HU227195B1 - Cryptophycin derivatives and pharmaceutical compositions containing them - Google Patents

Abstract

Cryptophycin compounds of formula (I) are new: Ar = e.g. Ph, simple optionally substituted aromatic or heteroaromatic, heterocyclic, 1-12C alkyl, 2-12C alkenyl, 2-12C alkynyl, NR51R52, COR52, OR5 etc.; R51, R52 = H or 1-3C alkyl; R53 = 1-12C alkyl; R93, R94 = 1-6C alkyl; R30 = H or 1-6C alkyl; or NR30 = 3-7 membered cyclic ring; R1, R2 = halo, mono- or di-alkylamino, trialkylammonium, alkylthio, dialkylsulphonium, sulphate, phosphate, OR31, SR31, NR31, OH, SH, NR92, R93, NR94 or NH2; provided that 1 of R1 and R2 is OR31, SR31, R31, OH or SH; R1CCR2 = epoxide, aziridine, episulphide, sulphate, cyclopropyl or mono(1-6C)alkyl phosphate rings; or R1+R2 = bond; R3 = lower alkyl; R4, R5 = H or OH; or R4 + R5 = bond; R6 = e.g. benzyl, hydroxybenzyl, alkoxybenzyl, mono- or di halohydroxybenzyl, mono- or di-haloalkoxybenzyl, B-ring heteroaromatic, substituted heteroaromatic, B-ring 1-6C alkyl, optionally substituted 3-8C cycloalkyl, etc.; R7 = NR51R52, R53NR51R52, OR53, H or 1-6C alkyl; R51-R53 = 1-3C alkyl; R8, R10, R14 = H or lower alkyl; or R7 + R8 = cyclopropyl; R9 = H, optionally unsaturated lower alkyl, lower alkyl-(3-5C)cycloalkyl or benzyl; R11 = H, OH, lower alkyl, optionally substituted Ph or optionally substituted benzyl; R31 = P, S, 1-12C alkyl, B, R32 or Si; R32 = e.g. aminoacid, carbohydrate, amino sugar, (saccharide)q etc.; R50 = H or =O; q = 2-4; X = O, C (sic), S, NH or alkylamino; Y = C (sic), O, NH, S, SO, SO2 or alkylamino; with provisos.

Description

The present invention relates to the field of pharmacology and organic chemistry and provides novel cryptophycin compounds which are useful as antimicrobial agents.

One of the major causes of death in humans and mammals is cancerous disease, which is characterized by the fact that cell division is not under the control of normal cell growth regulation. Clinical practice in cancer chemotherapy requires new and more effective drugs.

The microtubular population of eukaryotic cells is an important component of the cell structure, which is dynamically composed and disintegrates; the heterodimers of tubulin polymerize and form a microtubule. Microtubules play a key role in cellular structure, cellular metabolism and division. The dynamic state of microtubules is a prerequisite for the normal functioning of cells. In terms of cell division, tubulin polymerizes to form microtubules forming mitotic spindles.

When the mitotic spindle yarns have already played their role, the microtubules depolymerize. Consequently, agents that inhibit microtubule polymerization or depolymerization and thereby inhibit mitosis include some of the most potent agents that can be used clinically in cancer chemotherapy.

The compounds of the invention also have fungicidal activity. They can also be used for research purposes because of their ability to destroy the microtubule system.

Certain cryptophycin compounds are known in the literature but would require greater solubility and stability in order to be used as a drug. A broader range of cryptophycin compounds would provide additional options for treating cancer patients.

We have discovered novel compounds that have improved water solubility and are capable of disrupting the microtubular system. The compounds of the present invention are useful in the treatment of neoplasms. These compounds can be prepared by a full synthetic process and are thus useful in the development of pharmaceutical agents.

The present invention relates to compounds of formula (I), or pharmaceutically acceptable salts or solvates thereof, wherein:

Ar is phenyl, unsubstituted aromatic, aromatic substituted with halo or C 1-7 alkyl, unsubstituted C 3-8 heteroaromatic, C 1-12 alkyl, C 2-12 alkenyl, or C 2-12 alkynyl, NR ⁵¹ R ⁵² or OR ⁵³ or Ar ';

R ¹ is halogen or OH;

R ² is halogen or -OR ³¹ with the proviso that one of R ¹ and R ² , but not both, is halogen;

R ³ is C 1-6 alkyl;

R ⁴ is hydrogen or hydroxy;

^R5 is hydrogen or hydroxy; obsession

R ⁴ and R ⁵ together may form a second bond between C-13 and C-14;

R ⁶ is benzyl, hydroxybenzyl, (C 1-3 alkoxy) benzyl, halogenated hydroxybenzyl, dihalogenated hydroxybenzyl, (halogenated C 1-3 alkoxy) benzyl, or (dihalogenated 1). C3-C4 alkoxy) benzyl;

R ⁷ is hydrogen or C 1-6 alkyl;

R ⁸ is hydrogen or C 1-6 alkyl;

R ⁹ is hydrogen or C 1-6 alkyl;

R ¹⁰ is hydrogen or C 1-6 alkyl;

R ¹¹ is hydrogen, R ³¹ is R ³² ; R ³² is a naturally occurring amino acid,

C ₁₂ H ₂₂ O _ir or C ₆ H ₁₀ O ₅ carbohydrate, 1- ₁₂ Η ₂₂ Ο ₁₁ or C ₆ H ₁₀ O ₅ carbohydrate with 1 to 3 amino substituents, (saccharide) _q wherein the saccharide is lactose, maltose, sugar, fructose or starch; or C (O) R ³³ ;

R ³³ is R ³⁷ R ³⁸ ,

R ³⁷ is C 1-6 alkyl;

R ³⁸ is COOR ^39, or Vil, NH ₂ or a naturally occurring amino acid;

R ³⁹ is hydrogen or C 1-6 alkyl;

R ⁴⁰ , R ⁴¹ and R ⁴² are each independently hydrogen, halogen, OR ⁴³ , NH 2, NO 2, OPO (OR ⁴⁶ ) 2, -OR ⁴⁴ -phenyl, or R ⁴⁵ or the general formula;

R ⁴³ is C 1-6 alkyl;

R ⁴⁴ is C 1-6 alkylene;

R ⁴⁵ is an unsubstituted aromatic group or an aromatic group substituted with hydrogen or C 1-7 alkyl;

R ⁴⁶ is hydrogen, sodium or -C (CH ₃ ) ₃ ;

R ⁵¹ is hydrogen or C 1-3 alkyl;

R ⁵² is hydrogen or C 1-3 alkyl;

R ⁵³ is C 1 -C 12 alkyl; R ⁵⁴ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO 3 H, SO ³ H, SO ² R ⁵⁸ , N (R ⁵⁹ ) R ⁶⁰ , NHOR ⁶¹ , NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen, OR ⁶² or SR ⁶³ or general formula;

R ⁵⁵ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO 3 H, SO ³ H, SO ² R ⁵⁸ , NR ⁵⁹ R ⁶⁰ , NHOR ⁶¹ ', NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen OR ⁶² or SR ⁶³ or a group of the general formula;

R ⁵⁶ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO ₃ H, SO ₃ H, SO ₂ R ⁵⁸ , NR ⁵⁹ R ⁶⁰ , NHOR ⁶¹ ,

HU 227 195 Β1

NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen, OR ⁶² or

SR ⁶³ or general formula;

R ⁵⁷ is hydrogen or C 1 -C 12 alkyl;

R ⁵⁸ is hydrogen or C 1 -C 12 alkyl;

R ⁵⁹ is hydrogen, C 1-6 alkyl, or fluorenylmethoxycarbonyl (FMOC);

R ⁶⁰ is hydrogen or C 1-6 alkyl;

R ⁶¹ is hydrogen, OR ⁶⁴ , CH 2 NHR ⁶⁵ , NHR ⁶⁵ or fluorenylmethoxycarbonyl (FMOC); R ⁶¹ is hydrogen, OR ⁶⁴ , CH 2 NHR ⁶⁵ , NHR ⁶⁵ or fluorenylmethoxycarbonyl (FMOC);

R ⁶² is hydrogen or C 1-6 alkyl;

R ⁶³ is hydrogen or C 1-6 alkyl;

R ⁶⁴ is hydrogen, C 1-6 alkyl, or CH ₂ NR ⁶⁶ R ⁶⁷ ;

R ⁶⁵ is hydrogen, C 1-6 alkyl, NH _2, or fluorenylmethoxycarbonyl (FMOC);

R ⁶⁶ is hydrogen, C 1-6 alkyl, or fluorenylmethoxycarbonyl (FMOC);

R ⁶⁷ is hydrogen or C 1-6 alkyl;

q is 2, 3 or 4;

X is O, NH, or (C 1 -C 7) alkylamino; and

Y is carbon or oxygen, NH, sulfur, SO, SO ₂ or (C 1 -C 7) alkylamino, or a pharmaceutically acceptable salt or solvate thereof.

The invention also relates to the treatment of neoplasm and fungal infections of such compounds in mammals.

The invention also relates to the use of the compounds for the preparation of a medicament for the treatment of neoplasm or fungal infections in a mammal.

The present invention also provides a composition comprising the compounds of the invention together with one or more pharmaceutically acceptable carriers or diluents.

The term "straight alkyl" refers to a C 1-7 alkyl group which may be saturated, unsaturated, branched or unbranched. Examples include methyl, ethyl, η-propyl, isopropyl, tert-butyl, sec-butyl, methylated butyl, pentyl, tert-pentyl, secententyl, methylated pentyl and the like, limited to those listed below. "Alkenyl" means an alkyl group, as defined above, having from 1 to 3 double bonds. "Alkynyl" means an alkyl group as defined above containing at least one triple bond. Particularly preferred is an alkynyl group having only one triple bond.

"C 1 -C n -alkyl", where n 'is an integer from 2 to 12, means an alkyl group in which the number of carbon atoms is within the specified range. The 1-n 'alkyl group may be straight or branched.

The term "B-ring (C 1 -C 6) alkyl" refers to a saturated, unsaturated, branched or straight-chain alkyl group wherein the ring (C 1 -C 6) alkyl may have up to 3 non-carbon substituents which preferably OH, SCH ₂ -phenyl, NH ₂ , CO, CONH ₂ , CO ₂ H, PO ₃ H _2, or SO ₂ R ²¹ or a group of general formula wherein R ²¹ is hydrogen or C 1-3 alkyl. .

The term "amino acid" refers to an organic acid containing an amino group. The term includes natural and synthetic amino acids, so the amino group may, but is not necessarily, attached to a carbon atom adjacent to the acid group. The amino acid residue is attached to the parent molecule via the acid function.

The term "carbohydrate-derived group" refers to substituents consisting of carbon, hydrogen and oxygen in which the hydrogen / oxygen ratio is equal to or nearly equal to the hydrogen / oxygen ratio of water. The term "carbohydrate" also includes an aldehyde or ketone alcohol or a compound which, upon hydrolysis, produces an aldehyde or ketone. The term "carbohydrate" is commonly understood by those skilled in the art. For example, the term may include, but is not limited to, ΰ ₂ ιΗ ₂₂ 0ιι or C ₆ H ₁₀ O ₅ .

By "amino sugar moiety" is meant a carbohydrate moiety having from 1 to 3 amino groups at any available position on the carbohydrate molecule.

The term "saccharide" refers to carbohydrate subunits that form disaccharides or polysaccharides. Examples include, but are not limited to, lactose, maltose, sucrose, fructose, starch, and the like.

The term "substituted phenyl" refers to a phenyl group having 1 to 3 non-hydrogen substituents independently selected from the group consisting of simple alkyl, chlorine, bromine and iodine.

The term "substituted benzyl" refers to a benzyl group having 1 to 3 non-hydrogen substituents, which may be independently alkyl, chlorine, bromine, fluorine or iodine, and may be attached to any suitable carbon atom. Some preferably substituted benzyl groups are also described. The term "alkoxybenzyl" refers to a benzyl group having an alkoxy substituent on any suitable carbon atom of the benzyl ring. Most preferred are C 1 -C 3 -O-alkyl groups. Especially preferred is methoxy. The term "halogenated alkoxybenzyl" follows 3

195 Β 1 represents a benzyl group which, in addition to the alkoxy group, also has a halogen substituent. Both the halogen atom and the alkoxy group can be attached to any suitable carbon atom. Similarly, the term "halo-hydroxybenzyl" means a benzyl group substituted by a hydroxy group which also has a halogen atom on any available carbon atom of the benzyl ring. Finally, the term "dihalogenated alkoxybenzyl" refers to an alkoxy-substituted benzyl group which independently has two halogen atoms which can be attached to any available carbon atom on the benzyl ring.

"Ring B-heteroaromatic group" means an aromatic ring containing one or more non-carbon atoms, which may be oxygen, nitrogen or sulfur. Particularly preferred heterocyclic rings include, but are not limited to, X (XV) wherein R ²⁰ is hydrogen or C 1-6 alkyl.

A particularly preferred "B-ring heteroaromatic group" is that of formula (X) - (XVII).

The term "substituted aromatic group" refers to a group substituted with 1-3 substituents wherein the substituent is a simple alkyl group or a halogen atom.

The term "cycloalkyl" means a saturated herein means a C3-8 cycloalkyl group which may be substituted by 0-3 substituents, each substituent independently is C 1-3 alkyl, halo, or ^OR22 group of the formula in which R ²² is hydrogen or 1-3 C 1 -C 4 alkyl. These substituents can be attached to any available carbon atom. Particularly preferred cycloalkyl is unsubstituted or substituted cyclohexyl.

The term "lower alkoxy" as used herein, means any C 1 -C 5 alkyl group bonded through an oxygen atom. The term "lower alkyl" is used to denote C 1-6 linear or non-linear hydrocarbon chains such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl. but not limited to, methyl, butyl, pentyl, tert-pentyl, secententyl or methylated pentyl.

The terms "unsaturated lower alkyl" and "saturated lower alkyl" are used interchangeably with the terms unsaturated and saturated. The term "lower alkyl" may also include saturated and unsaturated lower alkyl. (For example, a saturated group does not have a double or triple bond.) The term "allyl-substituted alkene" refers to any C 2 -C 7 -alkene group substituted with alkyl.

The term "epoxide ring" refers to a three-membered ring whose backbone is composed of two carbon atoms and one oxygen atom. The term "aziridine ring" means a three-membered ring whose backbone is composed of two carbon atoms and one nitrogen atom. The term "sulfide ring" is used herein to refer to a three-membered ring having a framework consisting of two carbon atoms and one sulfur atom. An "episulfide ring" is a three-membered ring whose backbone is composed of two carbon atoms and one sulfur atom. The term "sulfate group" as used herein refers to a five-membered ring having a carbon-carbon-oxygen-sulfur backbone and two further oxygen atoms attached to the sulfur atom. The term "monoalkyl phosphate ring" refers to a five membered ring having a carbon-carbon-phosphorous oxygen backbone and two additional oxygen atoms attached to the phosphorus atom, one of which has a lower alkyl group.

The term "simple unsubstituted aromatic group" means, but is not limited to, a single or double ring conjugated system having 4 n + 2 electrons, such as, for example, afuryl, pyrrolyl, thienyl, pyridyl, indolyl or naphthyl. those listed.

The term "substituted simple aromatic group" refers to a simple aromatic ring which is singly substituted with a halogen atom or a lower alkyl group.

The term "heteroaromatic group" is used to refer to aromatic rings containing one or more non-carbon atoms, namely, oxygen, nitrogen, and sulfur. Most preferred heteroaromatic groups form a 3- to 8-membered ring. Particular preference is given to groups having 3-6 ring members. Particularly preferred are those having 1-3 non-carbon atoms in the ring. The five membered ring containing one oxygen atom is one of the preferred heteroatom rings; however, this list is not intended to be a limitation on heteroaromatic groups.

The term "heterocyclic group" is used to refer to rings which contain one or more non-carbon atoms, which may be oxygen, nitrogen or sulfur. The heterocyclic ring may be saturated or unsaturated. Further, the heterocyclic ring may be fused to another ring to form a two or three ring system. For example, a five-membered ring may contain one or two double bonds. The heterocyclic ring may be unsubstituted or substituted with 1 to 3 substituents selected from the group consisting of C 1 -C 6 alkyl, carbonyl, OR ⁶² , and halogen. Most preferred heterocyclic rings are 3-8 members. Particularly preferred are 3-6 membered rings. Particularly preferred are heterocyclic rings containing from 1 to 3 non-carbon atoms. One preferred heterocyclic ring is a five membered ring substituted with one methyl atom containing one nitrogen atom, one sulfur atom, and two double bonds.

A preferred heterocyclic group is the formula (XIX), but preferred groups are not limited thereto.

The term "substituted heteroaromatic group" refers to a group substituted with 1 to 3 substituents wherein the substituent is a simple alkyl group or a halogen atom.

The term "halogen" or "halo" has historically been known as halogens

We mean members of 195 Β1. Halogenation includes, but is not limited to, addition of hydrogen halides, high-temperature substitution, photohalogenation, etc., as are known in the art. Particularly preferred halogen atoms are, but are not limited to, chlorine, fluorine and bromine.

The term "aryl", as used in the art, is an organic group derived from the removal of a hydrogen atom from an aromatic hydrocarbon. Examples include, but are not limited to, phenyl, tolyl, salicyl, and the like.

The term "mammal" refers to beings of the mammalian class of higher vertebrates. The term "animal" includes mammals, birds, amphibians, and fish. The term "mammals" refers to both mammalian animals and man. By "treating" is meant preventing or, once the condition has developed, relieving or eliminating said condition. The cryptophycin compounds of the present invention are useful in human and veterinary medicine.

If the desired R ⁶ substituent on the compound contains an amino group, it must be protected with a protecting group. Those skilled in the art can readily select the appropriate amino protecting groups in conventional literature, such as "Protective Groups Organic Chemistry" (Plenum Press, London and New York, 1973); Greene, TW using Protecting Groups in Organic Synthesis (Wiley, New York, 1981).

The term "derivatization" refers to a conventional chemical conversion known to those skilled in the art to provide the desired compound of the invention.

The methods of the invention provide a method for the fully synthetic preparation of cryptophycin compounds.

Advantageously, commercially available amino acids can be ring-closed to form a cryptophycin molecule.

Several known cryptophycin compounds have been shown to have anticancer activity, but their poor water solubility may cause problems when administered intravenously. Problems include, for example, the use of solubility-enhancing surfactants, such as Gremophor, which can cause toxicity. The present invention provides novel cryptophycin compounds having anticancer activity which have improved water solubility. These compounds have the desired solubility properties and acceptable activity.

It is most advantageous to prepare the compounds of the invention in the presence of a solvent. One skilled in the art can select the appropriate solvent for conventional procedures. Suitable inert organic solvents include, but are not limited to, tetrahydrofuran (THF) and dimethylformamide (DMF). DMF is particularly preferred. Some of the methods used herein may also use aqueous solvents. The pH of these aqueous solvents can be adjusted to facilitate the process.

Some typical compounds of the invention are listed in Table 1, but the list of said compounds is not intended to limit the scope of the invention.

Table 1 [The substituents R in the table refer to the compound of formula (2)]

Compound R Compound R * Λ 13 ff NHC0m9u 4 »4 J? • 1 ' XMCO · »Qu OHOItO euii 1 ' · reasons 17 \ NHCOl »Qu 'Xo N •MONTH 1 '

HU 227 195 Β1

Table 1 (continued) Compound R Compound the · Ό ml 19 9  Jk ^ NKOi ^ ew \ ( 20 CT · CM 21 10 W ' · UW 32 11 CO-au 23 12 20

MMCQpfti

ΝΗ »· MCI

Compound R

NHW HCl

Other preferred compounds are, for example, those listed in Table 1, but are not limited to those in which the adjacent Cl atom and OR group are reversed, i.e., those in which R is one of those listed, but with respect to the compound of formula 2a.

Other preferred compounds are those listed in Table 1, wherein Ar is a group of formula XLIII instead of phenyl wherein R ⁵⁴ is a para-position of OCH ₃ and R ⁵⁵ and R ⁵⁶ are hydrogen.

Also preferred are the compounds listed in Table 1, wherein Y is not an oxygen atom but an NH group, i.e. compounds of formula 2b.

HU 227 195 Β1

Other preferred compounds include, but are not limited to, those listed in Table 1 and obtained by the exchange of Cl and OR, wherein a single methyl group is present at the 6-position of the crypto ring instead of the geminal dimethyl group. See, for example, compound (23).

Particularly preferred compounds of the invention are those wherein the cryptophytic ring has a dimethyl group at the 6-position.

Preferred compounds are those listed in Table 1, wherein Ar is (XLIII); Only one of R ⁵⁴ , R ⁵⁵ and R ⁵⁶ is OCH ₃ ; R ⁹ and R ¹⁰ are each methyl.

Silylating agents used in the preparation of the compounds of the invention are generally known; see, for example, Calvin E.W., "Silicon Reagents in Organic Synthesis" (Academic Press, London, 1988). Particularly suitable silylating agents are "tri (lower) alkylsilyl" agents such as triisopropylsilyl, trimethylsilyl, triethylsilyl, trimethylsilyl halides, silylated ureas such as bis (trimethylsilyl). silyl) urea (BSU); and silylated amides such as N, 0bis (trimethylsilyl) acetamide (BSA). Of these, BSA is preferred.

Preferred compounds of the invention are those wherein the compound is characterized by one of the properties listed below. These properties are independently advantageous for the invention, but are not intended to be limiting. It is therefore advantageous to:

A) R ⁸ is ethyl, propyl, isopropyl, butyl, isobutyl or isopentyl.

B) R ⁷ is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl;

C) R ⁷ is hydrogen, R ⁸ is methyl, R ³ is methyl, and X and Y are both oxygen;

D) R ³ is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl;

E) R ⁹ is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl;

F) R ¹⁰ is methyl, ethyl, propyl, butyl, isobutyl, pentyl or isopentyl;

G) at least one of the carbon atoms C-3, C-6, C-7, C-10, C-16, C-17 or C-18 in the cryptophycin molecule has the R configuration (numbering is given in formula (I));

H) at least one of the C-3, C-6, C-7, C-10, C-16, C-17 or C-18 carbon atoms in the cryptophycin molecule has the S configuration;

I) the group XLIII is phenyl substituted with NR ⁵⁹ R ⁶⁰ , NHOR ⁶¹ or NHCHR ⁶¹ ;

J) Y is alkylamino, NH or oxygen;

K) Y is O; R ⁷ and R ¹⁰ are each hydrogen; R ⁹ is lower alkyl; R ¹ is halogen;

L) R ⁷ and R ⁸ are each methyl;

M) R ⁷ is hydrogen;

N) ^R2 is glicinátcsoport;

O) R ² is an acylate;

P) R ¹ and R ² together form an epoxide ring;

Q) R ⁶ is benzyl, hydroxybenzyl, alkoxybenzyl, halogenated hydroxybenzyl, dihalogenated hydroxybenzyl;

R) R ⁴ and R ⁵ together form a double bond;

S) n is 0; R ⁶ is a substituted benzyl group, one of which is halogen and the other is OR ¹² , wherein R ¹² is lower alkyl;

T) the compound of formula I is used to disrupt the microtubular system;

U) the compound of formula I is used as an anti-neoplasmic agent;

V) a compound of formula I for use in the treatment of cancer in a mammal;

W) the compound of formula I is used as an antifungal agent;

X) R ⁶ is a group of formula III 'and is substituted at the para-position with a hydroxy group; R ⁶ is a group of formula (XX) - (XXXII);

Z) Z is - (CH ₂ ) _n -, wherein n is 0;

AA) Z is - (CH ₂ ) _n -, wherein n is 2;

BB) Z is - (CH ₂ ) _n -, wherein n is 1;

CC) ^{R15, R16} and ^R17, at least one is CH ₃ -phenyl, NH _2, CO, C0NH _2, C0 ₂ H, PO ₃ H _2, an R ²¹ group of formula, in which R ²¹ is hydrogen or 1 C 3 -C 3 alkyl;

DD) Ar is phenyl;

EE) Ar is phenyl, mono- or di-substituted with OH, OCH ₃ , halo or methyl;

FF) R ² is halogen, amino, monoalkylamino, dialkylamino, trialkylamino, alkylthio, dialkylsulfonium, sulfate, phosphate, OR ³¹ R ³² or SR ³¹ R ³² ;

GG) R positioned at the connection to the kriptoficinmolekulához by group Z in the first group ^{of 6} carbons (XXXIII) spatial structure;

HH) a compound of formula I for use in the treatment of a fungal infection;

II) R ⁵⁰ is a group of structure (XXXIV);

JJ) R ¹¹ is hydrogen;

KK) R ⁴ and R ⁵ are each hydrogen; LL) Ar is phenyl substituted with ethyl at the para-position;

MM) Ar is phenyl substituted with methyl at the para-position;

NN) Y is NH;

00) R ³ is methyl;

PP) ^R6 is (XXX), (XXXV) - (XLI) R.

The present invention provides a method for ameliorating a pathological condition in a mammalian cell due to cell proliferation comprising administering to the patient an effective amount of a human or veterinary drug, thereby preventing cell proliferation.

HU 227 195 Β1

In a preferred embodiment of the invention, the method is supplemented with at least one additional therapy to alleviate the pathological condition. In a preferred embodiment of the invention, the pathological condition is characterized by the formation of neoplasms. In a further preferred embodiment of the invention, the neoplasm is selected from the group consisting of breast cancer, small cell lung tumor, non-small cell lung cancer, colon and rectal cancer, leukemia, melanoma, pancreatic adenocarcinoma, central nervous system, neoplasmic disease, gastrointestinal, including pancreas and esophageal, gastric, gastric, myeloma, bladder, kidney, neuroendocrine neoplasms, including thyroid and non-Hodgkins disease.

The term "neoplasmic" refers to growth neoplasm, the abnormal growth of which is caused by the proliferation of cells that are out of normal growth restriction. By "neoplasmic agent" is meant any compound, composition, mixture, mixture that inhibits, abolishes, inhibits or reverses the neoplasmic phenotype of a cell.

Based on their mechanisms of action, mitotic inhibitors can be divided into three groups. The first group includes substances, including colchicine and colcemide, which inhibit microtubule formation by binding to tubulin. The second group of substances, such as vinblastine and vincristine, induce the formation of paracrystalline aggregates of tubulin. Vinblastine and vincristine are well known anticancer drugs; their effect destroys the microtubules of mitotic spindle filaments, thereby inhibiting the proliferation of cells. The third group includes drugs, including taxol, which promote tubulin polymerization and thereby stabilize microtubules.

The drug-resistant and multiple drug-resistant phenotypic expression of multiple tumor cells and the clinically proven mode of action of anti-microtubule agents against neoplasmic cells require the development of anti-microtubule agents that are equally cytotoxic to non-drug-resistant neoplasmic cells.

Chemotherapy, surgery, radiation, biological response modifiers, and immunotherapy are currently used to treat cancer. Each therapeutic mode has its own specific indication well known to those skilled in the art, and one or all of them can be used to attempt to destroy neoplasmic cells. Combination chemotherapy, when used in combination with other anti-neoplastic agents of the formula I, is also within the scope of the invention, since combined therapy is generally more effective than the use of an anti-neoplasm agent alone. Thus, the present invention also provides the above-mentioned beneficial compositions comprising a therapeutically effective amount of at least one compound of formula I or a non-toxic addition salt thereof. These compositions may also contain physiologically acceptable liquid, gel or solid carriers, diluents, additives, binders. Such carriers, additives and binders can be found in U.S. Pat. Pharmacopoeia, XXII. and National Formulary Volume XVII, US. Pharmacopoeia Convention, Inc., Rockville, MD (1989). Further treatments are described in the AHFS Drug Information (American Hospital Formulary Service, pages 522-660, 1993). All of these are well known and readily available to those skilled in the art.

It is a further object of the present invention to provide a pharmaceutical composition for treating a neoplasmic disease comprising at least one compound of formula (I) and at least one additional neoplasmic agent. The anti-neoplasmic agents that can be used in combination with the compound of formula (I) are listed in Merck Index 11, pages 16-17. (Merck & Co., Inc., 1989). The Merck Index is widely known and readily available to those skilled in the art.

In another embodiment of the invention, the antineoplasmic agents may be antimetabolites including methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine, arabinose, hydroxyurea, and 2-chlorodeoxyadenosine. may include, but is not limited to, those listed below. In another embodiment of the invention, the intended antineoplasmic agent is, but is not limited to, an alkylating agent such as cyclophosphamide, mephalan, busulfan, paraplatin, chlorambucil and nitrogen mustard. In another embodiment, the anti-neoplasmic agent is a plant alkaloid such as vincristine, vinblastine, taxol, and etoposide, but is not limited to those listed. In another embodiment, the intended anti-neoplastic agent is an antibiotic such as doxorubicin, daunorubicin, mitomycin C or bleomycin, but is not limited to those listed. In a further embodiment, the intended antineoplasmic agent is a hormone such as calusosterone, diomastavolone, propionate, epithiostanol, mepithiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, feldspar, or not.

In another embodiment, the intended anti-neoplasmic agent is an enzyme, which may be, but is not limited to, L-asparaginase or aminoacridine derivatives such as amsacrine. Other neoplasmic agents include those listed by Skeel, Roland T., in the Handbook of Cancer Chemotherapy Handbook of Antineoplastic Drugs and Biologic Response Modifier: Classification, Use and Toxicity of Clinically Useful Agents, 3rd Brown, Co., 1991 ).

These compounds and compositions may be administered to mammals for veterinary purposes. For example, pets can be used for treatment in the same way,

HU 227 195 Β1 as in human medicine. The dose required to produce a therapeutic effect will generally vary with the type of application, the route of administration, and the particular requirements of the organism being treated. The dosage will generally be in the range of 0.001 to 1000 mg / kg, preferably 0.01 to 10 mg / kg body weight. Within this range, doses may be administered by continuous infusion over a period of time necessary to achieve the desired therapeutic effect; this is usually 24 hours. Dosage and route of administration of the active ingredient based on relative efficacy, relative toxicity, tumor growth characteristics, cell cycle activity of the compound of formula (I), pharmacokinetics of the active ingredient, age, sex, physical condition of the subject being treated and preventive treatments. determine what a specialist can do.

The compounds of formula (I) may be formulated as such or in the form of their salts with or without other antineoplasmic agents. Pharmaceutically acceptable non-toxic salts include base addition salts which may be derived from inorganic bases such as sodium, potassium, ammonium, calcium or iron (III) hydroxides and organic bases such as isopropylamine, trimethylamine. , 2-ethylaminoethanol, histidine, procaine, etc. Such salts may also be acid addition salts with any free cationic group. Inorganic acids, such as hydrochloric or phosphoric acids, and organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like may be used in the salt formation. Examples of additives are described in U.S. Pat. Pharmacopoeia.

The preferred carrier for a particular pharmaceutical composition will be determined by the preferred mode of administration. For example, anti-neoplasmic compositions may be prepared for oral administration; such compositions are generally in the form of a liquid solution or suspension or in solid form. Additives for oral formulations generally include binders, fillers, carriers, preservatives, stabilizers, emulsifiers, buffers, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, and generally contain from 1% to 95%, more preferably from 2% to 70%, of the active ingredient.

The compositions of the present invention may be injectable; or in the form of a liquid solution, suspension or emulsion, or in the form of a solid preparation which is capable of being dissolved or suspended in a liquid before injection. These injectable formulations may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, or intrapleural. The active ingredient or compounds are often mixed with physiologically acceptable diluents, carriers or excipients which are compatible with the active ingredients. Suitable diluents and media include water, saline, dextrose, glycerol and the like, or combinations thereof. Further, the composition may, if necessary, contain minor amounts of additives, for example wetting or emulsifying agents, stabilizers or buffers.

The present invention further provides methods of inhibiting the proliferation of mammalian cells with a compound of formula (I), comprising contacting the mammalian cell with an amount of a compound of formula (I) necessary to inhibit its proliferation. A preferred embodiment is a method of inhibiting the proliferation of hyperproliferative mammalian cells. The term "hyperproliferative mammalian cell" refers to mammalian cells that are not subject to the typical limitations of growth (e.g., programmed cell death). In another preferred embodiment, the mammalian cell is a human cell. The invention further provides a contact between the mammalian cell and at least one compound of formula (I) and at least one antineoplasmic agent. Types of intentional anti-neoplasmic agents are discussed above.

The present invention further provides methods of inhibiting the proliferation of hyperproliferative cells of the drug-resistant phenotype, including cells having a multiple drug-resistant phenotype, by inhibiting the proliferation of hyperproliferative mammalian cells by the compound of formula (I). get in touch. In a preferred embodiment, the mammalian cell is a human cell. The invention further provides contact between the cell and a compound of Formula I and at least one additional neoplasmic agent discussed above.

The present invention provides a method of ameliorating pathological conditions caused by hyperproliferative mammalian cells, comprising administering to said organism a pharmaceutical composition comprising an amount of a compound of formula I that inhibits the proliferation of hyperproliferative cells. By "pathological condition" is meant any pathological condition resulting from the proliferation of mammalian cells excluded from normal growth restriction. Such cell division may be related to the neoplasms discussed above.

In a preferred embodiment of the invention, the neoplasmic cells are human cells. The present invention provides a method of alleviating such pathological conditions by combining the compounds of formula (I) with other therapies and other anti-neoplastic agents.

The efficacy of the compounds of the invention can be determined by known, conventional methods. The following are examples of such methods.

The compounds of the present invention have been found to be useful against pathogenic fungi. Thus, for example, the utility in the treatment of Cryptococcus neoformans infection is demonstrated by the results of an assay on Cryptococcus neoformans grown on yeast nitrogen-based dextrose / agar. For carrying out the assays, the compound of the present invention

HU 227 195 Β1

Dissolve in dimethylsulfoxide containing Tween 20. Dilute with 10% DMSO in sterile distilled aqueous solution to give a dilution series of 0.008-16.0 pg / ml for agar dilution assays. To demonstrate the desired antifungal activity, the minimum inhibitory concentration against 84 Cryptococcus neoformans isolates was determined.

Compounds are screened to determine minimum inhibitory concentrations against KB, a human nasopharyngeal carcinoma cell line, and LoVo, a colorectal adenocarcinoma cell line, using the Corbett assay (Corbett, TH et al., Models and Concepts for Drug Discovery and Development, pp. 35-87, Kluwer Academic Publishers, Norwell, 1992; Valeriote et al., "Discovery and Development of Anticancer Agents," Kluwer Academic Publisher, Norwell, 1993).

The most potent compounds are further tested for their cytotoxicity against four different cell types, such as murine leukemia, solid murine tumors, human solid tumors, and mildly malignant fibroblasts, using the Corbett assay.

Further evaluation of the compounds is assayed on a broad spectrum using murine tumors and human tumors implanted in mice, including drug resistant tumors.

Tumor load (T / C) (the tumor load of treated animals versus that of untreated conditions) is used for further evaluation. Treatments with a T / C of less than 42% are considered effective by the National Cancer Institute Standards, and those of less than 10% are considered excellent and have potential clinical activity.

Materials

Antibodies to vinblastine, cytocalazine B, tetramethyl rhodamine isothiocyanate (TRITC) -phalloidin, sulfurodamine B (SRB), and β-tubulin and vimentin are commercially available from known vendors. "Basal Medium Eagle" (BME) medium containing Earle's salts and Fetal Bovin Serum (FBS, bovine embryo serum) are also commercially available.

cell Lines

Jurkat T cell leukemia cell line and A-10 rat aortic smooth muscle cells are available from the American Type Culture Collection (ATCC) and can be cultured in BME containing 10% FBS and 50 pg / ml gentamicin sulfate. Human ovarian carcinoma cells (SKOV3) and the vinblastine-resistant subunit (SKVLB1) were dr. Gift from Victor Ling, Ontario Cancer Institute. Both cell lines were maintained in BME medium containing 10% FBST and 50 pg / ml gentamicin sulfate. Twenty-four hours after the passage of SKVLB1 cells, vinblastine was added to a final concentration of 1 pg / ml to maintain the cells overexpressing P-glycoprotein under selection pressure.

Investigation of cell division and cycle inhibition

Cell division assays were performed according to the method of Skehan et al. For Jurkat cells, the cells are treated with the respective agents as described by Skehan and the total number of cells is counted by counting the cells in a hemocytometer. The percentage of mitotic cells in PBS was determined by staining with 0.4% Giema and subsequent washing with fast PBS. To determine the mitotic state, at least 1000 cells per treatment are evaluated and the mitotic index is calculated as the number of cells in the mitotic state relative to the total cell number.

Immunofluorescence studies

A-10 cells were grown to near complete growth on a glass cover plate in BME containing 10% FBS. In PBS, the final concentration of compounds indicated was added to the cultures and incubated for an additional 24 hours. For staining of microtubules and intermediate filaments, cells were fixed with cold methanol and incubated with PBS containing 10% fetal bovine serum to block non-specific binding sites. The cells were then maintained at 37 ° C for 60 minutes in the presence of anti-T-tubulin or antivimentin at the concentrations specified by the manufacturers. The bound primary antibodies are then visualized by incubation with fluorescein-conjugated rabbit anti-mouse IgG for 45 minutes. The coverslips were placed on microscopic slides and examined for fluorescence using Zeiss Photomicroscope 111 with epifluorescence optics for fluroescein. For staining of the microfilaments, cells were fixed in 3% paraformaldehyde, made permeable to 0.2% Triton Χ-100, and reduced with sodium borohydride (1 mg / ml). 100 nM TRITC-phalloidin PBS solution was added to the cells and kept at 37 ° C for 45 minutes. The cells were washed quickly with PBS before placing the coverslips under the microscope and immediately photographed as described above.

Effect of cryptophycins and vinblastine on Jurkat cell proliferation and cell cycle

The dose-dependent effects of cryptophycin compounds and vinblastine on cell division and percentage of cells in mitosis were determined.

Effects of cytokalazine B, vinblastine and cryptophycins on cell structure

Aortic smooth muscle cells (A-10) were grown on glass coverslips and treated with PBS, 2 μΜ cytocalazine B, 100 nM vinblastine or 10 nM cryptophycin. After 24 hours, microtubules and vimentin intermediate filaments were visualized by indirect immunofluorescence, and the microfilaments were stained with TRITC-phalloidin. For each active substance, the morphological activity is examined. Untreated cells show a strong microtubule network with nucleus microtubule organizing centers. Vimentin Intermediate Filamentu10

The microfilaments are bundled along the principal axis of the cell. Cytokalazine B completely depolymerizes microfilaments with the accumulation of paracrystalline residues. This compound has no effect on the distribution of microtubules or intermediate filaments. As a result of cryptophycin, microtubule populations are cleared and vimentin intermediate filaments collapse.

Effect of cryptophycins and vinblastine on taxol-stabilized microtubules

A-10 cells were treated with 0 or 10 μΜ taxol for 3 hours and then PBS, 100 nM vinblastine or 10 nM cryptophycin were added. After 24 hours, microtubule organization is assayed by the immunofluorescence method described above. Compared to control cells, the microtubules of cells treated with taxol are strongly bundled, especially in the polar regions of the cells. Vinblastine, as above, causes complete depolymerization of microtubules in untreated cells. Taxol treatment, on the other hand, prevents vinblastine from depolymerizing microtubules. Similarly, taxol pre-treated microtubules can be observed after treatment with cryptophycin.

Reversibility of vinblastine or cryptophycin depolymerization of microtubules

Treatment of A-10 cells with 100 nM vinblastine or 10 nM cryptophycin for 24 hours results in complete depolymerization of the microtubules. The cells are then washed and kept in drug-free medium for 1 or 24 hours. After removal of vinblastine, microtubules rapidly repolymerize, reaching significant levels within one hour, and returning to full morphology within 24 hours. Microtubular status of cells treated with the cryptophycins of the invention can be visualized at 1 or 24 hours after cryptophycin removal.

Combined effect of vinblastine and cryptophycin on cell division

SKOV3 cells were treated with cryptophycins and vinblastine in combination for 48 hours. For each combination, the percentage of surviving cells is determined and the IC _{50S is} calculated.

Toxicity of cryptophycin, vinblastine and taxol is

SKOV3 and SKVLB1 cells

SKVLB1 cells are resistant to natural anticancer drugs due to overexpression of p-glycoprotein. Taxol, vinblastine and cryptophycins were observed to inhibit the growth of SKOV3 and SKVLB1 cells. Taxol both cell lines proliferation SKOV3 cells were dose-dependently inhibited by 1 nM to 8000 nM IC _50S SKVLB1 cells value. Vinblastine also inhibits the growth of both cell lines, SKOV3 cells at 0.35 nM, SKVLB1 cells at 4200 nM IC _50S . The cryptophycins of the invention are SKOV3 and

Activity against SKVLB1 cells characterized by using a value from 1 to 1000 pM IC _5.

Thus, it is demonstrated that the cryptophycins of the invention are potential inhibitors of cell proliferation, disrupting the microtubular network and inhibiting mitosis. These studies can demonstrate that cryptophycins disrupt the microbubular system and thereby normal cellular function, including mitotic functions.

Classical antimicrotubular drugs such as collicin and Vinca alkaloids prevent cell division in mitosis. It seems appropriate to compare the effects of one of these agents on cell division with that of cryptophycins. For this purpose, vinblastine, a class of Vinca alkaloids, was selected from the classical antimicrotubular agents. Thus, we compare the effects of cryptophycins and vinblastine on cell division and cell cycle progression of cells in the Jurka T leukemia cell line.

Because the anti-mitotic activity is usually accomplished through disruption of the mitotic spindle microtubules, the effect of cryptophycins on the cell structure is examined by fluorescence microscopy. Immunofluorescence staining of cells treated with cryptophycin or vinblastine demonstrates that both compounds cause complete loss of microtubules. Similar studies on SKOV3 cells show that the antimicrotubular effect of cryptophycins is not unique to the smooth muscle cell line.

Screening with GC3 Human Colon Cancer Cells One hour before the addition of the test compound, selected sites in the 96-well plate were inoculated with GC3 human colon cancer cells (1x10 cells / well in 100 μΙ assay medium). Cell-free media was added to other wells. Assay medium (RPMI1640 medium used, but any medium in which cells survive) was supplemented with 10% dialyzed bovine embryo serum and 25 mM HEPES buffer.

The test compound is stored in a yellow vial until use. Immediately before dilution with phosphate-buffered saline (PBS), a fresh stock solution of dimethylsulfoxide (200 pg / ml) was prepared. Dilute the dimethylsulfoxide solution in PBS 1:20 to give a final concentration of 10 pg / ml. Prepare a dilution series of 1: 3 dilutions using PBS for dilution. Falcon 2054 tubes are used for the assays.

In triplicate, 10 μΙ diluted test compound solution was added to GC3 cells and incubated at 37 ° C for 72 hours. 10 μl of a solution of 3- [4,5-dimethyl-2-yl] -2,5-diphenyltetrazolium bromide (5 mg "MTT" / ml in PBS) was added to each well. The plates were maintained at 37 ° C for one hour. After centrifugation of the plates, the medium was discarded and 10 μΙ acidic isopropanol (0.04 N hydrochloric acid in isopropanol) was added to each well. Color intensities were measured at 570 nm (SpectroMax reader) within one hour.

HU 227 195 Β1

The evaluation of the compounds of formula (I) demonstrates their usefulness in the treatment methods required by the present invention. Further, these compounds are useful for disrupting the microtubular system.

The compounds of the present invention may be prepared by a variety of methods using an activated ester followed by deprotection, if necessary, of an acid after chromatography (Figure 8).

For example, compound 1 (thick numbers of compounds in the examples) is reacted with acetic anhydride in the presence of triethylamine and 4-dimethylaminopyridine to give compound 3 after flash chromatography. Similarly, compound (4) is prepared from compound (1) using succinic anhydride and purification by reverse phase HPLC. Reaction of the pyridine solution of Compound (1) with commercially available nicotinoyl chloride hydrochloride in the presence of triethylamine and 4-dimethylaminopyridine afforded Compound (7) in high yield after chromatography. The pyridinium salt (8) was prepared in a 47% yield using the Nicolaou method [Angew. Chem. Int. Ed. Engl., 33 (1994)], wherein compound (1) is reacted with a commercial 2-fluoro-1-methylpyridinium / p-toluenesulfonate complex and purified by reverse phase HPLC to anion exchange. p-toluenesulfonate (exchanged for acetate) and lyophilized. The esters (5), (9), (11), (13), (15), (17), (19) and (21) can all be obtained in moderate or high yield from N- and commercially available N-. from a tert-Boc protected amino acid (except for (5) and (9)) via activation of 1,3-dicyclohexylcarbodiimide and in the presence of 4-dimethylaminopyridine.

The hydrochloride salts of (10), (12), (14), (16), (18), (20) and (22) are prepared in high yields from (9), (11), (13), (15), (17), (19) and (21) in 4.0 M hydrochloric acid in dioxane and the solvent was removed under reduced pressure. The disodium salt (6) is derived from compound (5) by cleaving the tert-butyl ester group with hydrochloric acid and reacting the resulting product with sodium hydroxide. The acid (24) required for the preparation of compound (5) was synthesized in 63% yield in a 5-step process using the phosphate function (Johns, Synthesis, 142, 1988).

The in vitro cytotoxicity of some new conjugates was tested on the GC3 tumor cell model. The results summarized in Table 2 clearly show that this series of compounds exhibits excellent activity relative to Cryptophycin-55 (1).

Any ester of type (2) (wherein R ¹ and R ^{2 are} derived from a carboxylic acid) can be prepared by a variety of processes using acid chlorides, anhydrides and commonly used activating agents (e.g. carbodiimides). Any solvent other than the alcohol involved may be used. Any mild base and / or catalyst (amines, carbonates) may be used to facilitate the esterification.

The carbamates (9), (11), (13), (15), (17), (19) and (21) can be converted into the corresponding salts with any strong acid, namely mineral acid, including hydrogen halides, hydrogen sulfates such as hydrogen phosphates, hydrogen nitrates, hydrogen perchlorates, or strong organic acids such as trifluoroacetic acid, p-toluenesulfonic acid and methanesulfonic acid. The same acids can be used to prepare the type (7) salts from free bases. Various counterions (cations) may be involved in the formation of the type (6) salts, including the cation of any alkali metal or alkaline earth metal. Various counterions (anions) may be used to form the salts of type (8), namely anions of any organic or inorganic acid.

Table 2

In vitro toxicity of cryptophycin derivatives (assays performed as described above)

We have a staff GC3 IC _50S (nM) First 0,065 Third 83 4th 31 6th 3.7 7th 116 8th 2.2 10th - 12th 0.10 14th 21 16th 230 18th 2.6 20th - 22nd 7.2 26th -

Other compounds of the invention have efficacy results in the GC3 assay:

Compound IC ₅₀ (μΜ) Cryptophycin-141 .7950 Cryptophycin-138 .0308 Cryptophycin-139 0.0034 Cryptophycin-140 0.863 Cryptophycin-143 0,850 Cryptophycin-142 0.0011 Cryptophycin-145 0.000015 Cryptophycin-129 -

Other compounds of the invention have IC ₅₀ values of less than 1 to 700 nM in GC3 assays, most commonly having IC ₅₀ values below 100 nM.

The solubilities of compounds (27), (1) and (12) were compared and the results show the increased solubility of compound (12). The results are shown in Table 3.

HU 227 195 Β1

Table 3

Comparison of solubilities of cryptophycin compounds (1), (12) and (27)

Additive to 0.05 M acetate buffer % (v / v) Solubility of Cryptophycins (mg / ml) (27) (1) (12) 1 propylene glycol ethanol 15 10 MHA MHA > 2.0 2 propylene glycol polyethylene glycol 300 15 20 MHA 0.0026 > 2.0 3 polysorbate 80 1 (w / v) MHA .3113 1-2 4 Emulphor EL719 1 (w / v) .0272 .3611 1.5 0.05 M acetate buffer pH 4.0 100 <0.001 <0.001 0.03

mha - below the measuring limit

The stability of the compounds (1) and (12) was tested at pH 4-8 with the aqueous solution maintained for 6 hours at room temperature. The results clearly demonstrate the excellent water solubility and stability of compound (12). For example, under pH 8 conditions, the solubility of Cryptophycin-55 at room temperature is 10 mg / ml, and that of Compound 12 is 30 mg / ml. At pH 4, the water solubility of Cryptophycin-55 is 50 mg / ml, however, the solubility of Cryptophycin-55 decreases as the pH becomes basic, while the water solubility (12) is substantially constant over the pH range tested.

On the basis of the solubility and stability studies, suitable parenteral carriers were selected to determine the absolute solubility / stability characteristics of Compound (12). Table 4 summarizes the solubility of Compound 12 in these supports. Preliminary studies indicate that Compound (12) is acceptable in formulation 6 for 3 weeks.

Table 4

Solubility of cryptophycin glycinate (12) in various parenteral vehicles

preparation Excipients % (v / v) (12) solubility in mg / ml First ethanol 10 0.11 propylene glycol 10 0.05 M citrate buffer pH 4.0 additional volume Second ethanol 10 1.99 propylene glycol 10 0.05 M acetate buffer pH 4.0 additional volume Third ethanol 10 1.29 0.05 M acetate buffer pH 4.0 additional volume 4th propylene glycol 10 1.58 0.05 M acetate buffer pH 4.0 additional volume 5th polysorbate 80 0.5 (v / v) 2.21 0.05 M acetate buffer pH 4.0 additional volume 6th ethanol 10 10.27 propylene glycol 25 0.05 M acetate buffer pH 4.0 additional volume

HU 227 195 Β1

Table 4 (continued)

preparation Excipients % (v / v) (12) solubility in mg / ml 7th ethanol 10 4.32 propylene glycol 15 0.05 M acetate buffer pH 4.0 additional volume

Accordingly, high concentrations of the compound of the present invention in a vehicle that does not contain a surfactant or emulsifier are achievable while allowing easy toxicological and clinical evaluation of these compounds. The glycinate ester (12) also exhibits improved stability in physiological aqueous media over a longer period, indicating an increased shelf life under normal conditions. Concentrates of compounds (1) and (27) must be prepared for storage and diluted immediately prior to administration. Ready-to-use solutions of the compounds of the invention can be prepared.

Compounds of formula (I) may be prepared from compounds of formula (II) wherein:

Ar, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁷ , R ⁸ , R ¹⁰ are as defined in formula (I);

R ¹³ is tert-butyl carbamate (BOC);

R ²⁴ is a group of formula (XLII) derived from N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide or salts thereof, 2-nitrophenyl, 4-nitrophenyl or 2-nitrophenyl; , 4-dichloro-phenyl;

X is O, NH or alkylamino; 35

Y is O, NH or alkylamino.

The compound of formula (III) wherein R and various substituents have the same definitions as described above may be prepared by reacting the compound of formula (IV) wherein R ²⁵ is an acid of formula (V) wherein R ²⁷ is hydrogen, C 1 -C 12 alkyl or aryl and is reacted with a silylating agent. A particularly preferred silylating agent is N, O-bis (trimethylsilyl) acetamide 45 (BSA).

The term "active ester substituent" refers to a substituent that makes the indicated substituent a good leaving group. Suitable substituents may be selected from commonly used manuals such as Protective Groups in Organic Chemistry (Plenum Press, London and New York, 1973); Greene T. W., Protecting Groups in Organic Synthesis (Wiley, New York, 1981); see in particular Greene's book 180-184. side. A particularly preferred active ester substituent is the group derived from N-hydroxysuccinimide (NHS). Other preferred groups include, but are not limited to, -ON-hydroxysuccinimide, -ON-hydroxysulfosuccinimide XLIV, and salts thereof, -O-2-nitrophenyl, -O- -nitro-60-phenyl and -0-2,4-dichlorophenyl, where - "O" - represents the oxygen atom required to form the ester functionality.

The term "amide" as used herein refers to an amide function which can be cleaved under alkaline conditions. Examples include, but are not limited to, -NMe ₂ . Further examples are found in Greene TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981).

The "active ester substituent" refers to a substituent that makes the substituent OR ^{24 a} good leaving group. Suitable substituents may be selected, for example, from the following manuals: "Protective Groups in Organic Chemistry" (Plenum Press, London and New York, 1973); Greene, TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981). A particularly preferred R ²⁵ group is the N-hydroxysuccinimide (NHS) group.

The processes described herein are preferably carried out in the presence of a solvent. One skilled in the art can select the appropriate solvent for the above procedures. Inert organic solvents are particularly preferred, but in some circumstances an aqueous solvent may be suitable. For example, when R ²⁷ is hydrogen and R ¹³ is BOC, it is effective as an aqueous solvent.

When the desired R ⁶ substituent in the compound of formula (I) contains an amine, the amine substituent on the R ⁶ group must be protected by an amino protecting group. One skilled in the art can readily select the appropriate amino protecting group from, for example, Protective Groups in Organic Chemistry (Plenum Press, London and New York, 1973); Greene, TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981).

The group R ²⁷ must be a group which allows the removal of the group -CO ₂ R ^{27 under} acidic, neutral or weakly basic conditions. Preferred R ²⁷ substituents include, but are not limited to, hydrogen, C 1-6 alkyl, trichloromethyl, trichloroethyl, or methylthiomethyl. It is particularly preferred that R ²⁷ is hydrogen.

Compounds of formula (I) may be prepared from compounds of formula (II) wherein:

Ar, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁷ , R ⁸ , R ¹⁰ are as defined in formula (I);

R ¹¹ is as defined above for R ¹¹ ;

R ¹³ is an amino protecting group;

HU 227 195 Β1

R ²⁴ is an active ester substituent, amide substituent, -O-2-nitrophenyl, -O-4-nitrophenyl, or -O-2,4-dichlorophenyl;

X is O, NH or alkylamino;

Y is carbon, sulfur, oxygen, NH, SO, SO ₂ or alkylamino.

The compound of formula (III) wherein R and the various substituents have the same definitions as described above may be prepared by reacting a compound of formula (IV) wherein R ²⁵ is an acid of formula (V) wherein R ²⁷ is hydrogen, C 1 -C 12 alkyl or aryl and is reacted with a silylating agent. A particularly preferred silylating agent is N, O-bis (trimethylsilyl) acetamide (BSA).

The term "active ester substituent" in R ²⁵ refers to a substituent that makes the substituent OR ^{24 a} good leaving group. Suitable substituents may be selected, for example, from the following manuals: "Protective Groups in Organic Chemistry" (Plenum Press, London and New York, 1973); Greene, TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981). A particularly preferred group R ^{25 is the} group derived from N-hydroxysuccinimide (NHS).

The processes described herein are preferably carried out in the presence of a solvent. One skilled in the art can select the appropriate solvent for the above procedures. Inert organic solvents are particularly preferred, but in some circumstances an aqueous solvent may be appropriate. For example, when R ²⁷ is hydrogen and R ¹³ is BOC, an aqueous base is effective as a solvent.

When the desired R ⁶ substituent in the compound of formula (I) contains an amine, the amine substituent on the R ⁶ group must be protected by an amino protecting group. One skilled in the art can readily select the appropriate amino protecting group from, for example, Protective Groups in Organic Chemistry (Plenum Press, London and New York, 1973); Greene, TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981).

The group R ²⁷ must be a group which allows the removal of the group -CO ₂ R ^{27 under} acidic, neutral or weakly basic conditions. Preferred R ²⁷ substituents include, but are not limited to, hydrogen, C 1-6 alkyl, trichloromethyl, trichloroethyl, or methylthiomethyl. It is particularly preferred that R ²⁷ is hydrogen.

9 and 10 are provided as further illustration.

In the formulas of Figure 10 and throughout this specification, R ¹ is halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, protected OH or protected SH; R ² is OH or SH; R ²⁶ is an alcohol protecting group which is added at an appropriate time in the synthesis to protect the alcohol group that would otherwise react during the reaction and then deprotected at a later stage of the synthesis. Numerous reactions for the deprotection and removal of such protecting groups have been described in commonly used literature, for example: "Protective Groups in Organic Chemistry" (Plenum Press, London and New York, 1973); Greene TW, Protecting Groups in Organic Synthesis (Wiley, New York, 1981). It is mainly through these jobs that the skilled person can select the appropriate group to protect the alcohol. One particularly suitable alcohol protecting group is tert-butyldimethylsilyl (TBS). Derivatives of the products of the reaction processes depicted in the drawings may be prepared by conventional methods to yield other cryptophycin compounds.

See steps 11, 12 and 11 for further reaction steps

13.

In the formulas of the figures, R ⁶ and R ¹¹ are as defined herein.

Further derivatives of the products of the reaction processes depicted in the figures may be prepared by conventional methods to yield additional cryptophycins.

From the drawings and the examples described below, one of ordinary skill in the art will be able to select appropriate starting materials and reagents.

For example, the starting ester can be prepared according to the reaction scheme of Figure 14.

Further guidance is given in the reaction sequence of Figure 1. Abbreviations commonly used in the figures and examples are:

DMAP: 4-dimethylaminopyridine

BOC: tert-butoxycarbonyl mcPBA: m-chloroperbenzoic acid

TMSCI: Chlorotrimethylsilane

HEW: Homer-Emmons-Wadsworth Reaction (a standard reaction for aldehyde olefin formation using phosphonate and base)

TMG: 1,1,3,3-tetramethylguanidine (base commonly used in the HEW reaction)

DIBAL: diisobutylaluminum hydride (the usual reagent for reducing an unsaturated ester to allyl alcohol)

SAE: "Sharpless" Asymmetric Epoxidation (for Enantioselective Epoxidation of Allylic Alcohols)

TBS: tert-butyldimethylsilyl

TBS-Otf: TBS trifluoromethanesulfonate (standard reagent for tert-butyldimethylsilylation of alcohols)

AIBN: 2,2'-azobis (isobutyronitrile) (common root initiator)

ACN: acetonitrile

DBU: 1,8-diazabicyclo [5.4.0] undec-7-ene (standard amine base)

EDCI: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

The preparation of the ester is shown in Figure 1.

The reaction illustrated in the figure is not intended to be the only synthesis of the indicated phenyl

EN 227 195 Β1 ring. One skilled in the art can use this process extensively to obtain a suitable starting material for the preparation of the compounds of the invention.

The time required for the reaction depends on the starting material and the temperature. The optimum time for a given process is a trade-off that takes into account the goals of throughput, which have a short reaction time and a maximum yield, while having a long reaction time.

Certain compounds of the present invention may also be prepared biosynthetically. For example, Cryptophycin-3 is treated with 65 mg of bromosuccinimide in DME / water 2: 1 at 0 ° C (about 0.24 mmol, 15.0 mL) and allowed to warm to room temperature. After 24 hours, the solvent is removed and the residue is dissolved in a solvent such as acetonitrile and chromatographed on reverse phase HPLC (eluent: acetonitrile / water 65:35, preferably 6 mLg / min). The mixture of cryptophycins was separated by standard phase HPLC (hexane / ethyl acetate, 1: 1, 3 ml / min) to give the desired cryptophycins.

Similarly, Cryptophycin-3 (40 mg, 0.063 mmol) was dissolved in DME / water (2: 1, 6 mL), N-chlorosuccinimide (0.075 mmol) was added and kept at 60-70 ° C for 24 hours. An additional 8 mg of NCS was added and the heating continued for another 24 hours. The solvent was removed and the residue was separated by HPLC chromatography (35% water in acetonitrile, 6 mL / min) to give the desired cryptophycins. The resulting mixture was further purified by HPLC eluting with 4: 1 methanol: water to give the cryptophycins of the desired purity.

The following are examples to illustrate what has been said. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

Step 1

Methyl-5-phenyl-pent-2 (E) -enoát

A solution of trimethylphosphonoacetate (376 g, 417 mL, 2.07 mol) in tetrahydrofuran (750 mL) was stirred in a 3 L round neck flask at 0 ° C. The flask is equipped with a mechanical stirrer and a nitrogen inlet. To the cooled solution, 239 g (260 mL, 2.07 mol) of tetramethylguanidine was added dropwise via an addition funnel. The cooled pale yellow solution was stirred for 25 minutes at 0 ° C. 90% Hydrocinnamaldehyde (250 g, 248 mL,

A 1.9 molar solution of tetrahydrofuran was added dropwise to the reaction mixture. After the addition is complete, the reaction mixture is stirred at room temperature for 10 hours. Gas chromatography studies show a 95: 5 ratio of product to starting material. Water (500 mL) was added to the reaction mixture and stirred for 1 hour and then the layers were allowed to separate. The organic phase is separated off and the aqueous phase is extracted with tert-butyl methyl ether. The organic layers were combined, dried over magnesium sulfate and evaporated under reduced pressure to give an orange oil.

The crude product was distilled at 0.3 ° C (129 mm Hg) to give 360.5 g (91.7%) of product as a clear, pale yellow oil.

EIMS m / z: 190 (13; M +), 159 (410), 158 (39), 131 (90),

130 (62), 117 (22), 104 (12), 95 (57), 91 (100), 77 (21), 65 (59).

HREIMS m / z 190.0998 (C ₁₂ H ₁₄ O ₂ D-0.4 mmu).

UV /. _max : (e) 210 (8400), 260 (230).

IR: 3027, 2949, 1723, 1658, 1454, 1319, 1203, 978,

700 cm ^-1 .

¹ H-NMR (CDCl ₃ ), δ:

7.15-7.3 (Ph-H5, bm), 7.00 (3-H, dt, 15.6 / 6.6), 5.84 (2-H, dt, 15.6 / 1, 2), 3.70 (OMe, s), 2.76 (5-H2, t,

7.2), 2.51 (4-H2, bdt, 6.6 / 7.2).

¹³ C-NMR (CDCl ₃ ), δ:

166.9 (1), 148.3 (3), 140.6 (Ph-T), 128.4 / 128.2 (Ph

2737576 '), 126.1 (Ph 4'), 121.4 (2), 51.3 (OMe),

34.2 / 33.8 (4/5).

Step 2

5-Phenyl-pent-2-en-1-ol

In a 12-neck four-necked flask equipped with thermocouple, mechanical stirrer and nitrogen inlet, 310.5 g (1.5 mol) of enoate ester

A solution of 1.5 L of tetrahydrofuran was cooled to -71 ° C in an isopropanol / carbon dioxide bath. To the reaction mixture was added dropwise a solution of 2.5 L (3.75 mol) of DIBAL in toluene at a rate such that the temperature remained below -50 ° C. After the addition was complete, the reaction mixture was stirred at -50 ° C overnight. TLC (silica gel; hexane: ethyl acetate = 3: 1) shows that starting material disappears from the reaction mixture within 16 hours. The reaction mixture was allowed to return to -15 ° C and 150 ml of 1 N hydrochloric acid was added slowly. The reaction mixture then forms a gelatinous solid. The semisolid was crushed with a spatula and 200 mL of 1N hydrochloric acid was added to make the mixture more fluid. Adding 625 ml of concentrated hydrochloric acid to this product separates it into two phases. The phases were separated and the product was extracted with t-BuOMe. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give 247.8 g of a pale yellow oil. The crude product was distilled at 145 ° C / 0.25 mm Hg to give 209.7 g (86.2%) of the desired product.

EIMS m / z 162 (1: M +) 144 (16), 129 (7), 117 (9), 108 (6), 92 (17), 91 (100), 75 (5), 65 (12) .

HREIMS m / z 162.1049 (C C HH ^ OO, D -0.4 mmu).

UV /. _max (e) 206 (9900), 260 (360). IR: 3356, 2924,

1603, 1496, 1454, 970, 746, 700 crrr ¹ . ¹ H-NMR (CDCl ₃ ), δ:

7.15-7.3 (Ph-H5, m), 5.70 (3-H, dt, 15.6 / 6.0), 5.61 (2-H, dt, 15.6 / 4, 8), 4.02 (1-H2, d, 4.8), 2.68 (5-H2, t,

7.2), 2.40 (OH, bs), 2.36 (4-H2, dt, 6.0 / 7.2). ¹³ C-NMR (CDCl ₃ ), δ:

141.6 (Ph 1 '), 131.8 (3), 129.5 (2), 128.3 / 128.2 (Ph 2737576'), 125.7 (Ph 4 '), 63.3 (1 ), 35.4 / 33.8 (4/5).

HU 227 195 Β1

Step 3 (2S, 3S) -2,3-Epoxy-5-phenyl-1-pentanol

To a 3 liter round-bottomed flask equipped with a mechanical stirrer, a thermocouple and an inlet of nitrogen are added 350 ml of dichloromethane and 30 g of 4 Angstroms molecular sieve and 7.62 g (0.037 mol) of L - (+) - diethyl tartrate are added. . The reaction mixture was cooled to -20 ° C and treated with titanium (IV) isopropoxide (2.9 mL, 0.031 mol) followed by 4.0 M tert-butyl hydroperoxide in dichloromethane (182 mL, 0.78 mol). The addition is carried out at such a rate that the temperature remains at -20 ° C. After the addition is complete, the reaction mixture is stirred for 30 minutes and then 50 g (0.31 mol) of allyl alcohol in 30 ml of dichloromethane are added at a rate such that the temperature of the reaction mixture is -20 ° C. The reaction mixture was stirred at this temperature for 5 hours and then filtered into a solution of 132 g of ferric sulfate and 40 g of tartaric acid in 400 ml of water at 0 ° C. The reaction mixture was stirred for 20 minutes then transferred to a separatory funnel and extracted with 2 x 200 mL of tert-butyl methyl ether. The combined organic phases were stirred with 30% sodium hydroxide in sodium chloride for 1 hour at 0 ° C. The phases are separated again and the aqueous phase is extracted with tert-butyl methyl ether. The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated to give 52.8 g of an amber oily product.

Step 4 (2R, 3R) -2-Hydroxy-3-methyl-5-phenylpentan-1-ol In a 5 L round neck flask equipped with mechanical stirrer, thermocouple and nitrogen inlet, 1 L of hexane and 0 ° C were added. Cool to C. 800 ml (1.6 mol) of a 2.0 M solution of trimethylaluminum in hexane are added, followed by a solution of epoxide (120 g, 6.77 mol) in hexane (250 ml) and dichloromethane (50 ml) at a temperature below 20 ° C. After the addition was complete, the cloudy reaction mixture was stirred at 5 ° C for 30 minutes, followed by the dropwise addition of 300 ml of 10% hydrochloric acid followed by 350 ml of concentrated hydrochloric acid. The phases are separated and the organic phase is washed with brine and dried over magnesium sulfate. The volatiles were evaporated under reduced pressure to give 122.1 g of an oily product.

Step 5 (2R, 3R) -2-Hydroxy-3-methyl-5-phenylpent-1-yl tosylate In a 2 L round neck flask equipped with mechanical stirrer and nitrogen inlet, 58 g (0.30 g) was weighed. mole) of diol, 1.5 g (0.006 mole, 2 mol%) of dibutyltin oxide, 57.5 g (0.30 mole) of toluenesulfonyl chloride, 580 ml of dichloromethane and 42.0 ml of 0.30 mole). mole) of triethylamine. The resulting reaction mixture was stirred at room temperature for 2 hours (although the reaction was complete within 1 hour), filtered, washed with water and dried over magnesium sulfate. Removal of volatile constituents under reduced pressure afforded 104.1 g of a pale amber oil.

Step 6 (2R, 3R) -2 - [(tert-Butyldimethylsilyl) oxy] -3-methyl-5-phenylpent-1-yl tosylate

To a solution of 100 g (0.29 mol) tosylate and 81.0 ml (0.58 mol) triethylamine in 1200 ml dichloromethane was added 99 ml (0.43 mol) TBS-OTf dropwise and the mixture was stirred for 20 minutes. to be continued. The reaction mixture was washed twice with brine, dried over magnesium sulfate and evaporated to dryness. The oily residue was dissolved in a minimal amount of hexane, filtered through a pad of silica gel, eluting with 9: 1 hexane: ethyl acetate to give 134 g of product as a light amber oil.

Step 7 (2R, 3R, 5RS) -2 - [(tert-Butyldimethylsilyl) oxy] -3-methyl-5-bromo-5-phenylpent-1-yl tosylate in a 5 L three-necked round bottom flask, equipped with a mechanical stirrer, a reflux condenser and a nitrogen inlet, 1680 ml of carbon tetrachloride, 140 g (0.30 mole) of TBS Ts, 65 g (0.365 mole) of NBS and 16.5 g (0.10 mole) are weighed. AIBN. The reaction mixture was degassed under reduced pressure and stirring and then saturated with nitrogen, repeating this process three times. The reaction mixture is then refluxed while the color changes to dark brown. After vigorous reflux for 15 minutes, the reaction mixture turned pale yellow and chromatographic analysis showed complete reaction. After cooling to room temperature, the reaction mixture is filtered and the filtrate is evaporated to dryness. The residue was dissolved in hexane, filtered again and evaporated to give 170.3 g of product as an amber oil.

Step 8 (2R, 3R) -2 - [(tert-Butyldimethylsilyl) oxy] -3-methyl-5-phenylpent-4 (E) -en-1-yl tosylate in a 2 L three-necked round bottom flask with mechanical stirrer, reflux and nitrogen inlet, weigh 100 g (0.186 mol) of bromide in 700 ml of acetonitrile, add 83.6 ml (0.557 mol) of DBU and reflux for 15 minutes. After the solution had cooled to room temperature, the solvent was removed under reduced pressure, the residue was dissolved in dichloromethane (200 mL) and filtered through a pad of silica gel. The volatiles were evaporated again and the residue was dissolved in ethyl acetate. The solution was washed with water, brine, dried over magnesium sulfate and evaporated to dryness under reduced pressure. Preparative HPLC (Prep 500) affords 50.3 g (60% based on 4 steps above) of the desired unsaturated compound.

Step 9 (3S, 4R) -3 - [(tert-Butyldimethylsilyl) oxy] -4-methyl-6-phenylhex-5 (E) -en-1-nitrile g (0.11 mol) tosylate was dissolved in 1 liter of dimethyl sulfoxide and 14.2 g (0.22 mol) of potassium cyanide and 25 ml of water were added. The reaction mixture was stirred at 60 ° C under nitrogen for 18 hours.

HU 227 195 Β1

After the reaction mixture has cooled to room temperature, it is partitioned between 1 L of ethyl acetate and 1 L of water. The aqueous layer was extracted with ethyl acetate (500 mL) and the combined organic layers were washed with brine and dried over sodium sulfate. Flash chromatography on silica gel, eluting with dichloromethane, afforded the desired nitrile in 92% yield.

Step 10

Methyl - [(5S, 6R) -5 - [(tert-butyldimethylsilyl) oxy] -6-methyl-8-phenyl-octa-2 (E), 7 (E) -dienoát]

Nitrile (14.67 g, 46.5 mmol) was dissolved in toluene (200 mL) and cooled to -78 ° C under nitrogen. Under vigorous stirring, 37.2 mL of 1.5M DIBAL in toluene (55.8 mmol) is added dropwise. After the addition was complete, the cooling bath was removed and the reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was carefully poured into 1N hydrochloric acid and stirred for 30 minutes at room temperature. The phases are separated and the organic phase is washed twice with a saturated aqueous solution of sodium potassium tartrate and then with brine and dried over sodium sulfate. The volatiles were removed under reduced pressure and the pale yellow oily product was used directly for the next condensation step. The above crude aldehyde was dissolved in 90 mL of tetrahydrofuran and 9.03 mL (55.8 mmol) of trimethyl phosphonoacetate and 7.0 mL (55.8 mmol) of tetramethyl guanidine were added at room temperature under nitrogen. The reaction mixture was stirred for 16 hours and then partitioned between ethyl acetate (200 mL) and water (100 mL). The aqueous layer was extracted with ethyl acetate (100 mL), and the organic layers were combined, washed with water, brine, and dried over sodium sulfate. Removal of volatile constituents under reduced pressure gave 17.0 g of a crude yellow oil. This oily product is chromatographed on silica gel using a dichloromethane / cyclohexane mixture for gradient elution with a 1: 1 to 2: 1 ratio. 13.67 g (78.5%) of the desired ester is obtained.

Example 2

The methyl ester (2.673 mmol) was dissolved in acetone and 26 mL of 1 N aqueous lithium hydroxide solution was added at room temperature. The cloudy mixture was diluted with acetone (20 mL) and the resulting yellow reaction mixture was stirred at room temperature for 23.5 hours. The reaction mixture was diluted with diethyl ether (400 mL), and the organic layer was washed with 1N hydrochloric acid (120 mL), brine (200 mL) and water (160 mL). The organic phase is dried, concentrated under reduced pressure and the yellow oily residue is purified by column chromatography. A yellow oil was obtained, which was purified by column chromatography (gradient elution: 5% acetic acid + 20-40% ethyl acetate / hexane). Yield: 960 mg (100%) of the carboxylic acid as a yellow oil.

¹ H-NMR (CDCl ₃ ) δ, ppm:

7.38-7.19 (m, PhH ₅ ), 7.09 (ddd, J = 15.2, 7.6 and

7.9 Hz, 3-H), 6.38 (d, J = 16 Hz, 8-H), 6.16 (dd,

J = 16 and 8 Hz, 7-H), 5.85 (d, J = 15.8 Hz, 2-H),

3.81-3.75 (m, 5-H), 2.49-2.37 (m, 6-H, 4-CH ₂ ),

1.12 (d, J = 6.7 Hz, 6-Me), 0.91 (s, SiCMe ₃ ), 0.065 (S, SiMe), 0.068 (S, SiMe).

IR (CHCl ₃ ): 2957, 2930, 2858, 1697, 1258, 1098,

838 cm ¹ .

MS (FD): 360.2 (M ⁺ , 100).

[α] _D = 87.6 ° (c = 10.5, CHCl ₃ ).

Analysis for C ₂₁ H ₃₂ O _3: Calc'd: C: 69.95, H: 8.95%; Found: C, 69.19; H, 8.39.

Example 3 A solution of carboxylic acid (mmol) in dry dimethylformamide (5.50 mL) was treated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (2.4 mmol) and N-hydroxysuccinimide (2.6 mmol) at room temperature. After stirring for 28 hours, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with 1N aqueous hydrochloric acid (2 x 50 mL), water (75 mL), dried and concentrated under reduced pressure. The resulting oily product was purified by column chromatography (gradient elution: 5-30% ethyl acetate / hexane). 724 mg (80%) of the active ester were obtained as a pale yellow oil.

¹ H-NMR (CDCl ₃ ) δ, ppm:

7.36-7.20 (m, PhH ₅ , 3-H), 6.38 (d, J = 16 Hz, 8-H),

6.14 (dd, J = 16.1 and 8.0 Hz, 7-H), 6.03 (d, J = 16 Hz,

2-H), 3.79 (q, J = 4.3 Hz, 5-H), 2.94 (br s, CH ₂ CH ₂ ),

2.58-2.42 (m, 6-H, 4-CH _2), 1.10 (d, J = 6.8 Hz,

6-Me), 0.90 (s, SiCMe ₃ ), 0.05 (s, SiMe ₂ ).

IR (CHCl ₃ ): 2957, 2931, 2858, 1772, 1741, 1648, 1364,

1254, 1092, 1069, 838 cm ^-1 .

MS (FD): 457 (M ⁺ , 100).

[α] _D = + 71.3 ° (c = 10.1, CHCl ₃ ).

Analysis for C ₂₅ H ₃₅ NO ₅ Calcd: C: 65.61, H: 7.71, N: 3.06%; Found: C, 65.51; H, 7.56; N, 3.02.

Example 4

To a solution of 2.50 g (5.47 mmol) of silyl ether in 130 ml of acetonitrile at 0 ° C is added 15 ml of 48% aqueous hydrogen fluoride. The solution was stirred at 0 ° C for 0.75 hours and then at room temperature for 4 hours. The reaction mixture was diluted with diethyl ether (300 mL) and washed with water until the pH of the wash water reached pH 7. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give a yellow product which was recrystallized from diethyl ether to give 1.46 g (78%) of alcohol as a white crystalline solid.

¹ H-NMR (CDCl ₃ ) δ, ppm:

7.41-7.20 (m, PhH ₅ , 3-H), 6.48 (d, J = 1.6 Hz, 8-H),

6.15-6.07 (m, 7-H, 2-H), 3.71-3.65 (m, 5-H), 2.83 (brs, CH ₂ CH ₂ ), 2.60-. 3.33 (m, 6-H, 4-CH _2), 1.95 (br s, 5-OH), 1.14 (d, J = 6.8 Hz, 6-Me).

IR (KBr): 3457, 1804, 1773, 1735, 1724, 1209, 1099,

1067, 1049, 975, 744, 694 cm in ^{the first}

UV (EtOH) /. _max : 250 (ε = 20535) nm.

MS (FD): 343.2 (M ⁺ , 100).

[α] _D = -57.8 ° (c = 10.56, CHCl ₃ ).

HU 227 195 Β1

Analysis calculated for C ₁₉ H ₂₁ N ₅ O ₅ S: C, 66.46; H, 6.16; N, 4.08. Found: C, 66.49; H, 6.16; N, 4.07.

Example 5

Preparation of Cryptophycin-55-acetate (3) To a solution of (1) mg (0.13 mmol) in methylene chloride (659 mL) at 0 ° C was added triethylamine (55 mL, 0.40 mmol), 1.6 mg (0.013 mmol). 4-dimethylaminopyridine and 19 mL (0.20 mmol) of acetic anhydride. After stirring at 0 ° C for 1 hour, methanol (19 mL) was added to the reaction mixture and the volume was evaporated to 0.5 volume. This reaction mixture was flash chromatographed directly on 19 g of silica gel using 3: 1 ethyl acetate: hexane as eluent. 88 mg (89%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.38-7.31 (m, 5H), 7.24 (d, 1H, J = 2.1 Hz),

7.22-7.18 (m, 1H), 7.10 (dd, 1H, J = 8.5, 2.1 Hz), 6.88 (d, 1H, J = 8.5 Hz), 6 , 75 (ddd, 1H, J = 15, 13,

4.6 Hz), 5.78 (dd, 1H, J = 15, 1.0 Hz), 5.55 (d, 1H, J = 7.9 Hz), 5.46 (dd, 1H, J = 9, Δ, 1.2 Hz), 4.95 (dd, 1H, J = 11, 2.9 Hz), 4.89 (ddd, 1H, J = 9.9, 9.9,

1.7 Hz), 4.81 (d, 1H, J = 9.8 Hz), 4.79-4.74 (m, 1H), 3.91 (s, 3H), 3.39 (dd, 1H, J = 13, 8.1 Hz), 3.22 (dd, 1H, J = 13.4.1 Hz), 3.16 (dd, 1H, J = 14, 5.1 Hz), 3.07 ( dd, 1H, J = 14, 7.6 Hz), 2.65-2.55 (m, 2H), 2.47-2.39 (m, 1H), 1.95 (ddd, 1H, J = 14, 13,

4.6 Hz), 1.86-1.77 (m, 1H), 1.73-1.66 (m, 1H), 1.68 (s, 3H), 1.27 (s, 3H), 1, 19 (s, 3H), 1.09 (d, 3H, J = 7.1 Hz), 1.03 (d, 3H, J = 6.7 Hz), 0.97 (d, 3H, J = 6) , 6 Hz).

Example 6

Preparation of Cryptophycin-55-Succinate (4) To a solution of (1) (27 mg, 0.038 mmol) and succinic anhydride (5.7 mg, 0.057 mmol) in methylene chloride (383 mL) was added triethylamine (16 mL, 0.115 mmol) and , 7 mg (0.038 mmol) of 4-dimethylaminopyridine. After stirring for 19 hours, additional succinic anhydride (5.7 mg, 0.057 mmol) and 4-dimethylaminopyridine (4.7 mg, 0.038 mmol) were added and stirring was continued for another 29 hours. To the reaction mixture was added 0.5 mL of 1 N aqueous hydrochloric acid and washed three times with 0.5 mL of methylene chloride. The combined organic phases were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a white foam. This product was purified by reverse phase HPLC to give 10 mg (32%) of the title compound as a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.36-7.31 (m, 6H), 7.22 (br, s, 1H), 7.08 (d, 1H, J = 8.4 Hz), 7.02 (br, s, 1H) , 6.87 (d, 1H, J = 8.4 Hz), 6.61 (m, 1H), 5.94-5.87 (m, 2H), 5.51 (d, 1H, J = 9 , 8 Hz), 4.95 (dd, 1H, J = 10, 2.8 Hz), 4.87-4.76 (m, 3H), 3.90 (s, 3H), 3.39 (dd) , 1H, J = 16, 5.6 Hz), 3.28 (dd, 1H, J = 16, 8.2 Hz), 3.17 (dd, 1H, J = 16,

5.6 Hz), 3.05 (dd, 1H, J = 16, 8.2 Hz), 2.68-2.62 (m,

1H), 2.60-2.46 (m, 2H), 2.45-2.28 (m, 3H),

2.01-1.93 (m, 2H), 1.88-1.70 (m, 2H), 1.26 (s, 3H),

1.18 (s, 3H), 1.12 (d, 3H, J = 7.0 Hz), 1.04 (d, 3H,

J = 6.6 Hz), 1.00 (d, 3H, J = 6.5 Hz).

Example 7

Preparation of Cryptophycin-55- (2'-di-tert-butylphosphatyl) phenylacetate (5)

To a solution of 0.102 mmol of Compound 1, 46 mg (0.134 mmol) of Compound 24 and 12 mg (0.102 mmol) of 4-dimethylaminopyridine in 250 mL of dry methylene chloride is added 27 mg (0.134 mmol) of 1.3. dicyclohexylcarbodiimide in 50 ml of methylene chloride. After stirring at room temperature for 6 hours, the reaction mixture was diluted with 1 mL of ethyl acetate / hexane (3: 1) and filtered through a pad of celite and washed with ethyl acetate / hexane (3: 1). The filtrate and the washings were evaporated under reduced pressure to give a red foam. Flash chromatography on silica gel (15 g) using ethyl acetate / hexane (4: 1) as eluent. 86 mg (82%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.35 (d, 1H, J = 8.3 Hz), 7.30-7.19 (m, 8H), 7.11 (dd,

1H, J = 8.4, 2.0 Hz), 7.02 (t, 1H, J = 7.5 Hz), 6.87 (d,

1H, J = 8.4 Hz), 6.84 (d, 1H, J = 7.5 Hz), 6.73 (ddd,

1H, J = 15, 13, 4.7 Hz), 5.92 (d, 1H, J = 7.9 Hz), 5.79 (dd, 1H, J = 15.1.0 Hz), 5, 43 (dd, 1H, J = 9.4.1.8 Hz),

4.98 (dd, 1H, J = 12, 3.1 Hz), 4.81 (ddd, 1H, J = 9.9,

9.9, 1.8 Hz), 4.75 (d, 1H, J = 9.4 Hz), 4.73-4.67 (m,

1H), 3.90 (s, 3H), 3.49 (d, 1H, J = 16Hz), 3.44-3.38 (m, 1H), 3.38 (d, 1H, J = 16 Hz), 3.27-3.17 (m, 2H),

3.10 (dd, 1H, J = 14, 8.2 Hz), 2.55-2.46 (m, 2H),

2.37-2.27 (m, 1H), 1.95 (ddd, 1H, J = 14, 12,

4.5 Hz), 1.83-1.70 (Μ, 2H), 1.49 (s, 18H), 1.27 (s,

3H), 1.20 (s, 3H), 1.03 (d, 3H, J = 6.5 Hz), 0.97 (d,

3H, J = 6.4Hz), 0.92 (d, 3H, J = 7.0Hz).

Example 8

Preparation of Cryptophycin-55- (2'-Phosphatyl) Phenylacetate Sodium Salt (6) To a solution of 5 (mg) (0.081 mmol) in 400 mL of methylene chloride was added 81 mL of a 4.0 M solution of hydrogen chloride in 1,4-dioxane. 0.33 mmol). The pale yellow solution was stirred at room temperature for 2 hours. After evaporation under reduced pressure, the off-white foam, the crude dihydrogen phosphate, was dissolved in 614 mL of tetrahydrofuran and 33 mL of 5.00 N aqueous sodium hydroxide solution (0.163 mmol) was added. After stirring for 10 minutes, the reaction mixture was concentrated to give a tan foam. The crude salt was taken up in a hot mixture of 1 mL of acetonitrile and 0.1 mL of water. The insoluble material was filtered off and the filtrate was concentrated under reduced pressure to give 69 mg (87%) of the title compound as a white solid. ¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.58 (d, 1H, J = 8.2 Hz), 7.38-7.32 (m, 2H),

7.32-7.28 (m, 3H), 7.28 (d, 1H, J = 2.1 Hz), 7.17 (dd,

1H, J = 8.5, 2.1 Hz), 7.09 (ddd, 1H, J = 7.8, 7.8,

1.7 Hz), 6.98 (d, 1H, J = 8.5 Hz), 6.79-6.70 (m, 2H),

HU 227 195 Β1

6.67 (d, 1H, J = 7.4 Hz), 5.91 (dd, 1H, J = 15.1.7 Hz),

5.45 (dd, 1H, J = 9.4, 1.6 Hz), 5.06 (dd, 1H, J = 10,

2.7 Hz), 5.01 (d, 1H, J = 9.4 Hz), 4.89-4.80 (m, 1H),

4.47 (dd, 1H, J = 11, 3.8 Hz), 3.84 (s, 3H), 3.67 (d,

1H, J = 16Hz), 3.45 (d, 1H, J = 14Hz), 3.42 (d, 1H,

J = 16 Hz), 3.18 (dd, 1H, J = 14, 3.8 Hz), 3.12 (d, 1H,

J = 14 Hz), 2.77 (dd, 1H, J = 14, 11 Hz), 2.67-2.60 (m, 1H), 2.56-2.48 (m, 1H), 2 , 31-2.22 (m, 1H),

1.96-1.88 (m, 1H), 1.85-1.77 (m, 2H), 1.22 (s, 3H),

1.20 (s, 3H), 1.03 (d, 3H, J = 6.2 Hz), 0.98 (d, 3H,

J = 6.1 Hz), 0.93 (d, 3H, J = 7.1 Hz).

Example 9

Preparation of Cryptophycin-55-Nicotinoate Hydrochloride (7) To a solution of 1 mg (0.071 mmol) in pyridine (354 mL) at room temperature was added 15 mg (0.085 mmol) of nicotinoyl chloride hydrochloride followed by 23 mL (0.170 mmol) of triethylamine. . After stirring for 1.5 hours, 4-dimethylaminopyridine (8.6 mg, 0.071 mmol) was added to the reaction mixture, and after stirring for 5 hours, another triethylamine (23 mL, 0.170 mmol), 8.6 mg (0.071 mmol) was added. dimethylaminopyridine and 15 mg (0.085 mmol) of nicotinoyl chloride hydrochloride were added, which was rinsed with 50 ml of pyridine. After stirring for 18 hours, 0.5 ml of a saturated aqueous sodium bicarbonate solution was added to the reaction mixture, which was washed with 4 x 1 ml of methylene chloride. The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to a light brown oil. The product was purified by flash chromatography on silica gel (14 g) eluting with 10: 1 ethyl acetate: hexane. 49 mg (85%) of the free base are obtained in the form of a white foam. The nicotinoate was dissolved in 1 mL of methylene chloride and 90 mL of a 1.0 M solution of hydrogen chloride in diethyl ether (0.090 mmol) was added. The clear, colorless solution was allowed to stand at room temperature for 5 minutes. Removal of the solvent under reduced pressure gave 51 mg of the title compound as a white foam. ¹ H-NMR (500 MHz, CDCl ₃ ), δ:

8.94 (s, 1H), 8.78 (br s, 1H), 8.29 (d, 1H, J = 7.0 Hz),

7.57 (br s, 1H), 7.38 (d, 2H, J = 7.1 Hz), 7.30-7.16 (m, 5H), 7.10 (dd, 1H, J = 8 , 4, 1.7 Hz), 6.88 (d, 1H,

J = 8.4 Hz), 6.71 (m, 1H), 5.80 (d, 1H, J = 15 Hz), 5.74 (d, 1H, J = 9.6 Hz), 5.56 (br s, 1H), 5.00 (d, 1H,

J = 9.6 Hz, 4.95 (t, 1H, J = 8.9 Hz), 4.84 (d, 1H,

J = 9.8 Hz), 4.77-4.72 (m, 1H), 3.91 (s, 3H), 3.39 (dd, 1H, J = 13.8.2 Hz), 3, 23-3.14 (m, 2H), 3.06 (dd,

1H, J = 14, 7.6 Hz), 2.81-2.74 (m, 1H), 2.62-2.45 (m, 2H), 1.93 (ddd, 1H, J = 14, 12, 4.8 Hz),

1.78-1.70 (m, 1H), 1.66-1.59 (m, 1H), 1.25 (s, 3H),

1.20 (d, 3H, J = 7.0 Hz), 1.19 (s, 3H), 0.98 (d, 3H,

J = 6.7 Hz), 0.84 (d, 3H, J = 6.5 Hz).

Example 10

Preparation of Cryptophycin-55-N-methylpyridinium acetate (8) To a solution of compound (1) (mg, 0.075 mmol) in methylene chloride (751 mL) at 0 ° C was added triethylamine (13 mL, 0.090 mmol), 0.083 mmol) of p-toluenesulfonate. The heterogeneous reaction mixture was warmed to room temperature and stirred for 3.5 hours, followed by the addition of 11 mg (0.039 mmol) of 2-fluoro-methylene-pyridinium p-toluenesulfonate.

After stirring for 14.5 hours, it is added again

2-Fluoro-1-methylpyridinium ptoluenesulfonate II (II) (0.039 mmol) followed by 2-fluoro-1-methylpyridinium ptoluenesulfonate (11 mg, 0.039 mmol) and triethyl (13 mL, 0.090 mmol) after 2.5 hours amine. After stirring for 1 hour, the reaction mixture was concentrated under reduced pressure to give an orange foam. Purification by reverse phase HPLC exchanged the p-toluenesulfonate anion for acetate anion and freeze-dried to give 40 mg (47%) of the title compound as a white solid.

¹ H-NMR (500 MHz, DMSO-d ₆ ), δ:

8.65-8.58 (m, 2H), 8.36 (t, 1H, J = 7.8 Hz), 7.68 (d, 1H, J = 8.9 Hz), 7.60 (d , 1H, J = 6.6 Hz), 7.48 (t, 1H, J = 6.6 Hz), 7.35-7.21 (m, 6H), 7.19 (dd, 1H, J = 8.5, 1.9 Hz), 7.05 (d, 1H, J = 8.5 Hz, 6.49 (ddd, 1H, J = 16, 13, 4.0 Hz), 5.91 (d , 1H, J = 16Hz), 5.72 (d, 1H, J = 8.0Hz), 5.66 (dd, 1H, J = 8.0, 1.9Hz), 5.32-5 , 27 (m, 1H), 4.73 (dd, 1H, J = 9.7, 4.3 Hz), 4.24 (ddd,

1H, J = 11, 9.8, 3.7 Hz), 3.93 (s, 3H), 3.81 (s, 3H), 3.32 (dd, 1H, J = 13, 9.3 Hz) ), 3.05-2.97 (m, 2H), 2.77-2.57 (m, 3H), 2.54-2.47 (m, 1H), 1.76 (s, 3H), 1.68-1.62 (m, 1H), 1.55-1.46 (m, 1H), 1.37-1.30 (m, 1H), 1.15 (d, 3H, J = 7 , 0 Hz), 1.13 (s, 3H), 1.00 (s, 3H), 0.88 (d, 3H, J = 6.7 Hz), 0.73 (d, 3H, J = 6 , 5 Hz).

II. example

Preparation of Cryptophycin-55-tert-BOC-3- (3-chloro-4-methoxyphenyl) - (D) -alaninate (9): mg (0.033 mmol) of compound (1), 16 mg (0.049 mmol) of N-tert- To a solution of BOC-3- (3-chloro-4-methoxyphenyl) - (D) alanine and some crystals of 4-dimethylaminopyridine in 143 ml of anhydrous methylene chloride was added 10 mg (0.049 mmol) of 1,3 -dicyclohexylcarbodiimide in 20 ml of methylene chloride. After stirring for 2 hours, the cloudy white reaction mixture was diluted with 1 mL of ethyl acetate / hexane 2: 1, stirred for 10 minutes, then filtered through celite, washed with ethyl acetate / hexane 2: 1. The filtrate and washings were evaporated under reduced pressure to give a colorless oil. This product was purified by flash chromatography on silica gel (14 g) eluting with ethyl acetate / hexane (2: 1). 29 mg (88%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.42-7.27 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz), 7.25-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 1.9 Hz), 6.91-6.86 (m, 2H), 6.84-6.70 (m, 3H), 5.75 (d, 1H, J = 15 Hz) ), 5.53 (d, 1H, J = 9.6 Hz), 5.47 (d, 1H, J = 7.6 Hz), 5.00 (dd, 1H, J = 10, 2.9 Hz) ), 4.90-4.80 (m, 2H), 4.78 ^ 4.71 (m, 1H), 4.63 (d, 1H, J = 8.3 Hz), 4.19-4, 12 (m, 1H), 3.91 (s, 3H), 3.88 (s, 3H), 3.40 (dd, 1H, J = 13, 8.1 Hz), 3.25-3.12 (m, 2H), 3.07 (dd, 1H, J = 14, 7.6 Hz), 2.67-2.57 (m, 2H), 2.39-2.27 (m, 2H), 2.15 (dd, 1H, J = 14, 8.0 Hz), 2.01 (ddd, 1H,

HU 227 195 Β1

J = 14, 12, 4.2 Hz), 1.87-1.76 (m, 2H), 1.39 (s, 9H),

1.27 (s, 3H), 1.20 (s, 3H), 1.09-1.04 (m, 6H), 1.01 (d, 3H, J = 6.3 Hz).

Example 12

Preparation of Cryptophycin-55-3- (3-chloro-4-methoxyphenyl) - (D) alaninate hydrochloride (10) To a solution of 9 (mg, 0.027 mmol) in methylene chloride (265 mL) was added 33 mL of 4, 0 M Hydrochloride in 1,4-dioxane (0.133 mmol). After stirring for 3 hours, the clear, colorless reaction mixture was concentrated under reduced pressure to give 26 mg (96%, corrected for 5% by weight of dioxane) of the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.3 Hz), 7.49-7.45 (m, 2H),

7.43-7.48 (m, 3H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd,

1H, J = 8.4, 2.1 Hz), 7.13 (d, 1H, J = 2.2 Hz),

7.07-6.95 (m, 3H), 6.71 (ddd, 1H, 15, 13, 3.8 Hz),

5.98 (dd, 1H, J = 15, 1.8 Hz), 5.69 (d, 1H, J = 10 Hz),

5.22 (d, 1H, J = 10 Hz), 5.18 (dd, 1H, J = 10, 2.5 Hz),

4.89 ^ 4.80 (m, 1H), 4.53 (dd, 1H, J = 11, 3.7 Hz),

4.16 (dd, 1H, J = 10, 4.4 Hz), 3.88 (s, 3H), 3.87 (s,

3H), 3.51 (dd, 1H, J = 13, 9.9 Hz), 3.20 (dd, 1H,

J = 14, 3.7 Hz), 3.14 (dd, 1H, J = 13, 2.3 Hz),

2.82-2.75 (m, 3H), 2.45 (dd, 1H, J = 15, 4.5 Hz),

2.42-2.34 (m, 1H), 2.08-2.00 (m, 1H), 1.97-1.86 (m, 3H), 1.27 (s, 3H), 1, 21 (s, 3H), 1.16 (d, 3H,

J = 7.1 Hz), 1.10 (d, 3H, J = 6.1 Hz), 1.06 (d, 3H,

J = 6.0 Hz).

Example 13

Preparation of Cryptophycin-55-N-tert-BOC-glycinate (11)

Compound (1) (118 mg, 0.167 mmol), N-tert-BOC-glycine (44 mg, 0.251 mmol) and 4-dimethylaminopyridine (2.0 mg, 0.0167 mmol) were prepared in 490 mL of anhydrous methylene chloride, to a solution of room temperature at room temperature was added a solution of 1,3-dicyclohexylcarbodiimide (52 mg, 0.251 mmol) in methylene chloride (67 mL). After stirring for 50 minutes, the cloudy white reaction mixture was diluted with 1 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes, filtered through celite, and washed with ethyl acetate / hexane (3: 1). The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was subjected to flash chromatography on silica gel (19 g), eluting with ethyl acetate / hexane (3: 1). 138 mg (96%) of the desired compound are obtained in the form of a white foam. ¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.34 (s, 5H), 7.24 (d, 1H, J = 2.0 Hz), 7.23-7.19 (m,

1H), 7.10 (dd, 1H, J = 8.4, 2.0 Hz), 6.88 (d, 1H,

J = 8.4 Hz), 6.79-6.70 (m, 1H), 5.77 (d, 1H,

J = 13 Hz), 5.50 (d, 1H, J = 8.0 Hz), 5.47 (d, 1H,

J = 9.8 Hz), 4.97 (dd, 1H, J = 11.2.7 Hz), 4.89 (t, 1H,

J = 10 Hz), 4.83 (d, 1H, J = 9.8 Hz), 4.79-4.72 (m,

1H), 4.68 (br s, 1H), 3.91 (s, 3H), 3.66 (dd, J = 18,

5.3 Hz), 3.42-3.35 (m, 2H), 3.21 (dd, 1H, J = 13,

4.0 Hz), 3.17 (dd, 1H, J = 15, 5.1 Hz), 3.08 (dd, 1H,

J = 15, 7.6 Hz), 2.66-2.57 (m, 2H), 2.47-2.38 (m,

1H), 1.95 (ddd, 1H, J = 14, 12.4,7 Hz), 1.85-1.77 (m,

1H), 1.75-1.67 (m, 1H), 1.43 (s, 9H), 1.27 (s, 3H),

1.20 (s, 3H), 1.08 (d, 3H, J = 7.0 Hz), 1.03 (d, 3H,

J = 6.7 Hz), 0.98 (d, 3H, J = 6.5 Hz).

Example 14

Preparation of Cryptophycin-55-glycinate hydrochloride (12)

To a solution of compound 11 (122 mg, 0.141 mmol) in methylene chloride (471 mL) at room temperature was added 178 mL of a 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.707 mmol). After stirring for 1 hour 20 minutes, the clear, colorless reaction mixture was evaporated under reduced pressure to give 120 mg (99%, 7% w / w dioxane) of the desired compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.81 (dd, 1H, J = 8.5, 2.2 Hz), 7.46-7.41 (m, 2H),

7.40-7.36 (m, 3H), 7.31 (d, 1H, J = 2.1 Hz), 7.20 (dd,

1H, J = 8.4, 2.1 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.70 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.97 (dd, 1H, J = 15,

1.7 Hz), 5.55 (d, 1H, J = 9.9 Hz), 5.18 (d, 1H,

J = 9.9 Hz), 5.14 (dd, 1H, J = 10, 2.8 Hz), 4.84 (t, 1H,

J = 10 Hz), 4.52 (dd, 1H, J = 11, 3.7 Hz), 3.87 (s, 3H),

3.78 (d, 1H, J = 18 Hz), 3.50 (dd, 1H, J = 13, 9.8 Hz),

3.23 (d, 1H, J = 18 Hz), 3.20 (dd, 1H, J = 14, 3.6 Hz),

3.13 (dd, 1H, J = 13, 2.4 Hz), 2.80-2.69 (m, 3H),

2.41-2.32 (m, 1H), 1.99-1.92 (m, 1H), 1.91-1.81 (m, 2H), 1.25 (s, 3H), 1.20 ( s, 3H), 1.12 (d, 3H,

J = 7.0 Hz), 1.06 (d, 3H, J = 6.2 Hz), 1.04 (d, 3H,

6.2 Hz).

Example 15

Preparation of Cryptophycin-55-N-tert-BOC - (> - alaninate)

Compound (1) (102 mg, 0.145 mmol), N-tert-BOC-β-alanine (41 mg, 0.217 mmol) and 4-dimethylaminopyridine (18 mg, 0.145 mmol) were prepared in 400 mL of anhydrous methylene chloride. to a solution of room temperature at room temperature was added a solution of 1,3-cyclohexylcarbodiimide (45 mg, 0.217 mmol) in methylene chloride (82 mL).

After stirring for 3.5 h, the cloudy white reaction mixture was diluted with 1 mL of ethyl acetate / hexane 3: 1, stirred for 10 min then filtered through celite, washed with ethyl acetate / hexane 3: 1. The filtrate and washings were evaporated under reduced pressure to give a colorless oil. The product was chromatographed on silica gel (21 g) eluting with ethyl acetate / hexane (2: 1 then 4: 1). 121 mg (95%) of the desired compound is obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.44-7.39 (m, 2H), 7.37-7.31 (m, 3H), 7.32 (d, 1H,

J = 2.1 Hz), 7.20 (dd, 1H, J = 8.4, 2.1 Hz), 7.01 (d, 1H,

J = 8.4 Hz), 6.72 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.96 (dd, 1H, J = 15, 1.6 Hz), 5.51 ( d, 1H, J = 9.8 Hz),

5.11-5.06 (m, 1H), 5.08 (d, 1H, J = 9.8 Hz),

4.90-4.83 (m, 1H), 4.50 (dd, 1H, J = 11, 3.6 Hz),

3.86 (s, 3H), 3.52-3.46 (m, 1H), 3.20 (dd, 1H, J = 14,

3.6 Hz), 3.13 (br d, 1H, J = 14 Hz), 3.05-2.92 (m,

2H), 2.79-2.63 (m, 3H), 2.45-2.37 (m, 1H), 2.24

HU 227 195 Β1 (dt, 1H, J = 16, 7.0 Hz), 2.08-1.99 (m, 1H),

1.96-1.79 (m, 3H), 1.43 (s, 9H), 1.25 (s, 3H), 1.21 (s, 3H), 1.12 (d, 3H), J = 7.0 Hz, 1.06 (d, 3H,

J = 6.2 Hz), 1.02 (d, 3H, J = 6.1 Hz).

Example 16

Preparation of Cryptophycin 55- (f) -alaninate hydrochloride (14)

To a solution of compound 13 (119 mg, 0.136 mmol) in methylene chloride (452 mL) at room temperature was added 170 mL of a 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.679 mmol). After stirring for 2 hours 15 minutes, the cloudy white reaction mixture was evaporated under reduced pressure to give 110 mg (96%, corrected for 4% by weight of dioxane) of the desired compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.80 (dd, 1H, J = 9.7, 2.3 Hz), 7.45-7.40 (m, 2H),

7.39-7.32 (m, 3H), 7.20 (dd, 1H, J = 8.4 2.1 Hz),

7.01 (d, 1H, J = 8.4 Hz), 6.68 (ddd, 1H, J = 15, 13,

3.8 Hz), 5.98 (dd, 1H, J = 15, 1.7 Hz), 5.48 (dd, 1H,

J = 9.4, 1.0 Hz), 5.15-5.11 (m, 1H), 5.13 (d, 1H)

J = 9.4 Hz), 4.82 (t, 1H, J = 10 Hz), 4.51 (dd, 1H,

J = 11, 3.7 Hz), 3.90 (s, 3H), 3.50 (dd, 1H, J = 14,

9.8 Hz), 3.20 (dd, 1H, J = 14, 3.7 Hz), 3.14 (dd, 1H,

J = 14, 2.4 Hz), 2.85 (t, 2H, J = 7.0 Hz), 2.80-2.65 (m,

5H), 2.54 (dt, 1H, J = 17, 7.4 Hz), 2.42-2.33 (m, 1H),

2.22 (dt, 1H, J = 17, 6.7 Hz), 1.90-1.81 (m, 3H), 1.25 (s, 3H), 1.20 (s, 3H), 1 , 13 (d, 3H, J = 7.1 Hz), 1.08 (d, 3H, J = 6.3 Hz), 1.04 (d, 3H, J = 6.2 Hz).

Example 17

Preparation of Cryptophycin-55-N-tert-BOC-N-butyrate (15) mg (0.068 mmol) of compound (1), 18 mg (0.088 mmol) of N-tert-BOC-4-aminobutyric acid and 8 mg To a solution of 4-dimethylaminopyridine (0.068 mmol) in anhydrous methylene chloride (150 mL) was added a solution of 1,3-dicyclohexylcarbodiimide (18 mg, 0.088 mmol) in methylene chloride (50 mL). After stirring for 45 minutes, the cloudy white reaction mixture was diluted with ethyl acetate / hexane (3: 1, 0.5 mL), stirred for 5 minutes and filtered through celite, washed with ethyl acetate / hexane (3: 1). The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was flash chromatographed on silica gel (15 g) eluting with ethyl acetate / hexane (3: 1). 55 mg (90%) of the desired compound is obtained in the form of a white foam. ¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.38-7.32 (m, 5H), 7.24 (d, 1H, J = 1.9 Hz),

7.22-7.19 (m, 1H), 7.10 (dd, 1H, J = 8.4, 1.9 Hz),

6.88 (d, 1H, J = 8.4 Hz), 6.75 (ddd, 1H, J = 15, 13,

3.9 Hz), 5.78 (d, 1H, J = 15 Hz), 5.60-5.55 (m, 1H),

5.49 (dd, 1H, J = 9.8, 1.4 Hz), 4.96 (dd, 1H, J = 11,

3.0 Hz), 4/89 (t, 1H, J = 9.2 Hz), 4.81 (d, 1H,

J = 9.8 Hz), 4.78-4.70 (m, 1H), 4.44 (br s, 1H), 3.91 (s, 3H), 3.40 (dd, 1H, J = 14, 8.1 Hz), 3.22 (dd, 1H,

J = 14, 4.1 Hz), 3.22-3.15 (m, 1H), 3.08 (dd, 1H,

J = 14, 7.8 Hz), 2.89-2.82 (m, 2H), 2.67-2.56 (m,

2H), 2.47-2.38 (m, 1H), 2.11-2.04 (m, 1H),

2.00-1.77 (m, 3H), 1.75-1.67 (m, 1H), 1.45 (s, 9H),

1.50-1.40 (m, 2H), 1.27 (s, 3H), 1.20 (s, 3H), 1.09 (d, 3H, J = 7.0 Hz), 1.04 (d, 3H, J = 6.6 Hz), 0.98 (d,

3H, J = 6.6 Hz).

Example 18

Preparation of Cryptophycin-55- _i '-aminobutyrate hydrochloride (16) To a solution of 15 (0.059 mmol) in methylene chloride (297 mL) at room temperature was added 297 mL of 1.0 M hydrogen chloride in diethyl ether (0.297). mmol). The starting material precipitates as a white ointment which is dissolved in 150 ml of methylene chloride. After stirring for 4 hours, another 59 ml (0.059 mmol) of hydrochloric acid was added. Stirring is continued for 14 hours and the reaction mixture is concentrated under reduced pressure to give 49 mg (100%) of the title compound as a white foam. ¹ H-NMR (500 MHz, DMSO-d ₆ ), δ:

8.49 (d, 1H, J = 8.0 Hz), 7.72 (br s, 3H), 7.44-7.33 (m, 5H), 7.32 (d, 1H, J = 1 , 9 Hz), 7.29 (dd, 1H, J = 9.4,

2.6 Hz), 7.20 (dd, 1H, J = 8.5, 1.9 Hz), 7.06 (d, 1H,

J = 8.5 Hz), 6.48 (ddd, 1H, J = 15, 13, 3.9 Hz), 5.87 (d, 1H, J = 15 Hz), 5.37 (d, 1H, J = 9.7 Hz), 5.33 (d,

1H, J = 9.7 Hz), 5.04-5.01 (m, 1H), 4.73 (t, 1H,

J = 11 Hz), 4.25 (ddd, 1H, J = 12, 9.8, 3.5 Hz), 3.82 (s,

3H), 3.40-3.30 (m, 1H), 3.07-3.01 (m, 2H), 2.72 (dd, 1H, J = 14, 12Hz), 2.65-2 , 47 (m, 4H),

2.38-2.28 (m, 1H), 2.21 (dt, 1H, J = 17, 7.5 Hz), 1.97 (dt, 1H, J = 17, 7.5 Hz), 1 , 80-1.70 (m, 3H),

1.54-1.46 (m, 2H), 1.17 (s, 3H), 1.03 (s, 3H), 1.01 (d, 3H, J = 7.0 Hz), 0.99 (d, 3H, J = 5.8 Hz), 0.95 (d,

3H, J = 5.8 Hz).

Example 19

Preparation of Cryptophycin-55-N-tert-BOC- (L) -alaninate (17)

Compound (1) (103 mg, 0.146 mmol), N-tert-BOC- (L) -alanine (41 mg, 0.219 mmol) and 4-dimethylaminopyridine (18 mg, 0.146 mmol) were prepared in 400 mL of anhydrous methylene chloride. to a solution of room temperature at room temperature was added a solution of 1,3-dicyclohexylcarbodiimide (45 mg, 0.219 mmol) in methylene chloride (87 mL). After stirring for 5 h 50 min, another 5.5 mg (0.029 mmol) of N-tert-BOC- (L) -alanine, 6.0 mg (0.029 mmol) of 1,3-dicyclohexylcarbodiimide and some crystals of 4-dimethyl are added to the cloudy white reaction mixture. aminopyridine is added. After stirring for 1 hour, the reaction mixture was diluted with 1 mL of ethyl acetate: hexane (3: 1), stirred for 10 minutes, filtered through celite, and washed with ethyl acetate / hexane (3: 1). The filtrate and washings were evaporated under reduced pressure to give a colorless oily product which was chromatographed on silica gel (22 g), eluting with ethyl acetate / hexane (1.5: 1, 2: 1 and finally 4: 1). 96 mg (75%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.35-7.30 (m, 5H), 7.26-7.21 (m, 2H), 7.10 (dd,

1H, J = 8.4, 1.9 Hz), 6.88 (d, 1H, J = 8.4 Hz), 6.76

HU 227 195 Β1 (ddd, 1H, J = 15, 13, 4.2 Hz), 5.77 (d, 1H, J = 15 Hz),

5.52 (d, 1H, 7.6 Hz), 5.44 (d, 1H, J = 9.7 Hz), 4.98 (dd, 1H, J = 11, 2.5 Hz), 4, 85-4.81 (m, 2H), 4.75 (q,

1H, J = 6.8Hz), 4.56 (d, 1H, J = 7.8Hz), 4.01-3.96 (m, 1H), 3.91 (s, 3H), 3, 41 (dd, 1H, J = 13, 8.3 Hz),

3.20 (dd, 1H, J = 13, 4.0 Hz), 3.16 (dd, 1H, J = 15,

5.9 Hz), 3.08 (dd, 1H, J = 15, 7.6 Hz), 2.65-2.57 (m,

2H), 2.40-2.31 (m, 1H), 2.02-1.96 (m, 1H),

1.87-1.73 (m, 2H), 1.43 (s, 9H), 1.27 (s, 3H), 1.20 (s, 3H), 1.11-1.02 (m, 9H), 0.99 (d, 3H, J = 6.3 Hz).

Example 20

Preparation of Cryptophycin-55- (L) -alaninate hydrochloride (18) To a solution of (17) (mg) (0.108 mmol) in methylene chloride (361 mL) at room temperature was added 135 mL of a 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.542). mmol). After stirring for 2.5 hours, the cloudy white reaction mixture was evaporated under reduced pressure to give 90 mg (96%, corrected for 6% dioxane) of the desired compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

8.54 (d, 1H, 7.6 Hz), 7.81 (br d, 1H, J = 9.7 Hz),

7.46-7.44 (m, 2H), 7.39-7.37 (m, 3H), 7.32 (d, 1H,

J = 2.0 Hz), 7.20 (dd, 1H, J = 8.4, 2.0 Hz), 7.01 (d, 1H,

J = 2.0Hz, 6.69 (ddd, 1H, J = 15, 11, 3.7Hz), 5.99 (d, 1H, 15Hz), 5.55 (d, 1H, J = 9.8 Hz), 5.20 (d, 1H,

J = 9.8 Hz), 5.15 (dd, 1H, J = 11.2.7 Hz), 4.78 (t, 1H,

J = 11 Hz), 4.53-4.50 (m, 1H), 3.87 (s, 3H), 3.65 (q,

1H, J = 7.3 Hz), 3.50 (dd, 1H, J = 13, 9.8 Hz), 3.20 (dd, 1H, J = 14, 3.5 Hz), 3.14 ( br d, 1H, J = 13 Hz),

2.81-2.71 (m, 3H), 2.41-2.34 (m, 1H), 1.98-1.93 (m, 1H), 1.88-1.82 (m, 2H) ), 1.41 (d, 3H, J = 7.3 Hz),

1.25 (s, 3H), 1.20 (s, 3H), 1.13 (d, 3H, J = 7.0 Hz),

1.06 (d, 3H, J = 6.2 Hz), 1.04 (d, 3H, 6.0 Hz).

Example 21

Preparation of Cryptophycin-44-N-tert-BOC- (D) -alaninate (19): mg (0.035 mmol) of compound (1), 10 mg (0.053 mmol) of N-tert-BOC- (D) -alanine and 0. To a solution of 4-dimethylaminopyridine (4 mg, 0.0035 mmol) in dry methylene chloride (130 mL) was added a solution of 1,3-dicyclohexylcarbodiimide (11 mg, 0.053 mmol) in methylene chloride (47 mL).

After stirring for 5.5 hours, the cloudy white reaction mixture was diluted with ethyl acetate / hexane (3: 1, 0.5 mL), stirred for 10 minutes, filtered through celite and washed with ethyl acetate / hexane (3: 1). The filtrate and the washings were evaporated under reduced pressure to give a colorless oily product which was flash chromatographed on silica gel (15 g, 2: 1 ethyl acetate / hexane). 26 mg (83%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.49-7.29 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.22-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 2.0 Hz),

6.87 (d, 1H, J = 8.4 Hz), 6.76 (ddd, 1H, J = 15, 13,

4.4 Hz), 5.77 (d, 1H, 15 Hz), 5.56 (d, 1H, J = 9.9 Hz),

5.48 (d, 7.7 Hz), 5.01 (dd, 1H, J = 10, 2.6 Hz), 4.9 (t,

1H, J = 9.4 Hz), 4.84 (d, 1H, J = 9.9 Hz), 4.81 ^ 1.73 (m, 2H), 3.99-3.93 (m, 1H). ), 3.91 (s, 3H), 3.39 (dd,

1H, J = 13.8.0 Hz), 3.22 (dd, 1H, J = 13, 3.6 Hz), 3.17 (dd, 1H, J = 14, 5.0 Hz), 3, 08 (dd, 1H, J = 14 Hz),

2.68-2.58 (m, 2H), 2.42-2.35 (m, 1H), 2.04-1.94 (m, 1H), 1.87-1.50 (m, 2H) ), 1.42 (s, 9H), 1.27 (s,

3H), 1.20 (s, 3H), 1.09 (d, 3H, J = 7.1 Hz), 1.04 (d,

3H, J = 6.4Hz), 0.99 (d, 3H, J = 6.3Hz), 0.65 (d, 3H,

J = 6.8 Hz).

Example 22

Preparation of Cryptophycin 55- (D) -alaninate hydrochloride (20) To a solution of (18) (mg, 0.027 mmol) in methylene chloride (274 mL) at room temperature was added 34 mL of a 4.0 M solution of hydrogen chloride in 1,4-dioxane (0.137). mmol). After stirring for 3.5 hours, the clear, colorless reaction mixture was evaporated under reduced pressure to give 24 mg (100%, corrected for 8% by weight of dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 9.5 Hz), 7.47-7.40 (m, 2H),

7.40-7.36 (m, 3H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd,

1H, J = 8.4, 2.0 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.71 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.98 (dd, 1H, J = 15,

1.6 Hz), 5.65 (d, 1H, J = 10 Hz), 5.20 (d, 1H,

J = 10 Hz), 5.17 (dd, 1H, J = 11, 2.5 Hz), 4.88-4.78 (m, 1H), 4.53 (dd, 1H, J = 11, 3 , 7 Hz), 3.95 (q, 1H,

J = 7.2 Hz), 3.87 (s, 3H), 3.51 (dd, 1H, J = 13, 9.8 Hz),

3.20 (dd, 1H, J = 14, 3.6 Hz), 3.14 (dd, 1H, J = 13,

2.3 Hz), 2.81-2.74 (m, 3H), 2.41-2.34 (m, 1H),

2.07-1.99 (m, 1H), 1.96-1.84 (m, 2H), 1.26 (s, 3H),

1.21 (s, 3H), 1.15 (d, 3H, J = 7.1 Hz), 1.09 (d, 3H,

J = 6.0 Hz), 1.05 (d, 3H, J = 6.0 Hz), 0.80 (d, 3H,

J = 7.4 Hz).

Example 23

Preparation of Cryptophycin-55-N: N-N; - di-tert-BOC- (L) -Lysinate (21)

Compound (1) (105 mg, 0.149 mmol), Na-N-di-tert-BOC- (L) -lysine (67 mg, 0.193 mmol) and 4-dimethylaminopyridine (18 mg, 0.149 mmol) in 400 mL of anhydrous methylene solution of 1,3-dicyclohexylcarbodiimide (40 mg, 0.193 mmol) in methylene chloride (96 ml) was added at room temperature. After stirring 4 hours, the cloudy white reaction mixture (10 mg, 0.030 mmol) _in N, _{N-e-di-t-BOC-} (L) -lizintés (0.030 mmol) of 1,3-dicyclohexylcarbodiimide were added 6.1 mg 100 dissolved in ml of methylene chloride. After stirring for 1 hour, the cloudy white reaction mixture was diluted with 1 mL of ethyl acetate / hexane 3: 1, stirred for 10 minutes, filtered through celite, and washed with ethyl acetate / hexane 3: 1. The filtrate and the washings were evaporated under reduced pressure to give a white foam, to which 34 mg (0.097 mmol) of Na-Ns-di-tert-BOC- (L) -lysine, 20 mg (0.097 mmol) were added under similar conditions. dicyclohexylcarbodiimide and

9.1 mg (0.075 mmol) of 4-dimethylaminopyridine. After stirring for 1.5 hours, the reaction mixture was worked up as above to give a white foamy product. The product

Flash chromatography on silica gel (195 g) using ethyl acetate / hexane (1: 1 then 4: 1) as eluent. 112 mg (73%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.42-7.37 (m, 2H), 7.36-7.29 (m, 3H), 7.27 (br s,

1H), 7.16 (br d, 1H, J = 8.5 Hz), 6.97 (d, 1H,

J = 8.5 Hz), 6.72 (ddd, 1H, J = 15, 13, 3.5 Hz), 5.92 (d, 1H, J = 15 Hz), 5.50 (d, 1H, J = 11Hz), 5.11-5.04 (m, 2H), 4.84 (t, 1H, J = 10Hz), 4.48 (dd, 1H, J = 11,

3.6 Hz), 3.84 (s, 3H), 3.75 (brs, 1H), 3.50-3.43 (m,

1H), 3.17 (dd, 1H, J = 14, 3.6 Hz), 3.11 (d, 1H,

J = 14 Hz), 2.97-2.91 (m, 2H), 2.76-2.58 (m, 3H),

2.36-2.27 (m, 1H), 1.98-1.80 (m, 3H), 1.48-1.38 (m, 2H), 1.43 (s, 9H), 1, 40 (s, 9H), 1.35-1.25 (m,

2H), 1.23 (s, 3H), 1.20 (s, 3H), 1.15-1.09 (m, 2H),

1.07 (d, 3H, J = 6.8 Hz), 1.06 (d, 3H, J = 6.0 Hz), 1.01 (d, 3H, J = 6.1 Hz).

Example 24

Preparation of Cryptophycin 55- (L) -Lysinate Dihydrochloride (22)

To a solution of compound 21 (107 mg, 0.103 mmol) in methylene chloride (345 mL) at room temperature was added a solution of 4.0 M hydrochloric acid in 1,4-dioxane (155 mL, 0.621 mmol). After stirring for 4 hours, the cloudy white reaction mixture was filtered. The collected white solid was washed twice with 1 mL of methylene chloride and dried under reduced pressure to give 87 mg (93%) of the desired compound.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

8.61 (d, 1H, J = 7.7 Hz), 7.81 (d, 1H, J = 7.7 Hz),

7.47-7.44 (m, 2H), 7.40-7.38 (m, 3H), 7.31 (d, 1H,

J = 2.2 Hz), 7.20 (dd, 1H, J = 8.4.2.2 Hz), 7.00 (d, 1H,

J = 8.4 Hz), 6.63 (ddd, 1H, J = 15, 13, 4.0 Hz), 6.00 (dd, 1H, J = 15, 1.6 Hz), 5.55 ( d, 1H, J = 9.8 Hz), 5.20 (d, 1H, J = 9.8 Hz), 5.15 (dd, 1H, J = 10, 2.9 Hz), 4.68 ( t, 1H, J = 11Hz), 4.55 ^ 1.49 (m, 1H), 3.87 (s, 3H),

3.79 (t, 1H, J = 5.6 Hz), 3.52 (dd, 1H, J = 14, 9.9 Hz),

3.20 (dd, 1H, J = 14, 3.6 Hz), 3.13 (dd, 1H, J = 13,

2.4 Hz), 3.06-2.98 (m, 1H), 2.94-2.87 (m, 1H),

2.85-2.74 (m, 3H), 2.45-2.38 (m, 1H), 1.98-1.76 (m, 5H), 1.71-1.64 (m, 2H) ), 1.39-1.30 (m, 2H),

1.25 (s, 3H), 1.18 (d, 3H, J = 8.2 Hz), 1.17 (s, 3H),

1.08 (d, 3H, J = 6.2 Hz); 1.05 (d, 3H, J = 6.1 Hz).

Example 25

Preparation of 2 '- (di-tert-butylphosphatyl) phenylacetic acid (24)

To a solution of 2'-hydroxyphenethyl alcohol (1.05 g, 7.60 mmol) in ,2, Ν-dimethylformamide (15.2 mL) at 0 ° C was added imidazole (621 mg, 9.11 mmol) and 1.26 g. g (8.34 mmol) of tert-butyldimethylsilyl chloride. After stirring at 0 ° C for 40 minutes and at room temperature for 45 minutes, another imidazole (155 mg, 2.28 mmol) and tert-butyldimethylsilyl chloride (229 mg, 1.52 mmol) were added to the reaction mixture. The reaction mixture was stirred for 15 minutes and then tert-butyl methyl ether (150 mL) was added. After washing with 14 ml of 1 N aqueous hydrochloric acid and 15 ml of water, the organic phase is dried over sodium sulphate, filtered and concentrated under reduced pressure. The oily product was flash chromatographed on silica gel (70 g), eluting with 5: 1 hexane: ethyl acetate. Silyl ether (1.81 g, 94%) was obtained as an off-white oil. To a solution of 506 mg (2.00 mmol) of silyl ether and 600 ml of 93% (2.00 mmol) of di-tert-butyl diethyl phosphoramidite in 2 ml of tetrahydrofuran was added 421 mg (6.01 mmol) of H-tetrazole. After stirring for 45 minutes, the reaction mixture was cooled to -10 ° C and a solution of m-chloroperbenzoic acid (450 mg, 99%, 2.61 mmol) in methylene chloride (3.6 mL) was added rapidly. The cloudy white reaction mixture was allowed to warm to room temperature and stirred for 15 minutes. 4 ml of 10% sodium bisulfite aqueous solution were added to the reaction mixture, vigorously stirred for 10 minutes, diluted with 15 ml of tert-butyl methyl ether and 2 x 10 ml of 10% sodium bisulfite aqueous solution and 2 x 10 ml. Wash with 0.5 N aqueous sodium hydroxide. The organic phase is dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting 941 mg colorless oily product was used directly for the next step. The crude phosphate was dissolved in tetrahydrofuran (10 mL), cooled to 0 ° C, and treated with 1M tetra-n-butylammonium fluoride (2.4 mL, 2.4 mmol) in tetrahydrofuran. After stirring for 20 minutes at 0 ° C and 1.5 hours at room temperature, the reaction mixture was diluted with tert-butyl methyl ether (60 ml), washed with water (10 ml) and brine (10 ml). The organic phase is dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting yellow oil was flash chromatographed on silica gel (50 g) eluting with ethyl acetate / hexane (3: 1). 525 mg (79%) of alcohol are obtained in the form of an off-white oil. 123 mg (0.372 mmol) of alcohol

To a solution of acetonitrile / carbon tetrachloride (1.49 mL, 1: 1) was added water (1.1 mL) at room temperature, followed by sodium periodate (239 mg, 1.12 mmol) and ruthenium (III) chloride hydrate (1.8 mg, 0.082 mmol). The brown reaction mixture was vigorously stirred at room temperature for 55 minutes. The reaction mixture was evaporated under reduced pressure and the product was flash chromatographed on silica gel (6 g) with ethyl acetate containing 10% methanol as eluent. 109 mg (85%) of the desired compound is obtained as a purple oil.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.49 (d, 1H, J = 7.53 Hz), 7.30-7.15 (m, 3H), 3.77 (s, 2H), 1.51 (s, 18H).

26-31. The compounds of Examples 1 to 8 are prepared as described above.

Example 26

Spectral data for Cryptophycin-129

EIMS m / z: (relative intensity) 654/656 (2.1 / 0.8, M-HBr), 412/414 (9.7 / 3.6), 280/282 (10.3 / 3.5) ), 227 (15.2), 195/197 (50.1 / 16.3), 105 (100), 91 (100); high resolution EIMS m / z 654.2687 (C ₃₅ H ₄₃ CIN ₂ O ₈ , D

2.1 mmu).

¹ H-NMR (CDCl ₃ ) amino or hydroxy acid moiety, δ, 7-bromo-5,8-dihydro-6-methyl-8-phenyl-2-octenoic acid (A): 5.76 (2d, 15.3), 6.68 (3, ddd, 15.3, 9.9 and 5.6), 2.29 (4, ddd, 14.2, 10.6 and 10.4), 2.67 (4 dd, 14.2 and

5.4), 4.95 (δ, ddd, 10.3, 10.3 and 1.8), 2.47 (6, m),

1.15 (6-Me, d, 6.5), 4.29 (7, dd, 9.1 and 2.1 Hz), 4.84 (8, d, 9.0), 7.34- 7.39 (10/11/12/13/14, m); 3-chloro-4-methoxyphenylalanine (B); 4.80 (2, m), 5.73 (2-NH, d,

8.8), 3.01 (3, dd, 14.6 and 7.6), 3.16 (3, dd, 14.3 and 5.6), 7.23 (5, d, 2.0) ), 3.87 (7-OMe, s), 6.84 (8, d,

8.4), 7.09 (9, dd, 8.4 and 2.0); 3-amino-2-methylpropionic acid (C): 2.73 (2, m), 1.23 (2-Me, d, 7.2),

3.25 (3, ddd, 13.5, 6.5 and 6.5), 3.53 (3, ddd, 13.4,

5.4 and 3.9), 6.93 (3-NH, brt, 6.1), 2-hydroxyisocaproic acid (D) 4.91 (2, dd, 10.5 and 2.8), 1.49 (3, m),

1.80 (3, m), 1.73 (4, m), 0.92 (4-Me, d, 6.5), 0.89 (5, d, 6.5).

¹³ C-NMR (CDCl3) units, δ (carbon position)

A: 165.4 (1), 125.2 (2), 141.3 (3), 36.6 (4), 75.7 (5),

37.9 (6), 11.8 (6-Me), 60.3 (7), 76.7 (8), 141.6 (9), 126.8 (10/14), 128.7 ( 11/13), 128.8 (12);

B: 171.0 (1), 53.6 (2), 35.0 (3), 130.0 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OMe), 112.3 (8), 128.4 (9);

C; 175.4 (1), 38.4 (2), 14.0 (2-Me), 41.3 (3);

D: 170.2 (1), 71.3 (2), 39.6 (3), 24.8 (4), 23.2 (4-Me), 21.4 (5).

Example 27

Spectral data for Cryptophycin-138

EIMS m / z 734/736 (0.3 / 0.1), 654/656 (0.6 / 0.2), 412/414 (12.6 / 4.7), 313/315 (20, 4 / 12.5), 280 (7.6), 227 (3.5), 195/197 (44.7 / 15.6), 155/157 (35.4 / 13.6), 105 (33 , 6), 91 (34.2), 80/82 (100/100); high resolution EIMS m / z 734.1985 (C ₃₅ H ₄₄ CIBrN ₂ O ₈ , D-1.6 mmu), 654.2722 (C ₃₅ H ₄₃ CIN ₂ O ₈ , D-1.4 mmu).

¹ H-NMR (CDCl ₃ ), δ: (A): 5.78 (2, d, 15.5), 6.69 (3, ddd,

15.1, 9.8 and 5.3), 2.33 (4, m), 2.71 (4, m), 4.97 (5, ddd, 10.4, 10.4 and 1.6) ), 2.45 (6, m), 1.17 (6-Me, d, 6.3), 4.40 (7, dd, 10.1 and 2.2 Hz), 5.06 (8, d, 10.1),

7.36-7.42 (10/11/12/13/14, m); (B): 4.82 (2, m),

5.69 (2-NH, d, 8.7), 3.02 (3, dd, 14.5 and 7.5), 3.17 (3, dd, 14.5 and 5.4), 7 , 23 (5, d, 2.1), 3.88 (7-OMe, s), 6.85 (8, d, 8.4), 7.09 (9, dd, 8.4 and 1, 9); (C): 2.73 (2, m), 1.23 (2-Me, d, 7.3), 3.25 (3, ddd, 13.4, 6.5 and

6.5), 3.54 (3, m), 6.92 (3-NH, br t, 5.7), 2-hydroxyisocaproic acid (D): 4.91 (2, dd, 10.6 and 2.7). ), 1.43 (3, m), 1.82 (3, m), 1.75 (4, m), 0.93 (4-Me, d, 6.6), 0.90 (5, d, 6.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.2 (2), 141.2 (3), 36.6 (4), 0 76.5 (5), 38.2 (6), 11.7 (6 -Me), 55.3 (7), 87.4 (8), 137.4 (9), 128.0 (10/14), 128.7 (11/13), 129.3 (12);

B: 171.0 (1), 53.6 (2), 35.0 (3), 129.9 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OMe), 112.2 (8), 128.4 (9);

C: 175.5 (1), 38.4 (2), 14.0 (2-Me), 41.2 (3);

D: 170.2 (1), 71.2 (2), 39.6 (3), 24.8 (4), 23.2 (4-Me), 21.4 (5).

Example 28

Spectral data for Cryptophycin-139

EIMS m / z: 732/734 (0.3 / 0.3), 652/654 (0.9 / 0.5), 533 (13.6), 445 (5.7), 195/197 (9 , 2 / 11.4), 105 (96.2), 80/82 (100/100); high resolution EIMS m / z 732.1783 (C ₃₅ H ₄₂ CIBrN ₂ O ₈ , E-3.0 mmu), 652.2573 (C ₃₅ H ₄₁ CIN ₂ O ₈ , E-2.1 mmu).

¹ H-NMR (CDCl ₃ ), δ: (A): 5.75 (2d, 15.4), 6.68 (3, ddd,

15.2, 10.1 and 5.0), 2.31 (4, ddd, 14.2, 10.7 and 10.7), 2.63 (4 dd, 14.4 and 5.1), 5.09 (δ, ddd, 9.7, 9.7 and

1.5), 2.46 (6, m), 1.18 (6, Me, d, 6.6), 5.41 (7, d,

3.5), 7.94 (10/14, brd, 8.6), 7.50 (11/13, t, 7.8), 7.63 (12, brt, 7.5); (B): 4.80 (2, m), 5.64 (2-NH, d, 8.3), 3.04 (3, dd, 14.6 and 7.2), 3.14 (3 , dd, 14.4 and 5.5),

7.22 (5, d, 2.0), 3.87 (7-OMe, s), 6.84 (8, d, 8.3), 7.08 (9, dd, 8.3 and 2.3) ); (C): 2.76 (2, m), 1.26 (2-Me, d, 7.1), 3.31 (3, ddd, 13.7, 6.8 and 6.6), 3 , 53 (3, m),

6.95 (3-NH, brt, 5.9), 2-hydroxyisocaproic acid (D): 4.93 (2, dd, 10.6 and 3.0), 1.51 (3, m), 1.95 (3, m),

1.80 (4, m), 0.97 (4-Me, d, 6.8), 0.91 (5, d, 6.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.2 (1), 125.3 (2), 141.0 (3), 36.2 (4), 76.0 (5),

39.3 (6), 13.8 (6-Me), 51.8 (7), 192.3 (8), 134.2 (9), 128.7 (10/14), 129.0 ( 11/13), 134.0 (12);

B: 170.9 (1), 53.7 (2), 35.0 (3), 129.8 (4), 131.0 (5), 122.4 * (6) (7, not observed) , 56.2 (7-OMe), 112.3 (8), 128.4 (9);

C: 175.6 (1), 38.3 (2), 14.2 (2-Me), 41.1 (3);

D: 170.2 (1), 71.1 (2), 39.8 (3), 24.7 (4), 23.2 (4-Me), 21.3 (5).

Example 29

Spectral data for Cryptophycin-145

EIMS m / z: (relative intensity) 734/736/738 (0.5 / 0.6 / 0.2), 654/656 (1.5 / 1.1), 412/414 (2.2 / 1) , 0) 313/315 (19.5 / 12.9), 227 (4.1, 195/197 (11.5 / 3.7), 105 (14.6), 91 (25.2), 80 / 82 (100/100; high resolution EIMS m / z: 736.1980 (Ο ₃₅ Η ₄₄ ΟΙΒγΝ ₂ Ο ₈ , E -3.1 mmu), 654.2714 (C ₃₅ H ₄₃ CIN ₂ O ₈ , E-0 , 6 mmu).

¹ H-NMR (CDCl ₃ ), δ: (A): 5.81 (2, d, 15.7), 6.72 (3, m), 2.31 (4, m), 2.77 ( 4, m), 5.62 (5, m), 2.42 (6, m),

1.22 (6-Me, d, 7.0), 4.23 (7, dd, 8.0 and 3.2), 4.94 (8, d, 7.9), 7.32-7.28 (10/11/12/13/14, m); (6 / -4.82 (2, m), 5.72 (2-NH, brs), 3.05 (3, m), 3.15 (3, m), 7.23 (5, brs) , 3.88 (7-OMe, s), 6.84 (8, brd, 7), 7.09 (9, m); (C): 2.75 (2, m), 1.25 (2 -Me, d, 7.0), 3.30 (3, m), 3.53 (3, m), 6.97 (3-N, brs); (D): 4.90 (2, dd). , 9.7 and

3.6), 1.58 δ, d, 6.6), 1.82 (3, m), 1.70 (4, m), 0.95 (4-Me, d, 6.6), 0, 89 (5, d, 6.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.14 (1), 125.2 (2), 141.3 (3), 35.8 (4), 75.8 (5), 41.2 (6), 13.4 (6- Me), 60.7 (7), 76.5 (8), 141.6 (9), 126.8 10/14), 128.5 (11/13), 128.6 (12);

6: 170.5 (1), 53.7 (2), 35.0 (3), 129.9 (4), 131.0 (5),

122.4 (6), 154.0 (7), 56.2 (7-OMe), 112.3 (8), 128.5 (9);

C: 175.4 (1), 38.4 (2), 14.1 (2-Me), 41.2 (3);

D: 170.5 (1), 71.3 (2), 39.7 (3), 24.6 (4), 22.9 (4-e), 21.5 (5).

HU 227 195 Β1

Example 30

Spectral data for Cryptophycin-140

EIMS m / z: 690/692 (2.3 / 1.8), 654/656 (3.8 / 2.2), 412/414 (5.4 / 2.1), 280/282 (5, 4 / 2.2), 227 (10.2), 195/197 (27.8 / 10/4), 155/157 (53.7 / 16/4), 105 (81.0), 91 (100 ); high resolution EIMS m / z 690.2427 (C ₃₅ H ₄₄ Cl ₂ N ₂ O ₈ , E 4.8 mmu), 654.2760 (C ₃₅ H ₄₃ CIN ₂ O ₈ , E -5.2 mmu).

¹ H-NMR (CDCl ₃ ), δ: (A): 5.77 (2, d, 15.4), 6.68 (3, ddd,

15.3, 9.8 and 5.6), 2.29 (4, m), 2.67 (4, m), 4.98 (5, ddd, 10.3, 10.3 and 1.7) ), 2.64 (6, m), 1.15 (6-Me, d,

6.6), 4.19 (7, dd, 9.2 and 2.1), 4.72 (8, d, 9.2), 7.33-7.39 (10/11/12/13 / 14, m); (B): 4.81 (2, m),

5.69 (2-NH, d, 8/8), 3.01 (3, dd, 14.5 and 7.5), 3.16 (3, dd, 14.5 and 5.5), 7 , 23 (5, d, 2.1), 3.87 (7-OMe, s), 6.84 (8, d, 8.3), 7.09 (9, dd, 8.4 and 2, 0); (C): 2.73 (2, m), 1.22 (2-Me, d, 7.3), 3.24 (3, ddd, 13.5, 6.8 and

6.8), 3.53 (3, m), 6.91 (3-NH, brt, 6.0); (D); 4.89 (2, dd, 10.4 and 2.9), 1.39 (3, m), 1.69-1.80 (3/4, m), 0.90 (4-Me, d) , 6.4), 0.87 (5, d, 6.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.2, 141.4 (3), 36.7 (4), 75.5 (5),

38.1 (6), 10.4 (6-Me), 65.0 (7), 75.8 (8), 141.4 (9), 126.8 (10/14), 128.7 ( 11/13), 128.8 (12);

B; 171.0 (1), 53.6 (2), 35.0 (3), 129.9 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OMe), 112.2 (8), 128.4 (9);

C: 175.5 (1), 38.4 (2), 14.1 (2-Me), 41.3 (3);

D: 170.3 (1), 71.3 (2), 39.5 (c), 24.7 (4), 23.2 (4-Me), 21.4 (5).

Example 31

Spectral data for Cryptophycin-141

EIMS m / z 654/656 (1.8 / 0.8, M-HCl), 412/414 (7.6 / 3.1), 280/282 (3.2 / 2.0), 227 ( 8.4), 195/197 (35.6 / 11.9), 155/157 (100 / 37.2), 105 (68.1), 91 (88.4); high resolution EIMS m / z 690.2468 (C ₃₅ H ₄₄ Cl ₂ N ₂ O ₈ , E 0.6 mmu), 654.2706 (C ₃₅ H ₄₃ CIN ₂ O ₈ , E 0.2 mmu).

¹ H-NMR (CDCl ₃ ), δ: (A): 5.69 (2, d, 15.4), 6.62 (3, ddd,

15.1, 9.9 and 5.2), 2.07 (4, m), 2.49 (4, dd, 14.4 and 5.4), 4.96 (5, ddd, 10.5) , 10.5 and 1.8), 1.88 (6, m), 1.07 (6-Me, d, 6.8, 4.24 (7, dd, 8.9 and 1.9), 4.73 (8, d, 8.8), 2.80 (8-OH, 2-hydroxyisocaproic acid), 7.32 (10/14, dd, 7.6 and 1.9), 7.36 -7.42 (11/12/13, m); (B); 4.78 (2, m), 5.65 (2-NH, d, 8.6), 3.00 (3, dd,

14.4 and 7.2), 3.13 (3, dd, 14.4 and 5.6), 7.20 (5, d, 2.3), 3.86 (7-OMe, s), 6, 82 (8, d, 8.3), 7.06 (9, dd,

8.3 and 2.2); (C): 2.73 (2), 1.23 (2-Me, d, 7.2), 3.27 (3, ddd, 13.5, 6.8 and 6.8), 3.51 (3, ddd, 13.5, 5.0 and

3.8), 6.91 (3-NH, brt, 6.0); (D): 4.83 (2, dd, 10.4 and

2.9), 1.48 (3, m), 1.70 (3, m), 1.76 (4, m), 0.99 (4-Me, d, 6.5), 0.95 (5 , d, 6.5).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.3 (1), 125.2 (s), 141.0 (3), 36.2 (4), 75.5 (5),

39.3 (6), 10.8 (6-Me), 70.0 (7), 76.2 (8), 138.9 (9), 126.7 (10/14), 129.0 (11 / 13), 129.0 (12);

B: 170.9 (1), 53.6 (2), 35.0 (3), 129.8 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-Me), 112.2 (8), 128.4);

C; 175.5 (1), 38.2 (2), 14.1 (2-Me), 41.1 (3);

D: 170.2 (1), 71.2 (2), 39.6 (3), 24.8 (4), 23.2 (4-Me), 21.4 (5).

Example 32

Examples of reaction processes in Figure 1

To a solution of the alkene (1) (1.15 g, 3.52 mmol) in dichloromethane (50 mL) at -78 ° C was added pyridine (0.3 mL, 3.9 mmol) and 0.1% (0.98 mL) Sudan red 7B in dichloromethane. Ozone was bubbled through the solution until the red color turned yellow. The progress of the reaction is monitored by thin layer chromatography. When the reaction is complete, 1.63 g (24.9 mmol) of zinc powder and 3.4 ml of glacial acetic acid are added. The cooling bath was removed and the reaction mixture was allowed to slowly warm to room temperature and stirred for 2 hours. The reaction mixture was filtered through a pad of celite and washed with 3 x 30 mL of copper sulfate solution, 3 x 20 mL of water, and 2 x 20 mL of saturated aqueous sodium bicarbonate. The organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure. 0.937 g (88%) of the desired aldehyde are obtained, which product is used without further purification in the next step.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

9.74-9.73 (d, 1H, J = 1.97 Hz), 7.0-6.82 (m, 1H), 5.91-5.86 (d, 1H, J = 15.4 Hz), 4.1-4.0 (m, 1H), 3.73 (s, 3H), 2.55-2.4 (m, 3H), 1.09-1.07 (d, 3H, J = 6.8 Hz), 0.87 (s, 9H), 0.08 (s, 3H), 0.06 (s, 3H).

A 1.5 M solution of 9.6 mL (14.4 mmol) of methyl lithium in diethyl ether / lithium bromide complex was added dropwise to 2,5-dimethylbenzyl triphenylphosphonium chloride in 120 mL of tetrahydrofuran at -78 ° C. added. The solution was allowed to slowly warm to 0 ° C and then cooled to -78 ° C. A solution of aldehyde (2) in tetrahydrofuran (40 mL) was added dropwise to the reaction mixture at -78 ° C. After stirring at -78 ° C for 15 minutes, the reaction mixture was allowed to slowly warm to room temperature. After stirring for 1 hour at room temperature, 50 ml of a saturated solution of ammonium chloride are added and the mixture is extracted with diethyl ether. The diethyl ether layer was washed with water (2 x 50 mL), brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure. The mixture of E / Z isomers of crude styrene was purified on silica gel using 2% ethyl acetate in hexane as eluent. 2.32 g (58%) of product are obtained in the form of a clear oil. The mixture of E / Z isomers was refluxed for 8 hours in benzene (120 ml) in the presence of 0.3 ml of thiophenol and 0.16 g of 1,1'-azobis (cyclohexane carbonitrile) (VAZO). The solution was cooled to room temperature, concentrated under reduced pressure and purified on a silica gel column using 2-5% ethyl acetate in hexane. 2.2 g (95%) of the pure E-isomer are obtained in the form of a clear oil.

[α] ²⁰ D = + 34.6 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.0 (m, 4H), 6.62-6.57 (d, 1H, J = 15.8 Hz),

6.11-6.03 (dd, 1H, J = 15.8, 8.1 Hz), 5.93-5.88 (d,

HU 227 195 Β1

1H, J = 15.5 Hz), 3.82-3.78 (m, 1H), 3.78 (s, 3H),

2.6- 2.34 (m, 3H), 2.37 (s, 3H), 2.34 (s, 3H),

1.18-1.16 (d, 3H, J = 6.8 Hz), 0.96 (s, 9H), 0.13 (s,

3H), 0.11 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

167.2, 146.9, 136.9, 135.7, 133.3, 132.4, 130.5,

128.8, 128.1, 126.5, 123.2, 75.5, 51.8, 43.4, 37.9,

26.2, 21.4, 19.7, 18.5, 16.7, -4.0, -4.2.

IR (CHCl 3): 2955, 2930, 2858, 1718, 1658, 1603, 1496,

1472, 1438, 1362, 1325, 1280, 1258, 1097,

1040 cm ^-1 .

Elemental Analysis for: C ₂₄ H ₃₈ O ₃ Si Calculated: C, 71.59; H, 9.51; Found: C, 71.38; H, 9.30.

To a solution of 2.2 g (5.46 mmol) (3) in 10 ml of tetrahydrofuran is added 11 ml of 2 M potassium hydroxide solution. Stir vigorously and keep the resulting mixture at 65 ° C for 24 hours. After cooling to room temperature, 11 mL of 2M hydrochloric acid was added and vigorously stirred for an additional 30 minutes. Ethyl acetate (50 mL) was added to the reaction mixture and the layers were separated. The aqueous phase was further extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with 50% brine (3 x 30 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure. 2.12 g (100%) of the desired acid are obtained in the form of a dense yellow oil.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-6.95 (m, 4H), 6.62-6.57 (d, 1H, J = 15.8 Hz),

6.1-6.02 (dd, 1H, J = 15.8, 8.0 Hz), 5.92-5.87 (d,

1H, J = 15.7 Hz), 3.9-3.8 (m, 1H), 2.6-2.2 (m, 3H),

2.36 (s, 3H), 2.34 (s, 3H), 1.18-1.15 (d, 3H,

J = 6.8 Hz), 0.94 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H).

To a solution of acid (4) (2.12 g, 5.46 mmol) in diisopropylethylamine (2.9 mL, 16.4 mmol) in dimethylformamide (7 mL) was added dropwise 1.1 mL (6). (01 mmol) diphenylphosphine chloride. After stirring for 5 minutes at 0 ° C and for 30 minutes at room temperature, the TFA salt of 3- (3-chloro-4-methoxyphenyl) -D-alanine-2,2,2-trichloroethyl ester (5) is added dropwise to 7 ml. dimethylformamide. After stirring for 2 hours at room temperature, the reaction mixture was poured into 100 ml of water and washed 3 times with 50 ml of diethyl ether. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered through celite and evaporated under reduced pressure. The product was purified by silica gel column chromatography (10-30% ethyl acetate / hexane). 2.86 g (72%) of the amide (6) are obtained as a white foam.

_[A] 2o = + 44 8 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.25 (s, 1H), 7.22 (s, 1H), 7.1-6.83 (m, 5H),

6.6-6.55 (d, 1H), J = 15.7Hz), 6.11-6.03 (dd, 1H,

J = 15.6, 8.1 Hz), 5.9-5.8 (m, 2H), 5.18-5.05 (m,

1 H), 4.83-4.75 (q, 2H, J = 12.0 Hz), 3.9 (s,

3H), 3.83-3.68 (m, 1H), 3.3-3.1 (m, 2H), 2.6-2.36 (m, 3H), 2.36 (s, 3H) , 2.32 (s, 3H), 1.16-1.14 (d,

3H, J = 6.7Hz), 0.95 (s, 9H), 0.11 (s, 3H), 0.05 (s,

3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

170.0, 165.1, 154.2, 143.1, 136.4, 135.3, 133.0,

132.0, 131.0, 130.9, 130.0, 128.4, 128.3, 127.7,

126.1, 124.6, 112.3, 112.1, 94.2, 75.0, 74.7, 56.0,

52.9, 42.8, 37.5, 36.5, 25.8, 20.9, 19.3, 18.0, 16.4,

-4.3, -4.7.

IR (CHCl ₃ ): 3428, 2957, 2930, 2857, 1759, 1676, 1645,

1606, 1503, 1464, 1442, 1380, 1349, 1281, 1259,

1173, 1067 cm ^-1 .

Example 33

The compound of Example 34 was prepared according to the procedure of Figure 1 through the following reaction steps.

2.1 g of the ester are prepared from 2.5 g of aldehyde 2 and 5.0 g of 3-methylbenzyl triphenylphosphonium chloride according to the procedure described above (65% yield).

[a] ² ° D = + 45.55 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.22-6.88 (m, 5H), 6.37-6.31 (d, 1H, J = 16.0 Hz),

6.18-6.10 (dd, 1H, J = 16.0, 8.0 Hz), 5.86-5.81 (d,

1H, J = 15.5 Hz), 3.8-3.7 (m, 4H), 2.5-2.3 (m, 6H),

1.11-1.08 (d, 3H, J = 6.9 Hz), 0.91 (s, 9H), 0.061 (s,

3H), 0.052 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

166.7, 146.4, 137.5, 135.9, 131.6, 130.4, 128.3,

127.7, 126.7, 123.1, 122.8, 75.0, 51.3, 42.7, 37.4,

33.8, 25.8, 21.9, 21.3, 18.0, 16.0, -4.5, -4.6.

IR (CHCl ₃ ): 2953, 2931, 2859, 1718, 1658, 1604, 1472,

1454, 1438, 1258 cm ^-1 .

1.93 g of acid were prepared from 2.0 g of ester according to the procedure described above (100% yield).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.22-7.0 (m, 5H), 6.38-6.33 (d, 1H, J = 16.0 Hz),

6.18-6.10 (dd, 1H, J = 16.0, 8.0 Hz), 5.87-5.82 (d,

1H, J = 15.7 Hz), 3.8-3.7 (m, 1H), 2.5-2.35 (m, 3H),

2.34 (s, 3H), 1.12-1.09 (d, 3H, J = 6.8 Hz), 0.9 (s,

9H), 0.068 (s, 3H), 0.061 (s, 3H).

2.9 g of amide were prepared from 1.93 g of acid according to the procedure described above (72% yield).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.22-7.1 (m, 4H), 7.1-7.0 (m, 2H), 6.9-6.8 (m, 2H),

6.36-6.30 (d, 1H, J = 16.0 Hz), 6.19-6.11 (dd, 1H,

J = 16.0, 8.0 Hz), 5.82-5.77 (m, 2H), 5.1-5.0 (m,

1H), 4.78 ^ 4.71 (q, 2H, J = 12.0 Hz), 3.86 (s, 3H),

3.8-3.7 (m, 1H), 3.25-3.15 (m, 2H), 2.5-2.3 (m,

6H), 1.1-1.08 (d, 3H, J = 6.8 Hz), 0.88 (s, 9H), 0.056 (s, 3H), 0.044 (s, 3H).

2.0 g of alcohol were prepared from 2.4 g of starting amide according to the procedure described above (yield 100). ¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.25-6.8 (m, 8H), 6.46-6.41 (d, 1H, J = 15.9 Hz),

6.16-6.07 (dd, 1H, J = 16.0, 8.8 Hz), 5.95-5.8 (m,

2H), 5.1-5.0 (m, 1H), 4.81-4.7 (q, 2H, J = 12.0Hz),

3.87 (s, 3H), 3.7-3.6 (m, 1H), 3.22-3.05 (m, 2H),

2.5-2.34 (m, 3H), 2.34 (s, 3H), 1.8-1.7 (bs, 1H),

1.15-1.13 (d, 3H, J = 6.8 Hz).

2.3 g of the substrate were prepared from 2.0 g of the starting alcohol according to the procedure described above (76% yield).

HU 227 195 Β1 [α]% 0 = + 31.6 ° (c = 1.08, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.2-7.0 (m, 6H), 6.84-6.81 (d, 1H, J = 8.4 Hz),

6.80-6.73 (m, 1H), 6.53-6.50 (bd, 1H, J = 7.3 Hz),

6.4-6.35 (d, 1H, J = 15.8Hz), 6.03-5.95 (dd, 1H,

J = 15.8, 8.5 Hz), 5.9-5.85 (d, 1H, J = 15.7 Hz),

5.42-5.35 (bt, 1H, J = 6.38 Hz), 5.1 ^ 4.82 (m, 3H),

4.8-4.67 (q, 2H, J = 12.0 Hz), 3.85 (s, 3H),

3.28-3.26 (d, 2H, J = 6.46 Hz), 3.23-3.16 (dd, 1H,

J = 14.3, 5.8 Hz), 3.1-3.03 (dd, 1H, J = 14.2, 6.7 Hz),

2.7-2.4 (m, 3H), 2.33 (s, 3H), 1.8-1.5 (m, 3H), 1.43 (s, 9H), 1.2 (s, 3H), 1.15 (s, 3H), 1.12-1.1 (d, 3H,

J = 6.7 Hz), 0.88-0.86 (d, 3H, J = 6.2 Hz), 0.84-0.82 (d, 3H, J = 6.4 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

170.6, 170.0, 165.2, 154.0, 139.2, 138.0, 136.7,

135.8, 131.8, 131.1, 129.7, 128.8, 128.4, 128.2,

126.8, 125.3, 123.3, 122.23, 112.1, 94.2, 78.9,

74.5, 71.3, 56.0, 53.1, 48.6, 43.9, 41.0, 39.4, 36.5,

33.3, 28.3, 24.7, 22.9, 22.7, 22.3, 21.3, 21.2, 16.5. IR (CHCl ₃ ): 3426, 3383, 2967, 2935, 2874, 2841, 1727,

1710, 1680, 1646, 1605, 1504, 1368, 1280, 1259,

1169.1151 cm ^-1 .

0.86 g of styrene was prepared from 2.2 g of the starting carbamate according to the procedure described above (54% yield). [a] ² ° D = + 33.1 ° (c = 1.03, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.24-7.0 (m, 7H), 6.85-6.82 (d, 1H, J = 8.4 Hz),

6.82-6.70 (m, 1H), 6.39-6.34 (d, 1H, J = 15.8 Hz),

6.03-5.95 (dd, 1H, J = 15.8, 8.7Hz), 5.78-5.73 (d,

1H, J = 15.2 Hz), 5.67-5.64 (d, 1H, J = 7.8 Hz),

5.1-5.0 (m, 1H), 4.87-4.83 (dd, 1H, J = 10.2,

3.5 Hz), 4.8-4.7 (m, 1H), 3.9 (s, 3H), 3.45-3.38 (dd,

1H, J = 13.4, 8.6 Hz), 3.2-3.0 (m, 3H), 2.6-2.3 (m,

3H), 2.32 (s, 3H), 1.75-1.25 (m, 3H), 1.22 (s, 3H),

1.15 (s, 3H), 1.13-1.11 (d, 3H, J = 6.8 Hz),

0.75-0.72 (t, 6H, J = 5.7 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

177.9, 170.5, 170.3, 165.1, 154.0, 142.1, 138.0,

136.6, 135.6, 131.8, 130.8, 129.9, 129.6, 128.4,

128.22, 128.17, 126.7, 124.5, 123.3, 122.5, 112.3,

71.4, 56.1, 54.3, 46.4, 42.7, 42.2, 39.4, 36.5, 35.3,

24.3, 22.8, 22.6, 22.5, 21.2, 21.1, 17.2.

IR (CHCl ₃ ): 3424, 3021, 3017, 2965, 1747, 1711, 1680,

1652,1528,1503, 1485, 1259, 1151, 1067 cm ^-1 . Analysis calculated for C ₃₇ H ₄₇ CIN ₂ O ₇ : 0: 66.60, H: 7.10, N: 4.20%; Found: C, 66.79; H, 7.03; N, 4.25.

Example 34

0.20 g of the indicated structure b-epoxide was prepared from 0.677 g of the starting styrene according to the procedure described above in 29% yield.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.0 (m, 7H), 6.90-6.87 (d, 1H, J = 8.4 Hz),

6.87-6.75 (m, 1H), 5.79-5.74 (d, 1H, J = 14.8 Hz),

5.54-5.51 (d, 1H, J = 7.8 Hz), 5.28-5.22 (m, 1H),

4.89 ^ 4.85 (dd, 1H, J = 10.4, 3.5 Hz), 4.82-4.75 (m,

1H), 3.92 (s, 3H), 3.69-3.68 (d, 1H, J = 1.63 Hz),

3.51-3.44 (dd, 1H, J = 13.4, 8.6 Hz), 3.2-3.1 (m,

2H), 2.98-2.95 (dd, 1H, J = 7.6, 1.6 Hz), 2.65-2.32 (m, 3H), 2.32 (s, 3H), 1 , 85-1.6 (m, 3H), 1.4-1.25 (m, 1H), 1.27 (s, 3H), 1.21 (s, 3H), 1.21-1.18 (d,

3H, J = 7.5 Hz), 0.90-0.86 (t, 6H, J = 6.13 Hz).

Example 35

To a solution of β-epoxide (0.1 g, 0.147 mmol) in chloroform (5.0 mL) at -60 ° C was added chlorotrimethylsilane (0.093 mL, 0.74 mmol). The solution was stirred at -60 ° C for 30 minutes and then at room temperature for 1.5 hours and finally concentrated under reduced pressure. The resulting residue contains 50:50 cis and trans chlorohydrins. The product was purified by reverse phase HPLC to give 0.028 g (27%) of the desired trans isomer.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.28-7.2 (m, 5H), 7.13-7.1 (dd, 1H, J = 8.32,

1.95 Hz), 6.91-6.88 (d, 1H, J = 8.5 Hz), 6.88-6.78 (m, 1H), 5.86-5.81 (d, 1H) , J = 15.0 Hz), 5.73-5.71 (d, 1H, J = 7.8 Hz), 5.24-5.17 (t, 1H, J = 9.4 Hz),

5.0-4.96 (dd, 1H, J = 9.58, 2.93 Hz), 4.81 ^ 4.74 (m,

1H), 4.67 ^ 4.64 (d, 1H, J = 9.73 Hz), 4.06 ^ 4.03 (dd,

1H, J = 9.6.1.1 Hz), 3.92 (s, 3H), 3.47-3.39 (dd, 1H,

J = 13.2, 8.3 Hz), 3.24-3.0 (m, 3H), 2.8-2.4 (m, 2H),

2.4 (s, 3H), 1.9-1.4 (m, 4H), 1.28 (s, 3H), 1.22 (s,

3H), 1.09-1.07 (d, 3H, J = 6.95Hz), 0.98-0.96 (d,

6H, J = 6.4 Hz).

Example 36

The compound of Example 36 having the structure given is prepared following the procedure of Example 1 through the following reaction steps.

2.75 g of ester are prepared from 339 g of aldehyde (2) and

7.7 g of 4-methoxybenzyl triphenylphosphonium chloride were obtained according to the procedure described above (54% yield).

[a] ² ° D = + 7185 ° (c = 1.03, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.25-7.21 (m, 2H, J = 9.0 Hz), 7.0-6.85 (m, 1H),

6.81-6.78 (d, 2H, J = 8.5 Hz), 6.30-6.23 (d, 1H,

J = 16.3 Hz), 6.0-5.9 (dd, 1H, J = 16.3, 8.2 Hz),

5.81-5.75 (d, 1H, J = 14.26 Hz), 3.75 (s, 3H), 3.67 (s, 4H), 2.42-2.22 (m, 3H), 1 , 05-1.02 (d, 3H,

J = 6.8 Hz), 0.86 (s, 9H), 0.005 (s, 3H), -0.001 (s,

3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

166.7, 158.8, 146.4, 130.4, 129.7, 129.7, 127.0,

122.7, 113.8, 75.0, 55.2, 51.3, 42.7, 37.4, 25.8,

18.0, 16.1, 4.5, 4.6.

IR (CHCl ₃ ): 3010, 2955, 2930, 2898, 2857, 1718, 1658,

1607, 1511 cm ^-1 .

1.58 g of acid are prepared from 1.7 g of ester according to the procedure described above (96%).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.27 (d, 2H, J = 8.7 Hz), 7.13-7.03 (m, 1H),

6.86-6.83 (d, 2H, J = 8.6 Hz), 6.35-6.29 (d, 1H,

J = 16.0 Hz), 6.04 to 5.96 (dd, 1H, J = 15.9, 8.0 Hz),

5.87-5.81 (d, 1H, J = 15.8Hz), 3.8 (s, 3H),

3.79-3.7 (m, 1H), 2.5-2.33 (m, 3H), 1.1-1.08 (d,

3H, J = 6.8Hz), 0.9 (s, 9H), 0.058 (s, 3H), 0.055 (s,

3H).

HU 227 195 Β1

2.0 g of the amide were prepared from 1.58 g of acid according to the procedure described above (70% yield).

[a] ² ° D = + 2.0 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.28-7.26 (d, 2H, J = 7.33 Hz), 7.18-7.17 (d, 1H,

J = 1.8 Hz), 7.04-7.0 (dd, 1H, J = 8.5, 1.8 Hz),

6.90-6.79 (m, 3H), 6.32-6.27 (d, 1H, J = 16.0 Hz),

6.05-5.97 (dd, 1H, J = 16.0, 8.1 Hz), 5.83-5.76 (m,

3H), 5.09-5.05 (m, 1H), 4.82-4.7 (q, 2H,

J = 11.9 Hz), 3.86 (s, 3H), 3.8 (s, 3H), 3.77-3.7 (m,

1H), 3.24-3.17 (dd, 1H, J = 14.2, 5.7 Hz), 3.13-3.07 (dd, 1H, J = 14.2, 5.94 Hz) , 2.5-2.3 (m, 3H),

1.09-1.06 (d, 3H, J = 6.8 Hz), 0.89 (s, 9H), 0.048 (s,

3H), 0.038 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

169.9, 165.1, 158.7, 154.2, 143.0, 135.5, 131.0,

130.4, 129.6, 128.4, 127.0, 124.6, 122.5, 113.8,

112.1, 94.2, 75.0, 74.7, 56.0, 55.2, 52,9,42.5, 37.4,

36.4,25.8, 18.0, 16.4, -4.4, -4.7.

IR (CHCL ₃ ): 2957, 2931, 2857, 1757, 1676, 1644,

1607.1511.1503 cm ^-1 .

1.35 g of alcohol were prepared from 1.63 g of the starting amide according to the procedure described above (98% yield).

[a] ² ° D = + 55.7 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.31-7.29 (d, 2H, J = 7.9 Hz), 7.18 (s, 1H),

7.05-7.02 (d, 1H, J = 8.3 Hz), 7.0-6.87 (m, 1H),

6.86-6.83 (d, 3H, J = 8.2 Hz), 6.44-6.38 (d, 1H,

J = 15.8 Hz), 6.0-5.82 (m, 3H), 5.10-5.0 (m, 1H),

4.81-4.7 (q, 2H, J = 11.8 Hz), 3.87 (s, 3H), 3.81 (s,

3H), 3.67-3.6 (m, 1H), 3.24-3.19 (dd, 1H, J = 14.1,

6.1 Hz), 3.12-3.07 (dd, 1H, J = 14.4, 5.9 Hz),

2.5-2.25 (m, 3H), 1.8-1.6 (bs, 1H), 1.14-1.12 (d,

3H), J = 6.6 Hz).

¹³ C-NMR (75 MHz, CDCl ₃ ), δ: 170.1, 165.2, 159.0,

154.2, 142.5, 131.2, 131.0, 129.8, 128.7, 128.4,

127.2, 125.0, 122.4, 113.9, 112.2, 94.2, 74.7, 73.8,

56.05, 55.2, 53.0, 43.2, 37.1, 36.4, 16.8.

IR (CHCl 3): 3428, 2964, 2936, 2912, 2874, 2840, 1758,

1677,1645,1607, 1512, 1503, 1258, 1175 crrr ¹ .

1.69 g of the substrate were prepared from 1.26 g of the starting alcohol according to the procedure described above (89% yield).

[.alpha.] D @ 20 = + 35.2 DEG (c = 1.02, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.23 (d, 21H, J = 8.0 Hz), 7.18-7.17 (d, 1H,

J = 1.7), 7.07-7.03 (dd, 1H, J = 8.4.1.6 Hz), 6.85-6.7 (m, 4H), 6.53-6, Δ (d, 1H, J = 7.9 Hz), 6.36-6.31 (d,

1H, J = 15.8 Hz), 5.9-5.81 (m, 2H), 5.42-5.35 (t,

1H), 5.1 ^ 4.95 (m, 2H), 4.94 ^ 1.90 (dd, 1H, J = 9.6,

3.4 Hz), 4.81 -4.67 (q, 2H, J = 11.9 Hz), 3.85 (s, 3H),

3.79 (s, 3H), 3.28-3.26 (d, 2H, J = 6.5 Hz),

3.23-3.16 (dd, 1H, J = 14.3, 5.8 Hz), 3.09-3.02 (dd, tH, J = 14.1, 6.7 Hz), 2, 61-2.4 (m, 3H), 1.8-1.5 (m, 3H), 1.43 (s, 9H), 1.20 (s, 3H), 1.15 (s, 3H) .

1.11-1.09 (d, 3H, J = 6.7Hz), 0.87-0.85 (d, 3H, J = 6.4Hz), 0.84-0.82 (d, 3H, J = 6.5 Hz).

¹³ C-NMR (75 MHz, CDCl 3), δ:

174.9, 170.6, 169.9, 165.3, 159.0, 154.0, 139.3,

135.4, 131.1, 131.0, 129.6, 128.8, 128.4, 127.8,

127.2, 125.2, 122.2, 113.9, 112.2, 94.3, 74.5, 71.3,

56.0, 55.2, 53.1, 48.6, 43.9, 41.0, 39.4, 36.5, 28.3,

24.7, 22.9, 22.7, 22.3, 21.4, 16.6.

IR (CHCl 3): 3426, 3383, 2965, 2936, 2874, 2840, 1728,

1711, 1680, 1646, 1607, 1512, 1465, 1367, 1254,

1175, 1067 cm ^-1 .

0.676 g of styrene product was prepared from 1.43 g of the starting carbamate according to the procedure described above (65% yield).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.23 (d, 3H, J = 8.4 Hz), 7.20-7.19 (d, 1H,

J = 1.8 Hz), 7.07-7.03 (dd, 1H, J = 8.4, 1.9 Hz),

6.84-6.81 (d, 3H, J = 8.5 Hz), 6.8-6.7 (m, 1H),

6.36-6.31 (d, 1H, J = 15.8 Hz), 5.89-5.81 (dd, 1H,

J = 15.8, 8.8 Hz), 5.78-5.73 (d, 1H, J = 13.7 Hz),

5.68-5.66 (d, 1H, J = 7.9Hz), 5.05-4.99 (ddd, 1H,

J = 10.6, 6.6, 1.6 Hz), 4.87 ^, 82 (dd, 1H, J = 9.7,

3.1 Hz), 4.78-4.7 (m, 1H), 3.86 (s, 3H), 3.79 (s,

3H), 3.45-3.37 (dd, 1H, J = 13.4, 8.6 Hz), 3.15-3.0 (m, 3H), 2.6-2.25 (m, 3H), 1.7-1.3 (m, 3H), 1.22 (s,

3H), 1.15 (s, 3H), 1.12-1.1 (d, 3H, J = 6.8 Hz),

0.76-0.75 (d, 3H, J = 2.9 Hz), 0.74-0.73 (d, 3H,

J = 2.8 Hz).

Example 37

To a solution of potassium tert-butoxide (1.17 g, 10.5 mmol) in tetrahydrofuran (120 mL) at 0 ° C was added 5.0 g (10.5 mmol) of p-nitrobenzyl triphenylphosphonium in small portions over 30 minutes. bromides. The reaction mixture was stirred at 0 ° C for 1 hour. A solution of aldehyde (2) in tetrahydrofuran (20 ml) was added dropwise, and the reaction mixture was stirred at 0 ° C for 15 minutes, then slowly allowed to warm to room temperature and stirred overnight. A saturated aqueous solution of ammonium chloride (50 mL) was added to the reaction mixture and extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and evaporated under reduced pressure. The crude styrene containing a mixture of E: Z isomers was purified by chromatography on silica gel using 2% ethyl acetate in hexane as eluent. 0.5 g of the Z-isomer and 1.1 g of the E-isomer (42%) were obtained as a yellow oil.

[a] ² ° D = ⁰ +63.81 (c = 1.05, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.18-8.15 (d, 2H, J = 8.6 Hz), 7.47-7.44 (d, 2H,

J = 8.7 Hz), 7.0-6.85 (m, 1H), 6.5-6.3 (m, 2H),

5.87-5.82 (d, 1H, J = 15.6Hz), 3.8-3.73 (m, 1H),

3.73 (s, 3H), 2.55-2.35 (m, 3H), 1.13-1.11 (d, 3H,

J = 6.9 Hz), 0.90 (s, 9H), 0.059 (s, 3H), 0.047 (s, 3H). ¹³ C-NMR (75 MHz, CDCl 3), δ:

166.5, 145.5, 143.9, 137.3, 128.7, 126.4, 123.9,

123.1,74.6, 51.3, 42.8, 37.8, 33.8, 25.7, 24.3, 21.9,

18.0, 16.3, -4.4, -4.7.

IR (CHCl 3): 2953, 2931, 2859, 1719, 1658, 1596, 1518,

1472, 1438, 1345, 1259, 1110 cm ^-1 .

3.06 g of amide were prepared from 2.55 g of acid according to the procedure described above (65% yield).

[a] ² ° D = + 70.67 ° (c = 1.05, MeOH).

HU 227 195 Β1 ¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.15-8.12 (d, 2H, J = 8.7 Hz), 7.45-7.42 (d, 2H,

J = 8.7 Hz), 7.17-7.16 (d, 1H, J = 2.0 Hz), 7.04-7.01 (dd, 1H, J = 8.45, 1.9 Hz) ), 6.88-6.82 (m, 2H),

6.43-6.3 (m, 2H), 5.93-5.91 (d, 1H, J = 7.5 Hz),

5.84-5.79 (d, 1H, J = 15.3Hz), 5.1-5.0 (m, 1H),

4.82-4.69 (q, 2H, J = 11.9Hz), 3.85 (s, 3H), 3.8-3.7 (m, 1H), 3.24-3.18 ( dd, 1H, J = 14.3, 5.7 Hz),

3.13-3.06 (dd, 1H, J = 14.2, 6.05 Hz), 2.52-2.3 (m,

3H), 1.11-1.09 (d, 3H, J = 6.8 Hz), 0.88 (s, 9H),

0.041 (s, 3H), 0.032 (s, 3H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

170.0, 165.0, 154.2, 146.5, 144.0, 142.2, 137.4,

136.0, 131.0, 128.7, 128.4, 126.4, 124.9, 123.9,

122.5, 112.1, 94.1, 74.72, 74.67, 56.0, 52.9, 42.7,

37.8, 36.4, 25.7, 18.0, 16.5, -4.7, 4.3.

IR (CHCl ₃ ), δ:

3429, 2957, 2931, 2858, 1757, 1677, 1645, 1597,

1517, 1503, 1345, 1259, 1180, 1110, 1067,

1026 cm ^-1 .

3.3 g of the substrate were prepared from 2.54 g of the starting alcohol according to the procedure described above (87% yield). [.alpha.] ^{2b J} = +38.2 (c = 1.07, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.23-8.2 (d, 2H, J = 8.7 Hz), 7.52-7.49 (d, 2H,

J = 8.7 Hz), 7.21 (s, 1H), 7.12-7.08 (dd, 1H, J = 8.2,

1.8 Hz), 6.89-6.86 (d, 1H, J = 8.4 Hz), 6.86-6.75 (m,

1H), 6.6-6.58 (d, 1H, J = 8.0 Hz), 6.55-6.5 (d, 1H,

J = 15.9 Hz), 6.31-6.23 (dd, 1H, J = 15.8, 8.5 Hz),

5.97-5.92 (d, 1H, J = 15.5Hz), 5.4-5.3 (bt, 1H),

5.2- 5.0 (m, 2H), 4.98-4.94 (dd, 1H, J = 9.5, 3.6 Hz),

4.86-4.72 (q, 2H, J = 12Hz), 3.9 (s, 3H), 3.33-3.3 (d, 1H, J = 6.6Hz), 3.27-3 , 22 (dd, 1H, J = 14.1,

5.7 Hz), 3.14-3.07 (dd, 1H, J = 14.0, 6.7 Hz),

2.8-2.5 (m, 3H), 2.9-1.47 (m, 3H), 1.47 (s, 9H),

1.3-1.2 (m, 9H), 0.91-0.89 (d, 3H, J = 6.4 Hz),

0.87-0.85 (d, 3H, J = 6.4 Hz).

¹³ C-NMR (75 MHz, CDCl ₃ ), δ; 176.8, 170.6, 169.9,

165.1, 156.2, 154.0, 146.8, 143.2, 138.8, 135.8,

135.4, 131.1, 129.7, 128.7, 128.4, 126.7, 126.6,

125.5, 123.9, 122.2, 112.1, 94.2, 79.0, 76.2, 74.5,

71.2, 56.0, 53.1, 48.5,43.9, 41.2, 39.4, 36.5, 33.3,

28.3, 24.7, 22.9, 22.7, 22.3, 21, 4, 16.3.

IR (CHCl ₃ ), δ:

3426, 3385, 2967, 2936, 2874, 2841, 1712, 1681,

1646, 1597, 1519, 1500, 1345, 1280, 1259, 1170,

1150,1067,1024 crrr ¹ .

0.55 g of styrene was prepared from 1.77 g of the starting carbamate according to the procedure described above (42% yield).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.23-8.21 (d, 2H, J = 8.7 Hz), 7.52-7.49 (d, 2H,

J = 8.7 Hz), 7.24-7.2 (m, 2H), 7.11-7.08 (dd, 1H,

J = 8.5, 1.92 Hz), 6.89-6.87 (d, 1H, J = 8.4 Hz),

6.86-6.72 (m, 1H), 6.56-6.51 (d, 1H, J = 15.9 Hz),

6.33-6.25 (dd, 1H, J = 15.9, 8.7 Hz), 5.94-5.91 (d,

1H, J = 7.8 Hz), 5.83-5.78 (d, 1H, J = 15.2 Hz),

5.17-5.12 (m, 1H), 4.92-4.88 (dd, 1H, J = 9.7,

3.6 Hz), 4.8-4.75 (m, 1H), 3.92 (s, 3H), 3.5-3.43 (dd, 1H, J = 13.5, 8.73 Hz) ), 3.2-3.1 (m, 3H),

2.7-2.35 (m, 3H), 1.8-1.6 (m, 2H), 1.4-1.19 (m,

1H), 1.2 (s, 3H), 1.16-1.14 (d, 6H), 0.82-0.81 (d,

3H, J = 3.73 Hz), 0.80-79 (d, 3H, J = 3.87 Hz).

Example 38

Analytical data for the compound of Example 38 having the following structure:

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.5 (s, 1H), 7.76-7.74 (d, 2H, J = 7.4 Hz), 7.58-7.56 (d, 2H, J = 7.4 Hz), 7.5 -7.1 (m, 8H), 7.03-7.0 (d, 1H,

J = 8.2 Hz), 6.81-6.78 (d, 1H, J = 8.5 Hz), 6.78-6.65 (m, 1H), 6.37-6.34 (d , 1H, J = 7.3 Hz), 5.84-5.81 (t,

1H, J = 5.2 Hz), 5.69-5.64 (d, 1H, J = 15.0 Hz),

5.18-5.15 (bd, 1H, J = 9.0 Hz), 4.83-4.6 (m, 2H),

4.45 ^ 4.43 (d, 2H, J = 6.7 Hz), 4.24 ^ 1.19 (t, 1H,

J = 6.6 Hz), 3.99 (bs, 2H), 3.81 (s, 3H), 3.61 (s, 1H),

3.46-3.39 (dd, 1H, J = 13.4, 8.8 Hz), 3.2-2.8 (m,

5H), 2.6-2.3 (m, 3H), 1.8-1.3 (m, 3H), 1.2 (s, 3H),

1.14 (s, 3H), 1.12-1.1 (d, 3H, J = 6.8 Hz), 0.86-0.81 (t, 6H, J = 7.3 Hz).

Example 39

Analytical data for the compound of Example 39 having the following structure:

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

9.5 (bs, 2H), 7.6-7.57 (d, 2H, J = 8.1 Hz), 7.24-7.18 (m, 5H), 7.05-7.03 (d, 1H, J = 8.3 Hz), 6.84-6.82 (d,

1H, J = 8.4 Hz), 6.82-6.65 (m, 1H), 6.02-6.0 (d, 1H,

J = 7.6Hz), 5.72-5.67 (d, 1H, J = 15.3Hz), 5.19-5.16 (bd, 1H, J = 10.5Hz), 4, 9-4.6 (m, 2H), 3.85 (s, 3H),

3.64 (s, 1H), 3.55-3.4 (m, 3H), 3.2-2.95 (m, 3H),

2.90-2.88 (d, 1H, 7.11Hz), 2.6-2.3 (m, 3H),

2.0-1.3 (m, 3H), 1.2 (s, 3H), 1.15 (s, 3H), 1.13-1.11 (d, 3H, J = 6.9 Hz) , 0.90-0.84 (t, 6H, J = 7.2 Hz).

Example 40

The desired compound is prepared according to the reaction scheme depicted in Figure 2, with the same numbering as that of Figure 2.

To a solution of 0.3 g (0.50 mmol) of alkene (17) in 9.0 mL of dichloromethane and 1.0 mL of methanol at -78 ° C was added 0.07 mL (0.074 mmol) of 2-picoline. This solution was reacted with 1.2 equivalent of ozone, and to the resulting ozonide was added 1.1 mL (2.2 mmol) of dimethyl sulfide. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solution was washed with water (2 x 20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. 0.251 g (18) of aldehyde (87%) are obtained. ¹ H-NMR (300 MHz, CDCl ₃ ), δ:

9.64-9.63 (d, 1H, J = 1.05 Hz), 7.25-7.2 (m, 2H),

7.08-7.05 (d, 1H, J = 8.2 Hz), 6.86-6.83 (d, 1H,

J = 8.4 Hz), 6.83-6.7 (m, 1H), 5.81-5.76 (d, 1H,

J = 15.1 Hz), 5.75-5.65 (bs, 1H), 5.38-5.3 (m, 1H),

4.85-4.81 (dd, 1H, J = 10.2, 3.2 Hz), 4.80-4.7 (m,

1H), 3.87 (s, 3H), 3.46-3.39 (dd, 1H, J = 13.5,

8.7 Hz), 3.2-3.0 (m, 3H), 2.7-2.4 (m, 3H), 1.8-1.6 (m, 2H), 1.4-1 , 3 (m, 1H), 1.23 (s, 3H), 1.18-1.16 (d, 6H), 0.94-0.92 (d, 3H, J = 6.5 Hz), 0.88-0.86 (d,

3H, J = 6.4 Hz).

HU 227 195 Β1

Example 41

The desired compound was prepared according to the reaction procedure described in Example 3, with the same numbering as that of Figure 3.

To a solution of Cryptophycin-53 (0.15 g, 0.23 mmol) in 3 mL of DME / 2.0 water was added 5 drops of concentrated sulfuric acid. The reaction mixture was stirred overnight and additional 5 drops of sulfuric acid were added and stirring continued for 24 hours. Saturated sodium bicarbonate solution was added slowly until the reactivity ceased, and the reaction mixture was extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and evaporated. The resulting evaporation residue was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent. 0.13 g of nuts are obtained. Sodium periodate (0.144 g, 0.675 mmol) was added to a solution of the nut mixture in a mixture of tetrahydrofuran (4.0 mL) and water (2.0 mL). After stirring at room temperature overnight, the reaction mixture was concentrated under reduced pressure and water (5 mL) was added to the residue and extracted with dichloromethane. The combined organic layers were dried over sodium sulfate, filtered and evaporated under reduced pressure to give 0.10 g (77%) of aldehyde (18).

To a solution of 0.23 g (0.4 mmol) of TIPS-protected 4-hydroxymethylbenzyl triphenylphosphonium chloride in 3.0 mL of tetrahydrofuran at -38 ° C was added dropwise 0.25 mL of 1.6 M n- butyl lithium. The reaction mixture was allowed to slowly warm to 0 ° C and stirred for 10 minutes. To a solution of aldehyde (18) in 4.0 ml of tetrahydrofuran at -78 ° C is added dropwise 2.5 ml of a 0.13 M solution of "ylide" prepared previously. The reaction mixture was stirred at -78 ° C for 2 hours and then at room temperature for 30 minutes. A saturated solution of ammonium chloride (20 mL) was added to the reaction mixture with ethyl acetate (10 mL), the layers were separated, and the organic layer was washed with water (3 x 10 mL) and brine. The organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (70% ethyl acetate / hexane). 0.09 g (62%) of the desired styrene is obtained.

Example 42

To a solution of Cryptophycin-53 (2.0 g, 2.99 mmol) in DME (30 mL) was added 2M aqueous perchloric acid (15 mL, 30 mmol) and the reaction mixture was stirred for 6 h. The reaction mixture was carefully neutralized with a saturated solution of sodium bicarbonate (50 mL), extracted with dichloromethane (4 x 100 mL), and the combined organic phases were dried over sodium sulfate, filtered and concentrated under reduced pressure. The evaporation product was purified by column chromatography over silica gel using 5% methanol in dichloromethane as eluent. 1.5 g (72%) of a mixture of nuts (19) containing 3: 1 anti / syn isomers were obtained. To a solution of 1.0 g (1.46 mmol) of walnut in 20 ml of tetrahydrofuran and 15 ml of water is added 1.9 g (8.9 mmol) of sodium periodate and the reaction mixture is stirred under nitrogen overnight. Most of the tetrahydrofuran was removed under reduced pressure, the residue was diluted with water (100 mL) and extracted with dichloromethane (4 x 50 mL). The combined organic extracts were washed with brine (25 mL), dried over sodium sulfate, filtered and evaporated under reduced pressure. The solid product was dissolved in toluene (100 mL) and evaporated at 40 ° C in a rotary evaporator to remove residual benzaldehyde. After two additional toluene additions and evaporation, 0.828 g (98%) of aldehyde (18) is obtained in the form of a yellow foam. The resulting aldehyde was used without further purification and stored at -23 ° C until use.

[a] ² ° D = + 23.0 ° (c = 0.565, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

9.64-9.63 (d, 1H, J = 1.4 Hz), 7.28-7.26 (m, 1H),

7.21-7.20 (d, 1H, J = 1.9 Hz), 7.08-7.05 (dd, 1H,

J = 7.1, 1.7 Hz), 6.87-6.84 (d, 1H, J = 8.5 Hz),

6.82-6.72 (m, 1H), 5.80-5.75 (d, 1H, J = 15.0 Hz),

5.54-5.51 (d, 1H, J = 7.7 Hz), 5.40-5.33 (m, 1H),

4.85-4.81 (dd, 1H, J = 9.7, 3.2 Hz), 4.78 ^ 1.71 (m,

1H), 3.88 (s, 3H), 3.46-3.39 (dd, 1H, J = 13.5,

8.6 Hz), 3.15-3.03 (m, 3H), 2.68-2.35 (m, 3H),

1.82-1.63 (m, 2H), 1.45-1.37 (m, 1H), 1.24 (s, 3H),

1.19-1.16 (d, 3H, J = 7.1 Hz), 1.18 (s, 3H),

0.94-0.92 (d, 3H, J = 6.5 Hz), 0.89-0.87 (d, 3H,

J = 6.5 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

200.7, 177.8, 170.6, 170.1, 165.1, 153.9, 141.1,

130.7, 129.8, 128.1, 124.9, 122.3, 112.3, 73.4,

71.1, 56.0, 54.6, 49.9, 46.4, 42.7, 39.2, 36.1, 35.2,

24.7,22.8, 22.7,21.3, 10.7.

IR (CHCl ₃ ): 3422, 2964, 2936, 1755, 1730, 1718, 1678,

1529, 1504, 1487, 1474, 1464, 1442, 1320, 1303,

1281, 1259, 1244, 1185, 1151, 1127, 1067 cm @ ^-1 . Elemental analysis for C29H29CIN2O6: C, Η, N.

Example 43

To a solution of 4- (triisopropylsiloxymethyl) benzyl triphenylphosphonium bromide (7.6 g, 12.2 mmol) in tetrahydrofuran (100 ml) was added dropwise 8.0 ml of 1.5 M n-butyllithium. solution (8.1 mL, 12.2 mmol). The reaction mixture was slowly warmed to room temperature and stirred for an additional 30 minutes.

To a solution of aldehyde (18) (2.95 g, 5.1 mmol) in tetrahydrofuran (100 mL) at -78 ° C was added the red solution of red ylide through a double-ended needle. The reaction mixture was stirred at -78 ° C for 3 hours and then at room temperature for 45 minutes, 100 ml of saturated ammonium chloride solution and 100 ml of ethyl acetate were added, the phases were separated and the aqueous phase was extracted twice with 50 ml of ethyl acetate. The combined organic layers were washed with water (3 x 40 mL), brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure. The resulting yellow evaporation residue was obtained

It was purified by column chromatography over silica gel, eluting with 10-20-50% ethyl acetate in hexane. 3.6 g (84%) of the desired styrene are obtained in the form of a white solid. The product is a mixture of E: Z isomers.

A mixture of 7.3 g (8.7 mmol) of isomer is dissolved in 240 mL of benzene and 0.32 g (0.87 mmol) of 1,1'-azobis (cyclohexane carbonitrile) (VAZO) and 3.7 mL (4, Reflux (0 mmol) in the presence of thiophenol. After refluxing for 5 hours, the solution was evaporated and the residue was purified by silica gel column chromatography using 5 to 50% ethyl acetate in hexane as eluent. 6.7 g (92%) of the E-isomer (20) are obtained in the form of a white solid.

[a] ² ° D = + 31.9 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.22 (m, 5H), 7.20-7.19 (d, 1H, J = 1.95 Hz),

7.07-7.04 (dd, 1H, J = 8.4, 2.0 Hz), 6.85-6.82 (d,

1H, J = 8.5 Hz), 6.8-6.7 (m, 1H), 6.4-6.38 (d, 1H,

J = 15.8 Hz), 6.02-5.94 (dd, 1H, J = 15.8, 8.8 Hz),

5.77-5.72 (d, 1H, J = 14.9 Hz), 5.56-5.54 (d, 1H,

J = 7.9 Hz), 5.1-4.7 (m, 5H), 3.9 (s, 3H), 3.45-3.37 (dd, 1H, J = 13.5, 8, 5 Hz), 3.2-3.0 (m, 3H), 2.6-2.3 (m, 3H), 1.7-1.5 (m, 2H), 1.4-1.0 (m, 31H),

0.75-0.71 (t, 6H, J = 6.1 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 165.2, 153.9, 142.1, 141.1,

135.2, 131.5, 130.8, 129.7, 129.6, 128.1, 125.9,

124.5, 122.4, 112.2, 77.0, 71.4, 64.7, 56.0, 54.4,

46.4, 42.7, 42.2, 39.4, 36.5, 35.3, 24.5, 22.8, 22.6,

22.5.21.2, 17.9, 17.2, 11.9.

IR (CHCl 3): 3423, 2962, 2945, 2867, 1746, 1712, 1681,

1652, 1528, 1503, 1485, 1473, 1464, 1303,

1259 cm ¹ .

Elemental analysis for C ₄₆ H ₆₇ CIN ₂ O ₈ Si: C, Η, N.

Example 44

To a solution of 3-chloroperoxybenzoic acid (0.27 g, 1.59 mmol) was added a solution of styrene (20) (1.25 g, 1.49 mmol) in dichloromethane (20 mL) at 0 ° C. The solution was stirred for 1 hour at 0 ° C and overnight at room temperature. After evaporation under reduced pressure, the resulting epoxides were separated by reverse phase HPLC to give 0.67 g of β-epoxide (22) as a white solid (57%).

[a] ² ° D = + 20.9 ° (c = 0.765, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.35-7.33 (d, 2H, J = 7.8 Hz), 7.26-7.2 (m, 4H),

7.05-7.02 (bd, 1H, J = 8.2 Hz), 6.84-6.81 (d, 1H,

J = 8.4 Hz), 6.81-6.65 (m, 1H), 5.8-5.65 (m, 2H),

5.25-5.15 (m, 1H), 4.9 ^ 1.7 (m, 4H), 3.9 (s, 3H), 3.7 (s, 1H), 3.46-3, 42 (dd, 1H, J = 13.4, 8.8 Hz),

3.15-3.0 (m, 3H), 2.93-2.9 (d, 1H, J = 7.3 Hz),

2.6-2.4 (m, 2H), 1.8-1.6 (m, 3H), 1.4-1.0 (m, 31H),

0.83-0.79 (t, 6H, J = 5.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.5, 170.4, 165.1, 153.9, 142.1, 141.6,

136.7, 135.1, 130.7, 129.8, 128.1, 125.9, 125.5,

124.6, 122.3, 112.2, 75.9, 71.0, 64.6, 63.0, 58.9,

56.0, 54.6, 46.3, 42.7, 40.5, 39.2, 36.8, 35.2, 24.2,

22.8.22.7.22.6, 18.0, 13.4, 11.9.

IR (CHCl 3): 3424, 2962, 2945, 2867, 1751, 1712, 1682,

1528, 1503, 1485, 1473, 1464 crrr ¹ .

Elemental analysis for C ₄₆ H ₆₇ CIN ₂ O ₉ Si: C, Η, N.

Example 45

A solution of 1.04 mL (0.14 mmol) of 1.0 M tetrabutylammonium fluoride in tetrahydrofuran was added dropwise to a solution of 0.1 g (0.117 mmol) of β-ethoxide (22) in 3.5 mL of tetrahydrofuran at 0 ° C. The solution was allowed to warm to room temperature and stirred for an additional 20 minutes, after which 10 ml of water and 20 ml of ethyl acetate were added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 x 20 mL). The combined organic phases are dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the free alcohol. The product was purified by silica gel column chromatography (70-100% ethyl acetate / hexane). Obtained 0.068 g (84%) of pure alcohol 23 as a white solid.

[a] ² ° D = + 26.2 ° (c = 0.435, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.39-7.36 (d, 2H, J = 7.8 Hz), 7.26-7.23 (d, 3H,

J = 9.1 Hz), 7.18 (s, 1H), 7.05-7.02 (d, 1H,

J = 8.5 Hz), 6.85-6.82 (d, 1H, J = 8.2 Hz), 6.82-6.7 (m, 1H), 5.72-5.67 (d , 1H, J = 15.1 Hz), 5.55-5.52 (d, 1H, J = 7.8 Hz), 5.22-5.17 (m, 1H), 4.85-4, 7 (m,

4H), 3.9 (s, 3H), 3.7 (s, 1H), 3.45-3.38 (dd, 1H,

J = 13.4, 9.3 Hz), 3.2-3.0 (m, 3H), 2.92-2.89 (d, 1H,

J = 7.6 Hz), 2.65-2.4 (m, 2H), 1.8-1.6 (m, 4H),

1.4-1.2 (m, 1H), 1.22 (s, 3H), 1.16 (s, 3H),

1.16-1.13 (d, 3H, J = 7.2 Hz), 0.86-0.82 (t, 6H,

J = 6.5 Hz).

¹³ C NMR (63 MHz, CDCl 3), δ:

177.8, 171.0, 170.4, 165.5, 153.8, 141.5, 141.4,

135.7, 133.5, 130.6, 130.0, 128.0, 127.1, 125.6,

124.6, 122.2, 112.3, 77.2, 76.5, 76.0, 71.0, 64.2,

63.1, 58.8, 56.0, 54.7, 46.3, 42.7, 40.5, 39.3, 36.9,

35.1, 24.5, 22.7, 22.5, 22.1, 13.4.

IR (CHCl 3): 3422, 2992, 2963, 2936, 2874, 1751, 1713,

1682, 1651, 1504, 1486, 1303, 1259, 1186, 1165,

1151, 1067 cm ^1st

FAB HRMS (M + H) calcd for C ₃₇ H ₄₈ N ₂ O ₉ : 699.3048, found: 699.3054.

Example 46

0.08 g (0.114 mmol) of alcohol (23), 0.034 g (0.194 mmol) of N- (tert-butoxycarbonyl) glycine and 0.004 g (0.034 mmol) of 4-dimethylaminopyridine (DMAP) 2.0 A solution of 1,3-dicyclohexylcarbodiimide (DCC) (0.040 g, 0.194 mmol) in dichloromethane (0 mL) was added. The reaction mixture was stirred at 0 ° C for 10 minutes and at room temperature for 45 minutes, filtered and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel using 70-80% ethyl acetate in hexane as eluent. 0.07 g (72%) of the ester was obtained as a white solid.

[a] ² ° D = + 18.5 ° (c = 0.65, _CHCl3).

HU 227 195 Β1 ¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.4-7.2 (m, 6H), 7.11-7.08 (dd, 1H, J = 8.4, 1.8 Hz),

6.9-6.87 (d, 1H, J = 8.4 Hz), 6.86-6.7 (m, 1H),

5.78-5.73 (d, 1H, J = 15.2 Hz), 5.64-5.62 (d, 1H,

J = 7.4 Hz), 5.3-5.22 (m, 1H), 5.22 (s, 2H), 5.1-5.0 (bs, 1H), 4.9-4.7 (m, 2H), 4.0-3.99 (d, 2H,

J = 5.4 Hz), 3.9 (s, 3H), 3.73-3.72 (d, 1H, J = 1.0 Hz),

3.5-3.43 (dd, 1H, J = 13.4, 8.6 Hz), 3.2-3.0 (m, 3H),

2.95-2.92 (d, 1H, J = 6.4 Hz), 2.65-2.4 (m, 2H),

1.8-1.6 (m, 3H), 1.5 (s, 9H), 1.45-1.3 (m, 1H), 1.26 (s, 3H), 1.2 (s, 3H), 1.2-1.17 (d, 3H, J = 8.7 Hz),

0.9-0.86 (t, 6H, J = 6.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.6, 170.3, 170.2, 165.1, 155.6, 153.8,

141.4, 137.1, 135.6, 130.6, 129.9, 128.6, 128.0,

125.7, 124.7, 122.2, 112.2, 79.9, 75.8, 70.9, 66.4,

63.1, 58.5, 56.0, 54.7, 48.9, 46.3, 42.7, 42.4, 40.5,

39.3, 36.8, 35.2, 28.2, 24.5, 22.8, 22.7, 22.6, 21.2,

13.5.

Elemental analysis for C ₄₄ H ₅₈ CIN ₃ O ₁₂ : C, Η, N.

Example 47

Trimethylsilyl chloride (0.09 mL, 0.75 mmol) was added to a solution of β-epoxide (24) (0.16 g, 0.187 mmol) in chloroform (5.0 mL) at -60 ° C. After stirring for 2 hours at -60 ° C to -40 ° C, an additional 0.09 ml of TMSCI is added and stirring is continued for 3 hours. The solution was allowed to warm to room temperature, evaporated and the residue was purified by reverse phase preparative HPLC using a 55:45 mixture of acetone in yttrium / water as eluent. Separation of the two chlorohydrin formed gave 0.058 g (35%) of the desired chlorohydrin (25).

[a] ² ° D = + 50.5 ° (c = 1.075, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.42-7.2 (m, 6H), 7.13-7.09 (dd, 1H, J = 8.4,

1.8 Hz), 6.9-6.87 (d, 1H, J = 8.4 Hz), 6.85-6.7 (m,

1H), 5.9-5.8 (m, 2H), 5.2 (s, 3H), 5.15-5.05 (m,

1H), 5.0-4.9 (m, 1H), 4.8-4.72 (m, 1H), 4.71-4.68 (d, 1H, J = 9.7 Hz), 4 , 07-4.03 (d, 1H, J = 9.3 Hz),

3.99-3.97 (d, 2H, J = 5.5Hz), 3.9 (s, 3H), 3.44-3.37 (dd, 1H, J = 13.6, 8.3 Hz), 3.23-3.14 (m, 2H),

3.08-3.0 (dd, 1H, J = 14.5, 8.0 Hz), 2.75-2.4 (m,

3H), 2.0-1.7 (m, 3H), 1.5 (s, 10H), 1.26 (s, 3H),

1.21 (s, 3H), 1.08-1.06 (d, 3H, J = 7.0 Hz),

0.977-0.963 (d, 3H, J = 4.0 Hz), 0.956-0.942 (d, 3H,

J = 4.1 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ): 177.5, 170.5, 170.2,

170.1, 165.3, 153.9, 142.2, 139.0, 138.3, 136.1,

130.8, 129.9, 128.7, 128.2, 128.1, 124.5, 122.3,

112.2, 80.0, 76.1,73,9, 71,1,66,2, 61.7, 56,1,54,6,

46.4, 42.7, 42.3, 39.6, 38.4, 36.3, 35.1, 28.2, 24.8,

23,0,22,9,22,7,21,5,8,6.

IR (CHCl ₃ ): 3428, 3009, 2966, 2935, 1750, 1714, 1683,

1504, 1486, 1369, 1259, 1193, 1162, 1127, 1067. FAB HRMS (M + H) calcd for C ₄₄ H ₆₀ CIN ₃ O ₂ :

Calculated: 892.3554, Found: 892.3565.

Example 48

4M Hydrochloride (0.08 mL, 0.33 mmol) in 1,4-dioxane was added to a solution of (25) glycinate (0.058 g, 0.065 mmol) in dichloromethane (0.2 mL). The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure, and the residue was kept under reduced pressure for 3 days to remove 1,4-dioxane. The desired hydrochloride salt (26) is obtained in quantitative yield.

[a] ² ° D = + 26.2 ° (c = 0.58 MeOH).

¹ H-NMR (500 MHz, CD ₃ OD), δ:

7.48-7.42 (q, 4H, J = 11.2 Hz), 7.31-7.3 (d, 1H,

J = 2.0 Hz), 7.21-7.19 (dd, 1H, J = 8.5, 2.0 Hz),

7.01-7.0 (d, 1H, J = 8.4 Hz), 6.8-6.7 (m, 1H),

6.0-5.95 (dd, 1H, J = 15.2, 1.5Hz), 5.3 (d, 2H,

J = 1.3 Hz), 5.16-5.1 (m, 1H), 5.09-5.07 (dd, 1H,

J = 10.0, 3.6 Hz), 4.84-4.82 (d, 1H, J = 9.8 Hz),

4.54 ^ 4.51 (dd, 1H, J = 11.3, 3.7 Hz), 4.05-4.03 (dd,

H, J = 9.5, 1.8 Hz), 3.9 (s, 2H), 3.86 (s, 3H),

3.5-3.47 (d, 1H, J = 13.5Hz), 3.22-3.18 (dd, 1H,

J = 14.5, 3.6 Hz), 3.14-3.11 (d, 1H, J = 13.5 Hz),

2.8-2.77 (d, 1H, J = 14.4Hz), 2.78-2.75 (m, 2H),

2.55-2.35 (m, 2H), 1.9-1.55 (m, 4H), 1.4-1.3 (m,

1H), 1.24 (s, 3H), 1.2 (s, 3H), 1.04-1.03 (d, 3H,

J = 7.0 Hz), 1.02-1.0 (t, 6H, J = 7.2 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

178.9, 173.8, 171.9, 168.3, 155.3, 144.2, 141.8,

136.7, 132.2, 131.5, 129.75, 129.7, 129.4, 125.2,

123.3, 113.5, 77.2, 74.7, 72.6, 68.5, 63.5, 57.6,

56.7, 47.6, 44.1,41,1,40.4, 37.9, 36.5, 26.3, 23.6,

23.5, 22.2, 9.0. IR (KBr) 3412, 2961, 2935, 1752,

1722, 1669, 1504, 1473, 1279, 1259, 1207, 1151,

1126, 1065 cm ^-1 .

Example 49

0.15 g (0.16 mmol) of (25) chlorohydrin, 0.041 g (0.24 mmol) of N- (tert-butoxycarbonyl) glycine and 0.002 g (0.016 mmol) of 4-dimethylaminopyridine (DMAP) To a solution of 0.7 mL of dichloromethane was added 0.049 g (0.24 mmol) of 1,3-dicyclohexylcarbodiimide. The reaction mixture was stirred at room temperature for 1 hour, filtered, washed with ethyl acetate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (50-60-70% ethyl acetate / hexane). The diglycinate (27) (0.158 g, 97%) was obtained as a white solid. [α] 20 = + 44.0 ° (c = 1.25, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.4-7.2 (m, 7H), 7.13-7.10 (bd, 1H, J = 9.8 Hz),

6.91-6.88 (d, 1H, J = 8.4Hz), 5.8-5.77 (d, 1H,

J = 15.3 Hz), 5.6-5.58 (m, 1H), 5.49-5.46 (d, 1H,

J = 9.6 Hz), 5.19 (s, 2H), 5.1 ^ t, 7 (m, 6H), 3.98-3.97 (d, 2H, J = 5.0 Hz), 3.93 (s, 3H), 3.69-3.62 (dd, 1H,

J = 18.2, 4.0 Hz), 3.5-3.0 (m, 5H), 2.7-2.35 (m, 3H),

2.0-1.7 (m, 3H), 1.49 (s, 9H), 1.44 (s, 9H), 1.28 (s,

3H), 1.22 (s, 3H), 1.1-1.08 (d, 3H, J = 7.0 Hz),

1.06-1.04 (d, 3H, J = 6.4 Hz), 1.0-0.98 (d, 3H,

J = 6.2 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.6, 170.4, 169.9, 168.5, 165.0, 155.2, 153.9,

HU 227 195 Β1

141.6, 138.6, 137.3, 136.3, 130.8, 129.8, 128.2,

128.1, 124.6, 122.3, 112.2, 79.9, 74.7, 71.1, 66.2,

60.0, 56.0, 54.5, 46.5, 42.8, 42.4, 41.8, 39.5, 38.0,

36.5, 35.2, 28.2, 28.15, 24.8, 23.1, 22.8, 22.6, 21.4,

9.9.

IR (CHCl ₃ ): 3431, 2982, 2966, 2935, 2872, 1756, 1713,

1685, 1504, 1369, 1258, 1193, 1161.

FAB HRMS (M-BOC + H) calcd for C ₄₆ H ₆₃ Cl ₂ N ₄ O ₁₃ :

calcd: 949.3769, found: 949.3777.

Example 50

A solution of 4 M hydrochloric acid in 1,4-dioxane (0.1 mL, 0.42 mmol) was added to a solution of diglycinate (27) (0.044 g, 0.042 mmol) in dichloromethane (0.2 mL). The reaction mixture was stirred at room temperature for 3 hours and then concentrated under reduced pressure and the residue was kept under reduced pressure for 3 days to remove the 1,4-dioxane residue. The desired hydrochloride salts (28) are obtained in quantitative yield. [α] ²⁰ D = + 33.1 ° (c = 0.865, MeOH).

¹ H-NMR (500 MHz, CD ₃ OD), δ:

7.77-7.74 (d, 1H), 7.46-7.41 (q, 4H, J = 20.3,

8.4 Hz), 7.29-7.28 (d, 1H, J = 2.1 Hz), 7.18-7.16 (dd, 1H, J = 8.6, 2.1 Hz), 6.99-6.97 (d, 1H,

J = 8.5 Hz), 6.7-6.6 (m, 1H), 5.95-5.92 (d, 1H,

J = 15.3 Hz), 5.54-5.52 (dd, 1H, J = 9.5, 1.4 Hz), 5.5 (s, 1H), 5.28 (s, 2H), 5.21-5.19 (d, 1H, J = 9.4 Hz),

5.12-5.09 (dd, 1H, J = 10.5, 3.1 Hz), 4.87 ^ 1.85 (d,

1H, J = 12.4 Hz), 4.5-4.47 (dd, 1H, J = 11.3, 3.7 Hz),

3.9 (s, 2H), 3.84 (s, 3H), 3.82-3.79 (d, 1H,

J = 17.8 Hz), 3.48-3.45 (d, 1H, J = 13.7 Hz), 3.35-3.3 (m, 1H), 3.19-3.15 (dd) , 1H, J = 14.5, 3.8 Hz),

3.11-3.09 (d, 1H, J = 13.8 Hz), 2.77-2.6 (m, 3H),

2.37-2.3 (m, 1H), 1.95-1.75 (m, 3H), 1.22 (s, 3H),

1.17 (s, 3H), 1.08-1.07 (d, 3H, J = 7.0 Hz),

1.03-1.02 (d, 3H, J = 6.4 Hz), 1.0-0.99 (d, 3H,

J = 6.2 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.8, 173.8, 171.9, 168.5, 168.2 167.8, 155.4,

143.4, 139.3 137.6, 132.2, 131.4, 129.7, 129.5,

129.4, 125.3, 123.3, 113.5, 77.9, 76.2, 72.4, 68.2,

60.8, 57.7, 56.7, 47.5, 44.2, 41.2, 40.5, 39.8, 37.9,

36,5,26,3,23,7, 23,4,22,0, 9,9.

IR (KBr): 3417, 3234, 2959, 2873, 2622, 1757, 1724,

1673, 1504, 1473, 1303, 1259, 1221, 1150,

1065 cm ^-1 .

Example 51

Fraction C '(0.22 g, 1.02 mmol) free acid, DMAP (0.032 g, 0.26 mmol) and DCC (0.21 g, 1.02 mmol)

A solution of 6.5 ml of dichloromethane at 0 ° C was stirred for 30 minutes. A solution of free alcohol (29) (0.35 g, 0.51 mmol) in dichloromethane (6.0 mL) was added dropwise to the reaction mixture. The reaction mixture was stirred at 0 ° C for 10 minutes and then at room temperature for 24 hours, then refluxed for 3 hours and allowed to cool to room temperature. The reaction mixture was concentrated under reduced pressure, filtered through a pad of celite, washed with ethyl acetate. The resulting residue was purified by silica gel column chromatography (60-70% ethyl acetate / hexane).

0.38 g (85%) of the ester of the formula is obtained as a white solid.

[α] ²⁰ D = + 23.2 ° (c = 1.0, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.4-7.2 (m, 6H), 7.12-7.08 (dd, 1H, J = 8.4, 2.0 Hz),

6.9-6.87 (d, 1H, J = 8.5Hz), 6.87-6.75 (m, 1H),

6.47-6.42 (d, 1H, J = 15.8 Hz), 6.11-6.03 (dd, 1H,

J = 15.8, 8.8 Hz), 5.82-5.77 (d, 1H, J = 14.9 Hz),

5.61-5.58 (d, 1H, J = 7.8 Hz), 5.12 (s, 2H),

5.12-4.75 (m, 4H), 3.9 (s, 3H), 3.49-3.42 (dd, 1H,

J = 13.4, 8.7 Hz), 3.29-3.27 (d, 1H, J = 6.5 Hz),

3.2-3.1 (m, 3H), 2.65-2.3 (m, 3H), 1.8-1.6 (m, 3H),

1.47 (s, 9H), 1.45-1.3 (m, 1H), 1.3-1.15 (m, 15H),

0.79-0.75 (t, 6H, J = 6.4 Hz).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

177.8, 170.4, 165.1, 153.9, 141.9, 136.7, 135.1,

131.1, 130.8, 129.7, 128.3, 128.2, 126.2, 124.6,

122.4, 112.2, 76.9, 71.3, 66.0, 56.0, 54.4, 48.2,

46.4, 43.7, 42.6, 42.2, 39.4, 36.4, 35.2, 28.3, 24.5,

22.8, 22.6, 21.2, 17.2. IR (CHCl ₃ ): 3426, 2968,

2935, 2874, 2841, 1746, 1713, 1684, 1652, 1504,

1486, 1474, 1368, 1318, 1304, 1259, 1244, 1165,

1151, 1067 cm ^-1 .

FAB HRMS (M-BOC + H) calcd for C ₄₂ H ₅₇ CIN ₃ O ₉ :

calcd: 782.3783, found: 782.3788.

Example 52

To a solution of the starting styrene (0.45 g, 0.51 mmol) in dichloromethane (9.0 mL) at 0 ° C was added 3-chloroperoxybenzoic acid (0.092 g, 0.54 mmol). The solution was stirred at 0 ° C for 1 hour and then at room temperature overnight. The solution was concentrated under reduced pressure, the resulting epoxides were dissolved in chloroform (10 mL) and cooled to -60 ° C. Freshly distilled TMSCI (0.25 mL, 1.95 mmol) was added to the -60 ° C solution and the reaction mixture was stirred for 1 hour. An additional 0.5 mL (3.9 mmol) of TMSCI was added to the reaction mixture and stirring was continued at -60 ° C to -40 ° C for 2 hours. The solution was allowed to warm to room temperature and another 0.25 mL (1.95 mmol) of TMSCI was added. After stirring at room temperature for 30 minutes, the solution is evaporated and the residue is chromatographed on preparative HPLC to isolate the chlorohydrins formed. Purification afforded 0.1 g (22%) of the desired chlorohydrin.

[a] ² ° D = + 47.9 ° (c = 0.75, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.5-7.2 (m, 6H), 7.13-7.10 (dd, 1H, J = 8.4,

1.9 Hz), 6.91-6.88 (d, 1H, J = 8.5 Hz), 6.87-6.75 (m, 1H), 5.85-5.8 (d, 1H) , J = 14.9 Hz), 5.75-5.6 (m,

1H), 5.22-5.1 (m, 3H), 5.0-4.9 (m, 2H), 4.8-4.72 (m, 1H), 4.71-4.68 ( d, 1H, J = 9.6 Hz), 4.07-4.03 (d,

1H, J = 9.5 Hz), 3.9 (s, 3H), 3.45-3.38 (dd, 1H,

HU 227 195 Β1

J = 13.4, 8.5 Hz), 3.31-3.26 (d, 1H, J = 6.4 Hz),

3.25-3.0 (m, 4H), 2.8-2.65 (bd, 1H), 2.6-2.35 (m,

2H), 1.9-1.7 (m, 3H), 1.47 (s, 10H), 1.27 (s, 3H),

1.25 (s, 6H), 1.22 (s, 3H), 1.09-1.07 (d, 3H,

J = 7.0 Hz), 0.98-0.96 (m, 6H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

177.5, 170.5, 165.2, 153.9, 142.5, 137.0, 130.8,

129.8, 128.2, 124.5, 122.4, 112.2, 79.1, 76.1, 73.9,

71.1, 65.6, 61.7, 56.1, 54.5, 48.2, 46.4, 43.7, 42.7,

39.6, 38.4, 36.3, 35.2, 28.3, 24.8, 23.0, 22.9,

22.8 22.7,21,5,8,6.

IR (CHCl 3): 3426, 2967, 2934, 2873, 2841, 1715, 1684,

1605, 1504, 1485, 1474, 1442, 1368, 1305, 1258,

1151 cm ^-1 .

FAB HRMS (M-BOC + H) calcd for C ₄₂ H ₅₈ Cl ₂ N ₃ O ₁₀ :

calcd: 834.3499, found: 834.3487.

Example 53

To a solution of chlorohydrin (0.08 g, 0.86 mmol) in dichloromethane (0.35 mL) was added a solution of 4M hydrogen chloride in 1,4-dioxane (0.11 mL, 0.43 mmol). The reaction mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure and the residue held under reduced pressure for 3 days to remove the 1,4-dioxane residue. 0.75 g of the desired hydrochloride salt is obtained in quantitative yield. [a] ² ° D = + 28.0 ° (c = 0.5, _CH3 OH).

¹ H-NMR (300 MHz, CD ₃ OH), δ:

8.52-8.49 (d, 1H, J = 7.5 Hz), 7.84-7.81 (d, 2H, J = 9,

Hz), 7.49-7.39 (q, 4H, J = 8.3 Hz), 7.32-7.31 (d,

1H, J = 1.9 Hz), 7.22-7.19 (dd, 1H, J = 8.4, 2.1 Hz),

7.02-7.0 (d, 1H, J = 8.4 Hz), 6.8-6.7 (m, 1H),

6.0-5.9 (dd, 1H, J = 15.4, 1.0 Hz), 5.22 (s, 2H),

5.2-5.0 (m, 2H), 4.85 ^ 1.81 (d, 1H, J = 9.6 Hz),

4.6-4.5 (m, 1H), 4.06-4.03 (dd, 1H, J = 9.6, 1.5 Hz),

3.9 (s, 3H), 3.54-3.46 (dd, 1H, J = 13.5, 9.7 Hz),

3.25-3.11 (m, 2H), 3.11 (s, 2H), 2.8-2.7 (m, 2H),

2.6-2.3 (m, 2H), 1.9-1.5 (m, 3H), 1.3 (s, 7H), 1.25 (s, 3H), 1.2 (s, 3H) , 1.06-1.03 (d, 3H, J = 6.9 Hz),

1.02-1.01 (d, 3H, J = 3.2 Hz), 1.0-0.99 (d, 3H,

J = 3.4 Hz).

¹³ C NMR (63 MHz, CD ₃ OH), δ:

178.9, 176.5, 173.8, 171.8, 168.3, 155.3, 144.2,

141.6, 137.4, 132.3, 131.5, 129.8, 129.4, 129.3,

125.2, 123.3, 113.5, 77.2, 74.7, 72.6, 67.8, 63.5,

57.6, 56.7, 47.7, 47.6, 44.1, 42.3, 41.1, 40.4, 37.9,

36.5,26,2,23.7, 23,4,22.9, 9.0.

IR (CHCl ₃ ): 3421, 2964, 2935, 2873, 2841, 1717, 1676,

1528, 1504, 1477, 1464, 1405, 1282, 1259, 1185,

1152.1067 cm ^-1 .

FAB HRMS (M-CI) calculated for C ₄₂ H ₅₈ Cl ₃ N ₃ O ₁₀ :

calcd: 834.3499, found: 834.3504.

Example 54

According to the above procedure for the preparation of styrene (20), 1.0 g (1.73 mmol) of aldehyde (18) and

3-Methylbenzyl triphenylphosphonium chloride (0.886 g, 2.2 mmol) afforded styrene of the formula (0.67 g, 58%).

[a] ² ° D = + 33.1 ° (c = 1.0, _CH3 OH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.24-7.0 (m, 7H), 6.85-6.82 (d, 1H, J = 8.4 Hz),

6.82-6.7 (m, 1H), 6.39-6.34 (d, 1H, J = 15.8 Hz),

6.03-5.95 (dd, 1H, J = 15.8, 8.7Hz), 5.78-5.73 (d,

1H, J = 15.2 Hz), 5.67-5.64 (d, 1H, J = 7.8 Hz),

5.1-5.0 (m, 1H), 4.87-4.83 (dd, 1H, J = 10.2,

3.5 Hz), 4.8 ^ 4.7 (m, 1H), 3.9 (s, 3H), 3.45-3.38 (dd,

1H, J = 13.4, 8.6 Hz), 3.2-3.0 (m, 3H), 2.6-2.3 (m,

3H), 2.32 (s, 3H), 1.75-1.25 (m, 3H), 1.22 (s, 3H),

1.15 (s, 3H), 1.13-1.11 (d, 3H, J = 6.8 Hz),

0.75-0.72 (t, 6H, J = 5.7 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.5, 170.3, 165.1, 154.0, 142.1, 138.0,

136.6, 135.6, 131.8, 130.8, 129.9, 129.6, 128.4,

128.22, 128.17, 126.7, 124.5, 123.3, 122.5, 112.3,

71.4, 56.1, 54.3, 46.4, 42.7, 42.2, 39.4, 36.5, 35.3,

24.5, 22.8, 22.6, 22.56, 21.2, 21.1, 17.2.

IR (CHCl ₃ ): 3424, 3021, 3017, 2965, 1747, 1711, 1680,

1652, 1528, 1503, 1485, 1259, 1151, 1067 ^cm-first

Example 55

To a solution of styrene (0.667 g, 1.0 mmol) in dichloromethane (0.5 mL) was added 3-chloroperoxybenzoic acid (0.19 g, 1.1 mmol). The solution was stirred overnight and then concentrated under reduced pressure. The product obtained contains a ratio of β- and α-epoxide of 1.8: 1. The two epoxides were separated by reverse phase HPLC, eluting with 70:30 acetonitrile / water. 0.20 g of β-epoxide are obtained in the form of a white solid.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.0 (m, 7H), 6.9-6.87 (d, 1H, J = 8.4 Hz),

0.87-6.75 (m, 1H), 5.79-5.74 (d, 1H, J = 14.8 Hz),

5.54-5.51 (d, 1H, J = 7.8 Hz), 5.28-5.22 (m, 1H),

4.89-4.85 (dd, 1H, J = 10.4, 3.5Hz), 4.82-4.75 (m,

1H), 3.9 (s, 3H), 3.69-3.68 (d, 1H, J = 1.6 Hz),

3.51-3.44 (dd, 1H, J = 13.4, 8.6 Hz), 3.2-3.1 (m,

2H), 2.98-2.95 (dd, 1H, J = 7.6, 1.6 Hz), 2.65-2.32 (m, 3H), 2.32 (s, 3H), 1 , 85-1.6 (m, 3H), 1.4-1.25 (s,

3H), 1.27 (s, 3H), 1.21 (s, 3H), 1.21-1.18 (d, 3H,

J = 7.5 Hz), 0.90-0.86 (t, 6H, J = 6.13 Hz).

Example 56

To a solution of the β-epoxide of the above formula (0.1 g, 0.147 mmol) in chloroform (5.0 mL) at -60 ° C was added chlorotrimethylsilane (0.093 mL, 0.74 mmol). The solution was stirred at -60 ° C for 30 minutes and then at room temperature for 1.5 hours and then concentrated under reduced pressure. The resulting evaporation residue contains 50:50 of sine and anti-chlorohydrin. Purification by reverse phase HPLC afforded 0.028 g (27%) of the desired trans isomer.

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.28-7.2 (m, 5H), 7.13-7.1 (dd, 1H, J = 8.3, 1.9 Hz),

HU 227 195 Β1

6.91-6.88 (d, 1H, J = 8.5Hz), 6.88-6.78 (m, 1H),

5.86-5.81 (d, 1H, J = 15.0 Hz), 5.73-5.71 (d, 1H,

J = 7.8 Hz), 5.24-5.17 (t, 1H, J = 9.4 Hz), 5.0-4.96 (dd, 1H, J = 9.6, 2.9 Hz) ), 4.81-4.74 (m, 1H),

4.67 ^ 4.64 (d, 1H, J = 9.7 Hz), 4.06 ^ 1.03 (dd, 1H,

J = 9.6, 1.1 Hz), 3.92 (s, 3H), 3.47-3.39 (dd, 1H,

J = 13.2, 8.3 Hz), 3.42-3.0 (m, 3H), 2.8-2.4 (m, 2H),

2.4 (s, 3H), 1.9-1.4 (m, 4H), 1.28 (s, 3H), 1.22 (s,

3H), 1.09-1.07 (d, 3H, J = 7.0Hz), 0.98-0.96 (d, 6H,

J = 6.4 Hz).

Example 57

According to the above procedure for the preparation of styrene (20), 0.095 g (63 g) of aldehyde (18) (0.2 g, 0.345 mmol) and 3,4-dimethylbenzyl triphenylphosphonium chloride (0.26 g, 0.62 mmol) were obtained. %) of styrene of the formula given.

[a] ² ° D = + 27.8 ° (c = 0.576, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.22-7.18 (d, 1H, J = 1.6 Hz), 7.12-7.02 (m, 5H),

6.87-6.81 (d, 1H, J = 8.3 Hz), 6.80-6.71 (m, 1H),

6.40-6.30 (d, 1H, J = 15.8 Hz), 6.00-5.88 (dd, 1H,

J = 15.8, 8.8 Hz), 5.80-5.70 (d, 1H, J = 15.1 Hz),

5.55-5.45 (d, 1H, J = 7.7Hz), 5.10-4.97 (m, 1H),

4.90-4.80 (dd, 1H, J = 9.4, 2.8 Hz), 4.80-4.70 (m,

1H), 3.88 (s, 3H), 3.50-3.35 (dd, 1H, J = 13.3,

4.8 Hz), 3.20-3.00 (m, 3H), 2.60-2.48 (m, 2H),

2.45-2.30 (m, 1H), 2.23 (s, 3H), 2.24 (s, 3H),

1.80-1.55 (m, 2H), 1.40-1.30 (m, 1H), 1.22 (s, 3H),

1.16 (s, 3H), 1.14-1.10 (d, 3H, J = 6.8 Hz),

0.80-0.73 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.5, 170.3, 165.1, 154.0, 142.2, 136.5,

135.9, 134.3, 131.6, 130.8, 129.7, 129.5, 128.8,

128.1, 127.2, 124.4, 123.6, 122.4, 112.2, 71.4,

56.0, 54.2, 46.4, 42.6, 42.1, 39.4, 36.4, 35.2, 24.5,

22.7, 22.6, 22.5, 21.1, 19.6, 19.3, 17.2.

IR (CHCl ₃ ): 3424, 2965, 2935, 1746, 1711, 1681, 1652,

1527, 1503, 1485, 1259, 1187, 1164, 1151, 1067,

970, 727 cm ^-1 .

Analysis for C ₃₈ H ₄ O ₇ gCIN ₂ O: C, Η N

Example 58

According to the procedure described above, 0.06 g (20%) of the above-mentioned styrene and 0.081 g (0.47 mmol) of 3-chloroperoxybenzoic acid are obtained in the same manner.

[a] ² ° D = + 20.0 ° (c = 1.43, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-6.96 (m, 6H), 6.98 (s, 1H), 6.84-6.82 (d, 1H,

J = 8.5 Hz), 6.79-6.65 (m, 1H), 5.73-5.69 (d, 1H,

J = 1.9 Hz), 5.68-5.67 (d, 1H, J = 4.6 Hz), 5.29-5.15 (m, 1H), 4.83-4.76 ( dd, 1H, J = 9.7, 2.8 Hz),

4.75-4.58 (m, 1H), 3.86 (s, 3H), 3.61-3.60 (d, 1H,

J = 1.6 Hz), 3.46-3.38 (dd, 1H, J = 13.4, 8.8 Hz),

3.14-2.97 (m, 3H), 2.92-2.89 (dd, 1H, J = 7.7,

1.6 Hz), 2.59-2.35 (m, 2H), 2.25 (s, 6H), 1.78-1.58 (m, 3H), 1.28-1.09 (m , 1H), 1.21 (s, 3H), 1.15 (s,

3H), 1.15-1.12 (d, 3H, J = 7.8 Hz), 0.85-0.77 (m,

6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.4, 170.3, 164.9, 154.1, 141.7, 137.0,

136.9, 134.0, 130.8, 129.9, 129.5, 128.1, 126.7,

124.6, 123.2, 122.4, 122.3, 75.9, 71.04, 62.8, 59.1,

56.1, 54.3, 46.4, 42.7, 40.7, 39.2, 36.8, 35.2, 24.4,

22.8, 22.6, 21.0, 19.7, 19.4, 13.6.

IR (KBr): 3419, 2962, 1752, 1721, 1681, 1654, 1534,

1504, 1473, 1442, 1302, 1282, 1259, 1192, 1126,

1066 cm ^-1 .

Elemental analysis for C ₃₈ H ₄₉ CIN ₂ O ₈ : C, Η, N.

Example 59

To a solution of styrene of the above formula (0.492 g, 0.72 mmol) in dichloromethane (2.4 mL) at 0 ° C was added 3-chloroperoxybenzoic acid (0.137 g, 0.79 mmol) and toluene (1.2 mL). The reaction mixture was stirred at 0 ° C for 30 minutes and then the ice bath was removed and stirred at room temperature for 24 hours. Diluted with dichloromethane (10 mL), the solution was washed with 10% sodium sulfate (10 mL), water (10 mL) and 10% sodium bicarbonate (10 mL), and dried over sodium sulfate. The solution was evaporated to give a 2: 1 mixture of crude epoxides.

The crude epoxide mixture (0.445 g, 0.638 mmol) was dissolved in dry chloroform (10 mL) and cooled to -60 ° C. Trimethylsilyl chloride (0.2 mL, 1.5 mmol) was added to the solution and stirred for 90 minutes and then concentrated under reduced pressure. The crude chlorohydrin was purified by reverse phase HPLC to give 0.115 g (21%) of the title compound as a white solid.

[a] ^> = - 45.9 ° _(C = O, 59, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.01 (m, 5H), 6.85-6.82 (d, 1H, J = 8.4 Hz),

6.80-6.71 (m, 1H), 5.71-5.66 (d, 1H, J = 15.1 Hz),

5.50-5.47 (d, 1H, J = 7.6Hz), 5.13-5.08 (t, 1H,

J = 8.8 Hz), 4.89-4.84 (m, 2H), 4.81 ^ 1.71 (m, 1H),

4.09-4.06 (d, 1H, J = 9.4 Hz), 3.87 (s, 3H),

3.44-3.37 (dd, 1H, J = 13.4, 8.4 Hz), 3.16-3.06 (m,

3H), 2.60-2.54 (m, 2H), 2.26 (s, 6H), 2.26-2.14 (m,

1H), 1.89-1.81 (m, 1H), 1.70-1.62 (m, 2H),

1.58-1.46 (m, 2H), 1.23 (s, 3H), 1.17 (s, 3H),

0.97-0.95 (d, 3H, J = 6.6Hz), 0.93-0.90 (m, 6H). ¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.5, 170.1, 165.2, 153.9, 142.3, 137.7,

137.3, 135.1, 130.7, 130.2, 129.8, 128.5, 128.1,

124.6, 124.4, 122.3, 112.2, 75.9, 74.2, 71.2, 68.8,

56.0, 54.4, 46.4, 42.7, 39.6, 38.4, 36.2, 35.2, 24.8,

22.9,22.8, 22.7,21.7, 19.8, 19.5,8,6.

IR (KBr): 3421, 2960, 1756, 1721, 1675, 1504, 1258,

1195, 1151, 1126, 1066 crrr ¹ .

Elemental analysis for C ₃₈ H ₅₀ Cl ₂ N ₂ O ₈ : C, Η, N.

Example 60

0.21 g of aldehyde (18) and 0.47 g (1.12 mmol) of 4-methoxybenzyl triphenylphosphonium chloride (0.5 g, 0.87 mmol) were prepared as described above for the preparation of styrene (20). Styrene (36%) was prepared.

HU 227 195 Β1 [α]% 0 = + 31.6 ° (c = 1.03, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.23 (d, 3H, J = 8.4Hz), 7.20-7.19 (d, 1H,

J = 1.8 Hz), 7.07-7.03 (dd, 1H, J = 8.4, 1.9 Hz),

6.84-6.81 (d, 3H, J = 8.5 Hz), 6.80-6.7 (m, 1H),

6.36-6.31 (d, 1H, J = 15.8 Hz), 5.89-5.81 (dd, 1H,

J = 15.8, 8.8 Hz), 5.78-5.73 (d, 1H, J = 13.7 Hz),

5.68-5.66 (d, 1H, J = 7.9 Hz), 5.05 ^ 1.99 (ddd, 1H,

J = 10.6, 6.6, 1.6 Hz), 4.87-4.82 (dd, 1H, J = 9.7,

3.1 Hz), 4.78-4.7 (m, 1H), 3.86 (s, 3H), 3.79 (s,

3H), 3.45-3.37 (dd, 1H, J = 13.4, 8.6 Hz), 3.15-3.0 (m, 3H), 2.6-2.25 (m, 3H), 1.7-1.3 (m, 3H), 1.22 (s,

3H), 1.15 (s, 3H), 1.12-1.1 (d, 3H, J = 6.8 Hz),

0.76-0.75 (d, 3H, J = 2.9 Hz), 0.74-0.73 (d, 3H,

J = 2.8 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 165.1, 159.1, 153.9, 142.1,

135.8, 131.0, 130.8, 129.7, 129.5, 128.2, 127.9,

127.2, 124.5, 122.4, 113.9, 112.3, 77.1, 71.4, 56.0,

55.2, 54.4, 46.4, 42.7, 42.1, 39.4, 36.4, 35.3, 24.5,

22.8.22.6.21.2, 17.3.

IR (CHCl ₃ ): 3422, 3003, 2964, 2936, 2873, 2840, 1746,

1712, 1681, 1651, 1607, 1527, 1512, 1504, 1485,

1465,1301,1251 crrr ¹ .

Elemental analysis for C ₃₇ H ₄₇ CIN ₂ O ₈ : C, Η, N.

Example 61

To 0.3 g (0.44 mmol) of styrene of the above formula are added 19 ml of acetone, 9 ml of water, 9 ml of dichloromethane and 1.2 g (14.5 mmol) of solid sodium bicarbonate. The reaction mixture was cooled to 0 ° C. Oxone (1.08 g, 1.8 mmol) was dissolved in water (9 mL) and 2 mL was added to the cooled reaction mixture. After vigorous stirring at 0 ° C for 30 minutes, an additional 2 ml of Oxone solution, then another 2 ml of solution were added and after 30 minutes all the Oxone solution was added. The progress of the reaction is monitored by reverse phase HPLC analysis and after 2.5 hours of stirring, the reaction is complete. At 0 ° C, 50 ml of a saturated aqueous solution of sodium bicarbonate and another 50 ml of dichloromethane were added. The layers were separated and the organic layer was washed with 50 mL of 10% sodium sulfate solution, 50 mL of saturated aqueous sodium bicarbonate and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The mixture of β and epoxides was separated by reversed phase HPLC using 45:55 acetonitrile / water as eluent. 0.12 g of β-epoxide (39%) are obtained in the form of a white solid.

[a]% = + 25.8 ° (c = 0.66, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26- 7.15 (m, 4H), 7.05-7.03 (d, 1H, J = 8.6 Hz),

6.9-6.87 (d, 2H, J = 8.6 Hz), 6.85-6.82 (d, 1H,

J = 8.5 Hz), 6.82-6.7 (m, 1H), 5.74-5.69 (d, 1H,

J = 15.1 Hz), 5.54-5.52 (d, 1H, J = 7.8 Hz), 5.22-5.16 (m, 1H), 4.84-4.7 (m , 2H), 3.87 (s, 3H), 3.81 (s,

3H), 3.63 (s, 1H), 3.46-3.38 (dd, 1H, J = 13.5,

8.8 Hz), 3.2-3.0 (m, 3H), 2.91-2.89 (d, 1H,

J = 7.4 Hz), 2.6-2.38 (m, 2H), 1.8-1.6 (m, 3H),

1.4-1.23 (m, 1H), 1.22 (s, 3H), 1.15 (s, 3H),

1.15-1.12 (d, 3H, J = 8.9 Hz), 0.84-0.80 (t, 6H,

J = Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.5, 170.4, 165.1, 159.8, 153.9, 141.6,

136.8, 130.7, 129.7, 128.6, 128.1, 126.9, 124.6,

122.3, 114.1, 112.2, 75.9, 71.0, 62.8, 58.9, 56.0,

55.2, 54.6, 46.3, 42.7, 40.6, 39.2, 36.8, 35.2, 24.4,

22.8.22.7, 22.6,21.1, 13.5.

IR (CHCl ₃ ): 3423, 3009, 2964, 2936, 2874, 2840, 1751,

1713, 1681, 1653, 1614, 1517, 1504, 1486, 1464,

1442, 1303, 1281, 1257.1183, 1173, 1152 cm ^1st Elemental analysis for C ₃₇ H ₄₇ CIN ₂ O ₉ : C, Η, N.

Example 62

Using the procedure described above for the preparation of styrene (20) from 0.911 g (1.57 mmol) of aldehyde (18) and 1.7 g (3.27 mmol) of 4- (tert-butyldimethylsiloxy) benzyltriphenylphosphonium chloride, 0.640 g (53%) of the following styrene were obtained.

[a] ² ° D = + 30.83 ° (c = 0.52, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.25 (s, 1H), 7.26-7.20 (d, 2H, J = 8.5 Hz),

7.12-7.06 (dd, 1H, J = 8.3, 1.6 Hz), 6.92-6.86 (d,

1H, J = 8.5 Hz), 6.84-6.78 (d, 2H, J = 8.5 Hz),

6.44-6.33 (d, 1H, J = 15.9 Hz), 5.95-5.85 (dd, 1H,

J = 15.8, 8.8 Hz), 5.85-5.77 (d, 1H, J = 15.5 Hz),

5.68-5.55 (bd, 1H, J = 7.9Hz), 5.15-5.00 (m, 1H),

4.95 ^ 4.80 (dd, 1H, J = 10.0, 3.0 Hz), 4.85 ^ 4.75 (m,

1H), 3.91 (s, 3H), 3.53-3.43 (dd, 1H, J = 13.4,

8.6 Hz), 3.23-3.08 (m, 3H), 2.65-2.50 (m, 2H),

2.50-2.35 (m, 3H), 1.75-1.60 (m, 2H), 1.45-1.36 (m, 1H), 1.27 (s, 3H), 1, 20 (s, 3H), 1.16-1.13 (d,

3H, J = 6.8Hz), 1.01 (s, 9H), 0.85-0.74 (m, 6H),

0.22 (s, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 165.1, 155.2, 153.9, 142.1,

131.1, 130.8, 130.1, 129.7, 128.2, 128.1, 127.1,

124.5, 122.4, 120.2, 112.2, 71.4, 56.0, 54.4, 46.4,

42.7, 42.2, 39.4, 36.5, 35.3, 25.6, 24.5, 22.8, 22.7,

21.2, 18.1, 17.3, -4.5.

IR (CHCl ₃ ): 3422, 3030, 3008, 2961, 2932, 2899, 2860,

1745, 1712, 1681, 1604, 1527, 1509, 1485, 1442,

1370, 1339, 1303, 1258, 1169, 1151, 1067, 1007,

970,912,841,822, 792 crrr ¹ .

Elemental analysis for C ₄₂ H ₅₉ CIN ₂ O ₈ Si: C,

Η, N.

Example 63

To a solution of silyl-protected phenol (0.084 g, 0.107 mmol) in dry tetrahydrofuran (4 mL) at -70 ° C was added a solution of tetrabutylammonium fluoride (TBAF) (0.11 mL, 0.11 mL) in tetrahydrofuran. The pale yellow solution was stirred at -70 ° C for 30 minutes, then 2 mL of a saturated aqueous solution of ammonium chloride was added and extracted with 3 x 25 mL of dichloromethane. The combined organic phases are dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by radial PLC on silica gel eluting with 50-100%

195 Β1 using ethyl acetate / hexane as eluent. 0.067 g (93%) of the desired alcohol is obtained in the form of a white solid. [a] ² ° D = + 30.83 ° (c = 0.52 _(CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.40-7.32 (m, 1H), 7.28-7.20 (m, 3H), 7.13-7.05 (dd, 1H, J = 8.3, 1.6 Hz), 6, 95-6.75 (m, 5H), 6.57 (s, 1H), 6.42-6.33 (d, 1H, J = 15.9 Hz), 5.93-5.83 (dd, 1H, J = 15.8, 8.8 Hz), 5.83-5.78 (d, 1H, J = 15.5 Hz),

5.75-5.73 (bd, 1H, J = 7.9Hz), 5.15-5.0 (m, 1H), 4.93-4.85 (dd, 1H, J = 10.0) , 3.0 Hz), 4.85-4.75 (m, 1H), 3.90 (s, 3H), 3.54-3.40 (dd, 1H, J = 13.4,

8.6 Hz), 3.25-3.02 (m, 3H), 2.65-2.35 (m, 3H),

1.80-1.60 (m, 2H), 1.45-1.36 (m, 1H), 1.27 (s, 3H),

1.20 (s, 3H), 1.17-1.12 (d, 3H, J = 6.8 Hz), 0.86-0.74 (d, 6H, J = 5.0 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.7, 170.6, 156.0, 154.0, 142.6, 137.1,

131.3, 130.8, 129.5, 128.9, 128.1, 127.4, 124.3,

122.5, 115.6, 112.3, 77.2, 71.5, 56.1, 54.5, 46.5,

42.7, 42.1, 39.4, 36.5, 35.3, 24.6, 22.8, 22.7, 21.2,

17.3.

IR (CHCl ₃ ): 3597, 3421, 3319, 2964, 2935, 2874, 2841, 1746, 1711, 1680, 1652, 1610, 1513, 1504, 1485, 1464, 1259, 1170, 1152, 1067 cm ^-1 .

Elemental analysis for C ₃₆ H ₄₆ CIN ₂ O ₈ : C, Η, N.

Example 64

A solution of 0.040 g (0.184 mmol) of the C 'acid fragment and 0.040 g (0.25 mmol) of carbonyldiimidazole (CDI) in 2 mL of toluene was heated at 45 ° C under nitrogen for 45 min. In toluene (1 mL), 0.10 g (0.15 mmol) of the alcohol of the above formula was added to the reaction mixture and heated at 45 ° C for 4 hours. After cooling to room temperature, the reaction mixture was diluted with 100 mL of ethyl acetate, washed with 10 mL of 0.1 N hydrochloric acid, 10 mL of water, 10 mL of saturated sodium bicarbonate, and 10 mL of brine. The organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The residual yellow foam was purified by radial PLC on silica gel using 50% ethyl acetate in hexane as eluent. 0.097 g (75%) of the pure ester of the formula is obtained as a yellow solid.

[a] ² ° D = + 17.2 ° (c = 0.58 _(CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.40-7.34 (d, 2H, J = 8.5 Hz), 7.29-7.22 (m, 3H),

7.13-7.00 (m, 3H), 6.92-6.86 (d, 1H, J = 8.8Hz), 6.86-6.76 (m, 1H), 6.50-. 6.38 (d, 1H, J = 15.9 Hz),

6.10-5.97 (dd, 1H, J = 15.8, 8.8 Hz), 5.85-5.75 (d, tH, J = 15.1 Hz), 5.55-5. 45 (d, 1H, J = 7.9 Hz),

5.15-5.06 (m, 1H), 5.06-4.96 (m, 1H), 4.95-4.85 (m, 1H), 4.83 ^ 4.72 (dd, 1H) , J = 10.0, 3.0 Hz), 3.92 (s, 3H), 3.53-3.35 (m, 3H), 3.22-3.06 (m, 3H), 2, 65-2.50 (m, 2H), 2.48-2.35 (m, 1H), 1.80-1.65 (m, 2H), 1.49 (s, 9H), 1.40 ( s, 6H). 1.27 (s, 3H),

1.21 (s, 3H), 1.20-1.15 (d, 3H, J = 6.9 Hz), 0.86-0.77 (d, 6H, J = 6.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 165.1, 156.0, 153.9, 150.0,

142.0, 134.5, 130.8, 130.6, 130.4, 129.6, 128.2,

126.9, 124.6, 121.5, 112.2, 79.2, 71.3, 56.0, 54.4,

48.2, 46.4, 44.0, 42.7, 42.1, 39.5, 36.5, 35.2, 28.3,

24.9, 24.5, 22.9, 22.8, 22.7, 21.3, 17.2.

IR (CHCl ₃ ): 3425, 2970, 2934, 2874, 1746, 1711, 1684,

1604, 1505, 1442, 1394, 1368, 1305, 1258, 1166,

1123, 1067, 1015, 971 crrr ¹ .

Elemental analysis for C ₄₁ H ₅₅ CIN ₃ O ₉ : C, Η, N.

Example 65

To the styrene of the above formula (0.276 g, 0.32 mmol) was added 12 ml of acetone, 6 ml of water, 6 ml of dichloromethane and 0.84 g (10 mmol) of solid sodium bicarbonate. The reaction mixture was cooled to 0 ° C. Oxone (0.78 g, 1.27 mmol) was dissolved in water (6 mL) and 2 mL of this solution was added to the cold reaction mixture. After vigorous stirring at 0 ° C, the reaction mixture is kept for 30 minutes, then 2 ml of Oxone solution, after 30 minutes another 2.0 ml of Oxone solution, and after 30 minutes all Oxone solution are added. The progress of the reaction is monitored by reverse phase HPLC analysis and after 2.5 hours of stirring, the reaction is complete. To the reaction mixture was added 50 mL of saturated aqueous sodium bicarbonate and 50 mL of dichloromethane at 0 ° C. The phases were separated and the organic phase was washed with 50 ml of 10% sodium sulfate solution, 50 ml of saturated aqueous sodium bicarbonate solution and brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. 0.72 g of crude epoxide is obtained in the form of a yellow foam.

To a solution of the epoxide in 4 mL of dichloromethane was added 0.2 mL (1.54 mmol) of trimethylsilyl chloride at -60 ° C. After standing for 3 hours at -60 ° C, 5 ml of 0.1 N hydrochloric acid was added to the reaction mixture to hydrolyze the trimethylsilyl ether and the reaction mixture was warmed to room temperature. The phases are separated and the organic phase is dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude chlorohydrin was purified twice by radial PLC on silica gel using 50-60-70-100% ethyl acetate / hexane as eluent. Final purification was by reverse phase HPLC using acetonitrile / water as eluent. Obtained as a white solid (0.090 g, 30%).

[w.] ² D = + 42.7 ° (c = 3.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.48-7.44 (d, 1H, J = 8.4 Hz), 7.31-7.26 (m, 4H),

7.16-7.10 (m, 3H), 6.92-6.89 (d, 1H, J = 8.4 Hz),

6.87-6.81 (m, 1H), 5.84-5.79 (d, 1H, J = 15.1 Hz),

5.58-5.55 (d, 1H, J = 7.8Hz), 5.24-5.19 (t, 1H,

J = 8.9 Hz), 5.05-4.95 (m, 2H), 4.79 ^ 1.75 (m, 1H),

4.72-4.69 (d, 1H, J = 9.5 Hz), 4.04-4.01 (dd, 1H,

J = 1.3, 9.3 Hz), 3.93 (s, 3H), 3.47-3.40 (m, 3H),

3.25-3.06 (m, 3H), 2.75-2.70 (d, 1H, J = 13.9 Hz),

2.55-2.41 (m, 2H), 1.90-1.71 (m, 2H), 1.63 (s,

1 H), 1.48 (s, 9 H), 1.39 (s, 6 H), 1.28 (s, 3 H), 1.22 (s, 3 H), 1.09-1.06 (d, 3 H) , J = 6.9 Hz), 1.0-0.96 (m,

6H).

HU 227 195 Β ¹³ C-NMR (63 MHz, CDCl ₃ ), δ:

177.6, 170.4, 170.2, 165.2, 153.9, 142.3, 136.1,

130.8, 129.7, 129.1, 128.2, 124.5, 122.4, 121.9,

112.2, 76.1.74.0, 71.1.61.5, 56.5, 54.4,46,4.44.1,

42.7, 39.6, 38.4, 36.3, 35.2, 28.3, 24.8, 22.9, 22.8,

22.7.21.5.8.6.

IR (CHCl ₃ ): 3417, 2974, 2934, 1755, 1720, 1677, 1505, 1473, 1368, 1320, 1258, 1205, 1167, 1153, 1123, 1066 cm ^-1 .

FAB HRMS (M-BOC) calculated for C ₄₁ H ₅₆ Cl ₂ N ₃ O ₁₀ :

calcd: 820.3343, found: 820.3354.

Example 66

0.070 g (0.067 mmol) of the above formula

To a solution of the BOC-protected amine in 0.25 mL of dichloromethane was added 0.1 mL (0.4 mmol) of 4 M hydrochloric acid in 1,4-dioxane. After stirring at room temperature for 2 hours, the solvent was removed under reduced pressure and the resulting evaporation residue was maintained under high vacuum for 2 days to give 0.062 g (95%) of the title compound as a white solid. [α] ²⁵ D = + 27.68 ° (c = 2.5, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.79-7.76 (d, 1H, J = 7.9 Hz), 7.48-7.38 (m, 2H),

7.27-7.26 (d, 1H, J = 1.2 Hz), 7.17-7.08 (m, 3H),

6.98-6.95 (d, 1H, J = 8.5Hz), 6.71-6.60 (m, 1H),

5.95-5.90 (d, 1H, J = 15.2Hz), 5.14-5.01 (m, 2H),

4.50-4.46 (dd, 1H, J = 3.0, 11.0 Hz), 3.99-3.96 (d, 1H, J = 9.1 Hz), 3.82 (s, 3H), 3.49-3.42 (m, 1H), 3.19 (s, 2H), 3.2-3.06 (m, 2H), 2.77-2.68 (m, 2H). .

2.49-2.46 (t, 1H, J = 6.8Hz), 2.44-2.31 (q, 1H, J = 11.4Hz), 1.85-1.65 (m, 2H), 1.60-1.50 (m, 1H), 1.46 (s, 6H), 1.20 (s, 3H), 1.16 (s, 3H), 1.08-0.94 (m, 9H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.9, 175.6, 173.8, 171.9, 155.3, 151.8, 144.2,

139.5, 132.2, 131.5, 130.7, 129.4, 125.2, 123.3,

122.6, 113.5, 77.2, 74.8, 72.6, 63.2, 57.6, 56.7,

50.1,48.3, 48.0, 47.4, 44.1, 42.7, 41.1,40.4, 37.8,

36.5, 28.8, 26.2, 23.6, 23.4, 22.2, 9.0.

IR (CHCl ₃ ): 3418, 2961, 2934, 1751, 1724, 1671, 1608, 1505, 1474, 1464, 1442, 1303, 1282, 1259, 1203, 1169, 1152, 1126, 1065, 1018 cm ^-1 .

FAB HRMS (M-Cl) calcd for C ₄₁ H ₅₆ Cl ₂ N ₃ O ₁₀ : 820.3343, found: 820.3354.

Example 67

A solution of 4.0 ml (4.1 mmol) of tetrabutylammonium fluoride (TBAF) was prepared in 1.0 M tetrahydrofuran. This solution was added dropwise to a solution of the protected (20) alcohol (3.1 g, 3.69 mmol) in tetrahydrofuran (120 mL) at -78 ° C. The solution was stirred at -78 ° C for 10 minutes and then the dry ice-cooling mixture was removed to allow it to warm to room temperature. After standing for 30 minutes at room temperature, 80 ml of water and 100 ml of ethyl acetate are added to the reaction mixture. The layers were separated and the aqueous layer was extracted with dichloromethane (3 x 50 mL). The combined organic phases are dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the free alcohol. The product was purified by silica gel column chromatography using 50-70-100% ethyl acetate / hexane. 2.51 g (99%) of pure alcohol (29) are obtained in the form of a white solid.

[α] ²⁰ D = + 30.0 ° (c = 1.0, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.31 (s, 4H), 7.26-7.21 (m, 1H), 7.2-7.19 (d, 1H,

J = 1.8 Hz), 7.07-7.03 (dd, 1H, J = 8.4, 1.7 Hz),

6.85-6.82 (d, 1H, J = 8.4 Hz), 6.82-6.7 (m, 1H),

6.43 - 6.37 (d, 1H, J = 15.9 Hz), 6.05 - 5.97 (dd, 1H,

J = 15.9, 8.7 Hz), 5.77-5.72 (d, 1H, J = 15.0 Hz),

5.58-5.55 (d, 1H, J = 7.9Hz), 5.08-5.02 (dd, 1H,

J = 9.4, 6.3 Hz), 4.87-4.83 (dd, 1H, J = 10.2, 3.1 Hz),

4.8 - 4.67 (m, 1H), 4.67 (s, 2H), 3.87 (s, 3H),

3.44-3.37 (dd, 1H, J = 13.5, 8.5 Hz), 3.2-3.0 (m,

3H), 2.6-2.3 (m, 3H), 1.8-1.6 (m, 3H), 1.4-1.25 (m,

1H), 1.22 (s, 3H), 1.15 (s, 3H), 1.14-1.12 (d, 3H,

J = 6.8 Hz), 0.76-0.73 (t, 6H, J = 5.5 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.7, 170.5, 165.4, 153.8, 142.0, 140.5,

135.9, 131.3, 130.7, 130.0, 129.9, 128.1, 127.1,

126.1, 124.5, 122.2, 112.2, 77.0, 71.3, 64.6, 56.0,

54.6, 46.4, 42.7, 42.1, 39.4, 36.4, 35.2, 24.5, 22.8,

22.6, 21.2, 17.2.

IR (CHCl ₃ ): 3423, 3011, 2965, 2935, 2874, 2841, 1747,

1712, 1681, 1652, 1528, 1503, 1485, 1442, 1371,

1303, 1259, 1151 cm ^-1 .

Elemental analysis for C ₃₇ H ₄₇ CIN ₃ O ₈ : C, Η, N.

Example 68

To a solution of alcohol (29) (0.13 g, 0.19 mmol) in tetrahydrofuran (2.0 mL) was added triphenylphosphine (0.065 g, 0.25 mmol), phthalimide (0.0367 g, 0.25 mmol), and finally 0.04 ml (0.25 mmol) of diethyl azodicarboxylate (DEAD). The resulting yellow solution was stirred at room temperature for 2 hours, then water (10 mL) and dichloromethane (10 mL) were added. The aqueous phase was extracted with dichloromethane (2 x 10 mL), and the combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by radial PLC on silica gel eluting with 50-60-70% ethyl acetate / hexane. Yield: 0.14 g (90%) of phthalimide (30) as a white solid.

[a] ² ° D = + 19.2 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.9-7.8 (m, 2H), 7.72-7.69 (m, 2H), 7.39-7.36 (d,

2H, J = 8.0 Hz, 7.28-7.26 (d, 2H, J = 5.3 Hz),

7.25-7.2 (m, 1H), 7.19-7.18 (d, 1H, J = 1.9 Hz),

7.06-7.03 (dd, 1H, J = 8.5, 1.9 Hz), 6.85-6.82 (d,

1H, J = 8.4 Hz), 6.81-6.7 (m, 1H), 6.39-6.33 (d, 1H,

J = 15.9 Hz), 6.01-5.93 (dd, 1H, J = 15.8, 8.8 Hz),

5.75-5.7 (d, 1H, J = 15.4Hz), 5.47-5.44 (d, 1H,

J = 7.9 Hz), 5.05-5.0 (dd, 1H, J = 9.4, 6.3 Hz), 4.8 (s,

2H), 4.83-4.7 (m, 2H), 3.87 (s, 3H), 3.4-3.36 (dd,

1H, J = 13.4, 8.6 Hz), 3.18-3.02 (m, 3H), 2.6-2.25

HU 227 195 Β1 (m, 3H), 1.65-1.5 (m, 2H), 1.35-1.22 (m, 1H), 1.21 (s, 3H), 1.14 (s) , 3H), 1.12-1.09 (d, 3H, J = 6.8 Hz),

0.71-0.69 (d, 3H, J = 6.4 Hz), 0.65-0.63 (d, 3H,

J = 6.4 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 167.8, 165.2, 153.9, 142.0,

136.3, 135.6, 133.9, 131.9, 131.1, 130.7, 130.6,

129.8, 128.9, 128.6, 128.5, 128.1, 126.3, 124.6,

123.2, 122.3, 112.2, 76.9, 71.3, 56.0, 54.5 46.4,

42.6, 42.1.41.2, 39.4, 36.4, 35.2, 24.4, 22.8, 22.6,

22.5,21.1, 17.2.

IR (CHCl ₃ ): 3421, 2967, 2935, 2873, 2840, 1747, 1716,

1682, 1527, 1503, 1485, 1433, 1395, 1259,

1151 cm ^-1 .

Example 69

To a solution of phthalimide (30) (0.1 g, 0.123 mmol) in ethanol (1.8 mL) was added n-butylamine (0.04 mL, 0.369 mmol). The solution was heated at 75 ° C for 2 days, concentrated under reduced pressure and purified by radial PLC on silica gel using 10-25% ethanol / dichloromethane as eluent. 0.048 g (57%) of free amine (31) are obtained.

Example 70

To a solution of N-tert-butoxycarbonyl sarcosine (0.07 g, 0.37 mmol) in dimethylformamide (1.5 mL) was added 1-hydroxybenzotriazole hydrate (HOBT) (0.05 g, 0.37 mmol). ) and 0.071 g (0.37 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI). The reaction mixture is stirred at room temperature for 45 minutes and a solution of (31) amine (0.17 g, 0.25 mmol) in dimethylformamide (2.5 mL) is added dropwise to the reaction mixture via a double-ended needle. After stirring for a further 3 hours, water (10 ml) was added and the mixture was extracted with dichloromethane (3 x 10 ml). The combined organic extracts were dried over magnesium sulfate, filtered and evaporated under reduced pressure. The resulting crude product was purified by radial PLC on silica gel eluting with 70-80-100% ethyl acetate / hexane. Yield: 0.15 g (71%) of (32) as a white solid.

[a] ² ° D = + 22.4 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.27-7.1 (m, 6H), 7.04-7.0 (dd, 1H, J = 8.5, 1.9 Hz),

6.82-6.79 (d, 1H, J = 8.5 Hz), 6.79-6.65 (m, 1H),

6.38- 6.32 (d, 1H, J = 15.9 Hz), 6.3-6.2 (bs, 1H),

6.01-5.93 (dd, 1H, J = 15.9, 8.7 Hz), 5.75-5.70 (d,

1H, J = 15.0 Hz), 5.65-5.6 (m, 1H), 5.0 ^ 1.99 (dd,

1H, J = 9.3, 6.1 Hz), 4.84 ^ 4.79 (dd, 1H, J = 9.6,

3.6 Hz), 4.74-4.67 (m, 1H), 4.42-4.4 (d, 2H,

J = 5.7 Hz), 3.87 (s, 2H), 3.84 (s, 3H), 3.42-3.34 (dd,

1H, J = 13.5, 8.6 Hz), 3.15-3.0 (m, 3H), 2.9 (s, 3H),

2.6-2.25 (m, 3H), 1.8-1.5 (m, 2H), 1.4 (s, 9H),

1.39-1.25 (m, 1H), 1.19 (s, 3H), 1.12 (s, 3H),

1.1-1.08 (d, 3H, J = 6.8 Hz), 0.73-0.72 (d, 3H,

J = 4.4 Hz), 0.71-0.69 (d, 3H, J = 4.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.5, 170.4, 169.2, 165.2, 153.9, 141.9,

137.3, 136.0, 131.1, 130.7, 130.3, 129.8, 128.6,

128.1, 127.7, 126.3, 124.6, 122.3, 112.2, 80.6,

76.9, 71.3, 56.0, 54.5, 53.1, 46.4, 42.8, 42.7, 42.0,

39.4, 36.4, 35.8, 35.2, 28.2, 24.5, 22.8, 22.6, 21.2,

17.1.

IR (CHCl ₃ ): 3427, 2967, 2935, 2874, 2841, 1747, 1680, 1526, 1504, 1484, 1464, 1442, 1393, 1369, 1302, 1281, 1259, 1151, 1067 cm ^-1 .

Example 71

According to the procedure described above, amide (32) (0.34 g, 0.398 mmol) was epoxidized with mCPBA (0.072 g, 0.42 mmol) in dichloromethane (1.2 mL). A 2: 1 mixture of β and epoxide is obtained. The crude epoxide mixture (0.3 g, 0.345 mmol) was dissolved in chloroform and cooled to -60 ° C. TMSCI (0.22 mL, 1.73 mmol) was added to the solution and stirred at -50 ° C to -20 ° C for 2 hours. An additional 0.44 mL (0.173 mmol) of TMSCI was added to the solution and allowed to warm to room temperature. The solution was evaporated under reduced pressure and the resulting product was purified twice by column chromatography using 70-80% ethyl acetate / hexane as eluent and once by radial PLC on silica gel using 2-5% methanol / dichloromethane as eluent. Trans-chlorohydrin (34) (0.1 g, 48%) was obtained as a white solid.

[α] D 20 = + 46.9 ° (c = 0.85, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.36-7.34 (d, 2H, J = 7.9 Hz), 7.29-7.26 (d, 2H, J = 8.3 Hz), 7.21 (s, 2H), 7 , 08-7.05 (d, 1H, J = 8.3 Hz), 6.86-6.83 (d, 1H, J = 8.5 Hz), 6.8-6.7 (m, 1H ), 6.5-6.2 (bs, 1H), 5.79-5.74 (d, 1H, J = 15.2 Hz), 5.64-5.62 (d, 1H, J = 7 , 7 Hz), 5.18-5.12 (t, 1H, J = 9.1 Hz, 4.94-4.9 (dd, 1H, J = 9.9, 3.5 Hz),

4.8-4.67 (m, 1H), 4.66-4.63 (d, 1H, J = 9.6 Hz),

4.47-4.45 (d, 2H, J = 5.3 Hz), 4.02-3.98 (d, 1H, J = 9.5 Hz), 3.89 (s, 2H), 3 , 88 (s, 3H), 3.41-3.34 (dd, 1H, J = 13.6, 8.5 Hz), 3.2-3.0 (m, 3H), 2.94 (s , 3H),

2.69-2.68 (bdd, 1H, J = 14.3, 2.1 Hz), 2.51-2.3 (m, 2H), 1.8-1.6 (m, 3H), 1.42 (s, 10H), 1.22 (s, 3H),

1.17 (s, 3H), 1.03-1.01 (d, 3H, J = 6.9 Hz), 0.94-0.9 (t, 6H, J = 5.5 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.6, 170.4, 170.2, 165.2, 153.9, 142.4, 139.1,

130.8, 129.7, 128.3, 128.2, 127.9, 124.4, 122.4,

112.2, 80.7, 76.1, 73.9, 71.2, 61.8, 56.1, 54.4, 53.1,

46.4, 42.7, 39.6, 38.4, 36.3, 35.9, 35.2, 28.2, 24.8, 23.0, 22.9, 22.7, 21.5, 8.5.

IR (KBr): 3419, 3317, 2964, 2932, 1755, 1670, 1538, 1504, 1473, 1392, 1368, 1301, 1258, 1151, 1066 cm ^-1 .

FAB HRMS (M + H) calcd for C 14 H 16 Cl 2 N ₃ O ₁ 905, 3870, found 905, 3876.

Example 72

The hydrochloride salt of BOC-protected amine (34) (0.045 g, 0.05 mmol) (34) was prepared according to the method described above in quantitative yield using 4M hydrochloric acid in 1,4-dioxane.

HU 227 195 Β1 ¹ H-NMR (300 MHz, MeOD), δ:

8.47-8.45 (d, 1H, J = 7.7Hz), 7.79-7.76 (d, 1H,

J = 8.9 Hz), 7.39-7.36 (d, 2H, J = 8.1 Hz), 7.3-7.27 (d, 3H, J = 9.0 Hz), 7, 17-7.14 (d, 1H, J = 8.5 Hz),

6.98-6.95 (d, 1H, J = 8.5 Hz), 6.75-6.6 (m, 1H),

5.94-5.89 (d, 1H, J = 15.1Hz), 5.2-5.0 (m, 2H),

4.78-4.75 (d, 1H, J = 9, 4 Hz), 4.5 ^ 1.42 (m, 1H),

4.41 (s, 2H), 4.01-3.98 (d, 1H, J = 9.5 Hz), 3.82 (s,

3H), 3.8 (s, 2H), 3.5-3.4 (m, 1H), 3.19-3.13 (dd,

1H, J = 14.4, 3.4 Hz), 3.11-3.06 (dd, 1H, J = 13.2,

1.9 Hz), 2.8-2.6 (m, 2H), 2.7 (s, 3H), 2.5-2.2 (m,

2H), 1.85-1.45 (m, 3H), 1.3-1.2 (m, 1H), 1.2 (s,

3H), 1.15 (s, 3H), 1.0-0.94 (q, 9H, J = 11.3, 6.0 Hz). ¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.9, 173.7, 171.9, 168.3, 166.3, 155.4, 144.2,

140.5, 139.7, 132.2, 131.5, 129.7, 129.4, 128.8,

125.2, 123.3, 113.5, 77.2, 74.7, 72.6, 63.7, 57.6,

56.6, 50.7, 47.4, 44,1,43,9, 41,1,40,4, 37,8, 36,5,

33.7.26.2.23.6, 23,4,22,1,9,0.

IR (KBr): 3410, 3058, 2961, 2933, 1752, 1721, 1675,

1539, 1504, 1463, 1440, 1282, 1259, 1196, 1154,

1127.1066 cm ^-1 .

Example 73

A suspension of 0.041 g (1.0 mmol) of sodium hydride (60% suspension) and 0.5 g (1.0 mmol) of 4-carbomethoxybenzyl-triphenylphosphonium bromide in 10 ml of tetrahydrofuran is heated at 65 ° C. After 1 hour, cool to room temperature. This orange reaction mixture was added dropwise to a solution of aldehyde (18) (0.46 g, 0.79 mmol) in tetrahydrofuran (10 mL) at -78 ° C. The resulting solution was warmed to room temperature, stirred for 2 hours, and then saturated ammonium chloride solution (30 mL) and ethyl acetate (30 mL) were added. The layers were separated and the aqueous layer was extracted with additional ethyl acetate (2 x 20 mL). The combined organic extracts were washed with brine (30 mL), dried over magnesium sulfate, filtered and evaporated under reduced pressure. The crude styrene was purified by silica gel column chromatography (50-65% ethyl acetate / hexane). 0.129 g (23%) of pure styrene are obtained. [a] ² D ⁰ = + 29.7 ° (c = 1.15, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.03-8.0 (d, 2H, J = 8.2 Hz), 7.44-7.41 (d, 2H,

J = 8.3 Hz), 7.3-7.25 (m, 1H), 7.24-7.23 (d, 1H,

J = 1.6 Hz), 7.11-7.08 (dd, 1H, J = 8.3, 1.9 Hz),

6.89-6.86 (d, 1H, J = 8.5 Hz), 6.86-6.8 (m, 1H),

6.52-6.46 (d, 1H, J = 15.9 Hz), 6.23-6.15 (dd, 1H,

J = 15.8, 8.8 Hz), 5.83-5.8 (d, 1H, J = 15.3 Hz),

5.64-5.61 (d, 1H, J = 7.9Hz), 5.14-5.09 (dd, 1H,

J = 9.4, 6.5 Hz), 4.91-4.87 (dd, 1H, J = 10.2, 3.6 Hz),

4.85-4.75 (m, 1H), 3.95 (s, 3H), 3.91 (s, 3H),

3.5-3.4 (dd, 1H, J = 13.5, 8.7 Hz), 3.2-3.1 (m, 3H),

2.9-2.3 (m, 3H), 1.8-1.6 (m, 3H), 1.4-1.3 (m, 1H),

1.26 (s, 3H), 1.2 (s, 3H), 1.2-1.18 (d, 3H, J = 6.9 Hz),

0.8-0.78 (d, 3H, J = 5.6 Hz), 0.78-0.76 (d, 3H,

J = 6.0 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.4, 166.7, 165.1, 153.9, 141.8, 141.1,

133.1, 130.8, 129.9, 129.7, 128.9, 128.2, 125.9,

124.6, 122.4, 112.2, 76.8, 71.3, 56.0, 54.4, 52.0,

46.4, 42.7, 42.2, 39.5, 36.5, 35.2, 24.5, 22.8, 22.6,

21.2, 17.1.

IR (CHCl ₃ ): 3424, 2964, 2936, 2874, 2841, 1748, 1716,

1681, 1608, 1528, 1503, 1485, 1437, 1283,

1259 cm ¹ .

Elemental analysis for C ₃₈ H ₄₇ CIN ₂ O ₉ : C, Η, N.

Example 74

To the styrene of the above formula (0.42 g, 0.59 mmol) was added 30 ml of acetone, 15 ml of water, 15 ml of dichloromethane and 1.7 g (20.2 mmol) of solid sodium bicarbonate. The reaction mixture was cooled to 0 ° C. Oxone (1.4 g, 2.3 mmol) was dissolved in water (12 mL) and 2 mL of the solution was added to the cooled styrene solution. After vigorous stirring at 0 ° C for 30 min, an additional 2 ml of Oxone solution was added to the reaction mixture and allowed to warm to room temperature. Every other hour, 2 ml of Oxone solution was added to the reaction mixture until exhausted. After stirring for a total of 5 hours, 50 ml of a saturated aqueous solution of sodium bicarbonate and 50 ml of dichloromethane are added. The phases are separated and the organic phase is washed with 50 ml of 10% sodium sulfate solution, 50 ml of saturated aqueous sodium bicarbonate solution and finally with brine, dried over sodium sulphate, filtered and concentrated under reduced pressure. The crude mixture was purified by reverse phase HPLC using 45:55 acetonitrile / water 1: water. 0.14 g (33%) of a mixture of β and epoxide and 0.14 g of a bis-epoxidation mixture are obtained.

0.14 g (0.19 mmol) of a β / α epoxide mixture was dissolved in 3.0 mL of chloroform and cooled to -60 ° C. Chlorotrimethylsilane (0.1 mL, 0.77 mmol) was added to the solution at -60 ° C and the reaction mixture was stirred for 1.5 h. An additional 0.1 mL (0.77 mmol) of TMSCI was added to the reaction mixture and the solution was allowed to warm to room temperature. After stirring at room temperature for 1 hour, the solution was concentrated and purified by radial PLC using 1-2% methanol / dichloromethane as eluent. 0.044 g (30%) of the desired chlorohydrin of the formula is obtained as a white solid.

[a] ² ° D = + 50.0 ° (c = 0.75, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.1-8.08 (d, 2H, J = 8.1 Hz), 7.54-7.51 (d, 2H,

J = 8.2 Hz), 7.3-7.25 (m, 1H), 7.25 (s, 1H),

7.13-7.09 (dd, 1H, J = 8.5, 1.5 Hz), 6.91-6.88 (d,

1H, J = 8.3 Hz), 6.87-6.78 (m, 1H), 5.86-5.8 (d, 1H,

J = 15.5 Hz), 5.7-5.6 (m, 1H), 5.24-5.18 (t, 1H,

J = 9.2 Hz), 4.99-4.95 (dd, 1H, J = 10.0, 3.6 Hz,

4.8-4.7 (m, 1H), 4.76-4.73 (d, 1H, J = 9.5 Hz),

4.09-4.06 (d, 1H, J = 9.6 Hz), 3.97 (s, 3H), 3.93 (s,

3H), 3.45-3.38 (dd, 1H, J = 13.6, 8.6 Hz), 3.25-3.0 (m, 3H), 2.75-2.62 (m, 1H), 2.6-2.4 (m, 2H),

1.9-1.6 (m, 3H), 1.5-1.4 (m, 1H), 1.27 (s, 3H), 1.22 (s, 3H), 1.1-1.07 ( d, 3H, J = 6.95 Hz), 0.98-0.95 (t,

6H, J = 5.3 Hz).

HU 227 195 Β ¹³ C-NMR (63 MHz, CDCl ₃ ), δ:

177.5, 170.6, 170.3, 166.3, 165.4, 153.9, 143.8,

142.2, 138.7, 130.7, 130.4, 129.9, 128.1, 128.08,

124.6, 122.3, 112.2, 76.1, 73.8, 71.1, 61.4, 56.0,

54.6, 52.2, 42.7, 39.6, 38.4, 36.3, 35.1, 24.8, 23.0,

22.9, 22.7, 21.5, 8.7.

IR (CHCl ₃ ): 3425, 2962, 2935, 2873, 2842, 1750, 1720,

1680, 1528, 1504, 1484, 1438, 1284, 1259, 1194,

1152, 1114, 1067 cm ^-1 .

Elemental analysis for C ₃₈ H ₄₈ Cl ₂ N ₂ O ₁₀ : C, Η, N.

Example 75

To a solution of alcohol (23) (0.32 g, 0.46 mmol) in dichloromethane (8.5 mL) at 0 ° C was added 0.19 g (2.29 mmol) of solid sodium bicarbonate, 0.18 g. (0.69 mmol) triphenylphosphine and finally 0.092 g (0.69 mmol) of N-chlorosuccinimide. The reaction mixture was stirred at 0 ° C for 20 minutes and then treated with 20 ml of a saturated aqueous solution of sodium bicarbonate. The layers were separated and the aqueous layer was extracted with dichloromethane (3 x 10 mL). The combined organic extracts were washed with brine, dried over sodium sulfate, filtered and evaporated under reduced pressure. The resulting yellow solid crude product was purified by radial PLC on silica gel, eluting with 20-50% ethyl acetate / hexane. 0.26 g (79%) of (36) benzyl chloride is obtained in the form of a white solid.

[α] ²⁵ D = + 25.6 ° (c = 0.9, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.4-7.38 (d, 2H, J = 7.9 Hz), 7.26-7.23 (d, 3H,

J = 8.3 Hz), 7.19-7.18 (d, 1H, J = 1.9 Hz), 7.06-7.03 (dd, 1H, J = 8.3, 1.9 Hz) ), 6.85-6.82 (d, 1H,

J = 8.4 Hz), 6.8-6.7 (m, 1H), 5.74-5.69 (d, 1H,

J = 15.4 Hz), 5.49-5.47 (d, 1H, J = 7.8 Hz), 5.22-5.17 (m, 1H), 4.85 ^ 4.8 (dd) , 1H, J = 9.7, 3.0 Hz), 4.78 ^ 4.7 (m, 1H), 4.6 (s, = 2H), 3.9 (s, 3H), 3.69 -3.68 (d, 1H,

J = 1.3 Hz), 3.45-3.38 (dd, 1H, J = 13.4, 8.6 Hz),

3.2-3.0 (m, 3H), 2.92-2.89 (dd, 1H, J = 7.6, 1.6 Hz),

2.6-2.4 (m, 2H), 1.8-1.6 (m, 3H), 1.4-1.3 (m, 1H),

1.22 (s, 3H), 1.16 (s, 3H), 1.16-1.13 (d, 3H,

J = 8.6 Hz), 0.86-0.82 (t, 6H, J = 6.8 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 175.1, 170.3, 164.9, 154.0, 141.6, 137.7,

137.1, 130.8, 129.5, 128.9, 128.1, 125.9, 124.6,

122.4, 112.3, 77.2, 75.8, 71.1, 63.1, 58.5, 56.1,

54.4, 46.4, 45.7, 42.7, 40.5, 39.3, 36.8, 35.2, 24.5,

22.8.22.6.21.2, 13.5.

IR (CHCl ₃ ): 3416, 3284, 2961, 2933, 2873, 2839, 1752,

1721, 1680, 1658, 1536, 1504, 1473, 1442, 1321,

1302, 1281, 1259, 1192, 1150, 1126, 1066 cm ^-1 . Elemental analysis for C ₃₇ H ₄₆ Cl ₂ N ₂ O ₈ : C, Η, N.

Example 76

To a solution of benzyl chloride (36) (0.03 g, 0.042 mmol) in tetrahydrofuran (0.3 mL) was added diethylamine (0.09 mL, 0.84 mmol). The reaction mixture was stirred at room temperature overnight and 5 mL of saturated aqueous sodium bicarbonate was added. The aqueous phase was extracted with dichloromethane (3 x 5 mL), and the combined organic extracts were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The yellow solid was purified by radial PLC on silica gel eluting with 50-70-80% ethyl acetate / hexane. 0.026 g (82%) of the amine (37) is obtained as a white solid.

[α] ²⁵ D = + 25.9 ° (c = 0.9, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.35-7.32 (d, 2H, J = 7.9 Hz), 7.25-7.12 (m, 4H),

7.06-7.02 (dd, 1H, J = 8.4, 1.6 Hz), 6.84-6.82 (d,

1H, J = 8.5 Hz), 6.82-6.7 (m, 1H), 5.74-5.69 (d, 1H,

J = 15.2 Hz), 5.57-5.55 (d, 1H, J = 7.8 Hz), 5.22-5.17 (m, 1H), 4.85-4.7 (m , 2H), 3.86 (s, 3H), 3.66 (s,

1H), 3.57 (s, 2H), 3.46-3.38 (dd, 1H, J = 13.4,

8.7 Hz), 3.2-3.0 (m, 3H), 2.93-2.91 (d, 1H,

J = 7.4 Hz), 2.6-2.4 (m, 6H), 1.8-1.6 (m, 3H),

1.4-1.3 (m, 1H), 1.22 (s, 3H), 1.15 (s, 3H),

1.15-1.12 (d, 3H, J = 9.2 Hz), 1.07-1.03 (t, 6H,

J = 7.1 Hz), 0.86-0.82 (t, 6H, J = 6.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.4, 170.3, 164.9, 154.0, 141.7 140.1,

135.2, 130.8, 129.6, 129.1, 128.1, 125.4, 124.6,

122.4, 112.3, 77.2, 75.8, 71.1, 62.9, 58.9, 57.1,

56.0, 54.4, 46.6, 46.4, 42.7, 40.6, 39.3, 36.8, 35.2,

24.5.22.8, 22.6.21.2, 13.5, 11.5.

IR (CHCl ₃ ): 3424, 2969, 2936, 2874, 1752, 1711, 1682,

1605, 1527, 1503, 1485, 1303, 1259, 1190, 1151,

1067 cm ^-1 .

Elemental analysis for C ₄₁ H ₅₆ CIN ₃ O ₈ : C, Η, N.

Example 77

To a solution of epoxide (37) (0.05 g, 0.066 mmol) in chloroform (0.8 mL) at -66 ° C was added dropwise a solution of 4 M hydrochloric acid (0.04 mL, 0.166 mmol) in 1,4-dioxane. The reaction mixture was stirred at -66 ° C for 10 minutes, then the dry ice coolant was removed and the solution was allowed to warm to room temperature. The solvent was removed under reduced pressure and the resulting salt was dried under high vacuum for 3 days to remove the dioxane residue. 0.054 g (100%) of the desired chlorohydrin (38) is obtained. [a] ² ° D = + 29.3 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, MeOD), δ:

8.5-8.47 (d, 1H, J = 7.5 Hz), 7.79-7.76 (d, 1H,

J = 8.8 Hz), 7.53 (s, 4H), 7.26-7.25 (d, 1H,

J = 1.6 Hz), 7.17-7.14 (dd, 1H, J = 8.6, 1.6 Hz),

6.97-6.94 (d, 1H, J = 8.4 Hz), 6.75-6.6 (m, 1H),

5.96-5.90 (d, 1H, J = 15.3Hz), 5.2-5.0 (m, 2H),

4.85 ^ 4.82 (d, 1H, J = 8.9 Hz), 4.5 ^ 1.4 (m, 1H), 4.33 (s, 2H), 4.02-3.98 ( d, 1H, J = 9.3 Hz), 3.8 (s, 3H),

3.49-3.42 (dd, 1H, J = 13.3, 9.9Hz), 3.2-3.0 (m,

6H), 2.8-2.6 (m, 2H), 2.5-2.2 (m, 2H), 1.8-1.5 (m,

3H), 1.34-1.3 (m, 7H), 1.2 (s, 3H), 1.15 (s, 3H),

1.01-0.99 (d, 3H, J = 7.2 Hz), 0.98-0.94 (t, 6H,

J = 5.4 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.9, 173.8, 171.8, 168.3, 155.4, 144.1, 143.4,

132.3, 132.2, 131.5, 131.1, 130.4, 129.4, 125.2,

123.3, 113.5, 77.2, 74.8, 72.6, 63.2, 57.6, 56.7,

HU 227 195 Β1

56.66, 48.0, 47.5, 44.1,41,1,40.4, 37.8, 36.5, 26.2,

23.6, 23.4, 22.2, 9.07, 9.0.

IR (KBr) 3414, 2960, 2934, 1751, 1721, 1671, 1521,

1504, 1463, 1443, 1259, 1197, 1155, 1127,

1065 cm ^-1 .

Example 78

According to the procedure described above, 0.147 g (81%) of benzyl chloride (36) (0.15 g, 0.21 mmol) and N-tert-butoxycarbonylpiperazine (0.195 g, 1.05 mmol) were obtained. we produce. [a] ² D ⁰ = + 25.4 ° (c = 0.65, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.37-7.23 (m, 5H), 7.11-7.08 (d, 1H, J = 8.6 Hz),

6.9-6.87 (d, 1H, J = 8.5 Hz), 6.86-6.72 (m, 1H),

5.78-5.73 (d, 1H, J = 15.2 Hz), 5.53-5.5 (d, 1H,

J = 7.7 Hz), 5.28-5.23 (m, 1H), 4.88 ^, 70 (m, 2H),

3.9 (s, 3H), 3.7 (s, 2H), 3.54 (s, 2H), 3.5-3.4 (m,

5H), 3.2-3.05 (m, 3H), 3.0-2.95 (d, 1H, J = 7.4 Hz),

2.65-2.4 (m, 6H), 1.85-1.6 (m, 3H), 1.5 (s, 9H),

1.45-1.4 (m, 1H), 1.27 (s, 3H), 1.2 (s, 3H), 1.2-1.18 (d, 3H, J = 8.3 Hz) , 0.91-0.87 (t, 6H, J = 6.1 Hz). ¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.3, 170.26, 164.9, 154.7, 154.0, 141.7,

138.4, 135.6, 130.8, 129.5, 129.3, 128.1, 125.5,

124.6, 122.4, 112.3, 79.5, 77.2, 75.8, 71.1, 62.9,

62.6, 58.8, 56.1, 54.4, 52.8, 46.3, 42.7, 40.6, 39.3,

36.7, 35.2, 28.3, 24.5, 22.8, 22.6, 21.2, 13.4.

IR (CHCl ₃ ): 3425, 3008, 2965, 2937, 2874, 2817, 1752,

1709, 1683, 1527, 1484, 1463, 1459, 1427, 1367,

1259, 1167, 1150 cm ^-1 .

FAB HRMS (M-BOC) calculated for C ₄₆ H ₆₄ CIN ₄ O ₁₀ :

calcd: 867.4311, found: 867.4300.

Example 79

To a solution of epoxy of the formula (0.135 g, 0.156 mmol) in chloroform (3.0 mL) at -66 ° C was added dropwise trimethylsilyl chloride (0.16 mL). The reaction mixture was stirred at -66 ° C for 2 hours and another 0.16 mL of TMSCI was added. After stirring for another hour at -66 ° C, the cooling bath was removed and the solution was allowed to slowly warm to room temperature. The solvent was removed under reduced pressure and the resulting solid was purified by radial PLC on silica gel eluting with 2-5% methanol / dichloromethane. 0.13 g (92%) of the chlorohydrin of the formula are obtained.

[α] ²⁰ D = + 50.0 ° (c = 1.0, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.34 (s, 4H), 7.21-7.2 (d, 2H, J = 1.4 Hz), 7.08-7.05 (dd, 1H, J = 8.6, 1.6 Hz), 6.86-6.83 (d, 1H,

J = 8.4 Hz), 6.82-6.7 (m, 1H), 5.8-5.75 d, 1H

J = 15.1 Hz), 5.65-5.62 (d, 1H, J = 7.8 Hz), 5.2-5.1 (m, 1H), 5.0-4.7 (m , 2H), 4.66-4.63 (d, 1H,

J = 9.7 Hz), 4.02-4.0 (d, 1H, J = 9.6 Hz), 3.88 (s, 3H),

3.49-3.48 (d, 2H, J = 4.2Hz), 3.45-3.3 (m, 5H),

3.2-3.0 (m, 3H), 2.7-2.3 (m, 7H), 1.8-1.6 (m, 3H),

1.45 (s, 10H), 1.23 (s, 3H), 1.17 (s, 3H), 1.04-1.02 (d, 3H, J = 6.9 Hz), 0.93 -0.91 (d, 6H, J = 6.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.5, 170.5, 170.2, 165.2, 154.6, 153.9, 142.3,

139.1, 137.3, 130.8, 129.8, 129.5, 128.1, 127.9,

124.5, 122.3, 112.2, 79.5, 76.0, 73.9, 71.1, 62.4,

61.9, 56.1, 54.5, 52.8, 46.4, 42.7, 39.6, 38.4, 36.4,

35.2, 28.3, 24.7, 23.0, 22.9, 22.7, 21.5, 8.6.

IR (CHCl ₃ ): 3424, 3007, 2966, 2936, 2872, 2820, 1751, 1712, 1682, 1528, 1504, 1483, 1426, 1367, 1259, 1168, 1150, 1127, 1067, 1006 cm ^-1 .

Elemental analysis for C ^ HH ^C ^N NO i: C, Η, N.

Example 80

According to the procedure described above, 0.112 g (0.13 mmol) of the BOC-protected piperazine salt were prepared using 0.116 g of the dihydrochloride salt using 4M hydrochloric acid in 0.32 ml (1.3 mmol) of 1,4-dioxane.

[α] ²⁰ D = + 26.3 ° (c = 0.7, MeOH).

¹ H-NMR (300 MHz, MeOD), δ:

8.47-8.45 (d, 1H, J = 7.5 Hz), 7.78-7.75 (d, 1H, J = 9.2 Hz), 7.6-7.52 (q, 4H, J = 16.9, 7.9 Hz),

7.27-7.26 (d, 1H, J = 1.15 Hz), 7.18-7.14 (dd, 1H, J = 8.6.1.8 Hz), 6.98-6, 95 (d, 1H, J = 8.4 Hz), 6.75-6.6 (m, 1H), 5.95-5.9 (d, 1H, J = 15.4 Hz), 5.2 -5.0 (m, 2H), 4.85-4.82 (d, 1H, J = 9.9 Hz), 4.5-4.4 (m, 1H),

4.4 (s, 2H), 4.0-3.98 (d, 1H, J = 9.3 Hz), 3.8 (s, 3H),

3.6-3.4 (m, 9H), 3.32-3.29 (d, 1H, J = 11.3 Hz), 3.19-3.13 (dd, 1H, J = 14.8 , 3.5 Hz), 3.1-3.06 (d, 1H, J = 13.7 Hz), 2.8-2.6 (m, 2H), 2.5-2.3 (m , 2 H),

1.85-1.5 (m, 3H), 1.3-1.2 (m, 1H), 1.2 (s, 3H), 1.15 (s, 3H), 1.02-0.95 ( q, 9H, J = 13.4, 6.3 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.8, 173.7, 171.9, 168.3, 155.4, 144.2, 143.7,

132.8, 132.2, 131.5, 130.4, 129.9, 129.4, 125.2,

123.3, 113.5, 77.2, 74.8, 72.6, 63.1, 61.2, 57.6,

56.7, 49.2, 47.5, 44.1,42,1,41,1,40,3, 37,8, 36,5,

26.3, 23.7, 23.4, 22.2, 9.0.

IR (KBr): 3415, 2960, 2933, 2455, 1749, 1721, 1671, 1504, 1475, 1442, 1304, 1258, 1197, 1152, 1126, 1065, 1012 cm ^-1 .

FAB HRMS (M-HCl ₂ ) calculated for C ₄₁ H ₅₇ Cl ₂ N ₄ O ₈ :

calculated: 803.3553, found: 803.3563.

Example 81

By the procedure described above, 0.15 g (0.22 mmol) of benzyl chloride (36) and 0.35 g (2.22 mmol) of tert-butyl N- (2-aminoethyl) -carbamate 0.15 g. g (78%) of the epoxide of the formula were obtained.

[a] ² ° D = + 22.3 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.41-7.38 (d, 2H, J = 7.8 Hz), 7.31 (s, 1H),

7.26-7.24 (d, 3H, J = 8.0 Hz), 7.11-7.08 (dd, 1H, J = 8.4, 1.7 Hz), 6.88-6, 86 (d, 1H, J = 8.4 Hz),

6.86-6.72 (m, 1H), 5.88-5.8 (bs, 1H), 5.78-5.73 (d, 1H, J = 15.2 Hz), 5.28-5, 22 (m, 1H), 5.2-5.08 (bs, 1H), 4.95-4.7 (m, 2H), 3.91 (s, 3H), 3.87 (s, 2H) .

3.7 (s, 1H), 3.45-3.38 (dd, 1H, J = 13.4, 8.4 Hz),

HU 227 195 Β1

3.35-3.0 (m, 5H), 2.96-2.93 (dd, 1H, J = 7.5,

1.2 Hz), 2.89-2.78 (m, 2H), 2.65-2.4 (m, 2H),

1.85-1.65 (m, 3H), 1.49 (s, 9H), 1.48-1.3 (m, 1H),

1.27 (s, 3H), 1.2 (s, 3H), 1.19-1.17 (d, 3H, 7.1H),

0.91-0.87 (t, 6H, J = 6.8 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.7, 170.4, 165.0, 156.0, 153.9, 141.5, 139.9,

135.6, 130.7, 129.7, 128.5, 128.1, 125.6, 124.7,

122.3, 112.2, 77.2, 75.7, 71.0, 63.0, 58.8, 56.0,

54.5, 52.9, 48.5, 46.3, 42.7, 40.5, 39.3, 36.8, 35.2,

28.3, 24.5, 22.83, 22.8, 22.6, 21.2, 13.5.

IR (CHCl ₃ ): 3425, 3009, 2967, 2936, 2874, 2841, 1751,

1709, 1685, 1504, 1368, 1280, 1259, 1165, 1153,

1067 cm ^-1 .

Elemental analysis based on the formula ΰ4 ₄ Η ₆₁ ΰΝ ₄ Ο ₁₀ : C, Η, N.

Example 82

To a solution of the epoxide (0.065 g, 0.076 mmol) in dichloromethane (0.9 mL) at -78 ° C was added dropwise a solution of 4 M hydrochloric acid (0.09 mL, 0.38 mmol) in 1,4-dioxane. The solution was stirred at -78 ° C for 30 minutes and then allowed to warm slowly to room temperature. After stirring at room temperature for a further 2 hours, the reaction mixture was concentrated under reduced pressure. 0.063 g of chlorohydrin of the given formula are obtained in quantitative yield.

[a] ² ° D = + 16.6 ° (c = 1.0, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

8.54-8.52 (d, 1H, J = 7.7Hz), 7.84-7.81 (dd, 1H,

J = 8.8, 1.7 Hz), 7.63-7.53 (q, 4H, J = 20.0, 8.2 Hz),

7.31-7.3 (d, 1H, J = 2.0 Hz), 7.22-7.18 (dd, 1H,

J = 8.4, 2.0 Hz), 7.02-6.99 (d, 1H, J = 8.5 Hz),

6.8-6.7 (m, 1H), 6.0-5.92 (d, 1H, J = 15.0 Hz),

5.2-5.0 (m, 2H), 4.9 ^ 4.8 (m, 1H), 4.6 ^ 4.4 (m, 1H),

4.3 (s, 2H), 4.07-4.03 (dd, 1H, J = 9.5, 1.4 Hz), 3.86 (s, 3H), 3.6-3.1 (m , 7H), 2.82-2.7 (m, 2H), 2.6-2.3 (m, 2H), 1.9-1.6 (m, 3H), 1.25 (s, 3H) ), 1.2 (s, 3H),

1.05-0.99 (m, 9H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.8, 173.8, 171.9, 168.3, 155.3, 144.2, 143.1,

132.2, 131.5, 131.4, 130.3, 129.4, 125.2, 123.2,

113.5, 77.2, 74.7, 72.6, 63.2, 57.6, 56.7, 52,4.47.5,

45.5, 44.1.41.1, 40.3, 37.8, 36.9, 36.5, 26.3, 23.7,

23.5.22.2, 9.0.

IR (KBr): 3412, 2961, 2933, 1749, 1721, 1663, 1504,

1462,1442,1259, 1199, 1152, 1126, 1065 crrr ¹ . FAB HRMS (M-CHCl ₂ ) calculated for C ₃₉ H ₅₅ Cl ₂ N ₄ O ₈ :

calcd: 777.3397, found: 777.3407.

Example 83

According to the procedure for the preparation of styrene (20), 1.0 g (1.73 mmol) of aldehyde (18) and 1.23 g (2.08 mmol) of 4- (ethyl-2-tert-butyldimethylsiloxy) - From benzyl triphenylphosphonium bromide, 1.2 g (86%) of a mixture of styrene E / Z isomers were obtained. The isomeric mixture was dissolved in 50 mL of toluene and 0.040 g (0.16 mmol) of azo-bis (cyclohexane carbonitrile) (VAZO) and

Reflux under reflux for 3 hours in the presence of 0.061 mL (0.59 mmol) of thiophenol. After evaporation, the resulting residue was purified by radial PLC, eluting with 20-75% ethyl acetate / hexane. 0.813 g (68%) of the E-isomer was obtained as a white foam. [.alpha.] D @ 20 = + 35.6 DEG (c = 0.56, MeOH).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.12 (m, 6H), 7.07-7.04 (d, 1H, J = 8.5 Hz),

6.85-6.82 (d, 1H, J = 8.4Hz), 6.83-6.70 (m, 1H),

6.40-6.35 (d, 1H, J = 15.8 Hz), 6.0-5.92 (dd, 1H,

J = 15.4, 8.7 Hz), 5.77-5.72 (d, 1H, J = 15.2 Hz),

5.46-5.43 (d, 1H, J = 7.7Hz), 5.07-5.02 (m, 1H),

4.86-4.83 (m, 1H), 4.82-4.74 (m, 1H), 3.88 (s, 3H),

3.78-3.74 (t, 2H, J = 7.1Hz), 3.44-3.37 (dd, 1H,

J = 12.5, 8.6 Hz), 3.15-3.08 (m, 3H), 2.81-2.77 (t,

2H, J = 7.1 Hz), 2.57-2.52 (m, 2H), 2.43-2.35 (m,

1H), 1.74-1.56 (m, 2H), 1.38-1.23 (m, 1H), 1.22 (s,

3H), 1.16 (s, 3H), 1.13-1.11 (d, 3H, J = 6.8 Hz), 0.88 (s, 9H), 0.84-0.72 (m , 6H), 0.0 (s, 6H).

¹³ C NMR (75 MHz, CDCl ₃ ), δ:

177.9, 170.5, 170.3, 165.1, 154.0, 142.2, 138.6,

134.6, 131.5, 130.9, 129.6, 129.4, 128.2, 126.0,

125.3, 124.5, 122.5, 112.3, 92.9, 77.0, 71.4, 64.4,

56.1, 54.3, 46.5, 42.7, 42.2, 39.4, 39.2, 36.5, 35.3,

25.9, 24.5, 22.8, 22.7, 22.6, 21.2, 17.2, -5.44.

IR (CHCl ₃ ): 3423, 2959, 2931, 2858, 1747, 1712, 1681,

1605, 1527, 1503, 1485, 1442, 1370, 1339, 1303,

1281, 1258, 1194, 1151, 1095, 1067, 1025, 1007,

838 cm ¹ .

Example 84

To a solution of the free alcohol of styrene (0.135 g, 0.189 mmol) in 0 mL of dichloromethane (4.5 mL) was added pyridine (0.06 mL, 0.76 mmol) followed by Dess-Martin (0.161 g, 0.379 mmol). reagent will. The reaction mixture was stirred at 0 ° C for 30 minutes and then at room temperature for 20 minutes, filtered through celite, washed with ethyl acetate and evaporated under reduced pressure. The crude product was purified by radial PLC on silica gel eluting with 80-100% ethyl acetate / dichloromethane. The desired aldehyde of the formula (0.08 g, 59%) was obtained as a white solid. ¹ H-NMR (300 MHz, CDCl ₃ ), δ:

9.77-9.76 (t; 1H, J = 2.0 Hz), 7.3-7.2 (m, 6H),

7.06-7.02 (dd, 1H, J = 8.3, 2.0 Hz), 6.85-6.82 (d,

1H, J = 8.4 Hz, 6.81-6.7 (m, 1H), 5.74-5.68 (d, 1H,

J = 15.3 Hz), 5.51-5.48 (d, 1H, J = 7.8 Hz), 5.22-5.17 (m, 1H), 4.85-4.81 (dd) , 1H, J = 10.3, 3.6 Hz),

4.77 ^ 4.71 (m, 1H), 3.87 (s, 3H), 3.72-3.71 (d, 2H,

J = 2.0 Hz), 3.69-3.68 (d, 1H, J = 1.5 Hz), 3.45-3.38 (dd, 1H, J = 13.5, 8.7 Hz) ), 3.17-3.0 (m, 3H),

2.92-2.89 (dd, 1H, J = 7.6, 1.8 Hz), 2.6-2.4 (m, 2H),

1.8-1.6 (m, 3H), 1.4-1.3 (m, 1H), 1.22 (s, 3H), 1.16 (s, 3H), 1.16-1, 13 (d, 3H, J = 8.3 Hz), 0.86-0.82 (t,

6H, J = 6.3 Hz).

Example 85

To the aldehyde of the above formula (0.08 g, 0.112 mmol) was added tetrahydrofuran (3.2 mL) and

HU 227 195 Β1

3.2 ml of water. The reaction mixture was cooled to 0 ° C and 3.2 ml of 2-methyl-2-butene, 0.081 g (0.896 mmol) of sodium perchlorate and 0.130 g (1.0 mmol) of sodium dihydrogen phosphate water were added sequentially. . The reaction mixture was allowed to warm to room temperature and stirred vigorously for 5 hours. The solution was diluted with dichloromethane (10 mL) and the layers were separated. The aqueous phase was extracted with dichloromethane (3 x 10 mL), and the combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified twice by radial PLC on silica gel eluting with 5-10-25% methanol / dichloromethane. Obtained as a white solid (0.03 g, 37%).

[α] D = + 24.5 ° (c = 0.33, MeOH).

¹ H-NMR (300 MHz, CD ₃ OD), δ:

7.57-7.75 (dd, 1H, J = 10.3, 1.9 Hz), 7.31-7.2 (m,

5H), 7.16-7.13 (dd, 1H, J = 8.4, 1.9 Hz), 6.97-6.95 (d, 1H, J = 8.4 Hz), 6.8 -6.6 (m, 1H), 5.87-5.81 (d,

1H, J = 15.3 Hz), 5.19-5.14 (dd, 1H, J = 11.0, 5.0 Hz),

4.9-4.8 (m, 2H), 4.48-4.43 (dd, 1H, J = 11.5,

3.5 Hz), 3.8 (s, 3H), 3.77 (s, 1H), 3.53 (s, 2H),

3.5-3.4 (m, 1H), 3.17-3.11 (dd, 1H, J = 14.3,

3.5 Hz), 3.05-3.0 (d, 1H, J = 13.6 Hz), 2.95-2.92 (dd, 1H, J = 7.7, 1.7 Hz), 2.8-2.6 (m, 2H), 2.5-2.3 (m, 1H), 1.8-1.6 (m, 3H), 1.4-1.2 (m, 1H) ), 1.17 (s,

3H), 1.13 (s, 3H), 1.13-1.1 (d, 3H, J = 9.2 Hz),

0.83-0.81 (d, 6H, J = 6.3 Hz).

¹³ C NMR (126 MHz, CDCl ₃ ), δ:

178.8, 173.7, 172.0, 168.2, 155.4, 143.4, 138.1,

136.8, 132.2, 131.2, 130.8, 129.3, 126.8, 125.4,

123.3, 113.5, 77.7, 72.4, 64.4, 60.0, 57.5, 56.6,

47.4, 44.1,41.7, 40.7, 38.6, 36.5, 25.9, 23.4, 23.3,

21.6, 14.0.

IR (KBr): 3417, 2961, 2934, 2874, 1750, 1721, 1674,

1561, 1504, 1464, 1441, 1300, 1259, 1194, 1151,

1066 cm ^-1 .

FAB HRMS (M + H) calcd for C ₃₈ H ₄₈ CIN ₂ O ₁₀ 727.2997, found 727.3005.

Example 86

To a mixture of [(2-methyl-4-thiazolyl) methyl] triphenylphosphonium chloride (0.496 g, 1.2 mmol) in tetrahydrofuran (10 mL) was added dropwise 1.6 mL (1.6 mmol) at -78 ° C. M N-butyllithium solution. The reaction mixture was allowed to slowly warm to room temperature and stirred for 45 minutes. To a solution of aldehyde (18) (0.5 g, 0.865 mmol) in tetrahydrofuran (15 mL) was added dropwise a solution of orange ylide at -78 ° C through a double-ended needle. The resulting reaction mixture was stirred at -78 ° C for 2 hours and then at room temperature for 1.5 hours. A saturated aqueous solution of ammonium chloride (30 mL) and ethyl acetate (30 mL) were added to the reaction mixture, the layers were separated and the aqueous layer was extracted with ethyl acetate (2 x 20 mL). The combined organic extracts were washed with water (2 x 20 mL), brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure. The resulting yellow evaporation residue was purified by silica gel column chromatography using 50-70-80% ethyl acetate / hexane as eluent. 0.4 g of the desired styrene is obtained with triphenylphosphinoxide. Triphenylphosphine oxide is easily removed by reverse phase HPLC using 50:50 acetonitrile / water as eluent. The pure thiazole (0.2 g, 34%) was obtained as a white solid.

[a] ² ° D = + 16.7 ° (c = 1.0, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.3-7.2 (m, 1H), 7.18-7.17 (d, 1H, J = 1.7 Hz),

7.06-7.03 (dd, 1H, J = 8.5, 1.8 Hz), 6.83 (s, 1H),

6.83-6.8 (d, 1H, J = 9.0 Hz), 6.8-6.67 (m, 1H),

6.37-6.35 (m, 2H), 5.85-5.82 (d, 1H, J = 7.9 Hz),

5.76-5.71 (d, 1H, J = 15.1 Hz), 5.05-5.0 (dd, 1H,

J = 9.0, 6.0 Hz), 4.86-4.82 (dd, 1H, J = 10.2, 3.6 Hz),

4.77-4.68 (m, 1H), 3.85 (s, 3H), 3.44-3.37 (dd, 1H,

J = 13.4, 8.6 Hz), 3.2-3.0 (m, 3H), 2.68 (s, 3H),

2.6-2.3 (m, 3H), 1.8-1.6 (m, 2H), 1.43-1.3 (m, 1H),

1.2 (s, 3H), 1.14 (s, 3H), 1.12-1.1 (d, 3H, J = 6.9 Hz),

0.79-0.78 (d, 3H, J = 3.1 Hz), 0.77-0.76 (d, 3H,

J = 3.1 Hz).

¹ H-NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 166.0, 165.2, 153.9, 153.0, 142.1,

136.7, 132.7, 130.8, 129.8, 128.1, 124.6, 124.4,

122.3, 114.1, 112.2, 76.9, 71.4, 56.0, 54.4, 46.4,

42.7, 41.9, 39.3, 36.5, 35.3, 24.5, 22.8, 22.7, 22.6,

21.2, 19.2, 17.1.

IR (CHCl ₃ ): 3423, 3027, 3008, 2965, 2935, 2874, 1747,

1712, 1681, 1652, 1604, 1528, 1504, 1485, 1259,

1181, 1152, 1067 cm ^-1 .

Elemental analysis for C ₃₄ H ₄₄ CIN ₃ O ₇ S: C, Η, N.

Example 87

To the styrene of the above formula (0.25 g, 0.37 mmol) was added 15 ml of acetone, 6 ml of water, 6 ml of dichloromethane and 1.0 g (11.9 mmol) of solid sodium bicarbonate. The reaction mixture was cooled to 0 ° C. Oxone (0.92 g, 1.5 mmol) was dissolved in water (8 mL) and 2 mL of the solution was added to the cold styrene reaction mixture. After vigorous stirring for 30 minutes at 0 ° C, another 2 ml of Oxone solution, then again 2.0 ml of Oxone solution and after another 30 minutes the whole Oxone solution is added to the reaction mixture. The progress of the reaction was monitored by reverse phase HPLC. The reaction was complete after stirring for 2.0 h. While still maintaining the reaction mixture at 0 ° C, 40 ml of a saturated aqueous solution of sodium bicarbonate and 40 ml of dichloromethane are added. The phases are separated and the organic phase is washed with 40 ml of 10% sodium sulfate solution, 40 ml of saturated sodium bicarbonate solution and brine, dried over sodium sulphate, filtered and concentrated under reduced pressure. The mixture of β and epoxides (54:46) was separated by reverse phase HPLC using 50:50 acetonitrile / water as eluent. 0.09 g (35%) of β-epoxide of the formula is obtained as a white solid.

HU 227 195 Β1 [α] D = + 26.0 ° (c = 1.0, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.19-7.18 (d, 2H, J = 1.8 Hz), 7.1 (s, 1H), 7.06-7.03 (dd, 1H, J = 8.5, 1.9 Hz), 6.85-6.82 (d, 1H,

J = 8.4 Hz), 6.82-6.7 (m, 1H), 5.76-5.71 (d, 1H,

J = 15.2 Hz), 5.49-5.47 (d, 1H, J = 7.8 Hz), 5.23-5.18 (m, 1H), 4.88-4.84 (dd) , 1H, J = 10.3, 3.6 Hz),

4.8-4.7 (m, 1H, 3.88 (s, 3H), 3.79 (d, 1H,

J = 0.93 Hz), 3.45-3.38 (dd, 1H, J = 13.4, 8.6 Hz),

3.35-3.32 (d, 1H, J = 7.2Hz), 3.2-3.0 (m, 3H), 2.7 (s, 3H), 2.6-2.4 ( m, 2H), 1.8-1.6 (m, 3H), 1.4-1.3 (m, 1H), 1.23 (s, 3H), 1.16 (s, 3H), 1 , 14-1.12 (d,

3H, J = 6.8Hz), 0.89-0.87 (d, 3H, J = 6.5Hz),

0.86-0.84 (d, 3H, J = 6.4 Hz).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.33, 170.3, 166.9, 165.0, 154.0, 151.9,

141.8, 130.8, 129.5, 128.2, 124.5, 122.4, 116.4,

112.3, 75.8, 71.1,61.3, 56.1.55.2, 54.3,46,4,42,7,

40.3, 39.3, 36.6, 35.2, 24.5, 22.85, 22.8, 22.6, 21.2,

19.1.13.3,

IR (CHCl 3): 3425, 3007, 2964, 2936, 2874, 2841, 1751,

1711, 1682, 1604, 1528, 1503, 1485, 1464, 1303,

1259, 1185, 1152, 1067 cm ^-1 .

Elemental analysis for C _{3 4} H 44 ClN ₃ O ₈ S: C, Η, N.

Example 88

By the same procedure as described above for the preparation of styrene (20), 0.5 g of aldehyde (18) (1.3 g, 2.2 mmol) and 2-fluorobenzyl triphenylphosphonium bromide (1.7 g, 3.8 mmol) were used. g (33%) of styrene of the formula were obtained. [a] ² ° D = + 17.0 ° (c = 1.16, CHCl _3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.40-7.34 (m, 1H), 7.23-6.96 (m, 6H), 6.83-6.80 (d, 1H, J = 8.4 Hz), 6.78-. 6.70 (m, 1H), 6.57-6.52 (d,

1H, J = 16.0 Hz), 6.10-6.02 (dd, 1H, J = 8.8.16.0 Hz),

5.76-5.71 (d, 1H, J = 15.3, Hz), 5.50-5.47 (d, 1H,

J = 7.8 Hz), 5.05-4.95 (m, 1H), 4.85-4.80 (dd, 1H,

J = 9.6, 3.1 Hz), 4.75 - 4.69 (m, 1H), 3.85 (s, 3H),

3.42-3.34 (dd, 1H, J = 13.5, 8.7 Hz), 3.13-3.05 (m,

3H), 2.56-2.51 (m, 2H), 2.37-2.33 (m, 1H),

1.68-1.58 (m, 2H), 1.34-1.23 (m, 1H), 1.20 (s, 3H),

1.13 (s, 3H), 1.13-1.11 (d, 3H, J = 7.3 Hz),

0.79-0.70 (m, 6H).

¹³ C NMR (63 MHz, CDCl 3), δ:

177.9, 170.5, 170.4, 165.1, 154.0, 142.0, 137.4,

133.0, 132.9, 130.8, 129.6, 128.8, 128.2, 127.0,

126.9, 124.6, 124.1, 115.9, 115.5, 112.3, 76.9,

71.4, 56.1, 54.4, 46.4, 42.7, 42.6, 39.4, 36.5, 35.3,

24,5,22,8,22,7, 22,6,21,1, 17.2.

IR (CHCl 3): 3423, 2965, 2935, 2874, 1747, 1711, 1681,

1605, 1527, 1503, 1487, 1457, 1441, 1370, 1340,

1321, 1280, 1259, 1151, 1093, 1067, 1009,

970 cm ^-1 .

Example 89

To a solution of styrene (0.26 g, 0.387 mmol) in 0 mL of dichloromethane (1.3 mL) was added 3-chloroperoxybenzoic acid (0.07 g, 0.41 mmol) and toluene (0.65 mL). Stirring was continued at 0 ° C for 30 minutes. After removing the ice bath, the reaction mixture was stirred at room temperature for 24 hours. After evaporation, the residue was purified by reverse phase HPLC using acetonitrile / water as eluent. 0.058 g (24%, corrected for recovered styrene) of the formula epoxide β is obtained in the form of a white foam.

[a] ² ° D = + 19.98 ° (c = 1.41, CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.30-7.00 (m, 6H), 6.86-6.83 (d, 1H, J = 8.5 Hz),

6.80-6.75 (m, 1H), 5.76-5.71 (d, 1H, J = 15.1 Hz),

5.47-5.45 (d, 1H, J = 7.8Hz), 5.23-5.20 (m, 1H),

4.86-4.82 (dd, 1H, J = 10.3, 3.5Hz), 4.78-4.70 (m,

1H), 4.01 (s, 1H), 3.88 (s, 3H), 3.45-3.38 (dd, 1H,

J = 13.4, 8.6 Hz), 3.12-3.08 (m, 3H), 2.91-2.88 (d,

1H, J = 7.8 Hz), 2.59-2.53 (m, 2H), 1.80-1.71 (m,

3H), 1.46-1.25 (m, 1H), 1.23 (s, 3H), 1.16 (s, 3H),

1.16-1.14 (d, 3H, J = 7.2 Hz), 0.87-0.83 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

178.1, 170.5, 170.3, 165.0, 142.0, 131.0, 129.7,

129.6, 128.3, 126.1, 126.08, 124.65, 124.5, 122.6,

115.5, 115.2, 112.4, 76.0, 71.2, 62.6, 56.2, 54.4,

53.5, 46.5, 42.8, 40.6, 39.4, 36.9, 35.3, 24.7, 22.9,

22.8.21.3, 13.8.

IR (CHCl 3): 3417, 2962, 2948, 1754, 1721, 1681, 1653,

1534, 1504, 1473, 1459, 1441, 1303, 1282, 1258,

1191, 1148, 1127, 1066 c- ¹ .

FAB HRMS (M + H) calcd for C ₃₆ H _{45 5} CIFN ₂ O ₈ : 687.2848, found: 687.2857.

Example 90

In the preparation of styrene (20) using the procedure described above, 0.85 g (2.0 g, 0.345 mmol) of aldehyde (18) and 1.92 g (4.25 mmol) of 3-fluorobenzyl triphenylphosphonium bromide were obtained. %) of the styrene E / Z isomeric mixture of the formula given. The isomeric mixture was dissolved in 25 mL of benzene and refluxed in the presence of 0.04 g (0.16 mmol) of 1,1'-azobis (cyclohexane carbonitrile) (VAZO) and 0.06 mL (0.58 mmol) of thiophenol. over. After evaporation, the residue was purified by radial PLC using 20-100% ethyl acetate / hexane as eluent. 0.652 g (77%) of the E-isomer was obtained as a white foam.

[a] ² ° D = + 30.55 ° (c = 0.98, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.30-7.01 (m, 8H), 6.95-6.88 (m, 1H), 6.86-6.83 (d, 1H, J = 8.5 Hz), 6.81-6, 72 (m, 1H), 6.40-6.35 (d,

1H, J = 15.9 Hz), 6.07 to 5.99 (dd, 1H, J = 8.8, 15.9 Hz),

5.78-5.73 (d, 1H, J = 15.1 Hz), 5.50-5.47 (d, 1H,

J = 7.9 Hz), 5.08-5.02 (dd, 1H, J = 9.7, 6.7 Hz),

4.87-4.82 (dd, 1H, J = 9.7, 3.0 Hz), 4.78-4.71 (m,

1H), 3.88 (s, 3H), 3.45-3.37 (dd, 1H, J = 13.5, 8,

Hz), 3.15-3.08 (m, 3H), 2.59-2.52 (m, 2H),

2.43-2.34 (m, 1H), 1.72-1.63 (m, 2H), 1.36-1.26 (m, 1H), 1.22 (s, 3H), 1, 16 (s, 3H), 1.15-1.12 (d,

3H, J = 6.9Hz), 0.80-0.73 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.4, 165.1, 165.0, 153.9, 141.9,

137.3, 131.7, 130.8, 130.6, 130.0, 129.6, 128.1,

124.6, 122.4, 114.4, 114.1, 112.6, 112.2, 76.8,

HU 227 195 Β1

71.3, 56.0, 54.4, 46.4, 42.7, 42.1, 39.5, 36.5, 35.2,

24,5,22,8,22,64,22,6,21,2,17.2.

IR (CHCl ₃ ): 3423, 3008, 2965, 2936, 2874, 1747, 1712,

1680, 1652, 1585, 1528, 1503, 1486, 1464, 1442,

1320, 1303, 1259, 1193, 1147, 1127, 1067,

973 cm ^-1 .

Analysis for C ₃₆ H44CIFN O2 ₂ O: C, Η N.

Example 91

To a solution of styrene (0.622 g, 0.927 mmol) in 0 mL of dichloromethane (3.0 mL) was added 3-chloroperoxybenzoic acid (0.170 g, 0.985 mmol) and toluene (1.5 mL). Stirring was continued at 0 ° C for 30 minutes. After removing the ice-cooling bath, the reaction mixture was stirred at room temperature for 22 hours. After evaporation, the resulting residue was purified by reverse phase HPLC using acetonitrile / water as eluent. 0.067 g (11%) of β-epoxide is obtained. [α] 25 D = 26.23 ° (c = 1.54, CHCl ₃ ).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.37-6.92 (m, 7H), 6.86-6.83 (d, 1H, J = 8.4 Hz),

6.82-6.73 (m, 1H), 5.75-5.70 (d, 1H, J = 15.5 Hz),

5.48-5.45 (d, 1H, J = 7.8Hz), 5.22-5.17 (m, 1H),

4.85-4.81 (dd, 1H, J = 3.1, 9.8 Hz), 4.76 ^, 70 (m,

1H), 3.88 (s, 3H), 3.68-3.67 (d, 1H, J = 0.89 Hz),

3.46-3.38 (dd, 1H, J = 8.6, 13.5 Hz), 3.13-3.07 (m,

3H), 2.90-2.87 (dd, 1H, J = 1.5, 7.4Hz), 2.60-2.37 (m, 2H), 1.82-1.64 (m, 3H), 1.36-1.25 (m, 1H),

1.22 (s, 3H), 1.16 (s, 3H), 1.15-1.12 (d, 3H,

J = 6.9 Hz), 0.88-0.83 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.9, 170.4, 165.0, 154.0, 141.6, 139.5, 130.8,

130.4, 130.2, 129.6, 128.2, 124.7, 122.4, 121.3,

115.6, 115.3, 112.3, 112.2, 75.8, 71.1, 63.2, 58.2,

56.1, 54.5, 46.4, 42.8, 40.4, 39.4, 36.8, 35.3, 24.5,

22.85, 22.82, 22.7, 21.2, 13.4.

IR (KBr): 3416, 3034, 2963, 2934, 2874, 1751, 1721,

1680, 1658, 1539, 1504, 1473, 1442, 1304, 1280,

1258, 1192, 1144, 1066 cnr ¹ .

Elemental analysis for C ₃₆ H ₄₄ CIFN ₂ O ₈ : C, Η, N.

Example 92

According to the procedure described above for the preparation of styrene (20), 1.5 g (2.6 mmol) of aldehyde (18) and

From 4-fluorobenzyl triphenylphosphonium bromide (1.4 g, 3.1 mmol), 1.24 g (71%) of the styrene E / Z isomeric mixture is obtained. The isomer mixture was dissolved in 40 mL of benzene and refluxed for 24 hours in the presence of 1,1'-azobis (cyclohexane carbonitrile) (VAZO) (0.050 g, 0.20 mmol) and thiophenol (0.076 mL, 0.74 mmol). After evaporation, the resulting residue was purified by radial PLC using 20-100% ethyl acetate / hexane as eluent. 1.06 g of the E-isomer are obtained in the form of a white foam which, according to NMR analysis, contains triphenylphosphine oxide. Purification by reverse phase HPLC (0.150 g) using acetonitrile / water gave 0.092 g of pure product as a solid.

[a] ² ° D = + 27.49 ° (c = 1.05, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.31-6.96 (m, 7H), 6.85-6.83 (d, 1H, J = 8.5 Hz),

6.81-6.74 (m, 1H), 6.39-6.34 (d, 1H, J = 15.8 Hz),

5.96-5.88 (dd, 1H, J = 8.8.15.8 Hz), 5.77-5.72 (d, 1H,

J = 15.2 Hz), 5.49-5.47 (d, 1H, J = 7.7Hz), 5.07-5.02 (m, 1H), 4.86-4.83 (dd) , 1H, J = 9.2, 2.5 Hz),

4.82-4.73 (m, 1H), 3.87 (s, 3H), 3.45-3.38 (dd, 1H,

J = 13.4, 8.5 Hz), 3.14-3.08 (m, 3H), 2.57-2.52 (m,

2H), 2.43-2.34 (m, 1H), 1.71-1.58 (m, 2H),

1.36-1.29 (m, 1H), 1.22 (s, 3H), 1.16 (s, 3H),

1.14-1.11 (d, 3H, J = 6.9 Hz), 0.78-0.73 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.5, 170.3, 165.1, 160.2, 154.0, 142.0,

137.6, 132.9, 132.8, 130.8, 130.4, 130.0, 129.6,

128.2, 127.6, 127.5, 124.6, 122.4, 115.6, 115.2,

112.2, 71.3, 56.0, 54.4, 46.4, 42.7, 42.1, 39.5, 36.4,

35.2, 24.5, 22.8, 22.6, 21.2, 17.2.

IR (KBr): 3421, 3289, 2862, 2933, 1751, 1722, 1678,

1604, 1534, 1509, 1259, 1228, 1149, 1066, 1024,

1011, 971, 815 cm ^-1 .

Elemental analysis for C ₃₆ H ₄₄ CIFN ₂ O ₇ : C, Η, N.

Example 93

To a solution of styrene (0.906 g, 1.35 mmol) in dichloromethane (4.5 mL) at 0 ° C was added 3-chloroperoxybenzoic acid (0.25 g, 1.45 mmol) and toluene (2.2 mL). Stirring was continued at 0 ° C for 30 minutes. After removing the ice-cooling bath, the reaction mixture was stirred at room temperature for 23 hours. After dilution with dichloromethane (20 mL), the reaction mixture was washed with 10 mL of 10% sodium pyrosulfite solution, 10 mL of water, 10 mL of saturated sodium bicarbonate, and finally 10 mL of brine, dried over sodium sulfate, filtered, and concentrated. evaporate under pressure. 0.814 g of product is obtained which is a mixture of β and epoxides. 0.23 g of product was purified by reverse phase HPLC to give 0.073 g of β-epoxide of the formula as a white foam. [ «] ?? = + 25.6 ° (c = 0.626, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.26-7.03 (m, 7H), 6.85-6.83 (d, 1H, J = 8.4 Hz),

6.82-6.72 (m, 1H), 5.74-5.69 (d, 1H, J = 15.2 Hz),

5.44-5.42 (d, 1H, J = 7.9Hz), 5.23-5.18 (m, 1H),

4.85-4.81 (dd, 1H, J = 9.7, 2.9 Hz), 4.77-4.73 (m,

1H), 3.88 (s, 3H), 3.66 (s, 1H), 3.46-3.39 (dd, 1H,

J = 13.5, 8.8 Hz), 3.12-3.07 (m, 3H), 2.89-2.87 (dd,

1H, J = 1.5, 7.7 Hz), 2.60-2.54 (m, 1H), 2.49-2.41 (m, 1H), 1.81-1.65 (m, 3H), 1.34-1.25 (m, 1H),

1.22 (s, 3H), 1.16 (s, 3H), 1.15-1.13 (d, 3H,

J = 7.0 Hz), 0.87-0.82 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.8, 170.3, 164.9, 164.7, 154.0, 141.6, 137.8,

132.4, 130.7, 129.6, 128.1, 127.3, 127.2, 124.6,

122.4, 115.8, 115.5, 112.2, 75.8, 71.0, 63.0, 58.2,

56.0, 54.4, 46.3, 42.7, 40.4, 39.3, 36.7, 35.2, 23.5,

22.8, 22.76, 22.6, 21.1, 13.3.

IR (CHCl ₃ ): 3426, 3030, 3006, 2964, 2936, 1752, 1711,

1683, 1608, 1514, 1485, 1442, 1303, 1281, 1259,

1188, 1155, 1067, 838 cm ^-1 .

Elemental analysis for C ₃₆ H ₄₄ CIFN ₂ O ₈ : C, Η, N.

HU 227 195 Β1

Example 94

To a solution of the epoxide β (0.44 g, 0.64 mmol) in dichloromethane (30 mL) at -70 ° C was added dropwise a solution of 4 M hydrochloric acid (0.4 mL, 1.6 mmol) in dioxane over 5 minutes. After stirring for a further 2 hours at -70 ° C, the solution was concentrated under reduced pressure. The crude product was purified by radial PLC on silica gel using 30-50-100% ethyl acetate / dichloromethane. Purification was accomplished by reverse phase HPLC using 50:50 acetonitrile / water as eluent. 0.152 g (33%) of the desired chlorohydrin is obtained as a white foam. [a] ² ° D = + 60.0 ° (c = 2.62, _CHCl3).

¹ H-NMR (300 MHz, CDCl ₃ ), δ:

7.41-7.05 (m, 7H), 6.87-6.84 (d, 1H, J = 8.4 Hz),

6.83-6.77 (m, 1H), 5.80-5.75 (d, 1H, J = 15.4 Hz),

5.52-5.49 (d, 1H, J = 7.8Hz), 5.13-5.21 (m, 1H),

4.94-4.90 (dd, 1H, J = 9.7, 3.2 Hz), 4.75-4.72 (m,

1H), 4.67 ^ 4.63 (d, 1H, J = 9.5 Hz), 4.00-3.95 (m,

1H), 3.89 (s, 3H), 3.42-3.35 (dd, 1H, J = 8.3,

13.5 Hz), 3.20-3.02 (m, 3H), 2.71-2.65 (m, 1H),

2.49-2.37 (m, 2H), 1.82-1.63 (m, 2H), 1.51-1.38 (m, 2H), 1.23 (s, 3H), 1, 17 (s, 3H), 1.04-1.02 (d,

3H, J = 7.0Hz), 0.97-0.85 (m, 6H).

¹³ C NMR (63 MHz, CDCl ₃ ), δ:

177.6, 170.5, 170.3, 165.3, 160.7, 153.9, 142.2,

137.5, 134.5, 130.8, 129.8, 129.7, 128.2, 124.6,

122.2, 76.1.74.0, 71.1.61.4, 56.1.54.5, 46.4, 42.7,

39.6, 38.4, 36.3, 35.1, 24.8, 23.0, 22.9, 22.7, 21.5,

8.6.

IR (CHCl ₃ ): 3423, 2965, 2935, 2873, 1751, 1715, 1679, 1607, 1528, 1504, 1485, 1464, 1442, 1302, 1281, 1193, 1159, 1152, 1127, 1067 cm ^-1 .

Elemental analysis for C ₃₆ H _{4 5} Cl ₂ N ₂ O ₈ : C, Η, N.

Example 95

Cryptophycin-151, -152, -153, -154, -155, -156,

-159, -160, -161, -166, -167, -172, -181, -188, -234, 20 -236, -238, -247, -251 and -255. (Tables 5 and 6).

Table 5

cryptophycin Ar or alkyl Yield (E + 2),% The% of the trans isomer is approximately 131 72 90 132 80 70 133 60 <10 13 < 31 70 160 ν ' 61 67 172 78 90 233 83 60

HU 227 195 Β1

Table 6

Cryptophycin Ar group Yield (E + 2)%% of E-isomer

153 '• V 80 90 154 «.ρΛ 63 90 159 75 90 WHO 61 80 Here 38 90 197 "ΖΓ ' «*. 48 90 111 of 73 75 18B -ρΛ 85 80 234 —MCT ¹ 81 85 239 59 90 247 51 > 90 251 43 > 90

The compounds were prepared by the reaction shown in Figure 15.

The following describes the procedure adapted for the coupling of Cryptophycin-108 and various triphenylphosphoranes, illustrated by an example of the preparation of Cryptophycin-152.

Preparation of Cryptophycin-152

To prepare the cinnamyl triphenylphosphorane in 5.7 mL of tetrahydrofuran at -78 ° C, 0.311 g (0.750 mmol) of cinnamyl triphenylphosphonium was reacted with 300 mL of 2.5 M butyl lithium hexane (0.750 mmol). The reaction mixture was allowed to slowly warm to room temperature. 1.46 mL (0.182 mmol) of the cinnamyl triphenylphosphine reaction mixture was slowly added to a solution of 68.6 mg (0.122 mmol) aldehyde in 3 mL tetrahydrofuran at -78 ° C and the reaction mixture was stirred for 2 hours. The reaction mixture was warmed to room temperature, washed with a saturated solution of ammonium chloride (5 mL), water (15 mL) and extracted with ethyl acetate (40 mL). The organic layer was washed with water, dried over magnesium sulfate and evaporated. The residue was chromatographed on a small ODS column using water / acetonitrile 1: 1 and 1: 3 as eluent. The latter fraction was evaporated to give a mixture of Cryptophycin-152 and its Z isomer (64.3 mg, 83%) (E / Z, 7: 3).

HU 227 195 Β1

Using the same procedure, Cryptophycin-108 is reacted with the following reagents:

naphthylmethyl triphenylphosphorane, 3-methoxybenzyl triphenylphosphorane, 3,5-dimethoxybenzyl triphenylphosphorane, methoxymethyl triphenylphosphorane, 2,4-dimethylbenzyl triphenylphosphorane, 3 -furan-methyl-triphenyl-phosphorane, p-trifluoromethyl-benzyl-triphenyl-phosphorane, 2-methyl-benzyl-triphenyl-phosphorane, 3,5-dimethyl-benzyl-triphenyl-phosphorane, 2-naphthylmethyl-triphenyl-phosphorane, 2 -fluorobenzyl triphenylphosphorane, 3,5-difluorobenzyl triphenylphosphorane, 4-hydroxymethylbenzyl triphenylphosphorane, 4- (tert-BOC-aminoethyl) benzyltriphenylphosphorane, 3- ( N-tert-BOC-amino) benzyl triphenylphosphorane and phenylthiomethyl triphenylphosphonium to give Cryptophycin-151, -153, -154, -156, -159, -160, -161, -166, - 167, -172, -181, -188, -234, -236, -247 and -255 respectively.

For the preparation of the ylides from the corresponding triphenylphosphonium chloride or bromide salts, butyl lithium is used as the base except for the preparation of methoxymethyl triphenylphosphorane, 3-furanomethyl triphenylphosphorane and 4-hydroxymethylbenzyl triphenylphosphorane, where the base used is phenyl lithium.

In a similar reaction, hydroxymethyltriphenylphosphonium bromide, n-butyllithium and Cryptophycin-108 are reacted to give an unexpected analog of Cryptophycin-155.

A slightly modified procedure was used to prepare the Cryptophycin-251 carboxymethyl analog. A solution of 4-carboxymethylbenzyl-triphenylphosphonium bromide (0.289 g, 0.59 mmol) in tetrahydrofuran (5 mL) was treated with 1.8 M phenyl lithium (653 µL, 1.18 mmol) for 5 min. C, and the reaction mixture is transferred to a vessel in an ice-water cooled bath. After 30 minutes, 0.9 ml of the reaction mixture was slowly added to a solution of 35 mg of Cryptophycin-108 in 3 ml of tetrahydrofuran at -78 ° C, and the reaction mixture was stirred for 2 hours. The reaction mixture was acidified with 1 ml 1 N hydrochloric acid, water (30 ml) was added and the mixture was extracted with ethyl acetate (2 x 30 ml). The organic layer was dried over magnesium sulfate and evaporated. The resulting residue was subjected to flash chromatography on ODS silica gel eluting with water / acetonitrile 1: 1 and 35:65. The latter fraction was evaporated and purified by reverse phase HPLC column (Econosil C18, 10 μ, 250 x 22 mm, 6 ml / min, eluent: water / acetonitrile 35:65). The resulting crude Cryptophycin-251 was further purified by reverse phase chromatography (Econosil C18, 10 μ, 250 x 10 mm, 3 ml / min, eluent: 0.5% acetic acid in water / acetonitrile 2: 3). 10 mg of Cryptophycin-251 are obtained.

Preparation of triphenylphosphonium salts

Cinnamyl triphenylphosphonium chloride, 1-naphthylmethyl triphenylphosphonium chloride, methoxymethyl triphenylphosphonium chloride and 2-methylbenzyl triphenylphosphonium bromide are commercially available. 3-Methoxybenzyl-triphenylphosphonium chloride, 3,5-dimethoxybenzyl-triphenylphosphonium chloride, 2,4-dimethylbenzyl-triphenylphosphonium chloride are prepared by preparing the appropriate triphenylphosphine. excess of chloride in toluene was refluxed for 4 hours. 4-trifluoromethyl-benzyl-triphenyl-phosphonium-bromide, 3-furon-methyl-triphenyl-phosphonium-bromide, 2-naphthylmethyl-triphenyl-phosphonium-bromide, 3,5-difluorobenzyl-triphenyl-phosphonium-bromide, 4 - (tert-butyldimethylsilyloxymethyl) benzyl triphenylphosphonium bromide, 4- (N-tert-BOC-aminomethyl) benzyl triphenylphosphonium bromide and 3- (N-tert-butyl). For the preparation of BOC-amino) benzyl triphenylphosphonium bromide, the corresponding bromide is reacted with triphenylphosphine in toluene at room temperature for 12 hours. The 4-carboxymethylbenzyl triphenylphosphonium bromide is prepared by reacting the bromide with triphenylphosphine in a 5: 1 mixture of toluene / tetrahydrofuran at room temperature for 48 hours. 3-Furon-methyl bromide, a

2-naphthylmethyl bromide and 3- (N-tert-BOC-amino) benzyl bromide from the corresponding 3-furon-methanol, 2-naphthylmethanol and 3- (N-tert-BOC-amino) - benzyl alcohol by reaction with phosphorus tribromide. The reaction was carried out in tetrahydrofuran at -78 ° C. 3-Furon-methanol and 2-naphthylmethanol are commercially available. 3- (N-tert-BOC-amino) -benzyl alcohol is commercially available

3-aminobenzyl alcohol. 4- (tert-Butyldimethylsilyloxymethyl) -benzyl bromide and 4- (N-tert-BOC-aminomethyl) -benzyl bromide are commercially available from 4-hydroxymethylbenzoic acid methyl ester, respectively. can be prepared from the aminomethylbenzoic acid using the following procedures.

Preparation of 4- (tert-butyldimethylsilyloxymethyl) benzyl bromide A solution of methyl (4-hydroxymethylbenzoate) g in triethylamine (3.36 ml) in dichloromethane (15.47 g) was prepared. with tert-butyldimethylsilyl triflate at -78 ° C. After 30 minutes, the reaction mixture was allowed to reach room temperature and stirred for 30 minutes more. Water (30 mL) and ethyl acetate (60 mL) were added to the reaction mixture, and the organic layer was washed with 0.3 M potassium bisulfate solution, water, brine, dried over magnesium sulfate and filtered. The solvent was evaporated and the residue was purified by flash column chromatography on silica gel using 10% ethyl acetate in hexane as eluent. Methyl 4- (tert-butyldimethylsilyloxymethyl) benzoate (3.3 g, 95%) was obtained.

Lithium aluminum hydride (0.21 g) was dispersed in diethyl ether (20 mL) and cooled to -78 ° C under argon, 2.05 g of methyl [4- (tert-butyldimethyl-1-yl) oxy] methyl benzoate] in diethyl ether (10 mL) was added dropwise. After 30 minutes, the excess hydride was decomposed by addition of ethyl acetate (2 mL) and a saturated solution of ammonium chloride (1.5 mL) was added. The precipitate was collected by filtration and washed with diethyl ether. The solvent was evaporated to give 1.71 g (93%) of 4- (tert-butyldimethylsilyloxymethyl) -benzyl alcohol.

1.7 g of 4- (tert-butyldimethylsilyloxymethyl) benzyl alcohol are dissolved in 15 ml of tetrahydrofuran and treated with 0.609 g of phosphorus tribromide at -78 ° C. After 30 minutes, the reaction mixture was treated with 80 ml of diethyl ether

Dilute 195 Β1 and wash with 30 ml of a saturated solution of sodium bicarbonate, water and brine. The organic solution was dried over magnesium sulfate, evaporated and the residue (2.0 g) was purified by flash chromatography on silica gel using 5% ethyl acetate in hexane as eluent. 1.05 g (49%) of 4- (tert-butyldimethylsilyloxymethyl) -benzyl bromide are obtained.

Preparation of 4- (N-tert-BOC-aminomethyl) -benzyl bromide

To a solution of di-tert-butyl dicarbonate (2.18 g, 10 mmol) in triethylamine / dimethylformamide (7.5 mL, 1: 9) was added 4- (aminomethyl) (0.75 g, 5 mmol) at room temperature. benzoic acid and the reaction mixture was heated at 40-50 ° C for 10 minutes. After the amino acid has dissolved, stirring is continued at room temperature for 1 hour. The solvent was removed under reduced pressure, and the residue was acidified with dilute hydrochloric acid (pH = 2) and immediately extracted with ethyl acetate. The organic phase is dried over magnesium sulfate. After removal of the solvent, the residue was reacted with excess diazomethane in diethyl ether for 30 minutes. The excess diazomethane was quenched by the addition of acetic acid. The solvent was removed and the residue was subjected to silica gel column chromatography (20 g, 9: 1 hexane: ethyl acetate). There was obtained 1.07 g (81%) of methyl [4- (N-tert-BOC-aminomethyl) benzoate].

To a suspension of lithium aluminum hydride (5 mg, 1.5 mmol) in diethyl ether (5 mL) at -78 ° C was added 0.8 g (3 mmol) of methyl [4- (N-tert-BOC-aminomethyl) -benzoate] - and allow the reaction mixture to warm to room temperature. After 3 hours, another 100 mg of lithium aluminum hydride was added to the reaction mixture and stirred at room temperature for 10 minutes. The excess lithium aluminum hydride was quenched by addition of ethyl acetate and 1.0 mL of a saturated solution of ammonium chloride was added to the reaction mixture. The precipitated solid is filtered off and washed with diethyl ether. The filtrate was evaporated to give 4- (N-tert-BOC-aminomethyl) -benzyl alcohol (250 mg, 35%).

To a solution of 4- (N-tert-BOC-aminomethyl) benzyl alcohol (250 mg, 1.1 mmol) in tetrahydrofuran (5 mL) was added phosphorus tribromide (30 µL, 0.32 mmol) at -78 ° C for 2 h. with. 50 mg of solid sodium bicarbonate are added to the reaction mixture, the solid is filtered off and the filtrate is evaporated to give 300 mg (95%) of 4- (tert-BOC-aminomethyl) -benzyl bromide.

The desired E-isomer is separated from the E / Z isomer mixture by crystallization in ethyl acetate / ethyl ether. In the case of cinnamyl and some other analogues with insufficient E / Z selectivity, the crude product is isomerized as described below. The illustrative procedure is described for the cinnamyl analogue, Cryptophycin-152.

isomerization

62 mg (0.10 mmol) of a mixture of the cis isomer of Cryptophycin-152 (E / Z 7: 3) was dissolved in 3 ml of benzene and 10 μΙ (0.10 mol) of thiophenol and 12 mg (0.05 mol) were added.

It is refluxed in the presence of 1,1'-azobis (cyclohexane carbonitrile). After 16 hours, the reaction mixture was cooled to room temperature and applied to a small silica gel column. The column was washed with dichloromethane and eluted with 1: 1 ethyl acetate: dichloromethane. Evaporation of the solvent afforded 53 mg (85%) of Cryptophycin-151, containing 5% cis isomeric impurity.

Cryptophycin-151: EIMS m / z (relative intensity) 688/690 (3.3 / 1.5), 412/414 (18/6), 377 (100), 233 (18), 195/197 (16 / 6), 193 (29), 141 (38); high resolution EIMS m / z 688.2900 (calculated for C ₃ gH ₄₅ CIN ₂ O ₇ , delta 1.5 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-152: EIMS m / z (relative intensity) 664/666 (24/7), 412/414 (21/7), 253 (100), 91 (85); high resolution EIMS m / z 664.2939 (calcd for C ₃₇ H ₄₅ CIN ₂ O ₇ , delta -2.3 mmu). ¹ H-NMR data in Table 3; ¹³ C-NMR data a

Table 8.

Cryptophycin-153: EIMS m / z (relative intensity) 668/670 (3.7 / 1.4), 412/414 (32/12), 257 (62), 198 (100), 195/197 36/11 ; high resolution EIMS m / z 668.2817 (calculated for C ₃₆ H ₄₅ CIN ₂ O ₈ , delta 4.7 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-154: EIMS m / z (relative intensity) 698/700 (2.5 / 0.8), 412/414 (18/5), 287 (28), 228 (35) 195/197 (18/6) ), 139 (100); high resolution EIMS m / z 698.2946 (calcd for C ₃₇ H ₄₇ CIN ₂ O ₉ , delta 2.5 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-155: EIMS m / z (relative intensity) 604/606 (20/6), 412/414 (24/8), 280/282 (24/8), 195/197 (100/33); high resolution EIMS m / z 604.2894 (calculated for C ₃₂ H ₄₅ CIN ₂ O ₇ , delta 2.1 mmu). ¹ H-NMR (CDCl ₃ ), δ:

(A): 5.76 (2, d, 15.5), 6.66 (3, ddd, 15.9, 9.5 and 5.9),

2.32-2.44 (4-H ₂ , m), 4.92 (5, m), 2.66-2.76 (6, m),

0.98 (6-Me, d, 6.8), 5.21 (7, t, 10.9), 5.46 (8, dt,

10.9 and 7.4), 2.00 (9-H _2, m), 1.38 (10-H _2, m), 0.91 (10-Me, t; 7.5); (B): 4.81 (2, m), 5.70 (2-NH, d, 8.5),

3.04 (3, dd, -14.5 and 7.2), 3.14 (3, dd, -14.5 and

5.6), 7.22 (5, d, 2.2), 3.87 (7-OMe, s), 6.84 (8, d,

8.5), 7.08 (9, dd, 8.5 and 2.2); (C): 2.66-2.76 (2, m),

1.22 (2-Me, d, 7.4), 3.27 (3, dt, 13.5 and 6.8), 3.52 (3, m), 6.93 (3-NH, br, t, 6.4); (D): 4.86 (2, dd, 9.8 and

3.5), 1.49 (3, m), 1.71-1.80 (3/4, m), 0.90 (4-Me, d,

6.0), 0.94 (5, d, 6.5).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.5 (1), 125.0 (2), 141.6 (3), 36.5 (4), 77.9 (5),

36.2 (6), 17.7 (6-Me), 130.0 (7), 131.6 (8), 29.6 _(9-H2), 22.8 _(10-H2), 13.8 (11-H ₃ );

B: 171.0 ^a (1), 53.5 (2), 35.1 (3), 129.9 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OCH ₃ ), 112.2 (8),

128.4 (9);

HU 227 195 Β1

C; 175.6 (1), 38.3 (2), 14.0 (2-Me), 41.2 (3);

D: 170.9 ^a (1), 71.6 (2), 39.5 (3), 24.7 (4), 21.4 (4-Me), 23.1 (5) ^a , interchangeable signals .

Cryptophycin-156: EIMS m / z (relative intensity) 412/414 (23/6), 381 (11), 280/282 (22/6), 195/197 (100/33); high resolution EIMS m / z 592.2568 (calculated for C ₃₀ H ₄₁ CIN ₂ O ₈ , delta -1.6 mmu). ¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-159: EIMS m / z (relative intensity) 666 (3), 412/414 (16/4), 396 (52), 255 (100), 195/197 (30/8), 91 (54); high resolution EIMS m / z 666.3060 (calculated for C ₃₇ H ₄₇ CIN ₂ O ₇ , delta 1.1 mmu). ¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-160: EIMS m / z (relative intensity) 628/630 (9/4), 412/414 (63/23), 280 (25), 217 (89), 195/197, (89/30); high resolution EIMS m / z 628.2532 (calculated for C ₃₃ H ₄₁ CIN ₂ O ₈ , delta 2.0 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-161: EIMS m / z (relative intensity) 706/708 (6/2), 412/414 (49/18), 295 (15), 280/282 (25/7), 195/197 (100 / 34); high resolution EIMS m / z 706.2623 (calculated for C ₃₆ H ₄₂ CIF ₃ N ₂ O ₇ , delta 1.0 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-166: EIMS m / z (relative intensity) 652/654 (1.1 / 0.4), 412/414 (18/5), 241 (37), 195/197 (64/20); high resolution EIMS m / z 652.2918 (calcd for C ₃₆ H ₄₅ CIN ₂ O ₇ , delta -0.2 mmu). ¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-167: EIMS m / z (relative intensity) 666/668 (3.4 / 1.1), 412/414 (26/9), 280 (11), 255 (67), 195/197 (46 / 15); high resolution EIMS m / z 666.3058 (calculated for C ₃₇ H ₄₇ CIN ₂ O ₇ , delta

1.4 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-172: EIMS m / z (relative intensity) 668/690 (3/2), 412/414 (12/4), 277 (67), 218 (100), 195/197 (29/10), 141 (63); high resolution EIMS m / z 668.2916 (calcd for C ₃₉ H ₄₅ CIN ₂ O ₇ , delta = 0.1 mmu). ¹ H-NMR data in Table 7; ¹³ C-NMR data in Table 8.

Cryptophycin-181: EIMS m / z (relative intensity) 656/658 (9.0 / 3.3), 412/414 (84/34), 245 (71), 195/197 (47/7); high resolution EIMS m / z 656.2674 (calculated for C ₃₅ H ₄₂ CIFN ₂ O ₇ , delta = 0.9 mmu). ¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-188: EIMS m / z (relative intensity) 674/676 (20/4), 412/414 (57/20), 280/282 (20/7), 263 (13), 195/197 (89 / 27); high resolution EIMS m / z 674.2551 (calculated for C ₃₅ H ₄₁ CIF ₂ N ₂ O ₇ , delta 4.7 mmu).

¹ H-NMR data in Table 7; ¹³ C-NMR data a

Table 8.

Cryptophycin-234: 58.3 mg of a mixture of Cryptophycin-234 and 15% Z isomer were recrystallized from ethyl acetate / diethyl ether to give 47 mg of Cryptophycin-234 and 11 mg of Z isomer. ¹ H-NMR (CDCl ₃ ), δ:

(A): 5.77 (2, d, 15.2), 6.68 (3, ddd, 15.2, 9.7 and 5.4),

2.37 (4, dt, 14.2 & 10.5), 2.52 (4, m), 4.99 (5, m),

2.55 (6, m), 1.13 (6-Me, d, 6.8), 5.99 (7, dd, 15.8 and

8.8), 6.40 (8, d, 15.8, 7.25 ^a (276 ', d, 8.2), 7.29 ^a (375', d, 8.2), 4.71 (4'-CH ₂ OTBDMS, brs), 0.08 (6H) and 0.93 (9H) (4'-CH ₂ O-TBDMS), (B) 4.81 (2, m), 5, 77 (2-NH, obscured by other signals), 3.03 (3, dd, -14.4 and 7.3), 3.14 (3, dd, -14.4 and 5.4), 7, 22 (5, d, 2.0), 3.86 (7-OMe, s), 6.83 (8, d, 8.4), 7.07 (9, dd,

8.4 and 2.0); (C): 2.71 (2 m), 1.22 (2-Me, d, 7.2), 3.29 (3, m), 3.49 (3, m), 6.98 (3- NH, br, t, 5.6); (D): 4.84 (2, m), 1.36 (3, m), 1.59-1.71 (3/4, m), 0.73 (4-Me, d, 6.4) ), 0.77 (5, d, 6.2).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.5 (1), 125.2 (2), 141.4 (3), 36.4 (4), 77.4 (5),

42.2 (6), 17.3 (6-Me), 129.6 (7), 131.6 (8), 136.4 (1 '), 126.0 ^a (276'), 126.3 ^? (375 '), 140.9 (4'), 64.7 (4H ₂ O-), 25.9 and -5.3 (4'-CH ₂ OTBDMS);

B: 171.0 ^b (1), 53.6 (2), 35.1 (3), 129.9 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OCH ₃ ), 112.2 (8),

128.4 (9);

C: 175.6 (1), 38.3 (2), 14.0 (2-Me), 41.1 (3);

D: 170.9 ^b (1), 71.5 (2), 39.5 (3), 24.5 (4), 22.7 (4-Me), 21.2 (5) ^a and ^{b are} interchangeable signs.

¹ H-NMR (CDCl ₃ ) of Cryptophycin-236, δ:

(A): 5.76 (2, d, 15.2), 6.68 (3, ddd, 15.2, 9.6 and 5.6),

2.37 (4, m), 2.51 (4, m), 5.00 (5, m), 2.54 (6, m),

1.13 (6-Me, d, 6.9), 6.00 (7, dd, 15.9 and 8.6), 6.39 (8, d, 15.9), 7.20-7 , 30 (2737576 ', br, m), 4.28 (4CH ₂ NH-tert-BOC, d, 4.9), 4.80 (4'-CH ₂ NH-tert-BOC, d, 4.9), 1.46 (4'-CH ₂ NH-tert-6OC, s); (B):

4.80 (2, m), 5.65 (2-NH, d, 8.5), 3.04 (3, dd,

-14.4 and 6.9), 3.13 (3, dd, -14.4 and 5.5), 7.22 (5, d,

2.2), 3.87 (7-OMe, s), 6.83 (8, d, 8.4), 7.08 (9, dd,

8.4 and 2.2); (C): 2.71 (2, m), 1.22 (2-Me, d, 7.3),

3.27 (3, m), 3.51 (3, m), 6.93 (3-NH, br t, 5.6); (D):

4.84 (2, dd, 9.9 and 3.5), 1.36 (3, m), 1.58-1.70 (3/4, m), 0.74 (4-Me, d) , 6.4), 0.78 (5, d, 6.4).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.2 (2), 141.4 (3), 36.5 (4), * (5),

42.2 (6), 17.3 (6-Me), 131.0 (7), 131.4 (8), 135.9 (1 '), 126.4 (276'), 127.8 ( 375 '), 138.4 (4'), 44.4 (4CH ₂ NH-tert-BOC), 28.4 (4'-CH ₂ NH-tert-BOQ);

B: 170.9 (1), 53.5 (2), 35.1 (3), 129.9 (4), 131.1 (5), (6), 154.0 (7), 56, 2 (7-OCH ₃ ), 112.3 (8), 128.4 (9);

C: 175.6 (1), 38.3 (2), 14.0 (2-Me), 41.2 (3);

D: 170.9 (1), 71.6 (2), 39.6 (3), 24.5 (4), 22.7 (4-Me), 21.3 (5) * solvent sign .

Cryptophycin-238: A solution of 14 mg Cryptophycin-234 in 1.5 ml tetrahydrofuran at 0 ° C

195 1951 μ11 M tetrabutylammonium fluoride was reacted with tetrahydrofuran solution. After 1 hour, saturated ammonium chloride solution (5 mL), water (15 mL) were added and the mixture was extracted with ethyl acetate (50 mL). The ethyl acetate layer was dried over magnesium sulfate and evaporated. The residue was applied to a small silica gel column and eluted with dichloromethane and ethyl acetate. The latter fraction was evaporated to give 11.8 mg of Cryptophycin-238.

¹ H-NMR (CDCl ₃ ), δ:

5.76 (2, d, 15.6), 6.68 (3, ddd, 15.6, 9.8 and 5.5), 2.36 (4, dt, 14.4 and 10.4) , 2.51 (4, br, dd, 14.4 and 5.1), 5.00 (5, m), 2.55 (6, m), 1.13 (6-Me, d, 6, 8), 6.01 (7, dd, 15.9 and 8.8), 6.40 (8, d, 15.9), 7.31 (2737576 ', br s), 4.47 (4'). -C / 7 ₂ OH, br s); (B): 4.80 (2, m), 5.78 (2-NH, d,

8.4), 3.01 (3, dd, -14.4 and 7.4), 3.13 (3, dd, -14.4 and

5.5), 7.21 (5, d, 2.0), 3.86 (7-OMe, s), 6.83 (8, d, 8.5), 7.07 (9, dd, 8, 5 and 2.0); (C); 2.71 (2, m), 1.21 (2-Me, d, 7.1), 3.28 (3, m), 3.49 (3, m), 6.98 (3-NH, brt, 6.0); (D): 4.84 (2, dd, 9.9 and 3.2) 1.35 (3, m), 1.58-1.71 (3/4, m), 0.74 (4- Me, d, 6.4), 0.77 (5, d, 6.3).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.5 (1), 125.2 (2), 141.4 (3), 36.4 (4), 77.4 (5),

42.2 (6), 17.3 (6-Me), 130.1 (7), 131.4 (8), 136.1 (1 '), 126.3 ^a (276'), 127.2 ^a (375 '), 140.4 (4'), 64.9 (4'CH ₂ OH);

B: 171.0 ^b (1), 53.7 (2), 35.0 (3), 129.9 (4), 131.0 (5), 122.3 (6), 153.9 (7 ), 56.1 (7-OCH ₃ ), 112.2 (8), 128.4 (9);

C: 175.6 (1), 38.2 (2), 14.0 (2-Me), 41.1 (3);

D: 170.8 ^b (1), 71.5 (2), 39.5 (3), 24.5 (4), 21.7 (4-Me), 22.3 (5) ^a and ^b a interchangeable signs. Cryptophycin-246: Cryptophycin-236 (9 mg, 0.012 mmol) in dichloromethane (50 μΙ) was reacted with 4N hydrochloric acid (20 μΙ, 0.08 mmol) in dioxane at room temperature. After 1 hour, the solvent was evaporated and the residue was flash chromatographed on a 500 mg C18 silica gel column (Alltech) using methanol / water 1: 1 as eluent. The first fraction (3 mL) was evaporated to give 7 mg (85%) of Cryptophycin-246. ¹ H-NMR (CD ₃ OD), δ:

(A): 5.92 (2, dd, 15.2 and 1.7), 6.69 (3, dddd, 15.2,

11.1 and 3.8), 2.35 (4, m), 2.68 (4, m), 5.06 (5, m), 2.63 (6, m), 1.15 (6 -Me, d, 7.4), 6.18 (7, dd, 15.9)

8.9), 6.50 (8, d, 15.9), 7.40 (276 'br d, 8.2), 7.46 (375', br d, 8.2), 4.08 (4'-CH ₂ NH ₂ HCl, s); (B): 4.51 (2, dd, 11.1 and 3.4) 2.75 (3, m), 3.17 (3, dd, -14.5 and 3.9), 7.27 (5, d, 2.0), 3.83 (7-OMe, s), 6.97 (8, d, 8.5), 7.16 (9, dd, 8.5 and 2.0) ; (C): 2.73 (2, m), 1.17 (2-Me, d, 8.0), 3.26 (3, m), 3.56 (3, m); (D): 4.92 (2, dd, 9.9 and 3.9), 1.35 (3, m), 1.54-1.65 (3/4, m), 0.71 (4 -Me, d, 6.4), 0.75 (5, d, 6.4).

¹³ C-NMR (CDCl ₃ ), δ:

A: 168.4 (1), 125.7 (2), 143.5 (3), 37.7 (4), 78.6 (5),

43.5 (6), 17.5 (6-Me), 133.6 (7), 131.9 (8), 139.5 ^a (1 '), 128.0 (276'), 130.3 (375 '), 133.3 ^a (4'), 44.0 (4'CH ₂ NH ₂ HCl);

B: 174.1 (1), 57.4 (2), 36.3 (3), 132.2 (4), 131.5 (5), 123.3 (6), 155.4 (7) , 56.6 (7-OCH ₃ ), 113.5 (8), 129.3 (9);

C; 177.5 (1), 39.0 (2), 15.1 (2-Me), 41.2 (3);

D: 172.3 (1), 72.8 (2), 41.0 (3), 25.7 (4), 21.7 (4-Me), 23.2 (5) ^are the interchangeable signals.

Cryptophycin-250: A solution of 5.1 mg of Cryptophycin-247 in 80 μΙ dichloromethane was reacted with 40 μΙ 4N hydrochloric acid in dioxane. After 2 hours, the reaction mixture was evaporated, diluted with water and passed through a short ODS column. The column was washed with water (5 mL) and eluted with acetonitrile (3 mL). The latter fraction was evaporated to give 5 mg of Cryptophycin-250 which was purified by reverse phase HPLC (Econosil C18, 25 cm x 10 mm, 10 μ, 35% water / acetonitrile, 4 ml / min). 4 mg of pure product are obtained (T _R : 18 minutes).

EIMS m / z (relative intensity) 653/655 (15/10), 533 (33), 242 (48), 195/197 (36/13); high resolution EIMS m / z 653.2865 (calculated for C ₃₅ H ₄₄ CIN ₃ O ₇ , delta 0.3 mmu).

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.78 (2, d, 15.1), 6.68 (3, ddd, 15,1,9,6 and 5.4),

2.35 (4, dt, 14.4 and 10.7), 2.48 (4, br dd, 14.4 and

5.1), 5.01 (5, m), 2.53 (6, m), 1.12 (6-Me, d, 6.8),

6.01 (7, dd, 15.9 and 8.7), 6.33 (8, d, 15.9), 6.81 (m),

6.89 (d), 6.94 (s) and 6.99 (m) (273'-NH _3/476 '), 7.15 (5', t; 7.8); (B); 4.79 (2, m), 5.95 (2-NH), 3.02 (3, dd,

-14.4 and 7.3), 3.15 (3, dd, -14.4 and 5.5), 7.22 (5, d,

2.0), 3.86 (7-OMe, s), 6.84 (8, d, 8.4), 7.08 (9, dd,

8.4 and 2.0); (C): 2.70 (2, m), 1.23 (2-Me, d, 7.1),

3.27 (3, m), 3.51 (3, m), 6.99 (3-NH, m); (D): 4.87 (2, dd, 9.8 and 3.2), 1.39 (3, m), 1.59-1.73 (3/4, m),

0.76 (4-Me, d, 5.4), 0.80 (5, d, 5.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.8 (1), 125.2 (2), 141.5 (3), 36.4 (4), 77.3 (5),

42.0 (6), 17.1 (6-Me), 131.0 (7), 131.3 (8), 138.3 (1 '), 116.0 (2'), 121.1 ( 4 '), 130.0 (5'), 118.0 (6 ');

B: 171.2 (1), 54.0 (2), 35.0 (3), 129.8 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OCH ₃ ), 112.3 (8),

128.4 (9);

C; 175.6 (1), 38.2 (2), 14.2 (2-Me), 41.0 (3);

D: 170.9 (1), 71.5 (2), 39.6 (3), 24.6 (4), 22.8 (4-Me), 21.4 (5).

Cryptophycin-251: EIMS m / z (relative intensity) 696 (0.7), 652 (M ⁺ - CO ₂ , 0.7), 412/414 (4.2), 285 (6), 241 (5). , 195/197 (11/3); high resolution EIMS m / z 653.2865 (calcd for C ₃₇ H ₄₅ CIN ₂ O ₉ , delta 0.3 mmu).

Cryptophycin-255:

¹ H-NMR (CDCl ₃ ), δ:

(4): 5.78 (2, d, 15.1), 6.67 (3, ddd, 15,1,9,5 and 5,9),

2.37 (4, dt, 14.9 & 10.3), 2.45 (4, m), 4.96 (5, m)

2.52 (6, m), 1.09 (6-Me, d 6.8), 5.74 (7, dd, 15.1 and

8.9), 6.24 (8, d, 15.1), 7.20-7.33 (273747576 ', m);

(B): 4.83 (2, m), 5.64 (2-NH, d, 8.8), 3.05 (3, dd,

-14.6 and 7.1), 3.14 (3, dd, -14.6 and 5.4), 7.22 (5, d,

2.0), 3.88 (7-OMe, s), 6.85 (8, d, 8.5), 7.08 (9, dd,

8.5 and 2.0); (C): 2.72 (2, m), 1.23 (2-Me, d, 7.3),

3.29 (3, m), 3.50 (3, m), 6.95 (3-NH, m); (D): 4.86 (2, dd, 10.0 and 4.3), 1.49 (3, m), 1.63-1.85 (3/4, m),

0.89 (4-Me, d, 6.4), 0.94 (5, d, 6.4).

HU 227 195 Β1

Table 7

K ¹ H NMR data of Cryptophycins at 500 MHz

She 3 151 1S7 153 154 154 159 140 161 164 167 172 181 188 251  J - THE 2 3.77am 3.78 5.77 5.77 5.77 5.76 5.76 5.77 5.78 5.77 5.77 5.78 5.78 5.78 5.76 3 6.68 6.71 6.67 6.68 6.68 6.66 6.68 6.68 6.68 6.68 6.68 6.70 6.68 6.6X 6.68 ddd 4K 2:37 2:43 2:37 2:37 2:37 2:34 2:38 2:35 2:36 2:39 2:37 2.41 2:36 2:35 2:35 Λ 2:51 45 2:54 2:58 247 2:52 2:52 2:41 2:51 2:49 2:53 2:52 2:51 2:55 2:54 2:52 btdd 5 5:01 5:10 4.97 5:00 5:00 4.88 5:02 4.96 5:02 5:03 4.99 5:04 5:00 5:01 5:00 ddd 6 2:36 2.71 2:47 2:54 2:54 2:27 2.56 2:49 2:59 2:50 2:54 2.61 2:57 2:58 2:55 ft-Me π 1.14 d 1:22 1:09 1:13 1:13 1:04 1:13 1:10 1:15 1:15 1:12 1:17 1:15 1:14 1:13 7 6:01 6:09 5.63 6:00 5.99 4:53 5.87 5.70 613 592 5.97 6 14 6:09 6:05 6:00 dd dd dd / ' 8 6:41 p.m. 7:19 6:23 6:38 6:34 6:31 6:39 6:26 6:45 6.63 6:34 6:57 6:58 6:34 6:39 dd d 9 6.71 dd 10 6:49 dd 7.67 8 / 10- · bt Α »· Γ 7:30 2 ' 7:28 7:41 7:36 6.85 6.68 2:31 7:37 7:42 2:33 6.94 6.83 tendon (0 br 1 d the brr d • br a m y 7:34 7:58 7:30 3.80 3.79 6.95 7:55 7:11 2:29 7:54 7:09 7:23 m m bri 1 1 bri d 10 1 m m d 4 ' 7:23 7.77 7:22 6.78 6:36 2:31 6:47 7:39 6.87 7.78 740 6.68 3.63 m d m br d l brr w tx a m m Ltd. 3 ' 7:28 7.94 7:30 7:21 3.79 6.95 7:35 7:55 * 229 7.78 7:02 7:23 U) d bri t s d tors d s m m 6 ' 7:34 7:41 7:36 6.92 6.6X 7.28 7:42 * 6.94 7:44 7:19 6.83 7:30 m Shoot br d d d d d br m m ffl d T 7:58 d 7:44 m 8 ' 8:07 d 7.7 ' m D2 4.84 4.85 4.87 4.84 4.84 4.87 4.84 4.83 4.84 4.85 4.85 4.86 4.85 4.84 484 dd 3 1.62 1.64 1.72 1.65 1.66 1.75 1.68 1.67 1.65 1.69 1.66 1.67 1 66 1.67 1:36 3 ' m 1:36 1:35 1:47 1:37 1:38 1:48 1:38 1:36 1:31 1:38 1.38 1:38 1:36 1:32 1:58 t <> 4 m 1.62 1.64 1.72 1.65 1.66 1.75 1.68 1.67 1.65 1.69 1.66 1.64 1.66 1.67 1.70 m 4-M " m 0.77 d 7:00. 0.87 0.77 0.79 0.94 0.79 0.80 0.76 0.79 0.78 0.74 0.7 » 0.79 0.77 5 0.73 0.64 0.86 0.74 nose 0.91 0.77 079 0.72 076 0.75 0.65 0 74 0.77 0.73

P = position

The spectra were recorded in deuterochloroform (CDCl ₃ ). In the table, the chemical shift of the protons or the methyl or methoxy or hydroxymethyl group at the indicated carbon atom! your details will appear. In units B and C, the chemical shift data for protons is ± 0.2 ppm and the coupling constants are ± 0.5 Hz for Cryptophycin-3. Data of coupling constants (J) in Hz, J (H, H): Chr.151: 3 ', 4' = 8.3 Hz); 5 ', 6' = 8.0 Hz;

7 ', 8' = 8.1 Hz; Chr.152: 6.7 = 8.7 Hz; 7.8 = 15.3 Hz;

HU 227 195 Β1

8.9 = 10.5 Hz; 9.10 = 15.7 2 ', 3' = 3 ', 4' = 4 ', 5' = 5 ', 6' = 7.4 Hz; Chr.153: 2 ', 4' = 2 ', 6' = 2.0 Hz; 4 ', 5' = 5 ', 6' = 7.9 Hz; Chr.154 = 2 ', 4' = 4 ', 6' = 2.2 Hz; Chr.156 the value δ for 8-OCH ₃ unity to 3.51, J: 6.7 His 9.3 Hz; 7.8 = 12.6 Hz; Chr.159: 5 ', 6' = 8.3 Hz, Chr. 161:

2 ', 3' = 4 ', 5' = 8.1 Hz; Chr. 251: 2 ', 3' = 5 ', 6' = 8.2 Hz. The observable coupling constants for the other protons in the table are within +0.5 Hz relative to the Cryptophy cin-3 data.

Table 8

¹³ C-NMR data of Cryptophycins at 125 MHz

HU 227 195 Β1

Table 8 (continued)

lake D 1 no. 1 133 .7 171 0 170 . 8 170 .9 170 .9 170 .9 170 .9 170 .9 170th 8 170 .9 Π1 .0 132 .9 170 .9 170 .8 170 .9 Π 0th 9 Ί 71st 71st 71st 71st 71st 71 71st 71st 71.5 71st 71st 71st 71st 71st 71 6 5 5 s 6 4 5 £ S 4 5 b 4 .5 3 39th 39th 39th 39th 39th 39th 39th 39th 39.5 39th 39th 39th 39th 39th 39 b b 6 5 b & 4 5 4 .5 1 b 4 . 4 4 24th 24th 24th 24th 24th 24th 24th 24th 24.5 24th 24th 24th 24th 24th 24 & 1 4 5 b 7 S S b b < b S b 4- 21st 21st 21st 21st 21st 21st 21st 21st 21.2 21st 21st 21st 9 » * Λ. 21st 21 H # i 2 t 4 2 • 9 THE 4 3 3 2 4 ♦ • 9 w 2 3 n 5 · » 22nd 22nd xx- 22nd 23rd 22nd » 4 »*« 22.7 22nd 22nd 22nd 22nd 22 6 7 7 7 0 7 B 8 7 4 7 8 .7

P = position

The spectra were recorded in deuterochloroform. The chemical shift data for the carbon atoms of units B and C are within ± 0.5 ppm of Cryptophycin-3.

* signs below CDCI ₃ sign ** signs could not be found t the 8-OCH ₃ sign ^of unit A the signs are interchangeable

The carbon atom of the 3'-OCH ₃ group of unit A resonates at δ 55.1 for Cryptophycin-153, the carbon atoms of the 3 'and 5'-OCH ₃ groups of Cr.154

They resonate at 55.3. The 2 'and 4'-CH ₃ carbon atoms of Cryptophycin-159 are found at δ 19.7 and 20.9, respectively, while the 2'-CH ₃ carbon at Cr 166 are at δ 19.8. Signs of the 3 'and 5'-CH ₃ carbon atoms of Cr.167 are observed at δ 21.2.

The carbon atoms of the methylene and carboxyl groups of the 4'-CH ₂ COOH give signals at δ 40.0 and 174.5 respectively.

Example 96

Epoxide Analogs: Cryptophycin-157, -158, -164, -165, -168, -169, -170, -171, -173, -174, -177, -178, -179, -180, -182, - 183, -200, -242, and -269 (Table 9). Prepared according to the reaction shown in Figure 16.

Table 9 (data refers to compounds of Figure 16)

Isztirén Cr. Price epoxide space- ed. RX / SS gold ISI Irish' 1S7 15 « SS 2.4 ; 1 153 148 1 » SS 2.4 : 1 139 170 M 2.9 1 ise 177 not M SS 2.0 1 1S7 "Pr * «Μ, $ 17 ISO M SS 2.7 i 1 X72 year 173 174 M SS 1.5 I 2

HU 227 195 Β1

Table 9 (continued)

181 of 182 183 JTF SS 2.0 : 1 188 \ R " f 200 M SS 1.4: 1 238 242 M 5S 2 1 238 289 M SS 2: 1 '·

* Λ Cr.188 requires 6 equivalents of MCPBA and produces an epoxide in 70% yield after 36 hours of reaction ** inseparable mixture

General procedure for the preparation of epoxides: A solution of 0.1 mmol of styrene analog and 0.3 ml of m-chloroperbenzoic acid (mCPBA) in 3 ml of dichloromethane was stirred at room temperature for 16 hours. The reaction mixture was diluted with 3 mL dichloromethane, washed with 5 mL 0.1 M phosphate buffer (pH 8) to remove 3-chlorobenzoic acid formed during the reaction. The organic phase was separated, the excess peroxide was destroyed by the addition of 20 μΙ of dimethyl sulfoxide and washed a second time with buffer.

The dichloromethane layer was separated, dried over magnesium sulfate, evaporated and the residue was kept under reduced pressure for 24 hours. The residue was chromatographed on reverse phase HPLC (Econosil C18, 10 μ, 250 x 22 mm, 35% water / acetonitrile, 6 mL / min) to yield the RR and SS epoxides.

The experiments and cytotoxic data are summarized in the table.

Cryptophycin-157 and -158: Cryptophycin-151 is reacted with m-CPBA and the products are separated by reverse phase HPLC. EIMS m / z (relative intensity) 704/706 (1 / 0.3), 195/197 (14/4), 141 (100), 215 (27), 195; high resolution EIMS m / z 704.2865 (calcd for C ₃₉ H ₄₅ CIN ₂ O ₈ , calculated = 1.0 mmu). ¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-164 and -165: A solution of 53 mg (0.08 mmol) of Cryptophycin-142 and 15 mg (0.087 mmol) of m-chloroperbenzoic acid in 3 mL of dichloromethane was stirred at room temperature for 16 hours. Work up and purification afforded 5-5 mg of 164 / 165A and 164 / 165B, 10 mg of starting material and decomposition products. The structure of the products is described for Cryptophycin-164 and Cryptophycin-165, but the bulk structure of the epoxide group has not been determined.

Cryptophycin-164 / 165A:

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.77 (2, d, 15.1), 6.67 (3, ddd, 15,1,9,7 and 5.4),

2.37 (4, m), 2.44 (4, m), 4.98 (5, m), 2.48 (6, m), 1.06 (6-Me, d, 6.7) , 5.83 (7, dd, 15.6 and 8.5), 5.40 (8, dd, 15.6 and 7.8), 3.32 (9, dd, 7.9 and 2.0) ), 3.75 (10, d, 2.0), 7.26 (276 ', m), 7.31-7.37 (37475', m); (B): 4.82 (2, m), 5.75 (2-NH, d, 9.2), 3.04 (3, dd, -14.5 and 7.3), 3.14 ( 3, dd, -14.5 and 5.5), 7.23 (5, d,

2.2), 3.87 (7-OMe, s), 6.84 (8, d, 8.2), 7.09 (9, dd, 8.2 and 2.2); (C): 2.73 (2, m), 1.23 (2-Me, d, 7.2), 3.29 (3, m), 3.51 (3, m), 6.97 ( 3-NH, brt, 6.3); (D): 4.88 (2, dd, 10.0 and 3.7), 1.52 (3, m), 1.79 (3/4, m), 0.92 (4-Me, d). , 6.5), 0.95 (5, d, 6.5).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.3 (2), 141.2 (3), 36.5 (4), 77.0 (5),

41.3 (6), 17.0 (6-Me), 136.2 (7), 129.4 (8), 62.5 (9),

60.1 (10), 136.9 (1 '), 125.4 (276'), 128.7 (375 '),

128.4 (4 ');

B: 171.0 (1), 53.6 (2), 35.0 (3), 129.9 (4), 131.0 (5),

122.4 (6), 154.0 (7), 56.1 (7-OMe), 112.3 (8), 128.3 (9);

C: 175.6 (1), 38.3 (2), 14.1 (2-Me), 41.2 (3);

D: 170.8 (1), 71.5 (2), 39.6 (3), 24.7 (4), 21.4 (4-Me), 22.9 (5).

¹ H-NMR (CDCl ₃ ) of Cryptophycin-164 / 165B, δ:

(A): 5.76 (2, d, 15.3), 6.66 (3, ddd, 15.3, 9.6 and 5.7), 2.36 (4, m), 2.40 (4, m), 5.00 (5, m), 2.47 (6, m), 1.08 (6-Me, d, 6.8), 5.85 (7, dd, 15.5) and 8.5), 5.43 (8, ddd, 15.5, 7.6 and 0.6), 3.35 (9, dd, 7.6 and 1.9), 3.75 (10, d, 1.9), 7.27-7.37 (273 '/ 475'6', m); (B): 4.82 (2, m), 5.65 (2-NH, d, 8.5), 3.04 (3, dd, -14.4 and 7.2), 3.14 ( 3, dd, -14.4 and 5.5), 7.22 (5, d, 2.1), 3.87 (7-OMe, s), 6.84 (8, d, 8.5) , 7.08 (9, dd,

8.5 and 2.1); (C): 2.72 (2, m), 1.22 (2-Me, d, 7.2),

3.27 (3, m), 3.52 (3, m), 6.90 (3-NH, br t, 6.3); (D): 4.87 (2, dd, 9.8 and 3.8), 1.48 (3, m), 1.77 (3, m), 1.73 (4, m), 0, 89 (4-Me, d, 6.4), 0.90 (5, d, 6.6).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.2 (2), 141.2 (3), 36.4 (4), 41.3 (6),

HU 227 195 Β1

16.9 (6-Me), 135.9 (7), 129.1 (8), 62.3 (9), 60.4 (10),

136.8 (1 '), 125.5 (276'), 128.6 (375 '), 128.4 (4');

B: 170.9 (1), 53.6 (2), 35.1 (3), 129.8 (4), 131.0 (5),

122.4 (6), 154.0 (7), 56.1 (7-OMe), 112.2 (8), 128.4 (9);

C: 175.5 (1), 38.3 (2), 14.0 (2-Me), 41.2 (3);

D: 170.8 (1), 71.4 (2), 39.7 (3), 24.7 (4), 21.5 (4-Me),

22.9 (5).

Cryptophycin-168 and -169: Cryptophycin-153 was reacted with m-CPBA and the product was separated by reverse phase HPLC.

Cryptophycin-168: EIMS m / z (relative intensity) 684/686 (7/2), 412/414 (17/6), 280/282 (12/6), 257 (20), 212/214 (14 / 4), 195/197 (69/24), 121 (100); high resolution EIMS m / z 684.2789 calculated for C36H45CIN2O9 - 1.0mmu error). ¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-169: ¹ NMR data in Table 10; ¹³ C-NMR data in Table 11.

Cryptophycin-170 and -171: Cryptophycin-159 was reacted with m-CPBA and the products were separated by reverse phase HPLC.

Cryptophycin-170: EIMS m / z (relative intensity) 682/684 (4/3), 412/414 (11/4), 280/282 (15/4), 255 (25), 195/197 (52 / 17); high resolution EIMS m / z 682.3043 (calculated for C ₃₇ H ₄₇ CIN ₂ O ₈ , 2.2 mmu).

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-171: EIMS m / z (relative intensity) 682/684 (2.0 / 0.7), 412/414 (7/4), 280/282 (14/4), 255 (19), 195 / 197 (50/17); high resolution EIMS m / z 682.3006 (calculated for C ₃₇ H ₄₇ CIN ₂ O ₈ ,

1.5mmu error);

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-173 and -174: Cryptophycin-171 was reacted with m-CPBA and the products were separated by reverse phase HPLC.

Cryptophycin-173: EIMS m / z (relative intensity) 704/706 (1.3 / 1.4), 412/414 (5.1 / 1.9), 277 (6.3), 141 (100); high resolution EIMS m / z 704.2839 (calculated for C ₃₉ H ₄₅ CIN ₂ O ₈ , 2.2 mmu). ¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-174:

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-177 and -178: Cryptophycin-166 was reacted with m-CPBA and the products were separated by reverse phase HPLC.

Cryptophycin-177: EIMS m / z (relative intensity) 668 (2/7), 412/414 (9/3), 280/282 (11/4), 241 (16), 195/197 (45/17) , 105 (100); high resolution EIMS m / z 668.2835 (calculated for C ₃₆ H ₄₅ CIN ₂ O ₈ , 2.9 mmu).

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-178: EIMS m / z (relative intensity) 668/670 (4.1 / 1.4), 412/414 (17/6), 280/282 (12/4), 241 (24), 195 / 197 (59/18), 105 (100); high resolution EIMS m / z 668.2836 (calculated for C ₃₆ H ₄₅ CIN ₂ O ₈ , 2.9 mmu);

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-179 and -180: Cryptophycin-167 was reacted with m-CPBA and the products were separated by reverse phase HPLC.

Cryptophycin-179: EIMS m / z (relative intensity) 682/684 (25/13), 412/414 (100/43), 280/282 (100/50), 255 (100), 211 (100), 195 / 197 (100/100), 121 (100); high resolution EIMS m / z 682.2992 (calculated for C ₃₇ H ₄₇ CIN ₂ O ₈ , 2.9 mmu). ¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-180: EIMS m / z (relative intensity) 682/684 (4/3), 412/414 (14/7), 280/282 (16/11), 255 (10), 195/197 (58 / 21), 119 (100); high resolution EIMS m / z 682.3021 (calcd for C ₃₇ H ₄₇ CIN ₂ O ₈ , 0 mmu);

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-182 and -183: Cryptophycin-181 is reacted with m-CPBA and the products are separated by reverse phase HPLC.

Cryptophycin-182: EIMS m / z (relative intensity) 672 (1), 412/414 (11/4), 280/282 (10/4), 245 (12), 195/197 (40/12), 109 (77); high resolution EIMS m / z 672.2590 (calculated for C ₃₅ H ₄₂ CIFN ₂ O ₈ , 2.3 mmu).

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-183: EIMS m / z (relative intensity) 672/674 (5/2), 412/414 (32/12), 280/282 (13/4), 245 (29), 195/197 (92 / 30); high resolution EIMS m / z 672.2620 (calcd for C ₃₅ H ₄₂ CIFN ₂ O ₈ , 0.7 mmu);

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-200: Cryptophycin-188 is reacted with m-CPBA, and the products are recovered by the general procedure, except that after 12 hours, another 3 equivalents of peracid are added to the reaction mixture and stirring is continued for 36 hours, -200 and SS epoxide. EIMS m / z (relative intensity) 690/692 (42/16), 412/414 (7/1), 263 (19), 195/197 (27); high resolution EIMS m / z 690.2494 (C ₃₅ H ₄₁ CIFN ₂ O ₈ for formula, reported

2.6mmu error).

¹ H-NMR data in Table 10; ¹³ C-NMR data a

See Table 11.

Cryptophycin-242: Cryptophycin-236 is reacted with m-CPBA and the products are recovered by the general procedure to obtain Cryptophycin-242 and its SS epoxide. THE

Cryptophycin-242 was purified by normal phase HPLC (Econosil Si, 250 x 10 mm, 10 μ, ethyl acetate / hexane 1: 1; T _r : 81 min).

HU 227 195 Β 11 ¹ H-NMR (CDCl ₃ ), δ:

(A): 5.73 (2, d, 15.4), 6.64 (3, ddd, 15.4, 10.2 and 5.0), 2.42 (4, m), 2.57 (4, m), 5.14 (5, m), 1.79 (6, m), 1.12 (6-Me, d, 6.7), 2.93 (7, dd, 8.4) and 1.6), 3.69 (8, d, 1.6), 7.26 ^a (276 ', d, 8.2), 7.22 ^a (375', d,

8.2), 4.28 (4'-C / 7 ₂ NH-t-BOC, d; 5.9), 4.96 (4'CH ₂ NH-t-BOC, brs), 1.44 (4'-CH ₂ NH-tert-BOC, s), (B): 4.66 (2, m), 5.66 (2-NH, d, 7.8), 2.92 (3, dd, - 14.5 and 7.2), 3.14 (3, dd, -14.5 and 5.0), 7.21 (5, d, 2.0), 3.85 (7-OMe, s) , 6.88 (8, d, 8.5), 7.09 (9, dd, 8.5 and 2.0); (C): 2.68 (2, m), 1.17 (2-Me, d, 7.4), 3.33 (3, m), 3.39 (3, m), 6.95 ( 3-NH, br t, 6.1); (D): 4.85 (2, dd, 10.0 and 3.3),

1.37 (3, m), 1.67-1.73 (3/4, m), 0.84 (4-Me, d, 6.7), 0.86 (5, d, 6.7) ).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.7 (1), 125.4 (2), 141.7 (3), 37.2 (4), 76.6 (5),

40.9 (6), 13.7 (6-Me), 63.4 (7), 59.0 (8), 136.4 (1 '), 126.2 ^b (276'), 127.9 ^b (375 '), 140.2 (4'), 44.5 (4'CH ₂ NH-tert-BOC), 28.5 (4'-CH ₂ NH-tert-BOC);

B; 171.2 (1), 54.6 (2), 35.5 (3), 130.5 (4), 131.2 (5),

122.5 (6), 154.4 (7), 56.5 (7-OCH ₃ ), 112.7 (8), 128.8 (9);

C * :( 1), 38.6 (2), 14.4 (2-Me), 41.1 (3);

D: 171.2 (1), 71.6 (2), 39.9 (3), 24.9 (4) 23.1 (4-Me),

21.4 (5).

^a and ^b interchangeable signals of the same index Cryptophycin-269: A solution of 6 mg of Cryptophycin-243 in 1.2 ml of acetone was reacted with solid potassium carbonate in a tube at room temperature with vigorous stirring. After 12 hours, the reaction mixture was filtered and the filtrate was evaporated. The residue was purified on a short silica gel column using dichloromethane / ethyl acetate as eluent.

5.1 mg of Cryptophycin-269 are obtained.

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.70 (2, d, 15.4), 6.67 (3 m), 2.42 (4, dt, 14.4, and 10.4), 2.53 (4, m ), 5.11-5.19 (5, m), 1.63-1.83 (6, m), 1.14 (6-Me, d, 6.8), 2.92 (7, d) , 7.6), 3.69 (8, br s), 7.37 ^a (276 ', d, 7.9), 7.24 ^a (375', d, 7.9), 4.71 ( 4'-CH ₂ OH, s), (B): 4.74-4.85 (2, m), 5.76 (2-NH, d,

8.3), 2.98 (3, dd, -14.5 and 7.7), 3.13 (3, dd, -14.5 and 5.3), 7.20 (5, br s) , 3.86 (7-OMe, s), 6.82 (8, d, 8.3), 7.05 (9, br d, 8.3); (C): 2.68 (2, m), 1.14 (2-Me, d, 7.3), 3.32 (3, m), 3.45 (3, m), 6.98 ( 3-NH, br m; (D): 4.74-4.85 (2, m), 1.35 (3, m), 1.63-1.83 (3/4, m), 0, 85 (4-Me, d, 6.1), 0.87 (5, d, 6.1) ^, the signals having the same index are interchangeable.

Table 10

The cryptophycin ¹ H-NMR data

P 157 158 148 149 (70 173 174 177 178 179 ISO 182 I8.1 200 THE 2 174 c 5.72 5.82 3.73 5.82 5.75 5.73 5:53 5.74 5x2 5 74 5 82 5.74 5.81 5.75 .1 4.68 6.70 4.72 4:47 4.70 4:48 4:43 4.72 Λ. 49 4.70 4:44 4.71 4:49 4.70 4.67 Tidd 4/1 145 154 172 2.4.3 2:47 2:49 2:44 170 2:51 2:38 2:45 2:57 2:55 157 2:42 ul ip m tendon m m ». *> ' 1:55 143 142 1:54 154 154 2.49 1ΛΟ 154 149 149 2:54 148 2J.3 hrdd m (P fP m m 1 .4.16 5:24 3:21 5:15 5:14 3:17 5:18 1:17 5:18 3.1.5 .1.14 5:13 5:27 3.1.5 5.J4 m w m 6 1.80 IfM 1.93 1.7 ' 1.78 1:16 1.85 1.83 187 1.77 1.74 1,7.5 1.Í2 1.79 l.íl m m 4-M " I.I4J 1:20 1:20 1:14 1.1) 1 1:14 1:18 1:08 1:14 1:10 1:14 1:14 1:14 1:08 l.i l 7 Wd (J 1.99 2.92 1911 2.87 2.86 103 3:01 137 2.80 2.89 190 2.87 f 18.5 11 3.49 J 441 4:24 3:44 158 3.85 3.85 3.77 3.88 3.74 3 61 3:53 4:01 3.8 ' 3:44 8 / 10- 1 7.77 7.7.1 Ar Γ HRI br i r 7:15 7:41 7:42 4.77 4.74 138 142 2:40 6.85 4.86 4.77 m J HRD 4r < tre k • br a br i m r 7:14 7:44 7:47 tendon 3.81 7.99 7:39 7:30 7:11 7:11 131 131 7.0.1 7:01 «» cf. 1 > R " 14 1 (1 fl l » n 4 ' 7:39 Γ.Κ 7.82 1.87 1.85 T.30 7.82 7.8.1 7.24 7:22 W6 5.93 L3.3 ml 4.77 t Irish d | bt d 14 1- c 1 u r n f π f br t ) r «1 m n, 1

HU 227 195 Β1

Table 10 (continued)

3 '(J ^- 7:11 ffl 7.91 d 7:33 id 7.91 d 7.33 ddd 7:27 ! 6.83 d 7:26 ! 6.83 d w 1 100 1 7.84 d 7.30 IFL 7.83 n, 7:46 4 Λ 2:31 w tjf í 2:31 » 6.86 br i - - Ó77 rn r 7:37 id 7J7 ddd 7.J0 m 7:32 m «· 8:08 d 8:07 d 7.84 d 7J3 <n D 2 4.83 dd 4.81 4.91 4.82 4.91 4.83 4.81 4.92 4 W 4.90 4.82 4.92 4.84 4.91 4 84 3 1J6 m 1:34 1:48 1:36 1:50 1:36 1:31 1:49 1:39 1-50 1:33 1:51 I.3V 1:56 1:36 3 ' 170 m 1.68 1.71 I 1.70 1.74 170 1.66 1.72 1.73 1.68 1.71 1.75 1.72 1.76 1.67 ιυ ⁴ 170 m 1.63 1.71 1.70 1.74 1.70 1.64 1.72 1.73 1.68 1.71 1.75 1.72 1.76 1:30 m 4-Mc 0.86 d 0.78 0:57 0.87 0.91 0:53 0.79 0.87 0.86 0.91 0.85 0.92 0.87 0.92 0.89 5 0.85 d 0.74 0.85 0.85 0.89 0.84 0.75 0.87 0.85 0.89 1 0.84 0.89 1 0.85 090 0.87 1

P = position

The spectra were obtained in _CDCl3. In the table, the chemical shift of the proton or methyl or methoxy group at the indicated carbon atom! your details will appear. In units "B" and "C", protons are chemical shifts! data are ± 0.2 ppm and the coupling constants are ± 0.5 Hz identical to those of Cryptophycin-1. The J (H, H) values for Hz at Cr-157 are 2 ', 3' = 3 ', 4' = 7.7 Hz; 5 ', 6' = 6 ', 7' = 7 ', 8' = 7.9 Hz;

5 ', 7' = 1.5 Hz; 6 ', 8' = 1.1 Hz; Cr-168: 4 ', 5' = 5 ', 6' = 8.0 Hz; for Cr.-733: 3 ', 4' = 8.5 Hz; 1 ', 3-1.4 Hz; 5 ', 6' = 7 ', 8' = 8.3 Hz. The coupling constants of the protons on the aryl portions of Cr.-158, -169 and 174 are within ± 0.5 Hz of those of Chr.-157, -169. and -173 respectively. The coupling constants of the other protons in the table are within ± 0.5 Hz of the Cryptophycin-1 data.

Table 11

¹³ C-NMR data for Crypotphycins

P 137 19 « 11 « 189 170 173 174 177 17 « 179 180 102 183 200 THE 165th 165 165 165 165 165 165 165 185 165th 165 165 165th 165 1 3 .5 .3 .5 .3 .3 .4 .4 .5 3 .5 .3 4 .3 2 125th 125 125 125 125 125 125 225 125 125th 125 125 125th 125 2 .2 .3 .2 .3 .3 .3 .3 .3 2 .2 .2 3 .4 3 Hl. 141 141 141 141 141 141 141 141 141st 141 141 141st 140 0 .4 .0 .4 .0 .0 .4 .1 . 4 2 .5 .2 3 .8 4 36.6 36th 36th 36th 36th 36th 36th 36th 36th 36.8 36th 36th 36.7 36th 8 7 7 6 8 B 5 8 7 7 7 5 76.1 • 76th • 76th 76th • 76th • 76.2 • 76th 76.8 75th 1 0 2 0 2 9 6 40.1 41st 40th 41st 40th 40th 41st 40th 41st 40. B 41st 40th 40.9 40th 0 6 0 _3 7 0 2 0 C 5 2 6- 13.1 13th 13th 13th 13th 13th 13th 13th 13th 13.7 13th 13th 13.3 13th 8 6 5 3 6 5 1 8 6 7 3 7 62.6 62nd 63rd 63rd 62nd 63rd 64th 62nd 62nd 62.9 63rd 62nd 62.6 63rd 7 0 1 4 2 3 4 7 1 5 3 8 56.3 54th 58th 56th 56th 59th 56th 56th 54th 59.2 56th 53rd 51.1 57th 7 9 3 3 | 3 5 * » 5 4 4 6

HU 227 195 Β1

0 / 132nd 133 138 138 10 Ac 1 ' 6 .0 .4 .8 * »» 122nd 122 110 110 4 .4 ^r .9 s * 3 125th 125 160 160 5 .5 .0 .0 4 ' 128th 128 114 111 S .3 .0 4.0 * 5 129th 128 129 129 0 .9 .8 , 7 6 ' 126th 126 118 117 7 ' B · 5 ' 10 1 0 126th 6 122nd 3 131st 4 133rd 4 .0 126 .5 122 .3 ' 131 .2 133 .3 .0 .9 D 170th 170 170 170 1 7 .8 .7 .9 2 71.4 71st 5 71st 3 71st 5 3 39.4 39th 3 39th 4 39th 3 4 24.5 24th 6 24th S 24th 7 4- 22.8 23rd 21st 21st Today 0 3 4 5 21.3 21st 3 22nd 9 23rd 1

Table 11 (continued)

137 125 124 134 135 .8 .3 .9 .9 .3 135 133 133 135 135 .8 .5 .3 .9 .6 131 122 122 130 129 .0 .6 .7 .2 .9 131 128 128 128 128 .8 .7 .5 .0 .8 124 127 127 124 124 .3 .8 .7 · .2 .0 127 126 126 126 126 .0 .3 ' .2 ^b .4 .3 126 12 6 . <· , 5 ^b 127 127 .8 · .8 133 133 .2 .2 134 134 .2 .5 170 170 170 170 170 .7 .8 .9 .7 .9 71st 71st 71st 71st 71st 3 3 5 4 5 39th 39th 39th 39th 39th 4 4 3 5 3 24th 24th 24th 24th 24th 5 5 7 6 7 21st 21st 21st 21st 21st 3 2 4 4 4 22nd 22nd 23rd 22nd 23rd 8 8 0 9 1

136th 6 137 .0 124 .3 • · 141 .1 n 123rd 123 161 • · 108 4 .1 .2 .4 d 138th 138 115 115th 163 4 .3 .3 2 .4 dd @ 1 JC. 128 129 129th 103 3 .4 .7 5 .9 t 138th 138 124 129th 163 4 .3 .5 5 .4 dd 123rd 123 126 124th 108 4 .1 .1 4 .4 170th 171 170 170th 170 8 .9 .7 8 .7 71.3 71st 5 71.5 71st 3 39.4 39th 3 39th 4 39.3 39th 5 24.5 24th 7 24th 6 24.7 24th 6 21.2 21st 4 21st 3 21.4 21st 3 22.9 23rd 1 22nd 9 23.1 22nd 9

P = position.

The spectra were obtained in _CDCl3. Chemical shifts for carbon atoms of units B and C, within ± 0.5 ppm, are identical to those for Cryptophycin-1.

* signs below CDCI ₃ ** signs could not be found a and ^b signs with the same index are interchangeable within a column

Cr.-168 and Cr.-169 have the 3'-OCH ₃ sign

At 55.3 (d). The 2 'and 4'-CH ₃ carbon atoms of Cr.-170 are at 18.9 (d) and 21.0 (d) respectively. The 2'-CH ₃ carbon atoms of Cr.-177 and Cr.-178 resonate at 19.0 (d) and 18.9 (d) respectively. The 3 'and 5'-CH ₂ signals of Cr.-179 at 21.3 (d) and the signals of the same groups at 21.2 (Cr.-180).

Example 97

Chlorohydrin analogs: Cryptophycin-163, -184, -185, -186, -187, -191, -192, -193, -194, -195, -212, -216, -217, -222, -223, - 224, -243, -252, -253, -263, -264, -265, -272 and -273 (Table 12).

The chlorohydrin analogs were prepared according to the reaction shown in Figure 17.

HU 227 195 Β1

Table 12 (data refers to compounds of Figure 17)

epoxide Cr. Price chlorine- ECH space- sfcerk. trans / cis? ai 157 191 192 Tsan cia 1.1: 1 16B '' Θ '· ¹ 195 Cranj Cl 3 1: < 0.1 170 216 217 Cranj cia 1: 2.6 179 -χτ ' ** ».. 222 Ciana eia <0.1: 1 172 * 03 193 194 tztna eia 1: 2 101 0? 223 Crana eia 1: < 0.1 200 • ρΛ f 212 T tan eia 1 i < 0.1 * · 104 Trana «« 224 106 CXJ 1: 2 ♦ * ♦ 243 cranj address 1: < 0.1

* Cryptophycin-200 changes only 50% under the circumstances. ** mixture of RR epoxides of 3,4- and 2,3-dimethylphenyl analogs.

*** A mixture of RR and SS epoxides.

Cryptophycin-163: To a solution of Cryptophycin-82 (12 mg, 0.018 mmol) in dichloromethane (1.5 mL) at 0 ° C was added m-CPBA (5 mg, 0.029) mmol, followed by 1.0 M hydrochloric acid (15 μΙ). , and allow the temperature to slowly rise to room temperature. After stirring under reflux, the solvent was evaporated and the residue was purified by flash chromatography on a 12 x 1 cm silica gel C18 column using water / acetonitrile as eluent. 12 mg (93%) of a mixture of chlorohydrin were obtained. Purification by reverse phase HPLC led to partial hydrolysis of the diols. However, 3.0 mg of Cryptophycin-163 (T _R : 37.2 min) were obtained by normal phase HPLC (Econosil silica gel, 250 x 10 mm, ethyl acetate / hexane, 1: 1). Proton and carbon NMR data are shown in Tables 13 and 14, respectively.

A 7: 3 mixture of Cryptophycin-184, -185, -186, -187 and -224: 3,4-dimethylbenzyl triphenylphosphonium chloride and 2,3-dimethylbenzyl triphenylphosphonium chloride in 3,4- and 2 Is converted to a mixture of 3-dimethylbenzyl triphenylphosphorane and reacted with Cryptophycin-108 as described previously for the preparation of Cryptophycin-152. An indivisible mixture of the four styrene isomers is obtained in a yield of 68%. 45 mg

The reaction mixture was reacted with 25 mg of m-CPBA in 3 ml of dichloromethane at room temperature with stirring for 15 hours. The solvent was removed and the residue was separated by HPLC (Econosil C18, 250 x 22 mm, water / acetonitrile 7: 3, 6 mL / min) into two fractions: I (T _R : 54.0 min) 20 mg and II ( T _R : 61.5 min) 12 mg. Fraction I (9 mg) in dichloromethane (2.0 mL) was treated with 40 μΙ TMSCI at -78 ° C and allowed to gradually rise to room temperature. After 3 hours, the solvent was evaporated and the residue was purified by reverse phase HPLC (Econosil C18, 10 μ, 250 x 22 mm, water / acetonitrile 35:65, 6 mL / min), 4.7 mg Cryptophycin-186 (T _R : 57.8 min), 2.3 mg of Cryptophycin-224 (T _R : 74.5 min) and 2.6 mg of Cryptophycin-184 (T _R : 78.2 min) are obtained. By the same procedure, the fraction II material was reacted with TMSCI and the compounds were separated, 0.7 mg Cryptophycin-185 (T _R : 65.0 min) and 8.0 mg Cryptophycin-187 (T _R :

62.2 minutes).

Cryptophycin-184: EIMS m / z (relative intensity) 682/684 (1.1 / 0.4), 236 (10), 195 (9), 135 (100), 119 (83); high resolution EIMS m / z 682.2999 (M ⁺ -HCl for C ₃₇ H ₄₇ CIN ₂ O ₈ calculated 2.2 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-185: EIMS m / z (relative intensity) 682/684 (6/2), 412/414 (14/5), 280/282 (14/5), 255 (10), 195/197 (50 / 16), 119 (100); high resolution EIMS m / z 682.3015 (M + -HCl, calcd for C ₃₇ H ₄₇ CIN ₂ O ₈ , 0.6 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-186: EIMS m / z (relative intensity) 682/684 (1.3 / 0.6), 412/414 (2.5 / 0.8), 254 (7), 195 (11), 119 ( 100); high resolution EIMS m / z 682.3015 (M ⁺ -HCl, calcd for C ₃₇ H ₄₇ CIN ₂ O ₈ , 0.6 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-187: EIMS m / z (relative intensity) 682/684 (0.9 / 0.2), 412/414 (3/1), 255 (3), 254 (7), 195 (11), 135 (100), 119 (94); high resolution EIMS m / z 682.3044 (M + -HCl, calcd for C ₃₇ H ₄₇ CIN ₂ O ₈ , -2.3 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-224: EIMS m / z (relative intensity) 682/684 (3/1), 412/414 (3/1), 255 (4), 254 (9), 195/197 (28/6), 119 (100); high resolution EIMS m / z 682.3064 (M ⁺ -HCl, calculated for C ₃₇ H ₄₇ CIN ₂ O ₈ , - 3.3 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-191, -192, -193, -194, -195, -212, -216, -217, -222 and -223:

General Procedure for the Preparation of Chlorohydrin: A solution of RR epoxide in chloroform is cooled to -60 ° C and reacted with 10 equivalents of trimethylsilyl chloride. After 2 hours, the solvent was evaporated and the residue was purified by reverse phase HPLC (Econosil C18, 10 μ 35% water / acetonitrile, 3 mL / min) to give the trans and cis chlorohydrin.

Experimental conditions and cytotoxicity data for the preparation of Cryptophycin-191, -192, -193, -194, -195, -212, -216, -217, -222 and -223 and -224 are summarized in the table.

Cryptophycin-195: EIMS m / z (relative intensity) 684/686 (3/2), 412/414 (12/4), 257 (11), 195/197 (46/15); high resolution EIMS m / z 684.2784 (M ⁺ -HCl, calcd for C ₃₆ H ₄₄ CIN ₂ O ₉ , delta 3.0 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-212: High Resolution EIMS m / z (M ⁺ -HCl calculated for C ₃₅ H ₄₁ CIF ₂ N ₂ O ₈ , delta ... mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-216: EIMS m / z (relative intensity) 682/684 (6/3), 412/414 (6/2), 255 (4), 195/197 (21/7); high resolution EIMS m / z 682.2990 (M ⁺ -HCl, calculated for C 37 H 47 ClN ² O 8, delta 3.1 mmu). ¹ H-NMR (CDCl 3), δ:

(A): 5.79 (2, d, 15.4), 6.70 (3, ddd, 15.4, 9.5 and 5.4),

2.36 (4, m), 2.67 (4, br dd, 14.9 and 4.9), 5.01 (5, m),

2.52 (6, m), 1.07 (6-Me, d, 7.1), 4.07 (7, br d, 9.8),

5.01 (8d, 9.8), 2.36 (2'-CH ₃ , s), 7.01 (2 ', br s), 2.31 (4'-CH ₃ , s), 7, 06 (5 ', brd, 8.1), 7.32 (6', d, 8.1); (B):

4.81 (2, m), 5.71 (2-NH, d, 8.5), 3.02 (3, dd,

-14.4 and 7.4), 3.16 (3, dd, -14.4 and 5.6), 7.23 (5, d,

2.0), 3.88 (7-OMe, s), 6.85 (8, d, 8.3), 7.09 (9, dd,

8.3 and 2.0); (C): 2.74 (2, m), 1.23 (2-Me, d, 7.1), 3.25 (3, m), 3.53 (3, m), 6.93 (3). -NH, br, m); (D): 4.93 (2, dd, 10.2 and 3.2), 1.49 (3, m), 1.68-1.85 (3/4, m), 0.95 (4 -Me, d, 6.4), 0.96 (5, d, 6.4).

Cryptophycin-217: EIMS m / z 682/984 (3/1), 412/414 (6/1), 255 (5), 195/197 (23/7); high resolution EIMS m / z 682.2920 (M ⁺ -HCl, calculated for C 37 H 47 ClN ² O 10, delta 10.0 mmu). ¹ H-NMR (CDCl 3), δ:

(A): 5.72 (2, d, 15.4), 6.66 (3, ddd, 15.4, 9.7 and 5.4),

2.17 (4, dt, 13.9 and 10.9), 2.53 (4, br dd, 13.9 and

5.0), 5.08 (5, m), 1.61 (6, m), 0.88 (6-Me, d, 6.8),

4.28 (7, br d, 9.6), 5.13 (8, d, 9.6), 2.38 (2'-CH ₃ s),

7.03 (2, brs), 2.34 (4'-CH ₃ , s), 7.03 (5 ', br d, 8.5),

7.17 (6 ', d, 8.5); (B): 4.80 (2, m), 5.66 (2-NH, d,

8.1), 3.03 (3, dd, -14.4 and 7.2), 3.14 (3, dd,

-14.4 and 5.6), 7.21 (5, d, 1.6), 3.87 (7-OMe, s), 6.83 (8, d, 8.3), 7.08 (9, dd, 8.3 and 1.7); (C): 2.74 (2, m),

1.24 (2-Me, d, 7.1), 3.29 (3, m), 3.50 (3, m), 6.96 (3-NH, br, m); (D): 4.89 (2, dd, 9.8 and 3.4), 1.52 (3, m), 1.87 (3, m), 1.72 (4, m), 0, 91 (4-Me, d, 7.3), 0.97 (5, d, 6.8).

Cryptophycin-222: EIMS m / z 718/720 (0.4 / 0.2), 682/684 (13/7), 412/414 (15/5), 280/282 (16/5), 255 ( 12), 195/197 (59/19), 119 (100); high resolution EIMS m / z 718.2767 (M + -HCl, calculated for C ₃₇ H ₄₈ Cl ₂ N ₂ O ₈ , delta 2.1 mmu). Cryptophycin-223:

HU 227 195 Β1

EIMS m / z 672/674 (1 / 0.8), 412/414 (3/1), 195/197 (15/3), 149 (100), 109 (35); high resolution EIMS m / z 708.2373 (calcd for C ₃₅ H ₄₃ FCl ₂ N ₂ O ₈ , delta 0.7 mmu).

^1H-NMR data in Table 13; ¹³ C-NMR data a

See Table 14.

Cryptophycin-243:

Epoxidation of Cryptophycin-238: Dissolve 11.8 mg of Cryptophycin-238 in 1.5 ml of dichloromethane and

9.6 mg m-CPBA was reacted. After 15 hours, the reaction mixture was diluted with 3 mL of dichloromethane and washed with 3 mL of 0.1 M phosphate buffer, pH 8. The organic phase is reacted with 10 μΙ of dimethylsulfide and washed again with phosphate buffer. The organic phase was separated and the evaporation residue (12 mg) was unsuccessfully purified by reverse phase HPLC (Econosil C18, 250 x 22 mm, 10 μ, 35% water / acetonitrile, 6 mL / min), RR and SS epoxide 6.2 mg. Reaction with TMSCI: The mixture was dissolved in 1 ml of chloroform and reacted with 15 μΙ of TMSCI at -60 ° C. After 2 hours, the solvent was evaporated. The residue (7 mg) was purified by normal phase HPLC (Econosil Si, 250 x 10 mm, 10 μ, 85% ethyl acetate / hexane, 4 mL / min). The fraction collected at 30 minutes was further purified by reverse phase HPLC (Econosil C18, 250 x 10 mm, 10 μ, 45% water / acetonitrile, 3 mL / min) to yield 2.4 mg of Cryptophycin-243.

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.76 (2, d, 15.0), 6.67 (3, ddd, 15.0, 9.6 and 5.4),

2.35 (4, dt, 14.4 and 10.4), 2.64 (4 br dd, 14.4 and

5.1), 5.07 (5, m), 2.50 (6, m), 1.04 (6-Me, d, 6.8),

4.02 (7, br d, 9.8), 4.68 (8, d, 9.8), 7.39 (273747576 ', br s), 4.70 (4'-CH ₂ OH, br s); (B): 4.74 (2, m), 5.72 (2-NH, d, 7.8), 2.99 (3, dd, -14.4 and

7.3), 3.14 (3, dd, -14.4 and 5.6), 7.22 (5, d, 2.0), 3.88 (7-OMe, s), 6.84 (8, d, 8.4), 7.08 (9, dd, 8.4 and 2.0);

(C / 2.73 (2, m), 1.21 (2-Me, d, 7.1), 3.23 (3, dt,

13.7 and 7.1), 3.50 (3, dt, 13.7 and 4.5), 6.93 (3-NH, br dd, 7.1 and 4.5); (D): 4.91 (2, dd, 10.0 and 3.4), 1.47 (3, m), 1.64-1.84 (3/4, m), 0.94 (4 -Me, d, 6.4), 0.95 (5, d, 6.3).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.6 (1), 125.0 (2), 141.9 (3), 36.3 (4), 76.4 (5),

38.3 (6), 8.7 (6-Me), 74.0 (7), 62.0 (8), 137.7 (T),

127.4 ^a (276 '), 128.2 ^a (375'), 142.2 (4 '), 64.6 (4'CH ₂ OH);

B 171.1 ^b (1), 53.6 (2), 35.1 (3), 129.9 (4) 131.0 (5) 122.4 (6) 154.0 (7 ), 56.2 (7-OCH ₃ ), 112.3 (8),

128.4 (9);

C: 175.4 (1), 38.5 (2), 14.1 (2-Me), 41.2 (3);

D 170.5 ^b (1) 71.4 (2), 39.7 (3), 24.8 (4), 23.2 (4-Me), 21.6 (5).

^a and ^b are interchangeable

Cryptophycin-252 and -253

Epoxidation of Cryptophycin-247: 12 mg of Cryptophycin-247 was dissolved in 1 ml of dichloromethane and reacted with 9.8 mg of m-CPBA. After 15 hours, the reaction mixture was diluted with 3 mL of dichloromethane and 3 mL

Wash with 0.1 M phosphate buffer, pH 8. The organic layer was separated, reacted with 15 mL of dimethyl sulfoxide and washed repeatedly with phosphate buffer. The organic phase was evaporated and the residue (11.5 mg) was purified by reverse phase HPLC (Econosil C18, 250 x 10 mm, 10 μ, 35% water / acetonitrile, 3 ml / min) to give 5 mg of RR epoxide and 3 mg of SS epoxide. RR-epoxide:

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.74 (2, d, 15.6), 6.67 (3, ddd, 15.6, 9.5, and

5.6), 2.44 (4, dt, 14.7 and 10.5), 2.50-2.58 (4 m),

5.15 (5, m), 1.62-1.81 (6, m), 1.12 (6-Me, d, 6.8),

2.91 (7, dd, 7.3 and 1.5), 3.66 (8, d, 1.5), 7.38 (2 ', br s), 6.54 (3'-NH, s), 1.51 (3'-NH-β-BOC / 7.21-7.30 (475 ', m), 6.91 (6', br d, 6.8); (B): 4 , 79 (2, m), 5.59 (2-NH, d, 8.5), 3.00 (3, dd, 14.5 and 7.3), 3.13 (3, dd, 14, 5 and 5.5), 7.20 (5, d, 2.0), 3.86 (7-OMe, s), 6.83 (8, d, 8.3), 7.06 (9, dd, 8.3 and 2.0) (C): 2.70 (2, m), 1.22 (2-Me, d, 7.1), 3.30 (3, m), 3, 46 (3, m), 6.97 (3-NH, m); (D): 4.83 (2, dd, 10.0 and 3.4), 1.36 (3, m), 62-1.81 (3/4, m), 0.85 (4-Me, d, 5.9), 0.87 (5, d, 5.6).

Deprotection of the amino group and preparation of the chlorohydrin: 8.0 mg of RR epoxide in 100 μΙ of dichloromethane was reacted with 50 μΙ of 4N hydrochloric acid in dioxane. After 30 minutes, the solvent was removed and the residue was purified by reverse phase HPLC (Econosil C18, 250 x 10 mm, 10 μ, 40% water / acetonitrile, 3.5 mL / min), 5 mg Cryptophycin-242 (T _R). : .... min) and 2 mg of Cryptophycin-238 (Tr: ... min) were obtained.

Cytotoxicity of Cryptophycin-252: 1.20-fold and 1.25-fold, respectively, of KB and LoVo activity against Cryptophycin-1.

Cryptophycin-252: EIMS m / z 670/672 (7.2 / 2.2), 669/671 (19/2 / 7.8), 280/282 (6.8 / 2.4), 242 (4 , 7), 195/197 (43/12); high resolution EIMS m / z 669.2856 (M ⁺ -2HCl-H calcd for C ₃₅ H ₄₄ CIN ₃ O ₈ , delta - 3.9 mmu).

¹ H-NMR (MeOH), δ:

(A): 5.93 (2, d, 15.1), 6.61-6.78 (3, m), 2.33 (4, dt,

14.7 and 9.8), 2.69-2.80 (4, m), 5.11 (5, m), 2.46 (6, m), 0.95-1.04 (6- Me, m), 3.99 (7, br d, 9.4), 4.62 (8, d, 9.4), 7.28 (2 ', brs), 7.17 (4', brd) , 8.1), 7.07 (5 ', t,

8.1), 6.98 (6 ', d, 8.1); (B): 4.50 (2, dd, 10.5 and 3.2),

2.69-2.80 (3, m), 3.18 (3, brd, 13.7), 6.61-6.78 (5 and 9, m), 3.86 (7-OMe, s) ; (C): 2.69-2.80 (2, m),

1.19 (2-Me, d, 7.3), 3.28 (3, br d, 13.4), 3.58 (3, br d, 13.4); (D): 5.01 (2, dd, 9.8 and 2.7), 1.53 (3, m),

1.70-1.84 (3/4, m), 0.95-1.04 (4/5-Me, m).

C ¹ H NMR (MeOH) of Cryptophycin-253, δ:

(A): 5.84 (2, dd, 15.3 and 1.6), 6.60-6.74 (3, m), 2.10 (4, dt, 14.9 and 11.2). , 2.64 (4, m), 5.05 (5, m), 1.50 (6, m), 0.90 (6-Me, d, 6.8), 4.01 (7, dd) , 9.5 and 1.2),

4.75 (8, d, 9.5), 6.60-6.74 (27476 ', m), 7.09 (5', t,

7.8); (B): 4.48 (2, dd, 11.4 and 3.9), 2.71 (3, dd,

14.4 and 11.4), 3.16 (3, dd, 14.4 and 3.7), 7.26 (5, d,

2.2), 3.86 (7-OMe, s), 6.96 (8, d, 8.4), 7.15 (9, dd,

8.4 and 2.2); (C): 2.76 (2, m), 1.18 (2-Me, d, 7.3),

HU 227 195 Β1

3.26 (3, m), 3.58 (3, dd, 13.5 and 3.1); (D): 4.94 (2, dd, 9.4 and 3.8), 1.58 (3, m), 1.80 (3, m), 1.70 (4, m), 0, 97 (4-Me, d, 6.1), 0.98 (5, d, 6.3).

Cryptophycin-263, -264, and -265: 26 mg of a mixture of Cryptophycin-242 and SS-epoxide was dissolved in 5 mL of dichloromethane and reacted with-120 ° C, 1.0 M TMSCI in dichloromethane at -78 ° C. After 3 hours, the solvent was evaporated and the 27 mg residue was purified by reverse phase HPLC (Econosil C18, 250 x 22 mm, 10 μ 10, acetonitrile / water 65:35, 6 mL / min), 6.0 mg Cryptophycin-264- t (T _R : 43 min) and

15.8 mg of a mixture of Cryptophycin-263 and -265 (T _R : 59 min) were obtained. Cryptophycin-263 and -265 were separated by normal phase HPLC (Econosil silica gel, 250 x 10 mm, 10 μ, ethyl acetate / hexane 55:45, 3 mL / min), 7.1 mg Cryptophycin-265- five (T _r : 51 min) and 7.8 mg of Cryptophycin-263 (T _r : 79.0 min) were obtained.

Cytotoxicity data for KB and LoVo cell lines relative to Cryptophycin-1 activity (1):

cryptophycin KB LoVo 263 1.67 0.56 264 0,044 0.023 265 .0033 0,021 Cryptophycin-263

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.79 (2, d, 15.6), 6.68 (3, m), 2.37 (4, m), 2.66 (4, brdd, 14.2 and 4.2). ), 5.10 (5, m), 2.49 (6, m), 1.03 (6-Me, d, 6.8), 4.00 (7, br d, 9.4), 4 , 64 (8, d, 9.4), 7.35 (276 ', br d, 7.9), 7.29 (375', br d, 7.9), 4.31 (4'CH ₂ NH-BOC, d; 4.8), 4.92 (4'-CH ₂ NH-t-BOC, brs), 1.45 (4'-CH ₂ NH-t-6 OC, s), (B ): 4.79 (2, m), 5.76 (2-NH, d, 8.6), 3.01 (3, dd, -14.4) and

7.3), 3.15 (3, dd, -14.4 and 4.9), 7.23 (5, d, 1.3), 3.87 (7-OMe, s), 6.84 (8, d, 8.4), 7.09 (9, br d, 8.4); (C): 2.73 (2, m), 1.22 (2-Me, d, 7.2), 3.24 (3, m), 3.53 (3, m), 6.93 ( 3-NH, br t, 5.5); (D): 4.92 (2, dd, 9.7 and 3.1), 1.37 (3, m), 1.48 (3, m), 1.78 (3, m), 1, 73 (4, m), 0.93 (4-Me, d, 6.4), 0.94 (5, d, 6.4).

³ C-NMR (CDCl 3), δ:

A: 165.6 (1), 125.3 (2), 141.6 (3), 36.4 (4), 76.5 (5),

38.3 (6), 8.7 (6-Me), 74.1 (7), 61.8 (8), 137.5 ^a (1 '),

128.3 (276 '), 128.0 (375'), 140.3 ^a (4 '), 44.2 (4'CH ₂ NH-tert-BOC), 28.4 and 155.9 (4'-CH). ₂ NH-tercBOQ;

B 171.1 ^b (1), 53.7 (2), 35.1 (3), 130.0 (4) 131.1 (5) 122.4 (6) 154.0 (7 ), 56.2 (7-OCH ₃ ), 112.3 (8), 128.5 (9);

C: 175.4 (1), 38.4 (2), 14.1 (2-Me), 41.3 (3);

D: 170.6 ^b (1), 71.4 (2), 39.8 (3), 24.8 (4) 23.2 (4-Me), 21.6 (5) ^a and ^{b are} the same indexes are interchangeable

¹ H-NMR (CDCl ₃ ) of Cryptophycin-264, δ:

(A): 5.71 (2, d, 15.4), 6.63 (3, ddd, 15.4, 9.9 and 5.5), 2.12 (4, m), 2.51 (4, brdd, 14.2 and 5.0), 5.05 (5, m),

1.47 (6, m), 0.92 (6-Me, d, 6.8), 4.06 (7, dd, 9.6 and

1.5), 4.87 (8, d, 9.6), 7.29 (276 ', br d, 8.4), 7.27 (375', br d, 8.4), 4, 33 (4'-CH ₂ NH-tert-BOC, br s),

1.47 (4'-CH ₂ NH-t-BOC, s); (6 / -4.80 (2, m), 5.69 (2-NH, br s), 3.01 (3, dd, -14.5 and 7.2), 3.13 (3, dd) .

-14.5 and 5.4), 7.21 (5, d, 2.0), 3.86 (7-OMe, s), 6.83 (8, d, 8.4), 7.07 (9, dd, 8.4 and 2.0); (C): 2.73 (2, m),

1.23 (2-Me, d, 7.3), 3.28 (3, m), 3.52 (3, m), 6.93 (3-NH, br t, 5.7); (D): 4.86 (2, m), 1.50 (3, m), 1.86 (3, m), 1.73 (4, m), 0.95 (4-Me, d, 6.6), 0.98 (5, d,

6.8).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.4 (1), 125.1 (2), 141.4 (3), 36.2 (4), 76.0 (5),

38.3 (6), 8.8 (6-Me), 74.3 (7), 68.4 (8), 136.6 ^a (1 '),

128.0 (276 '), 127.7 (375'), 140.3 ^a (4 '), 44.2 (4'CH ₂ NH-tert-BOC), 28.4 (4'-CH ₂ NH) -t-BOC /

B: 171.0 ^b (1), 53.6 (2), 35.0 (3), 129.9 (4), 131.0 (5),

122.4 (6), 153.9 (7), 56.1 (7-OCH ₃ ), 112.2 (8),

128.4 (9);

C: 175.4 (1), 38.5 (2), 14.1 (2-Me), 41.2 (3);

D: 170.4 ^b (1), 71.4 (2), 39.8 (3), 24.8 (4) 23.2 (4-Me), 21.7 (5) ^a and ^{b are} the same indexes are interchangeable

Cryptophycin-265:

¹ H-NMR (CDCl ₃ ), δ:

(A): 5.74 (2, d, 15.5), 6.68 (3, ddd, 15.5, 9.9 and

5.3), 2.36 (4, m), 2.54 (4, br dd, 14.2 and 4.9), 5.40 (5, m), 1.87 (6, m), 1.05 (6-Me, d, 7.0), 3.71 (7, brt,

5.8), 5.07 (8, d, 5.3), 7.36 (276 ', d, 8.2), 7.28 (375', br d, 8.2), 4.30 (4'-CH ₂ NH-tert-BOC, br s), 4.91 (4'-CH ₂ NH-tert-BOC, br s), 1.46 (4'-CH ₂ NH-tert-BOC, br) , (B): 4.78 (2, m), 5.70 (2-NH, br s), 3.03 (3, dd, -14.5 and 7.1), 3.12 (3, dd, -14.5 and 5.5), 7.21 (5, d, 1.8), 3.87 (7-OMe, s), 6.84 (8, d, 8.4), 7 , 07 (9, dd, 8.4 and 1.8); (C): 2.72 (2, m), 1.23 (2-Me, d, 7.1), 3.33 (3, m), 3.48 (3, m), 6.97 ( 3-NH, br t, 5.7); (D): 4.83 (2, dd, 9.7 and 4.3), 1.43 (3, m), 1.70 (3, m), 1.63 (4, m), 0, 93 (4-Me, d, 6.4), 0.88 (5, d, 6.4).

¹³ C-NMR (CDCl ₃ ), δ:

A: 165.5 (1), 125.0 (2), 142.0 (3), 34.6 (4), 74.4 (5),

39.8 (6), 12.9 (6-Me), 77.5 (7), 66.7 (8), 137.3 ^a (1 '),

127.9 (276 '), 127.8 (375'), 139.9 ^a (4 '), 44.5 (4'CH ₂ NH-tert-BOC), 28.4 (4'-CH ₂ NH-tert) -6OC / ·

B: 171.0 ^b (1), 53.6 (2), 35.1 (3), 129.9 (4), 131.0 (5),

122.4 (6), 154.0 (7), 56.1 (7-OCH ₃ ), 112.3 (8),

128.4 (9);

C: 175.6 (1), 38.4 (2), 14.1 (2-Me), 41.3 (3);

D: 170.3 ^b (1), 71.5 (2), 39.4 (3), 24.7 (4) 22.8 (4-Me), 21.6 (5) ^a and ^b indexes are interchangeable

Cryptophycin-272:

Cryptophycin-263 (4.9 mg, 0.006 mmol) in dichloromethane (30 μΙ) was treated with 4N hydrochloric acid in dioxane (30 μΙ, 0.012 mmol) at room temperature. After 3 hours, the solvent was removed and the residue was removed under reduced pressure for 2 days to remove traces of dioxane. 4.4 mg (98%) of Cryptophycin-272 are obtained.

HU 227 195 Β 11 ¹ H-NMR (CDCl ₃ ), δ:

(A): 5.94 (2, dd, 15.0, 1.8), 6.69 (3, ddd, 15.0,

11.1 and 4.9), 2.36 (4, m), 2.72 (4, m), 5.13 (5, m),

2.48 (6, m), 1.01 (6-Me, d, 7.1), 4.02 (7, dd, 9.4 and 2.0), 4.84 (8, d, 9). , 4), 7.45 (276 ', d, 8.4), 7.51 (375', d, 8.4), 4.11 (4'-CH ₂ NH ₂ HCl, s); (B): 4.51 (2, dd,

11.2 and 3.9), 2.74 (3, m), 3.18 (3, dd, -14.5 and 3.9), 7.28 (5, d, 2.2), 3.84 (7-OMe, s), 6.98 (8, d, 8.4),

7.17 (9, dd, 8.4 and 2.3); (C): 2.77 (2, m), 1.19 (2-Me, d, 7.5), 3.29 (3, m), 3.58 (3, dd, 13.7 and 3). , 3); (D): 5.02 (2, dd, 9.5 and 3.8), 1.56 (3, m), 1.78 (3, m), 1.78 (4, m), 0, 97 (4-Me, d, 6.6), 0.99 (5, d, 6.6).

¹³ C-NMR (CD3OD), δ:

A: 168.4 (1), 125.4 (2), 144.0 (3), 37.7 (4), 77.3 (5),

40.4 (6), 8.1 (6-Me), 74.8 (7), 63.3 (8), 142.5 ^a (1 '), 130.0 (276'), 130.3 (375 '), 134.6 ^a (4'), 44.0 (4'CH ₂ NH ₂ HCl).

B: 174.1 (1), 57.4 (2), 36.3 (3), 132.2 (4), 131.5 (5),

123.3 (6), 155.4 (7), 56.6 (7-OCH ₃ ), 113.5 (8), 129.3 (9);

C; 177.5 (1), 39.1 (2), 15.1 (2-Me), 41.2 (3);

D: 172.1 (1), 72.8 (2), 41.2 (3), 26.0 (4) 22.1 (4-Me),

23.6 (5).

interchangeable signals ^of the same index Cryptophycin-273: To 128 mg of 3-chloroperbenzoic acid at 0 ° C was added a solution of 146 mg of Cryptophycin-234 in 8 ml of dichloromethane. Allow the temperature of the solution to slowly reach room temperature and stir for 12 hours. The reaction mixture was diluted with dichloromethane (7 mL) and washed with 2x10 mL of 0.1 M phosphate buffer (pH 8). The organic layer was separated and the excess perbenzoic acid was decomposed with 50 mL of dimethyl sulfide and washed again with phosphate buffer. The organic phase was evaporated and the residue was purified by reverse phase HPLC (250 x 22 mm, 10 μ, 15% water / acetonitrile, 6 mL / min) to give Cryptophycin-269 precursor RR epoxide (74 mg, 50%).

of a solution of 3 mg of epoxide in 3 ml of THF was reacted with 115 μΙ of 1 M tetrabutylammonium fluoride in dichloromethane. After 1.5 hours, a saturated solution of ammonium chloride was added to the reaction mixture and extracted with ethyl acetate (2 x 30 mL). The organic extract was dried over magnesium sulfate and evaporated. The residue was purified on a short silica gel column using 50-100% ethyl acetate / dichloromethane as eluent. 62 mg (98%) of Cryptophycin-369 are obtained.

To a solution of 0 mg of Cryptophycin-269 mg, 30 ml of N- (tert-Boc) glycine and 8 mg of DMAP in 3 ml of dichloromethane was added 160 μΙ of 1 M DCC in dichloromethane. The reaction mixture was stirred at 0 ° C for 15 minutes, at room temperature for 2 hours, then filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on ODS silica gel using 75-30% water / acetonitrile as eluent. 64.4 mg (85%) of N-tert-Boc-glycinate ester of Cryptophycin-269 are obtained.

To a solution of 64.4 mg of glycinate ester in 3 ml of chloroform at -60 ° C is added 200 μΙ of 1 M TMSCI in dichloromethane. After 1 hour, the solvent was evaporated and the residue was purified on a short silica gel column using 40-90% ethyl acetate / hexane as eluent. 60 mg (89%) of the trans-chlorohydrin of the glycate of the given formula are obtained.

Chlorohydrin (mg) in dichloromethane (0.25 mL) was treated with 4 M hydrochloric acid in 1,4-dioxane (0.1 mL). The reaction mixture was stirred at room temperature for 2 hours, then concentrated under reduced pressure and freeze-dried for 48 hours. Cryptophycin-273 of the formula given is obtained as a white solid.

Table 13

The cryptophycin ¹ H-NMR data

f> fiction 14J itu TM5 184 187 t4t 192 143 144 143 212 222 223 224 THE 2 3.79 3.80 5.79 3.74 3.71 5.75 3.80 3.68 3.80 3.47 3.79 3.80 5.71 3.80 3.79 .1 6:49 ddd 4.70 4.70 4.68 4:44 4:49 6.70 6:43 4.70 4.62 6.69 6.68 .6.45 6.69 6.69 4Λ 137 133 (Π 138 m 136 m 113 m 137 itl 140 110 1140 2:09 137 140 117 2.39 137 45 167 m 173 m 170 m 133 m 133 m 1:39 m 170 147 169 2:31 168 144 134 2.60 168 5 3:10 ddd 3:00 3:12 3:32 m 106 RFI 3:40 m 3:14 3:07 3:13 bfi 5:07 5:11 5:10 5:07 5:11 5:11 6 UO m 2.83 2..U 1.9TD 1:30 m Ι.Λ3 m 143 1.64 2:33 1:57 149 2:47 1:31 2:48 2:30 6-Mc 1:03 d 1:13 1:09 1.09 0.93 t.Oó 1:18 0.95 1:09 0.90 1:04 1.Q3 0.93 1:04 j. »4

HU 227 195 Β1

Table 13 (continued)

The spectra were obtained in _CDCl3. The chemical shift data for protons or methyl or methoxy at the indicated carbon atoms are given in the table. The chemical shift data for the protons of units B and C are within ± 0.2 ppm and the coupling constants within ± 0.5 Hz are identical to those for Cryptophycin-8. Data for Cryptophycin-163 J (H, H) in Hz: 2 ', 3 -5', 6 '= 8.7 Hz; Cr-196: 2.6 = 2.0 Hz, 576 '= 7.7 Hz; Cr.-193 and 194: 3 ', 4' = 8.5 Hz; Cr-195: 2 ', 3' = 3 ', 4' = 7.9 Hz;

2 ', 6 -4', 6 -2.4 Hz The coupling constants of the other protons in the table are within ± 0.5 Hz of the data of Crypto50 phycin-8. Data for Cr-212 J (H, H) and J (H, F): 3'F, 4H = 4H, 5F = 8.7 Hz; 2 ', 4' = 4 ', 6' = 2.3 Hz.

Table 14

The cryptophycin ¹ H-NMR data

P β 1Í3 184 1β5 IAA 197 I91 ^f 192 ^f 194 195 212 222 223 224 THE 1 165 .5 ITIS . 4 165. 4 165 . 4 165 5 165.5 165.4 165th 4 165.5 165.5 165. 4 145.5 165.5 2 125 . 1 125 .3 125 .2 124 .9 t25 .0 125 .0 125.1 125.4 125th 1 125.1 125.4 125.1 125.3 125.2

HU 227 195 Β1

Table 14 (continued)

P = Position

The spectra were obtained in _CDCl3. The chemical shifts of the carbon atoms of units B and C are within ± 0.5 ppm of Cryptophycin-8.

* due to the small sample size, no signs were observed.

** the symbols below the solvent symbols are t test assignments

The 4-OMe carbon atoms of Cr.-163 and Cr.-195 resonate at δ 55.3. The Cr-195 2'-Me and 3'-Me carbon atoms show δ 15.0 and 20.9, respectively.

Signs of the 3'-Me and 4'-Me carbon atoms of Cr.-186 are observed at δ 19.6 and 19.9 respectively. The same carbon atoms appear at δ 19.5 and 19.8 at Cr.-187 and at 19.5 and 19.8 at Cr.-224, respectively. The 3 'and 5'-Me carbon atoms of Cr.-223 occur at 21.3.

HU 227 195 Β1

Example 98

The meanings of the R group of the compound of formula 2 '(Figure 18) are listed in Table 15, one being an ether group and the other being various acyl groups. The novel Cryptophycin analogs can be prepared by esterification in 5 medium or high yields; see Mulzer, J., Trost, Β. M., Fleming, I., edited by Comprehensive Organic Synthesis (Pergamon Press, Oxford, 1991), 6: 323-380; Benz, G., Trost, Β. M. and

Fleming, J., edited by Comprehensive Organic Synthesis (Pergamon Press, Oxford, 1991), 6: 381-417. The hydrochloride salts are prepared in high yield from the corresponding tert-butyl carbamates by treatment with hydrochloric acid; disodium salt (7 ') is prepared from compound (6') by cleavage of the tert-butyl ester and reaction with sodium hydroxide. The pyridinium salt (9 ') is prepared from compound (T) following the procedure of Nicolan et al., Angew. Chem. Int. Ed. Engl., 33: 1583 (1994)].

Table 15

The R group of the compound of formula 2 'is the compound ^R

jjAs-rx i HCl o

γΛ ^, ΝΜ, ΙΜΟ

Ο ο

HHO co>

ο θ mCO ^ Bu rXj Μ HQ ^ Μ-ΟΟ, Ι-βυ

Χ3

ΝΗ, I ΜΟ

ΟΗ

WC1

ΟΗ ^Ν Υ ~ ΝΜ, ΚΗΟ Ο ⁰

HU 227 195 Β1

The conjugates listed in Table 15 are prepared by a variety of methods, which generally consist of activated ester methodology followed by chromatography and, if necessary, deprotection. Compound (1 ') was reacted with acetic anhydride in the presence of triethylamine and 4-dimethylaminopyridine to give compound (3') in 89% yield after flash chromatography. Similarly, using succinic anhydride and subsequent reverse phase HPLC purification, compound (5 ') can be prepared from compound (1'). Alternatively, compound (5 ') can be prepared in high yield by acid treatment of mono-tert-butyl ester (4'). Reaction of the pyridine solution of compound (1 ') with commercially available nicotinoyl chloride hydrochloride in the presence of triethylamine and 4-dimethylaminopyridine followed by chromatography on hydrogen chloride afforded compound (8') in high yield. Compound (9 ') can be prepared in 47% yield by the aforementioned Nicolaou method, whereby compound (1') is treated with commercially available 2-fluoro-1-methylpyridinium p-toluenesulfonate followed by reverse phase HPLC with an anion exchange (replacing the p-toluenesulfonate group with acetate) and finally freeze-drying. The other esters can be obtained in moderate to high yields from Compound V and commercially available N-tert-Boc protected amino acids (except 4 ', 6', 10 ', 43' and 45 ') 1,3- activation with dicyclohexylcarbodiimide in the presence of 4-dimethylaminopyridine. The hydrochloride salts can be obtained in high yield by reaction with 4.0 M hydrochloric acid in dioxane and removal of the solvent under reduced pressure. The disodium salt (7 ') is prepared from compound (6') by cleaving the tert-butyl ester with hydrochloric acid and reacting the intermediate with sodium hydroxide. The (24 ') acid required for the preparation of (5') can be prepared in 63% yield using the five-step procedure of the Johns method for introducing the phosphate group (Perich, J.W., Johns, R.B., et al., Synthesis, 1988, 142).

Some of the novel conjugates were assayed for cytotoxicity in vitro in a Gc3 tumor cell model as described above. The results are summarized in Table 16.

Table 16

Results of in vitro cytotoxicity assays for cryptophycins

Compound Gc3 IC ₅₀ (nM) 1 ' 0,065 3 ' 83 5 ' 31 7 ' 3.7 8 ' 116 9 ' 2.2 11 ' 6.8

Compound Gc3 IC ₅₀ (nM) 13 ' 7.0 15 ' 60 17 ' 3.6 19 ' 0.10 21 ' 21 25 ' 230 28 ' 2.6 30 ' 30 32 ' 7.2 34 ' 6.5 36 ' 2.8 38 ' 1.6 40 ' 2.0 42 ' 15 44 ' 28 46 ' 50 47 ' 2.5 48 ' 10

Stability studies were performed in aqueous media at pH 4-8, determining the percentage of intact compound.

The tert-butyl carbamates can be converted to the corresponding salt by any strong acid. The acid may be a mineral acid such as hydrogen halide, hydrogen sulfate, hydrogen phosphate, hydrogen nitrate, hydrogen perchlorate or a strong organic acid such as trifluoroacetic acid, p-toluenesulfonic acid or methanesulfonic acid. The same acids can be used to prepare the type (8 ') salts from the corresponding free bases. Salts of the type (7 ') may be prepared with a variety of counterions (cations) including any alkali metal or alkaline earth metal cation. Various counterions (anions) may be involved in the formation of the (9 ') salts.

Preparation of Cryptophycin-55-acetate (3 ') (LSN 362376) To a solution of (1') (mg) (0.13 mmol) in methylene chloride (659 μ9) at 0 ° C was added triethylamine (55 μΙ, 0.40 mmol). 1.6 mg (0.013 mmol) of 4-dimethylaminopyridine and 19 μΙ (0.20 mmol) of acetic anhydride. After stirring for 1 hour at 0 ° C, 19 μΙ of methanol was added to the reaction mixture, concentrated to half volume, and the product was flash chromatographed on a 19 g silica gel column using ethyl acetate / hexane (3: 1). 88 mg (89%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.38-7.31 (m, 5H), 7.24 (d, 1H, J = 2.1 Hz),

7.22-7.18 (m, 1H), 7.10 (dd, 1H, J = 8.5, 2.1 Hz),

6.88 (d, 1H, J = 8.5 Hz), 6.75 (ddd, 1H, J = 15, 13,

4.6 Hz), 5.78 (dd, 1H, J = 15, 1.0 Hz), 5.55 (d, 1H,

J = 7.9 Hz), 5.46 (dd, 1H, J = 9.8, 1.2 Hz), 4.95 (dd,

HU 227 195 Β1

1H, J = 11, 2.9 Hz), 4.89 (ddd, 1H, J = 9.9, 9.9,

1.7 Hz), 4.81 (d, 1H, J = 9.8 Hz), 4.79-4.74 (m, 1H),

3.91 (s, 3H), 3.39 (dd, 1H, J = 13, 8.1 Hz), 3.22 (dd,

1H, J = 13.4.1 Hz), 3.16 (dd, 1H, J = 14, 5.1 Hz), 3.07 (dd, 1H, J = 14, 7.6 Hz), 2, 65-2.55 (m, 2H),

2.47-2.39 (m, 1H), 1.95 (ddd, 1H, J = 14, 13,

4.6 Hz), 1.86-1.77 (m, 1H), 1.73-1.66 (m, 1H), 1.68 (s, 3H), 1.27 (s, 3H), 1.19 (s, 3H), 1.09 (d, 3H,

J = 7.1 Hz), 1.03 (d, 3H, J = 6.7 Hz), 0.97 (d, 3H,

J = 6.6 Hz).

Preparation of Cryptophycin-55-succinate tert-butyl ester (4 ') (LSN 384665)

A solution of 133 mg (0.188 mmol) (1 '), 66 mg (0.377 mmol) of mono-tert-butyl succinate and 2.3 mg (0.019) mmol of 4-dimethylaminopyridine in 850 μΙ anhydrous methylene chloride was added at room temperature. a solution of 1,3-dicyclohexylcarbodiimide (78 mg, 0.377 mmol) in methylene chloride (92 μΙ) was added. After stirring for 1 hour at room temperature, celite (150 mg) was added to the reaction mixture, diluted with ethyl acetate / hexane (3: 1) (1 mL) and the mixture was filtered through celite, washed with ethyl acetate / hexane (3: 1). The filtrate and the washings were combined and concentrated under reduced pressure to give an off-white oily product. The product was chromatographed on 25 g of flash silica gel eluting with ethyl acetate / hexane (2: 1). 157 mg (97%) of the desired compound are obtained in the form of a white foam. ¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.38-7.30 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.22-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.5, 2.0 Hz),

6.88 (d, 1H, J = 8.5 Hz), 6.74 (ddd, 1H, J = 15, 10,

4.6 Hz), 5.76 (d, 1H, J = 15 Hz), 5.51 (d, 1H,

J = 8.0 Hz), 5.47 (d, 1H, J = 9.6 Hz), 4.97 (dd, 1H,

J = 11.3.1 Hz), 4.901, 1H, J = 9.3 Hz), 4.83 (d, 1H, J = 9.6 Hz), 4.79-4.73 (m, 1H) , 3.91 (s, 3H), 3.39 (dd,

1H, J = 13.8.0 Hz), 3.22 (dd, 1H, J = 13.4.0 Hz), 3.16 (dd, 1H, J = 14, 5.1 Hz), 3, 08 (dd, 1H, J = 14, 7.6 Hz),

2.63-2.55 (m, 2H), 2.44-2.37 (m, 1H), 2.32-2.21 (m, 2H), 2.19-2.06 (m, 2H) ), 1.99-1.92 (m, 1H),

1.85-1.68 (m, 2H), 1.42 (s, 9H), 1.27 (s, 3H), 1.19 (s, 3H), 1.08 (d, 3H, J = 7.0 Hz), 1.03 (d, 3H,

J = 6.6 Hz), 0.98 (d, 3H, J = 6.5 Hz).

Preparation of Cryptophycin-55 succinate (5) (LSN 377092) from Cryptophycin-55 succinate tert-butyl ester

To a solution of 127 mg (0.147 mmol) of (4 ') in 491 μΙ of methylene chloride at room temperature was added 184 μΙ (0.737 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. The reaction mixture was stirred at room temperature for 5 hours and then concentrated under reduced pressure to give 119 mg (100%) of the title compound as a foam.

Preparation of Cryptophycin-55-succinate (5) (LSN 377092) from succinic anhydride at room temperature was added 16 mg (0.038 mmol) (1 ') and 5.7 mg (0.057 mmol) succinic anhydride in 383 μΙ methylene chloride at room temperature. Triethylamine (0.115 mmol) and 4.7 mg (0.038 mmol)

4-dimethyl-aminopyridine. After 19 hours stirring, another

5.7 mg (0.057 mmol) of succinic anhydride and

4-Dimethylaminopyridine (4.7 mg, 0.038 mmol) was added and stirring was continued for 29 hours. To the reaction mixture was added 0.5 mL of 1 N aqueous hydrochloric acid and washed with methylene chloride (3 x 0.5 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated under reduced pressure to give a white foam. The product was purified by reverse phase HPLC to give 10 mg (32%) of the title compound as a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.36- 7.31 (m, 6H), 7.22 (br s, 1H), 7.08 (d, 1H, J = 8.4 Hz), 7.02 (br s, 1H), 6.87 (d, 1H, J = 8.4 Hz), 6.61 (m, 1H), 5.94-5.87 (m, 2H), 5.51 (d, 1H, J = 9.8 Hz) , 4.95 (dd, 1H, J = 10, 2.8 Hz), 4.87-4.76 (m, 3H), 3.90 (s, 3H), 3.39 (dd, 1H, J = 16, 5.6 Hz), 3.28 (dd, 1H, J = 16, 8.2 Hz), 3.17 (dd, 1H, J = 16,

5.6 Hz), 3.05 (dd, 1H, J = 16.8.2 Hz), 2.68-2.62 (m, 1H), 2.60-2.46 (m, 2H), 2.45-2.28 (m, 3H), 2.01-1.93 (m, 2H), 1.88-1.70 (m, 2H), 1.26 (s, 3H), 1, 18 (s, 3H), 1.12 (d, 3H, J = 7.0 Hz), 1.04 (d, 3H, J = 6.6 Hz), 1.00 (d, 3H, J = 6 , 5 Hz).

Preparation of Cryptophycin-55- (2'-di-tert-butylphosphatyl) phenylacetate (6) (LSN 379407)

To a room temperature solution of 0.102 mmol (1 '), 46 mg (0.134 mmol) of compound 24' and 12 mg (0.102 mmol) of 4-dimethylaminopyridine in 250 μΙ anhydrous methylene chloride was added 27 mg (0.134 mmol) of , 3-dicyclohexylcarbodiimide in 50 μΙ methylene chloride. After stirring at room temperature for 6 hours, the reaction mixture was diluted with ethyl acetate / hexane 3: 1 (1 mL), filtered through celite, and the filter washed with ethyl acetate / hexane (3: 1). The filtrate and washings were combined and concentrated under reduced pressure to give a purple foam which was flash chromatographed on silica gel (15 g) eluting with ethyl acetate / hexane (4: 1). 86 mg (82%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.35 (d, 1H, J = 8.3 Hz), 7.30-7.19 (m, 8H), 7.11 (dd, 1H, J = 8.4, 2.0 Hz), 7 , 02 (t, 1H, J = 7.5 Hz), 6.87 (d, 1H, J = 8.4 Hz), 6.84 (d, 1H, J = 7.5 Hz), 6.73 (ddd, 1H, J = 15, 13, 4.7 Hz), 5.92 (d, 1H, J = 7.9 Hz), 5.79 (dd, 1H, J = 15.1.0 Hz) , 5.43 (dd, 1H, J = 9.4.1.8 Hz), 4.98 (dd, 1H, J = 12, 3.1 Hz), 4.81 (ddd, 1H, J = 9 , 9.9, 9.9, 1.8 Hz), 4.75 (d, 1H, J = 9.4 Hz), 4.73-4.67 (m, 1H), 3.90 (s, 3H) , 3.49 (d, 1H, J = 16Hz), 3.44-3.38 (m, 1H), 3.38 (d, 1H, J = 16Hz), 3.27-3.17 ( m, 2H),

3.10 (dd, 1H, J = 14, 8.2 Hz), 2.55-2.46 (m, 2H),

2.37-2.27 (m, 1H), 1.95 (ddd, 1H, J = 14, 12,

4.5 Hz), 1.83-1.70 (m, 2H), 1.49 (s, 18H), 1.27 (s, 3H), 1.20 (s, 3H), 1.03 ( d, 3H, J = 6.5 Hz), 0.97 (d, 3H, J = 6.4 Hz), 0.92 (d, 3H, J = 7.0 Hz).

Preparation of 2 '- (di-tert-butylphosphatyl) phenylacetic acid (24) (LSN 379402)

1.05 g (7.60 mmol) of 2'-hydroxyphenethyl alcohol in 15.2 ml

M.p. 0-8 ° C in Ν, Ν-dimethylformamide

To the 195 µL data was added imidazole (621 mg, 9.11 mmol) and tert-butyldimethylsilyl chloride (1.26 g, 8.34 mmol). After stirring at 0 ° C for 40 minutes and stirring at room temperature for 45 minutes, another imidazole (155 mg, 2.28 mmol) and tert-butyldimethylsilyl chloride (229 mg, 1.52 mmol) were added to the reaction mixture. After stirring for a further 15 minutes, tert-butyl methyl ether (150 mL) was added. The reaction mixture was washed with 1N aqueous hydrochloric acid (15 mL) and water (15 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give a yellow oil. The product was chromatographed on flash silica gel (70 g), eluting with 5: 1 hexane: ethyl acetate. 1.81 g (94%) of the primary silyl ether was obtained as an off-white oil. To a room temperature solution of 506 mg (2.00 mmol) of silyl ether and 600 μΙ (93%, 2.00 mmol) of di-tert-butyl diethylphosphoramidite in 2 mL of tetrahydrofuran was added 421 mg (6.01 mmol) 1-H-tetrazole. After stirring for 45 minutes, the reaction mixture was cooled to -10 ° C and reacted rapidly with m-chloroperbenzoic acid (450 mg, 99%, 2.61 mmol).

Solution of 3.6 ml in methylene chloride. The white cloudy reaction mixture was allowed to warm to room temperature and stirred for 15 minutes. To the reaction mixture was added 4 ml of 10% aqueous sodium hydrogen sulfite solution, the mixture was stirred vigorously for 10 minutes, diluted with 15 ml of tert-butyl methyl ether and 2 x 10 ml of 10% aqueous sodium hydrogen sulphite solution, followed by washing with 2 x 10 ml of 0.5 N aqueous sodium hydroxide. The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure to give 941 mg of a colorless oil which was used directly for the next step. The crude phosphate was dissolved in tetrahydrofuran (10 mL), cooled to 0 ° C, and treated with 1M tetra-n-butylammonium fluoride (2.4 mL, 2.4 mmol) in tetrahydrofuran. After stirring at 0 ° C for 20 minutes and at room temperature for 1.5 hours, the reaction mixture was diluted with 60 ml of tert-butyl methyl ether, washed with 10 ml of water and 10 ml of brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give a yellow oil. The product was chromatographed on 50 g of flash silica gel eluting with ethyl acetate / hexane (3: 1). 525 mg (79%) of 1 ° alcohol are obtained as an off-white oil. To a room temperature solution of 123 mg (0.372 mmol) of alcohol in 1.49 ml of acetonitrile / carbon tetrachloride (1: 1) was added 1.1 ml of water followed by 239 mg (1.12 mmol) of sodium periodate and

Ruthenium (III) chloride hydrate (1.8 mg, 0.0082 mmol). The brown reaction mixture was stirred rapidly at room temperature for 55 minutes. Concentration under reduced pressure and flash chromatography on silica gel (8 g, 10% methanol / ethyl acetate) gave 109 mg (85%) of the title compound as a purple oil.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.49 (d, 1H, J = 7.53 Hz), 7.30-7.15 (m, 3H), 3.77 (s, 2H), 1.51 (s, 18H).

Preparation of Cryptophycin 55- (2'-Phosphatyl) Phenyl Acetate Sodium Salt (7) (LSN 374122) To a solution of mg (0.081 mmol) (6) in 400 μΙ methylene chloride was added 81 μΙ (0.33 mmol) 4 0 M hydrochloric acid in 1,4-dioxane. The pale yellow solution was stirred at room temperature for 2 hours. Concentration under reduced pressure afforded an off-white foam, dissolved in crude dihydrogen phosphate in 614 μΙ tetrahydrofuran and reacted with 33 μΙ (0.163 mmol) in 5.00 N aqueous sodium hydroxide. After stirring for 10 minutes, the reaction mixture was concentrated under reduced pressure to give a tan foam. The crude salt was dissolved in a mixture of 1 mL of hot acetonitrile and 0.1 mL of hot water. The insoluble material was filtered off and the filtrate was concentrated under reduced pressure to give 69 mg (87%) of the title compound as a white solid.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.58 (d, 1H, J = 8.2 Hz), 7.38-7.32 (m, 2H),

7.32-, 7.28 (m, 3H), 7.28 (d, 1H, J = 2.1 Hz), 7.17 (dd,

1H, J = 8.5, 2.1 Hz), 7.09 (ddd, 1H, J = 7.8, 7.8,

1.7 Hz), 6.98 (d, 1H, J = 8.5 Hz), 6.79-6.70 (m, 2H),

6.67 (d, 1H, J = 7.4 Hz), 5.91 (dd, 1H, J = 15, 1.7 Hz),

5.45 (dd, 1H, J = 9.4, 1.6 Hz), 5.06 (dd, 1H, J = 10,

2.7 Hz), 5.01 (d, 1H, J = 9 Hz), 4.89 ^ 4.80 (m, 1H),

4.47 (dd, 1H, J = 11, 3.8 Hz), 3.84 (s, 3H), 3.67 (d,

1H, J = 16Hz), 3.45 (d, 1H, J = 14Hz), 3.42 (d, 1H,

J = 16 Hz), 3.18 (dd, 1H, J = 14, 3.8 Hz), 3.12 (d, 1H,

J = 14 Hz), 2.77 (dd, 1H, J = 14, 11 Hz), 2.67-2.60 (m, 1H), 2.56-2.48 (m, 1H), 2, 31-2.22 (m, 1H),

1.96-1.88 (m, 1H), 1.85-1.77 (m, 2H), 1.22 (s, 3H),

1.20 (s, 3H), 1.03 (d, 3H, J = 6.2 Hz), 0.98 (d, 3H,

J = 6.1 Hz), 0.93 (d, 3H, J = 7.1 Hz).

Cryptophycin-55-nicotinoate hydrochloride (8) (LSN

Preparation of 368265) To a solution of mg (0.071 mmol) (1) in 354 μΙ pyridine at room temperature was added 15 mg (0.085 mmol) nicotinoyl chloride hydrochloride followed by 23 μΙ (0.170 mmol) triethylamine. After stirring for 1.5 hours

4-Dimethylaminopyridine (8.6 mg, 0.071 mmol) was added to the reaction mixture, stirred for 5 hours, followed by additional triethylamine (23 μΙ, 0.170 mmol), 4-dimethylamino (8.6 mg, 0.071 mmol). pyridine and 15 mg (0.085 mmol) of nicotinoyl chloride hydrochloride were added to the reaction mixture and washed with 50 μΙ of pyridine. After stirring for 18 hours, the reaction mixture was reacted with a saturated aqueous solution of sodium bicarbonate (0.5 mL) and extracted with methylene chloride (4 x 1 mL). The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a light brown oily product. The product was subjected to flash chromatography on silica gel (14 g), eluting with 10: 1 ethyl acetate: hexane. 49 mg (85%) of the free base are obtained in the form of a white foam. The nicotinoate was dissolved in 1 mL of methylene chloride and reacted with 90 μΙ (0.090 mmol) of 1.0 M hydrochloric acid in diethyl ether. The clear, colorless solution was stirred at room temperature for 5 minutes and then the solvent was removed under reduced pressure to give 51 mg of the title compound as a white foam.

HU 227 195 Β 11 ¹ H-NMR (500 MHz, CDCl ₃ ), δ:

8.94 (s, 1H), 8.78 (br s, 1H), 8.29 (d, 1H, J = 7.0 Hz),

7.57 (br s, 1H), 7.38 (d, 2H, J = 7.1 Hz), 7.30-7.16 (m, 5H), 7.10 (dd, 1H, J = 8 , 4, 1.7 Hz), 6.88 (d, 1H,

J = 8.4 Hz), 6.71 (m, 1H), 5.80 (d, 1H, J = 15 Hz), 5.74 (d, 1H, J = 9.6 Hz), 5.56 (br s, 1H), 5.00 (d, 1H,

J = 9.6 Hz, 4.95 (t, 1H, J = 8.9 Hz), 4.84 (d, 1H,

J = 9.8 Hz), 4.77-4.72 (m, 1H), 3.91 (s, 3H), 3.39 (dd, 1H, J = 13.8.2 Hz), 3, 23-3.14 (m, 2H), 3.06 (dd,

1H, J = 14, 7.6 Hz), 2.81-2.74 (m, 1H), 2.62-2.45 (m, 2H), 1.93 (ddd, 1H, J = 14, 12, 4.8 Hz),

1.78-1.70 (m, 1H), 1.66-1.59 (m, 1H), 1.25 (s, 3H),

1.20 (d, 3H, J = 7.0 Hz), 1.19 (s, 3H), 0.98 (d, 3H,

J = 6.7 Hz), 0.84 (d, 3H, J = 6.5 Hz).

Preparation of Cryptophycin-55-N-methylpyridinium acetate (9) (LSN 366550) To a solution of mg (0.075 mmol) (1 ') in 751 μΙ methylene chloride at 0 ° C was added 13 μΙ (0.090 mmol) triethyl and then 23 mg (0.083 mmol) of 2-fluoro-1-methylpyridinium p-toluenesulfonate. The heterogeneous reaction mixture is warmed to room temperature and

After stirring for 3.5 hours, another 2-fluoro-1-methylpyridinium p-toluenesulfonate (11 mg, 0.039 mmol) was added. After stirring for 14.5 hours, another 2-fluoro-1-methylpyridinium p-toluenesulfonate (11 mg, 0.039 mmol) was added to the reaction mixture, and after stirring for 2.5 hours, another 2-fluoro-1 - (11 mg, 0.039 mmol) was added. methylpyridinium ptoluenesulfonate and 13 μΙ (0.090 mmol) triethylamine were added. After stirring for 1 hour, the reaction mixture was concentrated under reduced pressure to give an orange foam. The product was purified by reverse phase HPLC while the p-toluenesulfonate group was replaced by the acetate group. The product was freeze-dried to give 30 mg (47%) of the title compound as a white solid.

¹ H-NMR (500 MHz, DMSO-d ₆ ), δ:

8.65-8.58 (m, 2H), 8.36 (t, 1H, J = 7.8 Hz), 7.68 (d,

1H, J = 8.9 Hz), 7.60 (d, 1H, J = 6.6 Hz), 7.48 (t, 1H,

J = 6.6 Hz), 7.35-7.21 (m, 6H), 7.19 (dd, 1H, J = 8.5,

1.9 Hz), 7.05 (d, 1H, J = 8.5 Hz), 6.49 (ddd, 1H,

J = 16, 13, 4.0 Hz), 5.91 (d, 1H, J = 16 Hz), 5.72 (d,

1H, J = 8.0 Hz), 5.66 (dd, 1H, J = 8.0, 1.9 Hz),

5.32-5.27 (m, 1H), 4.73 (dd, 1H, J = 9.7, 4.3 Hz),

4.24 (ddd, 1H, J = 11, 9.8, 3.7 Hz), 3.93 (s, 3H), 3.81 (s, 3H), 3.32 (dd, 1H, J = 13, 9.3 Hz), 3.05-2.97 (m,

2H), 2.77-2.57 (m, 3H), 2.54-2.47 (m, 1H), 1.76 (s,

3H), 1.68-1.62 (m, 1H), 1.55-1.46 (m, 1H),

1.37-1.30 (m, 1H), 1.15 (d, 3H, J = 7.0Hz), 1.13 (s,

3H), 1.00 (s, 3H), 0.88 (d, 3H, J = 6.7 Hz), 0.73 (d,

3H, J = 6.5 Hz).

Preparation of Cryptophycin-55-N-tert-Boc-3- (3-chloro-4-methoxyphenyl) - (D) -alaninate (10) (LSN 382049) 23 mg (0.033 mmol) (1), 16 mg (0.049) mmol)

To a solution of N-tert-Boc-3- (3-chloro-4-methoxyphenyl) - (D) -alanine and some crystals of 4-dimethylaminopyridine in 143 μΙ anhydrous methylene chloride was added 10 mg (0.049). mmol) of a solution of 1,3-dicyclohexylcarbodiimide in 20 μΙ of methylene chloride.

After stirring for two hours, the white cloudy reaction mixture was diluted with 1 mL of ethyl acetate / hexane 2: 1, stirred for 10 minutes, filtered through celite, and washed with ethyl acetate / hexane 2: 1. The filtrate and washings were combined and concentrated under reduced pressure to give a colorless oily product. The product was subjected to flash chromatography on silica gel (14 g) eluting with ethyl acetate / hexane (2: 1). 29 mg (88%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.42-7.27 (m, 5H), 7.24 (d, 1H, J = 2.0Hz), 7.25-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 1.9 Hz), 6.91-6.86 (m, 2H), 6.84-6.70 (m, 3H), 5.75 (d, 1H, J = 15 Hz), 5.53 (d, 1H, J = 9.6 Hz), 5.47 (d, 1H, J = 7.6 Hz), 5.00 (dd, 1H, J = 10, 2.9 Hz), 4.90-4.80 (m, 2H), 4.78-4.71 (m, 1H), 4.63 (d, 1H, J = 8.3 Hz), 4.19-4.12 ( m, 1H), 3.91 (s, 3H), 3.88 (s, 3H), 3.40 (dd, 1H, J = 13, 8.1 Hz), 3.25-3.12 (m , 2H), 3.07 (dd, 1H, J = 14, 7.6 Hz), 2.67-2.57 (m, 2H), 2.39-2.27 (m, 2H), 2, 15 (dd, 1H, J = 14, 8.0 Hz), 2.01 (ddd, 1H, J = 14, 12, 4.2 Hz), 1.87-1.76 (m, 2H), 1 , 39 (s, 9H), 1.27 (s, 3H), 1.20 (s, 3H), 1.09-1.04 (m, 6H), 1.01 (d, 3H, J = 6 , 3 Hz).

Preparation of Cryptophycin-55-3- (3-chloro-4-methoxyphenyl) - (D) alaninate hydrochloride (11) (LSN 382048) was prepared in methylene chloride (27 mg, 0.027 mmol) (10), To this solution at room temperature was added 33 μΙ (0.133 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 3 hours, the clear, colorless reaction mixture was evaporated under reduced pressure to give 26 mg (96%, corrected for 5% dioxane) as a white foam.

¹ H-NMR (500 MHz, MCOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.3 Hz), 7.49-7.45 (m, 2H),

7.43-7.48 (m, 3H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.4, 2.1 Hz), 7.13 (d, 1H, J = 2.2 Hz), 7.07-6.95 (m, 3H), 6.71 (ddd, 1H, J = 15, 13,

3.8 Hz), 5.98 (dd, 1H, J = 15, 1.8 Hz), 5.69 (d, 1H, J = 10 Hz), 5.22 (d, 1H, J = 10 Hz) ), 5.18 (dd, 1H, J = 10, 2.5 Hz), 4.89 ^ 4.80 (m, 1H), 4.53 (dd, 1H, J = 11, 3.7 Hz) , 4.16 (dd, 1H, J = 10, 4.4 Hz), 3.88 (s, 3H), 3.87 (s, 3H), 3.51 (dd, 1H, J = 3.7 Hz), 4.16 (dd, 1H, J = 13, 9.9 Hz), 3.20 (dd, 1H, J = 14, 3.7 Hz), 3.14 (dd, 1H, J = 13 , 2.3 Hz), 2.82-2.75 (m, 3H), 2.45 (dd, 1H, J = 15, 4.5 Hz), 2.42-2.34 (m, 1H) , 2.08-2.00 (m, 1H), 1.97-1.86 (m, 3H), 1.27 (s, 3H),

1.21 (s, 3H), 1.16 (d, 3H, J = 7.1 Hz), 1.10 (d, 3H, J = 6.1 Hz), 1.06 (d, 3H, J) = 6.0 Hz).

Preparation of Cryptophycin-55-N-tert-Boc- (L) -phenylalaninate (12) (LSN 382235) mg (0.041 mmol) (1), 16 mg (0.062 mmol)

To a room temperature solution of N-tert-Boc- (L) -phenylalanine and 0.5 mg (0.0041 mmol) of 4-dimethylaminopyridine in 165 μΙ of anhydrous methylene chloride was added 13 mg (0.062 mmol) of 1,3- dicyclohexylcarbodiimide in 42 μΙ methylene chloride. After stirring for 40 minutes, the reaction mixture was white

HU 227 195 Β1

Dilute with 0.5 mL of ethyl acetate / hexane (2: 1), stir for 10 minutes, filter through celite and wash the filter pad with ethyl acetate / hexane (2: 1). The filtrate and the washings were evaporated under reduced pressure to give a colorless oily product. The product was flash chromatographed on silica gel (12 g) eluting with ethyl acetate / hexane (2: 1). 20 mg (50 μΙ) of the desired compound is obtained in the form of a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.40-7.47 (m, 2H), 7.26-7.37 (m, 6H), 7.12-7.22 (m, 4H), 7.00 (d, 1H, J = 8 , 4 Hz), 6.78-6.71 (m, 1H),

5.94 (d, 1H, J = 15 Hz), 5.48 (d, 1H, J = 9.5 Hz),

5.15-5.10 (m, 1H), 5.06 (d, 1H, J = 9.5 Hz), 4.68 (t,

1H, J = 9.7 Hz), 4.52 (dd, 1H, J = 11, 3.7 Hz),

4.14-4.10 (m, 1H), 3.87 (s, 3H), 3.49 (d, 1H,

J = 13 Hz), 3.20 (dd, 1H, J = 14, 3.7 Hz), 3.14 (d, 1H,

J = 13 Hz), 2.77 (dd, 1H, J = 14, 11 Hz), 2.67 (dd, 1H,

J = 14, 6.2 Hz), 2.62-2.57 (m, 2H), 2.51 (dd, 1H,

J = 14, 9.1 Hz), 2.31-2.23 (m, 1H), 2.00-1.92 (m,

1H), 1.91-1.82 (m, 2H), 1.38 (s, 9H), 1.26 (s, 3H),

1.24 (s, 3H), 1.08 (d, 3H, J = 5.9 Hz), 1.03 (d, 3H,

J = 5.8 Hz), 0.87 (d, 3H, J = 7.0 Hz).

Preparation of Cryptophycin-55- (L) -phenylalaninate hydrochloride (13) (LSN 382236) To a solution of mg (0.019 mmol) (12) in 189 μΙ methylene chloride at room temperature was added 24 μΙ (0.094 mmol) 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 4 hours, the clear, colorless reaction mixture was concentrated under reduced pressure to give 15 mg (88%, corrected for 2% dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.6 Hz), 7.48-7.23 (m, 11H), 7.20 (dd, 1H, J = 8.6, 1.6 Hz), δ , 75-6.68 (m, 1H), 5.97 (d,

1H, J = 16Hz), 5.55 (d, 1H, J = 9.2Hz), 7.15-5.11 (m,

2H), 4.71 (t, 1H, J = 10Hz), 4.55 ^ 4.51 (m, 1H), 3.87 (s, 3H), 3.21 (dd, 1H, J = 14 , 3.8 Hz), 3.17-3.10 (m,

2H), 2.94 (dd, 1H, J = 15, 8.1 Hz), 2.78 (dd, 1H,

J = 14, 12 Hz), 2.69-2.63 (m, 2H), 2.34-2.27 (m,

1H), 1.98-1.92 (m, 1H), 1.89-1.82 (m, 2H), 1.25 (s,

3H), 1.22 (s, 3H), 1.06 (d, 3H, J = 6.2 Hz), 1.03 (d,

3H, J = 6.1 Hz), 0.89 (d, 3H, J = 7.0 Hz).

Preparation of Cryptophycin-55- (L) -histidinate dihydrochloride (15) (LSN 384046) N, N'-di-tert-Boc- (L) -histidine mg (0.027 mmol) (1), 18 mg (0.040 mmol) To a solution of benzene complex and 0.3 mg (0.0027 mmol) of 4-dimethylaminopyridine in 100 μΙ of anhydrous methylene chloride was added 8.3 mg (0.040 mmol) of 1,3-dicyclohexylcarbodiimide in 35 μΙ of methylene chloride solution. After stirring for 60 min, another 18 mg (0.040 pmol) of N, N'-di-tert-Boc- (L) -histidine-benzene complex and

1,3-Dicyclohexylcarbodiimide (8.3 mg, 0.040 mmol) was added to the reaction mixture. The white cloudy reaction mixture was stirred for 4 hours, diluted with ethyl acetate / hexane (3: 1, 0.5 mL), stirred for 10 minutes, filtered through celite and washed with ethyl acetate / hexane (3: 1). The filtrate and the washings were combined and concentrated under reduced pressure. The resulting colorless oily product was subjected to flash chromatography on silica gel (13 g), eluting with ethyl acetate / hexane (3: 1). 17 mg (61%) of N, N-diterc-Boc (14 ') were obtained as a white foam. To a solution of (14 ') (17 mg, 0.016 mmol) in methylene chloride (160 μΙ) at room temperature was added 24 μΙ (0.098 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 5 hours, the white cloudy reaction mixture was concentrated under reduced pressure to give 15.7 mg (10%, corrected for 4% dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

8.91 (s, 1H), 7.77 (d, 1H, J = 9.5 Hz), 7.51-7.37 (m,

6H), 7.31 (d, 1H, J = 2.1 Hz), 7.20 (dd, 1H, J = 8.4,

2.1 Hz), 6.66-6.58 (m, 1H), 5.99 (d, 1H, J = 15 Hz),

5.57 (d, 1H, J = 9.6 Hz), 5.20 (d, 1H, J = 9.6 Hz), 5.15 (dd, 1H, J = 10, 3.0 Hz), 4.58 (t, 1H, J = 11Hz), 4.53 (dd, 1H, 11.3.8Hz), 4.14 (t, 1H, J = 6.4Hz), 3.87 ( and,

3H), 3.55-3.47 (m, 1H), 3.27-3.12 (m, 3H),

2.81-2.68 (m, 3H), 2.40-2.31 (m, 1H), 1.98-1.81 (m, 3H), 1.25 (s, 3H), 1 , 21 (s, 3H), 1.10-1.04 (m,

6H), 1.04 (d, 3H, J = 6.1 Hz).

Preparation of Cryptophycin 55-N-tert-Boc- (L) -prolinate (16) (LSN 382926) mg (1), (0.027 mmol) 8.7 mg (0.040 mmol) N-tert-Boc- (L) -proline and 4-dimethylaminopyridine (0.3 mg, 0.027 mmol) in 100 μΙ anhydrous methylene chloride was added at room temperature.

Of a solution of 8.3 mg (0.040 mmol) of 1,3-dicyclohexylcarbodiimide in 35 μΙ of methylene chloride. After stirring for 45 minutes, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes and then filtered through a pad of celite, washing the pad with ethyl acetate / hexane (3: 1). The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was purified by flash chromatography on silica gel (15 g), eluting with 3: 1 ethyl acetate: hexane. 11 mg (46%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.39-7.30 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.21-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 2.0 Hz),

6.87 (d, 1H, J = 8.4), 6.80-6.68 (m, 1H), 5.77 (d, 1H,

J = 15 Hz), 5.61 (br s, 1H), 5.56 (t, 1H, J = 9.5 Hz),

5.04-4.68 (m, 4H), 4.19 ^ 4.15 (m, 1H), 3.90 (s, 3H),

3.41-3.34 (m, 2H), 3.27-3.02 (m, 4H), 2.64-2.50 (m, 2H), 2.35-2.25 (m, 1H) ), 2.10-2.00 (m, 1H),

1.90-1.72 (m, 3H), 1.70-1.50 (m, 3H), 1.44 (s, 9H),

1.28 (s, 3H), 1.20 (s, 3H), 1.08-0.90 (m, 9H).

Preparation of Cryptophycin-55- (L) -prolinate hydrochloride (17) (LSN 382927) To a solution of mg (0.012 mmol) (16) in 122 μ 122 methylene chloride at room temperature was added μΙ (0.061 mmol) 4.0 M, Hydrochloric acid in 4-dioxane.

After stirring for 1 h, the clear, colorless reaction mixture was concentrated under reduced pressure (10 mg, 100%)

The desired compound was obtained in the form of a white solid.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.77 (dd, 1H, J = 9.4, 2.3 Hz), 7.48-7.42 (m, 2H),

7.39-7.35 (m, 3H), 7.31 (d, 1H, J = 2.2 Hz), 7.20 (dd,

1H, J = 8.4, 2.2 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.68 (ddd, 1H, J = 15, 13, 3.8 Hz), 5.98 (dd, 1H, J = 15,

1.4 Hz), 5.52 (d, 1H, J = 9.8 Hz), 5.18 (d, 1H,

J = 9.8 Hz), 5.18-5.14 (m, 1H), 4.78 (t, 1H, J = 10 Hz),

4.52 (dd, 1H, J = 11, 3.9 Hz), 3.99 (t, 1H, J = 7.3 Hz),

3.87 (s, 3H), 3.48 (dd, 1H, J = 14, 9.7 Hz), 3.28-3.12 (m, 4H), 2.82-2.73 (m, 3H), 2.42-2.34 (m, 2h),

2.09-1.79 (m, 6H), 1.24 (s, 3H), 1.88 (s, 3H), 1.16 (d, 3H, J = 7.0 Hz), 1.07 (d, 3H, J = 6.3 Hz), 1.04 (d,

3H, J = 6.2 Hz).

Cryptophycin-55-N-tert-Boc-glycinate (18 ') (LSN

379403)

A solution of N-tert-Boc-glycine (118 mg, 0.167 mmol), 44 mg (0.251 mmol) and 4-dimethylaminopyridine (2.0 mg, 0.0167 mmol) in 490 µL of anhydrous methylene chloride was added at room temperature. A solution of 1,3-dicyclohexylcarbodiimide (52 mg, 0.251 mmol) in methylene chloride (67 μΙ). After stirring for 50 minutes, the white cloudy reaction mixture was diluted with 1 mL of ethyl acetate / hexane 3: 1, stirred for 10 minutes and then filtered through a pad of celite, washing the pad of celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was purified by flash chromatography on silica gel (19 g), eluting with 3: 1 ethyl acetate: hexane. 138 mg (96%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.34 (s, 5H), 7.24 (d, 1H, J = 2.0 Hz), 7.23-7.19 (m,

1H), 7.10 (dd, 1H, J = 8.4, 2.0 Hz), 6.88 (d, 1H,

J = 8.4 Hz), 6.79-6.7.0 (m, 1H), 5.77 (d, 1H,

J = 13 Hz), 5.50 (d, 1H, J = 8.0 Hz), 5.47 (d, 1H,

J = 9.8 Hz), 4.97 (dd, 1H, J = 11.2.7 Hz), 4.89 (t, 1H,

J = 10 Hz), 4.83 (d, 1H, J = 9.8 Hz), 4.79-4.72 (m,

1H), 4.68 (br s, 1H), 3.91 (s, 3H), 3.66 (dd, 1H,

J = 18, 5.3 Hz), 3.42-3.35 (m, 2H), 3.21 (dd, 1H,

J = 13, 4.0 Hz), 3.17 (dd, 1H, J = 15, 5.1 Hz), 3.08 (dd, 1H, J = 15, 7.6 Hz), 2.66- 2.57 (m, 2H),

2.47-2.38 (m, 1H), 1.95 (ddd, 1H, J = 14, 12,

4.7 Hz), 1.85-1.77 (m, 1H), 1.75-1.67 (m, 1H), 1.43 (s, 9H), 1.27 (s, 3H), 1.20 (s, 3H), 1.08 (d, 3H,

J = 7.0 Hz), 1.03 (d, 3H, J = 6.7 Hz), 0.98 (d, 3H,

J = 6.5 Hz).

Cryptophycin-55-glycinate hydrochloride (19) (LSN

368422)

To a solution of 122 mg (0.141 mmol) of (18 ') in 471 μΙ of methylene chloride at room temperature was added 178 μΙ (0.707 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 20 minutes, the clear, colorless reaction mixture was concentrated under reduced pressure to give 120 mg (99%, corrected for 7% dioxane) of the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.81 (dd, 1H, J = 8.5, 2.2 Hz), 7.46-7.41 (m, 2H),

7.40-7.36 (m, 3H), 7.31 (d, 1H, J = 2.1 Hz), 7.20 (dd,

1H, J = 8.4, 2.1 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.70 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.97 (dd, 1H, J = 15,

1.7 Hz), 5.55 (d, 1H, J = 9.9 Hz), 5.18 (d, 1H,

J = 9.9 Hz), 5.14 (dd, 1H, J = 10, 2.8 Hz), 4.84 (t, 1H,

J = 10 Hz), 4.52 (dd, 1H, J = 11, 3.7 Hz), 3.87 (s, 3H),

3.78 (d, 1H, J = 18 Hz), 3.50 (dd, 1H, J = 13, 9.8 Hz),

3.23 (d, 1H, J = 18 Hz), 3.20 (dd, 1H, J = 14, 3.6 Hz),

3.13 (dd, 1H, J = 13, 2.4 Hz), 2.80-2.69 (m, 3H),

2.41-2.32 (m, 1H), 1.99-1.92 (m, 1H), 1.91-1.81 (m, 2H), 1.25 (s, 3H), 1.20 ( s, 3H), 1.12 (d, 3H,

J = 7.0 Hz), 1.06 (d, 3H, J = 6.2 Hz), 1.04 (d, 3H,

6.2 Hz).

Preparation of Cryptophycin-55-N-tert-Boc-β-alaninate (20) (LSN 379404)

To a solution of N-tert-Boc-β-alanine (102 mg, 0.145 mmol), 41 mg (0.217 mmol) and 4-dimethylaminopyridine (18 mg, 0.145 mmol) in anhydrous methylene chloride at room temperature was added 45 mg. (0.217 mmol) of 1,3-dicyclohexylcarbodiimide in 82 μΙ of methylene chloride. After stirring for 3.5 hours, the white cloudy reaction mixture was diluted with 1 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes and then filtered through a pad of celite, washing the pad of celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was subjected to flash chromatography on silica gel (21 g) eluting with ethyl acetate / hexane (2: 1 then 4: 1). 121 mg (95%) of the desired compound is obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.44-7.39 (m, 2H), 7.37-7.31 (m, 3H), 7.32 (d, 1H,

J = 2.1 Hz), 7.20 (dd, 1H, J = 8.4, 2.1 Hz), 7.01 (d, 1H,

J = 8.4 Hz), 6.72 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.96 (dd, 1H, J = 15, 1.6 Hz), 5.51 ( d, 1H, J = 9.8 Hz),

5.11-5.06 (m, 1H), 5.08 (d, 1H, J = 9.8 Hz),

4.90-4.83 (m, 1H), 4.50 (dd, 1H, J = 11, 3.6 Hz),

3.86 (s, 3H), 3.52-3.46 (m, 1H), 3.20 (dd, 1H, J = 14,

3.6 Hz), 3.13 (br d, 1H, J = 14 Hz), 3.05-2.92 (m,

2H), 2.79-2.63 (m, 3H), 2.45-2.37 (m, 1H), 2.24 (dt, 1H, J = 16, 7.0 Hz), 2.08 -1.99 (m, 1H),

1.96-1.79 (m, 3H), 1.43 (s, 9H), 1.25 (s, 3H), 1.21 (s, 3H), 1.12 (d, 3H, J = 7.0 Hz), 1.06 (d, 3H,

J = 6.2 Hz), 1.02 (d, 3H, J = 6.1 Hz).

Preparation of Cryptophycin-55 - () - Alaninate Hydrochloride (21) (LSN 377718)

To a solution of 119 mg (0.136 mmol) of (20 ') in 452 μΙ of methylene chloride at room temperature was added 170 μΙ (0.679 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 2.15 minutes, the white cloudy reaction mixture was concentrated under reduced pressure to give 110 mg (96%, corrected for 4% dioxane) of the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.80 (dd, 1H, J = 9.7, 2.3 Hz), 7.45-7.40 (m, 2H),

7.39-7.32 (m, 3H), 7.20 (dd, 1H, J = 8.4, 2.1 Hz),

HU 227 195 Β1

7.01 (d, 1H, J = 8.4 Hz), 6.68 (ddd, 1H, J = 15, 13,

3.8 Hz), 5.98 (dd, 1H, J = 15, 1.7 Hz), 5.48 (dd, 1H,

J = 9.4, 1.0 Hz), 5.15-5.11 (m, 1H), 5.13 (d, 1H,

J = 9.4 Hz), 4.82 (t, 1H, J = 10 Hz), 4.51 (dd, 1H,

J = 11, 3.7 Hz), 3.90 (s, 3H), 3.50 (dd, 1H, J = 14,

9.8 Hz), 3.20 (dd, 1H, J = 14, 3.7 Hz), 3.14 (dd, 1H,

J = 14, 2.4 Hz), 2.85 (t, 2H, J = 7.0 Hz), 2.80-2.65 (m, 5H), 2.54 (dt, 1H, J = 17 , 7.4 Hz), 2.42-2.33 (m,

1H), 2.22 (dt, 1H, J = 17, 6.7 Hz), 1.90-1.81 (m,

3H, 1.25 (s, 3H), 1.20 (s, 3H), 1.13 (d, 3H,

J = 7.1 Hz), 1.08 (d, 3H, J = 6.3 Hz), 1.04 (d, 3H,

J = 6.2 Hz).

Preparation of Cryptophycin-55-N-Boc-N-aminobutyrate (22 ') (LSN 379401) mg (0.068 mmol) (1), 18 mg (0.088 mmol) N-tert-Boc-4-aminobutyric acid and 18 mg To a solution of 4-dimethylaminopyridine (0.088 mmol) in anhydrous methylene chloride (150 μΙ) at room temperature was added a solution of 1,3-dicyclohexylcarbodiimide (18 mg, 0.088 mmol) in methylene chloride (50 μΙ). After stirring for 45 minutes, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 5 minutes and then filtered through a pad of celite, washing the pad of celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was purified by flash chromatography on silica gel (15 g), eluting with 3: 1 ethyl acetate: hexane. 55 mg (90%) of the desired compound is obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.38-7.32 (m, 5H), 7.24 (d, 1H, J = 1.9 Hz),

7.22-7.19 (m, 1H), 7.10 (dd, 1H, J = 8.4, 1.9 Hz),

6.88 (d, 1H, J = 8.4 Hz), 6.75 (ddd, 1H, J = 15, 13,

3.9 Hz), 5.78 (d, 1H, J = 15 Hz), 5.60-5.55 (m, 1H),

5.49 (dd, 1H, J = 9.8, 1.4 Hz), 4.96 (dd, 1H, J = 11,

3.0 Hz), 4.89 (t, 1H, J = 9.2 Hz), 4.81 (d, 1H,

J = 9.8 Hz), 4.78-4.70 (m, 1H), 4.44 (br s, 1H), 3.91 (s, 3H), 3.40 (dd, 1H, J = 14, 8.1 Hz), 3.22 (dd, 1H,

J = 14.4, 4.1 Hz), 3.22-3.15 (m, 1H), 3.08 (dd, 1H,

J = 14, 7.8 Hz), 2.89-2.82 (m, 2H), 2.67-2.56 (m,

2H), 2.47-2.38 (m, 1H), 2.11-2.04 (m, 1H),

2.00-1.77 (m, 3H), 1.75-1.67 (m, 1H), 1.45 (s, 9H),

1.50-1.40 (m, 2H), 1.27 (s, 3H), 1.20 (s, 3H), 1.09 (d, 3H, J = 7.0 Hz), 1.04 (d, 3H, J = 6.6 Hz), 0.98 (d,

3H, J = 6.6 Hz).

Cryptophycin-55-i-aminohutyrate (25) (LSN

Preparation of 368513) To a solution of mg (0.059 mmol) (22) in 297 μΙ methylene chloride at room temperature was added 297 μΙ (0.297 mmol) in 1.0 M hydrochloric acid in diethyl ether. The starting material precipitates in the form of a white ointment which is dissolved in an additional 150 μΙ of methylene chloride. After stirring for 4 hours, another 59 μΙ (0.059 mmol) hydrochloric acid solution was added to the reaction mixture. After stirring for 14 hours, the reaction mixture was concentrated under reduced pressure to give 49 mg (100%) of the title compound as a white foam.

¹ H-NMR (500 MHz, DMSO-d ₆ ), δ:

8.49 (d, 1H, J = 8.0 Hz), 7.72 (br s, 3H), 7.44-7.33 (m, 5H), 7.32 (d, 1H, J = 1 , 9 Hz), 7.29 (dd, 1H, J = 9.4,

2.6 Hz), 7.20 (dd, 1H, J = 8.5, 1.9 Hz), 7.06 (d, 1H,

J = 8.5 Hz), 6.48 (ddd, 1H, J = 15, 03, 3.9 Hz), 5.87 (d, 1H, J = 15 Hz), 5.37 (d, 1H, J = 9.7 Hz), 5.33 (d,

1H, J = 9.7 Hz), 5.04-5.01 (m, 1H), 4.73 (t, 1H,

J = 11 Hz), 4.25 (ddd, 1H, J = 12, 9.8, 3.5 Hz), 3.82 (s,

3H), 3.40-3.30 (m, 1H), 3.07-3.01 (m, 2H), 2.72 (dd, 1H, J = 14, 12Hz), 2.65-2 , 47 (m, 4H),

2.38-2.28 (m, 1H), 2.21 (dt, 1H, J = 17, 7.5 Hz), 1.97 (dt, 1H, J = 17, 7.5 Hz), 1 , 80-1.70 (m, 3H),

1.54-1.46 (m, 2H), 1.17 (s, 3H), 1.03 (s, 3H), 1.01 (d, 3H, J = 7.0 Hz), 0.99 (d, 3H, J = 5.8 Hz), 0.95 (d,

3H), J = 5.8 Hz).

Preparation of Cryptophycin-55-N-tert-Boc- (L) -alaninate (26) (LSN 379405)

N-tert-Boc- (L) -alanine (103 mg, 0.146 mmol), 41 mg (0.217 mmol) and 4-dimethylaminopyridine (18 mg, 0.146 mmol) in 400 μΙ anhydrous methylene chloride were added at room temperature. To this solution was added a solution of 45 mg (0.219 mmol) of 1,3-dicyclohexylcarbodiimide in 87 μΙ of methylene chloride. After stirring for 5.50 hours, another 5.5 mg (0.029 mmol) of N-tert-Boc- (L) -alanine, 6.0 mg (0.029 mmol) of 1,3-dicyclohexylcarbodiimide and some crystals are added to the white cloudy reaction mixture. 4-dimethyl-aminopyridine. After stirring for 1 hour, the reaction mixture was diluted with 1 mL of ethyl acetate / hexane 3: 1, stirred for 10 minutes, and then filtered through a pad of celite, washing the pad of celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was subjected to flash chromatography on silica gel (22 g), eluting with 1.5: 1, then 2: 1 and finally 4: 1 ethyl acetate / hexane. 96 mg (75%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.35-7.30 (m, 5H), 7.26-7.21 (m, 2H), 7.10 (dd,

1H, J = 8.4, 1.9 Hz), 6.88 (d, 1H, J = 8.4 Hz), 6.76 (ddd, 1H, J = 15.13, 4.2 Hz), 5.77 (d, 1H, J = 15 Hz),

5.52 (d, 1H, J = 7.6 Hz), 5.44 (d, 1H, J = 9.7 Hz),

4.98 (dd, 1H, J = 11, 2.5 Hz), 4.85-4.81 (m, 2H),

4.75 (q, 1H, J = 6.8 Hz), 4.56 (d, 1H, J = 7.8 Hz),

4.01-3.96 (m, 1H), 3.91 (s, 3H), 3.41 (dd, 1H,

J = 13, 8.3 Hz), 3.20 (dd, 1H, J = 13, 4.0 Hz), 3.16 (dd, 1H, J = 15, 5.9 Hz), 3.08 ( dd, 1H, J = 15,

7.6 Hz), 2.65-2.57 (m, 2H), 2.40-2.31 (m, 1H),

2.02-1.96 (m, 1H), 1.87-1.73 (m, 2H), 1.43 (s,

9H), 1.27 (s, 3H), 1.20 (s, 3H), 1.11-1.02 (m, 9H),

0.99 (d, 3H, J = 6.3 Hz).

Preparation of Cryptophycin-55- (L) -alaninate hydrochloride (28) (LSN 377719) To a solution of mg (0.108 mmol) (26) in 361 μΙ methylene chloride was added at room temperature.

135 μΙ (0.542 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane.

After stirring for 2.5 hours, the white cloudy reaction mixture was stirred under reduced pressure for 2.5 hours

The white cloudy reaction mixture was evaporated under reduced pressure to give 90 mg (96%, corrected for 6% dioxane) of the title compound as a white foam.

¹ H-NMR (MeOH-d ₄ ), δ:

8.54 (d, 1H, 7.6 Hz), 7.81 (br d, 1H, J = 9.7 Hz),

7.46-7.44 (m, 2H), 7.39-7.37 (m, 3H), 7.32 (d,

1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.4, 2.0 Hz), 7.01 (d, 1H, J = 2.0 Hz), 6.69 ( ddd, 1H, J = 15, 11,

3.7 Hz), 5.99 (d, 1H, J = 15 Hz), 5.55 (d, 1H,

J = 9.8 Hz), 5.20 (d, 1H, J = 9.8 Hz), 5.15 (dd, 1H,

J = 11, 2.7 Hz), 4.78 (t, 1H, J = 11 Hz), 4.53-4.50 (m, 1H), 3.87 (s, 3H), 3.65 ( q, 1H, J = 7.3 Hz), 3.50 (dd, 1H, J = 13, 9.8 Hz), 3.20 (dd, 1H, J = 14,

3.5 Hz), 3.14 (br d, 1H, J = 13 Hz), 2.81-2.71 (m,

3H), 2.41-2.34 (m, 1H), 1.98-1.93 (m, 1H),

1.88-1.82 (m, 2H), 1.41 (d, 3H, J = 7.3 Hz), 1.25 (s,

3H), 1.20 (s, 3H), 1.13 (d, 3H, J = 7.0 Hz), 1.06 (d,

3H, J = 6.2 Hz), 1.04 (d, 3H, 6.0 Hz).

Preparation of Cryptophycin-55-N-tert-Boc- (D) -alaninate (29 ') (LSN 382426) Preparation of mg (0.035 mmol) (1), 10 mg (0.053 mmol) of N-tert-Boc- (D) - To a solution of alanine and 4-dimethylaminopyridine (0.4 mg, 0.0035 mmol) in dry methylene chloride (130 µL) at room temperature was added a solution of 1,3-dicyclohexylcarbodiimide (11 mg, 0.053 mmol) in methylene chloride (47 µL). After stirring for 5.5 hours, the white cloudy reaction mixture was diluted with ethyl acetate / hexane (3: 1, 0.5 mL), stirred for 10 minutes and then filtered through a pad of celite, washing the pad of celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was purified by flash chromatography on silica gel (15 g), eluting with 2: 1 ethyl acetate: hexane. 26 mg (83%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.49-7.29 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.22-7.18 (m, 1H), 7.09 (dd, 1H, 8.4, 2.0 Hz), 6.87 (d, 1H, J = 8.4 Hz), 6.76 (ddd, 1H, J = 15.13,4.4 Hz),

5.77 (d, 1H, 15 Hz), 5.56 (d, 1H, J = 9.9 Hz), 5.48 (d,

1H, J = 7.7 Hz), 5.01 (dd, 1H, J = 10, 2.6 Hz), 4.91 (t,

1H, J = 9.4 Hz), 4.84 (d, 1H, J = 9.9 Hz), 4.81-4.73 (m, 2H), 3.99-3.93 (m, 1H ), 3.91 (s, 3H), 3.39 (dd,

1H, J = 13, 8.0 Hz), 3.22 (dd, 1H, J = 13, 3.6 Hz), 3.17 (dd, 1H, J = 14, 5.0 Hz), 3, 08 (dd, 1H, J = 14 Hz),

2.68-2.58 (m, 2H), 2.42-2.35 (m, 1H), 2.04-1.94 (m, 1H), 1.87-1.50 (m, 2H) ), 1.42 (s, 9H), 1.27 (s,

3H), 1.20 (s, 3H), 1.09 (d, 3H, J = 7.1 Hz), 1.04 (d,

3H, J = 6.4Hz), 0.99 (d, 3H, J = 6.3Hz), 0.65 (d, 3H,

J = 6.8 Hz).

Preparation of Cryptophycin-55- (D) -alaninate hydrochloride (30) (LSN 382425) To a solution of mg (0.027 mmol) (29) in 274 μΙ methylene chloride at room temperature was added μΙ (0.137 mmol) 4.0 M, Hydrochloric acid in 4-dioxane.

After stirring for 3.5 hours, the clear, colorless reaction mixture was concentrated under reduced pressure (24 mg

100%, corrected for 8% dioxane) to give the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 9.5 Hz), 7.47-7.40 (m, 2H), 7.40-7.36 (m, 3H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.4, 2.0 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.71 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.98 (dd, 1H, J = 15,

1.6 Hz), 5.65 (d, 1H, J = 10 Hz), 5.20 (d, 1H, J = 10 Hz), 5.17 (dd, 1H, J = 11, 2.5 Hz), 4.88-4.78 (m, 1H), 4.53 (dd, 1H, J = 11, 3.7 Hz), 3.95 (q, 1H, J = 7.2 Hz), 3.87 (s, 3H), 3.51 (dd, 1H, J = 13, 9.8 Hz),

3.20 (dd, 1H, J = 14, 3.6 Hz), 3.14 (dd, 1H, J = 13,

2.3 Hz), 2.81-2.74 (m, 3H), 2.41-2.34 (m, 1H), 2.07-1.99 (m, 1H), 1.96-1. , 84 (m, 2H), 1.26 (s, 3H),

1.21 (s, 3H), 1.15 (d, 3H, J = 7.1 Hz), 1.09 (d, 3H, J = 6.0 Hz), 1.05 (d, 3H, J = 6) , 0 Hz), 0.80 (d, 3H, J = 7.4 Hz).

Preparation of Cryptophycin-55-Nu.-N'o-di-tert-Boc- (L) -Lysinate (31) (LSN 379406)

105 mg (0.149 mmol) (1), 67 mg (0.193 mmol) of NaNe-di-tert-Boc- (L) -lysine and 18 mg (0.149 mmol) of 4-dimethylaminopyridine were prepared in 400 μΙ of anhydrous methylene chloride. A solution of 1,3-dicyclohexylcarbodiimide (40 mg, 0.193 mmol) in methylene chloride (96 μl) was added at room temperature. After stirring for 4 hours, another 10 mg (0.030 mmol) of NaNe-di-tert-Boc- (L) -lysine and 6.1 mg (0.030 mmol) of 1,3-dicyclohexylcarbodiimide are dissolved in 100 μΙ of methylene chloride. . After stirring for 1 hour, the white cloudy reaction mixture was diluted with 1 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes and filtered through celite, washing the celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a white foamy product which was placed under the above reaction conditions and 34 mg (0.097 mmol, NaNe-di-tert-Boc- (L) -lysine, 20 mg (0.097 mmol) 1.3). -dicyclohexylcarbodiimide and 4-dimethylaminopyridine (9.1 mg, 0.075 mmol) were reacted again after 1.5 hours to give a white foam as a crude product, which was purified by flash chromatography on silica gel (21 g, ethyl acetate). hexane (1: 1 then 4: 1) to give 112 mg (73%) of the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.42-7.37 (m, 2H), 7.36-7.29 (m, 3H), 7.27 (br s, 1H), 7.16 (br d, 1H, J = 8.5 Hz), 6.97 (d, 1H, J = 8.5 Hz), 6.72 (ddd, 1H, J = 15, 13, 3.5 Hz), 5.92 (d, 1H, J = 15 Hz), 5.50 (d, 1H, J = 11Hz), 5.11-5.04 (m, 2H), 4.84 (t, 1H, J = 10Hz), 4.48 (dd, 1H, J = 11,

3.6 Hz), 3.84 (s, 3H), 3.75 (brs, 1H), 3.50-3.43 (m, 1H), 3.17 (dd, 1H, J = 14, 3.6 Hz), 3.11 (d, 1H, J = 14 Hz), 2.97-2.91 (m, 2H), 2.76-2.58 (m, 3H),

2.36-2.27 (m, 1H), 1.98-1.80 (m, 3H), 1.48-1.38 (m, 2H), 1.43 (s, 9H), 1, 40 (s, 9H), 1.35-1.25 (m, 2H), 1.23 (s, 3H), 1.20 (s, 3H), 1.15-1.09 (m, 2H) , 1.07 (d, 3H, J = 6.8 Hz), 1.06 (d, 3H, J = 6.0 Hz), 1.01 (d, 3H, J = 6.1 Hz).

HU 227 195 Β1

Preparation of Cryptophycin-55- (L) -Lysinate Dihydrochloride (32 ') (LSN 377562)

To a solution of 107 mg (0.103 mmol) (31) in 345 μΙ of methylene chloride at room temperature was added 155 μΙ (0.621 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 4 hours, the white cloudy reaction mixture was filtered. The collected white solid was washed with methylene chloride (2 x 1 mL) and dried at room temperature under reduced pressure to give 87 mg (93%) of the title compound.

¹ H-NMR (MeOH-d ₄ ), δ:

8.61 (d, 1H, J = 7.7 Hz), 7.81 (d, 1H, J = 7.7 Hz),

7.47-7.44 (m, 2H), 7.40-7.38 (m, 3H), 7.31 (d,

1H, J = 2.2 Hz), 7.20 (dd, 1H, J = 8.4, 2.2 Hz), 7.00 (d, 1H, J = 8.4 Hz), 6.63 ( ddd, 1H, J = 15, 13,

4.0 Hz), 6.00 (dd, 1H, J = 15, 1.6 Hz), 5.55 (d, 1H,

J = 9.8 Hz), 5.20 (d, 1H, J = 9.8 Hz), 5.15 (dd, 1H,

J = 10, 2.9 Hz), 4.68 (t, 1H, J = 11 Hz), 4.55-4.49 (m, 1H), 3.87 (s, 3H), 3.79 ( t, 1H, J = 5.6 Hz), 3.52 (dd, 1H, J = 14, 9.9 Hz), 3.20 (dd, 1H, J = 14,

3.6 Hz), 3.13 (dd, 1H, J = 13, 2.4 Hz), 3.06-2.98 (m, 1H), 2.94-2.87 (m, 1H), 2 , 85-2.74 (m, 3H),

2.45-2.38 (m, 1H), 1.98-1.76 (m, 5H), 1.71-1.64 (m, 2H), 1.39-1.30 (m, 2H), 1.25 (s, 3H), 1.18 (d,

3H, J = 8.2 Hz), 1.17 (s, 3H), 1.08 (d, 3H,

J = 6.2 Hz), 1.05 (d, 3H, J = 6.1 Hz).

Preparation of Cryptophycin-55-Na-N ?: -di- tert-Boc- (D) -Lysinate (33) (LSN 382504) mg (0.035 mmol) (1), 247 mg (0.069 mmol) Na-N? To a solution of di-tert-Boc- (D) lysine and 4-dimethylaminopyridine (0.4 mg, 0.0035 mmol) in anhydrous methylene chloride (140 μΙ) was added 1,3-dicyclohexyl (14 mg, 0.069 mmol). -carbodiimide in 30 μΙ methylene chloride. After stirring for 70 minutes, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes, and then filtered through celite, washing the celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give an off-white oily product. The product was flash chromatographed on silica gel 15 using 2: 1 ethyl acetate / hexane as eluent. 30 mg (87%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (MeOH-d ₄ ), δ:

7.46-7.40 (m, 2H), 7.39-7.31 (m, 3H), 7.31 (d,

1H, J = 1.8 Hz), 7.20 (dd, 1H, J = 8.4, 1.8 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.75 ( ddd, 1H, J = 16, 11,

3.7 Hz), 5.93 (d, 1H, J = 16 Hz), 5.59 (d, 1H,

J = 10 Hz), 5.13-5.09 (m, 2H), 4.92 (t, 1H,

J = 9.8 Hz), 4.52 (dd, 1H, J = 11, 3.6 Hz), 3.87 (s,

3H), 3.79-3.75 (m, 1H), 3.48 (d, 1H, J = 13Hz),

3.21 (dd, 1H, J = 14, 3.7 Hz), 3.12 (d, 1H, J = 13 Hz),

2.97-2.91 (m, 2H), 2.80-2.62 (m, 3H), 2.41-2.32 (m, 1H), 1.98-1.82 (m, 3H) ), 1.48-1.38 (m, 2H),

1.46 (s, 9H), 1.42 (s, 9H), 1.35-1.20 (m, 2H), 1.25 (s, 3H), 1.21 (s, 3H), 1 , 15-0.09 (m, 2H), 1.14 (d,

3H, J = 7.0Hz), 1.07 (d, 3H, J = 6.0Hz), 1.03 (d,

3H, J = 6.0 Hz).

Preparation of Cryptophycin-55- (D) -Lysinate Dihydrochloride (34) (LSN 377503) To a solution of mg (0.027 mmol) (33) in 181 μΙ methylene chloride at room temperature was added 41 μΙ (0.162 mmol) 4.0 M Hydrochloric acid in 4-dioxane.

After stirring for 5.5 hours, the white cloudy reaction mixture was concentrated under reduced pressure to give 25 mg (96%, 4.5% w / w dioxane) of the desired compound as a white solid.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

8.47 (d, 1H, J = 7.6 Hz), 7.76 (dd, 1H, J = 9.6, 2.1 Hz),

7.47-7.36 (m, 5H), 7.28 (d, 1H, J = 2.1 Hz), 7.17 (dd,

1H, J = 8/4, 2.1 Hz), 6.98 (d, 1H, J = 8.4 Hz), 6.68 (ddd, 1H, J = 15, 11, 3.7 Hz), 5.95 (dd, 1H, J = 15,

1.1 Hz), 5.67 (d, 1H, J = 10 Hz), 5.21 (d, 1H,

J = 10 Hz), 5.14 (dd, 1H, J = 10, 2.5 Hz), 4.83-4.77 (m, 1H), 4.52-4.49 (m, 1H), 3.92 (t, 1H, J = 6.1 Hz),

3.84 (s, 3H), 3.47 (dd, 1H, J = 14, 9.8 Hz), 3.18 (dd,

1H, J = 14, 3.6 Hz), 3.12 (dd, 1H, J = 14, 2.2 Hz), 2.83 (t, 2H, J = 7.6 Hz), 2.78- 2.70 (m, 3H), 2.38-2.30 (m,

1H), 2.02-1.82 (m, 3H), 1.52-1.43 (m, 2H), 1.23 (s,

3H), 1.22-1.00 (m, 4H), 1.18 (s, 3H), 1.13 (d, 3H,

J = 8.2 Hz), 1.06 (d, 3H, J = 6.2 Hz), 1.02 (d, 3H,

J = 6.1 Hz).

Preparation of Cryptophycin-55-N-tert-Boc-i-tert-butyl ester (L) -glutamate (35) (LSN 382366) 27 mg (0.038 mmol) (1), 17 mg (0.057 mmol)

To a solution of N-tert-Boc- (L) -glutamic acid γ-tert-butyl ester and 4-dimethylaminopyridine (0.5 mg, 0.0038 mmol) in 150 μΙ anhydrous methylene chloride was added 12 mg (0.057 mmol) of 1,3-dicyclohexylcarbodiimide in 41 μΙ of methylene chloride. After stirring for 60 minutes, the white cloudy reaction mixture was diluted with 2 mL of ethyl acetate / hexane (2: 1), stirred for 10 minutes and then filtered through a pad of celite, washing the pad of celite with 2: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give an off-white oily product. The product was purified by flash chromatography on silica gel (15 g) eluting with ethyl acetate / hexane (1: 1). 28 mg (74%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (CDCl ₃ ), δ:

7.39-7.29 (m, 5H), 7.24 (d, 1H, J = 1.9 Hz),

7.22-7.18 (m, 1H), 7.10 (dd, 1H, J = 8.5, 1.9 Hz),

6.87 (d, 1H, J = 8.5 Hz), 6.76 (ddd, 1H, J = 15, 13,

4.5 Hz), 5.78 (d, 1H, J = 15 Hz), 5.65 (d, 1H,

J = 7.6 Hz), 5.43 (d, 1H, J = 9.7 Hz), 4.99 (dd, 1H,

J = 10, 2.7 Hz), 4.90-4.80 (m, 1H), 4.83 (d, 1H,

J = 9.7 Hz), 4.69-4.61 (m, 1H), 4.55 (d, 1H,

J = 8.4 Hz), 4.01-3.96 (m, 1H), 3.90 (s, 3H), 3.40 (dd, 1H, J = 13.8, 8.0 Hz), 3.24 (dd, 1H, J = 13,

4.1 Hz), 3.18 (dd, 1H, J = 15, 5.0 Hz), 3.11 (dd, 1H,

J = 15, 7.9 Hz), 2.65-2.57 (m, 2H), 2.39-2.30 (m,

1H), 2.14-2.09 (m, 2H), 2.00-1.90 (m, 1H),

1.87-1.73 (m, 3H), 1.60-1.50 (m, 1H), 1.44 (s,

18H), 1.27 (s, 3H), 1.21 (s, 3H), 1.08 (d, 3H,

J = 7.0 Hz), 1.05 (d, 3H, J = 6.4 Hz), 1.00 (d, 3H,

J = 6.3 Hz).

HU 227 195 Β1

Preparation of Cryptophycin-55- (L) -α-glutamate hydrochloride (36 ') (LSN 382367) To a solution of mg (0.023 mmol) (35) in 232 µl of methylene chloride at room temperature was added μΙ (0.116 mmol) 4.0 M hydrochloric acid in 1,4-dioxane.

After stirring for 8.5 hours, the clear, colorless reaction mixture was evaporated under reduced pressure to give 20 mg (97%, corrected for 3% by weight of dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.6 Hz), 7.50-7.36 (m, 5H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd) , 1H, J = 8.6, 2.0 Hz), 7.01 (d, 1H, J = 8.6 Hz), 6.75-6.65 (m, 1H), 5.98 (d, 1H, J = 15 Hz), 5.57 (d, 1H, J = 9.7 Hz), 5.20 (d, 1H, J = 9.7 Hz), 5.15 (dd, 1H, J = 11, 3.0 Hz), 4.90 ^ 1.80 (m, 1H), 4.52 (dd, 1H, J = 11, 3.7 Hz), 3.87 (s, 3H), 3, 80-3.66 (m, 1H), 3.49 (dd, 1H, J = 13.10 Hz), 3.21 (dd, 1H, J = 14, 3.6 Hz), 3.15 (dd) , 1H, J = 14, 2.3 Hz), 2.82-2.72 (m, 3H), 2.41-2.30 (m, 3H), 2.05-1.81 (m, 5H). ), 1.25 (s, 3H), 1.20 (s, 3H), 1.15 (d, 3H, J = 7.1 Hz), 1.07 (d, 3H, J = 6.2 Hz) ), 1.05 (d, 3H, J = 6.1 Hz).

Cryptophycin A-55-N-t-Boc-ss-t-butyl ester- (L) aspartate (37) (LSN 382501) and cryptophycin 55 N-t-Boc \ ^J> -tert-butyl ester- ( Preparation of D-aspartate (39) (LSN 387040) 176 mg (0.249 mmol) (1), 144 mg (0.499 mmol)

To a solution of N-tert-Boc- (L) -aspartic acid p-tert-butyl ester and 4-dimethylaminopyridine (2.0 mg, 0.066 mmol) in 1.0 mL anhydrous methylene chloride was added 103 mg. (0.499 mmol) of 1,3-dicyclohexylcarbodiimide in 200 μΙ of methylene chloride. After stirring for 45 minutes, celite (88 mg) was added to the white cloudy reaction mixture and diluted with 2 mL of ethyl acetate / hexane (3: 1). After stirring for 10 minutes, the reaction mixture was filtered through a pad of celite, washing the celite with ethyl acetate / hexane 3: 1. The filtrate and the washings were evaporated under reduced pressure to give an off-white oily product. The product was purified by flash chromatography on silica gel (g: 2: 1 ethyl acetate: hexane). In addition to the fractions containing pure (37 '), mixed fractions (lower R _f ) are obtained. The mixed fractions were subjected to flash chromatography on silica gel (25 g) eluting with ethyl acetate / hexane (1: 1), then 2: 1, and finally 3: 1. In addition to pure compound (37 '), pure compound (39') (higher R _f ) is obtained. Fractions containing pure (37 ') were combined to give 149 mg (61%) total product as a white foam. Combining all fractions containing pure 39 'gave 56 mg (23%) of product as a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

(37 ') 7.39-7.30 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.24-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 2.0 Hz),

6.88 (d, 1H, J = 8.4 Hz), 6.76 (ddd, 1H, J = 15, 13,

4.3 Hz), 5.75 (d, 1H, J = 15 Hz), 5.50 (d, 1H, J = 7.9 Hz), 5.46 (d, 1H, J = 9.2 Hz) ), 5.01 ^ 1.98 (m, 2H), 4.93 (t, 1H, J = 9.9 Hz), 4.85 (d, 1H, J = 9.2 Hz),

4.78-4.71 (m, 1H), 4.17 ^ 1.10 (m, 1H), 3.91 (s, 3H),

3.41 (dd, 1H, J = 13, 8.2 Hz), 3.22 (dd, 1H, J = 13,

3.9 Hz), 3.17 dd, 1H, J = 14, 5.1 Hz), 3.09 (dd, 1H,

J = 14, 7.7 Hz), 2.61-2.50 (m, 4H), 2.36-2.28 (m,

1H), 2.02-1.97 (m, 1H), 1.83-1.76 (m, 2H), 1.45 (s,

9H), 1.41 (s, 9H), 1.28 (s, 3H), 1.20 (s, 3H),

1.06-1.01 (m, 6H), 0.99 (d, 3H, J = 6.2 Hz).

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

(39 ') 7.39-7.30 (m, 5H), 7.24 (d, 1H, J = 2.0 Hz),

7.24-7.18 (m, 1H), 7.09 (dd, 1H, J = 8.4, 2.0 Hz),

6.88 (d, 1H, J = 8.4 Hz), 6.73 (ddd, 1H, J = 15, 13,

4.3 Hz), 5.74 (d, 1H, J = 15 Hz), 5.47 (d, 1H,

J = 7.9 Hz), 5.42 (d, 1H, J = 9.1 Hz), 5.29 (d, 1H,

J = 9.0 Hz), 4.98 (dd, 1H, J = 10, 3.0 Hz), 4.92 (t, 1H,

J = 9.6 Hz), 4.85 (d, 1H, J = 9.1 Hz), 4.77 ^ 1.71 (m,

1H), 4.18-4.12 (m, 1H), 3.91 (s, 3H), 3.39 (dd, 1H,

J = 13, 8.1 Hz), 3.21 (dd, 1H, J = 13, 4.0 Hz), 3.16 (dd, 1H, J = 14, 5.2 Hz), 3.07 ( dd, 1H, J = 14, 7.6 Hz),

2.63-2.52 (m, 2H), 2.45 (dd, 1H, J = 17, 5.8 Hz),

2.36-2.26 (m, 1H), 2.22 dd, 1H, J = 17, 4.5 Hz),

1.99-1.90 (m, 1H), 1.86-1.69 (m, 2H), 1.47 (s, 9H),

1.43 (s, 9H), 1.25 (s, 3H), 1.19 (s, 3H), 1.11 (d, 3H,

J = 7.0 Hz), 1.03 (d, 3H, J = 6.5 Hz), 0.99 (d, 3H,

J = 6.4 Hz).

Preparation of Cryptophycin-55- (L) -aspartate hydrochloride (38) (LSN 382502)

To a solution of 146 mg (0.149 mmol) (37) in 498 μΙ methylene chloride at room temperature was added 374 μΙ (1.49 mmol) 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 19 hours, the clear, colorless reaction mixture was evaporated under reduced pressure to give 131 mg (100%, corrected for 2% dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.6 Hz), 7.45-7.36 (m, 5H), 7.31 (d,

1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.6, 2.0 Hz), 7.01 (d,

1H, J = 8.6 Hz), 6.75 (ddd, 1H, J = 15, 13, 3.7 Hz),

5.96 (dd, 1H, J = 15, 1.8 Hz), 5.54 (d, 1H, J = 10 Hz),

5.17 (d, 1H, J = 10 Hz), 5.14 (dd, 1H, J = 11, 2.1 Hz),

4.89 (t, 1H, J = 11Hz), 4.52 (dd, 1H, J = 11, 3.7Hz),

3.87 (s, 3H), 3.55 (t, 1H, J = 4.4 Hz), 3.52-3.47 (m,

1H), 3.21 (dd, 1H, J = 14, 3.6 Hz), 3.13 (d, 1H,

J = 14 Hz), 2.99 (dd, 1H, J = 18, 5.0 Hz), 2.83 (dd, 1H,

J = 18, 3.9 Hz), 2.81-2.68 (m, 3H), 2.35-2.28 (m,

1H), 2.02-1.84 (m, 3H), 1.26 (s, 3H), 1.21 (s, 3H),

1.08-1.02 (m, 9H).

Preparation of Cryptophycin-55- (D) aspartate hydrochloride (40) (LSN 387039) To a solution of mg (0.054 mmol) (39) in 271 μΙ methylene chloride was added 136 µl (0.542 mmol) 4.0 M 1.4 -dioxane hydrochloric acid solution. After stirring for 14 hours, the pure colorless reaction mixture was concentrated under reduced pressure to give 47 mg (94%, corrected for 6% by weight of dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.6 Hz), 7.45-7.36 (m, 5H), 7.31 (d,

1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.6, 2.0 Hz), 7.01 (d,

HU 227 195 Β1

1H, J = 8.6 Hz), 6.71 (ddd, 1H, J = 15, 13, 3.7 Hz),

5.96 (dd, 1H, J = 15, 1.8 Hz), 5.63 (d, 1H, J = 10 Hz),

5.19 (d, 1H, J = 10Hz), 5.15 (dd, 1H, J = 11, 2.1Hz),

4.86-4.80 (m, 1H), 4.55-4.50 (m, 1H), 4.24 (dd,

1H, J = 8.4,4.0 Hz), 3.87 (s, 3H), 3.50 (dd, 1H, J = 13,

9.7 Hz), 3.21 (dd, 1H, J = 14, 3.6 Hz), 3.14 (dd, 1H,

J = 13, 2.5 Hz), 2.80-2.70 (m, 3H), 2.40-2.32 (m,

1 H), 2.21 (dd, J = 18, 4.0 Hz), 2.09-1.97 (m, 2H),

1.92-1.84 (m, 2H), 1.26 (s, 3H), 1.21 (s, 3H), 1.15 (d, 3H, J = 7.1 Hz), 1.08 (d, 3H, J = 6.1 Hz), 1.04 (d,

3H, J = 6.0 Hz).

Preparation of Cryptophycin-55-N-tert-Boc-a-tert-butyl ester (L) -glutamate (41) (LSN 382572) 23 mg (0.033 mmol) (Γ), 15 mg (0.049 mmol)

To a solution of N-tert-Boc- (L) -glutamic acid-α-tert-butyl ester and 4-dimethylaminopyridine (0.4 mg, 0.0033 mmol) in 120 mL of anhydrous methylene chloride was added 10 mg ( 0.049 mmol) of 1,3-dicyclohexylcarbodiimide in 40 μΙ methylene chloride. After stirring for 45 minutes, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes, and then filtered through celite, washing the celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give an off-white oily product. The product was purified by flash chromatography on silica gel (15 g), eluting with 2: 1 ethyl acetate: hexane. 24 mg (75%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.39-7.30 (m, 5H), 7.24 (d, 1H, J = 2.1 Hz),

7.20-7.16 (m, 1H), 7.09 (dd, 1H, J = 8.4, 2.1 Hz),

6.87 (d, 1H, J = 8.4 Hz), 6.74 (ddd, 1H, J = 15, 13,

4.6 Hz), 5.78 (d, 1H, J = 15 Hz), 5.55 (br d, 1H,

J = 6.4 Hz), 5.46 (dd, 1H, J = 9.6, 1.0 Hz), 4.96 (dd,

1H, J = 11, 3.0 Hz), 4.91-4.80 (m, 2H), 4.83 (d, 1H,

J = 9.6 Hz), 4.72 (br s, 1H), 3.99 (br s, 1H), 3.91 (s,

3H), 3.39 (dd, 1H, J = 13, 7.9 Hz), 3.24 (dd, 1H,

J = 13, 3.9 Hz), 3.18 (dd, 1H, J = 14, 4.5 Hz), 3.09 (dd, 1H, J = 14, 7.8 Hz), 2.63- 2.55 (m, 2H),

2.44-2.37 (m, 1H), 2.10-1.92 (m, 3H), 1.85-1.78 (m, 1H), 1.76-1.61 (m, 3H) ), 1.46 (s, 9H), 1.45 (s,

9H), 1.27 (s, 3H), 1.20 (s, 3H), 1.08 (d, 3H,

J = 7.0 Hz), 1.04 (d, 3H, J = 6.6 Hz), 0.98 (d, 3H,

J = 6.4 Hz).

Preparation of Cryptophycin-55- (L) -? - glutamate hydrochloride (42) (LSN 382514) To a solution of mg (0.021 mmol) (41) in 212 μΙ methylene chloride at room temperature was added 53 μΙ (0.212 mmol) 4, 0 M hydrochloric acid in 1,4-dioxane.

After stirring for 23.5 hours, the reaction mixture was diluted with methanol and passed through a small pad of celite. The filtrate was concentrated under reduced pressure to give 20 mg (100%, corrected for 6% dioxane) of the desired compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.79 (d, 1H, J = 7.6 Hz), 7.50-7.29 (m, 6H), 7.20 (dd,

1H, J = 8.4, 2.0 Hz, 7.01 (d, 1H, J = 8.4 Hz), 6.66 (ddd, 1H, J = 15, 13, 3.7 Hz), δ , 97 (dd, 1H, J = 15,

1.5 Hz), 5.52 (dd, 1H, J = 10, 0.8 Hz), 5.14 (dd, 1H, J = 11, 3.0 Hz), 5.10 (d, 1H, J = 10 Hz), 4.90-4.80 (m, 1H), 4.51 (dd, 1H, J = 11, 3.7 Hz), 3.90-3.80 (m, 1H), 3.87 (s, 3H), 3.50 (dd, 1H, J = 13, 10Hz), 3.19 (dd, 1H, J = 14, 3.7Hz), 3.13 (dd, 1H) , J = 14, 2.3 Hz), 2.81-2.67 (m, 3H), 2.41-2.32 (m, 2H), 2.15-2.06 (m, 1H), 1.97-1.79 (m, 5H), 1.25 (s, 3H), 1.19 (s, 3H), 1.13 (d, 3H, J = 7.1 Hz), 1.08 (d, 3H, J = 5.9 Hz), 1.04 (d, 3H, J = 5.8 Hz).

Preparation of Cryptophycin-55-N, N'-di-tert-Boc- (S) -2,3-diaminopropionate (43) (LSN 382765) 21 mg (0.030 mmol) (1), 18 mg (0.060 mmol) N To a room temperature solution of N'-di-tert-Boc- (S) -2,3-diaminopropionic acid and 0.3 mg (0.0030 mmol) of 4-dimethylaminopyridine in 110 μΙ anhydrous methylene chloride was added (0.060 mmol) of 1,3-dicyclohexylcarbodiimide in 39 μΙ of methylene chloride. After stirring for 70 minutes, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes and then filtered through celite, washing the celite with 3: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give an off-white oily product. The product was flash chromatographed on silica gel (15 g), eluting with 2: 1 ethyl acetate: hexane. 24 mg (80%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.49-7.40 (m, 2H), 7.38-7.30 (m, 4H), 7.19 (dd, 1H, J = 8.5, 1.9 Hz), 7.00 (d, 1H, J = 8.5 Hz), 6.74 (ddd, 1H, J = 16, 11, 3.5 Hz), 5.95 (d, 1H, J = 16 Hz),

5.49 (d, 1H, J = 10Hz), 5.16-5.08 (m, 2H), 4.90-4.80 (m, 1H), 4.51 (dd, 1H, J = 11, 3.7 Hz), 4.06-4.10 (m, 1H), 3.87 (s, 3H), 3.48 (d, 1H, J = 13 Hz), 3.19 (dd, 1H, J = 14, 3.7Hz), 3.14 (d, 1H, J = 13Hz), 3.03 (dd, 1H, J = 14, 4.2Hz), 2.94-2, 87 (m, 1H), 2.80-2.59 (m, 3H), 2.39-2.30 (m, 1H),

1.98-1.81 (m, 3H), 1.43 (s, 18H), 1.25 (s, 3H), 1.21 (s, 3H), 1.10 (d, 3H, J = 7.0 Hz), 1.07 (d, 3H, J = 6.0 Hz), 1.03 (d, 3H, J = 5.8 Hz).

Preparation of Cryptophycin-55- (S) -2,3-diaminopropionate dihydrochloride (44) (LSN 382764) To a solution of 21 mg (0.021 mmol) (43) in 211 μΙ methylene chloride at room temperature was added 42 μ 0, (0.169 mmol). 4.0 M hydrochloric acid in 1,4-dioxane. After stirring for 5 hours, the reaction mixture was concentrated under reduced pressure to give 18.5 mg (100%, corrected for 3% dioxane) as a white solid.

¹ H-NMR (MeOH-D ₄ ), δ:

7.50-7.39 (m, 5H), 7.31 (d, 1H, J = 2.0 Hz), 7.20 (dd, 1H, J = 8.4, 2.0 Hz), 7.01 (d, 1H, J = 8.4 Hz), 6.68 (ddd, 1H, J = 15, 11, 3.8 Hz), 5.99 (dd, 1H, 15,

1.7 Hz), 5.56 (d, 1H, J = 9.6 Hz), 5.24 (d, 1H, J = 9.6 Hz), 5.16 (dd, 1H, J = 10, 3.1 Hz), 4.90-4.80 (m, 1H), 4.51 (dd, 1H, J = 11.3.8 Hz), 4.04-4.00 (m, 1H), 3.87 (s, 3H), 3.50-3.45 (m, 1H), 3.26 (dd, 1H,

HU 227 195 Β1

J = 14, 4.0 Hz), 3.22-3.12 (m, 3H), 2.81-2.75 (m,

3H), 2.42-2.32 (m, 1H), 1.98-1.78 (m, 3H), 1.25 (s,

3H), 1.19 (s, 3H), 1.22-1.16 (m, 3H), 1.07 (d, 3H,

J = 6.4 Hz), 1.04 (d, 3H, J = 6.3 Hz).

Preparation of Cryptophycin-55-N-tert-Boc- (L) -Serinate (45) (LSN 384340) N-tert-Boc-O-tert-butyl mg (0.053 mmol), 51 mg (0.160 mmol) of dimethylsilyl (L) -Serine and 4-dimethylaminopyridine (0.6 mg, 0.053 mmol) in 200 µl of anhydrous methylene chloride at room temperature was added 33 mg (0.160 mmol) of 1,3-dicyclohexylcarbodiimide 69 μΙ in methylene chloride. After stirring for 4 hours, the white cloudy reaction mixture was diluted with 1 mL of ethyl acetate / hexane 2: 1, stirred for 10 minutes and filtered through celite, washing the celite with 2: 1 ethyl acetate / hexane. The filtrate and washings were evaporated under reduced pressure to give a colorless oily product which was used directly for the next step. A solution of crude silyl ether (54 mg, 0.054 mmol) in 268 μΙ tetrahydrofuran at room temperature was treated with hydrogen fluoride / pyridine solution (prepared from 0.5 g hydrogen fluoride / pyridine complex; Aldrich, 4 ml tetrahydrofuran and 1 ml pyridine). After stirring for 4 hours, another 67 μΙ hydrogen fluoride / pyridine solution was added to the reaction mixture. After stirring for 30 minutes, 0.6 ml of a saturated aqueous solution of sodium bicarbonate are added to the reaction mixture, which is extracted with 3 ml of ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered and concentrated under reduced pressure to give a pale yellow foam. The product was flash chromatographed on silica gel (16 g) eluting with ethyl acetate / hexane (3: 1 then 6: 1). 26 mg (44%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (MeOH) -d ₄ ), δ:

7.79 (dd, 1H, J = 9.5, 2.4 Hz), 7.45-7.38 (m, 2H),

7.37-7.29 (m, 3H), 7.29 (d, 1H, J = 2.0 Hz), 7.18 (dd,

1H, J = 8.4, 2.0 Hz), 6.99 (d, 1H, J = 8.4 Hz), 6.72 (ddd, 1H, J = 15, 13, 3.7 Hz), 5.95 (dd, 1H, J = 15,

1.6 Hz), 5.53 (d, 1H, J = 9.5 Hz), 5.12-5.08 (m, 2H),

4.94 (t, 1H, J = 10 Hz), 4.52 (dd, 1H, J = 12, 3.6 Hz),

3.92 (t, 1H, J = 4.6 Hz), 3.85 (s, 3H), 3.52-3.46 (m,

3H), 3.20 (dd, 1H, J = 14, 3.5 Hz), 3.14 (dd, 1H,

J = 13, 3.0 Hz), 2.75 (dd, 1H, J = 14, 11 Hz),

2.73-2.66 (m, 1H), 2.65-2.59 (m, 1H), 2.38-2.29 (m, 1H), 2.00-1.82 (m, 3H) ), 1.43 (s, 9H), 1.25 (s,

3H), 1.22 (s, 3H), 1.10-1.06 (m, 6H), 1.03 (d, 3H,

6.0 Hz).

Preparation of Cryptophycin-55- (L) -Serinate Hydrochloride (46) (LSN 384339) To a solution of mg (0.029 mmol) (45) in 291 μΙ methylene chloride at room temperature was added 36 μΙ (0.146 mmol) 4.0 M Hydrochloric acid in 4-dioxane.

After stirring for 2.5 hours, the clear, colorless reaction mixture was concentrated under reduced pressure to give 23 mg (94%, corrected for 2% by weight of dioxane) as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.48-7.40 (m, 2H), 7.39-7.31 (m, 3H), 7.31 (d, 1H, J = 2.2 Hz), 7.20 (dd, 1H, J = 8.5.2.0 Hz), 7.01 (d, 1H, J = 8.5 Hz), 6.70 (ddd, 1H, J = 15, 13, 3.7 Hz), 5, 95 (dd, 1H, 15, 1.8 Hz), 5.59 (d, 1H, J = 10 Hz), 5.20 (d, 1H, J = 10 Hz), 5.14 (dd, 1H, J = 11, 2.1 Hz), 4.94 (t, 1H, J = 10 Hz), 4.52 (dd, 1H, J = 3.5 Hz), 3.96 (dd, 1H, J = 11.3.9 Hz), 3.83 (dd, 1H, J = 11.2.8 Hz), 3.66 (t, 1H, J = 3.2 Hz), 3.50 (d, 1H, J = 14Hz), 3.20 (dd, 1H, J = 14, 3.5Hz), 3.13 (d, 1H, J = 14Hz), 2.80-2.71 (m, 3H) , 2.38-2.28 (m, 1H), 2.01-1.82 (m, 3H), 1.26 (s, 3H), 1.21 (s, 3H), 1.13 (d , 3H, J = 7.0Hz), 1.07 (d, 3H, J = 6.1Hz), 1.05 (d, 3H, J = 6.1Hz).

Preparation of Cryptophycin-55-glycidylglycinate hydrochloride (47) (LSN 387750) mg (0.026 mmol) (1), 12 mg (0.051 mmol)

To a room temperature solution of N-tert-Boc-glycidylglycine and 0.3 mg (0.0026 mmol) in 4-dimethylaminofluoride and 28 μΙ N, N-dimethylformamide was added 11 mg (0.051 mmol) , 3-dicyclohexylcarbodiimide in 28 μΙ methylene chloride. After stirring for 3 hours, another 24 mg (0.102 mmol) of N-tert-Boc-glycidylglycine and 22 mg (0.102 mmol) of 1,3-dicyclohexylcarbodiimide in 30 μΙ Ν, dimet-dimethylformamide were added to the reaction mixture. After stirring for 1.5 hours, the white cloudy reaction mixture was diluted with 0.5 mL of ethyl acetate / hexane (3: 1), stirred for 10 minutes and then filtered through celite, washing the celite with ethyl acetate / hexane (3: 1). The filtrate and washings were evaporated under reduced pressure to give a colorless oily product. The product was chromatographed on 12 g of flash silica gel eluting with 1% methanol in ethyl acetate. 12 mg of product are obtained in the form of a white foam which is used directly for the next step. To a solution of 12 mg (0.013 mmol) of the above N-Boc-glycidylglycinate in 130 μΙ of methylene chloride was added 16 μΙ (0.065 mmol) of 4.0 M hydrochloric acid in 1,4-dioxane.

After stirring for 3.5 hours, the reaction mixture was concentrated under reduced pressure to give 11 mg (100%) of the title compound as a white foam.

¹ H-NMR (500 MHz, MeOH-d ₄ ), δ:

7.45-7.29 (m, 5H), 7.31 (d, 1H, J = 2.1 Hz), 7.20 (dd, 1H, J = 8.4, 2.1 Hz), 7 , 01 (d, 1H, J = 8.4 Hz), 6.72 (ddd, 1H, J = 15, 11, 3.8 Hz), 5.96 (dd, 1H, J = 15,

1.4 Hz), 5.49 (dd, 1H, J = 10, 0.8 Hz), 5.14-5.09 (m, 2H), 4.90-4.80 (m, 1H), 4.51 (dd, 1H, J = 11,

3.9 Hz), 3.91-3.84 (m, 1H), 3.87 (s, 3H), 3.70-3.65 (m, 2H), 3.50 (d, 1H, J = 14 Hz), 3.38 (d, 1H, J = 18 Hz), 3.19 (dd, 1H, J = 14, 3.6 Hz), 3.12 (d, 1H, J = 14 Hz) , 2.82-2.63 (m, 3H), 2.40-2.30 (m, 1H), 1.97-1.80 (m, 3H), 1.25 (s, 3H), 1 , 20 (s, 3H), 1.12 (d, 3H, J = 7.0 Hz), 1.06 (d, 3H, J = 6.0 Hz), 1.03 (d, 3H, J = 5.9 Hz).

Preparation of Cryptophycin-55-3,6,9-trioxadecanoate (48) (LSN 387414) mg (0.026 mmol) (1), 7.8 μΙ (0.051 mmol), 3,6,9-trioxadecanoic acid and 0.3 mg (0, 0026 mmol) 4-dimethyl81

To a solution of 195 µl of aminopyridine in 100 µl of anhydrous methylene chloride was added a solution of 11 mg (0.051 mmol) of 1,3-dicyclohexylcarbodiimide in 28 µl of methylene chloride. After stirring for 30 minutes, the white cloudy reaction mixture was diluted with ethyl acetate / hexane (3: 1, 0.5 mL), stirred for 10 minutes and then filtered through a pad of celite, washed with ethyl acetate / hexane (3: 1). The filtrate and the washings were evaporated under reduced pressure to give an off-white oily product. The product was purified by flash chromatography on 12 g of silica gel eluting with 2% methanol in ethyl acetate. 19 mg (86%) of the desired compound are obtained in the form of a white foam.

¹ H-NMR (500 MHz, CDCl ₃ ), δ:

7.49-7.29 (m, 5H), 7.24 (d, 1H, J = 1.8 Hz),

7.22-7.19 (m, 1H), 7.10 (dd, 1H, J = 8.4, 1.8 Hz),

6.87 (d, 1H, J = 8.4 Hz), 6.74 (ddd, 1H, J = 15, 10,

4.5 Hz), 5.32-5.28 (m, 2H), 5.56 (d, 1H, J = 9.7 Hz),

4.98-4.90 (m, 2H), 4.83 (d, 1H, J = 9.7 Hz), 4.77-4.71 (m, 1H), 3.90 (s, 3H) , 3.83 (d, 1H, J = 17Hz), 3.65-3.55 (m, 7H), 3.42-3.33 (m, 6H),

3.24-3.18 (m, 2H), 3.03 (dd, 1H, J = 15, 8.1 Hz), 2.69-2.61 (m, 1H), 2.61-2, 56 (m, 1H), 2.54-2.46 (m, 1H), 2.00-1.93 (m, 1H), 1.85-1.77 (m, 1H), 1.72-. 1.66 (m, 1H), 1.26 (s, 3H), 1.19 (s, 3H), 1.09 (d, 3H, J = 7.0 Hz), 1.02 (d, 3H) , J = 6.6 Hz), 0.97 (d, 3H, J = 6.5 Hz).

Claims (18)

PATENT CLAIMS A compound of formula (I) wherein Ar is phenyl, unsubstituted aromatic, aromatic substituted with halo or C 1-7 alkyl, unsubstituted C 3-8 heteroaromatic, C 1-12 alkyl, C 2-12 alkenyl or C 2-12 alkynyl, NR ⁵¹ R ⁵² or OR ⁵³ or Ar '; R ¹ is halogen or OH; R ² is halogen or -OR ³¹ with the proviso that one of R ¹ and R ² , but not both, is halogen; R ³ is C 1-6 alkyl; R ⁴ is hydrogen or hydroxy; ^R5 is hydrogen or hydroxy; obsession R ⁴ and R ⁵ together may form a second bond between C-13 and C-14; R ⁶ is benzyl, hydroxybenzyl, (C 1-3 alkoxy) benzyl, halogenated hydroxybenzyl, dihalogenated hydroxybenzyl, (halogenated C 1-3 alkoxy) benzyl, or (dihalogenated C3-C4 alkoxy) benzyl; R ⁷ is hydrogen or C 1-6 alkyl; R ⁸ is hydrogen or C 1-6 alkyl; R ⁹ is hydrogen or C 1-6 alkyl; R ¹⁰ is hydrogen or C 1-6 alkyl; R ¹¹ is hydrogen, R ³¹ is R ³² ; ^R32 is occurring naturally occurring amino acid, C ₁₂ H ₂₂ O or _ir C ₆ H ₁₀ O ₅ -carbohydrate 1-3 amino substituent Ci ₂ H ₂₂ 0h- or C ₆ H ₁₀ O ₅ carbohydrates, (saccharide) _q wherein the saccharide is lactose, maltose, sugar, fructose or starch; or C (O) R ³³ ; R ³³ is R ³⁷ R ³⁸ , R ³⁷ is C 1-6 alkyl; R ³⁸ is COOR ^39, or Vil, NH ₂ or a naturally occurring amino acid; R ³⁹ is hydrogen or C 1-6 alkyl; R ⁴⁰ , R ⁴¹ and R ⁴² are each independently hydrogen, halogen, OR ⁴³ , NH 2, NO 2, OPO (OR ⁴⁶ ) 2, -OR ⁴⁴ -phenyl, or R ⁴⁵ or the general formula; R ⁴³ is C 1-6 alkyl; R ⁴⁴ is C 1-6 alkylene; R ⁴⁵ is an unsubstituted aromatic group or an aromatic group substituted with hydrogen or C 1-7 alkyl; R ⁴⁶ is hydrogen, sodium or -C (CH ₃ ) ₃ ; R ⁵¹ is hydrogen or C 1-3 alkyl; R ⁵² is hydrogen or C 1-3 alkyl; R ⁵³ is C 1 -C 12 alkyl; R ⁵⁴ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO 3 H, SO ³ H, SO ² R ⁵⁸ , N (R ⁵⁹ ) R ⁶⁰ , NHOR ⁶¹ , NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen, OR ⁶² or SR ⁶³ or general formula; R ⁵⁵ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO 3 H, SO ³ H, SO ² R ⁵⁸ , NR ⁵⁹ R ⁶⁹ , NHOR ⁶¹ ', NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen OR ⁶² or SR ⁶³ or a group of the general formula; R ⁵⁶ is hydrogen, C 1-6 alkyl, aromatic, phenyl, COOR ⁵⁷ , PO 3 H, SO ³ H, SO ² R ⁵⁸ , NR ⁵⁹ R ⁶⁰ , NHOR ⁶¹ , NHCH ₂ R ⁶¹ , CN, NO ₂ , halogen, OR ⁶² or SR ⁶³ or a group of the general formula; R ⁵⁷ is hydrogen or C 1 -C 12 alkyl; R ⁵⁸ is hydrogen or C 1 -C 12 alkyl; R ⁵⁹ is hydrogen, C 1-6 alkyl, or fluorenylmethoxycarbonyl (FMOC); R ⁶⁰ is hydrogen or C 1-6 alkyl; R ⁶¹ is hydrogen, OR ^64, CH ₂ NHR ^65, NHR ⁶⁵ radical or fluorenyl-methoxycarbonyl (FMOC); HU 227 195 Β1 R ⁶¹ is hydrogen, OR ^64, CH ₂ NHR ^65, NHR ⁶⁵ radical or fluorenyl-methoxycarbonyl (FMOC); R ⁶² is hydrogen or C 1-6 alkyl; R ⁶³ is hydrogen or C 1-6 alkyl; R ⁶⁴ is hydrogen, C 1-6 alkyl, or CH ₂ NR ⁶⁶ R ⁶⁷ ; R ⁶⁵ is hydrogen, C 1-6 alkyl, NH _2, or fluorenylmethoxycarbonyl (FMOC); R ⁶⁶ is hydrogen, C 1-6 alkyl, or fluorenylmethoxycarbonyl (FMOC); R ⁶⁷ is hydrogen or C 1-6 alkyl; q is 2, 3 or 4; X is O, NH, or (C 1 -C 7) alkylamino; and Y is carbon or oxygen, NH, sulfur, SO, SC _2, or (C 1 -C 7) alkylamino, or pharmaceutically acceptable salts or solvates thereof. The compound of claim 1, wherein Y is oxygen. The compound of claim 2, wherein X is oxygen. The compound of claim 1, wherein R ⁶ is benzyl, hydroxybenzyl, halogenated hydroxybenzyl, dihalogenated hydroxybenzyl, halogenated alkoxybenzyl, or dihalogenated alkoxybenzyl, R ⁹ is isobutyl and R ¹⁰ is hydrogen. 5. A compound selected from the group consisting of Cryptophycin-55-acetate, Cryptophycin-55-succinate, Cryptophycin-55- (2'-di-tert-butyl-1-phosphatyl) -phenyl acetate, Cryptophycin-55- (2) '-phosphatyl) phenylacetate, Cryptophycin-55-nicotinoate, Cryptophycin-55-N-methylpyridinium, Cryptophycin-55-N-tert-Boc-3- (3-chloro-4-methoxyphenyl) - ( D) -alaninate, Cryptophycin-55-3- (3-chloro-4-methoxyphenyl) - (D) -alaninate, Cryptophycin-55-N-tert-Boc-glycinate, Cryptophycin-55-N-tert-Boc-p -alaninate, Cryptophycin-55-N-tert-Boc-Y-aminobutyrate, Cryptophycin-55-N-tert-Boc- (L) -alaninate, Cryptophycin-55-tert-Boc- (D) -alaninate, and Cryptophycin-55-Na-Nitrile-Boc- (L) -Lysinate or pharmaceutically acceptable salts thereof. 6. A compound selected from the group consisting of Cryptophycin-129, Cryptophycin-138, Cryptophycin-145, Cryptophycin-140 and Cryptophycin-141, or pharmaceutically acceptable salts thereof. 7. A compound selected from the group consisting of Cryptophycin-152, Cryptophycin-255, Cryptophycin-153, Cryptophycin-161, Cryptophycin-234, Cryptophycin-236, Cryptophycin-247, Cryptophycin-251, and Cryptophycin-238, or and pharmaceutically acceptable salts thereof. 8. A compound selected from the group consisting of Cryptophycin-55-succinate tert-butyl ester, Cryptophycin-55- (2'-di-tert-butylphosphatyl) phenylacetate, Cryptophycin-55- (2 ') -phosphatyl) -phenyl acetate, Cryptophycin-55-nicotinoate, Cryptophycin-55-N-methylpyridinium, Cryptophycin-55-N-tert-Boc-3- (3-chloro-4-methoxyphenyl) - (D ) -alaninate, Cryptophycin-55-N-tert-Boc- (L) -phenylalaninate, Cryptophycin-55- (L) -phenylalaninate, Cryptophycin-55- (L) -histidine, Cryptophycin-55-N- tert-Boc- (L) -prolinate, Cryptophycin-55- (L) -prolinate, Cryptophycin-55-N-tert-Bocglycinate, Cryptophycin-55-glycinate, Cryptophycin-55-Νterc-Boc-p-alaninate, Cryptophycin- 55-β-alaninate, Cryptophycin-55-N-tert-Boc-Y-aminobutyrate, Cryptophycin 55-Y-aminobutyrate, Cryptophycin-55-N-tert-Boc- (L) alaninate, Cryptophycin-55- (L) -alaninate, Cryptophycin-55-N-tert-Boc- (D) -alaninate, Cryptophycin-55- (D) alaninate, Cryptophycin-55-Na-N-di-tert-Boc- (L) lysinate, Cryptophycin- 55- (L) -Lysinate, Cryptophy cin-55-Na-N? -diterc-Boc- (D) lysinate, Cryptophycin-55- (D) -Lysinate, Cryptophycin-55-N-tert-Boc-Y-butyl ester (L) -glutamate , Cryptophycin-55- (L) -α-glutamate, Cryptophycin-55-tert-Boc-p-tert-butyl ester aspartate, Cryptophycin-55- (D) -aspartate, Cryptophycin-55-N-tert-Boc tert-butyl ester (L) -glutamate, Cryptophycin-55- (L) -Yglutamate, Cryptophycin-55-N, N'-di-tert-Boc- (S) -2,3-diaminopropionate, Cryptophycin 55- (S) -2,3-Diaminopropionate, Cryptophycin-55-N-tert-Boc (L) Serinate, Cryptophycin-55- (L) Serinate, and Cryptophycin-55 Succinate, or pharmaceutically acceptable salts thereof. 9. A compound according to any one of claims 1 to 6, wherein R ⁷ and R ⁸ are both methyl. 10. A compound according to any one of claims 1 to 9 and 9, wherein R ² is a glycinate. 11. Cryptophycin-55-glycinate or a pharmaceutically acceptable salt thereof. The compound of claim 12, wherein the compound is the hydrochloride salt of Cryptophycin-55-glycinate. 13. A compound according to any one of claims 1 to 6 for use in the treatment of the human or animal body. The compound of claim 13 for use in the treatment of neoplasm in a mammal. A compound according to claim 13 for use in the treatment of a fungal infection in a mammal. 16. Use of a compound according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of neoplasm in a mammal. 17. Use of a compound according to any one of claims 1 to 6 for the preparation of a medicament for the treatment of a fungal infection in a mammal. 18. A composition according to any one of claims 1-12. A compound according to any one of claims 1 to 6 together with one or more pharmaceutically acceptable carriers or diluents.