JP2003533533A - Synthetic method for the production of ectinacydin compounds - Google Patents

Abstract

  (57) [Summary] There is a need for alternative synthetic routes to ectinacydin compounds and related compounds. Such a synthetic route would provide a more economical route to known antitumor agents, as well as allow for the preparation of new active compounds. The present invention relates to synthetic methods for the formation of intermediates, derivatives and related structures of ectinacidin or other tetrahydroisoquinoline phenol compounds. In one aspect, the invention provides a method for preparing an ectinacydin product having a spiroamine-1,4-bridge. This method involves the formation of a 1,4 bridge using a 1-lavyl, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused ring compound, wherein the fused ring has the formula (XIV). In the present invention, C-18 protection is removed before spiroamine introduction.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to synthetic methods, and in particular, the present invention relates to synthetic methods for producing ecteinascidin compounds.

[0002]

[Prior art]

European Patent 309,477 relates to ecteinascidin 729, 743, 745, 759A, 759B and 770. The ecteinascidin compounds are disclosed to have antibacterial and other useful properties. Ectinacidin 743 is currently undergoing clinical trials as an antitumor agent.

Ectinacidin 743 has the complex tris (tetrahydroisoquinolinephenol) structure of formula (I):

[Chemical 6]

It is currently prepared by isolation from the extract of the marine caudate Ecteinascidin turbinata. Yields are low and alternative methods of preparation are sought.

Synthetic methods for producing ecteinascidin compounds are described in US Pat. No. 5,721,362.
, And also see WO 9812198, which is incorporated herein by reference in its entirety. The method described is long and complex, and there are 38 examples each describing one or more steps in the synthetic sequence to reach ecteinascidin 743.

Well-known synthetic methods include 1-labyl, 10-hydroxy, 18-protected hydroxy, di-6
The 1,4 bridge is formed using the 8,8-en-5-one fused ring compound. Compound (13) is converted to compound (14) as shown in Example 33:

[Chemical 7]

According to well known synthetic methods, spiroquinoline is formed in the 1,4 bridge and the 18-MOM protecting group is removed by the steps of Examples 34 to 36 to give ecteinascidin 7
70 is formed, which can then be converted to ecteinascidin 743.

Claim 25 of US Pat. No. 7,721,362 is directed to certain intermediate phenolic compounds of formula (11), which we also refer to as intermediate 11 or Int-11. It has the following bis (tetrahydroisoquinolinephenol) structure (II):

[Chemical 8] [Wherein MOM is a methoxymethyl substituent and TBDPS is a tert-butyldiphenylsilyl substituent].

From Intermediate 11, another interesting anti-tumor agent, Phthalacidin (Proc. Natl. Acad. Sci
USA, 96, 3496-3501, 1999) can be synthesized. Phthalacidin is a bis (tetrahydroisoquinolinephenol) derivative of formula (III):

[Chemical 9]

[0010]   In ecteinascidin 743 and 770, the 1,4 bridge has the structure of formula (IV):

[Chemical 10]

Other well-known ecteinascidin have a bridge having the structure of formula (V): ecteinascidin 722 and 736:

[Chemical 11] Ectinascidin 583 and 597 with a bridge having the structure of formula (VI):

[Chemical 12] Ectinascidin 594 and 596 with a bridge having the structure of formula (VII):

[Chemical 13] Compounds having different bridged cyclic ring systems, such as

The complete structures for these and related compounds are described in J. Am. Chem. Soc. (1
996) 118, 9017-9023. This document is incorporated by reference. )

Other references to ecteinascidin compounds include: Corey, E
J., J. Am. Chem. Soc., 1996, 118 pp. 9202-9203; Rinehart et al., Jounal of
Natural Products, 1990, "Bioactive Compounds from Aquatic and Terrestria
l Sources ", vol. 53, pp. 771-792; Rinehart et al., Pure and Appl. Chem., 1990.
, "Biologically active natural products", vol 62, pp. 1277-1280; Rinehar
t et al., J. Org. Chem., 1990, "Ecteinascidine 729, 743, 745, 759A, 759B, and
770: potent Antitumour Agents from the Caribbean Tunicate Ecteinascidin
e turninata ", vol. 55, pp. 4512-4515; Wright et al., J. Org. Chem., 1990," An
titumour Tetrahydroisoquinoline Alkanoids from the Colonial ascidian Ect
einascidia turbinata ", vol. 55, pp. 4508-4512; Sakai et al., Proc. Natl. Scad
. Sci. USA 1992, "Additional antitumor ecteinascidins from Caribbean tun
icate: 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, KE, "Asymmetric Synthesis", edited by Morrison,
Academic Press, Inc., Orlando, FL, vol. 5, 1985, p. 71: Barton et al., J. Ch.
em. Soc. Perkin Trans., 1, 1982, "Synthesis and Properties of a Series o
f Sterically Hindered Guanidine bases ", pp. 2085; Fukuyama et al., J. Am. Che
m. Soc., 1982, "Stereocontrolled Total Synthesis of (+)-Saframycin B", v
ol. 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 Inermed
iate to Saframycin A ", vol. 54, 5391; Still et al., J Org. Chem., 1978," Rapi
d Chromatographic Technique for Preparative Separations with Moderate Re
solution ", vol. 43, p. 2923; Kofron, WG; Baclawski, LM, J. Org. Ch
em., 1976, vol. 41, 1879; Guan et al., J. Biomolec. Struc. & Dynam., vo. 10,
pp. 793-817 (1993); Shamma et al., "Carbon-13 NMR Shift Assignments of Amines.
and Alkanoids ", p. 206 (1979); Lown et al., Biochemistry, 21, 419-428 (1982).
Zmijewski et al., Chem. Biol. Interactions, 52, 361-375 (1985); Ito, CRC Cr
it. Rev. Anal. Chem., 17, 65-143 (1986); Rinehart et al., "Topics in Pharmace.
utical Sciences 1989 ", pp. 613-626, DD Breimer, FJA Cromwelin, K
. K. Midha, Amsterdam Medical Press BV, Noordwijk, The Netherlands
(1989); Rinehart et al., "Biological Mass Spectrometry", 233-258, Burlingame.
Equal, 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 fom the Sea Proceedings" (1972)
, Marine Technology Society, Washington, DC 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 Prommier et al., Biochemistry, 1996, 35: 1330-13309.

Further compounds are known which lack a bridged cyclic ring system. They include the bis (tetrahydroquinoline quinone) antitumor-antimicrobial antibiotics safracin and saframycin, as well as the marine natural product renieramycin and zestomycin isolated from cultured microorganisms or sponges. They all share a common dimeric tetrahydroisoquinoline carbon skeleton. These compounds can be classified into four types, types I to IV, with respect to the oxidation pattern of the aromatic ring.

Type I dimeric isoquinoline quinones are the system of formula (VIII) most commonly present in this class of compounds and are referenced in Table I below.

[Table 1]

Type I aromatic rings have been observed in saframycins A, B and C; G and H; and S, isolated from Streptomyces lavendulae as a minor component. The cyano derivative of saframycin A, called cyanoquinone amine, is disclosed in Japanese Patent Laid-Open Publication No.
Known from 9/225189 and 60/084288. Saframycin Y _3, Yd _1, Ad ₁ and Yd ₂ is intended that suitably supplemented culture media were produced by S. lavendulae by directed biosynthesis. The saframycin Y _2b and Y _2b-d dimers formed by linking the nitrogen at one unit C-14 to another unit C-25 are also S.
It is produced in culture medium supplemented with lavendulae. Saframycin AR ₁ (= AH ₂ ), a microbial reduction product of saframycin A at C-25 produced by Rhodococcus amidophilus, also shows nonstereoselective saframycin A with sodium borohydride as a 1: 1 mixture of epimers Prepared by selective chemical reduction followed by chromatographic separation [other isomers AH ₁ are less polar]. The further reduction product saframycin AR ₃ , 21-decyano-25-dihydro-saframycin A (= 25-dihydrosaframycin B) was produced by the same microbial transformation. Another type of biotransformation of saframycin A using Nocardia sp. Produces saframycin B and My
Further reduction with cobacterium species produced saframycin AH ¹ Ac. The 25-O-acetates of saframycin AH ₂ and AH ₁ have also been chemically prepared for biological studies.

[0017]   Type I compounds of formula (X) have been isolated from marine sponges, see Table II.

[Table 2]

Renieramycin AD was isolated from an antimicrobial extract of the sponge, Reniera sp., Recovered in Mexico, with biogenically relevant monomeric isoquinolines and related compounds. The structure of Renieramycin A was first confirmed by the opposite stereochemistry at C-3, C-11 and C-13. However, careful observation of the ¹ H NMR data for novel related compounds, renieramycin E and F, isolated from the same sponge recovered in Parau, revealed that the ring junction of renieramycin was identical to that of saframycin. It was revealed that there is. This result is based on the previously certified renieramycin.
We conclude that the stereochemistry of A to D must be the same as that of saframycin.

Zestomycin was found in sponges, a Xestospongia species recovered from Sri Lankan water.

Type II compounds of formula (XI) having a reduced hydroquinone ring are saframycins D and F isolated from S. lavendulae and saframycins Mx-1 and Mx-2 isolated from Myxococcus xanthus. including. See Table III.

[Table 3]

The type III scaffold is found in the antibiotics safracin A and B isolated from cultured Pseudomonas fluorescens. These antibiotics of formula (XII) consist of a tetrahydroisoquinoline-quinone subunit and a tetrahydroisoquinoline phenolic subunit.

[Chemical 14] [Wherein R ²¹ is -H for safracin A and -OH for safracin B].

Saframycin R, the only compound classified as a Type IV scaffold, is S. la.
isolated from vendulae. This compound of formula (XIII), which consists of a hydroquinone ring with a glycolate side chain on one of the phenolic oxygens, is considered a prodrug of saframycin A because of its mild toxicity.

[Chemical 15]

All these known compounds have a five ring (A) to (E) fusion system as shown in the structure of formula (XIV) below:

[Chemical 16]

Rings A and E are phenolic in ecteinascidin and in some other compounds, especially saframycin among others, rings A and E are quinolic. In known compounds, rings B and D are tetrahydro and ring C is perhydro.

[0025]

[Problems to be Solved by the Invention]

There is a need for alternative synthetic routes to ecteinascidin compounds and related compounds. Such synthetic routes would provide a more economical route to known anti-tumor agents as well as allow the preparation of new active compounds.

[0026]

[Means for Solving the Problems]

The present invention relates to synthetic methods for the formation of intermediates, derivatives and related structures of ecteinascidin or other tetrahydroisoquinoline phenolic compounds.

[0027] In one aspect, the invention provides a method for preparing an ecteinascidin product having a spiroamine-1,4-bridge. This method involves the formation of a 1,4 bridge using a 1-labyl, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused ring compound, wherein the fused ring is of the formula (XIV). In the present invention, the C-18 protection is removed prior to spiroamine introduction.

Suitable starting materials for the novel synthetic methods include compounds relating to natural bis (tetrahydroisoquinoline) alkanoids. Such starting materials are described in WO 006986
It may be prepared either from various classes of saframycin and safracin available from various culture broths as described in 2 or by other synthetic or biochemical methods. In this regard, WO 0069862 is hereby fully incorporated by reference. This PCT application is based on application PCT / GB 00/018 published as WO 0069862.
Claim priority from 52. We incorporate the specification by reference to the extent there are disclosures there that are not present in this specification.

[0029]

DETAILED DESCRIPTION OF THE INVENTION

In one particular aspect, the invention relates to the use of intermediate 21 in a number of novel synthetic methods for the preparation of ecteinascidin 743 and related compounds:

[Chemical 17]

Intermediate 21 has a 5-allyloxy group, where the allyl group functions to protect the 5-hydroxy group. Other protecting groups are readily available and the present invention is directed to other such
It will be understood that it generally extends to the use of 5-protected hydroxy compounds.

Formation of ecteinascidin 743 and related compounds In general, conversion of intermediate 21 or related compounds to ecteinascidin products is
Includes the following key transformations: (a) conversion of NH ₂ to OH, for example by reaction with sodium nitrite in acetic acid; (b) E-ring phenol protection; (c) primary 1 with a protected cysteine side chain. -Esterification to protect hydroxy functional group; (d) Deprotection and oxidation of allyl machine; (e) Preparation of crosslinked ring by cyclization reaction; (f) Deprotection of E ring phenol and cysteine moiety; (g) Introduction of quinoline by transamination and Petter Spengler reaction.

The high functionality of intermediate compounds requires the use of protecting groups for the E-ring phenol and the cysteine side chain in order to avoid undesired side reactions.

As such, numerous alternative intermediates can be produced, depending on the particular choice of protecting group.

Another possible series is possible for combining these transformations, mainly depending on the protecting groups selected for the amines of the phenol ring and the cysteine side chain.

[0035]   The number of all synthetic transformations is also a function of the protecting group chosen.

For illustration purposes, the use of different combinations of protecting groups is described below for six exemplary routes for the preparation of ET-743 from intermediate 21, also referred to herein as SF21. Pathway Phenol protection Cysteine protection Number of steps 1 MOM Moc 12 2 MEM Boc 10 3 MEM Cbz 11 4 MOM Alloc 13 5 MEM Alloc 13 6 MOM Cbz 15

As one of ordinary skill in the art would readily be able to predict, the reaction schemes described herein may be modified and / or combined in a variety of ways to produce alternative sequences of steps and processes produced therefrom. Compounds which are part of the present invention.

Furthermore, the use of other protecting group strategies not described in detail is also part of the invention.

Details of methodologies for 6 exemplary synthetic routes. The complete reaction scheme for each route exists in Schemes 1-6 below. Scheme 1-ET-743: Semi-synthetic alternative route 1

[Chemical 18] Scheme 2-ET-743: Semi-synthetic alternative route 2

[Chemical 19] Scheme 3-ET-743: Semi-synthetic alternative route 3

[Chemical 20] Scheme 4-ET-743: Semi-synthetic alternative route 4

[Chemical 21] Scheme 5-ET-743: Semi-synthetic alternative route 5

[Chemical formula 22] Scheme 6-ET-743: Semi-synthetic alternative route 6

[Chemical formula 23]

In Route 1, protection of the E-ring phenol is accomplished in three steps, including protection / deprotection of the amine of SF21 at Troc.

For pathways 1 and 2, protection of the cysteine side chain with Boc allows the phenol and cysteine groups to be deprotected in a single step rather than two separate steps. The rest of the pathway requires additional deprotection steps.

For route 2, intermediate 25 is avoided through the use of a direct esterification methodology followed by protection of the phenol with the MEM group.

For Routes 2 and 3, protection of the E-ring phenol occurs after the diazotization and esterification steps, whereby the phenol is protected in a single step rather than by a series of three steps in Route 1. Be delayed.

For Routes 1, 2 and 3, direct esterification of the primary alcohol with a cysteine derivative eliminates the unproductive protection / deprotection step of the primary alcohol at the silyl group (route 4 and 5), thereby shortening the series by two steps.

Only route 6 contemplates the final step here from intermediate 161, which can easily be obtained from intermediate 21.

In Routes 4 and 5, the primary alcohol produced by the initial diazotization step is protected with silicon, allowing 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 is esterified with a cysteine derivative.

These changes are a direct result of the problems found in scaling up the pathway shown in WO 0069862. As a result of these changes, the entire route 2 will be shortened in 3 steps, thus allowing a more stable and / or cheaper routine manufacture.

Thus, with respect to Routes 1-6, the present invention extends to a method for preparing an ecteinascidin product having a spiroamine-1,4-bridge, the method comprising 1-labile, 10-hydroxy. , 18-protected hydroxy, di-6,8-en-5-one fused ring compounds are used to form 1,4 bridges, where the C-18 protection is removed prior to spiroamine introduction.

In one version of this method, the ecteinascidin product has a 21-hydroxy group, which involves conversion of the 21-cyano group to a 21-hydroxy group.

Typically the spiroamine is a spiroquinoline, especially the spiroquinoline of ecteinascidin 743.

Preferred methods include 1-labyl, 10-hydroxy, 18-protected hydroxy, di-6,8
The 18-protecting group of the -en-5-one fused ring compound is MOM: methoxymethyl; or MEM
: Protected by a methoxyethoxymethyl group.

A suitable 1-labyl group is an N-protected cysteinyloxymethylene group of the formula: -CH ₂ -O-CO-CNHProt ¹ -CH ₂ -SH.

In this formula, Prot ¹ is typically Boc: t-butyloxycarbonyl; Troc: 2,2,
2-trichloroethyloxycarbonyl; Cbz: benzyloxycarbonyl; or Alloc: allyloxycarbonyl.

In some embodiments of this method, Prot ¹ is removed in the same step as the C-18 protection.

The 1-labyl group can be purified from the 1-substituted form of the formula: -CH ₂ -O-CO-CNHProt ¹ -CH ₂ -S-Prot ²

In this formula, Prot ² is typically Fm: 9-fluoromethyl.

The 1-substitute of the formula: —CH ₂ —O—CO—CNHProt ¹ —CH ₂ —S-Prot ² can be formed by esterification of the —CH ₂ —OH substituent.

The esterification can be carried out before or after the formation of the 10-hydroxy, di-6,8-en-5-one structure.

In one version, the method of the present invention starts with 1-aminomethylene, 5-protected hydroxy, 7,8-dioxymethylene, 18-hydroxy, 21-cyano fused ring compounds.

The 1-aminomethylene group can be temporarily protected to allow protection at the 18-hydroxy group, with the temporary protection being removed.

Alternatively, the C-18 hydroxy group can be protected after formation of the 1-ester functional group.

In another version, the 1-aminomethylene group is converted to a 1-hydroxymethylene group and the 1-hydroxymethylene group is temporarily protected to allow protection at the 18-hydroxy group, The protection is removed.

[0063]   The fused ring structure is suitably of the formula:

[Chemical formula 24] In particular, R ¹⁵ is H. One or more or all of the remaining substituents are ecteinascidin
It can be of 743.

Semi-Synthesis The present invention allows the use of a compound known in semi-synthetic synthesis, safracin B, also referred to as quinone amine.

More generally, the present invention provides a semi-synthetic method for the formation of intermediates, derivatives and related structures of ecteinascidin or other tetrahydroisoquinoline phenolic compounds starting from natural bis (tetrahydroisoquinoline) alkanoids. Regarding Suitable starting materials for this semi-synthetic method include the classes of saframycin and safracin antibiotics available from various culture brass and the classes of reineramycin and zestomycin compounds available from marine sponges.

[0066]   The general formula (XV) for the starting compounds is as follows:

[Chemical 25] [Wherein R ¹ is an amidomethylene group such as —CH ₂ —NH—CO—CR ^25a R ^25b R ^25c , and R ^25a and R ^25b form a keto group, or one of them is —OH, —NH ₂ or -OCOCH ₃ , the other is -CH ₂ COCH ₃
, -H, -OH or -OCOCH ₃ , provided that R ^25a is -OH or -NH ₂ , then R ^25b is-.
Not OH, R ^25c is -H, -CH ₃ or -CH ₂ -CH ₃ , or R ¹ is an acyloxymethylene group such as -CH ₂ -O-CO-R, where R is- C (CH ₃ ) = CH-CH ₃ or -CH ₃ ; R ⁵ and R ⁶ are independently selected from -H, -OH or -OCOCH ₂ OH, or R ⁵ and R ⁸ are both keto And Ring A is a p-benzoquinone ring; R ^14a and R ^14b are both —H, or one is —H and the other is —OH, —OCH ₃ or —O.
CH ₂ CH ₃ or R ^14a and R ^14b together form a keto group; R ¹⁵ and R ¹⁸ are independently selected from —H or —OH, or R ⁵ and R ⁸ are both keto. And Ring A is a p-benzoquinone ring; and R ²¹ is -OH or -CN].

[0067]   More general formulas for these classes of compounds are provided below:

[Chemical formula 26] [Wherein, the substituents defined by R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ , R ₉ and R ₁₀ are independently H, OH and OCH ₃ , CN, ═O, CH ₃ ; wherein X is a different amide or ester functionality contained in the aforementioned natural product; wherein each dotted circle is 1, 2 or 3 Represents any double bond of].

Thus, according to the present invention there is provided a semi-synthetic route for the production of intermediates comprising intermediate 11 or 21, as well as a semi-synthetic route for the production of ecteinascidin compounds, as well as phthalacidine and further compounds. It Each of the semi-synthetic routes of the present invention involves a number of conversion steps to reach the desired product. Each step itself is the method according to the present invention. The present invention is not limited to the routes illustrated and alternative routes will be provided, eg by altering the order of the conversion steps as appropriate or by varying the protecting groups used.

[0069]   In particular, the invention includes providing a 21-cyano starting material of general formula (XVI):

[Chemical 27] [Wherein R ¹ , R ⁵ , R ⁸ , R ^14a , R ¹⁵ and R ¹⁸ are as described above].

Other compounds of formula (XVI) with different substituents at position 21 may also represent considered starting materials. In general, candidates include any derivative that can be produced by nucleophilic substitution of the 21-hydroxy group of compounds of formula (XV) where R ²¹ is a hydroxy group. Examples of suitable 21-substituents include, without limitation, the following: a mercapto group; an alkylthio group (an alkyl group having 1 to 6 carbon atoms); an arylthio group (an aryl group has 6 to 10 carbons). Substituted with 1 to 5 substituents having an atom, for example, an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a halogen atom, a mercapto group, and a nitro group. Unsubstituted); amino group; mono- or dialkylamino (whose or each alkyl group has from 1 to 6 carbon atoms); mono- or diarylamino group (whose or each aryl group is as defined above for arylthio groups) to) defined as; formula ^{-C (R a) (R b} ) -C (= O) α- carbonyl group (wherein the R ^c, R ^a and R ^b are hydrogen atom, 1 Etc. Alkyl groups having 20 carbon atoms, aryl groups (as described above for arylthio groups) and aralkyl groups (wherein alkyl groups having 1 to 4 carbon atoms are substituted by aryl groups as described above for arylthio groups). However, one of R ^a and R ^b is a hydrogen atom; R ^c is a hydrogen atom, 1 to 2
An alkyl group having 0 carbon atoms, an aryl group (as described above for an arylthio group), an aralkyl group (wherein an alkyl group having 1 to 4 carbon atoms is an aryl group as described above for an arylthio group). Has been replaced by)
Selected from alkoxy groups having 1 to 6 carbon atoms, amino groups, or mono- or dialkylamino groups as defined above).

Thus, in a more general aspect, the invention relates to a method wherein the first step is to use a nucleophile to form a 21-derivative. 21-Nuc such compounds
It is called a compound.

The presence of a 21-cyano group is required for some end products, especially ecteinascidin 770 and phthalacidin, while in other end products it is of the 21-hydroxy group of ecteinascidin 743 or 21-hydroxyphthalasidine. Such as a protecting group that can be easily converted into another substituent. Employing a 21-cyano compound as a starting material effectively stabilizes the molecule during subsequent synthetic steps until optionally removed. Other 21-Nuc compounds can provide this and other benefits.

In one important aspect, the invention relates to the use of 21-cyano compounds of general formula (XVI) in the preparation of bis- or tris- (tetrahydroisoquinolinephenol) compounds. The products that will be prepared include intermediates such as Intermediate 11 or 21, and ecteinascidin, as well as compounds of novel and known related structures.

Preferred starting materials include compounds of formula (XV) or (XVI) where R ^14a and R ^14b are both hydrogen. Preferred starting materials also include compounds of formula (XV) or (XVI) where R ¹⁵ is hydrogen. Furthermore, preferred starting materials include compounds of formula (XV) or (XVI) where E is a phenol ring. Preferred starting materials further include those of the formula (XV) or (X where at least one, and more preferably two or three of R ⁵ , R ⁸ , R ¹⁵ and R ¹⁸ are not hydrogen.
VI) compounds are included.

Examples of suitable starting materials for the present invention are saframycin A, saframycin B
, Saframycin C, saframycin G, saframycin H, saframycin S, saframycin Y ₃ , saframycin Yd ₁ , saframycin Ad ₁ , saframycin Yd ₂
, Saframycin AH ₂ , saframycin AH ₂ Ac, saframycin AH ₁ , saframycin AH ₁ Ac, saframycin AR ₃ , renieramycin A, renieramycin B, renieramycin C, renieramycin D, reniera Mycin E, renieramycin F,
Zestomycin, saframycin D, saframycin F, saframycin Mx-1, saframycin Mx-2, safracin A, safracin B, and saframycin R.
Preferred starting materials have a 21-position, i.e. a cyano group for R ^21.

In a particularly preferred aspect, the invention comprises a semi-synthetic method in which the conversion step is applied to safracin B:

[Chemical 28]

Safracin B represents a ring system closely related to ecteinascidin. This compound has the same five-membered ring structure and the same substituent pattern in the right-handed aromatic ring, ring E. Safracin B also offers great similarity to some of the synthetic intermediates in the total synthesis of ET-743, especially intermediates 11 or 21. Such an intermediate can be converted to Et-743 using well established methods. Therefore, synthetic conversion of safracin B to intermediate 11 or 21 would provide a semi-synthetic method to obtain ET-743.

Thus, provided is compound Safracin B, and intermediates 11 or 21, especially intermediates 11 or 21, made from compounds derived from ecteinascidin compounds. Further provided is phthalacidin made from safracin B. The present invention also relates to the use of safracin B in the production of intermediates 11 or 21, ecteinascidin compounds, and other intermediates of the invention. The invention also relates to the compounds described herein derived from other suggested starting materials, and the use of such compounds in the production of such compounds.

More preferred starting materials of the present invention have 21-cyano groups. The presently most preferred compound of the invention is the compound of formula 2. This compound is obtained directly from Safracin B and is considered a key intermediate in this semi-synthetic method.

[Chemical 29]

In a related aspect, fermentation of a safracin B-producing strain of PSurdomonas Fluorescens and manipulation of the culture broth with cyanide ion resulted in cyanosafracin B
Will be provided. A preferred strain of Pseudomonas fluorescens is A2-2 strain, FERM BP-14.
Which is used in the method of EP 055,299. A suitable source of cyanide ion is potassium cyanide. In a suitable operation, the broth is filtered and excess cyanide ion is added. After stirring at appropriate intervals, such as 1 hour, the pH is brought to alkaline, ie pH 9.5, and the organic extraction gives a crude extract which is further purified to give cyanosafracin B.

For the avoidance of doubt, the stereochemistry pointed out in this specification is based on our understanding of the exact stereochemistry of natural products. To the extent errors are found in the listed stereochemistry, appropriate corrections must be made in the formulas given throughout this specification. Furthermore, to the extent that the synthesis can be modified, the present invention extends to stereoisomers.

[0082]   The product of the invention typically has the formula (XVIIb):

[Chemical 30] [Wherein R ¹ and R ⁴ together form a group of formula (IV), (V), (VI) or (VII):

[Chemical 31] R ⁵ is -H or -OH; R ⁷ and R ⁸ together form a -O-CH ₂ -O- group; R ^14a and R ^14b are both -H, or one is -H The other is -OH, -OCH ₃ or -O
CH ₂ CH ₃ or R ^14a and R ^14b together form a keto group; and R ¹⁵ is —H or —OH; R ²¹ is —H, —OH or —CN] and especially R Derivatives, including acyl derivatives thereof, wherein ⁵ is acetyloxy or another acyloxy group of up to 4 carbon atoms.

In formula (XVIIb), R ¹ , which typically has R ⁴ , forms a (IV) or (V) group. R ¹⁸
The group is usually protected. Usually R ²¹ is cyano.

Preferably R ^14a and R ^14b are hydrogen. Preferably R ¹⁵ is hydrogen. The O-acyl derivative is suitably an aliphatic O-acyl derivative, in particular an acyl derivative of 1 to 4 carbon atoms, typically an O-acetyl group especially in the 5 position.

Suitable protecting groups for phenol and hydroxy groups are ethers and esters such as alkyl, alkoxyalkyl, aryloxyalkyl, alkoxyalkoxyalkyl, alkylsilylalkoxyalkyl, alkylthioalkyl, arylthioalkyl, azidoalkyl, cyanoalkyl. , Chloroalkyl, heterocycle, arylacyl, haloarylacyl, cycloalkylalkyl,
Alkenyl, cycloalkyl, alkylarylalkyl, alkoxyarylalkyl, nitroarylalkyl, haloarylalkyl, alkylaminocarbonylarylalkyl, alkylsulfinylarylalkyl, alkylsilyl, and other ethers, and arylacyl, arylalkylcarbonates, fats. Group carbonates, alkylsulfinyl arylalkyl carbonates, alkyl carbonates, aryl haloalkyl carbonates, arylalkenyl carbonates, aryl carbonates, alkyl phosphinyls, alkyl phosphinothionyls, aryl phosphinothionyls, aryl alkyl sulfonates, and others Including the ester of. Such groups may be optionally substituted with the groups described above for R ¹ .

Suitable protecting groups for amines include carbamates, amides, and other protecting groups,
For example alkyl, arylalkyl, sulfo- or halo-arylalkyl,
Haloalkyl, alkylsilylalkyl, arylalkyl, cycloalkylalkyl, alkylarylalkyl, heterocyclylalkyl, nitroarylalkyl, acylaminoalkyl, nitroaryldithioarylalkyl, dicycloalkylcarboxamidoalkyl, cycloalkyl, alkenyl, arylalkenyl, nitroaryl Alkenyl, heterocyclylalkenyl, heterocyclyl, hydroxyheterocyclyl, alkyldithio, alkoxy- or halo or alkylsulfinyl, arylalkyl, heterocyclylacyl,
And other carbamates, and alkanoyl, haloalkanoyl, arylalkanoyl, alkenoyl, heterocyclylacyl, aroyl, arylaroyl, haloaroyl, nitroaroyl, and other amides, and alkyl, alkenyl, alkylsilylalkoxyalkyl, alkoxyalkyl, cyanoalkyl, Heterocyclyl, alkoxyarylalkyl, cycloalkyl, nitroaryl, arylalkyl, alkoxy- or hydroxy-arylalkyl,
And many other groups. Such groups may be optionally substituted with the groups described above for R ¹ .

[0087]   Examples of such protecting groups are listed in the table below.

[0088] -OH group protection Ether abbreviation Methyl Methoxymethyl MOM Benzyloxymethyl BOM Methoxyethoxymethyl MEM 2- (Trimethylsilyl) ethoxymethyl 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 Propargil 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-Methylsulfinylbenzyl Msib 9-anthrylmethyl 4-picolyl Heptafluoro-p-tolyl Tetrafluoro-4-pyridyl Trimethylsilyl TMS t-Butyldimethylsilyl TBDMS t-Butyldiphenylsilyl TBDPS Triisopropylsilyl TIPS ester Aryl formate Aryl acetate Aryl Revlinate Aryl pivalonate ArOPv Aryl benzoate Aryl 9-fluorocarboxylate Aryl methyl carbonate 1-adamantyl carbonate t-Butyl carbonate BOC-OAr 4-Methylsulfinyl benzyl carbonate Msz-Oar 2,4-Dimethylpent-3-ylcarbonate Doc-Oar Aryl 2,2,2-trichloroethyl carbonate Aryl vinyl carbonate Aryl benzyl carbonate Aryl carbamate Dimethylphosphinyl Dmp-OAr Dimethylphosphinothionyl Mpt-OAr Diphenylphosphinothionyl Dpt-Oar Aryl methane sulfonate Aryltoluene sulfonate Aryl 2-formylbenzene sulfonate

Protected carbamate for the —NH ₂ group Abbreviation Methyl ethyl 9-fluorenylmethyl Fmoc 9- (2-sulfo) fluorenylmethyl 9- (2,7-dibromo) fluorenylmethyl 17-tetrabenzo [a , c, g, i] Fluorenylmethyl 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-chloroethyl 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-methylethyl t-Burmeoc 2- (2'- and 4 ' -Pyridyl) ethyl Pyoc 2,2-bis (4'-nitrophenyl) ethyl Bnpeoc n- (2-pivaloylamino) -1,1-dimethylethyl 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 alkyldithiobenzyl Cbz or Z p-methoxybenzyl Moz p-nitrobenzyl PNS p-bromobenzyl p-chlorobenzyl 2,4-dichlorobenzyl 4-methylsulfinylbenzyl Msz 9-anthrylmethyldiphenylmethylphenothiazinyl- (10) -carbonyl n'-p-toluenesulfonylaminocarbonyl n'-phenylaminothiocarbonylamide formamide Cetamide Chloroacetamide Trifluoroacetamide TFA Phenylacetamide 3-Phenylpropanamide Pent-4-enamide Picolinamide 3-Pyridylcarboxamide Benzamide p-Phenylbenzamide n-Tetrachlorophthalimide TCP 4-Nitro-n-phthalimide n-dithiasuccinimide Dts n-2,3-diphenylmaleimide n-2,5-dimethylpyrrole n-2,5-bis (triisopropylsiloxy) pyrrole BIPSOP n-1,1,4,4-STABASE tetramethyldi Syriazacyclopentane Theaduct 1,1,3,3-Tetramethyl-1,3-disilaisoindoline BSB Special -NH protecting group n-methylamine nt-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-dimethoxyben Dilamine Dmb 2-azanorbornene n-2,4-dinitrophenylamine n-benzylamine Bn n-4-methoxybenzylamine MPM n-2,4-dimethoxybenzylamine DMPM n-2-hydroxybenzylamine Hbn n- (diphenyl Methyl) amino DPM n-bis (4-methoxyphenyl) methylamine n-5-dibenzosuberylamine DBS n-triphenylmethylamine Tr n-[(4- MMTr methoxyphenyl) diphenylmethyl] amino n-9-phenylful Renylamine Pf n-Ferrocenylmethylamine Fcm n-2-picolylamine n'-oxide n-1,1-dimethylthiomethyleneamine n-benzylideneamine np-methoxybenzylideneamine n-diphenylmethyleneamine n- (5, 5-dimethyl-3-oxo-1-cyclohexenyl) amine n-nitroamine n-nitrosamine diphenylphosphine amide Dpp dimethylthiophosphine amide Mpt diphenylthio Ophosphinamide Ppt Dibenzylphosphoramidate 2-nitrobenzenesulfenamide Nps n-1- (2,2,2-trifluoro-1,1-TDE diphenyl) ethylsulfenamide 3-nitro-2-pyridinesulfinamide Phenamide Npys p-Toluenesulfoamide Ts Benzenesulfoamide

[0090]   A particular ecteinascidin product of the invention is a compound of formula (XVIII):

[Chemical 32] [Wherein, R ¹ and R ⁴ are each represented by the formula (IV), (V), (VI), or (VII):

[Chemical 33] R ²¹ is —H, —OH or —CN, especially —OH or —CN] and an acyl derivative thereof, especially Includes 5-acyl derivatives, including 5-acetyl derivatives.

In general, the conversion of a 21-cyano starting compound to an ecteinascidin product of formula (XVIII), for example, comprises the following steps: a) If necessary, a quinone system phenol system for ring E B) if necessary, conversion of the quinone system to the phenol system on ring A; c) conversion of the phenol system on ring A to the methylenedioxyphenol ring; d) position 1 on ring B And formation of a bridged spiro ring system of formula (IV), (VI) or (VII) connecting the 4 and 4 positions; and e) suitable derivatization such as acylation. Conversion of the quinone system to the phenol system for ring E, if necessary, in step (a) can be accomplished by conventional reduction methods. A suitable reagent system is hydrogen with palladium on carbon, but other reduction systems can be used. The conversion of the quinone system to the phenol system for ring A, if necessary, in step (b) is similar to step (a) and no further details are required. The conversion of the phenolic system for ring A in step (c) to a methylenedioxyphenol ring can be accomplished in several ways, presumably like step (b). For example, quinone ring A can be demethylated at the 7 position with a methoxy substituent, reduced to dihydroquinone, and a similar dihydroquinone that directly produces a CH ₂ Br ₂ , BrCH ₂ Cl, or methylenedioxy ring system. It can be trapped with a suitable electrophile such as a divalent reagent or with a divalent reagent such as thiocarbonyldiimidazole which produces a substituted methylenedioxy ring system that can be converted to the desired ring. Step (d) is typically affected by appropriate substitution at the 1-position with a cross-linking agent that can assist in forming the desired cross-link, forming an exendoquinone methide at the 4-position, the methide being
Reacts with 1-substituents to give crosslinked structures. Preferred crosslinkers are of formula (XIX):

[Chemical 34] [In the formula, Fu represents a protected functional group such as —NHProt ^4a , Prot ³ is a protective group, and a dotted line optionally represents a double bond].

Suitably, the methide is formed by first introducing a hydroxy group at position 10 at the junction of rings A and B to give a substructure of formula (XX):

[Chemical 35] Or more preferably provides a substructure of formula (XXI):

[Chemical 36] Wherein the R "group is selected for the desired group of formula (IV), (V), (VI) or (VII).
For the first two such groups, the R ″ group typically takes the form of —CHFu—CH ₂ —SProt ^3. The protecting group can then be removed and appropriately modified to give the desired compound. give].

A typical method for step (d) is provided in US Pat. No. 5,721,362, which is incorporated by reference. As a specific reference, column 8 of this US patent,
Part of step (I) and Example 33, and related parts are listed.

Derivatization in step (e) can include, for example, acylation with a R ^a —CO— group, where R ^a is alkyl, alkoxy, alkylene, arylalkyl, arylalkylene, amino acid acyl, Or can be various groups such as heterocyclyl, each of which is optionally halo, cyano, nitro, carboxyalkyl,
It may be substituted with alkoxy, aryl, aryloxy, heterocyclyl, heterocyclyloxy, alkyl, amino, or substituted amino. Other acylating agents include isothiocyanates, such as aryl isothiocyanates, especially phenyl isocyanate. The alkyl, alkoxy, or alkylene group of R ^a optionally has 1 to 6 or 12 carbon atoms and can be straight chain, branched, or cyclic. The aryl group is typically phenyl, biphenyl, or naphthyl.
A heterocyclyl group can be aromatic or partially or fully unsubstituted, optionally having 4 to 8 ring atoms, more preferably 5 or 6 ring atoms, nitrogen,
It may have one or more heteroatoms selected from sulfur and oxygen.

While not exhaustive, typical R ^a groups include alkyl, haloalkyl, alkoxyalkyl, haloalkoxyalkyl, arylalkylene, haloalkylarylalkylene, acyl, haloacyl, arylalkyl, alkenyl, and amino acids. For example, R ^a -CO- is acetyl, trifluoroacetyl, 2,2,2-trichloroethoxycarbonyl, isovalerylcarbonyl, trans-3- (trifluoromethyl) cinnamoylcarbonyl, heptafluorobutyrylcarbonyl, deca. Noylcarbonyl, trans-cinnamoylcarbonyl, butyrylcarbonyl, 3-chloropropylcarbonyl, cinnamoylcarbonyl, 4-methylcinnamoylcarbonyl, hydrocinnamoylcarbonyl, or trans-hexenoylcarbonyl,
Or alanyl, arginyl, aspartyl, asparagyl, cystyl, glutamyl, glutaminyl, glycyl, histidyl, hydroxyprolyl, isoleucyl, leucyl, lysyl, methionyl, phenylalanyl, prolyl, ceryl, threonyl, tyronyl, tryptophyll, tyrosyl, valyl, And other less common amino acid acyl groups, as well as phthalimide and other cyclic amides. Other examples may be found among the listed protecting groups. -CO-R ^a is derived from an amino acid, a compound containing an amino group can form itself acyl derivatives. Suitable N-acyl compounds include dipeptides that in turn can form N-acyl derivatives.

By way of illustration, it is possible to convert the cyanosafracin B compound of formula 2 into ET-743, which results in a shorter and more direct method of preparing ET-743 than previously described methods.

A retrosynthetic analysis for the preparation of ET-743 using compound 29 is represented in Scheme I. Scheme I

[Chemical 37]

Following Scheme I above, it is possible to obtain ET-743 in 21 consecutive steps. This method consists of (1) removal of the methoxy group located on ring A, (2) reduction of ring A and formation of methylene-dioxy group in one pot, (3) amide functional group located on carbon 1. Cyanosafracin B is converted to the intermediate 25 through a series of reactions including hydrolysis of (4) and conversion of the formed amine group to a hydroxyl group. In addition, this method uses cysteine residue 29 directly to form intermediate 27
Avoid protection and deprotection of the primary alcohol function at position 1 in ring B of 25. The cysteine derivative 29 has a compatibility with the existing allyl group and the MOM group, and therefore β-
Protected at the amine group with a β-β-trichloroethoxycarbonyl protecting group. Intermediate 27 is directly oxidized and cyclized. These environments, along with different deprotection strategies in the later steps of the synthesis, described the pathway in a novel, US Pat.
It is more suitable for industrial development than the method described in No.

Conversion of a 2-cyano compound to intermediate 25 usually involves the following steps (see Scheme II): Formation of a protected compound of formula 14 by reacting 2 with tert-butoxycarbonyl anhydride. Conversion of 14 to the di-protected compound of formula 15 by reaction with bromomethyl methyl ether in acetonitrile with diisopropylethylamine; at 15 to obtain the compound of formula 16 by reaction of sodium hydroxide with methanol Selective removal of the methoxy group of the quinone system; conversion of 16 to a methylene-dioxy compound of formula 18 by using the following preferred series: (1) The quinone group of compound 16 under hydrogen atmosphere at 10% Pd / Reduction with C; (2) reaction of bromochloromethane with cesium carbonate under hydrogen atmosphere to give a hydroquinone intermediate of formula 17 methylenedioxy compound Conversion; by protecting the free hydroxyl group as (3) OCH ₂ R group, to convert the 17 to a compound of formula 18. This reaction is
BrCH ₂ R and cesium carbonate, where R can be aryl, CH═CH ₂ , OR ′, etc .; to obtain the compound of formula 19 by reaction with a solution of HCl in dioxane, t
Removal of ert-butoxycarbonyl and methyloxymethyl protecting groups. This reaction is also accomplished by mixing 18 with a solution of trifluoroacetic acid in dichloromethane; by reacting 19 with phenylisothiocyanate to form a thiourea compound of formula 20; with a solution of hydrogen chloride in dioxane. Formula of compound of Formula 20 by reacting
Conversion of the compound of formula 21 to the amine compound by reaction of trichloroethyl chloroformate with pyridine.
Conversion to N-Troc derivative 22; Reaction of bromomethyl methyl ether with diisopropylethylamine 22 to form protected hydroxy compound of formula 23; Reaction of acetic acid with zinc to NH derivative 24 of compound of formula 23 Conversion of a compound of formula 24 to a hydroxy compound of formula 25 by reaction with sodium nitrite in acetic acid. Alternatively, nitric tetroxide can be used in a mixture of acetic acid and acetonitrile, followed by treatment with sodium hydroxide. It is also possible to use sodium nitrite in an acetic anhydride-acetic acid mixture and subsequently treat with sodium hydroxide. Scheme II

[Chemical 38]

[Chemical Formula 39]

Conversion of intermediate 25 to ET-743 using cysteine derivative 29 usually involves the following steps (see Scheme III): Scheme III

[Chemical 40]

[Chemical 41] (S) -N-2,2,2-Trichloroethoxycarbonyl-S- (9H-fluoren-9-ylmethyl)
Conversion of the compound of formula 24 into derivative 30 by protecting the primary hydroxyl function with cysteine 29; tributyltin hydride and dichloropalladium-bis (triphenylphosphine)
Conversion of the protected compound of formula 30 to the phenol derivative 31 by cleavage of the aryl group at; conversion of the phenol compound of formula 31 to the compound of formula 32 by oxidation with benzeneselenic hydride at low temperature; Conversion of a hydroxy compound of formula 32 to a lactone 33 by the following series: (1) reacting 2 equivalents of triflic anhydride with 5 equivalents of DMSO with a compound of formula 32; (2) followed by 8 equivalents of Reacting with diisopropylethylamine; (3) subsequently reacting with 4 equivalents of t-butyl alcohol; subsequently reacting with 10 equivalents of acetic anhydride; hydroxyl of lactone compound 33 by removal of MOM protecting group with TMS. Conversion to compound 34; Cleavage of N-trichloroethoxycarbonyl group of compound of formula 34 to compound 35 by reaction with Zn / AcOH; N-methylpyridiniumcarbo Conversion of the amino compound 35 to the corresponding α-ketolactone compound 36 by reaction with sulphido chloride followed by DBU; reaction of the compound of formula 36 with 3-hydroxy-4-methoxyphenylethylamine to form ET-770. Conversion of ET-770 to ET-743 by reaction with silver nitrate in a mixture of AcN / H2O.

[0101] Formation of intermediate 11 and related intermediates   Retrosynthetic analysis is described in the following series.

[Chemical 42]

In the present invention, a key class of intermediates includes intermediate 11 and has the general formula (XXI)
Have:

[Chemical 43] [Wherein Prot ¹ and Prot ² are hydroxy protecting groups and are preferably different; in the intermediate 11 itself, the Prot ¹ group is a methoxymethyl group and the Prot ² group is a t-butyldiphenylsilyl group] .

Conversion of the 21-cyano compound to intermediate 11 or related intermediates of formula (XXI) usually involves the following steps: a) If required, to the phenol system of the quinone system for ring E. B) formation of a -OProt ¹ group at the 18-position on ring E; c) formation of a -CH ₂ -OProt ² group at the 1-position on ring B; and d) on ring A, if necessary. Conversion of the quinone system to the phenol system; e) Conversion of the phenol system on ring A to the methylenedioxyphenol ring.

The formation of the —OProt ¹ group at position 18 in ring E of step (b) is a typical protecting reaction for phenol groups and need not be commented. Appropriate conditions are selected depending on the nature of the protecting group. Other steps are similar to other reactions.

Formation of a —CH ₂ —OProt ² group at the 1-position in ring B of step (c) forms a —CH ₂ NH ₂ group at the 1-position and then converts the amine functional group to a hydroxy functional group. It is usually carried out by protecting. Thus, if the starting material has a R ¹ group that is —CH ₂ —NH—CO—CR ^25a R ^25b R ^25c , it means to remove the N-acyl group. If the starting material had a R ¹ group that was —CH ₂ —O—CO—CR, then no change would be necessary for the ecteinascidin product with the same R ¹ substituent. For other products, this means removing the O-acyl group. Various methods are available for such deacylation. In one variation, deacylation and conversion to hydroxy functionality is carried out in one step. The hydroxy group can then be acylated and converted to give the otherwise applicable R ¹ group.

US Pat. No. 5,721,362 describes a synthetic method for making ET-743 through a long multi-step synthesis. One of the intermediates in this synthesis is Intermediate 11. Using cyanosafracin B as a starting material provides a much shorter method to make such intermediates to reach intermediate 11 and thus improve the method of making ET-743. Is possible.

Cyanosafracin B can be converted to intermediate 25 by the method described above. From intermediate 25 it is possible to reach intermediate 11 using the following steps of Scheme VII: Protection of formula 26 by reacting 25 with tert-butyldiphenylsilyl chloride in the presence of a base. Formation of a fluorinated hydroxy compound; the aryl group at 26 is replaced with tributyltin hydride with dichloropalladium-bis
Final cleavage with (triphenylphosphine) leads to the formation of intermediate 11. Scheme VII

[Chemical 44]

One embodiment of the synthetic method of the invention for converting safracin B to intermediate 11 is a modification and extension of Scheme VIII, which comprises the following sequential steps: reacting with KCN in acidic medium By stereoselective conversion of the compound safracin B to a compound of formula 2 by selective substitution of OH; formation of a thiourea compound of formula 3 by reacting a compound of formula 2 with phenylisothiocyanate; Conversion of the thiourea compound of formula 3 to the acetamide of formula 5 by hydrolysis in acidic medium, followed by addition of acetic anhydride; the intermediate amine compound of formula 4 is a hydrolyzate of sodium dicarbonate in acidic medium. Although it can be isolated by termination, this intermediate is very unstable and is rapidly converted to a 5-membered amine called compound 6; bromomethyl methyl ether in dichloromethane Of the quinone system of the compound of formula 7 to the compound of formula 8 by the reaction of sodium hydroxide with a methanolic solution of the compound of formula 7 by the reaction of diphenylethylamine with diisopropylethylamine. Methylation; Conversion of a compound of formula 8 to a methylenedioxy compound of formula 9 according to the following preferred sequence: (1) reducing the quinone group of compound 8 with 10% Pd / C under hydrogen atmosphere; (2 ) Converting the hydroquinone intermediate to a methylene-dioxy compound of formula 9 by reacting with bromochloromethane and cesium carbonate under hydrogen atmosphere; (3) BrCH ₂ R (wherein
R is aryl, CH = CH ₂ , OR ′, etc.) with cesium carbonate to protect the free hydroxyl group as an OCH ₂ R group to form a compound of formula 9
Conversion to the compound of formula 10; conversion of the acetamide group of the compound of formula 10 to the corresponding hydroxyl group of formula 25 by reaction with nitric oxide in a mixture of acetic acid and acetic anhydride, followed by treatment with sodium hydroxide. Alternatively, it can be achieved by using sodium nitrite in a mixture of acetic anhydride and acetic acid, followed by treatment with sodium hydroxide;
The acetamide group of the compound can be converted to a primary amine group by reacting with hydrazine, or Boc2O, DMAP, followed by hydrazine; 4-formyl-1-
With methylpyridinium benzenesulfonate or other pyridinium ion,
Oxidative conversion of a primary amine to the corresponding aldehyde, followed by treatment with DBU or other base and further hydrolysis, followed by reduction of the aldehyde to the corresponding hydroxyl group with lithium aluminum hydride or another reducing agent. Such primary amines can be converted to the corresponding hydroxyl groups by; reaction of t-butyldiphenylsilyl chloride with dimethylaminopyridine in dichloromethane to form a protected compound of formula 26 (Scheme VII). Conversion of the silylated compound of formula 26 to intermediate 11 by deprotecting the OCH ₂ R protecting group by reaction under reducing or acidic conditions. Typical methods use palladium black under a hydrogen atmosphere or in aqueous TFA, or tributyryl hydride and dichlorobis (triphenylphosphine palladium). Scheme VIII

[Chemical formula 45]

In yet another embodiment, the cyano compound of formula 2 can be converted to intermediate 11 using the extension of Scheme II, which includes the following additional steps: tert-Butyldiphenylsilyl chloride in the presence of base Formation of a protected hydroxy compound of formula 26 by reacting with 25; final cleavage of the alkyl in 26 with tributyryl hydride and dichloropalladium-bis (triphenylphosphine) leads to the formation of intermediate 11. Lead.

Thus, by these and other pathways, it is possible to convert cyanosafracin B into numerous intermediates and derivatives with potential antitumor therapeutic activity.
These intermediates can be made starting from the compounds described above or using alternative routes.

Novel Intermediate Compounds In light of the above description, it can be seen that the present invention provides novel intermediate compounds. Depending on ring A, the intermediate is of formula (XXIIa):

[Chemical formula 46] Or formula (XXIIb):

[Chemical 47] [Wherein R ¹ is —CH ₂ NH ₂ or —CH ₂ OH, or a protected or derivatized version of such a group, R ⁴ is —H; or both R ^1a and R ⁴ are Of formula (IV), (V), (VI) or (VII):

[Chemical 48] R ⁵ is —OH or a protected or derivatized version of such a group; R ^14a and R ^14b are both —H, or one is —H and the other is —OH, or such A protected or derivatized version of the group, -OCH ₃ or -OCH ₂ CH ₃ , or R ^{14 a} and R ^{14 b} together form a keto group; R ¹² is -H-, -CH ₃ -or -CH ₂ CH _3- ; R ¹⁵ is -H, -OH or a protected or derivatized version of such a group; and R ¹⁸ is -OH or a protected or derivatized version of such a group ].

In one embodiment, preferably at least one of R ¹ , R ⁵ , R ^14a , R ^14b , R ¹⁵ or R ¹⁸ is a protecting or derivatizing group.

In one variation of the invention, the R ¹ group is not a tert-butyldiphenylsilyl substituent and / or the R ¹⁸ group is not a methoxymethyl group.

Preferably R ¹ is —CH ₂ NH ₂ or —CH ₂ OH, or a protected or derivatized version of such a group, R ⁴ is —H; or R ^1a and R ⁴ are Both of the following groups:

[Chemical 49] To form.

Preferably R ^14a and R ^14b are both —H.

[0116]   One preferred class of intermediates has the formula:

[Chemical 50] Compounds identified as Compound 25 of.

This preferred class therefore has the general formula where the MOM group is replaced by any other protecting group.

Other preferred intermediates include compounds identified as compounds 45 and 43 (Scheme IX).

[Chemical 51]

Other N-acyl derivatives are readily prepared from compound 45 and are an important part of this invention. Suitable acyl groups include those mentioned above. The corresponding 21-hydroxy compounds are also useful and are among the active compounds found.

Novel Active Compounds We find that certain compounds of the invention, initially prepared as intermediates, have exceptional activity in the treatment of cancers such as leukemia, lung cancer, colon cancer, kidney cancer and melanoma. I found that further.

Thus, the present invention provides for any mammal afflicted with cancer comprising administering to a afflicted patient a therapeutically effective amount of a compound of the invention, or a pharmaceutical composition thereof,
In particular, a method of treating a human is provided.

The present invention also relates to pharmaceutical preparations, which contain a compound of the invention as active ingredient, as well as the process for their preparation.

Examples of pharmaceutical compositions have any suitable composition, either solid (tablets, pills, capsules, granules etc.) or liquid (solution, suspension) for oral, topical or parenteral administration. Or emulsions), which may include pure compounds or in combination with any carrier or pharmacologically active compound. These compositions will need to be sterile when administered parenterally.

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. 24 hours, more preferably
It is preferred to use an infusion time of 2-12 hours, most preferably 2-6 hours. A short infusion time without the need for overnight hospital treatment is particularly desirable. However, the infusion may be 12 to 24 hours, and may be longer if desired. Infusions may also be given at suitable intervals of 2 to 4 weeks. A pharmaceutical composition comprising a compound of the invention may be prepared by liposomes or nanospheres as a sustained release formulation.
Alternatively, it may be transported by other standard transportation means.

The exact dosage of compound will vary according to the particular formulation, mode of application, and particular condition, host, and tumor being treated. Other factors such as age, weight, sex, diet, time of administration, excretion rate, host status, drug combination, response sensitivity, and severity of disease will also be considered. Administration can be carried out continuously or intermittently at the maximum tolerated dose.

The compounds and compositions of this invention may also be used with other agents to provide a combined therapy. The other agents may form part of the same composition or may be provided as separate compositions that are administered at the same or different times. The identification of other agents is not specifically limited, and suitable candidates include: a) agents with anti-mitotic effects, especially those that target cytoskeletal elements, including microtubule modulating agents, such as taxan. Drugs (eg taxol, paclitaxel, taxotere, docetaxel), podophyllotoxin, or vinca alkanoid (
Vincristine, vinblastine); b) antimetabolites such as 5-fluorouracil, cytarabine, gencitarabin, purine analogues such as pentostatin, methotrexate; c) alkylating agents such as nitrogen mustards (eg cyclophosphamide or isofamide). ); D) drugs that target DNA, such as the anthracycline drugs adriamycin, doxorubicin, pharmorubicin, or epirubicin; e) drugs that target topoisomerases such as etoposide; f) hormones and hormone agonists or antagonists, such as estrogen, anti- Estrogens (tamoxifen and related compounds), androgens,
Flutamide, leuprorelin, goserelin, cyprotron, or octreotide; g) agents that target signal transduction in tumor cells, including antibody derivatives such as herceptin; h) alkylating agents, such as platinum agents (cisplatin, carboxylicplatin, oxaliplatin, Paraplatin) or nitrosoureas; i) agents that strongly influence tumor metastasis, such as matrix metalloproteinase inhibitors; j) gene and antisense agents; k) antibody therapeutics; l) other organisms of marine origin. Active compounds, in particular didemin such as Aplidine; m) steroid analogues, especially dexamethasone; n) anti-inflammatory agents, especially dexamethasone; and o) antiemetic agents, especially dexamethasone.

The present invention also extends to the compounds of the invention for use in a method of treatment and to the use of a compound in the preparation of a composition for the treatment of cancer.

Cytotoxic active cell culture: Earle's Balanced Salts, 2.0 mM L-glutamine, supplemented with non-essential amino acids, in Eagles minimal essential medium without sodium dicarbonate (EMEM / neaa), 10%
Cells were maintained in logarithmic growth phase supplemented with fetal calf serum (FCS), 10 ^-2 M sodium bicarbonate, 0.1 g / l penicillin-g + streptomycin sulfate.

A simple screening method was performed to measure and compare the anti-tumor activity of these compounds using an adaptation of the method described by Bergeron et al. (1984). The tumor cell lines used were P-388 (suspension culture of lymphoid neoplasm from DBA / 2 mouse), A-549 (monolayer culture of human lung carcinoma), HT-29 (human colon carcinoma). , And MEL-28 (human melanoma monolayer culture).

P-388 cells were seeded in 16 mm wells at 1 × 10 ⁴ cells per well in 1 ml aliquots of MEM 5FCS containing the indicated concentration of drug. Another set of cultures without drug was inoculated as a control growth to ensure that the cells were maintained in log phase. All measurements were performed in duplicate. After 3 days of incubation at 37 ° C, 10% CO ₂ in a 98% humidity environment, an approximate IC ₅₀ was determined by comparing the growth in wells with drug to the growth in control wells.

A-549, HT-29 and MEL-28 were seeded in 16 mm wells at 2 × 10 ⁴ cells per well in 1 ml aliquots of MEM 10FCS containing the indicated concentrations of drug. A separate set of cultures without drug was inoculated as a control growth to ensure that the cells were maintained in exponential growth phase. All measurements were performed in duplicate. 37 ° C in 98% humidity environment
After 3 days of incubation with 10% CO ₂ , cells were stained with 0.1% Crystal Violet. Approximate IC50s were determined by comparing growth in wells with drug to growth in control wells.

1. Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline. Robert
G. Hughes, Jr., Gary T. Elliot and Carl W. Potter. Antineoplastic and ant
iherpetic activity of spermidine catecholamide iron chelators. Biochem.
Bioph. Res. Comm. 1984, 121 (3), 848-854

2. Alan C. Schroeder, Robert G. Hughes, Jr. and Alexander Bloch. Effects
of Acyclic Pyrimidine Nucleoside Analoges. J. Med. Chem. 1981, 24 1078-1
083

[0134] Cytotoxic activity

[Table 4]

Thus, the active compounds of the present invention include compounds with a 10-hydroxy group and a 1-labyl group.

[0136]   Important methods of the invention include the following reactions:

[Chemical 52]

[0137]   Another important method of the present invention involves the following reactions:

[Chemical 53]

Another important method of the present invention involves a reaction in which the R ¹ group is an aminomethylene group and is converted to a hydroxymethylene group.

Another important method of the invention is that the R ¹ group is a hydroxymethylene group and the formula (XIX):

[Chemical 54] [Wherein Fu represents a protected functional group, Prot ³ represents a protective group, and a dotted line represents an arbitrary double bond].

[0140]   Another important method of the present invention is the formula (XV):

[Chemical 55] [Wherein R ¹ , R ⁵ , R ⁸ , R ^14a , R ^14b , R ¹⁵ and R ¹⁸ are as described, R ²¹ is a hydroxy group, and the desired 21-cyano group at the cyanide ion source is used. Yields a compound]
A reaction for the preparation of a 21-cyano compound of formula (XVI) comprising reacting a compound of

Further, methods of using another nucleophilic atom containing compound to produce a similar compound of formula (XVI) protected at the 21-position by another nucleophilic group, a 21-Nuc group, are also contemplated. Good. For example, a 21-Nuc compound of formula (XVI) having an alkylamino substituent at the 21-position is
It can be produced by reacting a compound of formula (XV) in which R ²¹ is a hydroxy group with an appropriate alkylamide. Alternatively, a 21-Nuc compound of formula (XVI) having a carbonylalkyl substituent at the 21-position is a compound of formula (XV) wherein R21 is a hydroxyl group,
It can be produced by reacting with a suitable carbonyl compound, typically in the presence of a base. Other synthetic routes are available for other 21-Nuc compounds.

Another important reaction of the present invention is the reaction of the present invention to form a 21-hydroxy compound.
Includes treatment of 1-cyano products. Such compounds have interesting in vivo properties.

[0143]

【Example】

  The invention is illustrated by the following examples.

[0144] Example 1

[Chemical 56] To a solution of 2 (21.53g, 39.17mmol) in ethanol (200ml) was added tert-butoxycarbonylanhydride (7.7g, 35.25mmol) and the mixture was stirred at 23 ° C for 7 hours. The reaction was then concentrated in vacuo, the residue was purified by flash column chromatography (SiO _2, hexane: ethyl acetate 6: 4) to give, as a yellow solid 14 (20
.6g, 81%).

[Equation 1]

[0145] Example 2

[Chemical 57] CH ₃ CN (159ml) solution of 14 (20.6g, 31.75mmol) To a stirred solution of diisopropylethylamine (82.96ml, 476.2mmol), methoxymethylene bromide (25.9 ml, 317.5
mmol) and dimethylaminopyridine (155 mg, 1.27 mmol) were added at 0 ° C. The mixture
The mixture was stirred at 23 ° C for 24 hours. The reaction was quenched with aqueous 0.1 N HCl (750 ml) (pH = 5) at 0 ° C. and extracted with CH ₂ Cl ₂ (2 × 400 ml). The organic layer was dried (sodium sulfate) and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , gradient hexane: ethyl acetate 4: 1 to hexane: ethyl acetate 3: 2) to give 15 (17.6 g, 83%) as a yellow solid.

[Equation 2]

[0146] Example 3

[Chemical 58] To a flask containing 15 (8 g, 1.5 ml) in methanol (1.6 l) was added 1M aqueous sodium hydroxide solution (3.2 l) at 0 ° C. The reaction was stirred at this temperature for 2 hours then 6MH.
Stopped with Cl to pH = 5. The mixture was extracted with ethyl acetate (3 x 11), the combined organic layers were dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , CHCl ₃ to CHCl ₃ : ethyl acetate 2: 1 gradient) to give 16 (5.3 mg, 68%).

[Equation 3]

[0147] Example 4

[Chemical 59] To a degassed solution of Compound 16 (1.8 g, 2.64 mmol) in DMF (221 ml), 10% Pd / C (360 m
g) was added and stirred under H ₂ (atmospheric pressure) for 45 minutes. The reaction product was treated with anhydrous Cs ₂ CO ₃ (2.5
To a flask containing 8 g, 7.92 mmol) was filtered through Celite under argon.
Bromochloromethane (3.40 ml, 52.8 mmol) was then added, the tube was sealed and 2
Stir at 100 ° C. for hours. The reaction was cooled, filtered through a pad of Celite and washed with CH ₂ Cl ₂ . The organic layer was concentrated and dried (sodium sulfate) to give a brown oil 17
Which was used in the next step without further purification.

[Equation 4]

[0148] Example 5

[Chemical 60] To a flask containing a solution of 17 (1.83 g, 2.65 mmol) in DMF (13 ml) was added Cs2CO3 (2
.6 g, 7.97 mmol) and alkyl bromide (1.15 ml, 13.28 mmol) were added at 0 ° C. The resulting mixture was stirred at 23 ° C for 1 hour. The reaction was filtered through a pad of Celite, washing with CH ₂ Cl ₂ . The organic layer was dried and dried (sodium sulfate). The residue was purified by flash column chromatography (SiO ₂ , CHCl ₃ : ethyl acetate 1: 4) to give 18 (1.08 mg, 56%) as a white solid.

[Equation 5]

[0149] Example 6

[Chemical formula 61] To a solution of 18 (0.1 g, 0.137 mmol) in dioxane (2 ml) was added 4.2M HCl / dioxane (1.46 ml) and the mixture was stirred at 23 ° C for 1.2 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (60 ml) at 0 ° C. and extracted with ethyl acetate (2 × 70 ml). The organic layer was dried (sodium sulfate) and concentrated in vacuo to give 19 (267 mg, 95%) as a white solid.
%), Which was used in subsequent reactions without further purification.

[Equation 6]

[0150] Example 7

[Chemical formula 62] To a solution of 19 (250 mg, 0.42 mmol) in CH ₂ Cl ₂ (1.5 mmol) was added phenylisothiocyanate (0.3 ml, 2.51 mmol) and the mixture was stirred for 1 h at 23 ° C. The reaction was vacuumed. Concentrate under and purify the residue by flash column chromatography (SiO ₂ , gradient hexane to hexane: ethyl acetate 5: 1) to give 20 as a white solid.
(270 mg, 87%) was obtained.

[Equation 7]

[0151] Example 8

[Chemical formula 63] To a solution of 20 (270 mg, 0.37 mmol) in dioxane (1 ml) was added 4.2N HCl / dioxane (3.5 ml) and the reaction was stirred at 23 ° C for 30 minutes. Then ethyl acetate (20 ml)
And H ₂ O (20 ml) were added and the organic layer was discarded. The aqueous phase was saturated with aqueous sodium bicarbonate (60 ml).
) (pH = 8) at 0 ° C. and then extracted with CH ₂ Cl ₂ (2 × 50 ml). The combined organic extracts were dried (sodium sulfate) and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , ethyl acetate: methanol 5: 1) to give compound 21 (158 mg, 82%) as a white solid.

[Equation 8]

[0152] Example 9

[Chemical 64] For a solution of 21 (0.64 g, 1.22 mmol) in CH ₂ Cl ₂ (6.13 ml), pyridine (0.104 ml,
1.28 mmol) and 2,2,2-trichloroethyl chloroformate (0.177 ml, 1.28 mmol)
Added at 10 ° C. The mixture was stirred at this temperature for 1 hour, then the reaction was run to 0.1N HCl (10 ml).
It was stopped by addition of and extracted with CH ₂ Cl ₂ (2 × 10 ml). The organic layer was dried over sodium sulfate and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , hexane: ethyl acetate 2: 1) to give 22 (0.84) as a white foamy solid.
g, 98%).

[Equation 9]

[0153] Example 10

[Chemical 65] CH ₃ CN (2.33ml) in 22 (0.32 g, 0.46 mmol) to a solution of diisopropylethylamine (1.62 ml, 9.34 mmol), bromomethyl ether (0.57 ml, 7.0 mmol), and dimethylaminopyridine (6 mg , 0.046 mmol) was added at 0 ° C. The mixture was heated at 30 ° C. for 10 hours. The reaction was then diluted with dichloromethane (30 ml) and HCl at pH = 5 (10 ml).
Of water. The organic layer was dried over sodium sulphate and the solvent removed under reduced pressure to give a residue which was purified by flash column chromatography (SiO ₂ , hexane: ethyl acetate 2: 1) to give 23 as a white foamy solid. (0.304g, 88%) was obtained.

[Equation 10]

[0154] Example 11

[Chemical formula 66] To a suspension of 23 (0.304 g, 0.41 mmol) in 90% aqueous acetic acid (4 ml) was added powdered zinc (0.2
g, 6.17 mmol) was added and the reaction was stirred at 23 ° C. for 7 hours. The mixture was filtered through a pad of Celite, which was washed with CH ₂ Cl _2. The organic layer was washed with an aqueous saturated solution of sodium dicarbonate (pH = 9) (15 ml) and dried over sodium sulfate. The solvent was removed under reduced pressure to give 24 (0.191 g, 83%) as a white solid.

[Equation 11]

[0155] Example 12

[Chemical formula 67] H ₂ O (0.7mmol) and 24 (20mg, 0.035mmol) in THF (0.7 mmol) to a solution of, NaNO2
(12 mg, 0.17 mmol) and 90% aqueous AcOH (0.06 ml) were added at 0 ° C. and the mixture was stirred at 0 ° C. for 3 hours. After diluting with CH ₂ Cl ₂ (5 ml), the organic layer was dried over water (1 ml) decene boss, sodium sulfate, and concentrated in vacuo. The residue was purified by flash column chromatography (SiO ₂ , hexane: ethyl acetate 2: 1) to give 25 (9.8 m as a white solid.
g, 50%) was obtained.

[Equation 12]

[0156] Example 13

[Chemical 68] The starting material (2.0 g, 5.90 mmol) was added to sodium hydroxide (354 mg,
8.86 mmol) and then the suspension at 23 ° C. with allyl chloroformate (1.
.135 ml, 8.25 mmol) and then refluxed for 3 hours. The suspension was cooled, filtered, the solid washed with ethyl acetate (100 ml) and the filtrate concentrated. The oily crude was spread with hexane (100 ml) and kept at 4 ° C. overnight. Then the solvent was discarded and the light yellow slurry was treated with CH ₂ Cl ₂ (20 ml) and precipitated with hexane (100 ml). After 10 minutes, the solvent was discarded again. The operation was repeated until a white solid appeared. The white solid was filtered and dried to give compound 29 (1.80 g, 65%) as a white solid.

[Equation 13]

[0157] Example 14

[Chemical 69] A mixture of compound 25 (585 mg, 1.03 mmol) and compound 29 (1.47 mg, 3.11 mmol) was azeotroped with anhydrous toluene (3 x 10 ml). For a solution of 25 and 29 in anhydrous CH ₂ Cl ₂ (40 ml), 2
DMAP (633 mg, 5.18 mmol) and EDC.HCl (994 mg, 5.18 mmol) were added in 3 times. The reaction mixture was stirred at 23 ° C. for 3 hours. The mixture was partitioned with a saturated aqueous solution of sodium dicarbonate (50 ml) and the layers separated. The aqueous phase was washed with CH ₂ Cl ₂ (50 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The crude was purified by flash column chromatography (ethyl acetate / hexane 1: 3) to give 30 (1.00 g, 95%) as a pearly cream yellow solid.

[Equation 14]

[0158] Example 15

[Chemical 70] 30 (845 mg, 0.82 mmol) to a solution of acetic acid in anhydrous _{CH 2 Cl 2 (20ml) (} 500mg, 8.2
8 mmol) and (PPh ₃ ) ₂ PdCl ₂ (29 mg, 0.04 mmol) at 23 ° C. and added dropwise to Bu3SnH (650 mg,
2.23 mmol) was added. The reaction mixture was stirred at this temperature for 15 minutes, causing bubbling.
The crude was quenched with water (50 ml) and extracted with CH ₂ Cl ₂ (3 x 50 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. Flash column chromatography (
Purification by ethyl acetate / hexanes (gradient 1: 5 to 1: 3) gave compound 31 (730 mg, 90%) as a pearly cream yellow solid.

[Equation 15]

[0159] Example 16

[Chemical 71] For a solution of 31 (310 mg, 0.32 mmol) in anhydrous CH ₂ Cl ₂ (15 ml) at −10 ° C., 70% benzeneselenic hydride in anhydrous CH ₂ Cl ₂ (7 ml) via cannula ( 165 mg,
0.32 mmol) solution was added and the temperature was maintained at -10 ° C. The reaction mixture was stirred at -10 ° C for 5 minutes. A saturated solution of sodium bicarbonate (30 ml) was added at this temperature. The aqueous phase was washed with additional CH ₂ Cl ₂ (40 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. Flash column chromatography of the crude product (1: 5 with ethyl acetate / hexane).
To a 1: 1 gradient) to give 32 (287 mg, 91%, HPLC: 91.3%) as a pearly cream-yellow solid and as a mixture of two isomers (65:35), which was Used in the process.

[Equation 16]

[0160] Example 17

[Chemical 72] The reaction flask was heat treated twice, vacuum / argon purged several times and maintained in an argon atmosphere for the reaction. DMSO in anhydrous CH ₂ Cl ₂ (4.5 ml) (39.1 ml, 0.55 mmol, 5
Solution of triflic hydride (37.3 ml, 0.22 mmol, 2
Equivalent) was added dropwise. The reaction mixture was stirred at -78 ° C for 20 minutes, then at -78 ° C anhydrous C
32 (110 mg, 0.11 mmol, HPL in H ₂ Cl ₂ (1 ml for main addition, and 0.5 ml for washing))
C: 91.3%) was added via cannula. During the addition, the temperature was -7 in both flasks.
The temperature was maintained at 8 ° C and the color changed from yellow to brown. The reaction mixture was stirred at -40 ° C for 35 minutes. During this period, the solution went from yellow to dark green. After this, ⁱ Pr ₂ NEt (153ml, 0
(.88 mmol, 8 eq) was added dropwise and the reaction mixture was kept at 0 ° C. for 45 min, during which time the color of the solution turned brown. Then t-butanol (41.6 ml, 0.44 mmol, 4 eq) and 2
- ^{t-butyl-1,1,3,3-tetramethylguanidine} (132.8ml, 0.77mmol, 7 eq) was added dropwise and the reaction mixture was stirred for 40 minutes at 23 ° C.. After this, acetic anhydride (104.3 ml, 1.
10 mmol, 10 eq) was added dropwise and the reaction mixture was kept at 23 ° C. for another hour.
The reaction mixture was then diluted with CH ₂ Cl ₂ (20 ml) and washed with an aqueous saturated solution of NH ₄ Cl (50 ml), sodium dicarbonate (50 ml) and sodium chloride (50 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (eluent: gradient 1: 3 to 1: 2 with ethyl acetate / hexane) to give compound 33 (54 mg, 58%) as a pearly yellow solid.

[Equation 17]

[0161] Example 18

[Chemical formula 73] 33 (1) in anhydrous dichloromethane (1.2 ml) and HPLC grade acetonitrile (1.2 ml).
2 mg, 0.014 mmol) solution at 23 ° C with sodium iodide (21 mg, 0.14 mmol) and freshly distilled (at atmospheric pressure with calcium hydroxide) trimethylsilyl chloride (1
5.4 mg, 0.14 mmol) was added. The reaction mixture turned orange. After 15 minutes, the solution was diluted with dichloromethane (10 ml) and washed with fresh saturated aqueous solution of Na ₂ S ₂ O ₄ (3 × 10 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. Compound 34 (13 mg, quantitative) was obtained as a pearlescent yellow solid and was used without further purification.

[Equation 18]

[0162] Example 19

[Chemical 74] For a solution of 34 (13 mg, 0.016 mmol) in a mixture of acetic acid / H ₂ O (90:10, 1 ml), 23
Powdered zinc (5.3 mg, 0.08 mmol) was added at ℃. The reaction mixture was heated at 70 ° C. for 6 hours. After this time, it was cooled to 23 ° C., diluted with CH ₂ Cl ₂ (20 ml) and washed with an aqueous saturated solution of sodium dicarbonate (15 ml) and an aqueous solution of Et ₃ N (15 ml). The organic layer was dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography on silica-NH ₂ (eluent: ethyl acetate / hexane gradient 0: 100 to 50:50),
Compound 35 (6.8 mg, 77% for two steps) was obtained as a pearly yellow solid.

[Formula 19]

[0163] Example 20

[Chemical 75] N-Methylpyridine-4-carboxaldehyde iodide (378 m in anhydrous DMF (5.8 mL)
(g, 1.5 mmol) was treated with anhydrous toluene and the amount of water was removed by azeotropic removal with toluene. A solution of 35 (134 mg, 0.21 mmol) pre-treated with anhydrous toluene (210 mL) in anhydrous CH ₂ Cl ₂ (CaH ₂ , distilled with 7.2 mL) was cannulated to this orange solution at 23 ° C. Added in. The reaction mixture was stirred at 23 ° C for 4 hours. After this, DBU (32
.2 mL, 0.21 mmol) was added dropwise at 23 ° C. and it was stirred at 23 ° C. for 15 minutes. Oxalic acid
(5.8 mL) fresh saturated aqueous solution was added to the reaction mixture and stirred at 23 ° C. for 30 minutes. The reaction mixture was then cooled to 0 ° C. and NaHCO ₃ was added in portions, followed by an aqueous saturated solution of NaHCO ₃ . The mixture was extracted with Et ₂ O. Add K ₂ CO ₃ to the aqueous phase and add it to Et ₂ O.
It was extracted with. The combined organic layers were dried over MgSO _4, the solvent was removed under reduced pressure. The crude was purified by flash column chromatography (AcOEt / hexane gradient 1/3 to 1/1) to give compound 36 (77 mg, 57%) as a pearly yellow solid.

[Equation 20]

[0164] Example 21

[Chemical 76] To a solution of 36 (49 mg, 0.08 mmol) and 2- (3-hydroxy-4-methoxyphenyl) ethylamine (46.2 mg, 0.27 mmol) in ethanol (2.5 ml) was added silica gel at 23 ° C. The reaction mixture was stirred at 23 ° C. for 14 hours. It was diluted with hexane and poured onto a chromatographic column (ethyl acetate / hexane 1/3 to 1/1 gradient) to afford Et-770 (55 mg, 90%) as a pearlescent yellow solid.

[Equation 21]

[0165] Example 22

[Chemical 77] To a solution of 21 (22 mg, 0.042 mmol) in CH ₂ Cl ₂ (0.8 ml) was added phthalic anhydride (6.4
4 mg, 0.042 mmol) was added and the reaction mixture was stirred at 23 ° C. for 2 hours. Carbonyldiimidazole (1 mg, 0.006 mmol) was then added and the mixture was stirred at 23 ° C for 7 hours. Carbonyldiimidazole (5.86 mg, 0.035 ml) was then added and the reaction was stirred at 23 ° C. for a further 17 hours. The solution was diluted with CH ₂ Cl ₂ (15 ml) and washed with 0.1N HCl (15 ml).
The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , hexane / ethyl acetate 2: 1) to give 27 (26.4 mg, 96%) as a white solid.

[Equation 22]

[0166] Example 23

[Chemical 78] CH ₂ Cl ₂ (11ml) solution of 27 (26 mg, 0.041 mmol) to a solution of acetic acid (11 ml), the _{(PPh 3) 2 PdCl 2 (} 2.36ng), and Bu ₃ SnH (28ml, 0.10mmol) Added at 23 ° C. After stirring at this temperature for 2 hours, the reaction was poured onto a pad of a flash column (SiO ₂ , hexane to hexane: ethyl acetate 2: 1 gradient) to give 28 (24.7 mg, 99%) as a white solid. .

[Equation 23]

[0167] Example 24

[Chemical 79] For a solution of 28 (357 mg, 0.058 mmol) in CH ₂ Cl ₂ , acetyl chloride (41.58 ml
, 0.58 mmol) and pyridine (47.3 ml, 0.58 ml) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (15 ml) and washed with 0.1N HCl (15 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography _{(RP-18, CH 3 CN} : H 2 O60: 40) to give the Futarashijin (354 mg, 94%) as a white solid.

[Equation 24]

[0168] Example 25

[Chemical 80] For a solution of 17 (300 mg, 0.432 mmol) in CH ₂ Cl ₂ (2 ml), acetyl chloride (30
0.7 ml, 0.432 mmol) and pyridine (34.9 ml, 0.432 mmol) were added at 0 ° C. The reaction mixture was stirred at this temperature for 2 h, then the solution was diluted with CH ₂ Cl ₂ (15 ml) and 0.1 N HCl (15 m
It was washed with l). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure to give 42 (318 mg, 100%) as a white solid, which was used in subsequent reactions without further purification.

[Equation 25]

[0169] Example 26

[Chemical 81] To a solution of 42 (318 mg, 0.432 mmol) in CH ₂ Cl ₂ (2.16 mmol) was added trifluoroacetic acid (1.33 ml, 17.30 mmol) and the reaction mixture was stirred at 23 ° C for 3.5 hours. The reaction was quenched with saturated aqueous sodium bicarbonate (60 ml) and CH ₂ Cl ₂ (2 × 70 ml). The combined organic layers were dried (sodium sulfate) and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , ethyl acetate: methanol 20: 1) to give 43 (154 mg, 60%) as a white solid.

[Equation 26]

[0170] Example 27

[Chemical formula 82] To a solution of 43 (154 mg, 0.26 mmol) in CH ₂ Cl ₂ (1.3 ml) was added phenylisothiocyanate (186 ml, 1.56 mmol) and the mixture was stirred at 23 ° C for 2 hours. The reaction was concentrated under vacuum and the residue was purified by flash column chromatography (SiO ₂ , hexane to hexane / ethyl acetate 1: 1 gradient) to afford 44 (1
20 mg, 63%) was obtained.

[Equation 27]

[0171] Example 28

[Chemical 83] To a solution of 44 (120 mg, 0.165 mmol) in dioxane (0.9 ml) was added 5.3N HCl / dioxane (1.8 ml) and the reaction was stirred at 23 ° C. for 2.5 hours. Then CH ₂ Cl ₂ (10 ml)
H ₂ O (5 ml) was added to the reaction and the organic layer was discarded. The aqueous phase was basified with saturated aqueous sodium bicarbonate (20 ml) (pH = 8) at 0 ° C. then extracted with CH ₂ Cl ₂ (2 × 15 ml). The combined organic layers were dried (sodium sulfate) and concentrated under vacuum to give 45 (75 mg, 87%) as a white solid, which was used in the next reaction without further purification.

[Equation 28]

[0172] Example 29

[Chemical 84] To a solution of 45 (10 mg, 0.02 mmol) in CH ₂ Cl ₂ (0.4 ml) was added phthalic anhydride (2.84
mg, 0.02 mmol) was added and the reaction mixture was stirred at 23 ° C. for 2 hours. Carbonyldiimidazole (0.5 mg, 0.003 mmol) was then added and the mixture was stirred at 23 ° C for 7 hours. Carbonyldiimidazole (2.61 mg, 0.016 mmol) was then added and the reaction was stirred at 23 ° C for an additional 7 hours. The solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography _{(RP-18, CH 3 CN} : H 2 O60: 40) to afford Futarashijin (11.7 mg, 93%) as a white solid.

[Equation 29]

[0173] Example 30

[Chemical 85] For a solution of 25 (18 mg, 0.032 mmol) in DMF (0.05 ml), cat.DMAP (0.5 mg, 0.0
04 mmol), imidazole (5 mg, 0.08 mmol) and tert-butyldiphenylsilyl chloride (12.5 ml, 0.048 mmol) were added at 0 ° C., and the reaction mixture was stirred at 23 ° C. for 6 hours.
(10 ml) was added at 0 ° C. and the aqueous layer was extracted with hexane: ethyl acetate 1:10 (2 × 10 mmol). The organic layer was dried (sodium sulfate), filtered and the solvent removed under reduced pressure. The crude was purified by flash column chromatography (SiO ₂ , hexane: ethyl acetate 3: 1) to give 26 (27 mg, 88%) as a white solid.

[Equation 30]

[0174] Example 31

[Chemical 86] To a solution of 26 (7 mg, 0.0087 mmol) in CH ₂ Cl ₂ (0.15 ml) was added acetic acid (2.5 ml, 0.044 ml).
mmol), (PPh ₃ ) ₂ PdCl ₂ (0.5 mg, 6.96 × 10 ^-4 mmol) and Bu ₃ SnH (3.5 ml, 0.013 mmol) 23
Added at ° C. The reaction mixture was stirred at this temperature for 1 hour. The solution was diluted with a mixture of hexane: ethyl acetate 5: 1 and flash column (SiO ₂ , 5: hexane: ethyl acetate 5:
Poured onto a (1 to 1: 1 gradient) pad to give ET-11 (5 mg, 75%) as a white solid.

[Equation 31]

[0175] Example 32

[Chemical 87] 2 (3.0 g, 5.46 mmol) in CH ₂ Cl ₂ (27 ml) and phenylisothiocyanate (3.92 mL,
A solution of 32.76 mmol) was stirred at 23 ° C. for 1.5 hours. The reaction mixture was added CH ₂ Cl ₂ (10 ml) and H ₂ O.
It fractionated between (5 ml). The organic layer was dried over sodium sulfate, filtered, and concentrated.
Flash column chromatography of the residue (SiO ₂ , hexane to hexane:
Purification by gradient of ethyl acetate (2: 3) gave 3 (3.29 g, 88%) as a yellow solid.

[Equation 32]

[0176] Example 33

[Chemical 88] A solution of 3 (0.143g, 0.208mmol) in 6.5M HCl / dioxane (150ml) was stirred at 23 ° C for 6 hours. Toluene (3 ml) was then added to this solution and the organic layer was discarded. The residue was partitioned between saturated aqueous sodium bicarbonate (3 ml) and CHCl _{3 (3} × 3ml). The organic layer was dried and concentrated to give the title compound as a mixture of 4 and 6 (4: 6 90:10), which was slowly cyclized to give the desired 6.

[Expression 33]

[0177] Example 34

[Chemical 89] A solution of 3 (0.143g, 0.208mmol) in 6.5M HCl / dioxane (150ml) was stirred at 23 ° C for 1 hour. Evaporation of 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.

[Equation 34]

[0178] Example 35

[Chemical 90] For a solution of 3 (2.38 g, 3.47 mmol) in dioxane (5 ml), dioxane (34 ml)
5.3M HCl in was added and the reaction was stirred at 23 ° C. for 45 minutes. Then Ac ₂ O (51 ml, 539.5 mm
ol) was added and the mixture was stirred for 4 hours. The reaction was cooled to 0 ° C. and saturated aqueous Na ₂ CO ₃ (
Partitioned between 300 ml) and ethyl acetate (300 ml) at this temperature. The organic layer was dried over sodium sulfate, filtered and concentrated. Flash column chromatography of the residue
Purification by (SiO ₂ , CH ₂ Cl ₂ to CH ₂ Cl ₂ : ethyl acetate 1: 2 gradient) gave 5 (1.75 g, 97%) as a yellow solid.

[Equation 35]

[0179] Example 36

[Chemical Formula 91] CH ₂ Cl ₂ (17ml) solution of 5 (1.75 g, 3.36 mmol) to a solution of diisopropylethylamine (11.71ml, 67.23mmol), DMAP ( 20mg, 0.17mmol) and bromomethyl methyl ether (4.11 mL, 50.42 mmol) at 0 ° C. After 6 hours at 23 ° C., the reaction was converted to CH ₂ Cl ₂ (
Partitioned between 50 ml) and saturated aqueous sodium bicarbonate (25 ml). The organic layer was dried over sodium sulfate and the solvent was removed under reduced pressure. The crude product was purified by flash column chromatography _{(RP-18, CH 3 CN} / H 2 O1 / 1), 7 as a yellow solid (1.32g, 70%)
Got

[Equation 36]

[0180] Example 37

[Chemical Formula 92] To a solution of 7 (0.37g, 0.65mmol) in methanol (74ml) at 0 ° C was added IM sodium hydroxide (130ml). The reaction was stirred for 15 minutes then quenched with 6M HCl at 0 ° C. to pH = 5. The mixture was extracted with ethyl acetate (3 x 50 ml) and the combined organic layers were dried over sodium sulfate and concentrated under vacuum. The remainder was purified by flash column chromatography and purified by _{(RP-C18, CH 3 CN} H 2 O1 / 1), 8 (232mg as a yellow solid,
65%).

[Equation 37]

[0181] Example 38

[Chemical formula 93] For a degassed solution of compound 8 (240 mg, 0.435 mmol) in DMF (30 ml), 10% Pd / C (48 m
g) was added and the reaction was stirred under hydrogen (atmospheric pressure) for 1 hour. The reaction was filtered through a pad of Celite into a Schlenk tube under argon to remove anhydrous Cs ₂ CO ₃ (240 mg, 0.739 mmol).
) Was obtained as a colorless solution. Then bromochloromethane (0.566ml, 8.71mmol)
Was added. The tube was sealed and stirred at 90 ° C for 3 hours. The reaction was cooled, filtered through Celite and washed with CH ₂ Cl ₂ . The organic layer was concentrated and dried (sodium sulfate),
9 was obtained as a brown oil which was used in the next step without further purification.

[Equation 38]

[0182] Example 39

[Chemical 94] To a flask containing 9 (245 mg, 0.435 mmol) in DMF (4 ml) was added cesium carbonate (42
5 mg, 1.30 mmol) and allyl bromide (376 ml, 4.35 mmol) were added at 0 ° C. and the mixture was added to 23
The mixture was stirred at 0 ° C for 1 hour. The reaction was filtered through a pad of Celite, CH ₂ Cl ₂ (25 ml) and H ₂ O (
It was fractionated between 10 ml). The organic layer is dried (sodium sulfate) and concentrated under reduced pressure,
Residue by flash column chromatography (SiO _2, CHCl3: ethyl acetate 1: 2) to afford 10 (113 mg, 43%) as a yellow oil.

[Formula 39]

[0183] Example 40

[Chemical 95] To a solution of 9 (22 mg, 0.039 mmol) in CH ₂ Cl ₂ (0.2 ml) was added acetyl chloride (2.
79 ml, 0.039 mmol) and pyridine (3.2 ml, 0.039 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure to give 46 (22 mg, 93%) as a white solid.

[Formula 40]

[0184] Example 41

[Chemical 96] To a solution of 46 (8 mg, 0.013 mmol) in dioxane (0.1 ml) was added 5.3N HCl / dioxane (0.5 ml) and the reaction was stirred at 23 ° C. for 1 hour. The solution was then CH ₂ Cl ₂ (5 ml)
It was diluted with and washed with 0.1 N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure to give 47 (5 mg, 70%) as a white solid.

[Formula 41]

[0185] Example 42

[Chemical 97] To a solution of 45 (10 mg, 0.0192 mmol) in CH ₂ Cl ₂ (0.3 ml) wasovaleryl chloride (2.34 ml, 0.0192 mmol) and pyridine (1.55 ml, 0.0192 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (5 ml) and washed with 0.1N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO ₂ , hexane: ethyl acetate 1
: 2) to give 48 (11 mg, 95%) as a white solid.

[Equation 42]

[0186] Example 43

[Chemical 98] To a solution of 45 (10 mg, 0.0192 mmol) in CH ₂ Cl ₂ (0.3 ml) wasovaleryl chloride (3.98 ml, 0.0192 mmol) and pyridine (1.55 ml, 0.0192 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (5 ml) and washed with 0.1N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO ₂ , hexane: ethyl acetate 1
: 2) to give 49 (12.4 mg, 96%) as a white solid.

[Equation 43]

[0187] Example 44

[Chemical 99] To a solution of 45 (14.5 mg, 0.0278 mmol) in CH ₂ Cl ₂ (0.3 ml) trans-3-trifluoromethylcinnamoyl chloride (4.76 ml, 0.0278 mmol) and pyridine (2.25 ml, 0
0.0278 mmol) was added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was added to CH ₂ Cl ₂ (5
ml) and washed with 0.1 N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. Flash column chromatography of the residue
Purified by (SiO ₂ , hexane: ethyl acetate 1: 1) as a white solid 50 (18.7 mg
, 94%).

[Equation 44]

[0188] Example 45

[Chemical 100] To a solution of 43 (33 mg, 0.0557 mmol) in CH ₂ Cl ₂ (0.4 ml) wasovaleryl chloride (6.79 ml, 0.0557 mmol) and pyridine (4.5 ml, 0.0557 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (5 ml) and washed with 0.1N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO ₂ , hexane: ethyl acetate 1
: 2) to give 51 as a white solid (34 mg, 91%).

[Equation 45]

[0189] Example 46

[Chemical 101] For a solution of 43 (33 mg, 0.0557 mmol) in CH ₂ Cl ₂ (0.4 ml) trans-3-trifluoromethylcinnamoyl chloride (9.52 ml, 0.0557 mmol) and pyridine (4.5 ml, 0.05
57 mmol) was added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was added to CH ₂ Cl ₂ (5 ml).
It was diluted with and washed with 0.1 N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. Flash column chromatography (S
iO ₂ , hexane: ethyl acetate 1: 2) and purified as a white solid 52 (40 mg, 92
%).

[Equation 46]

[0190] Example 47

[Chemical 102] To a solution of 43 (10 mg, 0.0169 mmol) in CH ₂ Cl ₂ (0.2 ml) was added trifluoroacetic anhydride (2.38 μl, 0.0169 mmol) at 23 ° C. The reaction mixture was stirred for 5 hours, then the solution was diluted with CH ₂ Cl ₂ (5 ml) and washed with 0.1N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , hexane: ethyl acetate 3: 2) to give 53 (10.7 mg, 93%) as a white solid.

[Equation 47]

[0191] Example 48

[Chemical 103] To a solution of 19 (11 mg, 0.0169 mmol) in CH ₂ Cl ₂ (0.2 ml) was added trifluoroacetic anhydride (2.38 ml, 0.0169 mmol) at 23 ° C. The reaction mixture was stirred for 5 hours, then the solution was diluted with CH ₂ Cl ₂ (5 ml) and washed with 0.1N HCl (3 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO _2, hexane: ethyl acetate 3: 2) to give the 54 as a white solid (10.7 mg, 93%).

[Equation 48]

[0192] Example 49

[Chemical 104] CH ₂ Cl ₂ (4ml) in 54 (100 mg, 0.415 mmol) to a solution of acetic acid _{(40ml), (PPh 3)} 2 PdCl 2 (8.4mg, 0.012mmol) and Bu ₃ SnH (157ml, 0.56mmol ) Was added at 23 ° C. After stirring at this temperature for 2 hours, the reaction was poured onto a pad of a flash column (SiO ₂ , hexane to hexane: ethyl acetate 2: 1 gradient) to afford 55 (90 mg, 96%) as a white solid.

[Equation 49]

[0193] Example 50

[Chemical 105] To a solution of 17 (200 mg, 0.288 mmol) in CH ₂ Cl ₂ (1.44 ml) was added trifluoroacetic acid.
(888 ml, 11.53 mmol) was added and the reaction mixture was stirred at 23 ° C. for 4 hours. The reaction was quenched at 0 ° C. with saturated aqueous sodium bicarbonate (60 ml) and extracted with ethyl acetate (2 × 70 ml).
The combined organic layers were dried (sodium sulfate) and concentrated in vacuo to give 56 (147 mg, 93%) as a white solid, which was used in the subsequent reaction without further purification.

[Equation 50]

[0194] Example 51

[Chemical formula 106] Phenylisothiocyanate (13 ml, 0.109 mmol) was added to a solution of 56 (10 mg, 0.018 mmol) in CH ₂ Cl ₂ (0.4 ml) and the reaction was stirred at 23 ° C. for 1.5 hours. The mixture was concentrated in vacuo, the residue was purified by flash column chromatography (hexane SiO _2, hexane: ethyl acetate gradient 1.1) to give as a white solid 57 (8
mg, 65%) was obtained.

[Equation 51]

[0195] Example 52

[Chemical formula 107] To a solution of 57 (45 mg, 0.065 mmol) in CH ₂ Cl ₂ (0.5 ml) was added acetyl chloride (4
.67 ml, 0.065 mmol) and pyridine (5.3 ml, 0.065 mmol) were added at 0 ° C. The reaction mixture was stirred for 3 hours, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography _{(RP-18, CH 3 CN} : H 2 O40: 60) to afford a white solid 58 (14mg, 28%).

[Equation 52]

[0196] Example 53

[Chemical 108] To a solution of 57 (130 mg, 0.189 mmol) in dioxane (1 ml) was added 5.3N HCl / dioxane (1.87 ml) and the reaction was stirred at 23 ° C. for 4 hours. CH ₂ Cl ₂ (15 ml) and H ₂ O (10 ml) were then added to this solution and the organic layer was discarded. The aqueous phase was basified at 0 ° C. with saturated aqueous sodium bicarbonate (60 ml) (pH = 8) then extracted with ethyl acetate (2 × 50 ml). The combined organic extracts were dried (sodium sulfate) and concentrated in vacuo to give 59 (63 mg, 70%) as a white solid.

[Equation 53]

[0197] Example 54

[Chemical 109] To a solution of 43 (20 mg, 0.0338 mmol) in CH ₂ Cl ₂ (0.3 ml) was added cinnamoyl chloride (5.63 ml, 0.0338 mmol) and pyridine (2.73 ml, 0.0338 mmol) at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and 0.1 N HCl (5 ml).
Washed with. The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 20: 1) to give 60 (22 mg, 90%) as a white solid.

[Equation 54]

[0198] Example 55

[Chemical 110] To a solution of 45 (19 mg, 0.0364 mmol) in CH ₂ Cl ₂ (0.3 ml) was added heptafluorobutyryl chloride (5.44 ml, 0.0364 mmol) and pyridine (2.95 ml, 0.0364 mmol) at 0 ° C. . The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N
Wash with HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO ₂ , EtOAc: MeO
Purification by H20: 1) gave 61 (11.7 mg, 45%) as a white solid.

[Equation 55]

[0199] Example 56

[Chemical 111] For a solution of 43 (24 mg, 0.04 mmol) in CH ₂ Cl ₂ (0.3 ml), butyryl chloride (4.
15 ml, 0.04 mmol) and pyridine (3.28 ml, 0.04 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 20: 1) to give 62 (24 mg, 90%) as a white solid.

[Equation 56]

[0200] Example 57

[Chemical 112] To a solution of 43 (19 mg, 0.0364 mmol) in CH ₂ Cl ₂ (0.3 ml) was added cinnamoyl chloride (6.06 ml, 0.0364 mmol) and pyridine (2.95 ml, 0.0364 mmol) at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and 0.1 N HCl (5 ml).
Washed with. The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 20: 1) to give 63 as a white solid (20.1 mg, 85%).

[Equation 57]

[0201] Example 58

[Chemical 113] To a solution of 43 (20 mg, 0.0338 mmol) in CH ₂ Cl ₂ (0.3 ml) was added 3-chloropropionyl chloride (3.22 ml, 0.0338 mmol) and pyridine (2.73 ml, 0.0338 mmol) at 0 ° C. . The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml), 0.1 NH
It was washed with Cl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO ₂ , EtOAc: MeOH ₂
Purification by 0: 1) gave 64 (20.5 mg, 89%) as a white solid.

[Equation 58]

[0202] Example 59

[Chemical 114] For a solution of 43 (19 mg, 0.0364 mmol) in CH ₂ Cl ₂ (0.3 ml), butyryl chloride (
3.78 ml, 0.0364 mmol) and pyridine (2.95 ml, 0.0364 mmol) were added at 0 ° C. The reaction mixture was stirred for 1 hour, then the solution was diluted with CH ₂ Cl ₂ (10 ml) and washed with 0.1N HCl (5 ml). The organic layer was dried over sodium sulfate, filtered and the solvent removed under reduced pressure.
The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 20: 1) to give 64 (19 mg, 87%) as a white solid.

[Equation 59]

[0203] Example 60

[Chemical 115] For a solution of 50 (31.7 mg, 0.044 mmol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (2
25 mg, 1.32 mmol) was added and the reaction was stirred at 23 ° C. for 17 hours. Brine (10 ml) and saturated aqueous NaHCO ₃ (10 ml) were added at 0 ° C., the mixture was stirred for 15 min, filtered through a pad of Celite and washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum.
The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 1) to give 66 (16 mg, 51%) as a white solid.

[Equation 60]

[0204] Example 61

[Chemical formula 116] For a solution of 53 (57 mg, 0.0828 mmol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (65
0 mg, 3.81 mmol) was added and the reaction was stirred at 23 ° C. for 24 hours. Brine (10 ml) and aqueous saturated
NaHCO ₃ (10 ml) was added at 0 ° C., the mixture was stirred for 15 minutes, filtered through a pad of Celite,
It was washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 1) to give 67 (28 mg, 50%) as a white solid.

[Equation 61]

[0205] Example 62

[Chemical 117] For a solution of 48 (32 mg, 0.0529 mmol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (27
0 mg, 1.58 mmol) was added and the reaction was stirred at 23 ° C. for 24 hours. Brine (10 ml) and aqueous saturated
NaHCO ₃ (10 ml) was added at 0 ° C., the mixture was stirred for 15 minutes, filtered through a pad of Celite,
It was washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 1) to give 68 (18 mg, 56%) as a white solid.

[Equation 62]

[0206] Example 63

[Chemical 118] For a solution of 51 (27 mg, 0.04 mmol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (204 m
g, 1.19 mmol) was added and the reaction was stirred at 23 ° C. for 24 hours. Brine (10 ml) and aqueous saturated Na
HCO ₃ (10 ml) was added at 0 ° C., the mixture was stirred for 15 min, filtered through a pad of Celite and washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 1) to give 69 (10 mg, 38%) as a white solid.

[Equation 63]

[0207] Example 64

[Chemical formula 119] For a solution of 63 (15 mg, 0.023 mmol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (118
mg, 0.691 mmol) was added and the reaction was stirred at 23 ° C. for 24 hours. Brine (10 ml) and aqueous saturated
NaHCO ₃ (10 ml) was added at 0 ° C., the mixture was stirred for 15 minutes, filtered through a pad of Celite,
It was washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum. The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 1) to give 70 (20.1 mg, 85%) as a white solid.

[Equation 64]

[0208] Example 65

[Chemical 120] For a solution of 65 (25 mg, 0.042 mol) in CH ₂ CN / H ₂ O (1.5 ml / 0.5 ml), AgNO ₃ (215.
56 mg, 1.269 mmol) was added and the reaction was stirred at 23 ° C. for 24 hours. Brine (10 ml) and saturated aqueous NaHCO ₃ (10 ml) were added at 0 ° C., the mixture was stirred for 15 min, filtered through a pad of Celite and washed with CH ₂ Cl ₂ (20 ml). The solution was discarded and the organic layer was dried and concentrated under vacuum.
The residue was purified by flash column chromatography (SiO ₂ , EtOAc: MeOH 5: 2) to give 71 (16 mg, 65%) as a white solid.

[Equation 65]

Fermentation Method Example A 1% glucose; 0.25% bovine extract; 0.5% bactopeptone; 0.25% NaCl; 0.8% Ca
Freezing stock of 0.1% microorganism, Pseudomanas fluores, in inoculum YMP3 containing CO _3.
Cens strain A2-2 was inoculated and incubated at 27 ° C on a rotary shaker (250 rpm). After 30 hours of incubation, 2% dextrose; 4% mannitol;
2% dried baker's yeast (Vitalevor® Biolux, Belgium); 1% (NH ₄ ) ₂ SO ₄ ; 0.04%
K ₂ HPO ₄ ; 0.8% KCl; 0.001% FeCl ₃ ; 0.1% L-Tyr; 0.8% CO ₃ Ca; 0.05% PPG-2000;
The seed culture was added to a stirred vessel fermenter with a production medium consisting of 0.2% antifoaming silicone (ASSAF-100, RHODIA UK). Sterilization was carried out at 122 ° C for 30 minutes. The inoculated volume was 2% (v / v). The temperature was 27 ° C from 0 to 16 hours and 24 ° C from 16 hours to the final step (41 hours). The pH was adjusted to 6.0 with diluted sulfuric acid from 28 hours to the final step. Pressurization was 0.5 bar. 1% mannitol or sorbitol was added in the final step from 16 hours (2 days uptime) and 2% was added in a 3 day fermentation step. After 41 or 64 hours, the fermentation broth must be extracted to recover safracin B and the clarified broth treated with KCN to recover safracin B-cyano.

Example B Obtaining Safracin B Cyano from Crude Extract Solids were removed from the clarified or filtrate from pH 6 fermentation broth. The clarified broth was adjusted to pH 9.5 with dilute sodium hydroxide and extracted twice with 2: 1 (v / v) ethyl acetate, methylene chloride or butyl acetate. Extraction was carried out for 20 minutes in a stirred vessel and the mixture was maintained at a temperature of 8-10 ° C. The two phases were separated by liquid-liquid centrifugation. The organic layer was dried over anhydrous sodium sulfate or frozen and then filtered to remove ice. The organic phase (ethyl acetate layer) was evaporated until a crude oily extract was obtained.

Example C Obtaining Safracin B Cyano from Clarified Broth Solids were removed from the clarified or filtrate from pH 6 fermentation broth. The clarified broth was adjusted to pH 3.9 with concentrated acetic acid. 0.5g / l KCN was added to the clarified broth and incubated for 1 hour at 20 ° C with agitation. Then the temperature was lowered to 15 ° C., the pH was adjusted to 9.5 with dilute sodium hydroxide and extracted with 2: 1.5 (v / v) ethyl acetate. Extraction was carried out for 20 minutes in a stirred vessel and the mixture was maintained at a temperature of 8-10 ° C. The two phases were separated by liquid-liquid centrifugation. The organic layer was dried over anhydrous sodium sulfate. The organic phase (ethyl acetate layer) was evaporated until a crude oily extract was obtained. The extract was purified by flash column chromatography (SiO2, gradient 20: 1 to 10: 1 to 5: 1 of ethyl acetate: methanol) to give quantitative compound 2 as a light yellow solid.

[Equation 66]

Example D Dextrose (2%), mannitol (4%), anhydrous brewer yeast (2%), ammonium sulfate (1%), dibasic potassium phosphate (0.04%), potassium chloride (0.8%), chloride Iron (II
I) Medium consisting of hexahydrate (0.001%), L-tyrosine (0.1%), calcium carbonate (0.8%), poly (polyethylene glycol) 2000 (0.05%), and anti-foaming ASSAF (0.2%) To 75l
Pour into a jar fermenter with a total volume of, and after sterilization, inoculate with a seed culture (2%) of A2-2 strain (FERM BP-14), aerobically cultivate with stirring, for 64 hours 27 To 24 ° C. (75 l aeration, 350-500 rpm stirring for 1 minute). The pH was controlled from 27 hours to the final step by automatic feeding of diluted sulfuric acid. 2% mannitol was added from 16 hours to the final step. The culture medium (45 l) thus obtained was centrifuged to remove the cells, adjusted to pH 9.5 with diluted sodium hydroxide and extracted twice with 25 liters of ethyl acetate. Mixing was carried out at 8 ° C for 20 minutes in a stirred vessel. The two phases were separated by liquid-liquid centrifugation. The organic phase is frozen at -20 ° C, filtered to remove ice,
The ice was evaporated and 40 g of oily dark extract were obtained. After introduction and purification of the cyanide group, 3.0 g of safracin B cyano was obtained.

Example E Dextrose (2%), mannitol (4%), anhydrous brewer yeast (2%), ammonium sulfate (1%), dibasic potassium phosphate (0.02%), potassium chloride (0.2%), chloride Iron (II
I) Medium consisting of hexahydrate (0.001%), L-tyrosine (0.1%), calcium carbonate (0.8%), poly (polyethylene glycol) 2000 (0.05%), and anti-foaming ASSAF (0.2%) To 75l
Pour into a jar fermenter having a total volume of 10%, sterilize, inoculate with a seed culture (2%) of A2-2 strain (FERM BP-14), and perform aerobic culture with stirring for 41 hours 27 To 24 ° C. (75 l aeration, 350-500 rpm stirring for 1 minute). The pH was controlled from 28 hours to the final step by automatic feeding of diluted sulfuric acid. 1% mannitol was added from 16 hours to the final step. The culture medium (45 l) thus obtained was adjusted to pH 3.9 with 200 ml concentrated acetic acid after removing the cells by centrifugation. After adding 25 grams of potassium cyanide 97% and stirring at 20 ° C. for 1 hour, the pH was adjusted to 9.5 with 1500 ml of 10% sodium hydroxide solution. It was then extracted with 35 liters of ethyl acetate. Mixing was carried out at 8 ° C for 20 minutes in a stirred vessel. The two phases were separated by liquid-liquid centrifugation. The organic phase was dried over anhydrous sodium sulfate and evaporated to give 60 g of oily dark crude extract. After chromatography, 4.9 grams of safracin B cyano was obtained.

[0214] Example 66

[Chemical 121] Commercially available Boc-C with 25 (7.83 g, 0.0139 mol) in dichloromethane (535 mL) under argon.
To a stirred solution of the ys (Fm) derivative (Bachem) (8.33g, 35.04mmol) was added dimethylaminopyridine (4.28g, 35.04mmol) and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimidehydro. Chloride (6.66g, 35.04mmol) was added at 23 ° C. Then the mixture
The mixture was stirred for 2.5 hours at 23 ° C. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate (500 mL), the organic phase separated and the aqueous phase back-extracted with dichloromethane (250 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The crude product was purified by flash column chromatography eluting with a mixture of ethyl acetate and hexanes in a 1: 4 to 2: 1 gradient format to afford 142 (12.21 g, 93%) as a light yellow solid. Rf = 0.35 Hex: EtOAc 1: 1

[Equation 67]

[0215] Example 67

[Chemical formula 122] Dichlorobis (triphenylphosphine) palladium (II) (0.71 g, 1.015 mmol) and acetic acid were added to a stirred solution of 142 (12.01 g, 0.0127 mol) in dichloromethane (318 mL).
(3.6 mL, 0.176 mol) was added at 23 ° C. under argon. Then tributyltin hydride (10.27 mL, 0.037 mol) was added dropwise. The mixture was stirred for 10 minutes at 23 ° C. The reaction was then filtered through a hexane pressed silica gel column. 1: 4, 1: 1 to 7:
By later eluting with a mixture of ethyl acetate and hexane in a gradient mode of 3,
143 (10.89 g, 95%) was obtained as a yellow solid. Rf = 0.25 Hex: EtOAc 2: 1

[Equation 68]

[0216] Example 68

[Chemical 123] 143 (10g, 0.011mol) in anhydrous dichloromethane (185mL) at -10 ℃ (bath temperature -15 ℃)
Solution of benzeneselenic hydride (5.7 g, 0.011 mol) was added to anhydrous dichloromethane (185 mL), and the white solid present in the solution was discarded.
The mixture was stirred at the same temperature for 10 minutes. Dilute the reaction with dichloromethane (200 mL),
Saturated aqueous sodium bicarbonate solution (500 mL) was added at -10 ° C. The organic phase was separated, dried over sodium sulphate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash column chromatography, eluting with a mixture of ethyl acetate and hexanes in a 1: 1, 3: 2, 7: 3 to 4: 1 gradient to give 144 (9.34 g, (92%)
Got The purified solid from chromatography was dissolved in dichloromethane (250 mL), charcoal (3.3 g) was added and the suspension was stirred at 23 ° C. for 1 hour. The mixture was filtered through Celite and the Celite was washed with dichloromethane (80 mL). The solvent was maintained at a temperature of 25-30 ° C. and evaporated under reduced pressure to give 144 (8.96 g, 88%) as a yellow solid. R
f = 0.30 and 0.25 (mixture of isomers) Hex: EtOAc 1: 1

[Equation 69]

[0217] Example 69

[Chemical formula 124] To a solution of DMSO (3.44 mL) in anhydrous dichloromethane (396 mL) was added triflic hydride (3.27 mL, 19.45 mmol) at -78 ° C under argon and the mixture was stirred at this temperature for 20 minutes. Then 144 (8.92 g, 9 mL in anhydrous dichloromethane (124 mL) at -78 ° C.
A solution of .6 mmol) was added and the mixture was stirred at −40 ° C. for 35 minutes under argon. Diisopropylethylamine (13.5 mL, 73.43 mmol) was added and the mixture was stirred at 0 ° C. for 45 minutes under argon. tert-Butanol (3.65 mL, 38.6 mmol) and tert-butyltetramethylguanidine (11.6 mL, 67.46 mmol) were added and the mixture was stirred at 23 ° C. for 40 minutes under argon. Acetic anhydride (9.15 mL, 96.78 mmol) was then added and the reaction was stirred at 23 ° C. for another hour. The reaction was diluted with dichloromethane (250 mL) and saturated aqueous ammonium chloride solution (500 mL) was added. The organic phase was separated and saturated aqueous sodium bicarbonate solution (500 m
L) and saturated aqueous sodium chloride solution (500 mL) were washed successively. The organic phase was separated, dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure maintaining the temperature at 25-30 ° C. The crude solid was then purified by flash chromatography, 1:
Elution with a mixture of ethyl acetate and hexane in the form of a solvent from 4 to 2: 3 gave 145 (4.99g, 68%) as a yellow solid. Rf = 0.44, Hex: EtOAc 3: 2

[Equation 70]

[0218] Example 70

[Chemical 125] Sodium iodide (1.52 g, 10.01 mmol) was added at 23 ° C. to a solution of 145 (1.0 g, 1.3 mmol) in acetonitrile (50 mL) and dichloromethane (25 mL). The mixture was then cooled to 0 ° C. and aluminum trichloride (1.33 g, 10.01 mmol) was added portionwise maintaining the temperature at 0 ° C. The mixture was then stirred at 0 ° C. for 2.5 hours. The reaction was diluted with dichloromethane (25 mL) and a saturated aqueous solution of sodium potassium tartrate (100 mL) was added. The aqueous phase was separated and extracted with dichloromethane (2 x 75 mL). Then saturated aqueous sodium bicarbonate solution (50 mL) was added to the aqueous phase, which was further extracted with dichloromethane (2 x 50 mL). The combined organic extracts were dried over sodium sulphate, filtered and evaporated to dryness under reduced pressure keeping the temperature at 25 ° C. The crude solid was then purified by column chromatography on amino silica gel, eluting with a mixture of ethyl acetate and hexane in a gradient fashion. 35 as a yellow solid (487 mg, 60
%) DMSO was obtained. The 35 experimental data were previously described in PCT / GB00 / 01852. 36, ET-770 and ET743 were prepared in the same way as previously described in PCT / GB00 / 01852.

[0219] Route 2 Example 71

[Chemical formula 126] A solution of 21 (9.84g, 18.97mmol) in THF (569mL) and H2O (285mL) at 0 ° C in an ice bath.
Cooled to. Then NaNO2 (1.96g, 28.45mmol) and 90% aqueous AcOH (18.97mL, 0.33mol)
Was added at 0 ° C. and the mixture was stirred at 23 ° C. for 18 hours. After cooling the reaction to 0 ° C., 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 x 300 mL). The combined organic extracts were dried over sodium sulphate and evaporated to dryness under reduced pressure. The crude solid was then dissolved in MeOH (379 mL) and IM NaOH (38 mL) was added at 0 ° C. 23 mixture
Stir at 4 ° C. for 4 hours. After diluting with EtOAc (600 mL) at 0 ° C., the organic phase was washed with a mixture of water (400 mL) and saturated aqueous sodium bicarbonate solution (100 mL, basic pH). After extraction, the aqueous phase was further extracted with EtOAc (3 x 300 mL). The combined organic extracts were dried over Na ₂ SO ₄ , filtered and concentrated under vacuum. The residue was purified by flash column chromatography (SiO2, Hex: EtOAc 3: 1 to 2: 1 gradient) to afford 146 (4.55 g, 46%) as a white solid. Rf: 0.33 (Hex: EtOAc 1: 1).

[Equation 71]

[0220] Example 72

[Chemical 127] Commercially available Boc-C with 146 (47.35 g, 0.091 mol) in dichloromethane (2.8 L) under argon.
For a stirred solution of the ys (Fm) derivative (54.6 g, 0.137 mol, dimethylaminopyridine (5
.6 g, 0.046 mol) and 1- [3- (dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (43.6 g, 0.227 mol) were added dropwise at 23 ° C. over 1.5 hours. The reaction was stirred at 23 ° C for an additional hour. The reaction was stopped by the addition of saturated aqueous sodium bicarbonate solution (1 L) and the organic phase was separated. The aqueous phase was back extracted with dichloromethane (2 x 500 mL). The combined organic extracts were dried over sodium sulfate and evaporated to dryness under reduced pressure. The crude product was purified by flash chromatography, 1: 4 to 3
Elute with a mixture of ethyl acetate and hexanes in a 1: 1 gradient format to give 14 as a white solid.
7 (74.3 g, 93%) was obtained. Rf = 0.5, Hex: EtOAc 1: 1

[Equation 72]

[0221] Example 73

[Chemical 128] CH ₃ CN (3.12mL) in the 147 (0.562g, 0.624mol) To a solution of, MEMCl (1.07mL, 9.
36 mmol), DIPEA (2.17 mL, 12.48 mmol) and DMAP (0.0076 g, 0.06 mmol) were added at 0 ° C. The mixture was stirred at 23 ° C for 5.5 hours. The reaction was diluted with CH ₂ Cl ₂ (50 mL) and 0.1N HC
Extracted with l (50 mL). The aqueous phase was extracted again with CH ₂ Cl ₂ (50 mL). The combined organic phases were dried over Na ₂ SO ₄ , filtered and concentrated in vacuo to give a residue which was purified by flash column chromatography (CH ₂ Cl ₂ : EtOAc 10: 1, 5: 1). Purification gave 148 (539 mg, 87%) as a white solid.

[Equation 73]

[0222] Example 74

[Chemical formula 129] 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) was added at 23 ° C. under argon. Then tributyltin hydride (3
6.5 mL, 0.136 mol) was added dropwise. The mixture was stirred at 23 ° C for 15 minutes. The reaction was then filtered through a silica gel column compressed with hexane. After that 0: 100, 1: 4, 1:
Elution with a mixture of ethyl acetate and hexane in the form of a gradient of 3, 2: 5, 2: 3, 1: 1, 2: 1, 3: 1 to 100: 0 gave 149 as a white solid (35.07 g, 95%). Rf = 0.25 He
x: EtOAc 2: 1

[Equation 74]

[0223] Example 75

[Chemical 130] 149 (15g, 0.0158mo) in anhydrous dichloromethane (185mL) at -10 ℃ (bath temperature -15 ℃)
To the solution of l) was added dropwise a solution of benzeneselenic hydride (7.4 g, 0.0143 mol) in anhydrous dichloromethane (265 mL) for 30 minutes. The mixture was stirred at the same temperature for another 10 minutes. The reaction was diluted with dichloromethane (200 mL) and saturated aqueous sodium bicarbonate solution (500 mL) was added at -10 ° C. The organic phase was separated, dried over sodium sulphate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash column chromatography, eluting with a mixture of ethyl acetate and hexanes in a gradient of 1: 2 to 100: 0 to give 150 (14.20 g, 89%) as a yellow solid. The purified solid from chromatography was dissolved in dichloromethane (250 mL) and charcoal (4.
95 g) was added. The suspension was then stirred at 23 ° 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 under reduced pressure to give 150 as a white solid (13.72g, 86%). Rf = 0.37 Hex: EtOAc1: 2

[Equation 75]

[0224] Example 76

[Chemical 131] The reaction flask was heat treated twice, vacuum / argon purged several times and maintained in an argon atmosphere for the reaction. To a solution of DMSO (385.0 μL) in anhydrous CH ₂ Cl ₂ (42 ml) was added triflic anhydride (366.5 μl, 2.16 mmol) dropwise at −78 ° C. The reaction mixture was stirred at −78 ° C. for 20 minutes, then at −78 ° C. anhydrous CH ₂ Cl ₂ (for main addition 1
A solution of 150 (1 g, 1.03 mmol) in 0 ml, and 5 ml for washing) was added via cannula. The temperature was maintained at −78 ° C. in both flasks during the addition and the color development changed from yellow to brown. The reaction mixture was stirred at -40 ° C for 35 minutes. During this period, the solution went from yellow to dark green. After this time ⁱ Pr ₂ NEt (1.51 ml, 9.55 mmol) was added dropwise and the reaction mixture was kept at 0 ° C. for 45 min, during which time the solution developed a brown color. Then t-butanol (409.5 μl, 4.33 mmol) and tert-butyltetramethylguanidine (1.31 ml, 7.61)
mmol) was added dropwise and the reaction mixture was stirred at 23 ° C. for 40 minutes. After this, acetic anhydride (
1.03 ml, 10.89 mmol) was added dropwise and the reaction mixture was kept at 23 ° C. for another hour. The reaction mixture was then diluted with CH ₂ Cl ₂ (25 ml), a saturated aqueous solution of NH ₄ Cl (50 ml), NaH.
It was washed with CO ₃ (50 ml) and NaCl (50 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. Flash column chromatography (
Inner diameter: 2.0 cm, silica height: 9 cm, eluent: ethyl acetate / hexane 20:80, 30:
70 to 40:60 gradient) to give compound 151 (832.6 mg, 99% as a white solid.
) Got. RF = 0.48 Hex: EtOAc 3: 2

[Equation 76]

[0225] Example 77

[Chemical 132] For a solution of 151 (2.9 g, 3.57 mmol) in CH ₂ Cl ₂ (120 mL), MeSO ₃ H (1.4 mL, 21.4
6 mmol) was added at 23 ° C. After stirring the reaction for 30 minutes at 23 ° C., saturated aqueous sodium bicarbonate solution (200 mL) was added at 0 ° C. The organic phase is separated, dried over sodium sulfate,
It was filtered and concentrated to dryness under reduced pressure. The residue was purified by flash column chromatography, eluting with a mixture of ethyl acetate and hexane in a 0: 1 to 1: 0 gradient to give 35 (1.43 g, 64%) as a pearly yellow solid. It was The 35 experimental data were previously described in PCT / GB00 / 01852. 36, ET-770 and ET-743 were prepared according to the same method as previously described in PCT / GB00 / 01852.

Route 3 The first step in this route (21 to 146 conversion) was that previously described in Example 71.

[Chemical 133] Commercially available HO.Cys (Fm) -H.HCl (Bachem) (40 g, 0.11 in acetone (500 mL) and water (500 mL).
0 mol of 1M Na ₂ CO ₃ solution (238 mL) and BnCO ₂ Cl (18.7 mL, 0.131 mol)
Added at ° C. After stirring the reaction for 30 minutes at 60 ° C., the mixture was quenched with 1N HCl (pH 0 1) and extracted with ether (3 × 400 mL). The organic phase was separated, dried over magnesium sulphate, filtered and concentrated under reduced pressure to dryness. The crude solid was dissolved in a mixture of EtOAc / CH2Cl2 1: 1, precipitated with hexane and kept at 4 ° C. overnight. The suspension was then filtered, the solid washed with hexane (200 mL) and the filtrate dried under vacuum to give a white solid.
(50.16g, 97%) was obtained.

[Equation 77]

[0227] Example 79

[Chemical 134] Dimethylaminopyridine (705 mg, 5.77 mmol), 1- [3- (dimethylamino) propyl was added to a stirred solution of 146 (10 g, 19.2 mmol) and 152 (12.5 g, 28.8 mmol) under argon.
] -3-Ethylcarbodiimide hydrochloride (9.2g, 48.1mmol) and diisopropylethylamine (7.4mL, 42.3mmol) were added dropwise at 0 ° C over 1 hour. The reaction was quenched by the addition of saturated aqueous sodium bicarbonate solution (600 mL). Separate the organic phase,
Saturated aqueous ammonium chloride solution (500 mL) and saturated aqueous sodium chloride solution (500 m
It was washed with L). The organic extract was dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The crude product was purified by flash column chromatography _{(RP18, CH 3 CN; H} 2 O 4: 1) to afford 153 as a pearl-like yellow solid (13.89g, 77%).

[Equation 78]

[0228] Example 80

[Chemical 135] For a solution of 153 (13.89 g, 14.85 mmol) in CH ₃ CN (74.3 mL), MEMCl (25.4 mL, 2
23 mmol), DIPEA (52 mL, 297 mmol) and DMAP (0.181 g, 0.15 mmol) were added at 0 ° C.
The mixture was stirred at 23 ° C for 5.5 hours. The reaction was diluted with CH ₂ Cl ₂ (400 mL) and 0.1N HCl (3
It was extracted with 00 mL) and 3N HCl (pH = 3). The aqueous phase was extracted again with CH ₂ Cl ₂ (2 x 50 mL). The combined organic phases were dried over Na ₂ SO ₄ , filtered and concentrated under vacuum to give a residue which was flash column chromatographed (SiO ₂ , CH ₂ Cl ₂ : EtOAc 10: 1, 5: 1). ) To give 154 (13.47 g, 88%) as a white solid. Rf = 0.27 CH ₂ Cl ₂ : AcOEt
6: 1

[Equation 79]

[0229] Example 81

[Chemical 136] 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) was added under argon at 23 ° C. Then tributyltin hydride (2
7.44 mL, 0.1 mol) was added dropwise. The mixture was stirred at 23 ° C for 15 minutes. The reaction was then filtered through a silica gel column compressed with hexane. After that 1: 4, 1: 1, 3: 2
Elution with a mixture of ethyl acetate and hexanes in a 7: 3 gradient to gave 155 (18.78 g, 94%) as a pearlescent yellow solid.

[Equation 80]

[0230] Example 82

[Chemical 137] 155 (18.5g, 18.82m) in anhydrous dichloromethane (530mL) at -10 ° C (bath temperature -15 ° C)
mol) solution of benzene seleninic hydride (9.68 g, 18.82 mmol) in anhydrous dichloromethane (290 mL) was added dropwise and the white solid present in the solution was discarded. The mixture was stirred at the same temperature for another 10 minutes. The reaction was quenched with saturated aqueous sodium bicarbonate solution (600 mL). The organic phase was separated and the aqueous phase was replaced with CH ₂ Cl ₂ (2 x 300 mL
). The combined organic phases were dried over sodium sulphate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash column chromatography, eluting with a mixture of ethyl acetate and hexanes in a 1: 1, 3: 2, 7: 3 to 4: 1 gradient format to give 156 ( 17.62g, 88%) was obtained.

[Equation 81]

[0231] Example 83

[Chemical 138] The reaction flask was heat treated twice, vacuum / argon purged several times and maintained in an argon atmosphere for the reaction. To a solution of DMSO (178 μL) in anhydrous CH ₂ Cl ₂ (20 ml) was added triflic hydride (169 μl, 1 mmol) dropwise at −78 ° C. The reaction mixture was stirred at -78 ° C for 20 min, then at -78 ° C a solution of 156 (0.5 g, 0.5 mmol) in anhydrous CH ₂ Cl ₂ (4 ml for main addition, and 1.5 ml for washing) was added. Added via cannula.
The temperature was maintained at −78 ° C. in both flasks during the addition and the color development changed from yellow to brown. The reaction mixture was stirred at -40 ° C for 35 minutes. During this period, the solution went from yellow to dark green. After this, ⁱ Pr ₂ NEt (0.7 ml, 4.42 mmol) was added dropwise and the reaction mixture was heated to 0 ° C.
For 45 minutes, the color of the solution turned brown during this time. Then, t-butanol (189
μl, 2 mmol) and tert-butyltetramethylguanidine (0.6 ml, 3.49 mmol) were added dropwise and the reaction mixture was stirred at 23 ° C. for 40 minutes. After this, acetic anhydride (0.47 ml, 4.97 m
mol) was added dropwise and the reaction mixture was kept at 23 ° C. for another hour. The reaction mixture was then diluted with CH ₂ Cl ₂ (15 ml), an aqueous saturated solution of NH ₄ Cl (25 ml), NaHCO ₃ (25 ml) and N 2.
It was washed with aCl (25 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography (internal diameter: 2.0 cm, silica height: 9 cm, eluent: ethyl acetate / hexane 1: 4, 1: 3, 1: 2 to 1: 1 gradient), Compound 157 (128 mg, 30%) was obtained as a light yellow solid. RF = 0.3
7 Hex: EtOAc 3: 2

[Equation 82]

[0232] Example 84

[Chemical 139] For a solution of 157 (100 mg, 0.118 mmol) in CH ₂ Cl ₂ (2 mL) and CH ₃ CN (2 mL), NaI (71
mg, 0.472 mmol) and TMSCI (60 mL, 0.472 mmol) were added at 0 ° C. After stirring the reaction for 50 min at 23 ° C., the mixture was quenched with water (30 mL) and extracted with CH ₂ Cl ₂ (2 × 20 mL). The combined organic phases were washed successively with a saturated solution of NaCl (20 mL) and a saturated solution of sodium dithionite (20 mL), dried over Na ₂ SO ₃ , filtered and concentrated under vacuum. The residue was flash column chromatographed (eluent :) ethyl acetate / hexane 1: 4
, Gradient 1: 2 to 1: 1) to give 158 (62 mg, 70%) as a white solid. Rf = 0.21 Hex: EtOAc 1: 1

[Equation 83]

[0233] Example 85

[Chemical 140] To a solution of 158 (100 mg, 0.132 mmol) in MeOH (6.8 mL), HCO ₂ H (360 μL) and 10%.
Pd / C (140 mg, 0.132 mmol) was added at 23 ° 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. Azeotropic distillation with toluene 3
Repeated 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 x 10 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was then purified by flash chromatography on amino-silica gel, eluting with a mixture of ethyl acetate and hexanes in a 1: 2, 1: 1 to 2: 1 gradient format to give 35 (57 mg, 70%). The 35 experimental data were previously described in PCT / GB00 / 10852. 36, ET-770 and ET-743 were prepared according to the same method as previously described in PCT / GB00 / 01852.

Route 4 The first step in this route (21 to 146 conversion) was as described above in Example 71. Example 86

[Chemical 141] To a solution of 146 (18 mg, 0.032 mmol), catalytic amount of DMAP and imidazole (5 mg, 0.08 mmol) in DMF (0.05 mL) at 0 ° C., tert-butyldiphenylsilyl chloride (12.5
μL, 0.048 mmol) was added and the reaction was stirred at 23 ° C. for 4 hours. Then add water (30 mL) to 0
C was added and the mixture was extracted with Hex: EtOAc 1:10 (2 x 40 mL). The combined organic phases were dried over sodium sulphate, filtered and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO ₂ , Hex: EtOAc 3: 1) to give 159 (27 mg, 88%) as a white solid. Rf = 0.29 Hex: EtOAc 3: 1

[Equation 84]

[0235] Example 87

[Chemical 142] For a solution of 159 (2.4 g, 3.17 mmol) in CH3CN (16 mL), MOMBr (2.6 mL, 31.75 mm
ol), DIPEA (8.3 mL, 47.6 mmol) and DMAP (16 mg, 0.127 mmol) were added at 0 ° C. The mixture was stirred at 23 ° C for 6 hours. The reaction was diluted with CH ₂ Cl ₂ (50 mL) and extracted with 0.1N HCl (50 mL). The aqueous phase was extracted again with CH ₂ Cl ₂ (50 mL). The combined organic phases are dried over Na ₂ SO ₄ , filtered and concentrated under vacuum to give a residue which is flash column chromatography (SiO ₂ , CH ₂ Cl ₂ : EtOAc 15: 1, 5: Purification by 1) gave 26 (1.78g, 70%) as a white solid. The 26 experimental data were previously described in PCT / GB00 / 01852. Experimental methods for Int. 11, 160, 161, 162 and 163 are described in US Pat.
As previously described in.

[0236] Example 88

[Chemical 143] Anhydrous CH ₂ Cl ₂ (250mL) and acetonitrile (300 mL) solution of 163 (15.8 g, 0.02 mol) to a solution of newly diluted with NaI (31.5 g, 0.21 mol) and CITMS (CaH _2, 26.7 mL, 0.2
1 mol) was added at 23 ° C. under an argon atmosphere. The reaction mixture was stirred for 40 minutes. The reaction was then partitioned between CH ₂ Cl ₂ (200 mL) and water (300 mL). The organic phase is a saturated aqueous solution of NaCl.
Washed with (2 x 300 mL). The organic phase was dried over Na ₂ SO ₄ , filtered and the solvent removed under reduced pressure. The crude was purified by flash column chromatography using ethyl acetate / hexane 2: 3 as eluent to give 164 (10.74 g as a pearly yellow solid).
, 76%). Rf = 0.25 Hex: EtOAc 3: 2

[Equation 85]

[0237] Example 89

[Chemical 144] 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) was added at 23 ° C. under argon. Then tributyltin hydride (4.51m
L, 17.02 mmol) was added dropwise over 25 minutes. After the addition of HsnBu ₃ , the mixture was stirred at 23 ° C for another 20 minutes. The reaction was filtered through a silica gel column compressed with hexane.
Subsequent elution with a mixture of ethyl acetate and hexanes in a 1: 2 to 15: 1 gradient format gave 35 (1.38 g, 78%). The 35 experimental data were previously described in PCT / GB00 / 01852. 36, ET-770 and ET-743 were prepared according to the same method as previously described in PCT / GB00 / 01852.

Route 5 The first step in this route (21 to 146 conversion) was as described above in Example 71. Example 90

[Chemical 145] To a solution of 146 (8.372 g, 16.78 mmol) in DMF (20.1 mL) was added imidazole (3.43
g, 50.34 mmol), tert-butyldimethylchlorosilane (7.58 mL, 50.34 mmol) and DMA
P (0.2 g, 1.7 mmol) was added at 0 ° C. After stirring at 23 ° C for 3.5 hours, the reaction mixture was washed with water (
It was quenched with 100 mL) and extracted with EtOAc / Hex 1: 3 (2 x 75 mL). The combined organic phases
It was 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 under vacuum. The residue was purified by flash column chromatography (Hex: EtOAc 10: 1, 3: 1) to give 165 as a white solid (9.85g, 93%). Rf = 0.39 Hex: AtOEc 2: 1

[Equation 86]

[0239] Example 91

[Chemical 146] For a solution of 165 (7.62 g, 12.02 mmol) in THF (87.46 mL) and water (0.24 mL), MEMCl
(2.33 mL, 20.43 mmol) was added at -6 ° C. 60% NaH little by little over 45 minutes (0.72g, 18.03m
(mol) was added and the mixture was stirred at this temperature for 1.5 hours. The reaction was quenched with water (150 mL) and extracted with CH ₂ Cl ₂ (2 x 100 mL). The combined organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under vacuum to give 166 (8.69 g, 100%) as a white solid, which was used in the following step without further purification. did. Rf = 0.24 Hex: AcOEt 2: 1

[Equation 87]

[0240] Example 92

[Chemical 147] For a solution of 166 (10.76 g, 14.90 mmol) in anhydrous CH ₂ Cl ₂ (275 mL), Pd (PPh ₃ P) ₂ C
l ₂ (837 mg, 1.19 mmol), acetic acid (4.26 mL, 74.5 mmol) and tributyltin hydride (11
.85 mL, 44.7 mmol) was added at 23 ° C. under an argon atmosphere. The reaction mixture was stirred at 23 ° C for 15 minutes. (TLC AcOEt / hexane 1: 1 showed no starting material). Hexane (100m
L) and the mixture is flash chromatographed (SiO ₂ , 0: 100, 1: 4, 2: 3 to.
Poured into EtOAc: Hexanes) in a 1: 1 gradient to give 167 as a yellow solid (9.95 g, 98%).
Got Rf = 0.42 Hex: EtOAc 3: 7

[Equation 88]

[0241] Example 93

[Chemical 148] To a solution of 167 (9.95 g, 14.6 mmol) in anhydrous CH ₂ Cl ₂ (300 mL) was added anhydrous CH ₂ Cl ₂ (12
A solution of benzeneselenic hydride (7.51 g, 14.6 mmol, 70% reagent purity) in 0 mL) was added dropwise at -15 ° C under an argon atmosphere (discarded residual white solid). The solution was then stirred at −15 ° 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 ₂ Cl ₂ (500 mL). The combined extracts were dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The reaction crude was purified by flash chromatography (SiO ₂ , EtOAc: Hexanes in a 2: 3 to 3: 1 gradient mode) to afford 168 (9.86 g, 97%) as a yellow solid. Rf = 0.3
3 Hex: EtOAc 3: 7

[Equation 89]

[0242] Example 94

[Chemical 149] To a solution of 168 (16.38g, 23.47mmol) in anhydrous THF (727mL, 0.03M) was added 1M THF.
A solution of TBAF in (59 mL, 59 mmol) was added dropwise at 23 ° C. The reaction mixture at 45 ° C at 45
Stir for minutes. The mixture was partitioned between saturated aqueous NaCl solution (850 mL) and CH ₂ Cl ₂ (950mL). Both phases were separated, the organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO _2, EtOAc: hexane 40: 60, 50: 50,70: 30, 90: 10 to 100: 0 gradient fashion) to afford 169 as a light yellow solid (12.17g, 89%) was obtained. Rf = 0.1 Hex: EtOAc 3: 7

[Equation 90]

[0243] Example 95

[Chemical 150] 169 (11.49 g, 19.69 mmol) and Alloc-Cys- (Fm) (11.32 g, 29 in anhydrous CH ₂ Cl ₂ (688 mL).
.53 mmol) (for preparation, Kruse, CH; Holden, KG, J. Org. Chem., 2985, 50,
pp. 2792-2794), DMAP (2.4 g, 19.69 mmol) and EDCHCl (9.4
4 g, 49.22 mmol) was added at 23 ° C. DIPEA (5.14 mL, 29.53 mmol) was then added at 0 ° C. and the reaction was stirred at 23 ° C. for 3 hours. The mixture was washed with a saturated aqueous solution of NaHCO ₃ (500 mL), NaC.
Washed successively with 1 (400 mL) and NH ₄ Cl (2 × 300 mL). The organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. Flash column chromatography (S
iO ₂ , EtOAc: hexane 1: 1, 6: 4 to 7: 3 gradient) to give 170 (14.76 g, 79%) as a pearly yellow solid. Rf = 0.31 and 0.40 Hex: EtOAc 3:
7 (mixture of isomers)

[Formula 91]

[0244] Example 96

[Chemical 151] The reaction flask was heat treated twice, vacuum / argon purged several times and maintained in an argon atmosphere for the reaction. To a solution of DMSO (5.4 mL) in anhydrous CH ₂ Cl ₂ (554 ml) was added triflic hydride (5.11 ml, 30.4 mmol) dropwise at -78 ° C. The reaction mixture was stirred for 20 minutes at -78 ° C., then 170 in anhydrous CH ₂ Cl ₂ (188ml) at -78 ° C. (14.43
g, 15.2 mmol) was added via cannula. The temperature was maintained at −78 ° C. in both flasks during the addition and the color of the reaction was yellow. The reaction mixture was stirred at -40 ° C for 35 minutes. During this period, the solution went from yellow to dark green. After this, ⁱ Pr ₂ NEt (21.1 ml
, 121.6 mmol) was added dropwise and the reaction mixture was kept at 0 ° C. for 45 minutes, during which time the solution developed a pearly brown color. Then t-butanol (5.8 mL, 60.8 mmol) and ter
t-Butyltetramethylguanidine (18.3 ml, 106.4 mmol) was added dropwise and the reaction mixture was stirred at 23 ° C for 40 minutes. After this time acetic anhydride (14.34 ml, 152 mmol) was added dropwise and the reaction mixture was maintained at 23 ° C. for another hour. The reaction mixture was then added to CH ₂ Cl ₂ (3
It was diluted with 8 ml) and washed with a saturated aqueous solution of NH ₄ Cl (500 ml), NaHCO ₃ (500 ml) and NaCl (500 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated.
The residue was flash column chromatographed (SiO ₂ , EtOAc: Hexane from 3: 7.
171 (6.24 g, 5 as a pearly yellow solid).
2%). Rf = 0.38 Hex: EtOAc 1: 1

[Equation 92]

[0245] Example 97

[Chemical 152] Anhydrous CH ₂ Cl ₂ (74mL) and acetonitrile (74 mL) in 171 (2.26 g, 2.85 mmol) to a solution of (newly diluted with _{CaH 2) NaI (3.42g, 22.8mmol} ) and TMSCl ( 2.6mL, 22.8m
mol) was added at 23 ° C. and the reaction was stirred for 35 minutes. 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 replaced with CH ₂ Cl ₂ (2 x 100 mL
). The combined organic phases were dried over sodium sulphate, filtered and the solvent removed under reduced pressure to give 164 (2.4 g, 100%) as a pearlescent yellow solid which was used without further purification. Used in the reaction. The experimental data for 164 is shown in Example 88.
It was as described above. The conversion from 164 to 35 was as described above in Example 89. Intermediate 36, ET-770 and ET-743 were prepared according to the same method as previously described in PCT / GB00 / 01852.

[0246] Route 6 Example 98

[Chemical 153] To a solution of 144 (7 g, 7.6 mmol) in MeOH (140 mL) was added IM NaOH (15.1 mL),
The reaction was stirred at 23 ° C for 10 minutes. A saturated aqueous solution of NH ₄ Cl (100 ml) was added to the reaction mixture. The organic phase was separated and washed with 5% HCl until the color developed to yellow. The organic extract was dried over sodium sulfate, filtered and the solvent removed under reduced pressure. The residue was flash column chromatographed (SiO2, EtOAc: hexane 0: 1, 1: 3, 1: 2, 1: 1, 1: 1 to 1: 1.
Purification (in a 3: 1 gradient format) to give 161 (3.76 g, 85%). Experimental data for 161 was previously described in US Pat. No. 5,721,362.

[0247] Example 99

[Chemical 154] 161 (200 mg, 0.37 mmol) and cysteine 152 (240 mg, 0.55 mmo) in anhydrous CH ₂ Cl ₂ (20 mL)
l) solution of DMAP (110 mg, 0.925 mmol) and EDC.HCl (170 mg, 0.925 mmol) 23
The reaction was stirred at this temperature for 1.5 hours. The mixture was then saturated aqueous NaHCO3 (15mL), NaCl (15mL ), and washed successively with _{NH 4 Cl (2 × 10mL)} . The organic phase was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (SiO ₂ , AcOEt / hexanes in a 1: 4 to 1: 2 gradient mode) to afford 172 (285 mg, 80%) as a white solid. Rf = 0.3 Hex: EtOAc 2: 1

[Equation 93]

[0248] Example 100

[Chemical 155] The reaction flask was heat treated twice, vacuum / argon purged several times and maintained in an argon atmosphere for the reaction. To a solution of DMSO (977 μL) in anhydrous CH ₂ Cl ₂ (118 ml) was added triflic hydride (930 μl, 5.5 mmol) dropwise at −78 ° C. The reaction mixture was stirred at -78 ° C for 20 min, then at -78 ° C anhydrous CH ₂ Cl ₂ (26 mL for the main addition.
, A solution of 172 (2.63 g, 2.75 mmol) in 13 mL for washing was added via cannula (addition time: 5 min). The temperature was maintained at −78 ° C. in both flasks during the addition and the color development changed from yellow to brown. The reaction mixture was stirred at -40 ° C for 35 minutes. During this period
The solution went from yellow to dark green. After this time ⁱ Pr ₂ NEt (3.48 ml, 22 mmol) was added dropwise and the reaction mixture was kept at 0 ° C. for 45 minutes, during which time the color of the solution turned brown. Then, t-butanol (1.04 mL, 11 mmol) and tert-butyltetramethylguanidine (3.
31 ml, 19.25 mmol) was added dropwise and the reaction mixture was stirred at 23 ° C. for 40 minutes. After this,
Acetic anhydride (2.6 ml, 27.5 mmol) was added dropwise and the reaction mixture was maintained at 23 ° C for an additional hour. The reaction mixture was then diluted with CH ₂ Cl ₂ (70 ml) and a saturated aqueous solution of NH ₄ Cl (18
It was washed successively with 0 ml), NaHCO ₃ (180 ml) and NaCl (180 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. Flash column chromatography of the residue (SiO ₂ , 4: 1, Hex: EtOAc, in a gradient of 3: 1 to 2: 1).
Purify by to give 173 (1.145 g, 52%) as a white solid. Rf = 0.31 Hex: EtOA
c 3: 2

[Equation 94]

[0249] Example 101

[Chemical 156] To a solution of 173 (100 mg, 0.125 mmol) in CH ₂ Cl ₂ (2 mL) and CH ₃ CN (2 mL) was added NaI (75 mL).
mg, 0.5 mmol) and TMSCl (63 mL, 0.5 mmol) were added at 0 ° C. After stirring the reaction at 23 ° C. for 50 minutes, the mixture was quenched with water (30 mL) and extracted with CH ₂ Cl ₂ (2 × 20 mL). The combined organic phases were washed successively with a saturated aqueous solution of NaCl (20 mL) and sodium dithionite (20 mL), dried over Na ₂ SO _4, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (SiO _2, EtOAc: 1 with Hex: 4, 1: 2 to 1: 1 gradient fashion) to give a white solid 158 (66 mg, 70%) . Rf = 0.21 Hex: EtOAc 1:
The experimental data for 1.158 was as described above in Example 19. The conversion of 158 to 35 was as described above in Example 85. Intermediate 36, ET-770 and ET-743 were prepared according to the same method as previously described in PCT / GB00 / 01852.

[0250]

[References]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE, TR), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, G M, KE, LS, MW, MZ, SD, SL, SZ, TZ , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, B Z, CA, CH, CN, CO, CR, CU, CZ, DE , DK, DM, DZ, EC, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, I N, IS, JP, KE, KG, KP, KR, KZ, LC , LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, P L, PT, RO, RU, SD, SE, SG, SI, SK , SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Marta Perez             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Andres Frances, the inventor             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Carolina Fernandez             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Jose Luis Cicarlo             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Pirall Gallego             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Maria Sarsuelo             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Ignacio Manzanares             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Maria Jesus Martin             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima (72) Inventor Simon Munt             Spain E-28760 Madrid Torre             Su Cantos Polygono Industri             Al de Torres Cantos Calle             De La Carrera 3. Pharma Mar.             Sociedade Anonima F-term (reference) 4C071 AA04 AA07 BB03 BB06 CC03                       CC13 CC21 EE24 FF14 GG01                       HH05 HH08 HH09 HH17 HH28                       KK01 LL01

Claims (29)

[Claims] 1. A method for preparing an ecteinascidin product having a spiroamine-1,4-bridge, which comprises a 1-labyl, 10-hydroxy, 18-protected hydroxy, di-6,8-en-5-one fused ring. A method of forming a 1,4 bridge using a compound, wherein the 18-C protection is removed prior to spiroamine introduction. 2. The method of claim 1, wherein the ecteinascidin product has a 21-hydroxy group and comprises converting a 21-cyano group to a 21-hydroxy group. 3. The method according to claim 1, wherein the spiroamine is spiroquinoline. 4. The 1-labyl, 10-hydroxy, 18-protected hydroxy, di-6
4. The 1,8-en-5-one fused ring compound according to claim 1, wherein the 18-protecting group is protected by MOM, methoxymethyl group; or MEM, methoxyethoxymethyl group. the method of. 5. The 1-labyl group is an N-protected cysteinyloxymethylene group of the formula: —CH ₂ —O—CO—CNHProt ¹ —CH ₂ —SH. The method according to any one of 1. 6. Prot ¹ is Boc, t-butyloxycarbonyl; Troc, 2,2,2-trichloroethyloxycarbonyl; Cbz, benzyloxycarbonyl; or All
The method according to claim 5, wherein oc is allyloxycarbonyl. 7. The method according to claim 5 or 6, wherein Prot ¹ is removed in the same step as the C-18 protection. 8. The 1-labyl group is produced from a 1-substituted form of the formula: —CH ₂ —O—CO—CNHProt ¹ —CH ₂ —S-Prot ² 7. The method according to 7. 9. The method of claim 8, wherein Prot ² is Fm, 9-fluorenylmethyl. 10. The ¹ -substituted form of the following formula: —CH ₂ —O—CO—CNHProt ¹ —CH ₂ —S-Prot ² is formed by esterification of the —CH ₂ —OH substituent. The method according to claim 8 or 9. 11. The method of claim 10, wherein the esterification is performed prior to formation of the 10-hydroxy, di-6,8-en-5-one structure. 12. The method of claim 10, wherein the esterification is carried out after the introduction of the 10-hydroxy, di-6,8-en-5-one structure. 13. A method according to any one of claims 1 to 12, starting from 1-aminomethylene, 5-protected hydroxy, 7,8-dioxymethylene, 18-hydroxy, 21-cyano fused ring compounds. the method of. 14. The 1-aminomethylene group is temporarily protected to provide the 18-aminomethylene group.
14. The method of claim 13, wherein protection with a hydroxy group is allowed and the temporary protection is removed. 15. The method of claim 13, wherein the C-18 hydroxy group is protected after formation of the 1-ester functional group. 16. The 1-aminomethylene group is converted into a 1-hydroxymethylene group, and the 1-hydroxymethylene group is temporarily protected to allow protection at the 18-hydroxy group, 14. The method of claim 13, wherein the protection is removed. 17. The 1-labyl, 10-hydroxy, 18-protected hydroxy, di-
The -6,8-en-5-one fused ring compound has the following formula (XIV): A method according to claim 1 prepared by starting from a 21-Nuc compound having the structure: wherein at least one ring A or E is quinoline and Nuc represents a nucleophile residue. Method. 18. The method of claim 17, wherein the compound of formula (XIV) is cyanosafracin B. 19. The product is of the formula (XXIIb): [Wherein R ¹ and R ⁴ are both the following formulas (IV), (V), (VI) or (VII): R ⁵ is —OH or a protected or derivatized version of such a group; R ^14a and R ^14b are both —H, or one is —H and the other is —OH, or A protected or derivatized version of such a group, -OCH ₃ or -OCH ₂ CH ₃ , or R ^14a and R ^14b together form a keto group; R ¹² is -NCH _3- ; R ¹⁵ is -OH or a protected or derivatized version of such a group; and
R ¹⁸ is —OH or a protected or derivatized version of such a group]. 20. The method of claim 19, wherein R ⁵ is alkanoyloxy of 1 to 5 carbon atoms. 21. R ⁵ is acetyloxy. 21. The method of claim 20. 22. The method according to claim 19, 20 or 21, wherein R ^14a and R ^14b are hydrogen. 23. The method according to any one of claims 19 to 22, wherein R ¹⁵ is hydrogen. 24. The method of any one of claims 19-23, wherein R ²¹ is -OH or -CN. 25. The method of claim 11, wherein R ⁷ and R ⁸ together form a —O—CH ₂ —O— group. 26. R ¹ and R ⁴ are both of the formula (IV): 26. The method according to any one of claims 19 to 25, wherein the group of 27. The method of any one of claims 1-26, wherein the ecteinascidin product is ecteinascidin 743. 28. The following scheme: A method step in the preparation of an ecteinascidin compound comprising removing both protecting groups in a single step according to [wherein Prot ^NH is an amino protecting group and Prot ^OH is a hydroxy protecting group]. 29. The method according to any one of claims 1 to 27, comprising the method step according to claim 28.