 23.degree. C. for 7 h. Then, carbonyldiimidazole
(2.61 mg, 0.016 mmol) was added and the reaction was stirred at
23.degree. C. for an additional 17 h. The solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (RP-18, CH.sub.3CN:H.sub.2O
60:40) to afford phthalascidin (11.7 mg, 93%) as a white solid.
[0432] Rf: 0.37 (CH.sub.3CN:H.sub.2O 7:3, RP-18).
[0433] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.72-7.68 (m,
2H), 7.67-7.63 (m, 2H), 6.38 (s, 1H), 5.69 (d, J=1.2 Hz, 1H), 5.64
(d, J=1.2 Hz, 1H), 5.30 (bs, 1H), 4.25-4.21 (m, 2 h), 4.02 (d,
J=2.1 Hz, 1H), 3.64-3.62 (m, 5H), 3.33 (d, J=68.4 Hz, 1H),
3.21-3.16 (m, 1H), 3.02 (dd, J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H),
2.76 (dd, J.sub.1=1.8 Hz, J.sub.2=15.6 Hz, 1H), 2.63 (d, J=17.7 Hz,
1H), 2.29 (s, 3H), 2.28 (s,3H), 2.21 (s, 3H), 2.0 (s, 3H), 1.73
(dd, J.=12.0 Hz, J=15.3 Hz, 1H)).
[0434] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 168.5, 167.6,
146.2, 144.2, 142.5, 141.0, 140.5, 133.4, 131.8, 130.7, 128.2,
120.9, 120.8, 117.9, 116.4, 113.6, 101.1, 60.4, 60.0, 57.0, 56.3,
55.6, 55.4, 41.6, 41.5, 26.5, 25.2, 20.2, 15.7, 9.4.
[0435] ESI-MS m/z: Calcd. for C.sub.36H.sub.34N.sub.4O.sub.8: 650.
Found (M+H).sup.+: 651.2.
Example 30
[0436] 171
[0437] To a solution of 25 (18 mg, 0.032 mmol) in DMF (0.05 ml),
cat. DMAP (0.5 mg, 0.004 mmol), imidazole (5 mg, 0.08 mmol) and
tert-Butyldiphenylsilyl chloride (12.5 ml, 0.048 mmol) were added
at 0.degree. C. and the reaction mixture was stirred for 6 h at
23.degree. C. Water (10 ml) was added at 0.degree. C. and the
aqueous phase was extracted with hexane:ethyl acetate 1:10
(2.times.10 mmol). The organic layer was dried (sodium sulphate),
filtered, and the solvent was removed under reduced pressure. The
crude was purified by flash column chromatography (SiO.sub.2,
hexane:ethyl acetate 3:1) to afford 26 (27 mg, 88%) as a white
solid.
[0438] Rf: 0.29 (hexane:ethyl acetate 3:1).
[0439] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.61-7.58 (m, 2
h), 7.42-7.28 (m, 8H), 6.71 (s, 1H), 6.19-6.02 (m, 1H), 5.78 (d,
J=1.2 Hz, 1H), 5.64 (d, J=1.2 Hz, 1H), 5.40 (dd, J.sub.1=1.2 Hz,
J.sub.2=17.1 Hz, 1H), 5.27 (dd, J.sub.1=1.2 Hz, J.sub.2=10.2 Hz,
1H), 5.13 (s, 2 h), 4.45 (d, J=2.4 Hz, 1H), 4.24 (d, J=2.1 Hz, 1H),
4.17-4.06 (m, 3H), 3.75 (s, 3H), 3.64 (dd, J.sub.1=2.4 Hz,
J.sub.2=9.9 Hz, 1H), 3.59 (s, 3H), 3.42-3.21 (m, 4H), 3.10 (dd,
J.sub.1=8.1 Hz, J.sub.2=17.7 Hz, 1H), 2.70 (d, J=17.7 Hz, 1H), 2.33
(s, 3H), 2,26 (s, 3H), 2.11 (s, 3H), 2.08-1.89 (m, 1H), 0.87 (s,
9H);
[0440] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 148.5, 148.3,
148.1, 144.0, 139.0, 135.6, 135.4, 133.8, 133.1, 132.6, 130.5,
130.3, 129.6, 129.4, 127.5, 127.4, 125.1, 124.3, 121.6, 118.5,
117.5, 112.9, 111.7, 100.8, 99.2, 74.0, 67.7, 61.5, 59.6, 59.0,
57.7, 57.1, 55.4, 41.6, 29.6, 26.6, 25.5, 18.8, 15.8, 9.2.
[0441] ESI-MS m/z: Calcd. for C.sub.47H.sub.55N.sub.3O.sub.7Si:
801.3. Found (M+H).sup.+: 802.3.
Example 31
[0442] 172
[0443] To a solution of 26 (7 mg, 0.0087 mmol) in CH.sub.2Cl.sub.2
(0.15 ml), acetic acid (2.5 ml, 0.044 mmol),
(PPh.sub.3).sub.2PdCl.sub.2 (0.5 mg, 6.96.times.10.sup.-4 mmol) and
Bu.sub.3SnH (3.5 ml, 0.013 mmol) were added at 23.degree. C. The
reaction mixture was stirred at that temperature for 1 h. The
solution was diluted with a mixture of hexane:ethyl acetate 5:1
(0.5 ml) and poured into a pad of flash column (SiO.sub.2, gradient
5:1 to 1:1 hexane:ethyl acetate) affording ET-11 (5 mg, 75%) as a
white solid.
[0444] Rf: 0.36 (hexane:ethyl acetate 1:5, silica).
[0445] .sup.1H NMR (300 MHz, CDC): .delta. 7.56 (m, 2 h), 7.41-7.25
(m, 8H), 6.67 (s, 1H), 5.72 (d, J=1.0 Hz, 1H), 5.58 (d, J=1.0 Hz,
1H), 5.51 (s, 1H), 5.38 (d, J=5.75 Hz, 1H), 5.16 (d, J=5.7 Hz, 1H),
4.57 (d, J=2.9 Hz, 1H), 4.21 (m, 1H), 4.09 (m, 1H), 3.72 (s, 3H),
3.71 (s, 3H), 3.68 (dd, J.sub.1=2.1 Hz, J.sub.2=10.4 Hz, 1H),
3.38-3.26 (m, 3H), 3.11 (dd, J.sub.1=2.5 Hz, J.sub.2=15.7 Hz, 1H),
3.01 (dd, J.sub.1=8.9 Hz, J.sub.2=17.9 Hz, 1H), 2.70 (d, J=17.9 Hz,
1H), 2.31 (s, 3H), 2.25 (s, 3H), 2.06 (s, 3H), 1.89 (dd,
J.sub.1=12.1 Hz, J.sub.2=15.7 Hz, 1H), 0.9 (s, 9H).);
[0446] .sup.13C NMR (75 MHz, CDC): .delta. 149.0, 147.4, 145.3,
144.3, 136.3, 135.7, 135.4, 133.2, 130.9, 130.5, 129.6, 129.5,
127.5, 125.0, 118.6, 112.5, 112.1, 105.7, 100.5, 99.8, 68.5, 61.5,
59.7, 58.8, 57.7, 56.9, 56.5, 55.4, 41.7, 26.6, 26.2, 25.5, 18.9,
15.8, 14.2, 8.7.
[0447] ESI-MS m/z: Calcd. for C.sub.44H.sub.51N.sub.3O.sub.7Si:
761. Found (M+H).sup.+: 762.
Example 32
[0448] 173
[0449] A solution of 2 (3.0 g, 5.46 mmol) and phenyl isothiocyanate
(3.92 mL, 32.76 mmol) in CH.sub.2Cl.sub.2 (27 ml) was stirred at
23.degree. C. for 1.5 h. The reaction mixture was partitioned
between CH.sub.2Cl.sub.2 (10 ml) and H.sub.2O (5 ml). The organic
layer was dried over sodium sulphate, filtered and concentrated.
The residue was purified by flash column chromatography (SiO.sub.2,
gradient Hex to 2:3 hexane:ethyl acetate) to give 3 (3.29 g, 88%)
as a yellow solid.
[0450] Rf: 0.27 (ACN:H.sub.2O 3:2, RP-C18);
[0451] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.77 (bs, 1H),
7.42-7.11 (m, 5H), 6.65 (d, 1H), 6.29 (s, 1H), 5.6-5.5 (m, 1H),
4.19-4.14 (m, 2 h), 4.08 (d, 1H), 3.92 (s, 3H), 3.87-3.65 (m, 6H),
3.77 (s, 3H), 3.37-2.98 (m, 8H), 2.50 (d, 1H), 2.31 (s, 3H), 2.20
(s, 3H), 1.96 (d, 1H), 1.87 (s, 3H), 1.81-1.75 (m, 1H), 0.96 (d,
3H);
[0452] .sup.13C NMR (75 MHz,
[0453] CDCl.sub.3): 6185.7, 180.9, 178.9, 172.0, 155.7, 147.1,
143.2, 142.4, 136.0, 135.1, 130.5, 129.9, 129.3, 128.5, 126.9,
124.4, 120.2, 117.4, 116.3, 77.1, 60.9, 58.6, 56.2, 55.8, 55.0,
54.6, 53.5, 41.7, 40.3, 25.1, 24.5, 18.4, 15.8, 8.7
[0454] ESI-MS m/z: Calcd. for C.sub.36H.sub.40N.sub.6O.sub.6S:
684.8. Found (M+H).sup.+: 685.2.
Example 33
[0455] 174
[0456] A solution of 3 (0.143 g, 0.208 mmol) in 6.5 M HCl/dioxane
(150 ml) was stirred at 23.degree. C. for 6 h. Then, toluene (3 ml)
was added to this reaction and the organic layer was decanted. The
residue was partitioned between saturated aqueous sodium
bicarbonate (3 ml) and CHCl.sub.3 (3.times.3 ml) The organic layers
were dried and concentrated to afford title compound as a mixture
of 4 and 6 (4:6 90:10) which slowly cyclizes to 6 on standing.
[0457] Rf: 0.4 (ethyl acetate:methanol5:1, silica);
[0458] .sup.1H NMR (300 MHz, CDCl.sub.3): 6.45 (s, 1H), 4.16 (m,
1H), 4.02 (d, 1H), 3.96 (s, 3H), 3.79 (m, 2 h), 3.75 (s, 3H), 3.35
(m, 1H), 3.20-3.00 (m, 3H), 2.87 (d, 1H), 2.75 (d, 1H), 2.43 (d,
1H), 2.34 (s, 3H), 2.30 (s, 3H), 1.93 (s, 3H), 1.72-1.5 (m,
3H).
[0459] ESI-MS m/z: Calcd. for C.sub.26H.sub.30N.sub.4O.sub.5:
478.5. Found (M+H).sup.+: 479.2
Example 34
[0460] 175
[0461] A solution of 3 (0.143 g, 0.208 mmol) in 6.5M HCl/dioxane
(150 ml) was stirred at 23.degree. C. for 1 h. Evaporation of the
solvent gave a residue which was purified by flash column
chromatography (ethyl acetate/methanol/triethylamine 100:25:0.1) to
give 6 (80 mg, 83%) as a yellow solid.
[0462] Rf: 0.26 (ACN:H.sub.2O 3:2, RP-C18);
[0463] .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 6.46 (s, 1H), 5.9
(bs, 1H) 4.67 (dd, J=18.3 Hz, J=7.8 Hz, 1H), 4.24 (d, 1H), 4.16 (s,
3H), 3.93 (d, J=2.7 Hz, 1H), 3.8 (m, 2 h), 3.77 (s, 3H), 3.45 (m, 2
h), 3.08 (dd, J=17.9 Hz, J=3.6 Hz, 1H), 2.78 (m, 1H), 2.55 (d, 1H),
2.3 (m, 1H), 2.3 (s, 3H), 2.28 (s, 3H), 1.90 (s, 3H);
[0464] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 186.2, 162.1,
154.9, 146.9, 145.3, 143.0, 130.1, 129.4, 128,1, 125.0, 121.4,
116.4, 116.2, 66.6, 60.7, 60.7, 60.1, 59.6, 58.8, 55.6, 54.9, 41.9,
25.3, 24.7, 15.7, 8.9.
[0465] ESI-MS m/z: Calcd. for C.sub.26H.sub.28N.sub.4O.sub.4:
460.5. Found (M+H).sup.+: 461.1
Example 35
[0466] 176
[0467] To a solution of 3 (2.38 g, 3.47 mmol) in dioxane (5 ml)
5.3M HCl in dioxane (34 mrl) was added and the reaction was stirred
at 23.degree. C. for 45 minutes. Then Ac.sub.2O (51 ml, 539.5 mmol)
was added and the mixture was stirred for 4 h. The reaction was
cooled at 0.degree. C. and partitioned between aqueous saturated
Na.sub.2CO.sub.3 (300 ml) and ethyl acetate (300 ml) at this
temperature. The organic phase was dried over sodium sulphate,
filtered and concentrated. The residue was purified by flash column
chromatography (SiO.sub.2, gradient CH.sub.2Cl.sub.2 to
CH.sub.2Cl.sub.2:ethyl acetate 1:2) to give 5 (1.75 g, 97%) as a
yellow solid.
[0468] Rf: 0.53 (ACN:H.sub.2O 3:2, RP-C18);
[0469] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.51 (s, 1H),
5.98 (bs, 1H), 4.84 (dd, 1H), 4.17 (d, 1H), 4.00 (d, 1H), 3.99 (s,
3H), 3.85 (bs, 1H), 3.81 (m, 1H), 3.74 (s, 3H), 3.70 (d, 1H), 3.23
(m, 1H), 3.11 (dd, 1H), 3.09 (m, 1H), 2.93 (m, 2 h), 2.44 (d, 1H),
3.67 (s, 3H), 2.25 (s, 3H), 1.70 (s, 3H), 1.60-1.50 (m, 2 h), 1.29
(s, 3H);
[0470] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 185.9, 180.8,
169.9, 160.2, 156.2, 147.0, 143.1, 140.4, 136.1, 130.6, 129.6,
127.9, 120.4, 117.2, 61.0, 60.7, 58.6, 56.1, 55.7, 55.1, 54.3,
41.8, 41.1, 25.7, 23.9, 22.2, 15.7, 8.7.
[0471] ESI-MS m/z: Calcd. for C.sub.28H.sub.32N.sub.4O.sub.6:
520.6. Found (M+H).sup.+: 521.1
Example 36
[0472] 177
[0473] To a solution of 5 (1.75 g, 3.36 mmol) in CH.sub.2Cl.sub.2
(17 ml) diisopropylethylamine (11.71 ml, 67.23 mmol), DMAP (20 mg,
0.17 mmol) and bromomethyl methyl ether (4.11 ml, 50.42 mmol) were
added at 0.degree. C. After 6 h at 23.degree. C. the reaction was
partitioned between CH.sub.2Cl.sub.2 (50 ml) and aqueous saturated
sodium bicarbonate (25 ml). The organic layer was dried over sodium
sulphate and the solvent was eliminated under reduced pressure. The
crude was purified by flash column chromatography (RP-18,
CH.sub.3CN/H.sub.2O 1/1) to give 7 (1.32 g, 70%) as a yellow
solid.
[0474] Rf: 0.34 (ACN:H.sub.2O 2:3, RP-C18);
[0475] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.74 (s, 1H),
5.14 (s, 2 h), 4.82 (m, 1H), 4.22 (d, 1H), 4.00 (s, 3H), 4.0 (m,
1H), 3.83 (m, 2 h), 3.7 (s, 3H), 3.58 (s, 3H), 3.4 (m, 1H),
3.2-2.95 (m, 6H), 2.43 (d, 1H), 2.37 (s, 3H), 2.22 (s, 3H), 1.89
(s, 3H), 1.5-1.4 (m, 2 h), 1.31 (s, 3H);
[0476] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 185.9, 180.7,
169.6, 156.2, 148.9, 148.5, 140.3, 136.2, 131.3, 130.1, 127.7,
124.6, 123.7, 117.3, 99.5, 99.2, 60.9, 59.7, 58.8, 57.7, 56.4,
55.7, 55.0, 54.2, 51.0, 41.6, 41.0, 40.5, 25.5, 23.9, 22.3, 19.3,
15.6, 14.6, 8.6.
[0477] ESI-MS m/z: Calcd. for C.sub.30H.sub.36N.sub.4O.sub.7:
564.6. Found (M+H).sup.+: 565.3
Example 37
[0478] 178
[0479] To a solution of 7 (0.37 g, 0.65 mmol) in methanol (74 ml)
at 0.degree. C. was added 1M sodium hydroxide (130 ml). The
reaction was stirred for 15 minutes and then, quenched at 0.degree.
C. with 6M HCl to pH=5. The mixture was extracted with ethyl
acetate (3.times.50 ml) and the combined organic layers were dried
over sodium sulphate and concentrated in vacuo. The residue was
purified by flash column chromatography (RP-C18 CH.sub.3CN:H.sub.2O
1/1) to afford 8 (232 mg, 65%) as a yellow oil.
[0480] Rf: 0.5 (ACN:H.sub.2O 3:2, RP-C18);
[0481] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.75 (s, 1H),
5.15 (s, 2 h), 4.86 (m, 1H), 4.26 (d, 1H), 4.01 (d, 1H), 3.88-3.81
(m, 2 h), 3.70 (s, 3H), 3.58 (s, 3H), 3.39 (m, 1H), 3.27-3.21 (m,
1H), 3.18-3.08 (m, 2 h), 3.03-2.97 (m, 1H) 2.47 (d, 1H), 2.37 (s,
3H), 2.22 (s, 3H), 1.90 (s, 3H), 1.57-1.46 (m, 2 h), 1.33 (s,
3H);
[0482] .sup.13C NMR (75 MHz, .quadrature.CDCl.sub.3):.delta. 185.3,
180.6, 175.9, 170.1, 151.5, 148.9, 148.6, 143.3, 133.7, 131.5,
129.9, 124.7, 123.5, 117.1, 117.0, 99.2, 59.8, 58.7, 57.8, 56.3,
55.3, 54.9, 54.3, 41.5, 40.7, 29.6, 25.5, 24.4, 22.2, 20.7, 15.7,
8.0.
[0483] ESI-MS m/z: Calcd. for C.sub.29H.sub.34N.sub.4O.sub.7:
550.6. Found (M+H).sup.+: 551.2
Example 38
[0484] 179
[0485] To a degassed solution of compound 8 (240 mg, 0.435 mmol) in
DMF (30 ml) 10% Pd/C (48 mg) was added and the reaction was stirred
under H.sub.2 (atmospheric pressure.) for 1 h. The reaction was
filtered through a pad of celite under Argon to a Schlenk tube, as
a colourless solution, containing anhydrous Cs.sub.2CO.sub.3 (240
mg, 0.739 mmol). Then, bromochloromethane (0.566 ml, 8.71 mmol) was
added. The tube was sealed and stirred at 90.degree. C. for 3 h.
The reaction was cooled and filtrated through celite and washed
with CH.sub.2Cl.sub.2. The organic layer was concentrated and dried
(sodium sulphate) to afford 9 as a brown oil that was used in the
next step with no further purification.
[0486] Rf: 0.36 (SiO.sub.2, hexane:ethyl acetate 1:5)
[0487] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.71 (s, 3H),
5.89 (d, 11H), 5.81 (d, 1H), 5.63 (bs, 1H), 5.33 (d, 1H), 5.17 (d,
1H), 4.97 (m, 1H), 4.20 (d, 1H), 4.09 (m, 1H), 3.99 (m, 1H), 3.68
(m, 1H), 3.65 (s, 6H), 3.59-3.47 (m, 4H), 3.37-3.27 (m, 2 h),
3.14-2.97 (m, 2 h), 2.62 (d, 1H), 2.32 (s, 3H), 2.20 (s, 3H), 2.08
(s, 3H), 1.72 (m, 1H), 1.36 (s, 3H);
[0488] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 169.8, 149.1,
147.4, 145.5, 136.2, 130.9, 130.8, 125.0, 122.9, 117.7, 112.6,
111.8, 106.4, 100.8, 99.8, 59.8, 58.9, 57.7, 56.6, 56.4, 55.5,
55.2, 41.6, 40.1, 29.6, 25.9, 25.0, 22.6, 15.6, 8.8.
[0489] ESI-MS m/z: Calcd. for C.sub.30H.sub.36SiN.sub.4O.sub.7:
564.6. Found (M+H).sup.+: 565.3.
Example 39
[0490] 180
[0491] To a flask containing 9 (245 mg, 0.435 mmol) in DMF, (4 ml),
cesium carbonate (425 mg, 1.30 mmol) and allyl bromide (376 ml,
4.35 mmol) were added at 0.degree. C. and the mixture was stirred
at 23.degree. C. for 1 h. The reaction was filtered though a pad of
celite and partitioned between CH.sub.2Cl.sub.2 (25 ml) and
H.sub.2O (10 ml). The organic phase was dried (sodium sulphate) and
concentrated at reduced pressure to afford a residue that was
purified by flash column chromatography (SiO.sub.2,
CHCl.sub.3:ethyl acetate 1:2) to give 10 as a yellow oil. (113 mg,
43%).
[0492] Rf: 0.36 (hexane:ethyl acetate 1:5)
[0493] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.74 (s, 1H),
6.3-6.0 (m, 1H), 5.94 (d, 1H), 5.87 (d, 1H), 5.43-5.36 (m, 2 h),
5.22 (s, 2 h), 5.00 (m, 1H), 4.22 (m, 1H), 4.17-4.01 (m, 1H), 3.98
(m, 2 h), 3.71-3.67 (m, 1H), 3.69 (s, 3H), 3.62-3.51 (m, 3H), 3.58
(s, 3H), 3.39-3.37 (m, 1H), 3.31-3.26 (m, 3H), 3.09 (dd, 1H), 2.56
(d, 1H), 2.36 (s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 2.24-2.10 (m,
1H), 1.82-1.73 (m, 1H), 1.24 (bs, 3H)
[0494] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 169.4, 148.8,
148.3, 139.1, 133.7, 130.9, 130.3, 125.2, 120.2, 117.7, 113.1,
112.6, 101.3, 99.3, 74.1, 59.7, 59.3, 57.8, 57.0, 56.1, 56.1, 55.2,
41.6, 41.0, 40.9, 29.7, 26.3, 22.5, 15.6, 9.3
[0495] ESI-MS m/z: Calcd. for C.sub.33H.sub.40N.sub.4O.sub.7:
604.7. Found (M+H).sup.+: 605.3.
Example 40
[0496] 181
[0497] To a solution of 9 (22 mg, 0.039 mmol) in CH.sub.2Cl.sub.2
(0.2 ml), acetyl chloride (2.79 ml, 0.039 mmol) and pyridine (3.2
ml, 0.039 mmol) were added at 0.degree. C. The reaction mixture was
stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure to afford 46 (22 mg,
93%) as a white solid.
[0498] Rf: 0.4 (hexane:ethyl acetate 1:5).
[0499] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 6.74 (s, 1H),
5.97 (d, J=0.9 Hz, 1H), 5.91 (d, J=0.9 Hz, 1H), 5.12 (d, J=5.7 Hz,
2 h), 5.04 (d, J=5.7 Hz, 1H) 4.90 (t, J=6 Hz, 1H), 4.17 (d, J=2.7
Hz, 1H), 4.05 (d, J=2.7 Hz, 1H), 4.01 (bs, 1H), 3.71 (s, 3H), 3.57
(s, 3H), 3.50-3.44 (m, 2 h), 3.38-3.36 (m, 1H), 3.30-3.26 (m, 1H),
3.00 (dd, J.sub.1=7.8 Hz, J.sub.2=18.0 Hz, 1H), 2.79 (d, J=12.9 Hz,
1H), 2.60 (d, J=18.0 Hz, 1H), 2.35 (s, 3H), 2.32 (s, 3H), 2.21 (s,
3H), 2.00 (s, 3H), 1.68 (dd, J.sub.1=11.7 Hz, J.sub.2=15.6 Hz,
1H).
[0500] ESI-MS m/z: Calcd. for C.sub.32h38N.sub.4O.sub.8: 606.67.
Found (M+H).sup.+: 607.3.
Example 41
[0501] 182
[0502] To a solution of 46 (8 mg, 0.013 mmol) in dioxane (0.1 ml),
5.3N HCl/dioxane (0.5 ml) was added and the reaction was stirred at
23.degree. C. for 1 h. Then, the solution was diluted with
CH.sub.2Cl.sub.2 (5 ml) and washed with 0.1 N HCl (3 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure to afford 47 (5 mg,
70%) as a white solid.
[0503] Rf: 0.4 (hexane:ethyl acetate 1:5).
[0504] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 6.51 (s, 1H),
5.97 (d, J=1.2 Hz, 1H), 5.91 (d, J=1.2 Hz, 1H), 4.97 (bs, 1H), 4.11
(bs, 1H), 4.04-4.02 (m, 2 h), 3.75 (s, 3H),), 3.65 (d, J=2.1 Hz, 2
h), 3.56-3.30 (m, 2 h), 3.04 (dd, J.sub.1=7.5 Hz, J.sub.2=18 Hz,
1H), 2.80 (d, J=14.4 Hz, 1H), 2.59 (d, J=18.3 Hz, 1H), 2.33 (s,
3H), 2.24 (s, 3H), 2.00 (s, 3H), 1.76 (dd, J.sub.1=12.0 Hz,
J.sub.2=15.9 Hz, 1H), 1.33 (s, 3H), 1.25 (s, 3H).
[0505] ESI-MS m/z: Calcd. for C.sub.30H.sub.34N.sub.4O.sub.7:
562.61. Found (M+H).sup.+: 563.3.
Example 42
[0506] 183
[0507] To a solution of 45 (10 mg, 0.0192 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), isovaleryl chloride (2.34 ml, 0.0192 mmol) and pyridine
(1.55 ml, 0.0192 mmol) were added at 0.degree. C. The reaction
mixture was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (5 ml) and washed with 0.1 N HCl (3 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, Hex: ethyl
acetate 1:2) to afford 48 (11 mg, 95%) as a white solid.
[0508] Rf: 0.12 (Hex: ethyl acetate 1:2).
[0509] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.50 (s, 1H),
5.98 (d, J=1.5 Hz, 1H), 5.91(d, J=1.5 Hz, 1H), 5.75 (s, 1H), 5.02
(t, J=5.4 Hz, 1H), 4.10 (d, J=1.5 Hz, 1H), 4.06 (d, J=2.7 Hz, 1H),
4.02 (d, J=2.7 Hz, 1H), 3.77 (s, 3H), 3.76-3.71 (m, 1H), 3.86-3.28
(m, 3H), 3.04 (dd, J.sub.1=8.1 Hz, J.sub.2=18.3 Hz, 1H), 2.78 (d,
J=15.9 Hz, 1H), 2.55 (d, J=18 Hz, 1H), 2.32 (s, 6H), 2.26 (s, 3H),
1.98 (s, 3H), 1.84-1.68 (m, 2 h), 1.36 (d, J=7.2 Hz, 2 h), 0.69 (d,
J=6.6 Hz, 3H), 0.62 (d, J=6.6 Hz, 3H).
[0510] ESI-MS m/z: Calcd. for C.sub.33H.sub.40N.sub.4O.sub.7:
604.69. Found (M+H).sup.+: 605.3.
Example 43
[0511] 184
[0512] To a solution of 45 (10 mg, 0.0192 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), isovaleiyl chloride (3.98 ml, 0.0192 mmol) and pyridine
(1.55 ml, 0.0192 mmol) were added at 0.degree. C. The reaction
mixture was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (5 ml) and washed with 0.1 N HCl (3 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, Hex: ethyl
acetate 1:2) to afford 49 (12.4 mg, 96%) as a white solid.
[0513] Rf: 0.7 (ethyl acetate:methanol10:1).
[0514] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.50 (s, 1H),
5.98 (d, J=1.5 Hz, 1H), 5.91 (d, J=1.5 Hz, 1H), 5.73 (s, 1H), 5.08
(t, J=5.4 Hz, 1H), 4.10 (d, J=1.5 Hz, 1H), 4.05 (m., 1H), 4.01 (m,
1H), 3.76 (s, 3H), 3.65-3.61 (m, 1H), 3.40-3.27 (m, 3H), 3.03 (dd,
J.sub.1=8.1 Hz, J.sub.2=18.6 Hz, 1H), 2.78 (d, J=13.2 Hz, 1H), 2.57
(d, J=18.3 Hz, 1H), 2.32 (s, 3H), 2.31 (s, 3H), 2.25 (s, 3H), 1.99
(s, 3H), 1.79 (dd, J.sub.1=12.0 Hz, J.sub.2=16.5 Hz, 1H), 1.73-1.42
(m, 4H), 1.33-1.18 (m, 10H), 1.03 (m, 2 h), 0.87 (t, J=6.6 Hz,
3H).
[0515] ESI-MS m/z: Calcd. for C.sub.38H.sub.50N.sub.4O.sub.7:
674.83. Found (M+H).sup.+: 675.5.
Example 44
[0516] 185
[0517] To a solution of 45 (14.5 mg, 0.0278 mmol) in
CH.sub.2Cl.sub.2 (0.3 ml), trans-3-trifluoromethyl cinnamoyl
chloride (4.76 ml, 0.0278 mmol) and pyridine (2.25 ml, 0.0278 mmol)
were added at 0.degree. C. The reaction mixture was stirred for 1 h
and then, the solution was diluted with CH.sub.2Cl.sub.2 (5 ml) and
washed with 0.1 N HCl (3 ml). The organic layer was dried over
sodium sulphate, filtered, and the solvent was eliminated under
reduced pressure. The residue was purified by flash column
chromatography (SiO.sub.2, Hex: ethyl acetate 1:1) to afford 50
(18.7 mg, 94%) as a white solid.
[0518] Rf: 0.64 (ethyl acetate:methanol5:1).
[0519] .sup.1H NMR (300 MHz, CH.sub.3OD). .delta. 7.74-7.55 (m,
4H), 7.23 (d, J=16.0 Hz, 1H), 6.34 (s, 1H), 6.12 (d, J=16.0 Hz,
1H), 6.07 (d, J=0.9 Hz, 1H), 5.96 (d, J=0.9 Hz, 1H), 4.39 (d, J=2.4
Hz, 1H), 4.07-4.05 (m, 1H), 3.81 (bs, 1H), 3.46-3.51 (m, 3H), 3.42
(s, 3H), 3.09 (br d, J=12.0 Hz, 1H), 2.94-2.85 (m, 2 h), 2.74 (d,
J=18.3 Hz, 1H), 2.38 (s, 3H), 2.23 (s, 3H), 2.02 (s, 3H), 1.80 (s,
3H), 1.84-1.75 (m, 1H).
[0520] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 168.7, 165.3,
146.5, 144.7, 142.6, 140.6, 138.0, 135.9, 131.0, 130.9, 129.1,
128.6, 125.8, 125.7, 124.5, 124.4, 122.7, 121.2, 117.8, 116.5,
113.0, 112.0, 101.7, 60.4, 59.1, 56.5, 56.4, 55.6, 55.3, 41.8,
40.3, 26.6, 25.1, 20.3, 15.4, 9.3.
[0521] ESI-MS m/z: Calcd. for
C.sub.38H.sub.37F.sub.3N.sub.4O.sub.7: 718.72. Found (M+H).sup.+:
719.3.
Example 45
[0522] 186
[0523] To a solution of 43 (33 mg, 0.0557 mmol) in CH.sub.2Cl.sub.2
(0.4 ml), isovaleryl chloride (6.79 ml, 0.0557 mmol) and pyridine
(4.5 ml, 0.0557 mmol) were added at 0.degree. C. The reaction
mixture was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (5 ml) and washed with 0.1 N HCl (3 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, Hex: ethyl
acetate 1:2) to afford 51 (34 mg, 91%) as a white solid.
[0524] Rf: 0.09 (Hex: ethyl acetate 1:2).
[0525] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 6.46 (s,1H), 6.10
(bs, 1H), 5.99 (d, J=0.9 Hz, 1H), 5.90 (d, J=0.9 Hz, 1H), 5.30 (t,
J=6.0 Hz, 1H), 4.10-4.05 (m, 3H),3.81 (bs, 1H), 3.74 (s, 3H), 3.54
(bs,1H), 3.38-3.36 (m, 1H), 3.29-3.21 (m, 1H), 3.00 (dd,
J.sub.1=8.0 Hz, J.sub.2=18.0 Hz, 1H), 2.25 (s, 3H), 2.20 (s, 3H),
2.00 (s, 3H), 1.95-1.90 (m, 3H), 0.87 (d, J=6.6 Hz, 6H), 0.76 (d,
J=6.0 Hz, 3H).
[0526] ESI-MS m/z: Calcd. for C.sub.36H.sub.45N.sub.5O.sub.8:
675.77. Found (M+H).sup.+: 676.3.
Example 46
[0527] 187
[0528] To a solution of 43 (33 mg, 0.0557 mmol) in CH.sub.2Cl.sub.2
(0.4 ml), trans-3-trifluoromethyl cinnamoyl chloride (9.52 ml,
0.0557 mmol) and pyridine (4.5 ml, 0.0557 mmol) were added at
0.degree. C. The reaction mixture was stirred for 1 h and then, the
solution was diluted with CH.sub.2Cl.sub.2 (5 ml) and washed with
0.1 N HCl (3 ml). The organic layer was dried over sodium sulphate,
filtered, and the solvent was eliminated under reduced pressure.
The residue was purified by flash column chromatography (SiO.sub.2,
Hex: ethyl acetate 1:2) to afford 52 (40 mg, 92%) as a white
solid.
[0529] Rf: 0.21 (hexane:ethyl acetate 1:2).
[0530] .sup.1H NMR (300 MHz, CD.sub.3OD). .delta. 7.74-7.47 (m,
4H), 6.49 (s, 1H), 6.40 (d, J=15.6 Hz, 1H), 6.00 (d, J=1.5 Hz, 1H),
5.90 (d, J=1.5 Hz, 1H), 5.47 (t, J=6 Hz, 1H), 4.12-4.09 (m, 3H),
3.93 (bs, 1H), 3.71 (s, 3H), 3.59-3.58 (m, 1H), 3.38 (d, J=7.8 Hz,
1H), 3.29 (d, J=12.0 Hz, 1H), 3.00 (dd, J.sub.1=8.1 Hz,
J.sub.2=18.3 Hz, 1H), 2.79-2.78 (m, 1H), 2.65 (d, J=18.3 Hz, 1H)
2.29 (s, 6H), 2.28 (s, 3H), 2.22 (s, 3H), 1.84-1.80 (m, 1H),
0.85-0.84 (m, 3H).
[0531] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 171.9, 168.8,
164.4, 146.9, 144.6, 143.0, 140.5, 140.5, 139.3, 135.7, 131.1,
131.0, 129.4, 129.1, 126.0, 124.1, 124.0, 122.4, 121.1, 120.7,
120.6, 117.7, 116.9, 112.8, 112.0, 101.6, 60.6, 59.3, 57.1, 56.3,
55.9, 55.2, 49.0, 41.7, 49.9, 26.5, 25.1, 20.2, 18.4, 15.7,
9.3.
[0532] ESI-MS m/z: Calcd. for
C.sub.41H.sub.42F.sub.3N.sub.5O.sub.8: 789.8. Found (M+H).sup.+:
790.3.
Example 47
[0533] 188
[0534] To a solution of 43 (10 mg, 0.0169 mmol) in CH.sub.2Cl.sub.2
(0.2 ml) trifluoroacetic anhydride (2.38 .mu.l, 0.0169 mmol) was
added at 23.degree. C. The reaction mixture was stirred for 5 h and
then, the solution was diluted with CH.sub.2Cl.sub.2 (5 ml) and
washed with 0.1 N HCl (3 ml). The organic layer was dried over
sodium sulphate, filtered, and the solvent was eliminated under
reduced pressure. The residue was purified by flash column
chromatography (SiO.sub.2, Hex: ethyl acetate 3:2) to afford 53
(10.7 mg, 93%) as a white solid.
[0535] Rf: 0.57 (ethyl acetate:methanol5:1).
[0536] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.45 (s, 1H), 6.00
(d, J=1.2 Hz, 1H), 5.90 (d, J=1.2 Hz, 1H), 5.87 (bs, 1H), 5.32 (bs,
1H), 4.12(d, J=2.1 Hz, 1H), 4.08 (d, J=1.8 Hz, 1H), 3.78-3.56 (m,
3H), 3.72 (s, 3H), 3.40 (d, J=8.1 Hz, 1H), 3.25 (d, t 9.3 Hz, 1H),
3.00 (dd, J.sub.1=8.4 Hz, J.sub.2=18.0 Hz, 1H), 2.77 (dd,
J.sub.1=2.1 Hz, J.sub.2=15.9 Hz, 1H), 2.68 (d, J=18.6 Hz, 1H), 2.30
(s, 3H), 2.28 (s, 3H), 2.22 (s, 3H), 2.00 (s, 3H), 1.75 (dd,
J.sub.1=11.4 Hz, J.sub.2=15.9 Hz, 1H), 0.69 (d, J=6.3 Hz, 3H).
[0537] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.1, 168.6,
156.0, 147.0, 144.6, 143.0, 140.6, 140.4, 131.0, 129.4, 120.9,
120.7, 117.6, 116.8, 112.4, 112.1, 101.6, 60.5, 59.0, 57.1, 56.3,
55.6, 55.2, 48.7, 41.6, 39.4, 26.5, 24.9, 20.2, 17.8, 15.4,
9.2.
[0538] ESI-MS m/z: Calcd. for
C.sub.33H.sub.36F.sub.3N.sub.5O.sub.8: 687.63. Found (M+H).sup.+:
688.66.
Example 48
[0539] 189
[0540] To a solution of 19 (11 mg, 0.0169 mmol) in CH.sub.2Cl.sub.2
(0.2 ml) trifluoroacetic anhydride (2.38 ml, 0.0169 mmol) was added
at 23.degree. C. The reaction mixture was stirred for 5 h and then,
the solution was diluted with CH.sub.2Cl.sub.2 (5 ml) and washed
with 0.1 N HCl (3 ml). The organic layer was dried over sodium
sulphate, filtered, and the solvent was eliminated under reduced
pressure. The residue was purified by flash column chromatography
(SiO.sub.2, Hex: ethyl acetate 3:2) to afford 54 (10.7 mg, 93%) as
a white solid.
[0541] Rf: 0.6 (ethyl acetate:methanol5:1).
[0542] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.33 (d, J=6.3 Hz,
1H), 6.45 (s, 1H), 6.04 (m, 1H), 5.95 (d, J=1.5 Hz, 1H), 5.84 (d,
J=1.5 Hz, 1H), 5.32 (m, 2 h), 5.21 (m, 1H), 4.11 (m, 4H), 3.73 (s,
3H), 3.64 (m, 2 h), 3.51 (m, 1H), 3.37 (d, J=7.8 Hz, 1H), 3.22 (m,
2 h), 3.03 (dd, 1H, J.sub.1=8.1 Hz, J.sub.2=18.3 Hz, 1H), 2.60 (d,
J=18.3 Hz, 1H), 2.29 (s, 3H), 2.24 (s, 3H), 2.08 (s, 3H), 1.86 (dd,
J.sub.1=12 Hz, J.sub.2=16.2 Hz, 1H), 0.82 (d, J=7.2 Hz, 3H).
[0543] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.0, 156.0,
148.4, 147.1, 144.3, 143.0, 138.7, 133.8, 130.5, 129.4, 120.6,
120.4, 117.6, 117.5, 117.0, 113.5, 112.5, 112.4, 101.1, 74.1, 66.8,
60.4, 59.3, 56.9, 56.6, 56.3, 55.4, 48.7, 41.6, 40.1, 26.2, 25.0,
17.6, 15.4, 9.1.
[0544] ESI-MS m/z: Calcd. for
C.sub.35H.sub.39F.sub.3N.sub.5O.sub.7: 685.69. Found (M+H).sup.+:
686.3.
Example 49
[0545] 190
[0546] To a solution of 54 (100 mg, 0.415 mmol) in CH.sub.2Cl.sub.2
(4 ml), acetic acid (40 ml), (PPh.sub.3).sub.2PdCl.sub.2 (8.4 mg,
0.012 mmol) and Bu.sub.3SnH (157 ml, 0.56 mmol) were added at
23.degree. C. After stirring at that temperature for 2 h the
reaction was poured into a pad of flash column (SiO.sub.2, gradient
Hex to hexane:ethyl acetate 2:1) to afford 55 (90 mg, 96%) as a
white solid.
[0547] Rf: 0.6 (hexane:ethyl acetate 1:2).
[0548] .sup.1H NMR (300 MHz, CDCB) .delta. 7.55 (d, J=7.2 Hz, 1H),
6.45 (s, 1H), 5.90 (d, J=1.2 Hz, 1H), 5.82 (d, J=1.2 Hz, 1H), 5.37
(t, J=6.0 Hz, 1H), 4.15 (d, J=2.1 Hz, 1H), 4.04 (d, J=1.8 Hz, 1H),
3.70 (s, 3H), 3.66-3.53 (m, 2 h), 3.37-3.31 (m, 2 h), 3.19-3.15 (d,
J=11.7 Hz, 1H), 3.08-3.00 (m, 2 h), 2.56 (d, J=18.3 Hz, 1H), 2.30
(s, 3H), 2.24 (s, 3H), 2.04 (s, 3H), 1.91 (dd, J.sub.1=12.0 Hz,
J.sub.2=15.6 Hz, 1H), 0.84 (d, J=6.9 Hz, 3H).
[0549] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.1, 156.3,
147.3, 144.9, 144.4, 143.3, 136.7, 130.7, 129.3, 120.6, 117.6,
117.4, 114.4, 112.1, 107.7, 101.0, 85.8, 60.5, 59.3, 56.5, 56.4,
56.2, 55.2, 48.9, 41.6, 40.9, 25.7, 25.3, 18.0, 15.6, 8.7.
[0550] ESI-MS m/z: Calcd. for C.sub.32h35F.sub.3N.sub.5O.sub.7:
645.63. Found (M+H).sup.+: 646.2.
Example 50
[0551] 191
[0552] To a solution of 17 (200 mg, 0.288 mmol) in CH.sub.2Cl.sub.2
(1.44 ml), trifluoroacetic acid (888 ml, 11.53 mmol) was added and
the reaction mixture was stirred for 4 h at 23.degree. C. The
reaction was quenched at 0.degree. C. with saturated aqueous sodium
bicarbonate (60 ml) and extracted with ethyl acetate (2.times.70
ml). The combined organic layers were dried (sodium sulphate) and
concentrated in vacuo to afford 56 (147 mg, 93%) as a white solid
that was used in subsequent reactions with no further
purification.
[0553] Rf: 0.19 (ethyl acetate:methanol5:1).
[0554] .sup.1H NMR (300 MHz, CD.sub.3OD). .delta. 6.48 (s, 1H),
5.88, d, J=0.9 Hz, 1H), 5.81 (d, J=0.9 Hz, 1H), 4.35 (d, J=2.4 Hz,
1H),4.15 (d, J=1.8 Hz, 1H), 3.99-3.98 (m, 1H), 3.70 (s, 3H),
3.52-2.96 (m, 7H), 2.68 (d, J=18.3 Hz, 1H), 2.24 (s, 3H), 2.23 (s,
3H), 2.06 (s, 3H), 1.85 (dd, J.sub.1=11.7 Hz, J.sub.2=15.6 Hz, 1H),
0.91 (d, J=6.6 Hz, 3H).
[0555] .sup.13C NMR (75 MHz, CD.sub.3OD): .delta. 173.2, 149.1,
145.6, 144.9, 138.0, 132.2, 130.6, 121.4, 119.6, 117.4, 114.3,
109.2, 102.5, 82.3, 60.4, 58.4, 58.3, 57.8, 56.6, 50.1, 42.3, 41.6,
27.8, 26.2, 19.5, 15.5, 9.8.
[0556] ESI-MS m/z: Calcd. for C.sub.29H.sub.35N.sub.5O.sub.6:
549.62. Found (M+H).sup.+: 550.3.
Example 51
[0557] 192
[0558] To a solution of 56 (10 mg, 0.018 mmol) in CH.sub.2Cl.sub.2
(0.4 ml), phenyl isothiocyanate (13 ml, 0.109 mmol) was added and
the reaction was stirred at 23.degree. C. for 1.5 h. The mixture
was concentrated in vacuo and the residue was purified by flash
column chromatography (SiO.sub.2, gradient Hexane to 1:1
hexane:ethyl acetate) to afford 57 (8 mg, 65%) as a white
solid.
[0559] Rf: 0.57 (ethyl acetate:methanol10:1).
[0560] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta. 7.88 (bs, 1H),
7.41-7.36 (m, 2 h), 7.27-7.22 (m, 1H), 7.02-7.00 (d, J=7.8 Hz, 2
h), 6.71 (d, J=7.2 Hz, 1H), 6.31 (s, 1H), 6.17 (bs, 1H), 5.93 (d,
J=1.2 Hz, 1H), 5.83 (d, 1.2 Hz, 1H), 5.55 (bs, 1H), 5.20-5.17 (m,
1H), 4.16 (d, J=1.8 Hz, 1H), 4.05 (bs, 1H), 4.02 (d, J=2.4 Hz, 1H),
3.79 (s, 3H), 3.75-3.71 (m, 1H), 3.35 (d, J=7.8 Hz, 1H), 3.28-3.19
(m, 2 h), 3.12-2.97 (m, 2 h), 2.50 (d, J=18.3 Hz, 1H), 2.32 (s,
3H), 2.21 (s, 3H), 2.15-2.09 (dd, J.sub.1=11.4 Hz, J.sub.2=15.9 Hz,
1H), 1.95 (s, 3H), 0.88 (d, J=6.9 Hz, 3H).
[0561] .sup.13C NMR (75 MHz, CDCl.sub.3): .delta. 178.5, 171.7,
147.2, 145.0, 144.3, 143.3, 137.0, 135.7, 130.6, 130.4, 129.6,
127.5, 124.3, 120.6, 117.7, 117.2, 115.3, 112.1, 108.3, 100.9,
60.9, 59.5, 56.7, 56.5, 56.2, 55.2, 54.1, 41.7, 41.1, 26.3, 25.4,
18.5, 15.8, 9.0.
[0562] ESI-MS m/z: Calcd. for C.sub.36H.sub.40N.sub.6O.sub.6S:
684.81. Found (M+H).sup.+: 685.3.
Example 52
[0563] 193
[0564] To a solution of 57 (45 mg, 0.065 mmol) in CH.sub.2Cl.sub.2
(0.5 ml), acetyl chloride (4.67 ml, 0.065 mmol) and pyridine (5.3
ml, 0.065 mmol) were added at 0.degree. C. The reaction mixture was
stirred for 3 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (RP-18, CH.sub.3CN:
H.sub.2O 40:60) to afford 58 (14 mg, 28%) as a white solid.
[0565] Rf: 0.34 (CH.sub.3CN: H.sub.2O 7:15).
[0566] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 11.90 (d, J=6.6
Hz, 1H), 7.45-7.40 (m, 3H), 7.18-7.15 (m, 2 h), 6.58 (s, 1H), 6.00
(d, J=1.2 Hz, 1H), 5.89 (d, J=1.2 Hz, 1H), 5.70 (s, 1H), 5.37 (t,
J=4.8 Hz, 1H), 4.48 (m, 1H), 4.23 (bs, 1H), 4.07 (bs, 2 h),
3.85-3.75 (m, 1H), 3.70 (s, 3H), 3.46-3.41 (m, 2 h), 3.24-3.20 (m,
1H), 3.00-2.95 (m, 1H), 2.87-2.75 (m, 1H), 2.31 (s, 3H), 2.28 (s,
3H), 2.24 (s, 3H), 2.00 (s, 3H), 1.85 (dd, J.sub.1=11.4 Hz,
J.sub.2=15.6 Hz, 1H), 1.66 (s, 3H), 0.82 (d, J=6.0 Hz, 3H).
[0567] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 182.6, 174.3,
171.0, 146.6, 144.6, 142.7, 142.3, 140.7, 140.2, 131.3, 129.8,
129.3, 128.9, 128.8, 121.5, 120.4, 117.3, 116.6, 112.8, 112.0,
111.3, 101.5, 60.5, 59.0, 57.6, 56.2, 55.9, 55.3, 55.1, 41.6, 39.4,
27.8, 26.5, 24.8, 20.2, 17.1, 15.5, 9.3.
[0568] ESI-MS m/z: Calcd. for C.sub.40H.sub.44N.sub.6O.sub.8S:
768.88. Found (M+H).sup.+: 769.2.
Example 53
[0569] 194
[0570] A solution of 57 (130 mg, 0.189 mmol) in dioxane (1 ml),
5.3N HCl/dioxane (1.87 ml) was added and the reaction was stirred
at 23.degree. C. for 4 h. Then, CH.sub.2Cl.sub.2 (15 ml) and
H.sub.2O (10 ml) were added to this reaction and the organic layer
was decanted. The aqueous phase was basified with saturated aq
sodium bicarbonate (60 ml) (pH=8) at 0.degree. C. and then,
extracted with ethyl acetate (2.times.50 ml). The combined organic
extracts were dried (sodium sulphate), and concentrated in vacuo to
afford 59 (63 mg, 70%) as a white solid.
[0571] Rf: 0.15 (ethyl acetate:methanol5:1).
[0572] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 6.67 (s, 1H),
5.99 (d, J=0.9 Hz, 1H), 5.91 (d, J=1.2 Hz, 1H), 5.10 (bs, 1H), 4.32
(d, J=7.2 Hz, 1H), 4.25 (dd, J.sub.1=3.6 Hz, J.sub.2=9.3 Hz, 1H),
3.7 (s, 3H), 3.71-3.64 (m, 2 h), 3.50 (dd, J.sub.1=2.4 Hz,
J.sub.2=15.9 Hz, 1H), 3.42-3.37 (m, 2 h), 3.16 (dd, J.sub.1=3.6 Hz,
J.sub.2=12.9 Hz, 1H), 2.57 (dd, J.sub.1=9.3 Hz, J.sub.2=12.9 Hz,
1H), 2.27 (s, 3H), 2.11 (s, 3H), 1.91 (dd, J.sub.1=12.0 Hz,
J.sub.2=15.9 Hz, 1H).
[0573] ESI-MS m/z: Calcd. for C.sub.26H.sub.30N.sub.4O.sub.5:
478.5. Found (M+H).sup.+: 479.3.
Example 54
[0574] 195
[0575] A solution of 43 (20 mg, 0.0338 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), cinnamoyl chloride (5.63 mg, 0.0338 mmol) and pyridine
(2.73 ml, 0.0338 mmol) were added at 0.degree. C. The reaction
mixture was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 60 (22 mg, 90%) as a white solid.
[0576] Rf: 0.56 (EtOAc:MeOH 5:1).
[0577] .sup.1H NMR (300 MHz, CDCl.sub.3). .delta. 7.51 (s, 1H),
7.50-7.47 (m, 2H), 7.36-7.35 (m, 2H), 6.43 (s, 1H), 6.36 (brd,
J=15.9 Hz, 2H), 6.01 (d, J=1.5 Hz, 1H), 5.90 (brd, J=1.5 Hz, 2H),
5.42 (t, J=6.0 Hz 1H), 4.12-4.07 (m, 3H), 3.96-3.95 (m, 1H), 3.73
(bs, 3H), 3.58 (bs, 2H), 3.39 (d, J=8.7 Hz, 1H), 3.25 (d, J=11.7
Hz, 1H), 3.0 (dd, J.sub.1=7.5 Hz, J.sub.2=17.7 Hz, 1H), 2.78 (d,
J=15.9 Hz, 1H), 2.67 (d, J=16.5 Hz, 1H), 2.29 (s, 6H), 2.23 (s,
3H), 1.99 (s, 3H), 1.82 (dd, J.sub.1=11.4 Hz, J.sub.2=15.6 Hz, 1H),
0.83 (d, J=6.0 Hz, 3H).
[0578] .sup.13C NMR (75 MHz, CDCl.sub.3)): .delta. 172.0, 165.0,
146.9, 144.6, 143.1, 141.0, 140.5, 134.8, 131.0, 129.7, 129.1,
128.8, 127.8, 125.5, 123.8, 123.0, 121.1, 120.5, 117.7, 116.9,
112.8, 112.0, 101.9, 60.6, 59.2, 57.1, 56.4, 55.9, 55.3, 48.8,
41.7, 40.0, 26.5, 25.1, 20.3, 18.5, 15.7, 9.3.
[0579] ESI-MS m/z: Calcd. for C.sub.40H.sub.43N.sub.5O.sub.8:
721.8. Found (M+H).sup.+: 722.3.
Example 55
[0580] 196
[0581] A solution of 45 (19 mg, 0.0364 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), heptafluorobutyryl chloride (5.44 ml, 0.0364 mmol) and
pyridine (2.95 ml, 0.0364 mmol) were added at 0.degree. C. The
reaction mixture was stirred for 1 h and then, the solution was
diluted with CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5
ml). The organic layer was dried over sodium sulphate, filtered,
and the solvent was eliminated under reduced pressure. The residue
was purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 61 (11.7 mg, 45%) as a white solid.
[0582] Rf: 0.76 (EtOAc:MeOH 5:1).
[0583] .sup.1H NMR (300 MHz, CDCb.sub.3) .delta. 6.46 (s, 1H), 6.12
(bs, 1H), 5.98 (d, J=1.2 Hz, 1H), 5.93 (d, J=1.2 Hz, 1H), 5.72 (bs,
1H), 4.13-4.11 (m, 2H), 4.0 (d, J=2.4 Hz, 1H), 3.98-3.96 (m, 1H),
3.73 (s, 3H), 3.39 (d, J=7.5 Hz, 1H), 3.39-3.28 (m, 2H), 3.09 (dd,
J.sub.1=8.1 Hz, J.sub.2=18.0 Hz, 1H), 2.80 (d, J=16.2 Hz, 1H), 2.46
(d, J=18.3 Hz, 1H), 2.32 (s, 6H), 2.21 (s, 3H), 1.99 (s, 3H), 1.80
(dd, J.sub.1=12.0 Hz, J.sub.2=16.2 Hz, 1H).
[0584] ESI-MS m/z: Calcd. for
C.sub.32H.sub.31F.sub.7N.sub.4O.sub.7: 716.6. Found (M+H).sup.+:
717.2.
Example 56
[0585] 197
[0586] A solution of 43 (24 mg, 0.04 mmol) in CH.sub.2Cl.sub.2 (0.3
ml), butyryl chloride (4.15 ml, 0.04 mmol) and pyridine (3.28 ml,
0.04 mmol) were added at 0.degree. C. The reaction mixture was
stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 62 (24 mg, 90%) as a white solid.
[0587] Rf: 0.35 (EtOAc:MeOH 5:1).
[0588] .sup.1H NMR (300 MHz, CDCb) 86.47 (s, 1H), 6.10 (d, J=6.5
Hz, 1H), 6.0 (d, J=1.5 Hz, 1H), 5.91 (d, J=1.5 Hz, 1H), 5.86 (bs,
1H), 5.31 (d, J=6.9 Hz, 1H), 4.11-4.06 (m, 3H), 3.85-3.81 (m, 1H),
3.75 (s, 3H), 3.59-3.53 (m, 2H), 3.38 (d, J=7.5 Hz, 1H), 3.27-3.22
(m, 1H), 3.0 (dd, J.sub.1=7.8 Hz, J.sub.2=17.4 Hz, 1H), 2.79 (d,
J=15.3 Hz, 1H), 2.63 (d, J=17.7 Hz, 1H), 2.31 (s, 3H), 2.0 (s, 3H),
1.80 (dd, J.sub.1=12.0 Hz, J.sub.2=15.9 Hz, 1H), 1.58 (q, J=7.2 Hz,
2H), 0.89 (t, J=7.2 Hz, 3H), 0.76 (d, J=6.6 Hz, 3H).
[0589] ESI-MS m/z: Calcd. for C.sub.35H.sub.43N.sub.5O.sub.8:
661.64. Found (M+H).sup.+: 662.3
Example 57
[0590] 198
[0591] A solution of 43 (19 mg, 0.0364 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), cinnamoyl chloride (6.06 mg, 0.0364 mmol) and pyridine
(2.95 ml, 0.0364 mmol) were added at 0.degree. C. The reaction
mixture was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 63 (20.1 mg, 85%) as a white solid.
[0592] Rf: 0.65 (EtOAc:MeOH 5:1).
[0593] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.39-7.29 (m, 5H),
6.42, (s, 1H), 6.01 (d, J=1.5 Hz, 1H), 5.92 (d, J=1.5 Hz, 1H), 5.73
(bs, 1H), 5.24 (t, J=6.8 Hz, 1H), 4.12-4.08 (m, 3H), 3.66-3.64 (m,
2H), 3.58 (bs, 3H), 3.36 (d, J=8.7 Hz, 1H), 3.29 (d, J=12.0 Hz,
1H), 2.98 (dd, J.sub.1=8.1 Hz, J.sub.2=18 Hz, 1H), 2.33 (s, 6H),
2.29 (s, 3H), 2.01 (s, 3H), 1.84 (dd, J.sub.1=12.0 Hz, J.sub.2=15.9
Hz, 1H).
[0594] ESI-MS m/z: Calcd. for C.sub.37H.sub.38N.sub.4O.sub.7:
650.72. Found (M+H).sup.+: 651.2.
Example 58
[0595] 199
[0596] A solution of 43 (20 mg, 0.0338 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), 3-chloropropionyl chloride (3.22 ml, 0.0338 mmol) and
pyridine (2.73 ml, 0.0338 mmol) were added at 0.degree. C. The
reaction mixture was stirred for 1 h and then, the solution was
diluted with CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5
ml). The organic layer was dried over sodium sulphate, filtered,
and the solvent was eliminated under reduced pressure. The residue
was purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 64 (20.5 mg, 89%) as a white solid.
[0597] Rf: 0.32 (EtOAc:Hexane 5:1).
[0598] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.48 (s, 3H), 6.28
(m, 1H), 5.99 (d, J=1.2 Hz, 1H), 5.91 (d, J=1.2 Hz, 1H), 5.86 (bs,
1H), 5.31 (m, 1H), 4.08-4.07 (m, 3H), 3.75 (s, 3H), 3.72-3.53 (m,
5H), 3.39 (d, J=8.1 Hz, 1H), 3.24 (d, J=12.0 Hz, 1H), 3.00 (dd,
J.sub.1=8.1 Hz, J.sub.2=18.0 Hz, 1H), 2.79 (d, J=13.5 Hz, 1H), 2.50
(t, J=6.3 Hz, 2H), 2.32 (s, 3H), 2.28 (s, 3H), 2.25 (s, 3H), 2.0
(s, 3H), 1.79 (dd, J.sub.1=12.3 Hz, J.sub.2=14.8 Hz, 1H), 0.81 (d,
J=6.3 Hz, 3H).
Example 59
[0599] 200
[0600] A solution of 43 (19 mg, 0.0364 mmol) in CH.sub.2Cl.sub.2
(0.3 ml), butyl chloride (3.78 ml, 0.0364 mmol) and pyridine (2.95
ml, 0.0364 mmol) were added at 0.degree. C. The reaction mixture
was stirred for 1 h and then, the solution was diluted with
CH.sub.2Cl.sub.2 (10 ml) and washed with 0.1 N HCl (5 ml). The
organic layer was dried over sodium sulphate, filtered, and the
solvent was eliminated under reduced pressure. The residue was
purified by flash column chromatography (SiO.sub.2, EtOAc:MeOH
20:1) to afford 64 (19 mg, 87%) as a white solid.
[0601] Rf: 0.60 (EtOAc:MeOH 5:1).
[0602] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.50 (s, 1H), 5.98
(d, J=1.5 Hz, 1H), 5.91 (d, J=1.5 Hz, 1H), 5.75 (s, 1H), 5.01 (t,
J=6.4 Hz, 1H), 4.10-4.09 (m, 1H), 4.06 (d, J=2.1 Hz, 1H), 4.03-4.02
(m, 1H), 3.76 (s, 3H), 3.67-3.60 (m, 1H), 3.42-3.35 (m, 2H), 3.29
(d, J=12.0 Hz, 1H), 3.02 (dd, J.sub.1=7.8 Hz, J.sub.2=17.7 Hz, 1H),
2.79 (d, J=14.1 Hz, 1H), 2.56 (d, J=18.3 Hz, 1H), 2.32 (s, 3H),
2.31 (s, 3H), 2.25 (s, 3H), 1.78 (dd, J.sub.1=12.0 Hz, J.sub.2=15.9
Hz, 1H), 1.63 (s, 3H), 1.53-1.46 (m, 2H), 1.28-1.16 (m, 2H), 0.68
(t, J=7.2 Hz, 3H).
[0603] ESI-MS m/z: Calcd. for C.sub.32H.sub.38N.sub.4O.sub.7:
590.67. Found (M+H).sup.+: 591.2.
Example 60
[0604] 201
[0605] To a solution of 50 (31.7 mg, 0.044 mmol) in
CH.sub.3CN/H.sub.2O (1.5 ml/0.5 ml), AgNO.sub.3 (225 mg, 1.32 mmol)
was added and the reaction was stirred at 23.degree. C. for 17 h.
Then brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added at
0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:1) to afford 66 (16 mg,
51%) as a white solid.
[0606] Rf: 0.26 (EtOAc:MeOH 5:1).
[0607] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.66-7.42 (m, 4H),
7.20 (bs, 1H), 6.44 (s, 1H), 5.97 (b, J=1.2 Hz, 1H), 5.90 (d, J=1.2
Hz, 1H), 5.76 (bs, 1H), 5.28 (bs, 1H), 4.54 (bs, 1H), 4.43 (bs,
1H), 4.00 (bs, 1H), 3.68-3.57 (m, 4H), 3.47 (d, J=3.3 Hz, 1H), 3.40
(d, J=11.7 Hz, 1H), 3.17 (d, H=6.9 Hz, 1H), 2.92 (dd, J.sub.1=8.1
Hz, J.sub.2=17.7 Hz, 1H), 2.74 (d, J=17.1 Hz, 1H), 2.48 (d, J=18.6
Hz, 1H), 2.32 (s, 6H), 2.28 (s, 3H), 1.99 (s, 3H), 1.76 (dd,
J.sub.1=12.0 Hz, J.sub.2=16.2 Hz, 1H).
[0608] ESI-MS m/z: Calcd. for
C.sub.37H.sub.38F.sub.3N.sub.3O.sub.8: 709. Found (M.sup.+-17):
692.3.
Example 61
[0609] 202
[0610] To a solution of 53 (57 mg, 0.0828 mmol) in
CH.sub.3CN/H.sub.2O (1.5 mL/0.5 ml), AgNO.sub.3 (650 mg, 3.81 mmol)
was added and the reaction was stirred at 23.degree. C. for 24 h.
Then, brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added at
0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:1) to afford 67 (28 mg,
50%) as a white solid.
[0611] Rf: 0.28 (EtOAc:MeOH 10:1).
[0612] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.47 (s, 1H), 5.97
(s, 1H), 5.88 (s, 1H), 5.35 (bs, 1H), 4.51 (bs, 1H), 4.41 (bs, 1H),
4.12-4.05 (m, 1H), 4.00 (d, J=2.7 Hz, 1H), 3.77 (s, 3H), 3.64 (bs,
1H), 3.46 (d, J=3.3 Hz, 1H), 3.34 (d, J=11.4 Hz, 1H), 3.18 (d,
J=7.5 Hz, 1H), 2.95 (dd, J.sub.1=8.4 Hz, J.sub.2=18.3 Hz, 1H), 2.70
(d, J=15.6 Hz, 1H), 2.48 (d, J=17.7 Hz, 1H), 2.28 (s, 3H), 2.27 (s,
3H), 2.26 (s, 3H), 1.98 (s, 3H), 1.68 (dd, J.sub.1=12 Hz,
J.sub.2=15.6 Hz, 1H), 0.86 (d, J=6.3 Hz, 3H).
[0613] ESI-MS m/z: Calcd. for
C.sub.32H.sub.37F.sub.3N.sub.4O.sub.9: 678.66. Found (M.sup.+-17):
661.2.
Example 62
[0614] 203
[0615] To a solution of 48 (32 mg, 0.0529 mmol) in
CH.sub.3CN/H.sub.2O (1.5 ml/0.5 ml), AgNO.sub.3 (270 mg, 1.58 mmol)
was added and the reaction was stirred at 23.degree. C. for 24 h.
Then, brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added at
0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:1) to afford 68 (18 mg,
56%) as a white solid.
[0616] Rf: 0.40 (EtOAc:MeOH 5:1).
[0617] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.50 (s, 1H), 5.95
(d, J=1.2 Hz, 1H), 5.88 (d, J=1.2 Hz, 1H), 5.23 (d, J=6.9 Hz, 1H),
4.45 (d, J=3.3 Hz, 1H), 4.38 (s, 1H), 4.01 (d, J=2.4 Hz, 1H), 3.78
(m, 1H), 3.77 (s, 3H), 3.41-3.37 (m, 1H), 3.17-3.15 (m, 1H), 2.96
(dd, J.sub.1=7.8 Hz, J.sub.2=18.0 Hz, 1H), 2.70 (d, J=15.3 Hz, 1H),
2.40 (d, J=18.0 Hz, 1H), 2.30 (s, 6H), 2.27 (s, 3H), 1.76-1.65 (m,
1H), 1.35-1.25 (m, 2H), 0.89-0.82 (m, 1H), 0.69 (d, J=6.6 Hz, 3H),
0.58 (d, J=6.6 Hz, 3H)
Example 63
[0618] 204
[0619] To a solution of 51 (27 mg, 0.04 mmol) in
CH.sub.3CN/H.sub.2O (1.5 ml/0.5 ml), AgNO.sub.3 (204 mg, 1.19 mmol)
was added and the reaction was stirred at 23.degree. C. for 24 h.
Then, brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added at
0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:1) to afford 69 (10 mg,
38%) as a white solid.
[0620] Rf: 0.38 (EtOAc:MeOH 5:1).
[0621] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.48 s, 1H), 6.16
(bs, 1H), 5.98 (d, J=1.5 Hz, 1H), 5.89 (d, J=1.5 Hz, 1H), 5.33 (t,
J=6.0 Hz, 1H), 4.50 (m, 1H), 4.40 (m, 1H), 4.11-4.09 (m, 1H), 4.00
(d, J=2.6 Hz, 1H), 3.78 (s, 3H), 3.41-3.32 (m, 3H), 3.18 (d, J=8.4
Hz, 1H), 2.94 (dd, J.sub.1=8.4 Hz, J.sub.2=18.3 Hz, 1H), 2.70 (d,
J=14.4 Hz, 1H), 4.45 (d, J=18.3 Hz, 1H), 2.31 (s, 3H), 2.28 (s,
3H), 2.27 (s, 3H), 2.04 (s, 3H), 2.00-1.86 (m, 3H), 1.73 (m, 1H),
0.87 (d, J=6.3 Hz, 6H).
Example 64
[0622] 205
[0623] To a solution of 63 (15 mg, 0.023 mmol) in
CH.sub.3CN/H.sub.2O (1.5 ml/0.5 ml), AgNO.sub.3 (118 mg, 0.691
mmol) was added and the reaction was stirred at 23.degree. C. for
24 h. Then, brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added
at 0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:1) to afford 70 (20.1 mg,
85%) as a white solid.
[0624] Rf: 0.43 (EtOAc:MeOH 5:1).
[0625] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.38-7.28 (m, 5H),
6.48 (s, 1H), 5.98 (d, J=1.5 Hz, 1H), 5.91 (d, J=1.5 Hz, 1H), 5.75
(bs, 1H), 5.38 (brd, 1H), 5.30 (bs, 1H), 4.53 (m, 1H), 4.42 (m,
1H), 4.02 (d, J=2.7 Hz, 1H), 3.78-3.65 (m, 5H), 3.46-3.40 (m, 2H),
3.17 (d, J=7.8 Hz, 1H), 2.94 (dd, J.sub.1=7.8 Hz, J.sub.2=17.7 Hz,
1H), 2.73 (d, J=16.8 Hz, 1H), 2.45 (d, J=18.0 Hz, 1H), 2.31 (s,
6H), 2.28 (s, 3H), 1.97 (s, 3H), 1.77 (dd, J.sub.1=12.0 Hz,
J.sub.2=15.3 Hz, 1H).
Example 65
[0626] 206
[0627] To a solution of 65 (25 mg, 0.042 mmol) in
CH.sub.3CN/H.sub.2O (1.5 ml/0.5 ml), AgNO.sub.3 (215.56 mg, 1.269
mmol) was added and the reaction was stirred at 23.degree. C. for
24 h. Then, brine (10 ml) and Aq sat NaHCO.sub.3 (10 ml) were added
at 0.degree. C. and the mixture was stirred for 15 min, filtered
through a pad of celite and washed with CH.sub.2Cl.sub.2 (20 ml).
The solution was decanted and the organic layer was dried and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, EtOAc:MeOH 5:2) to afford 71 (16 mg,
65%) as a white solid.
[0628] Rf: 0.0.5 (EtOAc:MeOH 5:2).
[0629] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.50 (s, 1H), 5.95
(d, J=1.5 Hz, 1H), 5.78 (s, 1H), 5.19 (bs, 1H), 4.45 (d, J=3.3 Hz,
1H), 4.37 (bs, 1H), 4.11 (brd, J=4.8 Hz, 1H), 4.01 (d, J=2.1 Hz,
1H), 3.76 (s, 1H), 3.71-3.69 (m, 1H), 3.49-3.35 (m, 1H), 3.24 (d,
J=13.5 Hz, 1H), 3.15 (d, J=9.3 Hz, 1H), 2.95 (dd, J.sub.1=8.1 Hz,
J.sub.2=17.7 Hz, 1H), 2.70 (d, J=15.6 Hz, 1H), 2.40 (d, J=18.0 Hz,
1H), 2.31 (s, 3H), 2.29 (s, 3H), 2.26 (s, 3H), 1.96 (s, 3H),
1.75-1.66 (m, 1H), 1.52-1.17 (m, 2H), 0.66 (t, J=7.2 Hz, 3H).
[0630] Fermentation Procedures
Example A
[0631] Seed medium YMP3 containing 1% glucose; 0.25% beef extract;
0.5% bacto-peptone; 0.25% NaCl; 0.8% CaCO.sub.3 was inoculated with
0.1% of a frozen vegetative stock of the microorganism, strain A2-2
of Pseudomonas fluorescens, and incubated on a rotary shaker (250
rpm) at 27.degree. C. After 30 h of incubation, the seed culture
was added to a agitated-vessel fermentor with a production medium
composed of 2% dextrose; 4% mannitol, 2% dried brewer's yeast
(Vitalevo Biolux, Belgium); 1% (NH.sub.4).sub.2SO.sub.4; 0.04%
K.sub.2HPO.sub.4; 0.8 KCl; 0.001% FeCl.sub.3; 0.1% L-Tyr; 0.8%
CO.sub.3Ca; 0.05% PPG-2000; 0.2% anti-foam silicone (ASSAF-100,
RHODIA UK). The sterilisation was carried out at 122.degree. C. 30
minutes. The volume inoculated was a 2% (v/v). The temperature was
27.degree. C. (0 to 16 h) and 24.degree. C. from 16 h to final
process (41 hours). The dissolve oxygen-pressure was upper to 25%.
The pH was controlled at 6.0 with diluted sulphuric acid since 28
hours till final process. The overpressure was 0.5 bar. A 1%
mannitol or sorbitol was added from 16 h to final process (for two
days running) and 2% for three days fermentation-process.
[0632] After 41 or 64 hours, the fermentation broth must be
extracted for recovery safracin B or KCN treatment in the clarified
broth for recovery safracin B--cyano.
Example B
[0633] Obtention of Safracin B Cyano from the Crude Extract.
[0634] A clarification or filtration from the fermentation broth at
pH 6 removes the solids. The clarified broth was adjusted a pH 9.5
with diluted sodium hydroxide and extracted twice with 2:1 (v/v)
ethyl acetate, methylene chloride or butyl acetate. The extraction
was carried out into an agitated-vessel during 20', the temperature
of the mixture was maintained at 8 to 10.degree. C. The two phases
were separated by a liquid-liquid centrifuge. The organic phase was
dried with sodium sulphate anhydrous or frozen and then filtered
for removing ice. This organic phase (ethyl acetate layer) was
evaporated until obtention of an oil-crude extract.
Example C
[0635] Obtention of Safracin B Cyano from the Clarified Broth.
[0636] A clarification or filtration from the fermentation broth at
pH 6 removes the solids. The clarified broth was adjusted at pH 3.9
with concentrated acetic acid. 0.5 grams per litre of KCN are added
to the clarified broth an incubated at 20.degree. C. during 1 hour
with agitation. Then, the temperature was decreased at 15.degree.
C. and the pH was adjusted at 9.5 with diluted sodium hydroxide and
extracted with 2:1.5 (v/v) ethyl acetate. The extraction was
carried out into an agitated-vessel during 20 minutes, the
temperature of the mixture was maintained at 8 to 10.degree. C. The
two phases were separated by a liquid-liquid centrifuge. The
organic phase was dried with sodium sulphate anhydrous. This
organic phase (ethyl acetate layer) was evaporated until obtention
of an oil-crude extract. This extract was purified by flash column
chromatography (SiO.sub.2, gradient 20:1 to 10: to 5:1 ethyl
acetate:methanol) to afford quantitatively compound 2 as a light
yellow solid.
[0637] Rf: 0.55 (ethyl acetate:methanol5:1); .t.sub.R=19.9 min
[HPLC, Delta Pack C4, 5 .mu.m, 300 A, 150.times.3 mm, .lambda.=215
nm, flow=0.7 ml/min, temp=50.degree. C., grad.: CH.sub.3CN-aq.
NaOAc (10 mM) 85%-70% (20')];
[0638] .sup.1H NMR (300 Mhz, CDCl.sub.3): .delta. 6.54 (dd,
J.sub.1=4.4 Hz, J.sub.2=8.4 Hz, 1H),6.44 (s, 1H), 4.12 (d, J=2.4
Hz, 1H), 4.04 (d, J=2.4 Hz, 1H), 4.00 (s, 3H), 3.87 (bs, 1H), 3.65
(ddd, J.sub.1=1.5 Hz, J.sub.2=8.7 Hz, J.sub.3=9.9 Hz, 1H), 3.35
(br. D, J=8.4 Hz, 1H), 3.15-2.96 (m, 4H), 2.92 (q, J=7.2 Hz, 1H),
2.47 (d, J=18.3 Hz, 1H), 2.29 (s, 3H), 2.18 (s, 3H) 1.83 (s, 3H),
1.64 (ddd, J.sub.1=2.7 Hz, J.sub.2=11.1 Hz, J.sub.3=14.1 Hz, 1H),
0.79 (d, J=7.2 Hz, 3H);
[0639] .sup.13C NMR (75 Mhz, CDCl.sub.3): .delta. 186.0 (q), 175.9
(q), 156.2 (q), 146.8 (q), 142.8 (q), 140.7 (q), 136.6 (q), 130.5
(q), 128.8 (q), 127.0 (q), 120.5 (s), 117.4 (q), 116.5 (q), 60.8
(t), 60.4 (s), 58.7 (t), 56.2 (s), 55.7 (s), 54.8 (s), 54.8 (s),
54.4 (s), 50.0 (s), 41.6 (t), 39.8 (d), 25.2 (d), 24.4 (d), 21.2
(t), 15.5 (t), 8.4 (t).
[0640] ESI-MS m/z: Calcd for C.sub.29H.sub.35N.sub.5O.sub.6: 549.6.
Found (M+Na).sup.+: 572.3.
Example D
[0641] A medium (50 l) composed of dextrose (2%), mannitol (4%),
dry brewer's yeast (2%), ammonium sulphate (1%), potassium
secondary phosphate (0.04%), potassium chloride (0.8%), iron (III)
chloride 6-hydrate (0.001%), L-tyrosine (0.1%), calcium carbonate
(0.8%), poly-(propylene glycol) 2000 (0.05%) and antifoam ASSAF
1000 (0.2%) was poured into a jar-fermentor with 75 l total
capacity and, after sterilisation, inoculated with seed culture
(2%) of A2-2 strain (FERM BP-14) and aerated cultivation under
agitation was carried out at 27.degree. C. to 24.degree. C. for 64
hours (aeration of 75 l per minute and agitation from 350 to 500
rpm). The pH was controlled by automatic feeding of diluted
sulphuric acid from 27 hours to final process. A 2% mannitol was
added from 16 hours to final process. The cultured medium (45 l)
thus obtained was, after removal of cells by centrifugation,
adjusted to pH 9.5 with diluted sodium hydroxide, extracted with 25
litres of ethyl acetate twice. The mixture was carried out into an
agitated-vessel at 8.degree. C. for 20 minutes. The two phases were
separated by a liquid-liquid centrifuge. The organic phases were
frozen at -20.degree. C. and filtered for removing ice and
evaporated ice and evaporated until obtention of a 40 g
oil-dark-crude extract. After introduction of the cyanide group and
purification, 3.0 grams of safracin B cyano were obtained.
Example E
[0642] A medium (50 l) composed of dextrose (2%), mannitol (4%),
dry brewer's yeast (2%), ammonium sulphate (1%), potassium
secondary phosphate (0.02%, potassium chloride (0.2%), Iron (III)
chloride 6-hydrate (0.001%, L-tyrosine (0.1%), calcium carbonate
(0.8%, poly-(propylene glycol) 2000 (0.05%) and antifoam ASSAF 1000
(0.2%) was poured into a jar-fermentor with 75 l total capacity
and, after sterilisation, inoculated with seed culture (2%) of A2-2
strain (FERM BP-14) and aerated cultivation under agitation was
carried out at 27.degree. C. to 24.degree. C. for 41 hours
(aeration of 75 l per minute and agitation from 350 to 500 rpm).
The pH was controlled by automatic feeding of diluted sulphuric
acid from 28 hours to final process. A 1% mannitol was added from
16 hours to final process. The cultured medium (45 l) thus obtained
was, after removal of cells by centrifugation, adjusted to pH 3.9
with 200 ml of conc. acetic acid. 25 grams of potassium cyanide 97%
were added and after 1 hour of agitation at 20.degree. C., the pH
was adjusted to 9.5 with 1500 ml of a solution 10% sodium
hydroxide. Then, extracted with 35 litres of ethyl acetate. The
mixture was carried out into an agitated-vessel at 8.degree. C. for
20 minutes. The two phases were separated by a liquid-liquid
centrifuge. The organic phase was dried by sodium sulphate
anhydrous and evaporated until obtention of a 60 g oil-dark-crude
extract.
[0643] After chromatography, 4.9 grams of safracin B cyano were
obtained.
Example 66
[0644] 207
[0645] To a stirred solution of 25 (7.83 g, 0.0139 mol) and the
commercial available Boc-Cys (Fm) derivative (Bachem) (8.33 g,
35.04 mmol) in dichloromethane (535 mL) under argon,
dimethylaminopyridine (4.28 g, 35.04 mmol) and
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (6.66
g, 35.04 mmol) were added at 23.degree. C. The mixture was then
stirred at 23.degree. C. for 2.5 hours. The reaction was quenched
by addition of a saturated aqueous sodium bicarbonate solution (500
mL), the organic phase separated and the aqueous layer
back-extracted with dichloromethane (250 mL). The combined organic
extracts were dried over sodium sulphate, filtrated and evaporated
to dryness under reduced pressure. The crude product was purified
by flash column chromatography eluting with mixtures of ethyl
acetate and hexane in a gradient manner, from 1:4 to 2:1 to yield
142 (12.21 g, 93%) as a light yellow solid. Rf=0.35 Hex:EtOAc
1:1.
[0646] .sup.1H-NMR (300 MHz, CDCl.sub.3): .delta. 7.72 (d, J=7.3,
2.7 Hz 2H), 7.55 (dd, J.sub.1=14.6, J.sub.2=7.6 2H), 7.40-7.34 (m,
2H), 7.30-7.24 (m, 2H), 6.63 (s, 1H), 6.08-5.99 (m, 1H), 5.91 (d,
J=1.5 Hz, 1H), 5.80 (d, J=1.5 Hz, 1H), 5.39 (dd, J.sub.1=17.3,
J.sub.2=1.7 Hz 1H), 5.24 (dd, J.sub.1=10.5, J.sub.2=1.7 Hz, 1H),
5.09 (AB, J=4.48 Hz, 2H), 5.07 (t, J=7.8 Hz, 1H), 4.34-4.29 (m,
2H), 4.17 (d, J=1.9 Hz, 1H), 4.16-4.04 (m, 4H), 4.02-3.96 (m, 2H),
3.93 (t, J=5.3 Hz, 1H), 3.70 (s, 3H), 3.56 (s, 3H), 3.32 (d, J=8.0,
1H), 3.23-3.17 (m, 2H), 3.0-2.89 (m, 3H), 2.65-2.57 (m, 2H), 2.29
(s, 3H), 2.20 (s, 3H), 2.03 (s, 3H), 1.76 (dd, J.sub.1=16.3,
J.sub.2=12.7 Hz, 1H), 1.45, 1.44 (s, 9H).
[0647] .sup.13C-NMR (75 MHz, CDCl.sub.3): .delta. 170.9, 155.3,
148.9, 148.6, 146.1, 146.0, 144.7, 141.2, 141.1, 139.4, 134.0,
131.0, 130.1, 127.8, 127.2, 125.2, 125.0, 124.3, 121.3, 121.2,
120.1, 118.1, 117.6, 112.9, 101.4, 99.5, 80.3, 74.2, 65.6, 60.4,
60.1, 57.9, 57.4, 57.2, 57.1, 56.9, 55.6, 53.2, 47.0, 41.8, 41.7,
36.7, 35.3, 28.5, 26.6, 25.3, 15.9, 9.4.
[0648] ESI-MS m/z: Calcd. For C.sub.53H.sub.60N.sub.4O.sub.10S:
945.13. Found (M+1).sup.+: 946.3.
Example 67
[0649] 208
[0650] To a stirred solution of 142 (12.01 g, 0.0127 mol) in
dichloromethane (318 mL), dichlorobis(triphenylphosphine) palladium
(II) (0.71 g, 1.015 mmol) and acetic acid (3.6 mL, 0.176 mol) were
added under argon at 23.degree. C. Then, tributyl tin hydride
(10.27 mL, 0.037 mol) was added in a dropwise manner. The mixture
was stirred at 23.degree. C. for 10 minutes. The reaction was then
filtered through a silica gel column compacted with hexane. 143
(10.89 g, 95%) was obtained as a yellow solid by subsequent elution
with mixtures of ethyl acetate and hexane in a gradient manner,
from 1:4, 1:1 to 7:3. Rf=0.25 Hex:EtOAc 2:1.
[0651] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.72 (d, J=7.6 Hz,
2H), 7.61 (d, J=6.6 Hz, 1H), 7.52 (d, J=7.3 Hz, 1H), 7.37 (t, J=7.8
Hz, 2H), 7.28 (m, 2H), 6.63 (s, 1H), 5.87 (d, J=1.5 Hz, 1H), 5.76
(d, J=1.5 Hz, 1H), 5.58 (bs, 1H), 5.31 (d, J=5.8 Hz, 1H), 5.17 (d,
J=5.6 Hz, 1H), 4.91 (d, J=8.3 Hz, 1H), 4.17-4.06 (m, 4-6H), 3.85
(t, J=5.7 Hz, 1H), 3.70 (s, 3H), 3.68 (s, 3H), 3.34 (brd, J=6.6 Hz,
1H), 3.23 (brd, J=11.2 Hz, 1H), 3.06 (brd, J=12.9 Hz, 1H),
3.04-2.86 (m, 3H), 2.65-2.54 (m, 2H), 2.28 (s, 3H), 2.21 (s, 3H),
1.94 (s, 3H), 1.80 (dd, J.sub.1=11.5 Hz, J.sub.2=15.8 Hz, 1H), 1.45
(s, 9H).
[0652] .sup.13C NMR (75 MHz, CDCl.sub.3) 6175.3, 170.5, 154.9,
149.1, 147.6, 145.9, 145.8, 145.7, 144.5, 140.9, 140.8, 136.1,
130.9, 127.4, 126.9, 124.3, 124.7, 122.9, 119.7, 117.6, 112.3,
111.4, 106.6, 100.7, 99.7, 80.0, 64.2, 60.3, 59.8, 57.6, 57.0,
56.5, 56.4, 55.2, 52.7, 46.7, 46.5, 41.4, 41.3, 36.9, 36.6, 34.9,
28.2, 26.0, 24.9, 20.9, 20.7, 15.7, 14.1, 8.5.
[0653] ESI-MS m/z: Calcd. For C.sub.50H.sub.56N.sub.4O.sub.10S:
905.5. Found (M+1).sup.+: 906.3.
Example 68
[0654] 209
[0655] To a solution of 143 (10 g, 0.01 .mu.mol) in anhydrous
dichloromethane (185 mL) at -10.degree. C. (bath temperature
-15.degree. C.), a solution of benzeneseleninic anhydride (5.7 g,
0.011 mol) was added in anhydrous dichloromethane (185 mL),
discarding any white solid present in the solution. The mixture was
stirred for 10 minutes at the same temperature. The reaction was
diluted with dichloromethane (200 mL) and a saturated aqueous
sodium bicarbonate solution (500 mL) was added at -10.degree. C.
The organic phase was separated, dried over sodium sulphate,
filtered and concentrated to dryness at reduced pressure. The
residue was purified by flash column chromatography, eluting with
mixtures of ethyl acetate and hexane in a gradient manner, from
1:1, 3:2, 7:3 to 4:1 to obtain 144 (9.34 g, 92%) as a yellow solid.
The purified solid from chromatography was dissolved in
dichloromethane (250 mL), charcoal (3.3 g) was added and the
suspension was stirred at 23.degree. C. for 1 hour. The mixture was
filtered through celite and the celite was washed with
dichloromethane (80 mL). The solvent was evaporated at reduced
pressure maintaining the temperature at 25-30.degree. C. to yield
144 (8.96 g, 88%) as a yellow solid. Rf=0.30 and 0.25 (mixture of
isomers) Hex:EtOAc 1:1.
[0656] .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 7.73-7.61 (m, 4H), 7.37-7.30 (m, 4H), 6.62 (s, 1H), 6.59
(s, 1H), 6.53 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.61 (s, 1H),
5.55 (bs, 1H), 5.34 (m, 2H), 5.08 (AB sist., J.sub.AB=6.7 Hz, 1H),
5.00 (AB sist., J.sub.AB=5.9 Hz, 1H), 4.67 (m, 1H), 4.50 (m, 1H),
4.38 (dd, J.sub.1=4.9 Hz, J.sub.2=12.9 Hz, 1H), 4.21 (dd,
J.sub.1=6.3 Hz, J.sub.2=12.9 Hz, 1H), 4.11 (t, J=5.9 Hz, 1H), 4.02
(m, 3H), 3.87 (m, 1H), 3.83 (s, 3H), 3.72 (m, 1H), 3.61 (s, 3H),
3.49 (s, 3H), 3.27 (m, 1H), 3.15 (dd, J.sub.1=1.8 Hz, J.sub.2=6.2
Hz, 2H), 3.07 (d, J=6.3 Hz, 1H), 2.94 (m, 4H), 2.86 (m, 2H), 2.42
(m, 2H), 2.25 (s, 3H), 2.20 (s, 3H), 2.15 (s, 3H), 2.08 (dd,
J.sub.1=2.4 Hz, J.sub.2=13.9 Hz, 1H), 1.77 (s, 3H), 1.76 (s, 3H),
1.43 (s, 9H).
[0657] .sup.13C NMR (75 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 200.6, 171.2, 160.4, 155.6, 148.9, 148.8, 148.3, 145.9,
145.8, 141.3, 141.2, 138.7, 130.9, 127.9, 127.4, 127.3, 127.3,
125.3, 125.1, 124.2, 120.1, 117.1, 111.9, 108.5, 105.0, 104.7,
101.7, 101.3, 99.5, 99.4, 80.5, 72.5, 70.8, 60.5, 60.1, 58.4, 58.0,
57.9, 56.9, 56.8, 56.3, 55.9, 55.5, 55.4, 53.8, 53.7, 47.1, 42.0,
41.8, 41.5, 37.4, 37.3, 35.6, 35.5, 28.5, 25.8, 25.7, 16.1, 16.0,
7.7, 7.3.
[0658] ESI-MS m/z: Calcd. for C.sub.50H.sub.56N.sub.4O.sub.11S:
921.3. Found (M+1).sup.+: 922.3.
Example 69
[0659] 210
[0660] To a solution of DMSO (3.44 mL) in anhydrous dichloromethane
(396 mL), triflic anhydride (3.27 mL, 19.45 mmol) was added under
argon at -78.degree. C. and the mixture was stirred at-that
temperature for 20 minutes. Then, a solution of 144 (8.92 g, 9.6
mmol) in anhydrous dichloromethane (124 mL) at -78.degree. C. was
added and the mixture was stirred under argon at -40.degree. C. for
35 minutes. Diisopropylethylamine (13.5 mL, 73.43 mmol) was added
and the mixture was stirred under argon for 45 minutes at 0.degree.
C. Tert-butanol (3.65 mL, 38.6 mmol) and tert-butyl tetramethyl
guanidine (11.6 mL, 67.46 mmol) were added and the mixture was
stirred under argon for 40 minutes at 23.degree. C. Acetic
anhydride (9.15 mL, 96.78 mmol) was then added and the reaction
stirred for a further 1 hour at 23.degree. C. The reaction was
diluted with dichloromethane (250 mL) and a saturated aqueous
ammonium chloride solution (500 mL) was added. The organic layer
was separated and washed sequentially with a saturated aqueous
sodium bicarbonate solution (500 mL) and a saturated aqueous sodium
chloride solution (500 mL). The organic layer was separated, dried
over sodium sulphate, filtered and concentrated to dryness at
reduced pressure, maintaining the temperature at 25-30.degree. C.
The crude solid was then purified by flash column, chromatography,
eluting with mixtures of ethyl acetate and hexane in a gradient
manner, from 1:4 to 2:3 to give 145 (4.99 g, 68%) as a yellow
solid. Rf=0.44 Hex:EtOAc 3:2.
[0661] .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 6.79 (s, 1H), 6.09 (s, 1H), 6.00 (s, 1H), 5.20 (d, J=5.4
Hz, 1H), 5.14 (d, J=5.6 Hz, 1H), 5.02 (d, J=11.7 Hz, 1H), 4.63 (d,
J=9.0 Hz, 1H), 4.50 (m, 1H), 4.33 (d, J=5.4 Hz, 1H), 4.30 (m, 1H),
4.25 (bs, 1H), 4.18 (d, J=2.4 Hz, 1H), 4.17 (dd, J.sub.1=1.3 Hz,
J.sub.2=11.7 Hz, 1H), 3.78 (s, 3H), 3.57 (s, 3H), 3.42 (m, 2H),
2.93 (m, 2H), 2.35 (m, 1H), 2.31 (s, 3H), 2.29 (s, 3H), 2.22 (s,
3H), 2.09 (m, 1H), 2.05 (s, 3H), 1.45 (s, 9H).).
[0662] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 207.3, 170.9,
168.8, 155.4, 149.8, 148.6, 146.0, 141.1, 140.7, 131.7, 130.6,
125.1, 120.6, 118.3, 113.7, 102.2, 99.4, 80.0, 61.6, 60.4, 59.8,
59.4, 59.2, 57.7, 55.0, 54.7, 54.0, 41.9, 41.6, 33.1, 31.8, 28.7,
23.9, 20.6, 16.1, 14.3, 9.8.
[0663] ESI-MS m/z: Calcd. for C.sub.38H.sub.46N.sub.4O.sub.11S:
766.86. Found (M+1).sup.+: 767.3.
Example 70
[0664] 211
[0665] To a solution of 145 (1.0 g, 1.3 mmol) in acetonitrile (50
mL) and dichloromethane (25 mL), sodium iodide (1.52 g, 10.01 mmol)
was added at 23.degree. C. The mixture was then cooled to 0.degree.
C. and aluminium trichloride (1.33 g, 10.01 mmol) was added
portionwise maintaining the temperature at 0.degree. C. The mixture
was then stirred for 2.5 hours at 0.degree. C. The reaction was
diluted with dichloromethane (25 mL) and a saturated aqueous
solution of sodium potassium tartrate (100 mL) was added. The
aqueous phase is separated and extracted with dichloromethane
(2.times.75 mL). A saturated aqueous sodium bicarbonate solution
(50 mL) was then added to the aqueous phase which was further
extracted with dichloromethane (2.times.50 mL). The combined
organic extracts were dried over sodium sulphate, filtered and
evaporated to dryness under reduced pressure, maintaining the
temperature below 25.degree. C. The crude solid was then purified
by column chromatography on amino-silicagel and eluting with
mixtures of ethyl acetate and hexane in a gradient manner. 35 (487
mg, 60%) was obtained as a yellow solid. Experimental data of 35
were previously described in PCT/GB00/01852.
[0666] 36, ET-770 and ET-743 were prepared following the same
procedures than those previously described in PCT/GB00/01852.
[0667] Route 2
Example 71
[0668] 212
[0669] A solution of 21 (9.84 g, 18.97 mmol) in THF (569 mL) and
H.sub.2O (285 mL) was cooled at 0.degree. C. with an ice bath.
Then, NaNO.sub.2 (1.96 g, 28.45 mmol) and 90% aq. AcOH (18.97 mL,
0.33 mol) were added at 0.degree. C. and the mixture was stirred at
23.degree. C. for 18 h. After cooling down the reaction to
0.degree. C., a saturated aqueous sodium bicarbonate solution (300
mL, basic pH) and dichloromethane (500 mL) were added. After
extraction, the aqueous phase was further extracted with
dichloromethane (2.times.300 mL). The combined organic extracts
were dried over sodium sulphate and evaporated to dryness under
reduced pressure. The crude solid was then disolved in MeOH (379
mL), and 1M NaOH (38 mL) was added at 0.degree. C. The mixture was
stirred at 23.degree. C. for 4 h. After dilution with EtOAc (600
mL) at 0.degree. C., the organic layer was washed with a mixture of
water (400 mL) and, a saturated aqueous sodium bicarbonate solution
(100 mL, basic pH). After extraction, the aqueous phase was further
extracted with EtOAc (3.times.300 mL). The combined organic
extracts were dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo. The residue was purified by flash column
chromatography (SiO.sub.2, Hex:EtOAc gradient from 3:1 to 2:1) to
afford 146 (4.55 g, 46%) as a white solid. Rf: 0.33 (Hex:EtOAc
1:1).
[0670] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.48 (s, 1H),
6.15-6.02 (m, 1H), 5.92 (d, J=1.5 Hz, 1H), 5.86 (d, J=1.5 Hz, 1H),
5.77 (s, 1H), 5.39 (dd, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.26
(dd, J.sub.1=1.5 Hz, J.sub.2=10.5 Hz, 1H), 4.24-4.15 (m, 3H), 4.04
(d, J=2.4 Hz, 1H), 3.97 (t, J=3.3 Hz, 1H), 3.74 (s, 3H), 3.64 (dt,
J.sub.1=3.3 Hz, J.sub.2=11.1 Hz, 1H), 3.43 (dd, J.sub.1=3.3 Hz,
J.sub.2=10.5 Hz, 1H), 3.38-3.34 (m, 2H), 3.31 (t, J=2.7 Hz, 1H),
3.22 (dd, J.sub.1=2.4 Hz, J.sub.2=15.6 Hz, 1H), 3.10 (dd,
J.sub.1=8.1 Hz, J.sub.2=18.3 Hz, 1H), 2.49 (d, J=18.3 Hz, 1H), 2.34
(s, 3H), 2.24 (s, 3H), 2.11 (s, 3H), 1.88 (dd, J.sub.1=12 Hz,
J.sub.2=15.9 Hz, 1H).
[0671] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.6, 146.7,
144.4, 143.0, 138.9, 133.9, 130.2, 129.1, 121.1, 120.9, 117.7,
117.4, 116.8, 113.3, 112.3, 101.1, 74.3, 63.7, 60.6, 60.1, 58.1,
56.9, 56.7, 55.4, 41.7, 26.2, 25.7, 15.7, 9.3.
[0672] ESI-MS m/z: Calcd. for C.sub.29H.sub.33N.sub.3O.sub.6:
519.59. Found (M+1).sup.+: 520.5.
Example 72
[0673] 213
[0674] To a stirred solution of 146 (47.35 g, 0.091 mol) and the
commercial available Boc-Cys (Fm) derivative (54.6 g, 0.137 mol) in
dichloromethane (2.8 L) under argon, dimethylaminopyridine (5.6 g,
0.046 mol) and 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride (43.6 g, 0.227 mol) were added dropwise during 1.5 h
at 23.degree. C. The mixture was then stirred at 23.degree. C. for
1 more hour. The reaction was quenched by addition of a saturated
aqueous sodium bicarbonate solution (1 L) and the organic phase was
separated. The aqueous layer was back-extracted with
dichloromethane (2.times.500 mL). The combined organic extracts
were dried over sodium sulphate and evaporated to dryness under
reduced pressure. The crude product was purified by flash column
chromatography eluting with mixtures of ethyl acetate and hexane in
a gradient manner, from 1:4 to 3:1 to yield 147 (74.3 g, 93%) as a
white solid. Rf=0.5 Hex: EtOAc 1:1.
[0675] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.73 (d, J=7.8 Hz,
2H), 7.63-7.55 (m, 2H), 7.39-7.35 (m, 2H), 7.29-7.25 (m, 2H), 6.41
(s, 1H), 6.07-5.97 (m, 1H), 5.92 (d, J=1.2 Hz, 1H), 5.80 (d, J=1.2
Hz, 1H), 5.67 (s, 1H), 5.34 (dd, J.sub.1=1.8 Hz, J.sub.2=17.4 Hz,
1H), 5.23 (dd, J=1.8 Hz, J.sub.2=10.5 Hz, 1H), 5.04 (d, J=9.3 Hz,
1H), 4.32-4.29 (m, 1H), 4.13-3.91 (m, 9H), 3.72 (s, 3H), 3.31 (d,
J=7.2 Hz, 1H), 3.26-3.17 (m, 2H), 2.96-2.87 (m, 3H), 2.68-2.54 (m,
2H), 2.27 (s, 3H), 2.24 (s, 3H), 2.05 (s, 3H), 1.83 (dd,
J.sub.1=12.6 Hz, J.sub.2=15.9 Hz, 1H), 1.45 (s, 9H).
[0676] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.9, 155.4,
149.0, 147.1, 146.2, 146.0, 144.7, 143.0, 141.1, 139.4, 134.1,
131.5, 129.1, 127.8, 127.2, 125.0, 121.3, 120.9, 120.1, 118.2,
117.6, 117.2, 112.9, 112.4, 101.4, 80.3, 76.6, 74.4, 65.3, 61.0,
60.4, 57.4, 56.9, 56.7, 55.6, 53.0, 46.9, 41.8, 36.7, 35.3, 31.8,
28.5, 26.6, 25.2, 22.9, 16.0, 14.4, 9.5.
[0677] ESI-MS m/z: Calcd. for CsiH.sub.56N.sub.4O.sub.9S: 900.3.
Found (M+1).sup.+: 901.3.
Example 73
[0678] 214
[0679] To a solution of 147 (0.562 g, 0.624 mol) in CH.sub.3CN
(3.12 mL), MEMCl (1.07 mL, 9.36 mmol), DIPEA (2.17 mL, 12.48 mmol)
and DMAP (0.0076 g, 0.06 mmol) were added at 0.degree. C. The
mixture was stirred for 5.5 h at 23.degree. C. The reaction was
diluted with CH.sub.2Cl.sub.2 (50 mL) and extracted with 0.1N HCl
(50 mL). The aqueous phase was extracted again with
CH.sub.2Cl.sub.2 (50 mL). The combined organic phases were dried
over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give a
residue which was purified by flash column chromatography
(CH.sub.2Cl.sub.2:EtOAc 10:1, 5:1) to give 148 (539 mg, 87%) as a
white solid. Rf=0.50 CH.sub.2Cl.sub.2:AcOEt 6:1.
[0680] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.73-7.71 (m, 2H),
7.57 (dd, J.sub.1=7.2 Hz, J.sub.2=15.3 Hz, 2H), 7.40-7.34 (m, 2H),
7.29-7.26 (m, 2H), 6.62 (s, 1H), 6.08-5.99 (m, 1H), 5.91 (d, J=1.2
Hz, 1H), 5.79 (d, J=1.2 Hz, 1H), 5.35 (dd, J.sub.1=1.2 Hz,
J.sub.2=17.1 Hz, 1H), 5.23 (d, J=6.3 Hz, 1H), 5.21 (bs, 1H), 5.13
(d, J=6.3 Hz, 1H), 5.04 (brd, J=9 Hz, 1H), 4.33-4.29 (m, 2H),
4.16-3.90 (m, 8H), 3.85-3.78 (m, 1H), 3.69 (s, 3H), 3.60-3.55 (m,
2H), 3.38 (s, 3H), 3.31 (brd, J=8.1 Hz, 1H), 3.21-3.17 (m, 2H),
2.98-2.88 (m, 3H), 264-2.56 (m, 2H), 2.29 (s, 3H), 2.20 (s, 3H),
2.02 (s, 3H), 1.75 (dd, J.sub.1=11.7 Hz, J.sub.2=15.6 Hz, 1H), 1.47
(s, 9H).
[0681] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.5, 155.0,
148.6, 148.5, 148.2, 145.77, 145.6, 144.4, 140.8, 140.7, 139.0,
133.6, 130.7, 130.5, 127.4, 126.9, 124.8, 124.6, 123.8, 120.8,
119.7, 117.8, 117.2, 122.5, 111.9, 101.0, 98.1, 80.0, 77.4, 77.0,
76.6, 73.8, 71.6, 69.2, 65.0, 60.2, 60.0, 59.8, 59.0, 56.8, 56.7,
56.6, 55.2, 52.7, 46.6, 41.3, 36.2, 34.9, 29.6, 28.2, 26.3, 24.9,
15.6, 14.1, 9.0.
[0682] ESI-MS m/z: Calcd. for C.sub.55H.sub.64N.sub.4O.sub.1S:
988.4. Found (M+1).sup.+: 989.3.
Example 74
[0683] 215
[0684] To a stirred solution of 148 (38.32 g. 0.039 mol) in
dichloromethane (1 L), dichlorobis(triphenylphosphine) palladium
(II) (2.17 g, 0.0031 mol) and acetic acid (11.1 mL, 0.195 mol) were
added under argon at 23.degree. C. Then, tributyl tin hydride (36.5
mL, 0.136 mol) was added in a dropwise manner. The mixture was
stirred at 23.degree. C. for 15 minutes. The reaction was then
filtered through a silica gel column compacted with hexane. 149
(35.07 g, 95%) was obtained as a white solid by subsequent elution
with mixtures of ethyl acetate and hexane in a gradient manner,
from 0:100, 1:4, 1:3, 2:5, 2:3, 1:1, 2:1, 3:1 to 100:0. Rf=0.25
Hex:EtOAc 2:1.
[0685] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.74 (d, J=7.2 Hz,
2H), 7.63-7.53 (m, 2H), 7.39-7.34 (m, 2H), 7.30-7.27 (m, 2H), 6.62
(s, 1H), 5.87 (m, 1H), 5.75 (s, 1H), 5.69 (bs, 1H), 5.37 (d, J=6
Hz, 1H), 5.23 (d, J=5.7 Hz, 1H), 4.96 (d, J=8.1 Hz, 1H), 4.44 (brd,
J=8.7 Hz, 1H), 4.18-3.70 (m, 11H), 3.69 (s, 3H), 3.38 (s, 3H),
3.34-3.18 (m, 3H), 2.99-2.88 (m, 3H), 2.63-2.58 (m, 2H), 2.28 (s,
3H), 2.21 (s, 3H), 2.05 (s, 3H), 1.78 (dd, J.sub.1=12.9 Hz,
J.sub.2=15.6.3 Hz, 1H), 1.41 (s, 9H).
[0686] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.8, 155.2,
149.0, 148.0, 146.2, 146.0, 144.8, 141.1, 136.4, 131.3, 131.2,
127.8, 127.2, 125.1, 125.0, 123.2, 120.0, 118.1, 112.6, 111.6,
107.2, 101.0, 98.9, 98.8, 80.3, 71.8, 69.8, 64.9, 60.6, 60.2, 59.2,
57.1, 56.9, 55.5, 53.0, 47.0, 46.9, 41.8, 37.0, 35.3, 28.5, 26.2,
25.2, 21.9, 21.3, 16.1, 14.4, 9.0.
[0687] ESI-MS m/z: Calcd. for C.sub.52H.sub.60N.sub.4O.sub.11S:
948.4. Found (M+1).sup.+: 949.3.
Example 75
[0688] 216
[0689] To a solution of 149 (15 g, 0.0158 mol) in anhydrous
dichloromethane (265 mL) at -10.degree. C. (bath temperature
-15.degree. C.), a solution of benzeneseleninic anhydride (7.4 g,
0.0143 mol) in anhydrous dichloromethane (265 mL) was added
dropwise during 30 minutes, discarding any white solid present in
the solution. The mixture was stirred for a further 10 minutes at
the same temperature. The reaction was diluted with dichloromethane
(200 mL) and a saturated aqueous sodium bicarbonate solution (500
nL) was added at -10.degree. C. The organic phase was separated,
dried over sodium sulphate, filtered and concentrated to dryness at
reduced pressure. The residue was purified by flash column
chromatography eluting with mixtures of ethyl acetate and hexane in
a gradient manner, from 1:2 to 100:0 to obtain 150 (14.20 g, 89%)
as a yellow solid. The purified solid from chromatography is
dissolved in dichloromethane (250 mL) and charcoal (4.95 g) was
added. The suspension was then stirred at 23.degree. C. for 1 hour.
The mixture was filtered through a pad of celite and the celite was
washed with dichloromethane (80 mL). The solvent was evaporated at
reduced pressure to yield 150 (13.72 g, 86%) as a white solid.
Rf=0.37 Hex:EtOAc 1:2.
[0690] .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 7.73 (t, J=6.7 Hz, 4H), 7.63 (m, 2H), 7.54 (d, J=7.6 Hz,
2H), 7.40-7.34 (m, 4H), 7.31-7.27 (m, 4H), 6.62 (s, 2H), 5.86 (s,
1H), 5.81 (s, 1H), 5.75 (s, 1H), 5.72 (s, 1H), 5.70 (s, 1H), 5.35
(d, J=5.9 Hz, 1H), 5.30 (d, J=8.4 Hz, 1H), 5.23 (d, J=5.9 Hz, 1H),
5.22 (d, J=5.9 Hz, 1H), 5.13 (d, J=5.9 Hz, 1H), 4.97 (d, J=8.8 Hz,
1H), 4.43 (m, 2H), 4.20-4.01 (m, 8H), 3.97-3.86 (m, 4H), 3.82 (s,
3H), 3.80-3.74 (m, 1H), 3.69 (s, 3H), 3.66-3.64 (m, 4H), 3.54 (m,
2H), 3.38 (s, 3H), 3.35 (s, 3H), 3.34-2.90 (m, 8H), 2.60-2.31 (m,
4H), 2.27 (s, 3H), 2.25 (s, 3H), 2.21 (s, 3H), 1.97 (s, 3H),
1.94-1.81 (m, 2H), 1.77 (s, 3H), 1.43 (s, 9H), 1.41 (s, 9H).
[0691] .sup.13C NMR (75 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 200.2, 198.3, 170.7, 170.5, 160.0, 155.2, 154.9, 148.5,
148.4, 145.5, 142.1, 140.9, 138.3, 130.9, 130.5, 130.0, 129.8,
127.5, 126.9, 125.0, 124.9, 124.7, 123.8, 122.5, 119.8, 117.2,
116.7, 111.5, 108.1, 104.6, 104.3, 101.3, 100.9, 98.0, 80.1, 72.1,
71.5, 70.5, 69.2, 69.0, 66.4, 63.5, 60.7, 60.1, 59.6, 58.9, 58.8,
58.0, 56.7, 56.4, 56.2, 55.9, 55.5, 55.0, 53.5, 46.7, 41.7, 41.3,
41.1, 36.9, 35.2, 35.1, 31.4, 28.1, 25.4, 25.3, 22.5, 15.7, 15.6,
14.0, 7.2.
[0692] ESI-MS m/z: Calcd. for C.sub.52H.sub.60N.sub.4O.sub.12S:
964.4. Found: 965.3 (M+1).sup.+, 987.3 (M+23).sup.+.
Example 76
[0693] 217
[0694] The reaction flask was flamed twice, purged vacuum/Argon
several times and kept under Argon atmosphere for the reaction. To
a solution of DMSO (385.0 .mu.L) in anhydrous CH.sub.2Cl.sub.2 (42
mL) was dropwise added triflic anhydride (366.5 .mu.L, 2.16 mmol)
at -78.degree. C. The reaction mixture was stirred at -78.degree.
C. for 20 minutes. Then, a solution of 150 (1 g, 1.03 mmol) in
anhydrous CH.sub.2Cl.sub.2 (10 mL, for the main addition and 5 mL
for washing) was added via canula (addition time: 5 min) at
-78.degree. C. During the addition the temperature was kept at
-78.degree. C. in both flasks and the color changed from yellow to
brown. The reaction mixture was stirred at -40.degree. C. for 35
minutes. During this period of time the solution was turned from
yellow to dark green. After this time, .sup.iPr.sub.2NEt (1.51 mL,
9.55 mmol) was dropwise added and the reaction mixture was kept at
0.degree. C. for 45 minutes, the color of the solution turned brown
during this time.
[0695] Then, tBuOH (409.5 L, 4.33 mmol) and tert-butyl tetramethyl
guanidine (1.31 mL, 7.61 mmol) were dropwise added and the reaction
mixture was stirred at 23.degree. C. for 40 minutes. After this
time, acetic anhydride (1.03 mL, 10.89 mmol) was dropwise added and
the reaction mixture was kept at 23.degree. C. for 1 hour more.
Then, the reaction mixture was diluted with CH.sub.2Cl.sub.2 (25
mL) and washed with aqueous saturated solution of NH.sub.4Cl (50
mL), NaHCO.sub.3 (50 mL), and NaCl (50 mL). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and concentrated.
The residue was purified by flash column chromatography (inner
diameter: 2.0 cm, height of silica: 9 cm; eluent: ethyl
acetate/hexane in a gradient manner, from 20:80, 30:70 to 40:60) to
afford 151 (832.6 mg, 99%) as a white solid. Rf=0.48 Hex:EtOAc
3:2.
[0696] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.78 (s, 1H), 6.09
(d, J=1.2 Hz, 1H), 5.99 (d, J=1.2 Hz, 1H), 5.32 (d, J=5.8 Hz, 1H),
5.19 (d, J=5.6 Hz, 1H), 5.01 (d, J=11.7 Hz, 1H), 4.62 (d, J=9.8 Hz,
1H), 4.50 (bs, 1H), 4.34 (d, J=5.1 Hz, 1H), 4.28 (dd, J.sub.1=2.4
Hz, J.sub.2=6.8 Hz, 1H), 4.24 (s, 1H), 4.17 (m, 2H), 3.90 (m, 2H),
3.76 (s, 3H), 3.58 (t, J=4.8 Hz, 2H), 3.42-3.37 (m, 2H), 3.37 (s,
3H), 2.91 (m, 2H), 2.36-2.08 (m, 2H), 2.30 (s, 3H), 2.28 (s, 3H),
2.21 (s, 3H), 2.04 (s, 3H), 1.44 (s, 9H).
[0697] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.9, 168.9,
168.0, 155.4, 149.8, 148.6, 146.0, 141.1, 140.6, 131.6, 131.1,
130.6, 129.0, 125.1, 120.6, 118.3, 102.2, 98.4, 79.9, 71.9, 69.4,
61.6, 60.4, 59.8, 59.4, 59.2, 54.9, 54.7, 54.0, 41.6, 30.6, 29.1,
28.7, 23.9, 23.2, 20.6, 16.1, 14.2, 11.2, 9.8.
[0698] ESI-MS m/z: Calcd. for C.sub.40H.sub.50N.sub.4O.sub.12S:
810.91. Found (M+1).sup.+: 811.3.
Example 77
[0699] 218
[0700] To a solution of 151 (2.9 g, 3.57 mmol) in CH.sub.2Cl.sub.2
(120 mL), MeSO.sub.3H (1.4 mL, 21.46 mmol) was added at 23.degree.
C. After stirring the reaction for 30 minutes at 23.degree. C., a
saturated aqueous sodium bicarbonate solution (200 mL) was added at
0.degree. C. The organic phase was separated, dried over sodium
sulphate, filtered and concentrated to dryness at reduced pressure.
The residue was purified by flash column chromatography, eluting
with mixtures of ethyl acetate and hexane in a gradient manner,
from 0:1 to 1:0 to obtain 35 (1.43 g, 64%) as a pale yellow solid.
Experimental data of 35 was previously decribed in
PCT/GB00/01852.
[0701] 36, ET-770 and ET-743 were prepared following the same
procedures than those previously described in PCT/GB00/01852.
[0702] Route 3
[0703] The first step of this Route (transformation of 21 into 146)
was described above in Example 71.
Example 78
[0704] 219
[0705] To a solution of the commercial available HO.Cys(Fm)--H.HCl
(Bachem) (40 g, 0.119 mol) in acetone (500 mL) and water (500 mL),
1M Na.sub.2CO.sub.3 solution (238 mL) and BnCO.sub.2Cl (18.7 mL,
0.131 mol) were added at 0.degree. C. After stirring the reaction
at 60.degree. C. for 30 minutes, the mixture was quenched with 1N
HCl (pH 0 1) and extracted with eter (3.times.400 mL). The organic
phase was separated, dried over magnesium sulphate filtered and
concentrated to dryness at reduced pressure. The crude solid was
disolved in a mixture of EtOAc/CH.sub.2Cl.sub.2 1:1, precipitated
with hexane and kept at 4.degree. C. overnight. Then, the
suspension was filtered off, the solid washed with hexane (200 mL)
and the filtrate was dried in vacuo to afford 152 (50.16 g, 97%) as
a white solid.
[0706] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 10.66 (bs, 1H),
7.74 (d, J=7.5 Hz, 2H), 7.69-7.64 (m, 2H), 7.62-7.29 (m, 9H), 5.67
(d, J=7.5 Hz, 1H), 5.14 (bs, 2H), 4.70-4.64 (m, 1H), 4.09-4.05 (m,
1H), 3.12-3.09 (m, 2H).
[0707] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 175.2, 155.9,
145.5, 141.0, 135.8, 128.5, 128.2, 128.1, 127.5, 127.0, 124.7,
119.8, 84.8, 67.3, 46.8, 37.0.
[0708] ESI-MS m/z: Calcd. for C.sub.25H.sub.23NO.sub.4S: 433.52.
Found (M+1).sup.+: 434.4.
Example 79
[0709] 220
[0710] To a stirred solution of 146 (10 g, 19.2 mmol) and 152 (12.5
g, 28.8 mmol) in dichloromethane (800 mL) under argon,
dimethylaminopyridine (705 mg, 5.77 mmol),
1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (9.2
g, 48.1 mmol) and diisopropylethy amine (7.4 mL, 42.3 mmol) were
added dropwise during 1 h at 0.degree. C. The mixture was then
stirred at 23.degree. C. for 1.5 more hour. The reaction was
quenched by addition of a saturated aqueous sodium bicarbonate
solution (600 mL). The organic phase was separated and washed again
with a saturated aqueous amonium chloride solution (500 mL) and a
saturated sodium chloride solution (500 mL). The organic extract
were dried over sodium sulphate, filtered and evaporated to dryness
under reduced pressure. The crude product was purified by flash
column chromatography (RP18, CH.sub.3CN:H.sub.2O 4:1) to yield 153
(13.89 g, 77%) as a pale yellow solid.
[0711] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.74-7.72 (m, 2H),
7.61-7.53 (m, 2H), 7.37-7.24 (m, 9H), 6.39 (s, 1H), 6.09-5.96 (m,
1H), 5.90 (s, 1H), 5.84 (s, 1H), 5.78 (s, 1H), 5.34 (dd,
J.sub.1=1.5 Hz, J.sub.2 17.4 Hz, 1H), 5.32 (bs, 1H), 5.24 (dd,
J.sub.1=1.5 Hz, J.sub.2=10.2 Hz, 1H), 5.17-5.07 (m, 2H), 4.40 (dd,
J.sub.1=3.6 Hz, J.sub.2=10.8 Hz, 1H), 4.30 (m, 1H), 4.18-4.01 (m,
6H), 3.92 (brt, J=6.3 Hz, 1H), 3.71 (s, 3H), 3.30-3.19 (m, 3H),
2.99-2.85 (m, 3H), 2.65 (dd, J.sub.1=4.5 Hz, J.sub.2=14.4 Hz, 1H),
2.55 (d, J=18.3 Hz, 1H), 2.26 (s, 3H), 2.21 (s, 3H), 2.06 (s, 3H),
1.86 (dd, J.sub.1=11.7 Hz, J.sub.2=15.9 Hz, 1H).
[0712] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.2, 155.6,
148.6, 146.8, 145.7, 145.6, 144.3, 142.6, 140.7, 139.0, 133.7,
131.1, 128.8, 128.4, 128.1, 128.0, 127.4, 126.9, 124.7, 124.6,
121.0, 120.5, 119.7, 117.8, 117.3, 116.8, 112.5, 112.0, 101.0,
74.1, 67.0, 64.7, 60.7, 59.9, 57.0, 56.6, 56.3, 55.2, 53.1, 46.5,
41.4, 36.4, 34.8, 26.2, 24.8, 15.6, 9.2.
[0713] ESI-MS m/z: Calcd. for C.sub.54H.sub.54N.sub.4O.sub.9S:
934.36. Found (M+1).sup.+: 935.4.
Example 80
[0714] 221
[0715] To a solution of 153 (13.89 g, 14.85 mmol) in CH.sub.3CN
(74.3 mL), MEMCl (25.4 mL, 223 mmol), DIPEA (52 mL, 297 mmol) and
DMAP (0.181 g, 0.15 mmol) were added at 0.degree. C. The mixture
was stirred for S h at 23.degree. C. The reaction was diluted with
CH.sub.2Cl.sub.2 (400 mL) and extracted with 0.1N HCl (300 mL) and
3N HCl (pH=3). The aqueous phase was extracted again with
CH.sub.2Cl.sub.2 (2.times.50 mL). The combined organic phases were
dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo to
give a residue which was purified by flash column chromatography
(SiO.sub.2, CH.sub.2Cl.sub.2:EtOAc 10:1, 5:1) to give 154 (13.47 g,
88%) as a white solid.
[0716] Rf=0.27 CH.sub.2Cl.sub.2:AcOEt 6:1.
[0717] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.73-7.70 (m, 2H),
7.58-7.50 (m, 2H), 7.38-7.22 (m, 9H), 6.59 (s, 1H), 6.08-5.98 (m,
1H), 5.89 (s, 1H), 5.77 (s, 1H), 5.35 (d, J=17.1 Hz, 1H), 5.31-5.28
(m, 1H), 5.23 (d, J=6.9 Hz, 1H), 5.13 (d, J=6.9 Hz, 1H), 5.12-5.05
(m, 2H), 4.37-4.29 (m, 2H), 4.15-3.77 (m, 9H), 3.68 (s, 3H),
3.58-3.55 (m, 2H), 3.37 (s, 3H), 3.30-3.27 (m, 1H), 3.21-3.16 (m,
2H), 2.96-2.84 (m, 4H), 2.64-2.58 (m, 1H), 2.55 (d, J=18 Hz, 1H),
2.27 (s, 3H), 2.16 (s, 3H), 2.02 (s, 3H), 1.75 (dd, J.sub.1=12.3
Hz, J.sub.2=16.2 Hz, 1H).
[0718] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 171.9, 170.2,
155.5, 148.7, 148.6, 148.3, 145.8, 145.7, 144.5, 142.1, 140.9,
139.1, 136.1, 133.8, 130.8, 130.5, 128.5, 128.3, 128.1, 127.6,
127.0, 124.9, 124.7, 123.9, 122.2, 120.9, 119.8, 117.8, 117.3,
112.6, 112.0, 101.1, 98.2, 74.0, 71.7, 69.3, 67.1, 65.1, 60.1,
59.8, 59.0, 56.9, 56.8, 56.7, 55.3, 53.3, 46.7, 41.4, 36.5, 35.0,
31.6, 29.7, 26.4, 25.0, 22.6, 15.7, 14.1, 9.2.
[0719] ESI-MS m/z: Calcd. for C.sub.58H.sub.62N.sub.4O.sub.11S:
1023.2. Found (M+23).sup.+: 1046.3.
Example 81
[0720] 222
[0721] To a stirred solution of 154 (20.84 g. 0.02 mol) in
dichloromethane (530 mL), dichlorobis(triphenylphosphine) palladium
(II) (1.14 g, 1.63 mmol) and acetic acid (11.64 mL, 0.2 mol) were
added under argon at 23.degree. C. Then, tributyltin hydride (27.44
mL, 0.1 mol) was added in a dropwise manner. The mixture was
stirred at 23.degree. C. for 15 minutes. The reaction was then
filtered through a silica gel column compacted with hexane. 155
(18.78 g, 94%) was obtained as a pale yellow solid by subsequent
elution with mixtures of ethyl acetate and hexane in a gradient
manner, from 1:4, 1:1, 3:2 to 7:3.
[0722] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.71 (d, J=7.2 Hz,
2H), 7.59 (d, J=7.5 Hz, 1H), 7.53 (d, J=7.5 Hz, 1H), 7.41-7.23 (m,
9H), 6.60 (s, 1H), 5.87 (bs, 2H), 5.74 (s, 1H), 5.40 (d, J=6.3 Hz,
1H), 5.33 (d, J=5.8 Hz, 1H), 5.18 (d, J=9 Hz, 1H), 5.09 (d, J=12
Hz, 1H), 4.97 (d, J=12 Hz, 1H), 4.56 (dd, J.sub.1=3 Hz,
J.sub.2=11.1 Hz, 1H) 4.19 (d, J=2.1 Hz, 1H), 4.16-3.87 (m, 9H),
3.66 (s, 3H), 3.38 (s, 3H), 3.32-3.20 (m, 3H), 2.96-2.87 (m, 3H),
2.62-2.54 (m, 2H), 2.28 (s, 3H), 2.19 (s, 3H), 1.97 (s, 3H), 1.82
(dd, J.sub.1=13.2 Hz, J.sub.2=15.6 Hz, 1H).
[0723] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.0, 155.4,
149.0, 147.5, 145.7, 145.6, 144.4, 140.8, 135.9, 130.9, 128.4,
128.1, 128.0, 127.4, 126.9, 124.7, 124.6, 122.7, 119.7, 117.7,
112.4, 111.4, 100.6, 98.7, 71.5, 69.4, 67.0, 64.9, 63.9, 59.7,
59.6, 58.8, 57.0, 56.5, 56.4, 55.1, 54.9, 53.1, 52.5, 46.5, 41.4,
36.8, 34.9, 25.8, 24.7, 15.7, 8.7.
Example 82
[0724] 223
[0725] To a solution of 155 (18.5 g, 18.82 mmol) in anhydrous
dichloromethane (530 mL) at -10.degree. C. (bath temperature
-15.degree. C.), a solution of benzeneseleninic anhydride (9.68 g,
18.82 mmol) in anhydrous dichloromethane (290 mL) was added
dropwise, discarding any white solid present in the solution. The
mixture was stirred for 10 minutes at the same temperature. The
reaction was then quenched with a saturated aqueous sodium
bicarbonate solution (600 mL). The organic phase was separated, and
the aqueous phase was extracted with CH.sub.2Cl.sub.2 (2.times.300
mL). The combined organic extracts were dried over sodium sulphate,
filtered, and concentrated to dryness under reduced pressure. The
residue was purified by column chromatography, eluting with
mixtures of ethyl acetate and hexane in a gradient manner, from
1:1, 3:2, 7:3 to 4:1 to obtain 156 (17.62 g, 88%) as a pale yellow
solid.
[0726] .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of isomers)
.delta. 7.73 (d, J=7.5 Hz, 2H), 7.63 (d, J=7.5 Hz, 2H), 7.40-7.29
(m, 9H), 6.59 (s, 1H), 6.52 (s, 1H), 5.68 (s, 1H), 5.66 (s, 1H),
5.58 (s, 1H), 5.56 (s, 1H), 5.23 (d, J=6 Hz, 1H), 5.15-5.05 (m,
4H), 4.76-4.68 (m, 1H), 4.64-4.55 (m, 1H), 4.40-4.37 (m, 1H),
4.15-3.68 (m, 8H), 3.60 (s, 3H), 3.57 (s, 3H), 3.39 (s, 3H), 3.36
(s, 3H), 3.25-2.78 (m, 7H), 2.38-2.24 (m, 2H), 2.20 (s, 3H), 2.18
(s, 3H), 2.15 (s, 3H), 2.09 (m, 1H), 2.04 (s, 3H), 1.77 (s, 3H),
1.58 (s, 3H).,
[0727] ESI-MS m/z: Calcd. for Cs.sub.5H.sub.58N.sub.4O.sub.12S:
999.13. Found (M+1).sup.+: 1000.0.
Example 83
[0728] 224
[0729] The reaction flask was flamed twice, purged vacuum/Argon
several times and kept under Argon atmosphere for the reaction. To
a solution of DMSO (178 .mu.L) in anhydrous CH.sub.2Cl.sub.2 (20
mL) was dropwise added triflic anhydride (169 .mu.L, 1 mmol) at
-78.degree. C. The reaction mixture was stirred at -78.degree. C.
for 20 minutes. Then, a solution of 156 (0.5 g, 0.5 mmol) in
anhydrous CH.sub.2Cl.sub.2 (4 mL, for the main addition and 1.5 mL
for washing) was added via canula (addition time: 5 min) at
-78.degree. C. During the addition the temperature was kept at
-78.degree. C. in both flasks and the color changed from yellow to
brown. The reaction mixture was stirred at -40.degree. C. for 35
minutes. During this period of time the solution was turned from
yellow to dark green. After this time, .sup.iPr.sub.2NEt (0.7 mL,
4.42 mmol) was dropwise added and the reaction mixture was kept at
0.degree. C. for 45 minutes, the color of the solution turned brown
during this time. Then tBuOH (189 .mu.L, 2 mmol) and tert-butyl
tetramethyl guanidine (0.6 mL, 3.49 mmol) were dropwise added and
the reaction mixture was stirred at 23.degree. C. for 40 minutes.
After this time, acetic anhydride (0.47 mL, 4.97 mmol) was dropwise
added and the reaction mixture was kept at 23.degree. C. for 1 hour
more. Then, the reaction mixture was diluted with CH.sub.2Cl.sub.2
(15 mL) and washed with aqueous saturated solution of NH.sub.4Cl
(25 mL), NaHCO.sub.3 (25 mL), and NaCl (25 mL). The combined
organic layers were dried over Na.sub.2SO.sub.4, filtered and
concentrated. The residue was purified by flash column
chromatography (inner diameter: 2.0 cm, height of silica: 9 cm;
eluent: ethyl acetate/hexane in a gradient manner, from 1:4, 1:3,
1:2 to 1:1) to afford 157 (128 mg, 30%) as a light yellow solid.
Rf=0.37 Hex:EtOAc 3:2.
[0730] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.37 (bs, 5H),
6.66 (s, 1H), 6.09 (s, 1H), 5.99 (s, 1H), 5.30 (d, J=5.4 Hz, 1H),
5.17 (d, J=6 Hz, 1H), 5.06 (d, J=7.8 Hz, 1H), 5.00 (s, 1H), 4.83
(d, J=9.3 Hz, 1H), 4.50 (s, 1H), 4.34-4.17 (m, 7H), 3.90-3.87 (m,
2H), 3.66 (s, 3H), 3.65-3.56 (m, 2H), 3.37 (s, 3H), 2.89-2.90 (m,
2H), 2.28 (s, 3H), 2.18 (s, 3H), 2.15-2.04 (m, 2H), 2.03 (s, 3H),
1.99 (s, 3H).
[0731] ESI-MS m/z: Calcd. for C.sub.43H.sub.48N.sub.4O.sub.12S:
844.93. Found (M+1).sup.+: 845.8.
Example 84
[0732] 225
[0733] To a solution of 157 (100 mg, 0.118 mmol) in
CH.sub.2Cl.sub.2 (2 mL) and CH.sub.3CN (2 mL), NaI (71 mg, 0.472
mmol) and TMSCl (60 .mu.L, 0.472 mmol) were added at 0.degree. C.
After stirring the reaction at 23.degree. C. for 50 minutes, the
mixture was quenched with water (30 mL) and extracted with
CH.sub.2Cl.sub.2 (2.times.20 mL). The combined organic phases were
washed successively with a saturated solution of NaCl (20 mL) and a
saturated solution of sodium ditionite (20 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue
was purified by flash column chromatography (eluent: ethyl
acetate/hexane gradient from 1:4, 1:2 to 1:1) to afford 158 (62 mg,
70%) as white solid. Rf=0.21 Hex:EtOAc 1:1.
[0734] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.36 (bs, 5H),
6.44 (s, 1H), 6.07 (d, J=1.2 Hz, 1H), 5.97 (d, J=1.2 Hz, 1H), 5.81
(bs, 1H), 5.10-5.00 (m, 3H), 4.82 (d, J=9.3 Hz, 1H), 4.49 (bs, 1H),
4.35-4.30 (m, 1H), 4.21-4.17 (m, 2H), 4.16-4.14 (m, 2H), 3.65 (s,
3H), 3.41-3.36 (m, 2H), 2.88-2.85 (m, 2H), 2.28 (s, 3H), 2.24-2.03
(m, 2H), 2.17 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H).
[0735] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 170.5, 168.8,
155.9, 148.3, 146.0, 143.1, 141.2, 140.6, 136.6, 130.6, 130.0,
128.8, 128.7, 128.5, 121.0, 120.3, 118.3, 118.2, 113.7, 113.6,
102.2, 67.2, 61.5, 60.8, 60.3, 59.6, 59.5, 54.8, 54.7, 54.1, 41.9,
41.6, 32.9, 23.9, 20.8, 15.5, 9.8.
[0736] ESI-MS m/z: Calcd. for C.sub.39H.sub.40N.sub.4O.sub.10S:
756.82. Found (M+1).sup.+: 757.3.
Example 85
[0737] 226
[0738] To a solution of 158 (100 mg, 0.132 mmol) in MeOH (6.8 mL),
HCO.sub.2H (360 .mu.L) and 10% Pd/C (140 mg, 0.132 mmol) were added
at 23.degree. C. and the mixture was stirred for 15 minutes. Then,
toluene (7 mL) was added to the reaction and the solvent was
evaporated under reduced pressure. The azeotropic destination with
toluene was repeated 3 times. The residue was then diluted with
dichloromethane (15 mL) and a saturated aqueous solution of sodium
bicarbonate (15 mL) was added. The aqueous phase was separated and
extracted with dichloromethane (2.times.10 mL). The combined
organic extracts were dried over sodium sulphate, filtered and
evaporated to dryness under reduced pressure. The residue was then
purified by flash column chromatography on amino-silicagel and
eluting with mixtures of ethyl acetate and hexane in a gradient
manner, from 1:2, 1:1 to 2:1 to give 35 (57 mg, 70%) as a yellow
solid. Experimental data of 35 were previously described in
PCT/GB00/01852.
[0739] 36, ET-770 and ET-743 were prepared following the same
procedures than those previously described in PCT/GB00/01852.
[0740] Route 4
[0741] The first step of this Route (transformation of 21 into 146)
was described above in Example 71.
Example 86
[0742] 227
[0743] To a solution of 146 (18 mg, 0.032 mmol), cat. DMAP and
imidazole (5 mg, 0.08 mmol) in DMF (0.05 mL) at 0.degree. C.,
tert-buthyldiphenylsilyl chloride (12.5 .mu.L, 0.048 mmol) was
added and the reaction was stirred for 4 hours at 23.degree. C.
Then, water (30 mL) was added at 0.degree. C. and the mixture was
extracted with Hex:EtCAc 1:10 (2.times.40 mL). The combined organic
phases were dried over sodium sulphate, filtered, and the solvent
was removed under reduced pressure. The residue was puified by
flash column chromatography (SiO.sub.2, Hex:EtOAc 3:1) to afford
159 (27 mg, 88%) as a white solid. Rf=0.29 Hex:EtOAc 3:1.
[0744] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 7.72-7.41 (m, 2H),
7.40-7.20 (m, 8H), 6.46 (s, 1H), 6.16-6.00 (m, 1H), 5.77 (d, J=1.5
Hz, 1H), 5.71 (s, 1H), 5.63 (d, J=1.5 Hz, 1H), 5.24 (dd,
J.sub.1=1.2 Hz, J.sub.2=17.1 Hz, 1H), 5.23 (dd, J.sub.1=1.2 Hz,
J.sub.2=10.2 Hz, 1H), 4.18 (d, J=2.4 Hz, 1H), 4.13-4.00 (m, 4H),
3.77 (s, 3H), 3.63 (dd, J.sub.1=2.4 Hz, J.sub.2=7.5 Hz, 1H),
3.39-3.19 (m, 4H), 2.99 (dd, J.sub.1=8.1 Hz, J.sub.2=18.0 Hz, 1H),
2.68 (d, J=17.7 Hz, 1H), 2.30 (s, 3H), 2.28 (s, 3H), 2.08 (s, 3H),
1.99 (dd, J.sub.1=12.6 Hz, J.sub.2=16.3 Hz, 1H), 0.89 (s, 9H).
[0745] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.3, 146.6,
144.0, 142.5, 139.0, 135.7, 135.4, 133.9, 133.6, 132.2, 131.2,
129.5, 129.4, 128.3, 127.5, 127.4, 121.8, 120.9, 118.7, 117.3,
117.2, 112.9, 111.7, 100.8, 74.2, 68.0, 61.6, 60.6, 60.3, 59.0,
57.4, 56.7, 55.4, 41.7, 29.6, 26.6, 26.5, 25.5, 18.9, 15.8,
9.3.
[0746] ESI-MS m/z: Calcd. for C.sub.45H.sub.51N.sub.3O.sub.6Si:
757.9. Found (M+1).sup.+: 758.4.
Example 87
[0747] 228
[0748] To a solution of 159 (2.4 g, 3.17 mmol) in CH.sub.3CN (16
mL), MOMBr (2.6 mL, 31.75 mmol), DIPEA (8.3 ML, 47.6 mmol) and DMAP
(16 mg, 0.127 mmol) were added at 0.degree. C. The mixture was
stirred for 6 h at 23.degree. C. The reaction was diluted with
CH.sub.2Cl.sub.2 (50 mL) and extracted with 0.1 N HCl (50 mL). The
aqueous phase was extracted again with CH.sub.2Cl.sub.2 (50 mL).
The combined organic phases were dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo to give a residue which was
purified by flash column chromatography (SiO.sub.2,
CH.sub.2Cl.sub.2:EtOAc 15:1, 5:1) to give 26 (1.78 g, 70%) as a
white solid. Experimental data of 26 were described previously in
PCT/GB00/01852.
[0749] Experimental procedures for Int. 11, 160, 161, 162, and 163
were previously described in U.S. Pat. No. 5,721,362.
Example 88
[0750] 229
[0751] To a solution of 163 (15.8 g, 0.02 mol) in anhydrous
CH.sub.2Cl.sub.2 (250 mL) and acetonitrile (300 mL), NaI (31.5 g,
0.21 mol) and ClTMS (freshly distilled over CaH.sub.2, 26.7 mL,
0.21 mol) were added under argon atmosphere at 23.degree. C. The
reaction mixture was stirred for 40 minutes. Then the reaction was
partitioned between CH.sub.2Cl.sub.2 (200 mL) and water (300 mL).
The organic layer was washed with a saturated aqueous solution of
NaCl (2.times.300 mL). The organic phase was dried over
Na.sub.2SO.sub.4, filtered and the solvent was eliminated under
reduced pressure. The crude was purified by flash column
chromatography using ethyl acetate/hexane 2:3 as eluent to afford
164 (10.74 g, 76%) as a pale yellow solid. Rf=0.25 Hex:EtOAc
3:2.
[0752] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.57 (s, 1H), 6.08
(d, J=1.5 Hz, 1H), 5.98 (d, J=1.5 Hz, 1H), 5.96-5.85 (m, 1H), 5.76
(bs, 1H), 5.30 (dd, J.sub.1=1.5, J.sub.2=17.3 Hz, 1H), 5.23 (dd,
J.sub.1=1.5, J.sub.2=10.2 Hz, 1H), 5.00 (d, J=12.1 Hz, 1H), 4.81
(d, J=9.8 Hz, 1H), 4.58-4.45 (m, 3H), 4.34-4.28 (m, 1H), 4.23 (m,
2H), 4.17-4.00 (m, 2H), 3.76 (s, 3H), 3.40-3.38 (m, 2H), 2.91-2.85
(m, 2H), 2.30 (s, 3H), 2.29 (s, 3H), 2.24-2.23 (m, 2H), 2.19 (s,
3H), 2.02 (s, 3H).
[0753] .sup.13C NMR (75 MHz, CDCl.sub.3) 170.1, 168.4, 155.2,
148.0, 145.5, 142.8, 140.7, 140.1, 132.7, 130.2, 129.6, 120.7,
119.9, 117.8, 113.3, 101.9, 65.6, 61.0, 60.4, 59.9, 59.2, 59.0,
54.3, 53.6, 41.5, 41.2, 32.6, 29.5, 23.5, 20.4, 15.6, 9.4.
[0754] ESI-MS m/z: Calcd. for C.sub.35H.sub.38N.sub.4O.sub.10S:
706.76. Found (M+1).sup.+: 707.2.
Example 89
[0755] 230
[0756] To a stirred solution of 164 (2 g. 2.85 mmol) in
dichloromethane (142 mL), dichlorobis(triphenylphosphine) palladium
(II) (0.2 g, 0.28 mmol) and acetic acid (0.65 mL, 11.4 mmol) were
added under argon at 23.degree. C. Then, tributyltin hydride (4.51
mL, 17.02 mmol) was added in a dropwise manner during 25 minutes.
After addition of HsnBu.sub.3, the mixture was stirred at
23.degree. C. for 20 minutes more. The reaction was filtered
through a silical gel column compacted with hexane. 35 (1.38 g,
78%) was obtained by subsequent elution with mixtures of ethyl
acetate and hexane in a gradient manner, from 1:2 to 15:1.
Experimental data of 35 were previously described in
PCT/GB00/01852.
[0757] 36, ET-770 and ET-743 were prepared following the same
procedures than those previously described in PCT/GB00/01852.
[0758] Route 5
[0759] The first step of this Route (transformation of 21 into 146)
was described above in Example 71.
Example 90
[0760] 231
[0761] To a solution of 146 (8.72 g, 16.78 mmol) in DMF (20.1 mL),
imidazol (3.43 g, 50.34 mmol), tert-butyl dimethyl chlorosilane
(7.58 mL, 50.34 mmol) and DMAP (0.2 g, 1.7 mmol) were added at
0.degree. C. After being stirred at 23.degree. C. for 3.5 h, the
reaction mixture was quenched with water (100 mL) and extracted
with EtOAc/Hex 1:3 (2.times.75 mL). The combined organic phases
were washed with 0.1 M HCl (50 mL) and the aqueous phase was
extracted again with EtOAc/Hex 1:3 (40 mL). The combined organic
phases were dried over sodium sulphate, filtered and concentrated
in vacuo. The residue was purified by flash column chromatography
(Hex:EtOAc 10:1, 3:1) to obtain 165 (9.85 g, 93%) as a white solid.
Rf=0.39 in Hex:AcOEt 2:1.
[0762] .sup.1H NMR (300 MHz, CDCl.sub.3) .quadrature. 6.43 (s, 1H),
6.15-6.03 (m, 1H), 5.92 (d, J=1.2 Hz, 1H), 5.84 (d, J=1.2 Hz, 1H),
5.67 (s, 1H), 5.41 (dd, J.sub.1=1.5, J.sub.2=17.1 Hz, 1H), 5.26
(dd, J.sub.1=1.5, J.sub.2=10.5 Hz, 1H), 4.44 (d, J=2.7 Hz, 1H),
4.20-4.08 (m, 3H), 3.97 (dd, J.sub.1=2.7, J.sub.2=8.1 Hz, 1H), 3.75
(s, 3H), 3.61 (dd, J.sub.1=2.71, J.sub.2=9.9 Hz, 1H), 3.18 (brd,
J=8.7 Hz, 1H), 3.22-3.16 (m, 2H), 2.99 (dd, J.sub.1=8.1,
J.sub.2=17.4 Hz, 1H), 2.65 (d, J=17.4 Hz, 1H), 2.28 (s, 3H), 2.25
(s, 3H), 2.11 (s, 3H), 1.89 (dd, J.sub.1=12, J.sub.2=15.6 Hz, 1H),
0.8 (s, 9H), -0.05 (s, 3H), -0.09 (s. 3H).
[0763] .sup.13C NMR (75 MHz, CDCl.sub.3) .quadrature. 148.2, 146.5,
143.8, 142.4, 138.9, 133.8, 131.0, 128.0, 121.5, 120.4, 118.4,
117.1, 112.8, 111.6, 100.7, 74.0, 68.2, 61.5, 60.2, 58.6, 57.1,
56.5, 55.2, 41.3, 26.2, 25.4, 25.2, 20.6, 17.8, 15.3, 13.8, 9.0,
-3.9, -6.0.
[0764] ESI-MS m/z: Calcd. for C.sub.35H.sub.47N.sub.3O.sub.6Si:
633.85. Found (M+1).sup.+: 634.2.
Example 91
[0765] 232
[0766] To a solution of 165 (7.62 g, 12.02 mmol) in THF (87.64 mL)
and water (0.24 mL), MEMCl (2.33 mL, 20.43 mmol) was added at
-6.degree. C. After addition of 60% NaH (0.72 g, 18.03 mmol) in
portions over 45 min, the mixture was stirred for 1.5 h at that
temperature. The reaction was quenched with water (150 mL) and
extracted with CH.sub.2Cl.sub.2 (2.times.100 mL). The combined
organic phases were dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuo to give 166 (8.69 g, 100%) as a white solid
which was used in following steps with no further purification.
Rf=0.24 Hex:AcOEt 2:1.
[0767] .sup.1H NMR (300 MHz, CDCl.sub.3) .delta. 6.64 (s, 1H),
6.16-6.05 (m, 1H), 5.92 (d, J=1.2 Hz, 1H), 5.85 (d, J=1.2 Hz, 1H),
5.41 (dd, J.sub.1=1.51, J.sub.2=17.1 Hz, 1H), 5.29-5.24 (m, 2H),
5.14 (d, J=6 Hz, 1H), 4.42 (d, J=2.7 Hz, 1H), 4.21-4.06 (m, 3H),
4.01-3.95 (m, 2H), 3.88-3.82 (m, 1H), 3.72 (s, 3H), 3.64-3.57 (m,
3H), 3.39 (s, 3H), 3.29 (brd J=7.5 Hz, 1H), 3.25-3.15 (m, 2H), 3.00
(dd, J.sub.1=8.1, J.sub.2=17.4 Hz, 1H), 2.65 (d, J=18 Hz, 1H), 2.30
(s, 3H), 2.21 (s, 3H), 2.11 (s, 3H), 1.82 (dd J.sub.1=12,
J.sub.2=15.6 Hz, 1H),-0.79 (s, 9H), -0.06 (s, 3H), -0.11 (s,
3H).
[0768] .sup.13C NMR (75 MHz, CDCl.sub.3) .delta. 148.4, 148.1,
144.1, 139.2, 133.9, 130.9, 130.8, 130.2, 128.8, 125.1, 124.2,
121.5, 118.8, 117.45, 113.0, 111.9, 101.0, 98.2, 74.1, 71.7, 69.3,
68.3, 61.7, 59.6, 59.0, 58.9, 57.3, 57.1, 55.5, 41.6, 29.7, 26.4,
25.8, 25.5, 25.4, 15.7, 9.2, -5.6, -5.6.
[0769] ESI-MS m/z: Calcd. for C.sub.39H.sub.55N.sub.3O.sub.8Si:
721.3. Found (M+1).sup.+: 722.3.
Example 92
[0770] 233
[0771] To a solution of 166 (10.76 g, 14.90 mmol) in anhydrous
CH.sub.2Cl.sub.2 (275 mL), Pd(PPh.sub.3P).sub.2Cl.sub.2 (837 mg,
1.19 mmol), acetic acid (4.26 mL, 74.5 mmol) and tributyltin
hydride (11.85 mL, 44.7 mmol) were added under Argon atmosphere at
23.degree. C. The reaction mixture was stirred at 23.degree. C. for
15 minutes. (TLC AcOEt/Hexane 1:1 showed no starting material).
Hexane (100 mL) was added and the mixture was poured into a flash
column chromatography, (SiO.sub.2, EtOAc:Hexane in a gradient
manner, from 0:100, 1:4, 2:3 to 1:1) to afford 167 (9.95 g, 98%) as
a yellow solid. Rf=0.42 Hex:EtOAc 3:7.
[0772] .sup.1H-RMN (300 MHz, CDCl.sub.3): .delta. 6.63 (s, 1H),
5.89 (d, J=1.4 Hz, 1H), 5.79 (d, J=1.4 Hz, 1H), 5.76 (m, 1H), 5.38
(d, J=5.6 Hz, 1H), 5.23 (d, J=5.9 Hz, 1H), 4.53 (d, J=2.7 Hz, 1H),
4.17 (dd, J.sub.1=1.95 Hz, J.sub.2=6.05 Hz, 1H), 4.11 (dd,
J.sub.1=7.0 Hz, J.sub.2=12.5 Hz, 1H), 4.01-3.92 (m, 2H), 3.70 (s,
3H), 3.67 (m, 3H), 3.40 (s, 3H), 3.29 (m, 1H), 3.24-3.13 (m, 3H),
2.99 (dd, J.sub.1=8.0 Hz J.sub.2=17.5 Hz, 1H), 2.67 (d, J=17.5 Hz,
1H), 2.28 (s, 3H), 2.09 (s, 3H), 2.05 (s, 3H), 1.80 (dd,
J.sub.1=11.2 Hz, J.sub.2=14.9 Hz, 1H), 0.82 (s, 9H), -0.03 (s, 3H),
-0.07 (s, 3H).
[0773] .sup.13C-RMN (75 MHz, CDCl.sub.3): .delta. 148.4, 147.3,
145.5, 144.1, 136.2, 134.9, 134.8, 130.9, 130.2, 124.8, 123.1,
118.6, 112.8, 112.1, 106.2, 100.4, 98.4, 71.5, 69.2, 68.9, 61.7,
59.6, 58.7, 58.6, 56.9, 56.6, 55.3, 41.5, 29.5, 25.7, 25.3, 17.9,
15.5, 8.7, -5.7, -5.8.
[0774] ESI-MS m/z: Calcd. for C.sub.36H.sub.51N.sub.3O.sub.8Si:
681.89. Found (M+1).sup.+: 682.3. HPLC: Conditions: Column:
Symmetry C18; mobile phase: AcN--buffer phosphate 25 mM, pH=5,
isocratic of AcN (65%) in 5 minutes and gradient in AcN from 65-92%
in 31 minutes, .O slashed.: 0.6 mL/min, t.sup.a: 40.degree. C.
Retention time: 27.89 minutes. HPLC purity in area: 89.62%.
Example 93
[0775] 234
[0776] To a solution of 167 (9.95 g, 14.6 mmol) in anhydrous
CH.sub.2Cl.sub.2 (300 mL), a solution of benceneseleninic anhydride
(7.51 g, 14.6 mmol, reagent purity 70%) in anhydrous
CH.sub.2Cl.sub.2 (120 mL) was dropwise added, under Argon
atmosphere at -15.degree. C. (the remaning white solid was
discarded). The solution was then stirred at -15.degree. C. for 15
minutes (TLC EtOAc/Hexane 2:3, showed no starting material). A
saturated aqueous solution of sodium bicarbonate (500 mL) was added
to the reaction mixture at this temperature. The organic phase was
separated and the aqueous phase was extracted with CH.sub.2Cl.sub.2
(500 mL). The combined organic extracts were dried over sodium
sulphate, filtered and the solvent was eliminated under reduced
pressure. The crude of the reaction was purified by flash column
chromatography, (SiO.sub.2, EtOAc:Hexane in a gradient manner, from
2:3 to 3:1) to afford 168 (9.86 g, 97%) as a yellow solid. Rf=0.33
Hex:EtOAc 3:7).
[0777] .sup.1H-RMN (300 MHz, CDCl.sub.3) (Isomers
ratio:.apprxeq.3:2): .delta. 6.59 (s, 1H), 6.57 (s, 1H), 5.77 (s,
1H), 5.76 (s, 1H), 5.68 (s, 1H), 5.63 (s, 1H), 5.19 (d, J=6.0 Hz,
1H), 5.09 (d, J=6.0 Hz, 1H), 5.07 (d, J=6.1 Hz, 1H), 5.00 (d, J=6.1
Hz, 1H), 4.40 (d, J=2.7 Hz, 1H), 4.27 (d, J=2.44 Hz, 1H), 4.22 (d,
J=10.5 Hz, 1H), 3.95 (d, J=1.7 Hz, 1H), 3.86-3.75 (m, 2H), 3.81 (s,
3H), 3.72-3.68 (m, 2H), 3.65 (m, 2H), 3.54 (s, 3H), 3.50 (m, 3H),
3.31 (s, 3H), 3.29 (s, 3H); 3.24 (m, 1H), 3.09 (dt, J=3.2 Hz, J=7.6
Hz, 1H), 3.02 (d, J=11.2 Hz, 1H), 2.92 (m, 2H), 2.48 (d, J=9.5 Hz,
1H), 2.43 (d, J=9.3 Hz, 1H), 2.21 (s, 3H), 2.14 (s, 3H), 2.13 (s,
3H), 2.03 (m, 2H), 1.73 (s, 3H), 1.71 (s, 3H), 0.86 (s, 9H), 0.77
(s, 9H), 0.04 (s, 3H), 0.02 (s, 3H).
[0778] .sup.13C-RMN (75 MHz, CDCl.sub.3): 200.5, 197.2, 159.8,
157.7, 148.4, 148.2, 147.7, 140.0, 137.6, 130.5, 130.2, 129.9,
129.4, 124.9, 124.7, 124.0, 122.7, 117.1, 116.9, 113.4, 110.8,
103.9, 103.8, 101.0, 100.4, 97.8, 72.8, 71.3, 69.7, 68.9, 68.8,
65.4, 64.1, 60.2, 59.9, 59.3, 59.1, 59.0, 58.6, 58.5, 56.8, 56.5,
56.2, 55.5, 54.9, 54.8, 42.5, 41.1, 40.9, 35.8, 25.6, 25.5, 25.4,
25.3, 20.6, 17.9, 17.8, 15.5, 15.3, 13.8, 7.0, 6.7, -5.7, -6.0,
-6.1.
[0779] ESI-MS m/z: Calcd. for C.sub.36H.sub.51N.sub.3O.sub.9Si:
697.89. Found (M+1).sup.+: 698.8 HPLC: Conditions: Column: Symmetry
C18; mobile phase: AcN, buffer phosphate 25 mM, pH=5, gradient in
AcN from 30-100% in 50 minutes. .O slashed.: 1.2 mL/min, t.sup.a:
40.degree. C. Retention time: 30.70 minutes and 30.95 minutes (the
two isomers). HPLC purity in area: 60.77% and 31.99%.
Example 94
[0780] 235
[0781] To a solution of 168 (16.38 g, 23.47 mmol) in anhydrous THF
(727 mL, 0.03 M), a solution of TBAF in 1M THF (59 mL, 59 mmol) was
dropwise added at 23.degree. C. The reaction mixture was stirred at
23.degree. C. for 45 minutes. Then, the mixture was partitioned
between a saturated aqueous NaCl solution (850 mL) and
CH.sub.2Cl.sub.2 (950 mL). Both layers were separated and the
organic layer was dried over anhydrous Na.sub.2SO.sub.4, filtered
and concentrated under reduced pressure. The residue was purified
by flash column chromatography (SiO.sub.2, EtOAc:Hexane in a
gradient manner, from 40:60, 50:50, 70:30, 90:10 to 100:0) to
afford 169 (12.17 g, 89%) as a light yellow solid. Rf=0.1 Hex:EtOAc
3:7.
[0782] .sup.1H-RMN (300 MHz, CDCl.sub.3) (Isomers ratio: 3:2):
.delta. 6.63 (s, 1H), 6.57 (s, 1H), 5.79 (s, 1H), 5.77 (s, 1H),
5.75 (s, 1H), 5.62 (s, 1H), 5.23 (s, 1H), 5.18 (d, J=6.1 Hz, 1H),
5.08 (d, J=6.1 Hz, 1H), 5.01 (d, J=6.1 Hz, 1H), 4.22 (d, J=2.7 Hz,
1H), 4.09 (d, J=2.4 Hz, 1H), 4.00 (m, 4H), 3.82 (s, 3H), 3.87-3.64
(m, 6H), 3.55 (s, 3H), 3.51-3.44 (m, 2H), 3.30 (s, 3H), 3.29 (s,
3H), 3.26 (m, 1H), 3.18 (dt, J.sub.1=2.9 Hz, J.sub.2=7.3 Hz, 1H),
2.94 (m, 4H) 2.50 (m, 4H), 2.22 (s, 3H), 2.16 (s, 3H), 2.15 (s,
3H), 2.11 (s, 0.3H), 2.02 (d, J=7.3 Hz, 2H), 1.72 (s, 3H), 1.69 (s,
3H).
[0783] .sup.13C-RMN (75 MHz, CDCl.sub.3): 200.2, 200.1, 159.6,
158.5, 148.5, 148.4, 148.1, 147.9, 140.5, 137.4, 130.9, 130.4,
130.1, 130.0, 125.1, 124.9, 123.8, 122.7, 116.9, 116.6, 113.3,
110.7, 104.5, 103.9, 101.4, 100.7, 98.1, 97.9, 71.9, 71.5, 71.4,
70.1, 69.0, 69.0, 62.0, 60.1, 59.5, 58.7, 58.5, 58.1, 57.4, 56.9,
56.8, 56.4, 55.9, 55.1, 55.0, 41.3, 41.0, 36.1, 31.3, 25.3, 25.2,
22.4, 15.6, 15.5, 13.8, 7.0, 6.8.
[0784] ESI-MS m/z: Calcd. for C.sub.30H.sub.37N.sub.3O.sub.9:
583.63. Found (M+1).sup.+: 584.2.
Example 95
[0785] 236
[0786] To a solution of 169 (11.49 g, 19.69 mmol) and
Alloc-Cys-(Fm) (11.32 g, 29.53 mmol) (for its preparation see
Kruse, C. H.; Holden, K. G., J. Org. Chem., 1985, 50, pp.
2792-2794) in anhydrous CH.sub.2Cl.sub.2 (688 mL), DMAP (2.4 g,
19.69 mmol) and EDC-HCl (9.44 g, 49.22 mmol) were added at
23.degree. C. Then, DIPEA (5.14 mL, 29.53 mmol) was added at
0.degree. C. and the reaction was stirred at 23.degree. C. for 3
hour. The mixture was washed successively with a saturated aqueous
solution of NaHCO.sub.3 (500 mL), NaCl (400 mL) and NH.sub.4Cl
(2.times.300 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The residue was purified by flash column chromatography (SiO.sub.2,
AcOEt:Hex in a gradient manner, from 1:1, 6:4 to 7:3) to afford 170
(14.76 g, 79%) as a pale yellow solid. Rf=0.31 and 0.40 Hex:EtOAc
3:7 (mixture of isomers).
[0787] .sup.1H-RMN (300 MHz, CDCl.sub.3): 7.74 (d, J=7.6 Hz, 4H),
7.63 (dd, J=7.0 Hz, J=15.3 Hz, 4H), 7.38 (t, J=7.3 Hz, 4H), 7.29
(m, 4H), 6.61 (s, 1H), 6.54 (s, 1H), 5.89 (m, 2H); 5.73 (s, 1H),
5.70 (s, 1H), 5.69 (s, 1H), 5.62 (s, 1H), 5.55 (m, 1H), 5.32 (d,
J=15.1 Hz, 1H), 5.23 (d, J=6.1 Hz, 1H), 5.22 (d, J=10.6 Hz, 1H),
5.14 (d, J=5.9 Hz, 1H), 5.13 (d, J=6.0 Hz, 1H), 5.07 (d, J=6.3 Hz,
1H), 4.68 (m, 1H), 4.56 (m, 4H), 4.51 (m, 2H), 4.38 (dd,
J.sub.1=4.5 Hz, J.sub.2=12.6 Hz, 1H), 4.22 (dd, J.sub.1=6.2 Hz,
J.sub.2=11.1 Hz, 1H), 4.14-3.88 (m, 12H), 3.83 (s, 3H), 3.79-3.69
(m, 4H), 3.61 (s, 3H), 3.56 (m, 4H), 3.39 (s, 3H), 3.36 (s, 3H),
3.23 (m, 2H), 3.16 (d, J=6.0 Hz, 2H), 3.07 (d, J=6.1 Hz, 2H),
3.00-2.81 (m, 6H), 2.46-2.34 (m, 4H), 2.25 (s, 3H), 2.20 (s, 3H),
2.16 (s, 3H), 2.07 (m, 1H), 1.83 (dd, J.sub.1=9.5 Hz, J.sub.2=15.1
Hz, 1H), 1.78 (s, 3H), 1.77 (s, 3H).
[0788] .sup.13C-RMN (75 MHz, CDCl.sub.3): .delta. 200.3, 198.4,
170.3, 160.0, 158.1, 148.7, 148.7, 148,5, 148.2, 145.6, 145.6,
145.5, 142.2, 141.1, 141.0, 141.0, 138.5, 132.4, 132.3, 131.1,
130.6, 130.1, 129.8, 128.8, 127.6, 127.1, 127.1, 125.1, 125.0,
124.8, 124.7, 124.7, 124.0, 122.7, 119.9, 118.1, 118.0, 117.2,
116.8, 111.6, 108.3, 104.8, 104.5, 101.5, 101.0, 98.2, 98.2, 72.3,
71.7, 71.7, 70.6, 69.3, 69.2, 66.4, 66.0, 66.0, 65.5, 63.8, 60.8,
60.2, 59.8, 59.0, 58.9, 58.1, 56.8, 56.6, 56.5, 56.3, 56.1, 55.7,
55.3, 55.2, 53.9, 46.9, 41.9, 41.4, 41.2, 37.2, 36.9, 35.4, 31.5,
29.6, 25.6, 25.4, 22.6, 15.8, 15.7, 14.1, 7.3, 7.0.
[0789] ESI-MS m/z: Calcd. for C.sub.51H.sub.96N.sub.4O.sub.12S:
948.36. Found (M+1).sup.+: 949.3.
Example 96
[0790] 237
[0791] The reaction flask was flamed twice, purged vacuum/Argon
several times and kept under Argon atmosphere for the reaction. To
a solution of DMSO (5.4 mL) in anhydrous CH.sub.2Cl.sub.2 (554 mL)
was dropwise added triflic anhydride (5.11 mL, 30.4 mmol) at
-78.degree. C. The reaction mixture was stirred at -78.degree. C.
for 20 minutes. Then, a solution of 170 (14.43 g, 15.2 mmol) in
anhydrous CH.sub.2Cl.sub.2 (188 mL) at -78.degree. C. was added via
canula. During the addition the temperature was kept at -78.degree.
C. in both flasks and the color of the reaction was yellow. The
reaction mixture was stirred at -40.degree. C. for 35 minutes.
During this period of time the solution was turned from yellow to
dark green. After this time, .sup.iPr.sub.2NEt (21.2 mL, 121.6
mmol) was dropwise added and the reaction mixture was kept at
0.degree. C. for 45 minutes. The color of the solution turned to
pale brown during this time. Then, tBuOH (5.8 mL, 60.8 mmol) and
tert-butyl tetramethyl guanidine (18.3 mL, 106.4 mmol) were
dropwise added and the reaction mixture was stirred at 23.degree.
C. for 40 minutes. After this time, acetic anhydride (14.34 mL, 152
mmol) was dropwise added and the reaction mixture was kept at
23.degree. C. for 1 hour more. Then, the reaction mixture was
diluted with CH.sub.2Cl.sub.2 (38 mL) and washed with a saturated
aqueous solution of NH.sub.4Cl (500 mL), NaHCO.sub.3 (500 mL), and
NaCl (500 mL). The combined organic layers were dried over
Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by flash column chromatography (SiO.sub.2, EtOAc:Hex in a
gradient manner, from 3:7 to 4:6) to afford 171 (6.24 g, 52%) as a
pale yellow solid. Rf=0.38 Hex:EtOAc 1:1.
[0792] .sup.1H-RMN (CDCl.sub.3): 6.78 (s, 1H), 6.07 (d, J=1.2 Hz,
1H), 5.98 (d, J=1.2 Hz, 1H), 5.92 (m, 1H), 5.32 (d, J=5.9 Hz, 1H),
5.31 (dd, J.sub.1=1.5 Hz, J.sub.2=17.1 Hz, 1H), 5.23 (dd,
J.sub.1=1.5 Hz, J.sub.2=10.4 Hz, 1H), 5.19 (d, J=5.6 Hz, 1H), 5.01
(d, J=11.5 Hz, 1H), 4.81 (d, J=9.8 Hz, 1H), 4.53-4.51 (m, 3H),
4.35-4.27 (m, 2H), 4.24 (s, 1H), 4.18-4.13 (m, 2H), 3.94-3.84 (m,
2H), 3.73 (s, 3H), 3.58 (t, J=4.7 Hz, 2H), 3.43-3.37 (m, 2H), 3.36
(s, 3H), 2.91 (m, 2H), 2.27 (s, 3H), 2.26 (s, 3H), 2.20 (s, 3H),
2.36-2.06 (m, 2H), 2.02 (s, 3H).
[0793] .sup.13C-RMN (CDCl.sub.3): 170.23, 168.49, 155.26, 149.62,
148.26, 145.63, 140.85, 140.24, 132.74, 131.60, 130.11, 124.89,
124.70, 120.14, 117.89, 117.84, 113.21, 101.89, 98.03, 92.67,
71.60, 69.04, 65.70, 61.20, 60.35, 59.36, 59.01, 58.89, 54.71,
54.42, 53.79, 41.53, 41.19, 32.68, 29.53, 23.57, 20.26, 15.62,
9.45.
[0794] ESI-MS m/z: Calcd. for C.sub.39H.sub.46N.sub.4O.sub.12S:
794.87. Found: 796 (M+1).sup.+, 817 (M+23).sup.+. HPLC: Conditions:
Column: Simmetiy C18, Mobile phase: AcN/buffer phosphate (pH: 5) in
gradient from 45 to 65% in 15 minutes and 65-90% in 36 minutes. .O
slashed.=0.8 ml/min, t.sup.a=40.degree. C. Retention time: 19.734
minutes. HPLC purity in area: 83.17%
Example 97
[0795] 238
[0796] To a solution of 171 (2.26 g, 2.85 mmol) in anhydrous
CH.sub.2Cl.sub.2 (74 mL) and acetonitrile (74 mL), NaI (3.42 g,
22.8 mmol) and TMSCl (freshly distilled over CaH.sub.2) (2.6 mL,
22.8 mmol) were added at 0.degree. C. and the reaction was stirred
for 35 minutes. A saturated aqueous solution of sodium bicarbonate
(150 mL) was added to the reaction mixture at this temperature. The
organic phase was separated and the aqueous phase was extracted
with CH.sub.2Cl.sub.2 (2.times.100 mL). The combined organic
extracts were dried over sodium sulphate, filtered and the solvent
was eliminated under reduced pressure to give 164 (2.4 g, 100%) as
a pale yellow solid which was used in subsequent reactions with no
further purification. Experimental data of 164 were described above
in Example 88.
[0797] Transformation of 164 into 35 was previously described above
in Example 89.
[0798] Intermediates 35, 36, ET-770 and ET-743 were prepared
following the same procedures than those previously described in
PCT/GB00/01852.
[0799] Route 6
Example 98
[0800] 239
[0801] To a solution of 144 (7 g, 7.6 mmol) in MeOH (140 mL), 1M
NaOH (15.1 mL) was added and the reaction was stirred for 10
minutes at 23.degree. C. A saturated aqueous solution of NH.sub.4Cl
(100 mL) was added to the reaction mixture. The organic phase was
separated and washed with 5% HCl until the colour turned into
yellow. The organic extract was dried over sodium sulphate,
filtered and the solvent was eliminated under reduced pressure. The
residue was purified by flash column chromatography (SiO.sub.2,
EtOAc:Hexane in a gradient manner, from 0:1, 1:3, 1:2, 1:1, 1:1 to
3:1) to afford 161 (3.76 g, 85%). Experimental data of 161 were
previously described in U.S. Pat. No. 5,721,362.
Example 99
[0802] 240
[0803] To a solution of 161 (200 mg, 0.37 mmol) and the cysteine
152 (240 mg, 0.55 mmol) in anhydrous CH.sub.2Cl.sub.2 (20 mL), DMAP
(110 mg, 0.925 mmol) and EDC.HCl (170 mg, 0.925 mmol) were added at
23.degree. C. and the reaction was stirred at that temperature for
1.5 hours. The mixture was then washed successively with a
saturated aqueous solution of NaHCO.sub.3 (15 mL), NaCl (15 mL) and
NH.sub.4Cl (2.times.10 mL). The organic layer was dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure.
The residue was purified by flash column chromatography with silica
gel (SiO.sub.2, AcOEt/Hexane in a gradient manner, from 1:4 to 1:2)
to afford 172 (285 mg, 80%) as a white solid. Rf=0.3 Hex:EtOAc
2:1.
[0804] .sup.1H RMN (CDCl.sub.3) .delta. 7.73 (d, J=7.5 Hz, 2H),
7.59-7.57 (m, 2H), 7.40-7.28 (m, 9H), 6.60 (s, 1H), 5.69 (s, 1H),
5.65 (s, 1H), 5.54 (d, J=7.8 Hz, 1H), 5.11-5.08 (m, 4H), 4.52-4.49
(m, 1H), 4.21-3.90 (m, 6H), 3.83 (s, 3H), 3.49 (s, 3H), 3.21 (d,
J=6.6 Hz, 1H), 3.09-2.90 (m, 6H), 2.41 (d, J=18 Hz, 1H), 2.34-2.31
(m, 1H), 2.25 (s, 3H), 2.19 (s, 3H), 1.88-1.83 (m, 1H), 1.77 (s,
3H).
[0805] .sup.13C-RMN (CDCl.sub.3) .delta. 198.7, 170.5, 158.4,
155.9, 148.9, 148.8, 145.8, 142.5, 141.3, 136.2, 131.4, 130.0,
128.8, 128.6, 128.4, 127.9, 127.3, 125.3, 125.0, 124.9, 123.0,
120.1, 117.5, 108.5, 104.8, 101.7, 99.5, 70.8, 67.4, 60.5, 57.8,
57.0, 56.5, 56.0, 55.5, 47.1, 41.6, 37.4, 37.1, 31.8, 25.8, 22.8,
15.9, 14.3, 7.6.
[0806] ESI-MS m/z: Calcd. for C.sub.53H.sub.4N.sub.4O.sub.11S:
954.35. Found (M+23).sup.+: 977.8.
Example 100
[0807] 241
[0808] The reaction flask was flamed twice, purged vacuum/Argon
several times and kept under Argon atmosphere for the reaction. To
a solution of DMSO (977 .mu.L) in anhydrous CH.sub.2Cl.sub.2 (118
mL) was dropwise added triflic anhydride (930 .quadrature.L, 5.5
mmol) at -78.degree. C. The reaction mixture was stirred at
-78.degree. C. for 20 minutes. Then, a solution of 172 (2.63 g,
2.75 mmol) in anhydrous CH.sub.2Cl.sub.2 (26 mL, for the main
addition and 13 mL for washing) was added via canula (addition
time: 5 min) at -78.degree. C. During the addition the temperature
was kept at -78.degree. C. in both flasks and the color changed
from yellow to brown. The reaction mixture was stirred at
-40.degree. C. for 35 minutes. During this period of time the
solution was turned from yellow to dark green. After this time,
.sup.iPr.sub.2NEt (3.48 mL, 22 mmol) was dropwise added and the
reaction mixture was kept at 0.degree. C. for 45 minutes, the color
of the solution turned brown during this time. Then, tBuOH (1.04
mL, 11 mmol) and tert-butyl tetramethyl guanidine (3.31 mL, 19.25
mmol) were dropwise added and the reaction mixture was stirred at
23.degree. C. for 40 minutes. After this time, acetic anhydride
(2.6 mL, 27.5 mmol) was dropwise added and the reaction mixture was
kept at 23.degree. C. for 1 hour more. Then, the reaction mixture
was diluted with CH.sub.2Cl.sub.2 (70 mL) and washed successively
with a saturated aqueous solution of NH.sub.4Cl (180 mL),
NaHCO.sub.3 (180 mL), and NaCl (180 mL). The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and concentrated
at reduced pressure. The residue was purified by flash column
chromatography (SiO.sub.2, Hex:EtOAc in a gradient manner, from
4:1, 3:1, to 2:1) to afford 173 (1.145 g, 52%) as a white solid.
R.sup.f-0.31 Hex:EtOAc 3:2.
[0809] .sup.1H RMN (CDCl.sub.3) .delta. 7.37 (bs, 5H), 6.67 (s,
1H), 6.08 (d, J=1.2 Hz, 1H), 5.99 (d, J=1.2 Hz, 1H), 5.19-5.00 (m,
4H), 4.82 (d, J=9.3 Hz, 1H), 4.49 (bs, 1H), 4.32-4.15 (m, 5H), 3.67
(s, 3H), 3.55 (s, 3H), 3.44 (d, J=4.8 Hz, 1H), 3.39 (d, J=6 Hz,
1H), 2.90-2.87 (m, 2H), 2.28 (s, 3H), 2.19 (s, 3H), 2.15-2.07 (m,
2H), 2.03 (s, 3H), 2.00 (s, 3H).
[0810] .sup.13C-RMN (CDCl.sub.3) .delta. 170.6, 168.8, 155.8,
149.9, 148.5, 146.0, 141.2, 140.6, 136.6, 132.0, 130.4, 128.8,
128.7, 128.5, 125.2, 124.9, 120.5, 118.2, 113.7, 113.6, 102.2,
99.4, 67.2, 61.6, 60.7, 59.7, 59.3, 57.6, 55.1, 54.8, 54.2, 41.9,
41.6, 33.0, 29.9, 23.9, 20.6, 15.6, 9.8.
[0811] ESI-MS m/z: Calcd. for C.sub.41H.sub.44N.sub.4O.sub.11S:
800.87. Found (M+23).sup.+: 823.7.
Example 101
[0812] 242
[0813] To a solution of 173 (100 mg, 0.125 mmol) in
CH.sub.2Cl.sub.2 (2 mL) and CH.sub.3CN (2 mL), NaI (75 mg, 0.5
mmol) and TMSCl (63 .quadrature.L, 0.5 mmol) were added at
0.degree. C. After stirring the reaction at 23.degree. C. for 50
minutes, the mixture was quenched with water (30 mL) and extracted
with CH.sub.2Cl.sub.2 (2.times.20 mL). The combined organic phases
were washed successively with a saturated aqueous solution of NaCl
(20 mL) and sodium ditionite (20 mL), dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (SiO.sub.2, EtOAc:Hexane in a gradient
manner, from 1:4, 1:2 to 1:1) to afford 158 (66 mg, 70%)-as white
solid. Rf=0.21 Hex:EtOAc 1:1. Experimental data of 158 was
described above in Example 19.
[0814] Transformation of 158 into 35 was described above in Example
85.
[0815] Intermediates 36, ET-770 and ET-743 were prepared following
the same procedures than those previously described in
PCT/GB00/01852.
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* * * * *