Cephalotaxane derivatives and their processes of preparation and purification

Abstract

The present invention concerns a new general process for asymmetric hemisynthesis of harringtonines and their analogs, that are alkaloids used in chemotherapy. This process comprises direct esterification of a natural cephalotaxine with an acylating compound constituted of a side chain precursor which backbone and functionalization are entirely preformed. The invention also concerns a natural, synthetic or semi-synthetic harringtonines including their tautomeric forms and their salts of the following formula:

##STR00001##
wherein n=2 (i.e. harringtonine) or n=3 (i.e. homoharringtonine),
in which the total content of impurities, possibly including enantiomeric forms, is lower than 1%, and/or the content of the major impurity is lower than 0.9%, and/or the chromatographic assay exhibits a harringtonines content higher than 97.5%.

Description

This applicationtypical tertiaire
where
Ω (“omega”) is a representative radical of the chain terminal moiety and —CO— is the carbonyl of the ester group bonded to cephalotaxane;
the Ω-CO— radical is corresponding:

either to the following substituted heterocycloalkane formula:

##STR00029##
where n is included between 0 and 8;
Z is oxygen, nitrogen or sulfur heteroatom;

R⁵, R⁶ and R⁸ are independently hydrogen;

hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s); R⁶ and R⁸ may be included in a cycle;

oxygen ether bearing one of the former radicals;

or to the following linear alkene formula:

##STR00030##
where m is included between 1 and 8, R⁵, R⁶ and R⁸ are as defined above;

or to the following formula:

##STR00031##
where n, R⁵, R⁶ and R⁸ are as defined above;
Z and Q² are independently oxygen, nitrogen or sulfur heteroatom;
Q1 is carbon, silicium or phosphorus atom;
R⁹ and R¹⁰ are independently hydrogen, alkoxy, hydrocarbon radical, including or not heteroatom(s), saturated, unsaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl;
R⁹ and/or R¹⁰ having the ability to be null or taken together to make an heteroatom and/or make a multiple bond with Q¹, R⁹ and R¹¹ having the ability to be null to make a multiple bond between the two atoms of carbon bearing them; and
R¹¹ is hydrogen, arylcarbonyl, alkoxycarbonyl, aryloxycarbonyl or alkylcarbonyl;
where

##STR00032##
—O—CTX is cephalotaxine moiety of the following formula a salt thereof:
where p is equal to 1 or 2;
the two types of radicals -Ω and —CTX above-mentioned being bonded with an ester bond —CO—O—
the said process bringing together:

• • either carboxylic acid with general formula Ω-CO—OH or a salt thereof;

##STR00033##

• • or an activated form of an acid with general formula Ω-CO-A or a salt thereof, with Ω-CO of the following formula:
    where n, Z, R⁵, R⁶ and R⁸ are as defined above;
    where Ω-CO of the following formula:

##STR00034##
m is included between 1 and 8, Z, R⁵, R⁶ and R⁸ are as defined above;
where Ω-CO of the following formula:

##STR00035##
and where n, Z, Q¹, Q², R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above A represents:

either cyclic anhydride of the following formula:

##STR00036##
where n, R⁶ and R⁸ are as defined above;
the reaction has been completed by methylation of the primary carboxyl thus formed, with:

either a cephalotaxane or a salt thereof, bearing at least a free hydroxyl group, of the formula H—O—CTX, where CTX are as defined above;

or a metallic alcoxide of the formula M-O—CTX, where CTX are as defined above and M is a metal;

or an activated form of its hydroxyl group of the formula Y—O—CTX, where —O—CTX is as defined above and Y is, either a leaving group to allow a negative charge on oxygen atom by cleavage between Y— and —O—CTX, or to allow a carbocation by cleavage between Y—O— and —CTX;

with the possible presence of one or several reaction additives to form said sidechain-bearing cephalotaxane and/or a salt thereof.

Most preferably, Z is an oxygen atom and the cephalotaxane H—O—CTX is a cephalotaxine of the following formula, or a salt thereof:

##STR00037##
where R¹, R², R³ and R⁴ are independently hydrogen, hydroxyl group or alkoxide.

A cephalotaxane H—O—CTX, as defined above, is cephalotaxine, or a salt thereof, where R¹ is hydroxyl, R² is methoxyl, R³ and R⁴ are hydrogen.

##STR00038##
R⁵ is preferably hydrogen or —CH₂—CO—O-Me.

The Ω-CO radical is preferably such as n=1 to 4, R⁶ and R⁸ are methyl.

The Ω-CO radical may be too such as n=1 or 2, R⁶ is phenyl and R⁸ is hydrogen.

When R⁵ is —CH₂—CO—O-Me, R¹═OH, R2=OMe, R³═R⁴═H, the cephalotaxane is preferably such as n=0, Z is a nitrogen atom and R⁸ is hydrogen.

A may be Ω-CO—CO where Ω is defined as above, or an halide.

A may also be a radical of compound Ω-CO-A having the ability to generate cleavage of the bond between carbonyl group and substituent A to provide Ω-CO⁺ and A⁻.

In addition, A is a radical selected from substituents:

Méthoxyformyloxy of formula MeOCOO—,

trifluoroacétyloxy of formula CF₃COO—,

alkylsulfonoxy of formula RSO₃—,

phosphoxy of formula (RO)₂PO—,

halophosphoxy of formula ROP(Cl)O—,

trialkylsilyloxy of formula R₃SiO—,

formulas wherein R is alkyl,

diméthyl-formamidinium chloride of formula

##STR00039##
or acyloxy-pyridinium bromide of formula

##STR00040##

A may also be 2,4,6-trichlorobenzoyloxy or a radical corresponding to the following formula:

##STR00041##

In the case where A is a carbonyl-diimidazole, where A is 2,4,6-trichlorobenzoyloxy, the reagent of formula Ω-CO-A is obtained by contacting an acid Ω-CO—OH, as defined above, with 2,4,6-trichlorobenzoyloxy carbonyl-diimidazole in presence of a strong base such as an alkoxide.

According carbodiimide method, the coupling additive is a substituted carbodiimide and/or a basic additive such as tertiary amine for example. For example, the substituted carbodiimide is selected from cyclohexylcarbodiimide (DCC), 1,3-diisopropylcarbodiimide (DIC) and chlorhydrate of 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.

The cephalotaxine alcoxide, corresponding to the formula M-O—CTX where M and CTX are as defined above, may be obtained by contacting a cephalotaxine of formula H—O—CTX with metal himself, an amidure, a metallic hydride or an alkyl-metal.

M may be an alkaline metal such as lithium, potassium or sodium.

The aim of the present invention is also the preparation of new compounds such as:

the lithium alcoxide of cephalotaxine corresponding to the following formula:

##STR00042##

the sodium alcoxide of cephalotaxine corresponding to the following formula:

##STR00043##

a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

##STR00044##
where
n is included between 0 and 8;
Z is oxygen, nitrogen or sulfur heteroatom;

R⁵, R⁶ and R⁸ are independently hydrogen;

hydrocarbon radical, saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocycloalkyl, of said radical including or not heteroatom(s);

oxygen ether bearing one of the former radicals;

CTX is as defined above;

except for compounds where Z is oxygen atom and,

1°) n=2 or 3, and simultaneously R⁶═R⁸=methyl and R⁵═OMe or hydroxyl,

1°) n=2 and simultaneously R⁶═R⁸=methyl and R⁵═OMe or hydroxyl;

3°) n=3 and simultaneously R⁶ is hydroxyl, when R⁸ is methyl and R⁵ is —CH₂CO₂CH₃ radical.

a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

##STR00045##
where
m, R⁵, R⁶, R⁸ and CTX are as defined above;
except compound where m=2, R⁵═CH₂CO₂CH₃, R⁶═R⁸=methyl and CTX is as defined above.

R⁵ is preferably the —CH₂—CO—O—CH₃ radical.

a sidechain-bearing cephalotaxane corresponding to the following formula and/or a salt thereof:

##STR00046##
where n, Z, Q¹, Q², R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹ and CTX are as defined above.

Preferably, Q² is oxygen atom and/or Z is nitrogen atom and the cephalotaxane such as n=0.

a sidechain-bearing cephalotaxane corresponding to the following formula:

##STR00047##

a sidechain-bearing cephalotaxane corresponding to the following formula:

##STR00048##

a sidechain-bearing cephalotaxane corresponding to the following formula:

##STR00049##

When the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula:

##STR00050##

where n, R⁵, R⁶, R⁸, CTX and Z are as defined above, the said chain is open with an agent and/or a protonic or not protonic electrophilic radical E in aqueous or not aqueous medium, to provide an intermediate compound of the following formula:

##STR00051##
where n, CTX, R⁵, R⁶ and R⁸ are as defined above, E is either hydrogen or the provisionally or definitively fixed eletrophilic radical;
the aforementioned intermediate compound may be attacked with an agent or a nucleophilic radical Z′, deliberately added or possibly present in the medium, and
when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula:

##STR00052##
where n, R⁵, R⁶, R⁸, R⁹, R¹⁰ and R¹¹ are as defined above, and Z′ is an heteroatom;
the said chain is open by hydrolysis or carefully solvolysis with possibly presence of activation and/or opening additive.

In addition, to provide an open sidechain-bearing cephalotaxane of the following formula:

##STR00053##
where n, CTX, R⁵, R⁶ and R⁸ are as defined above;
Z′ is:

either a halogen or an heteroatom bearing a hydrogen or a radical R¹¹ such as defined above;

or an hydrogen, hydrocarbon radical, the said radical bearing or not heteroatom(s), saturated, insaturated or aromatic, linear or ramified and/or cyclic, especially alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or heterocycloalkyl.

For example, cephalotaxine esters of the following formulas:

##STR00054##

Where R⁵, R⁶, R⁸, Z′, X and CTX are as defined above;

bromodeoxyharrintonine (n=2) and bromodeoxyhomoharintonine (n=3)

##STR00055##
where CTX is as defined above;

aminodeoxyharrintonine (n=2) and aminodeoxyhomoharrintonine (n=3)

##STR00056##
where CTX is as defined above;

In addition, when the cyclic side-chain of sidechain-bearing cephalotaxane, and/or a salt thereof, presents the following formula:

##STR00057##
where n, R⁵, R⁶, R⁸, CTX and Z are as defined above,
the said chain is open by treatment with a solution of hydrobromic acid in acetic acid, in an halogenated solvent, preferably dichloromethane, followed by in situ hydrolysis to provide, without isolation of the intermediate, a sidechain-bearing cephalotaxane of the following formula:

##STR00058##
where n, CTX, R⁵, R⁶ et R⁸ are defined above.

Acids corresponding to the following formula:
Ω-CO—OH
where Ω radical is as defined according above;
the said formula equivalent to racemic mixture containing compounds of the formulas (+)-Ω-CO—OH and (−)-Ω-CO—OH such as (+)-Ω-CO—OH represents its dextrogyre enantiomer and (−)-Ω-CO—OH represent its levogyre enantiomer, were obtained
a) by contacting of said racemic mixture or one of its activated form of the formula
Ω-CO-A
which is as defined above;
the said racemic mixture or said activated form generating respectively:

either an anion corresponding to the formula (Ω-CO—O)⁻;

or a cation corresponding to the formula (Ω-CO)⁺;

with a pure enantiomeric form of chiral entity, said “resolution agent” symbolized by Δ*(delta stella), having the ability to form:

either a stable combination, by covalent bonding;

or an easily reversible labil combination, by hydrogen bonding or by hydrophobic interaction;

or intermediate lability bonding by electrostatic interaction;

to provide a diastereomeric mixture of Ω-CO—O-Δ* and de Ω-CO-Δ*;

b) then by physical separation of the mixture of two diastereomers or two complex compounds or more generally of two new entities physically and/or chemically different then obtained;

c) then by regeneration and finally separation of each one of enantiomers of the generic formula Ω*-CO—OH, where Ω* (<<oméga stella>>) represents the generic symbol of the same chiral radical in the either one or the other pure enantiomeric forms corresponding to the following formulas (+)-Ω-CO—OH and (−)-Ω-CO—OH which are as defined above.

##STR00059##

Preferably, Ω-CO— is:

either a radical corresponding to the following formula:

where n, Z, R⁶, R⁸, and R⁵ are as defined above;

or a radical corresponding to the following formula:

##STR00060##
where m, Z, R⁶, R⁸, and R⁵ are as defined above;

or a radical corresponding to the following formula:

##STR00061##
where n, R⁵, R⁶, R⁸, Z, Q², Q¹, R⁹, R¹⁰ and R¹¹ are as defined above.

The stable combination may be represented by an ester of the following formula Ω-CO—O-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with a chiral alcohol corresponding to the formula HO-Δ* such as Δ* is as defined above, according the process of invention.

The stable combination may be represented by an amide corresponding to the either one or the other formulas Ω-CO—NH-Δ* or Ω-CO—N-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with primary or secondary chiral amine corresponding to formulas H₂N-Δ* or NN=Δ* such as Δ* is as defined above, according the process of the invention.

The stable combination may be represented by an thioester of the following formula Ω-CO—S-Δ* such as Ω and Δ* are as defined above, the said stable combination is obtained by contacting acid with a chiral thiol corresponding to the formula HS-Δ* such as Δ* is as defined above, according the process invention.

Finally, the ionic combination may be represented by a salt just prepared by contacting of acid with a chiral amine corresponding to the either one or the other of the three following formulas:
Ω-CO—O⁻[NH-Δ*]⁺
Ω-CO—O⁻[NH₂-Δ*]⁺
Ω-CO—O⁻[NH₃-Δ*]⁺
where Ω and Δ*. are as defined above.

The bringing into play of a labil bonding based combination is achieved in the form of chromatography with the help of a chiral stationary phase.

The bringing into play of an interatomic or intermolecular labil bonding based combination, within crystalline lattice, is achieved in the form of fractionated crystallization initiated by a chiral precursor.

The chiral alcohol HO-Δ* is:

either (−)-quinine corresponding to the following formula:

##STR00062##

or (−)- or (+)-methyl mandelate corresponding to the following formulas:

##STR00063##

or (−)- or (+)-menthol corresponding to the following formulas:

##STR00064##

The chiral amine H₂N-Δ* is (−)- or (+)-ephedrine corresponding to the following formulas:

##STR00065##

The present invention concerns the following new compounds:

the (−)-quinidyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-quinidyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

##STR00066##

the (−)-menthyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-menthyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

##STR00067##
the (−)-methylmandelyl (2′R)-(−)-anhydro-homoharringtonate and the (−)-methylmandelyl (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

##STR00068##

the (−)-ephedrinium (2′R)-(−)-anhydro-homoharringtonate and the (−)-ephedrinium (2′S)-(−)-anhydro-homoharringtonate corresponding respectively to the two following formulas:

##STR00069##

According the process of invention, when the carboxylic acid is the tertiary heterocycloalcane carboxylic acid corresponding to the following formula:

##STR00070##
where n, Z, R⁵, R⁶ and R⁸ are as defined above, the said acid is obtained by treatment in aprotic or protic solvant, eventually in the presence of cyclization additive and/or dehydrating agent, the said treatment eventually supported with physical carrying of the water formed.

or open tertiary ethylenic acid corresponding to the following formula:

##STR00071##
where m, Z, R⁵, R⁶ and R⁸ are as defined above.

or open tertiary ethylenic acid corresponding to the following formula:

##STR00072##
where m is included between 1 and 8, Z, R⁵, R⁶ and R⁸ are as defined above, R¹² is not a CTX radical as defined above and represents R⁵ and/or a protective group of acids and/or a chiral group;
then R¹² is removed later, either just by saponification, or by hydrogenolysis, or more generally by the method of the state of art to remove protective groups of acids.

In the absence of cyclization additive, the reaction of cyclization just take place by heating.

Preferably, the cyclization additive is a protic acid such as sulfonic or formic acid, or an aprotic acid, included in immobilized form.

In the step of preparation of the acid described above, Z is an oxygen atom.

The aim of the present invention is also the preparation of the following new compounds:

the tertiary heterocycloalcane carboxylic acid, included its salts and each one of its pure enantiomeric forms or in racemic mixture or in variable composition, corresponding to the following formula:

##STR00073##
where n, Z, R⁵, R⁶ and R⁸ are as defined above, and R⁵ is not hydrogen;
except for compounds where Z is oxygen atom and,
1°) n=0 and R⁵ is not —CH₂CO₂H or —CH₂CO₂CH₃ radical;
2°) n=0 and R⁵ is —CH₂CO₂H or —CH₂CO₂CH₃ radical, and R⁶═R⁸=methyl or —CH₂CO₂H or —CH₂CO₂CH₃ radical;
3and 14 show a chromatographic profiles of harringtonines coming from various sources.

Phase III clinical trial with an HHT drug substance exhibiting a non-reliable impurity profile. [He et al., 2000] The NCI got finally an HHT suitable for use in phase III clinical trial but, despite of its effort, the product they use contains a non-removable impurities of the which contain is higher than 1%. [He et al., 2000]. In addition to the process described for the purification of the NCI production. [He et al., 2000]

Our recent semi-synthesis of harringtonines, including harringtonine and homoharringtonine, by attachment of entirely prior formed acyl side-chains to the cephalotaxine moieties, changed dramatically this situation dramatically: chromatographic purity of the final harringtonine and homoharringtonine drug substance is consistently higher then than 99.8% versus 98.5% for the above cited NCI products (the purest ever previously described) versus 95%-97% for Chinese products, corresponding to 0.2%, 1.5 1.5% and 3-5% of impurities (see FIGS. 6, 7 and 12 8, 9 and 14). In addition, since cephalotaxine, as a precursor of semi-synthetic HA and HHT, is abundant in a renewable part of the tree, this semi-synthetic process overcome overcomes the serious environmental concern induce concerns induced by the destroying destruction of a rare and endanger endangered plant.

A well definite crystalline form of a drug substance is a very important condition, to have reliable solid final form of drugs useful for example for oral administration.

Although HHT and HA would be very promising drugs for the treatment of patients with CML, the current mode of administration by continuous intravenous central infusion (CIVI) is a strong handicap for the administration of this therapy during several years. In addition, while extra-hematologic toxicity of HHT/HA is very mild, the occurrence of infection due to catheter is the main toxicity of this regimen. The use of an oral form of these drugs could be change completely this situation and would extend widely the market of this product.

The present invention provides natural, synthetic or semi-synthetic harringtonines including their tautomeric forms and their salts of the following formula:

##STR00101##
wherein n=2 (i.e. harringtonine) or n=3 (i.e. homoharringtonine),
in which:

the total content of impurities, possibly including enantiomeric forms, is lower than 1%, and/or

the content of the major impurity is lower than 0.9%, and/or

the chromatographic assay exhibits a harringtonines content higher than 97.5%.

A preferred embodiment of the invention provides a natural, synthetic or semi-synthetic homoharringtonine including its tautomeric forms and its salts in which:

the total content of impurities, possibly including enantiomeric forms, is lower than 1%, and/or

the content of the major impurity is lower than 0.9%, and/or

the chromatographic assay exhibits a homoharringtonines content higher than 97.5%.

A further preferred embodiment of the invention provides a natural, synthetic or semi-synthetic harringtonine including its tautomeric forms and its salts in which:

the total content of impurities, possibly including enantiomeric forms, is lower than 1%, and/or

the content of the major impurity is lower than 0.9%, and/or

the chromatographic assay exhibits a harringtonine content higher than 97.5%.

A further preferred aspect of the invention is a crystalline natural, synthetic or semi-synthetic homoharringtonine having substantially the same DSC curve as set out in FIG. 3.

Yet, a further embodiment of the invention provides a crystalline natural, synthetic or semi-synthetic homoharringtonine having substantially the same X-ray diffractogram as set out in FIG. 4, and substantially the same IR spectrum, in KBr as set out in FIG. 5.

Yet, another embodiment of the invention provides a crystalline natural, synthetic or semi-synthetic homoharringtonine having substantially the same DSC curve as set out in FIG. 3, and substantially the same X-ray diffractogram as set out in FIG. 4, and substantially the same IR spectrum, in KBr as set out in FIG. 5.

Yet, another preferred embodiment of the invention provides a crystalline natural, synthetic or semi-synthetic harringtonine having substantially the same DSC curve as set out in FIG. 6.

Yet, a preferred aspect of this invention provides a pharmaceutical composition comprising an effective antitumor amount of a natural, synthetic or semi-synthetic homoharringtonine having substantially the same X-ray diffractogram as set out in FIG. 4, and substantially the same IR spectrum, in KBr as set out in FIG. 5, and substantially the same DSC curve as set out in FIG. 3, together with one or more pharmaceutically acceptable inactive components such as carriers, excipients, adjuvants or diluents.

Another aspect of the invention provides a pharmaceutical composition comprising an effective antitumor amount of a natural, synthetic or semi-synthetic harringtonine having substantially the same IR spectrum, in KBr as set out in FIG. 7, and substantially the same DSC curve as set out in FIG. 6, together with one or more pharmaceutically acceptable inactive components such as carriers, excipients, adjuvants or diluents.

Another preferred aspect of the invention provide a process of purification of natural, synthetic or semi-synthetic crude harringtonines for the preparation of pure harringtonines exhibiting the above included features including for eventual enantiomeric enrichment, and comprising the successive steps:

• (i) at least one chromatographic purification, preferably in reverse phase in aqueous mobile phase such as a lower alkanol or tetrahydrofurane or acetonitrile, purified water, and an acidic buffer, preferably based on phosphoric acid and is salt. Stationary phase may be any standard chemically bound phase preferably an alkylsilane or an alkylnitrile, bounded on an inert core, preferably silicagel;
• (ii) at least one crystallization in water or aqueous solvent containing an organic solvent, preferably a lower C_1-4 alkanol,

The progression of the process of purification is monitored by HPLC analyses and several termal analysis at the solid state. The progression of enantiomeric purity is monitored by optical rotation checking of the dried solid form.

A preferred embodiment provides a new method of monitoring of enantiomeric purity of cephalotaxanes using an HPLC with a chiral stationary phase preferably based upon beta-cyclodextrine

Another preferred embodiment of the invention is the above process of purification in which the lower C_1-4 alkanol is methanol and the cephalotaxane is harringtonine

A further preferred aspect of the invention is the above process of purification in which the lower C_1-4 alkanol is methanol and the cephalotaxane is homoharringtonine

This invention include also a pharmaceutical composition which comprises an antitumor effective amount of at least one above described harringtonine or homoharringtonine with one or more pharmaceutically acceptable carriers, excipients or diluents therefore, including the process for preparing the said solid pharmaceutical composition such as, for examples, tablet, capsule, implant or suppository.

Another aspect of the invention is the use of at least the above solid form of one harringtonine or homoharringtonine described in the invention for preparing the above pharmaceutical composition as (i) chemotherapeutic agent, (ii) enhancer of other chemotherapeutic agents (iii) for inhibiting tumors growth, (iv) for inhibiting mammalian parasites, (v) as immunosuppressive agent, or (vi) as reversal agent.

The present invention further describes a method for treating mammalian tumors which comprises administering to a mammal an antitumor effective amount of the solid form of at least one harringtonine or homoharringtonine described in this invention, by parenteral, topic, subcutaneous or anal mode.

A preferred embodiment of the invention describes a method for treating mammalian tumors which comprises oral administering to a mammal an antitumor effective amount of the solid form of at least one harringtonine or homoharringtonine described in this invention.

A further preferred embodiment of the invention describes a method for treating mammalian tumors which comprises implantable pharmaceutical preparation administering to a mammal an antitumor effective amount of the solid form of at least one harringtonine or homoharringtonine described in this invention.

Finally, the invention is also concerned with the use of purified and/or solid harringtonines as defined above, for the preparation of pharmaceutical compositions for the treatment of cancers and leukemias particularly acute myelod leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative disorders including chronic myelogenous leukemia.

The following examples, which are given without implied limitation, illustrate the present invention.

EXAMPLE 1

Preparation of ethyl 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate or ethyl 6-desoxy-5,6-dehydrohomoharringtonate

##STR00102##
1°) Preparation of the Intermediate Oxalate

5-Bromo-2-methyl-pent-2-ene (15.6 g, 95.6 mmol) was added dropwise to a stirred mixture of magnesium (2.32 g, 95.5 mmol) (activated with further crystal of iodine) in anhydrous tetrahydrofurane (75 ml). The onset of the reaction is accompanied with a vigorous overheating and refluxing of the reaction mixture. The reflux was maintained until most of magnesium had reacted and the reaction mixture was diluted with anhydrous tetrahydrofurane (150 ml). To a stirred mixture of diethyl oxalate (10.8 ml, 80 mmol) in anhydrous tetrahydrofurane (75 ml) was added at −78° C. the resulting Grignard reagent over a period of 20 minutes. The stirring was maintained at −78° C.±5° C. for 30 minutes and then the temperature was raised to −10° C. over a period of 1.5 hours. The mixture was quenched with 15% ammonium chloride solution (300 ml). The separated organic layer was washed with 15% ammonium chloride solution (300 ml) and evaporated to dryness. The aqueous layer was extracted with ether (2×300 ml). The organic layers were combined with the concentrate and washed with brine (300 ml), dried over magnesium sulfate and evaporated to dryness. The crude product, after purification with a bulb-to-bulb distillation apparatus, afforded colorless oil (10.3 g, 70%). The intermediate α-cetoester showed the following characteristics:

##STR00103##

IR (ATR) (cm⁻¹): 2790; 2916; 1725; 1068.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.08 (1H, t, J=7.2, HC═); 4.32 (2H, q, J=7.1, OCH₂); 2.86 (2H, t, J=7.2, CH₂CO); 2.32 (2H, q, J=7.2, CH₂—C═); 1.68 (3H, s, CH₃); 1.62 (3H, s, CH₃); 1.37 (3H, t, J=7.1, OCH₂CH₃).

2°) Preparation of the Title Product

Anhydrous methyl acetate (0.6 ml, 7.5 mmol) was added to a stirred commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane tetrahydrofuran (7.5 ml, 7.5 mmol) at −78° C. over a period of 1 minute and this was allowed the reaction was allowed to proceed at −78±5° C. for 20 minutes. To a stirred mixture of ethyl 2-oxo-6-methylhept-5-enoate prepared above (480 mg, 2.6 mmol) in anhydrous tetrahydrofurane tetrahydrofuran (10 ml) at −78° C. was added the lithium enolate over a period of 5 minutes, and the resulting mixture was stirred at −78±5° C. for 30 minutes. After monitoring in CCM, the freezing cooling bath was removed and the mixture was quenched with 15% ammonium chloride solution (10 ml). The separated organic layer was washed again with 15% ammonium chloride solution (10 ml) and evaporated to dryness. The aqueous layers were extracted with ether (2×10 ml). The organic layers were combined with the concentrate and washed with brine (10 ml), dried over magnesium sulfate and evaporated to dryness. The resulting crude product (1.13 g) was purified by column chromatography (cyclohexane/ethyl acetate (95:5), silica (15-40 □m) (15-40 μm) 38 g) to provide a colorless oil (482 mg, 72%). The product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 3508; 2969; 2919; 1732; 1438; 1193.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.05 (1H, t, J=7.1, HC═); 4.27 (2H, q, J=7.1, OCH₂); 3.70 (1H, s, OH); 3.68 (3H, s, OCH₃); 2.92 and 2.70 (2H, 2d, J_AB=16.1, CH₂CO₂); 2.12 (1H, m); 1.88 (1H, m); 1.72 (2H, m); 1.67 (3H, s, CH₃); 1.58 (3H, s, CH₃); 1.31 (3H, t, J=7.1, OCH₂CH₃).

EXAMPLE 2

Preparation of ethyl 2-methoxycarbonylmethyl-2-hydroxy-6,6-dimethyl-2-tetrahydropyrane carboxylate or ethyl anhydrohomoharringtonate

##STR00104##
1°) Method A

p-Toluenesulfonic acid (2.06 g, 10.8 mmol) was added to a stirred solution of ethylenic ester resulting from Example 1 (2.8 g, 10.8 mmol) in toluene (30 ml) and the resulting mixture was stirred at 65° C. for 5 hours. After cooling at room temperature, the mixture was hydrolyzed with saturated sodium hydrogen carbonate solution. The aqueous layer was extracted with ether (3×50 ml), and the organic layers were combined, washed with brine (100 ml), dried over magnesium sulfate and evaporated to dryness. The resulting crude product (2.8 g) was purified by column chromatography (cyclohexane/ether (95:5), silica (15-40 μm) 10 g) to provide a colorless oil (1.94 g, 69%). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 4.21 (2H, m, O CH₂CH₃); 3.64 (3H, s, OCH₃); 2.85 and 2.60 (2H, 2d, J_AB=14.0, CH₂CO₂); 2.30 (1H, dt, J=13.3 and 3.7); 1.87 (1H, qt, J=13.8 and 3.6); 1.62 (1H, m); 1.51 (2H, m); 1.43 (1H, m); 1.31 (3H, t, J=7.1, OCH₂CH₃); 1.22 (3H, s, CH₃); 1.13 (3H, s, CH₃).

2°) Method B

To a stirred solution of ethylenic ester resulting from Example 1 (50 mg, 0.19 mmol) in methanol (30 ml) was added hydrochloric acid 1N (0.5 ml) and the resulting mixture was stirred at 65° C. for 15 hours. After dilution with dichloromethane, the organic layer was dried over magnesium sulfate and evaporated to dryness. The resulting crude product (32 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (9:1), silica (15-40 μm) 2.2 g) to provide the expected intermediate diol (20 m g, 37%). The product thus obtained showed the following characteristics:

##STR00105##

IR (ATR) (cm⁻¹): 3490; 2966; 1731; 1193; 1177; 1152.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 4.28 (2H, q, J=7.2, OCH₂); 3.75 (1H, s, OH); 3.68 (3H, s, OCH₃); 2.93 and 2.69 (2H, 2d, J_AB=16.2, CH₂CO₂); 1.70 (2H, m); 1.53 (1H, m); 1.44 (1H, m); 1.30 (3H, t, J=7.1, OCH₂CH₃); 1.20 (3H, s, CH₃); 1.19 (3H, s, CH₃).

To a stirred solution of diol prepared above (19 mg, 0.069 mmol) in 1,2-dichloroethane (1.4 ml) was added anhydrous zinc chloride (10 mg, 0.069 mmol) and the resulting mixture was stirred at 80° C. for 1.5 hours. After cooling at ambient temperature, the mixture was washed with water, then with brine, and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford cyclic diester (7 mg, 40%). The product thus obtained showed identical characteristics to this obtained with method A.)

3°) Method C

A solution of ethylenic ester resulting from Example 1 (400 mg, 1.55 mmol) in a mixture of formic acid (4 ml) and water (4 ml) was stirred at 50° C. for 15 hours. After removal of formic acid in vacuo, the residue was treated with 5% sodium hydrogen carbonate solution. The aqueous layer was extracted three times with dichloromethane then the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (375 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 μm) 16 g) to provide a colorless oil (235 mg, 55%). The product thus obtained showed identical characteristics to this obtained with method A. The cyclisation of the diol thus obtained with zinc chloride, like Example 2 method B above, afforded cyclic diester showing identical characteristics to this obtained with method A.

EXAMPLE 3

Preparation of 2-carboxymethyl-2-hydroxy-6-methylhept-5-enoic acid or O-demethyl-6-desoxy-5,6-dehydrohomoharringtonic acid

##STR00106##

A mixture of potassium hydroxide (14.2 g, 252 mmol) in water (170 ml) was added to a stirred solution of ethylenic ester resulting from Example 1 (10.95 g, 42 mmol) in methanol (300 ml) and the resulting mixture was stirred at reflux for 1.5 hours. After cooling at room temperature, and removal of methanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (250 ml). After acidification (pH 1) with 10% hydrochloric acid, the aqueous layer was extracted with ether (3×250 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a white solid (8.66 g, 95%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 3500; 3019; 2966; 2931; 1716; 1691; 1656; 1219; 1199; 1111.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.06 (1H, t, J=6.9, HC═); 3.04 and 2.78 (2H, 2d, J_AB=17.1, CH2CO₂); 2.25-1.20 (4H, m, 2×CH₂); 1.67 (3H, s, CH₃); 1.60 (3H, s, CH₃).

EXAMPLE 4

Preparation of 2-carboxymethyl-6,6-dimethyl-2-tetrahydro-pyranecarboxylic acid or O-demethylanhydrohomoharringtonic acid

##STR00107##
1°) Method A

A mixture of potassium hydroxide (4.2 g, 75 mmol) in water (45 ml) was added to a stirred solution of cyclic diester resulting from Example 2 (1.94 g, 7.5 mmol) in ethanol (75 ml) and the resulting mixture was stirred at reflux for 5 hours. After cooling at room temperature, and removal of ethanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (2×50 ml). After acidification with hydrochloric acid 2N (35 ml), the aqueous layer was saturated with sodium chloride then was extracted with ether (3×50 ml). The combined organic layers were washed with brine (2×100 ml) dried over magnesium sulfate and evaporated to dryness to afford a pale yellow oil (1.66 g, 98%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 2974; 2941; 1709; 1215.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 3.01 and 2.95 (2H, 2d, J_AB=16.1, CH₂CO₂); 1.89 (1H, m); 1.75 (2H, m, CH₂); 1.58 (3H, m); 1.31 (6H, s, 2×CH₃).

2°) Method B

To a stirred solution of ethylenic diacid resulting from Example 3 (50 mg, 23 mmol) in anhydrous toluene (500 μl) was added zinc chloride (6 mg, 0.04 mmol) and the resulting mixture was stirred at 80° C. for 15 hours. After cooling at room temperature, the mixture was hydrolyzed with 10% hydrochloric acid, and the resulting aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a pale yellow solid (38 mg, 76%). The crude product thus obtained showed identical characteristics to this obtained with method A.

3°) Method C

A solution of ethylenic diacid resulting from Example 3 (50 mg, 0.23 mmol) in a mixture of formic acid (500 μl) and water (500 μl) was stirred at 60° C. for 3 hours. After cooling at room temperature and removal of formic acid in vacuo, the residue was treated with ethyl acetate. The resulting organic layer was washed with 10% hydrochloric acid and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a pale yellow solid (50 mg, 100%). The crude product thus obtained showed identical characteristics to this obtained with method A.

EXAMPLE 5

Preparation of 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoic acid or 6-desoxy-5,6-dehydrohomoharringtonic acid

##STR00108##

A mixture of ethylenic diacid resulting from Example 3 (500 mg, 2.3 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (4.5 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 16 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (50 ml), the resulting aqueous layer was washed with ether (50 ml), acidified (pH 1) with hydrochloric acid 2N (0.5 ml and extracted with ether (3×50 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a viscous yellow oil (310 mg, 58%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 3483; 2954; 1731; 1197; 1173.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.06 (1H, m, HC═); 4.12 (2H, br.s, CO₂H+OH); 3.73 (3H, s, OCH₃); 2.99 and 2.74 (2H, 2d, J_AB=16.7, CH₂CO₂); 2.16 (1H, m); 1.98 (1H, m); 1.85-1.60 (4H, m); 1.67 (3H, s, CH₃); 1.60 (3H, s, CH₃).

EXAMPLE 6

Preparation of 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydro-pyranecarboxylic acid or anhydrohomoharringtonic acid)

##STR00109##
1°) Preparation from Cyclic Diacid

A mixture of cyclic diacid resulting from Example 4 (1.6 mg, 7.4 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (15.5 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 15 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (50 ml), the resulting aqueous layer was washed with ether (2×50 ml) (to see annex preparation below), acidified (pH 1) with hydrochloric acid 2N (15 ml) and extracted with ether (3×75 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow oil (1.17 g, 69%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 2974; 2951; 1740; 1718; 1437.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 3.70 (3H, s, OCH₃); 3.03 and 2.98 (2H, 2d, J_AB=16.1, CH₂CO₂); 1.82 (1H, m); 1.74 (3H, m); 1.62 (1H, m); 1.48 (1H, m); 1.31 (3H, s, CH₃); 1.26 (3H, s, CH₃).

Annex Preparations:

a) Obtaining of Diester

##STR00110##

The combined organic layers above-mentioned was dried over magnesium sulfate and evaporated to dryness to afford a mixture of diester and monoester (396 mg). After treatment of this mixture with saturated sodium hydrogen carbonate solution, the aqueous layer was extracted with ether, and the resulting organic layer was dried over magnesium sulfate and evaporated to dryness to afford an oil (292 mg, 17%). The crude product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 3.75 (3H, s, OCH₃); 3.65 (3H, s, OCH₃); 2.85 and 2.61 (2H, 2d, J_AB=14.1, CH₂CO₂); 1.85 (1H, m); 1.62 (1H, m); 1.50 (2H, m); 1.43 (1H, m); 1.21 (3H, s, CH₃); 1.11 (3H, s, CH₃).

b) Obtaining of Regio-Hemiester by Mono Saponification of Diester Above-Mentioned

##STR00111##

To a stirred solution of cyclic diester above-mentioned (285 mg, 1.17 mmol) in methanol (11 ml) was added a mixture of potassium hydroxide (654 mg, 11.7 mmol) in water (7 ml) and the resulting mixture was stirred at room temperature for 30 minutes. After removal of methanol in vacuo, the residue was treated with water (7 ml) and the resulting aqueous layer was extracted three times with ether. After acidification (pH 1) with 10% hydrochloric acid solution, the aqueous layer was extracted with. The combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (236 mg) was purified by column chromatography (dichloromethane/methanol (95:5), silica (15-40 μm) 6.5 g) to provide a pale yellow solid (220 mg, 82%). The product thus obtained showed the following characteristics:

IR (KBR) (cm⁻¹): 3421; 2960; 2929; 1744; 1705; 1209.

¹H NMR 400 MHz (CDCl₃) δ ppm, J Hz): 3.76 (3H, s, OCH₃); 2.76 and 2.67 (2H, 2d, J_AB=15.3, CH₂CO₂); 2.36 (1H, m, J_AB=13.7, J_3-4=3.5, J_3-5=1.2, H-3_eq); 1.85 (1H, m, J_AB˜J_ax-ax=14.0, J_ax-eq=3.7, H-4_ax); 1.67 (1H, m, J_AB=14.1, J_4-3,5=3.9, H-4_eq); 1.59 (1H, m, J_AB=13.4, J_5-4=3.6, J_5-3=1.0, H-5_eq); 1.49 (1H, m, J_AB˜J_ax-ax=13.2, J_ax-eq=4.0, H-3_ax); 1.42 (1H, m, J_AB˜J_ax-ax=13.2, J_ax-eq=4.5, H-5_ax); 1.33 (3H, s, CH₃); 1.16 (3H, s, CH₃).

2°) Preparation from Ethylenic Hemiester

To a stirred solution of ethylenic hemiester resulting from Example 5 (4.6 g, 20 mmol) in toluene (125 ml) was added p-toluenesulfonic acid (3.8 g, 20 mmol) and the resulting mixture was stirred at 65° C. for 5 hours. After cooling at room temperature, the mixture was hydrolyzed with saturated sodium hydrogen carbonate solution (100 ml). The aqueous layer was washed with ether (2×100 ml) and the organic layers were discarded (to eliminate resulting diester of the reaction). After acidification (pH 1) with hydrochloric acid 1N (35 ml), the aqueous layer was saturated with sodium chloride then was extracted with ether (3×100 ml). The combined organic layers were washed with brine (100 ml) dried over magnesium sulfate and evaporated to dryness.

The resulting crude product (3.9 g) was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 μm) 160 g) to provide a yellow oil (3.1 g, 67%). The crude product thus obtained showed identical characteristics to this obtained above.

EXAMPLE 7

Preparation of cyclic anhydride of 2-carboxymethyl-2-hydroxy-6-methylhept-5-enoic acid

##STR00112##

A mixture of cyclic diacid resulting from Example 4 (245 mg, 1.1 mmol) and acetic anhydride (4 ml) was stirred at reflux for 16 hours. After evaporation of reaction mixture in vacuo, the residue was treated with toluene and evaporated again in high vacuum to afford a viscous yellow oil (189 mg, 84%). The product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 2976; 2951; 1732; 1188; 1170

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 3.02 (2H, s, CH₂CO₂); 1.98 (2H, m, CH2); 1.8-1.5 (4H, m, CH₂); 1.31 (3H, s, CH₃); 1.22 (3H, s, CH₃).

EXAMPLE 8

Preparation of (−)-Cephalotaxyl Pivalate

##STR00113##
1°) Method Via Mixed Anhydride

To a stirred mixture of pivalic acid (100 mg, 0.98 mmol) in anhydrous toluene (2 ml) was added at room temperature triethylamine (dried over potassium hydroxide) (138 μl, 0.98 mmol) and 2,4,6-trichlorobenzoyl chloride (153 μl, 0.98 mmol). After stirring at 18±5° C. for 1.5 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (139 mg, 1.14 mmol) was added the reaction mixture was allowed to react for 5 minutes and cephalotaxine (103 mg, 0.33 mmol) was added. After stirring at 18±5° C. for 15 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml) then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm)) to provide a solid (130 mg, 93%). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.60 (1H, s, H-17*); 6.58 (1H, s, H-14*); 5.84 et 5.83 (2H, 2d, J_AB=1.5, OCH₂O); 5.83 (1H, d, H-3); 5.02 (1H, s, H-1); 3.77 (1H, d, J_4-3=9.6, H-4); 3.69 (3H, s, OCH₃); 3.21 (1H, m, J_AB=14.0, J=12.5, 7.8, H-11b); 3.09 (1H, m, H-8a); 2.94 (1H, td, J=11.5, 7.1, H-10a); 2.57 (2H, m, H-8b+H-10b); 2.35 (1H, dd, J_AB=14.5, J=6.9, H-11a); 2.03 (1H, td, J_AB=12.1, J=9.7, H-6_A); 1.89 (1H, m, J_AB=12.1, J=7.9, 4.0, H-6_B); 1.75 (2H, m, CH₂-7); 0.83 (9H, s, C(CH₃)₃).

2°) Method Using DCC

To a stirred mixture of pivalic acid (50 mg, 0.49 mmol) in anhydrous toluene (2 ml) maintained in an inert atmosphere was added 1,3-dicyclohexylcarbodiimide (130 mg, 0.63 mmol). After stirring for 10 minutes at room temperature, cephalotaxine (50 mg, 0.16 mmol) and pyrrolidinopyridine (24 mg, 0.16 mmol) were added. After stirring at 18±5° C. for 2 hours, then at 50° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product (130 mg) was purified by column chromatography (dichloromethane/methanol (9:1), silica (15-40 μm) 3g) to provide a white solid (36 mg, 57%). The crude product thus obtained showed identical characteristics to this obtained above via mixed anhydride.

EXAMPLE 9

Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate or anhydrohomoharringtonine and methyl 2-cephalotaxyloxy-carbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate, from cyclic anhydride resulting from Example 7

##STR00114##

To a stirred mixture of anhydride resulting from Example 7 (50 mg, 0.24 mmol) in anhydrous dichloromethane (0.5 ml) at room temperature were successively added pyridine (250 μl, 3.1 mmol), pyrrolidinopyridine (10 mg, 0.07 mmol) and cephalotaxine (76.4 mg, 0.24 mmol). After stirring at 18±5° C. for 48 hours, were successively added 1,3-dicyclohexylcarbodiimide (100 mg, 0.48 mmol), methanol (60 □l 60 μl, 1.5 mmol), pyrrolidinopyridine (10 mg, 0.07 mmol) and toluene (1 ml). After stirring at 18±5° C. for 24 hours (with control of reaction in CCM), the reaction mixture was filtered and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 μm)) to provide expected product (12 mg, two diastereomers) contaminated with regioisomer* (two diastereomers) resulting from the opening of anhydride. The expected product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.61 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.91 (J_3-4=9.8) and 5.84 (2H, 2d, H-3); 5.84 et and 5.79 (2d, J_AB=1.4, OCH₂O); 5.84 and 5.82 (2d, J_AB=1.4, OCH₂O); 5.04 and 5.01 (1H, 2s, H-1); 3.79 and 3.78 (1H, 2d, J_4-3=9.6, H-4); 3.70 and 3.65 (3H, 2s, OCH₃); 3.59 (3H, s, OCH₃); 3.15 (1H, m, H-11 □ H-11β); 3.09 (1H, m, H-8□ H-8α); 2.94 (1H, m, H-10□ H-10α); 2.58 (2H, m, H-8□+H-10□ H-8β+H-10β); 2.37 (1H, m, H-11□ H-11α); 2.16 and 1.81 (2d, J_AB=14.4, CH₂CO₂); 2.13 and 1.66 (2d, J_AB=14.3, CH₂CO₂); 2.02 (1H, m, H-6_A); 1.88 (1H, m, H-6_B); 1.75 (2H, m, CH₂-7); 1.8-1.2 (6H, m, 3×CH₂); 1.11 and 1.02 (2s, 2×CH₃); 1.10 and 1.04 (2s, 2×CH₃). *The regioisomer above-mentioned was also obtained from the following conditions:

To a stirred mixture of hemiester resulting from Example 6 method C (100 mg, 0.43 mmol) in anhydrous toluene (1 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (120 mg, 0.58 mmol). After stirring for 5 minutes, cephalotaxine (45 mg, 0.15 mmol) and pyrrolidinopyridine (21 mg, 0.14 mmol) were added. After stirring at 35° C. for 45 minutes, then at 8° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 4 g) to provide expected product (23 mg, 30%, two diastereomers). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.61 and 6.58 (1H, 2s, H-17*); 6.57 and 6.53 (1H, 2s, H-14*); 5.89 and 5.86 (2d, J_AB=1.5, OCH₂O); 5.87 and 5.85 (2d, J_AB=1.5, OCH₂O); 5.76 (1H, d, J_3-4=9.4, H-3); 5.02 (1H, 2s, H-1); 3.73 and 3.72 (1H, 2d, J_4-3=9.4, H-4); 3.70 and 3.68 (3H, 2s, OCH₃); 3.69 and 3.65 (3H, 2s, OCH₃); 3.15 (1H, m, H-11□ H-11α); 3.07 (1H, m, H-8□ H-8α); 2.90 (1H, m, H-10□ H-10α); 2.74 and 1.95 (2d, J_AB=15.3, CH₂CO₂); 2.56 (2H, m, H-8□+H-10□ H-8β+H-10β); 2.33 (1H, m, H-11□ H-11α); 2.28 and 2.23 (2d, J_AB=15.4, CH₂CO₂); 2.16 (m, H-3′_eq); 1.97 (1H, m, H-6_A); 1.9-1.1 (5H, m, CH₂); 1.86 (1H, m, H-6_B); 1.73 (2H, m, CH₂-7); 1.14 (3H, s, CH₃); 1.03 (3H, s, CH₃).

EXAMPLE 10

Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2 tetrahydropyrane carboxylate or anhydrohomoharringtonine, from tetrahydropyranecarboxylic acid resulting from Example 6

Formula of Example 9)

1°) Method Via Mixed Anhydride

To a stirred mixture of hemiester resulting from Example 6 (50 mg, 0.22 mmol) in anhydrous toluene (1 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (29.4 μl, 0.22 mmol) and 2,4,6-trichlorobenzoyl chloride (32.7 μl, 0.22 mmol). After stirring at 25° C. for 20 hours (with control of disappearing of starting acid in infrared), 4-dimethylaminopyridine (29 mg, 0.24 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (16.5 mg, 0.05 mmol) was added. After stirring at 25° C. for 24 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml), then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm)) to provide expected product (16 mg, 56%, two diastereomers). The product thus obtained showed identical characteristics to this obtained in Example 9.

2°) Method Using DCC

To a stirred mixture of hemiester resulting from Example 6 (100 mg, 0.43 mmol) in anhydrous toluene (1 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (180 mg, 0.87 mmol). After stirring for 10 minutes, cephalotaxine (165 mg, 0.52 mmol) and pyrrolidinopyridine (77 mg, 0.52 mmol) were added. After stirring at 18±5° C. for 18 hours, was added ether, the reaction mixture was filtered on ground-glass filter, and the cake was washed with ether. The resulting organic layer was successively washed with 10% sodium hydrogen carbonate solution, with water, then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 9 g) to provide a solid (110 mg, 48%). The product thus obtained showed identical characteristics to this obtained in Example 9.

EXAMPLE 11

Preparation of (−)-cephalotaxyl(2′RS)-2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate or anhydrohomoharringtonine, from ethylenic acid resulting from Example 6

Formula of Example 9

1°) Method A: Via Mixed Anhydride, Coupling with Cyclisation

To a stirred mixture of ethylenic ester resulting from Example 5 (50 mg, 0.22 mmol) in anhydrous toluene (1 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (29 μl, 0.22 mmol) and 2,4,6-trichlorobenzoyl chloride (34 μl, 0.22 mmol). After stirring for 30 minutes (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (30 mg, 0.25 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (31 mg, 0.1 mmol) was added. After stirring at 18±5° C. for 65 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml), then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm)) to provide expected product (46 mg, 96%, two diastereomers 40/60). The product thus obtained showed identical characteristics to this obtained in Example 9.

2°) Method B: Using DCC, Coupling with Cyclisation

To a stirred mixture of ethylenic acid resulting from Example 5 (50 mg, 0.22 mmol) in anhydrous toluene (2 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (270 mg, 1.31 mmol). After stirring for 5 minutes, cephalotaxine (70 mg, 0.22 mmol) and pyrrolidinopyridine (32 mg, 0.22 mmol) were added. After stirring at 18±5° C. for 65 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 9 g) to provide a solid (40 mg, 35%). The product thus obtained showed identical characteristics to this obtained in Example 9.

EXAMPLE 12

Preparation of Purified (−) Cephalotaxine from Total Alkaloidic Extract of Cephalotaxus sp

##STR00115##
1°) Preparation of Total Alkaloids Extract:

In a 30 liters-tank, crushed leaves (fresh or dry) of Cephalotaxus sp (10 kg) were mixed with methanol (201) and steed during 65 hours, then percolated (501). Solution was filtered and concentrated under vacuum to a volume of 51. Concentrated solution was acidified with a 6% aqueous solution of tartaric acid. Then hydro-alcoholic solution was washed by dichloromethane (5×51) for removing fatty materials and pigments. Aqueous solution was basified with aqueous ammonia (2.5%) until pH 9, then extracted with dichloromethane (5×51). After concentration under reduced pressure, crude alkaloids extract was recovered as a white crystalline solid (24.5 g). Cephalotaxine contain was 71% (HPLC).

2°) Isolation and Chromatographic Purification of (−)-cephalotaxine from Crude Alkaloids Extract:

Above crude extract was dissolved in mobile phase (triethylamine (1.55/1000) in deionised water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 m, porosity 100, 1 kg). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was monitored by U.V. detector and TLC. Retained fraction were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml), After concentration under reduced pressure a resin was obtained which on trituration with methanol gave (−)-cephalotaxine (18g) as a white crystalline solid (HPLC purity=99.8%). The product thus obtained showed the following characteristics:

[α]_D²⁰: −174.1 (c=0.20; CHCl₃)

¹NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.68 (1H, s, H-17*); 6.65 (1H, s, H-14*); 5.91 and 5.90 (2H, 2d, J_AB=1.5, OCH₂O); 4.93 (1H, s, H-1); 4.77 (1H, dd, J_3-4=9.4, J_3-OH=3.4, H-3); 3.73 (3H, s, OCH₃) 3.68 (1H, d, J_4-3=9.4, H-4); 3.35 (1H, m, J_AB=14.3, J=12.2 and 7.9, H-11β); 3.08 (1H, m, J=9.1 and 4.9, H-8α); 2.92 (1H, td, J=11.6 and 7.1, H-10α); 2.59 (2H, m, H-8α+H-10α); 2.35 (1H, dd, J_AB=14.4, J=6.9, H-11α); 2.02 (1H, td, J_AB=12.1, J=9.7, H-6_A); 1.87 (1H, m, J_AB=12.1, J=7.9 and 4.4, H-6_B); 1.74 (2H, m, CH₂-7); 1.62 (1H, d, J_3-OH=3.5, 3-OH).

EXAMPLE 13

Preparation of Lithium Alcoolate of (−) cephalotaxine (Trapped Like 3-O-acetyl Derivative)

1°) Butyllithium Method

A commercial solution of butyllithium in hexane (0.44 ml, 1.6 M in hexane, 0.70 mmol) was added to a stirred mixture of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane (6.8 ml). The reaction mixture was maintained at −60° C. for 20 minutes, then at −48° C. for 30 minutes, acetic anhydride (90 μl, 0.095 mmol) was added over a period of 8 minutes and the stirring was maintained at −48° C. for 20 minutes then at 0° C. for 1 hour. The mixture was quenched with saturated ammonium chloride solution (5 ml) then extracted with ethyl acetate (3×8 ml). The combined organic layers were washed with brine (15 ml) dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 6 g) to provide a white solid (60 mg, 26%). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.60 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.89 and 5.86 (2H, 2d, J_AB=1.4, OCH₂O); 5.80 (1H, d, J_3-4=9.3, H-3); 5.05 (1H, s, H-1); 3.77 (1H, d, J_4-3=9.4, H-4); 3.72 (3H, s, OCH₃); 3.23 (1H, m, J_AB=14.3, J=12.3 and 7.9, H-11β); 3.08 (1H, m, H-8α); 2.92 (1H, td, J=11.5 and 7.1, H-10α); 2.57 (2H, m, H-8β+H-10β); 2.36 (1H, dd, J_AB=14.4, J=7.0, H-11β); 2.02 (1H, td, J_AB=12.1, J=9.7, H-6_A); 1.88 (1H, m, J_AB=12.1, J=8.0 and 4.0, H-6_B); 1.74 (2H, m, CH₂-7); 1.57 (3H, s, OAc).

2°) Lithium bis-(trimethylsilyl)amide (LHDS) Method

A commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (0.95 ml, 0.95 mmol) was added to a stirred solution of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane at −40° C. After stirring for 5 minutes, acetic anhydride (90 μl, 0.95 mmol) was added, and the reaction mixture was treated like method above-mentioned in 1°). The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

3°) Lithium Diisopropylamide (LDA) Method

A commercial solution of lithium diisopropylamide 2M in tetrahydrofarane (0.35 ml, 0.70 mmol) was added to a stirred solution of (−)-cephalotaxine (200 mg, 0.63 mmol) in anhydrous tetrahydrofurane (6.8 ml) at −60° C. over a period of 20 minutes After stirring at −60° C. for 20 minutes, then at −48° C. for 30 minutes, acetic anhydride (90 μl, 0.95 mmol) was added. The solution was stirred at −48° C. for 20 minutes, then at 0° C. for 1 hour and the reaction mixture was treated like method above-mentioned in 1°). The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

4°) Sodium Hydride Method

To a stirred mixture of sodium hydride (1.5 g) in freshly distilled dimethylformamide (3 ml) were added at −60° C. a solution of cephalotaxine (200 mg, 0.63 mmol) in dimethylformamide (3 ml) and acetic anhydride (90 μl, 0.95 mmol). After stirring at ambient temperature for 24 hours, the reaction mixture was treated at 0° C. with water (3 ml) and extracted with ether (3×5 ml). The combined organic layers were dried over magnesium sulfate and evaporated on vacuum. The product thus obtained showed identical characteristics to this obtained above in butyllithium method.

EXAMPLE 14

Preparation of (−)-cephalotaxyl 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate or anhydrohomoharringtonine, via Lithium Alcoolate of Cephalotaxine

Formula of Example 9

To a stirred solution of lithium alcoolate of (−)-cephalotaxine (158 mg, 0.5 mmol) in anhydrous tetrahydrofurane prepared according to Example 13 was added mixed anhydride resulting from Example 10 (0.75 mmol) at −50° C. over a period of 10 minutes. After stirring at −50° C. for 30 minutes, then at 0° C. for 2 hours, the reaction mixture was quenched with saturated ammonium chloride solution (5 ml) and extracted with ethyl acetate (3×10 ml). The combined organic layers were washed with brine (15 ml) dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 7 g) to provide a white solid (48 mg). The product thus obtained showed identical characteristics to this obtained in Example 9.

EXAMPLE 15

Preparation of Diastereomeric Mixture of (−)-quinidyl 2-methoxycarbonyl-methyl-6,6-dimethyl-2-tetrahydropyrane carboxylates, from tetrahydropyranecarboxylic acid Resulting from Example 6

##STR00116##
1°) Method A: Via Mixed Anhydride

To a stirred mixture of acid resulting from Example 6 (458 mg, 1.99 mmol) in anhydrous toluene (8 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (270 μl, 1.92 mmol) and 2,4,6-trichlorobenzoyl chloride (300 μl, 1.91 mmol). After stirring for 3 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (352 mg, 2.88 mmol) was added, the reaction mixture was allowed to react for 5 minutes and quinine (936 mg, 2.88 mmol) was added. After stirring at 18±5° C. for 65 hours, the reaction mixture was filtered on paper and diluted with ether (15 ml). The resulting organic layer was successively washed with water (15 ml), with saturated sodium hydrogen carbonate solution (15 ml), with water again (15 ml) then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 □m μm) 32 g) to provide expected product (930 mg, 84%, two diastereomers 50/50). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 8.73 (1H, m, H-2_qn), 8.0 and 7.98 (1H, 2d, J=9.2, H-8_qn); 7.63 and 7.50 (1H, 2br.s); 7.45 (br.s) and 7.39 (d, J=4.5) (1H, H-3_qn); 7.36 (1H, dd, J=9.1 and 2.6, H-7_qn); 6.50 (1H, br.s); 5.89 (1H, m, ═CH_qn); 5.03 (2H, m, ═CH_2qn); 3.99 and 3.97 (3H, 2s, OCH₃); 3.54 and 3.33 (3H, 2br.s, OCH₃); 3.2-1.0 (m, 7□ 7×CH₂+3CH); 2.92 and 2.67 (2d, J_AB=14.9, CH₂CO₂); 2.87 (d, J_AB=14.8, CH₂CO₂); 1.17 and 0.99 (2s, 2×CH₃); 1.03 and 0.42 (2br.s, 2×CH₃).

2°) Method B: DCC

To a stirred mixture of tetrahydrocarboxylic acid resulting from Example 6 (200 mg, 0.87 mmol) in anhydrous toluene (4 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (239 mg, 1.16 mmol). After stirring for 5 minutes, quinine (94 mg, 0.29 mmol) and pyrrolidinopyridine (43 mg, 0.29 mmol) were added. After stirring at 18±5° C. for 65 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (9:1), silica (15-40 μm)) to provide the expected product (96 mg, 60%, two diastereomers 50/50). The product thus obtained showed identical characteristics to this obtained above.

EXAMPLE 16

Preparation of (−)-menthyl 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydro-pyrane carboxylate, tetrahydropyrane-carboxylic acid resulting from Example 6

##STR00117##

To a stirred mixture of acid resulting from Example 6 (50 mg, 0.22 mmol) in anhydrous toluene (1 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (90 mg, 0.44 mmol). After stirring for 5 minutes, (−)-menthol (68 mg, 0.44 mmol) and pyrrolidinopyridine (64 mg, 0.44 mmol) were added. After stirring at 30° C. for 1 hour, then at 8° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (cyclohexane/ethyl acetate (95:5 then 90:10), silica (15-40 μm) 4 g) to provide the expected product (40 mg, 50%, two diastereomers 60/40). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 4.68 (1H, m, H-1_men); 3.64 (3H, s, OCH₃); 2.84 and 2.64 (2d, J_AB=14.6, CH₂CO₂); 2.83 and 2.63 (2d, J_AB=14.3, CH₂CO₂); 2.29 (1H, m, H-3_eq); 2.1-0.8 (m, CH and CH₂); 1.21 (3H, 2s, CH₃); 1.17 and 1.16 (3H, 2s, CH₃); 0.9 and 0.88 (6H, 2d, J=6.4, 2□ 2×CH_3men); 0.74 and 0.72 (3H, 2d, J=6.8, CH_3men).

EXAMPLE 17

Preparation of (−)-methyl mandelate 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydropyrane carboxylate from tetrahydropyranecarboxylic acid Resulting from Example 6

##STR00118##

To a stirred mixture of tetrahydropyranecarboxylic acid resulting from Example 6 (226 mg, 0.98 mmol) in anhydrous toluene (4 ml) maintained in an inert atmosphere at room temperature was added 1,3-dicyclohexylcarbodiimide (261 mg, 1.2 mmol). After stirring for 5 minutes, menthyl mandelate (53 mg, 0.32 mmol) and pyrrolidinopyridine (47 mg, 0.32 mmol) were added. After stirring at 18±5° C. for 12 hours, the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (9:1), silica (15-40 μm)) to provide a colorless oil (64 mg, 17%, two diastereomers). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δppm, J Hz): 7.47 (2H, m, Ph); 7.38 (3H, m, Ph); 5.96 (1H, s, CH); 3.73 and 3.72 (3H, 2s, OCH₃); 3.54 (3H, 2s, OCH₃); 2.88 and 2.72 (2d, J_AB=14.4, CH₂CO₂); 2.85 and 2.65 (2d, J_AB=14.2, CH₂CO₂); 2.35 (1H, m, H-3_eq); 2.0-1.15 (5H, m, CH₂); 1.23 and 1.22 (3H, 2s, CH₃); 1.19 and 1.07 (3H, 2s, CH₃).

EXAMPLE 18

Separation of (−)-quinyl(2′R)-anhydrohomoharringtonate and (−)-quinyl(2′S)-anhydrohomoharringtonate from Diastereomeric Mixture Resulting from Example 15

Diastereomeric A diastereomeric mixture of (−)-quinyl(2′R)-anhydrohomoharringtonate and of (−)-quinyl(2′S)-anhydrohomoharringtonate (5 g) was submit submitted to preparative HPLC. Above The mixture was dissolved in buffer (triethylamine (1.55/1000) in deionised deionized water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on onto a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 □m 15 μm, porosity 100, 1 kg; mobile phase: buffer/acetonitrile 70/30). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was were monitored by U.V. detector and TLC. Retained fraction fractions were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml),. After concentration under reduced pressure the two separated isomers were obtained as white crystalline solids corresponding to (−)-quinyl(2′R)-anhydrohomoharringtonate (2 g) and (−)-quinyl(2′S)anhydrohomoharringtonate (2.2 g). The products thus obtained showed the following characteristics:

1°) Diastereomer 2′R

##STR00119##

IR (film NaCl) (cm⁻¹): 2947; 2871; 1743; 1626; 1509.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 8.73 (1H, d, J=4.4, H-2_qn), 8.0 (1H, d, J=9.2, H-8_qn); 7.50 (1H, br.s); 7.39 (1H, d, J=4.5, H-3_qn); 7.36 (1H, dd, H-7_qn); 6.39 (1H, br.s); 5.88 (1H, m, ═CH_qn); 5.03 (2H, m, ═CH_2qn,); 3.97 (3H, s, OCH₃); 3.31 (3H, br.s, OCH₃); 3.5-1.2 (m, 7×CH₂+3CH); 2.86 and 2.64 (2H, 2d, J_AB=15.0, CH₂CO₂); 1.17 (3H, s, CH₃); 0.99 (3H, s, CH₃).

2°) Diastereomer 2′S

##STR00120##

IR (film NaCl) (cm⁻¹): 2947; 2871; 1743; 1626; 1509.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 8.74 (1H, d, J=4.4, H-2_qn), 7.99 (1H, d, J=9.2, H-8_qn); 7.65 (1H, br.s, H-3_qn); 7.44 (1H, br.s, H-5_qn); 7.36 (1H, dd, J=9.2 and 2.7, H-7_qn); 6.55 (1H, br.s); 5.89 (1H, m, ═CH_qn); 5.05 (2H, m, ═CH_2qn); 3.99 (3H, s, OCH₃); 3.54 (3H, s, OCH₃); 3.1-1.0 (m, 7×CH₂+3CH); 2.91 and 2.67 (2H, 2d, J_AB=15.0, CH₂CO₂); 1.03 (3H, br.s, CH₃); 0.44 (3H, br.s, CH₃).

EXAMPLE 19

Preparation of (2R)-anhydrohomoharringtonic acid from (−)-quinyl(2′R)-anhydrohomoharringtonate resulting from Example 18

##STR00121##
1°) Obtaining Via Hydrogenolysis

To a stirred solution of quinyl (2′R)-anhydroharringtonate (100 mg, 0.19 mmol) in ethyl acetate (11 ml) was added 10% palladium on charcoal (40 mg). The resulting mixture was stirred at room temperature under hydrogen pressure (50 p.s.i.) for 20 hours, and after CCM control the reaction mixture was filtered and the resulting organic layer was treated with saturated sodium hydrogen carbonate solution. The aqueous layer was washed with ethyl acetate, and after acidification with hydrochloric acid 1N was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated to dryness to afford a yellow solid (20 mg, 50%). The product thus obtained showed the following characteristics:

• • [α]_D²⁰: −23 (c=0.38; CHCl₃).

IR (film NaCl) (cm⁻¹): 2974; 2951; 1740; 1718; 1437.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): ¹H NMR spectra of the product thus obtained was identical to this described in Example 6-1.

2°) Obtaining Via Total Saponification then Selective Methylation

A mixture of potassium hydroxide (396 mg, 7.1 mmol) in water (8 ml) was added to a stirred solution of quinyl(2′R)-anhydroharringtonate (396 g. 0.72 mmol) in ethanol (15 ml) and the resulting mixture was stirred at reflux for 24 hours. After cooling at room temperature and removal of ethanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (4×15 ml). After acidification (pH 1) with hydrochloric acid 2N and saturation with sodium chloride, the aqueous layer was extracted with ethyl acetate (3×15 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow solid (110 mg, 72%). The intermediate diacid thus obtained showed the following characteristics:

[α]_D²⁰: −14 (c=0.54; CHCl₃)

IR (film NaCl) (cm⁻¹): 2975; 2941; 1716; 1217.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): ¹H NMR spectra of the product thus obtained was identical to this described in Example 4.

A mixture of (2R)-cyclic diacid above-mentioned (110 mg, 0.5 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (1.1 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 15 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (20 ml), the resulting aqueous layer was washed with ether (3×15 ml), acidified (pH 1) with hydrochloric acid 2N, and extracted with ether (3×15 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a viscous yellow oil (69 mg, 59%). The product thus obtained showed identical characteristics to this obtained from method 1° above.

EXAMPLE 20

Preparation of (2S)-anhydrohomoharringtonic acid from (−)-quinyl (2′R)-anhydrohomoharringtonate resulting from Example 18)

##STR00122##
1°) Obtaining Via Hydrogenolysis

To a stirred solution of quinyl (2′S)-anhydroharringtonate (100 mg, 0.19 mmol) in ethyl acetate (11 ml) was added 10% palladium on charcoal (40 mg). The resulting mixture was stirred at room temperature under hydrogen pressure (50 p.s.i.) and after CCM control the reaction mixture was filtered and the resulting organic layer was treated with saturated sodium hydrogen carbonate solution. The aqueous layer was washed with ethyl acetate and after acidification with hydrochloric acid 1N was extracted with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate and evaporated to dryness to afford a yellow solid (23 mg, 53%). The product thus obtained showed the following characteristics:

• • [α]_D²⁰: +(c=0.36; CHCl₃)

IR (film NaCl) (cm⁻¹): 2975; 2951; 1740; 1718; 1439.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): ¹H NMR spectra of the product thus obtained was identical to this described in Example 6-1.

2°) Obtaining Via Total Saponification then Selective Methylation

A mixture of potassium hydroxide (430 mg, 7.7 mmol) in water (9 ml) was added to a stirred solution of quinyl(2′S)-anhydroharringtonate (447 g, 0.81 mmol) in ethanol (16 ml) and the resulting mixture was stirred at reflux for 24 hours. After cooling at room temperature and removal of ethanol in vacuo, the residue was treated with water (10 ml) and the resulting aqueous layer was extracted with ether (4×15 ml). After acidification (pH 1) with hydrochloric acid 2N and saturation with sodium chloride, the aqueous layer was extracted with ethyl acetate (3×15 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow solid (140 mg, 80%). The cyclic diacid thus obtained showed the following characteristics:

• • [α]_D²⁰: +8 (c=0.19; CHCl₃)

IR (film NaCl) (cm⁻¹): 2975; 2945; 1717.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): ¹H NMR spectra of the product thus obtained was identical to this described in Example 4.

A mixture of (2S)-cyclic diacid above-mentioned (136 mg, 0.62 mmol) and a commercial solution of boron trifluoride-methanol complex in methanol (1.3 ml, BF₃ 12% w/w) was stirred at 18±5° C. for 15 hours. After careful addition of the reaction mixture at saturated sodium hydrogen carbonate solution (20 ml), the resulting aqueous layer was washed with ether (3×15 ml), acidified (pH 1) with hydrochloric acid 2N and extracted with ether (3×15 ml). The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a viscous yellow oil (81 mg, 63%). The product thus obtained showed identical characteristics to this obtained from method 1° above.

EXAMPLE 21

Preparation of Anhydrohomoharringtonine, Via Esterification of Cephalotaxine with (2R)-(+)-Anhydrohomoharringtonic Acid

##STR00123##

To a stirred mixture of hemiester (R) resulting from Example 19 (65 mg, 0.28 mmol) in anhydrous toluene (1 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (38 μl, 0.28 mmol) and 2,4,6-trichlorobenzoyl chloride (43 □l 43 μl, 0.28 mmol). After stirring at 30° C. for 1.5 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (50 mg, 0.41 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (129 mg, 0.41 mmol) was added. After stirring at 30° C. for 18 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml) then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 μm)) to provide expected product (65 mg, 43%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.61 (1H, s, H-17*); 6.58 (1H, s, H-14*); 5.92 (1H, d, J_3-4=9.6, H-3); 5.87 and 5.79 (2H, 2s, OCH₂O); 5.04 (1H, br.s, H-1); 3.80 (1H, d, J_4-3=9.2, H-4); 3.70 (3H, s, OCH₃); 3.59 (3H, s, OCH₃); 3.12 (2H, m, H-11β+H-8α); 2.95 (1H, m, H-10α); 2.60 (2H, m, H-8β+H-10β); 2.38 (1H, m, H-11α); 2.13 and 1.66 (2H, 2d, J_AB=14.3, CH₂CO₂); 2.02 (1H, m, H-6_A); 1.90 (1H, m, H-6_B); 1.76 (2H, m, CH₂-7); 1.8-1.2 (6H, m, 3×CH₂); 1.10 (3H, s, CH₃); 1.04 (3H, s, CH₃).

EXAMPLE 22

Preparation of Anhydroepihomoharringtonine, Via Esterification of Cephalotaxine with (2S)-(+)-anhydrohomoharringtonic acid

##STR00124##

To a stirred mixture of hemiester (S) resulting from Example 20 (87 mg, 0.38 mmol) in anhydrous toluene (1.7 ml) at room temperature was added triethylamine (dried over potassium hydroxide) (52 μl, 0.38 mmol) and 2,4,6-trichlorobenzoyl chloride (57 μl, 0.38 mmol). After stirring at 30° C. for 1.5 hours (with control of disappearing of starting acid in infra-red), 4-dimethylaminopyridine (70 mg, 0.57 mmol) was added, the reaction mixture was allowed to react for 5 minutes and cephalotaxine (180 mg, 0.57 mmol) was added. After stirring at 30° C. for 18 hours, the reaction mixture was filtered on paper and diluted with ether (5 ml). The resulting organic layer was successively washed with water (5 ml), with saturated sodium hydrogen carbonate solution (5 ml), with water again (5 ml), then was dried over magnesium sulfate and evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 μm)) to provide expected product (101 mg, 50%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.61 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.84 (3H, m, H-3)+OCH₂O); 5.04 (1H, m, H-1); 3.78 (1H, d, J_4-3=9.7, H-4); 3.65 (3H, s, OCH₃); 3.59 (3H, s, OCH₃); 3.23 (1H, m, H-11β); 3.09 (1H, m, H-8α); 2.93 (1H, m, H-10α); 2.58 (2H, m, H-8β+H-10β); 2.39 (1H, dd, J_AB=14.4, J=7.0, H-11α); 2.16 and 1.83 (2H, 2d, J_AB=14.5, CH₂CO₂); 2.06 (1H, m, H-6_A); 1.88 (1H, m, H-6_B); 1.74 (2H, m, CH₂-7); 1.5-1.2 (6H, m, 3×CH₂); 1.11 (3H, s, CH₃); 1.02 (3H, s, CH₃).

EXAMPLE 23

Preparation of 6′-bromo-6′-desoxy-homoharringtonine, from anhydro-homoharringtonine resulting from Example

##STR00125##

To a stirred solution of product resulting from Example 21 (60 mg, 0.114 mmol) in anhydrous dichloromethane (300 μl) was added at −10° C. a commercial solution of hydrobromic acid in acetic acid (205 μl, 1.02 mmol, HBr 30% w/w). After stirring at −10° C. for 3 hours, was added saturated sodium hydrogen carbonate solution up to pH 8. The resulting aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow oil (60 mg, 87%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 2957; 1744; 1653; 1487; 1223.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.63 (1H, s, H-17*); 6.54 (1H, s, H-14*); 5.99 (1H, d, J_3-4=9.8, H-3); 5.87 (2H, m, OCH₂O); 5.05 (1H, s, H-1); 3.78 (1H, d, J_4-3=9.8, H-4); 3.69 (3H, s, OCH₃); 3.58 (3H, s, OCH₃); 3.54 (1H, s, 2′-OH); 3.10 (2H, m, H-11β+H-8α); 2.94 (1H, m, H-10α); 2.60 (2H, m, H-8β+H-10β); 2.39 (1H, dd, J_AB=14.0, J=6.8, H-11α); 2.26 and 1.89 (2H, 2d, J_AB=16.5, CH₂CO₂); 2.03 (1H, m, H-6_A); 1.91 (1H, m, H-6_B); 1.75 (2H, m, CH₂-7); 1.74 (3H, s, CH₃); 1.72 (3H, s, CH₃); 1.6-1.2 (6H, m, 3×CH₂).

EXAMPLE 24

Preparation of 6′-bromo-6′-desoxy-epihomoharringtonine, from anhydro-epihomoharringtonine resulting from Example 22

##STR00126##

A commercial solution of hydrobromic acid in acetic acid (205 μl, 1.02 mmol, HBr 30% w/w) was added to a stirred solution of product resulting from Example 22 (60 mg, 0.114 mmol) in anhydrous dichloromethane (300 μl) at −10° C. After stirring at −10° C. for 3 hours, was added a saturated sodium hydrogen carbonate solution up to pH 8 and the resulting aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a yellow oil (63 mg, 91%). The crude product thus obtained showed the following characteristics:

IR (ATR) (cm⁻¹): 2957; 1744; 1653; 1487; 1223.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.64 (1H, s, H-17*); 6.59 (1H, s, H-14*); 5.97 and 5.87 (2H, 2d, J_AB=1.1, OCH₂O); 5.95 (1H, d, J_3-4=9.7, H-3); 5.04 (1H, s, H-1); 3.78 (1H, d, J_4-3=9.7, H-4); 3.67 (3H, s, OCH₃); 3.66 (3H, s, OCH₃); 3.49 (1H, s, 2′-OH); 3.10 (2H, m, H-11β+H-8α); 2.93 (1H, m, H-10α); 2.62 and 2.54 (2H, 2d, J_AB=16.5, CH₂CO₂); 2.60 (2H, m, H-8β+H-10β); 2.40 (1H, m, H-11α); 2.03 (1H, m, H-6_A); 1.89 (1H, m, H-6_B); 1.74 (2H, m, CH₂-7); 1.72 (3H, s, CH₃); 1.70 (3H, s, CH₃); 1.6-0.7 (6H, m, 3×CH₂).

EXAMPLE 25

Preparation of homoharringtonine, from 6′-bromo-6′-desoxy-homoharringtonine resulting from Example 23

##STR00127##
1) Method A:

A 5% sodium hydrogen carbonate solution (3 ml) was added to a stirred solution of product resulting from Example 23 (60 mg, 0.099 mmol) in acetone (1.5 ml). After stirring at room temperature for 2 hours, the reaction mixture was evaporated in vacuo and the residual aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (55 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (99:1 then 95:5), silica (15-40 μm) 2.75 g) to provide homoharringtonine (29 mg, 47%). The product thus obtained showed following characteristics:

[α]_D²⁰: −110 (c=0.24; CHCl₃)

IR(film NaCl) (cm⁻¹): 3468; 2961; 1745; 1656; 1487; 1224; 1033.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.63 (1H, s, H-17*); 6.55 (1H, s, H-14*); 6.01 (1H, d, J_3-4=9.8, H-3); 5.87 (2H, m, OCH₂O); 5.05 (1H, s, H-1); 3.78 (1H, d, J_4-3=9.8, H-4); 3.68 (3H, s, OCH₃); 3.58 (3H, s, OCH₃); 3.54 (1H, s, 2′-OH); 3.10 (2H, m, H-11β+H-8α); 2.95 (1H, m, H-10α); 2.59 (2H, m, H-8β+H-10β); 2.38 (1H, dd, J_AB=14.0, J=6.7, H-11α); 2.27 and 1.90 (2H, 2d, J_AB=16.5, CH₂CO₂); 2.02 (1H, m, H-6_A); 1.90 (1H, m, H-6_B); 1.76 (2H, m, CH₂-7); 1.5-1.15 (6H, m, 3×CH₂); 1.30 (1H, s, 6′-OH); 1.19 (6H, 2s, 2×CH₃).

2) Method B:

A saturated calcium carbonate solution (3 ml) was added to a stirred solution of product resulting from Example 23 (60 mg, 0.099 mmol) in acetone (3 ml). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

3) Method C:

A saturated barium carbonate solution (9 ml) was added to a stirred solution of product resulting from Example 23 (60 mg, 0.099 mmol) in acetone (3 ml). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

4) Method D:

To a stirred solution of product resulting from Example 23 (60 mg, 0.099 mmol) in a mixture acetone/water (3/2, 2.15 ml) was added silver nitrate (25 mg, 0.149 mmol). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

EXAMPLE 26

Preparation of Epihomoharringtonine 2′-epi-homoharringtonine (2′-epi HHT)

##STR00128##
1°) from 6′-bromo-6′-desoxy-epihomoharringtonine resulting from Example 24:
a) Method A:

A 5% sodium hydrogen carbonate solution (3 ml) was added to a stirred solution of product resulting from Example 24 (60 mg, 0.099 mmol) in acetone (1.75 ml). After stirring at room temperature for 2 hours, the reaction mixture was evaporated in vacuo and the residual aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (60 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (99:1 then 97:3), silica (15-40 μm) 3 g) to provide epihomoharringtonine (29 mg, 47%). The product thus obtained showed following characteristics:

[α]_D²⁰: −92 (c=0.29; CHCl₃)

IR (film NaCl) (cm⁻¹): 3514; 2961; 1744; 1655; 1488; 1223; 1035. ¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 6.65 (1H, s, H-17*); 6.60 (1H, s, H-14*); 5.95 (1H, d, H-3); 5.95 and 5.86 (2H, 2d, OCH₂O); 5.05 (1H, s, H-1); 3.78 (1H, d, J_4-3=9.7, H-4); 3.68 (3H, s, OCH₃); 3.66 (3H, s, OCH₃); 3.52 (1H, br.s, 2′-OH); 3.13 (2H, m, H-11β+H-8α); 2.97 (1H, m, H-10α); 2.63 (2H, m, H-8β+H-10β) 2.61 and 2.52 (2H, 2d, J_AB=16.5, CH₂CO₂); 2.40 (1H, dd, J_AB=13.8, J=6.3, H-11□ H-11α); 2.04 (1H, m, H-6_A); 1.94 (1H, m, H-6_B); 1.78 (2H, m, CH₂-7); 1.45-0.7 (6H, m, 3×CH₂); 1.16 (3H, s, CH₃); 1.15 (3H, s, CH₃).

b) Method B:

A saturated calcium carbonate solution (3 ml) was added to a stirred solution of product resulting from Example 24 (60 mg, 0.099 mmol) in acetone (3 ml). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

c) Method C:

A saturated barium carbonate solution (9 ml) was added to a stirred solution of product resulting from Example 24 (60 mg, 0.099 mmol) in acetone (3 ml). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

d) Method D:

To a stirred solution of product resulting from Example 24 (60 mg, 0.099 mmol) in a mixture acetone/water (3/2, 2.15 ml) was added silver nitrate (25 mg, 0.149 mmol). After stirring at room temperature for 2 hours, obtaining of product resulting from method A was specified by CCM.

2°) from Anhydroepihomoharringtonine Resulting from Example 22

To a stirred solution of anhydroepihomoharringtonine resulting from Example 22 (58 mg, 0.109 mmol) in anhydrous dichloromethane (0.3 ml) was added at −10° C. a commercial solution of hydrobromic acid in acetic acid (0.195 ml, 0.98 mmol, HBr 30% w/w). After stirring at −10° C. for 3 hours, was added water (2.8 ml) and then the temperature was raised to 20° C. After stirring at 20° C. for 3 hours, was added a sodium carbonate solution (0.76M; 6 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3×10 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to provide epihomoharringtonine (45 mg brut, 75%). The crude product thus obtained showed identical characteristics to this obtained with method A.

EXAMPLE 27

Preparation of Homoharringtonine as a Pharmaceutical Use from Crude Semi-Synthetic Homoharringtonine Resulting from Example 25 by Preparative High-Performance Liquid Chromatography

##STR00129##
homoharringtonine or cephalotaxine, 4-methyl (2R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester)

##STR00130##
epi-homoharrintonine 2′-epi-homoharringtonine or 2′-epi-HHT
1°) Method A

Crude homoharringtonine (35 g) is dissolved in buffer (triethylamine (1.55/1000) in deionised water and orthophosphoric acid to adjust pH to 3. The solution was filtered then injected on a preparative high-performance liquid chromatograph equipped with axial compression and high pressure pump (stationary phase: n-octadecylsilane, 15 μm, porosity 100, 1 kg; mobile phase: buffer/tetrahydrofurane 85/15). Elution was performed at a flow rate of 0.2 l/min. Fractions contain was monitored by U.V. detector and TLC. Retained fraction were finally checked by HPLC then combined, alkalinised with 2.5% aqueous ammonia and extracted with dichloromethane (4×400 ml), After concentration under reduced pressure homoharringtonine is obtained as a pale yellow resin which on trituration in a 8/2 water-methanol mixture gave pure homoharringtonine as a white crystalline solid (mp=127° C.), HPLC purity was higher than 99.8%.

2°) Method B

Same procedure of purification as method A was performed but mobile phase buffer/methanol (68/32) was used instead buffer/tetrahydrofurane.

3°) Method C

Same procedure of purification as method A was performed but mobile phase buffer/acetonitrile (85/15) was used instead buffer/tetrahydrofurane.

EXAMPLE 28

Preparation of Homoharringtonine as a Pharmaceutical Use from Semi-Purified Natural Cephalotaxine

Crude homoharringtonine, prepared according to Example 25 from a partially racemized natural cephalotaxine and purified by chromatography and crystallisation according to the method A of Example 27, gave an homoharringtonine showing a non natural enantiomeric epi-homoharringtonine content less than 0.05%.

EXAMPLE 29

Preparation of 2′-de-(methoxycarbonylmethyl)-(2-O-3′)-dehydroneoharringtonine or cephalotaxyl phenylglycidate, via esterification of cephalotaxine with phenylglycidic acid

##STR00131##

To a stirred mixture of (2R,3R)-cis-phenylglicidic acid (78 mg, 0.48 mmol) in anhydrous toluene (2 ml) was added 1,3-dicyclohexylcarbodiimide (130 mg, 0.63 mmol). After stirring for 10 minutes at room temperature, cephalotaxine (50 mg, 0.16 mmol) and pyrrolidinopyridine (24 mg, 0.16 mmol) were added. After stirring at 18±5° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 8:2), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product (200 mg) was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 4 g) to provide expected product (19 mg, 27%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.27 (3H, m, Ph); 7.18 (2H, m, Ph); 6.63 (1H, s, H-17*); 6.40 (1H, s, H-14*); 5.96 and 5.85 (2H, 2d, J_AB=1.5, OCH₂O); 5.73 (1H, d, J_3-4=9.4, H-3); 5.01 (1H, s, H-1); 4.01 (1H, d, J_3′-2′=4.6, H-3′); 3.65 (3H, s, OCH₃); 3.62 (1H, d, J_4-3=9.3, H-4); 3.40 (1H, d, J_2′-3′=4.5, H-2′); 3.27 (1H, m, J_AB=14.3, J=12.1 and 7.8, H-11β); 3.05 (1H, m, H-8α); 2.91 (1H, td, J=11.7 and 7.4, H-10α); 2.57 (2H, m, H-8β+H-10β); 2.43 (1H, dd, J_AB=14.5, J=7.0, H-11α); 1.93 (1H, m, H-6_A); 1.84 (1H, m, H-6_B); 1.68 (2H, m, CH₂-7).

EXAMPLE 30

Preparation of 2′-de-(methoxycarbonylmethyl)-neo-harringtonine, via hydrosenolysis of cephalotaxyl phenylglycidate resulting from Example 29

##STR00132##

To a stirred solution of cephalotaxyl phenylglicidate resulting from Example 29 (200 mg, 0.433 mmol) in methanol (10 ml) was added 10% palladium on charcoal (100 mg). The resulting mixture was stirred at room temperature under hydrogen pressure (50 p.s.i.) for 4 hours, and the reaction mixture was filtered and evaporated to dryness. The resulting crude product (175 mg) was purified by column chromatography (dichloromethane/methanol (99:1 then 98:2), silica (15-40 μm) 5.5 g) to provide an amber solid (86 mg, 43%). The product thus obtained showed following characteristics:

IR (pastille KBr) (cm⁻¹): 3436; 2937; 1747; 1655; 1487; 1224 et 1035.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.25 (3H, m, m, p-Ph); 7.0 (2H, m, o-Ph); 6.65 (1H, s, H-17*); 6.63 (1H, s, H-14*); 5.98 (1H, d, J_3-4=9.3, H-3); 5.85 (2H, 2d, J_AB=1.2, OCH₂O); 5.09 (1H, s, H-1); 4.17 (1H, m, H-2′); 3.85 (1H, d, J_4-3=9.6, H-4); 3.71 (3H, s, OCH₃); 3.20 (1H, m, H-11β); 3.10 (1H, m, H-8α); 2.95 (1H, m, H-10α); 2.60 (2H, m, H-8α+H-10β); 2.39 (2H, m, H-11α+H-3′_A); 2.04 (1H, m, H-6_A); 2.0 (1H, dd, J_AB=14.3, J_3′B-2′=9.5, H-3′B); 1.91 (1H, m, H-6_B) 1.77 (2H, m, CH₂-7).

EXAMPLE 31

Preparation of 2′R-de-(methoxycarbonylmethyl)-3′S-azido-neoharringtonine, from cephalotaxyl phenylglycidate resulting from Example 29

##STR00133##

To a stirred solution of cephalotaxyl phenylglicidate resulting from Example 29 (100 mg, 0.217 mmol) in a mixture of methanol/water (8/1, 1.27 ml) was added sodium azide (70 mg, 1.08 mmol) and methyl formate (174 μl, 2.82 mmol). After stirring at 50° C. for 68 hours, and cooling at ambient temperature, was added 5% sodium hydrogen carbonate solution up to pH 8. The resulting aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (120 mg) was purified by column chromatography (dichloromethane/methanol (99:1), silica (15-40 μm) 3.5 g) to provide a viscous yellow oil (84 mg, 76%). The product thus obtained showed following characteristics:

IR (ATR) (cm⁻¹): 3488; 2935; 2105; 1748; 1654; 1486; 1223; 1034.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.38 (3H, m, m, p-Ph); 7.29 (2H, m, o-Ph); 6.74 (1H, s, H-17*); 6.67 (1H, s, H-14*); 6.08 (1H, d, J_3-4=9.8, H-3); 5.90 (2H, 2d, J_AB=1.4, OCH₂O); 5.08 (1H, s, H-1); 4.07 (1H, d large, H-2′); 3.85 (1H, d, J_4-3=9.7, H-4); 3.78 (1H, br.s, H-3′); 3.69 (3H, s, OCH₃); 3.23 (1H, m, H-11β): 3.11 (1H, m, H-8α); 2.98 (1H, m, H-10α); 2.90 (1H, d, J_2′-OH=8.2, 2′-OH); 2.63 (2H, m, H-8β+H-10β); 2.47 (1H, dd, J_AB=14.2, J=6.9, H-11α); 2.05 (1H, m, H-6_A); 1.92 (1H, m, H-6_B); 1.78 (2H, m, CH₂-7).

EXAMPLE 32

Preparation of 2′R-de(methoxycarbonylmethyl)-3′S-amino-neoharringtonine, via hydrogenolysis of azide resulting from Example 31

##STR00134##

To a stirred solution of product resulting from Example 29 (80 mg, 0.158 mmol) in a mixture ethyl acetate-methanol (9/1, 10 ml) was added 10% palladium on charcoal (40 mg). The resulting mixture was stirred at room temperature under hydrogen pressure (50 p.s.i.) for 15 hours and after CCM control the reaction mixture was filtered and evaporated to dryness to provide a white solid (67 mg, 88%). The crude product thus obtained showed following characteristics:

IR (ATR) (cm⁻¹): 3299; 2935; 1740; 1654; 1486; 1222 et 1034.

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.27 (5H, m, Ph); 6.69 (1H, s, H-17*); 6.67 (1H, s, H-14*); 6.0 (1H, d, J_3-4=9.7, H-3); 5.85 (2H, m, OCH₂O); 5.09 (1H, br.s, H-1); 4.01 (1H, d, J=1.2, H-2′); 3.86 (1H, d, J_4-3=9.5, H-4); 3.72 (3H, s, OCH₃); 3.38 (1H, br.s); 3.25 (1H, m, H-11β); 3.14 (1H, m, H-8α); 2.99 (1H, m, H-10α); 2.64 (2H, m, H-8β+H-10β); 2.49 (1H, m, H-11α); 2.05 (1H, m, H-6_A); 1.94 (1H, m, H-6_B); 1.79 (2H, m, CH₂-7).

EXAMPLE 33

Preparation of acetonide of 2′-de-(methoxycarbonylmethyl)-3′-hydroxy-neoharringtonine, via esterification of cephalotaxine

##STR00135##

To a stirred mixture of isopropylidene-2,3-dihydroxy-3-phenylpropionic acid (17.5 mg, 0.078 mmol) in anhydrous toluene (1 ml) was added 1,3-dicyclohexylcarbodiimide (25 mg, 0.12 mmol). After stirring for 10 minutes at room temperature, cephalotaxine (75 mg, 0.24 mmol) and pyrrolidinopyridine (12 mg, 0.08 mmol) were added, After stirring at 18±5° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 8:2), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 μm)) to provide expected product (22 mg, 53%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.27 (5H, Ph); 6.63, 6.62, 6.60 and 6.57 (1H, 4s, H-14*); 6.51, 6.49, 6.42 and 6.41 (1H, 4s, H-17*); 5.93 (J_3-4=9.6), 5.89, 5.43 (J_3-4=9.5) and 5.31 (J_3-4=9.3 (1H, 4d, H-3); 5.89 (s), 5.87+5.84 (2d, J_AB=1.5), 5.85+5.80 (2d) and 5.84+5.77 (2d, J_AB=1.5) (2H, OCH₂O); 5.23 (J_5′-4′=7.3), 5.20 (J_5′-4′=7.4), 4.58 (J_5′-4′=8.0) and 4.49 (J_5═-4′=6.2) (1H, 4d, H-5′); 5.07, 5.03 and 4.83 (1H, 3s, H-1); 4.32 (J_4′-5′=7.4), 4.21 (J_4′-5′=6.2), 4.18 (J_4′-5′=7.4) and 3.75 (1H, 4d, H-4′); 3.86 (J_4-3=9.6), 3.76 and 3.60 (J_4-3=9.5) (1H, 4d, H-4); 3.76, 3.75, 3.70 and 3.43 (3H, 4s, OCH₃); 3.3-1.6 (10H, m); 1.66+1.41, 1.65+1.37, 1.51+1.44 and 1.47+1.22 (6H, 8s, 2×CH₃).

EXAMPLE 34

Preparation of cephalotaxyl N-benzyl-3-phenyl-aziridine-1-carboxylate via esterification of cephalotaxine

##STR00136##

To a stirred mixture of (2S,3S)-cis-N-benzyl-3-phenylaziridine-1-carboxylic acid (360 mg, 1.42 mmol) in anhydrous toluene (5 ml) was added 1,3-dicyclohexylcarbodiimide (390 mg, 1.9 mmol). After stirring for 5 minutes at room temperature, cephalotaxine (150 mg 0.47 mmol) and pyrrolidinopyridine (70 mg, 0.47 mmol) were added. After stirring at 18±5° C. for 2 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 8:2), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (15 ml) and the filtrate was evaporated in vacuo. The resulting crude product (785 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 μm) 23 g) to provide a solid (240 mg, 92%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.24 (10H, m, 2×Ph); 6.63 (1H, s, H-17*); 6.60 (1H, s, H-14*); 5.85 and 5.80 (2H, 2d, J_AB=1.4, OCH₂O); 5.64 (1H, d, J_3-4=9.3, H-3); 4.97 (1H, s, H-1); 3.92 and 3.20 (2H, 2d, J_AB=13.7, CH₂Ph); 3.71 (1H, d, J_4-3=9.4, H-4); 3.56 (3H, s, OCH₃); 3.25 (1H, m, H-11β); 3.07 (1H, m, H-8α); 2.93 (1H, m, H-10α); 2.86 (1H, d, J_3′-2′=6.8, H-3′); 2.57 (2H, m, H-8β+H-10β); 2.38 (1H, dd, J_AB=14.4, J=7.0, H-11α); 2.07 (1H, d, J_2′-3′=6.8, H-2′); 1.96 (1H, m, H-6_A); 1.82 (1H, m, H-6_B); 1.70 (2H, m, CH₂-7).

EXAMPLE 35

Preparation of N,O-anhydro-2′-de-(methoxycarbonylmethyl)-3′-benzamidoneo-harringtonine or cephalotaxyl N,O-anhydro-N-benzoyl-phenylisoserinate via esterification of cephalotaxine

##STR00137##

To a stirred mixture of (4S,5R)-2,4-diphenyl-4,5-dihydrooxazole-5-carboxylic acid (510 mg, 1.91 mmol) in anhydrous toluene (7 ml) was added 1,3-dicyclohexylcarbodiimide (525 mg, 2.54 mmol). After stirring for 15 minutes at room temperature, cephalotaxine (200 mg, 0.63 mmol) and pyrrolidinopyridine (95 mg, 0.64 mmol) were added. After stirring at 18±5° C. for 3.5 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (15 ml) and the filtrate was evaporated in vacuo. The resulting crude product (1 g) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 μm)) to provide a yellow solid (330 mg, 91%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 8.0 (2H, d, J=7.3, o-PhC═N); 7.52 (1H, t, J=7.4, p-PhC═N); 7.44 (2H, t, J=7.5, m-PhC═N); 7.32 (2H, t, J=7.2, m-Ph); 7.26 (1H, m, p-Ph); 7.15 (2H, d, J=7.1, o-Ph); 6.58 (1H, s, H-17*); 6.51 (1H, s, H-14*); 5.98 (1H, d, J_3-4=9.5, H-3); 5.85 and 5.76 (2H, 2d, J_AB=1.3, OCH₂O); 5.08 (1H, s, H-1); 4.67 (1H, d, J_4′5′=5.6, H-4′); 4.52 (1H, d, J_5′-4′=5.6, H-5′) 3.85 (1H, d, J_4-3=9.6, H-4); 3.70 (3H, s, OCH₃); 3.17 (1H, m, H-11β); 3.08 (1H, m, H-8α); 2.93 (1H, m, H-10α); 2.59 (2H, m, H-8β+H-10β); 2.31 (1H, dd, J_AB=14.2, J=6.8, H-11α); 2.04 (1H, m, H-6_A); 1.91 (1H, m, H-6_B); 1.75 (2H, m, CH₂-7).

EXAMPLE 36

Preparation of N,O-methoxymethylene-2′-de-(methoxycarbonylmethyl)-3′-benz-amidoneoharringtonine or cephalotaxyl N,O-methoxymethylene-N-benzoylphenylisoserinate via esterification of cephalotaxine

##STR00138##

To a stirred mixture of (4S,5R)-3-N-benzoyl-2p-methoxyphenyl-4-phenyloxazolidine-5-carboxylic acid (165 mg, 0.5 mmol) in anhydrous toluene (2 ml) was added 1,3-dicyclohexylcarbodiimide (140 mg, 0.68 mmol). After stirring for 5 minutes at room temperature, cephalotaxine (53 mg, 0.17 mmol) and pyrrolidinopyridine (25 mg, 0.17 mmol) were added. After stirring at 18±5° C. for 15 hours (with control of reaction in CCM, eluant dichloromethane/methanol; 9:1), the reaction mixture was filtered on ground-glass filter, the cake was washed with toluene (5 ml) and the filtrate was evaporated in vacuo. The resulting crude product (230 mg) was purified by column chromatography (dichloromethane, then dichloromethane/methanol (98:2), silica (15-40 μm) 7 g) to provide expected product (90 mg, 86%). The product thus obtained showed following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.66 (2H, br.s, o-BzN); 7.41 (4H, m, BzN+Ph); 7.32 (2H, m, Ph); 7.26 (2H, m, Ph); 6.56 (1H, s, H-17*); 6.54 (1H, s, H-14*); 5.89 (1H, d, J_3-4=9.5, H-3); 5.83 and 5.80 (2H, 2m, OCH₂O); 5.76 (1H, br.s, H-2′); 5.10 (1H, s, H-1); 4.85 (1H, br.s, H-4′); 4.42 (1H, br.s, H-5′); 3.84 (1H, d, J_4-3=9.5, H-4); 3.72 (3H, s, OCH₃); 3.28 (3H, br.s, 2′-OCH₃); 3.19 (1H, m, H-11β); 3.09 (1H, m, H-8α); 2.93 (1H, m, H-10α); 2.60 (2H, m, H-8β+H-10β); 2.37 (1H, dd, J_AB=14.4, J=6.6, H-11α); 2.03 (1H, m, H-6_A); 1.90 (1H, m, J_AB=12.2, J=7.8 and 4.4, H-6_B); 1.76 (2H, m, CH₂-7).

EXAMPLE 37

Preparation of 2′-de-(methoxycarbonylmethyl)-3′-benzamido-neoharringtonine or cephalotaxyl N-benzoyl-phenylisoserinate

##STR00139##
1°) Via Acidic Hydrolysis of Product Resulting from Example 35

To a stirred solution of cephalotaxyl (4S,5R)-2,4-diphenyl-4,5-dihydrooxazole-5-carboxylate resulting from Example 35 (300 mg, 0.53 mmol) in a mixture of methanol/tetrahydrofurane 50/50 (10 ml) was added at room temperature hydrochloric acid 1N (3.2 ml). After stirring at 18±5° C. for 3 hours (with control of reaction in CCM), a saturated sodium hydrogen carbonate solution (19 ml) and a mixture of methanol/tetrahydrofurane 50/50 (50 ml) were added. After stirring at 18±5° C. for 20 hours (with control of reaction in CCM), the reaction mixture was treated with ethyl acetate and water. The resulting aqueous layer was extracted with ethyl acetate and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (170 mg) was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 8 g) to provide a white solid (180 mg, 58%; HPLC purity 92.3%). The product thus obtained showed the following characteristics:

[α]_D²⁰: −119.2 (c=0.141; CHCl₃)

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.75 (2H, d, J=7.3, o-BzN); 7.51 (1H, t, J=7.3, p-BzN); 7.13 (2H, t, J=7.3, m-BzN); Ph); 7.27 (5H, m, Ph); 6.88 (1H, d, J_3′-NH=7.9, 3′-NH); 6.59 (1H, s, H-17*); 6.57 (1H, s, H-14*); 5.93 (1H, d, J_3-4=9.7, H-3); 5.78 and 5.69 (2H, 2d, JAB=1.5, OCH₂O); 5.06 (1H, s, H-1); 4.98 (1H, dd, J_3′-NH=7.9, H-3′); 4.22 (1H, br.s, H-2′); 3.81 (1H, d, J_4-3=9.6, H-4); 3.58 (3H, s, OCH₃); 3.19 (1H, m, J=12.8, 7.9, H-11b); 3.07 (1H, m, H-8a); 2.93 (1H, m, H-10a); 2.72 (1H, br.s, 2′-OH), 2.58 (2H, m, H-8b+H-10b); 2.43 (1H, dd, J_AB=14.2, J=7.0, H-11a); 2.01 (1H, m, H-6_A); 1.88 (1H, m, J_AB=12.0, J=7.8, 3.8, H-6_B); 1.75 (2H, m, CH₂-7)

2°) Via Amidification of Product Resulting from Example 32

To a stirred solution of product resulting from Example 32 (60 mg, 0.125 mmol) in ethyl acetate (850 μl ) were added a saturated sodium hydrogen carbonate solution (850 μl) and benzoyl chloride (19 μl, 0.163 mmol). A white precipitate was formed during the course of reaction. After stirring at room temperature for 1 hour, the reaction mixture was diluted with ethyl acetate and the organic layer was washed with a saturated sodium hydrogen carbonate solution. The resulting aqueous layer was extracted three times with ethyl acetate and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (65 mg) was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 2.5 g) to provide a white solid (41 mg, 56%). The product thus obtained showed identical characteristics to this obtained from method above-mentioned.

EXAMPLE 38

Preparation of N-terbutoxycarbamoyl-2′-de-(methoxycarbonylmethyl)-3′-amino-neoharringtonine or cephalotaxyl N-terbutoxycarbamoyl-phenylisoserinate via amidification of product resulting from Example 32

##STR00140##

To a stirred solution of product resulting from Example 32 (60 mg, 0.125 mmol) in dichloromethane (850 μl) were added a saturated sodium hydrogen carbonate solution (850 μl) and diterbutyldicarbonate (27 mg, 0.125 mmol). After stirring at room temperature for 1 hour, the reaction mixture was diluted with dichloromethane and the organic layer was washed with brine. The resulting aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product (70 mg) was purified by column chromatography (dichloromethane/methanol (98:2), silica (15-40 μm) 3 g) to provide a white solid (40 mg, 55%). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.27 (3H, m, m, p-Ph); 6.94 (2H, d, J=6.6, o-Ph); 6.71 (1H, s, H-17*); 6.66 (1H, s, H-14*); 6.01 (1H, d, J_3-4=9.7, H-3); 5.90 (2H, s, OCH₂O); 5.06 (1H, s, H-1); 5.05 (1H, m, NH); 4.56 (1H, m, H-3′); 4.15 (1H, m, H-2′); 3.81 (1H, d, J_4-3=9.7, H-4); 3.69 (3H, s, OCH₃); 3.19 (1H, m, H-11β); 3.10 (1H, m, H-8α); 2.93 (1H, m, H-10α); 2.61 (2H, m, H-8β+H-10β); 2.51 (1H, m, H-11α); 2.05 (1H, m, H-6_A); 1.89 (1H, m, H-6_B); 1.77 (2H, m, CH₂-7); 1.44 (9H, s, OC(CH₃)₃).

EXAMPLE 39

Preparation of tert-butyl 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate

##STR00141##
1°) Preparation of Intermediate Oxalate

5-Bromo-2-methyl-pent-2-ene (1.34 g, 8.2 mmol) was added dropwise to a stirred mixture of magnesium (240 g, 10 mmol) (activated with further crystal of iodine) in anhydrous tetrahydrofurane (8 ml). The onset of the reaction is accompanied with a vigorous overheating and refluxing of the reaction mixture. The reflux was maintained until most of magnesium had reacted and the reaction mixture was diluted with anhydrous tetrahydrofurane (16 ml). To a stirred mixture of tert-butyl ethyl oxalate (1.4 g, 8 mmol) in anhydrous tetrahydrofurane (8 ml) was added the resulting Grignard reagent at −78° C. over a period of 20 minutes. The temperature was allowed to rise to −15° C. over a period of 2 hours and the mixture was quenched with hydrochloric acid 1N. The separated organic layer was washed three times with brine, dried over magnesium sulfate and evaporated to dryness. The resulting crude product (2 g) was purified by column chromatography (cyclohexane/ethyl acetate (98:2), silica (15-40 μm) 80 g) to provide an oil (660 mg, 39%). The intermediate α-cetoester showed the following characteristics:

##STR00142##

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.08 (1H, m, H-5); 2.80 (2H, t, J=7.3, CH2-3); 2.28 (2H, m, CH₂-4); 1.68 (3H, s, CH₃); 1.62 (3H, s, CH₃); 1.54 (9H, s, O-tertBu).

2°) Preparation of the Title Compound

To a stirred solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (9 ml, 9 mmol) was added anhydrous methyl acetate (0.7 ml, 8.75 mmol) at −78° C. over a period of 1 minute and this was allowed the reaction to proceed at −78±5° C. for 20 minutes. To a stirred mixture of tert-butyl 2-oxo-6-methylhept-5-enoate prepared above (640 mg, 3 mmol) in anhydrous tetrahydrofurane (10 ml) was added the lithium enolate at −78° C. over a period of 5 minutes and the resulting mixture was stirred at −78±5° C. for 30 minutes. After monitoring in CCM, the freezing bath was removed and the mixture was quenched with 15% ammonium chloride solution (10 ml). The separated organic layer was washed with 15% ammonium chloride solution (10 ml) and evaporated to dryness. The aqueous layers were extracted with ether (2×10 ml). The organic layers were combined with the concentrate and washed with brine (10 ml), dried over magnesium sulfate and evaporated to dryness. The resulting crude product (1.3 g) was purified by column chromatography (cyclohexane/ethyl acetate (95:5), silica (15-40 μm) 60 g) to provide an oil (222 mg, 26%). The product thus obtained showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.07 (1H, m, HC═); 3.67 (3H, s, OCH₃); 3.66 (1H, s, OH); 2.86 et 2.67 (2H, 2d, J_AB=15.8 CH₂CO₂); 2.13 (1H, m, CH₂); 1.85 (1H, m, CH₂); 1.67 (3H, s, CH₃) et (2H, m, CH₂); 1.59 (3H, s, CH₃); 1.51 (9H, s, tert-BuO).

EXAMPLE 40

Preparation of ethyl 2-N-[(4′S)-isopropyl-2′-oxazolidino]carbonylmethyl-2-hydroxy-6-methylhept-5-enoate

##STR00143##

A commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (1.28 ml, 1.28 mmol) was added to a stirred solution of (4S)-3-acetyl-4-isopropyl-2-oxazolidinone (200 mg, 1.17 mmol) in anhydrous tetrahydrofurane at −78° C. and this was allowed the reaction to proceed at −78° C. for 30 minutes. Then a solution of ethyl 2-oxo-6-methylhept-5-enoate (323 mg, 1.75 mmol) in anhydrous tetrahydrofurane (5 ml) was added and the resulting mixture was stirred at −78° C. for 1 hour. After monitoring in CCM, the mixture was quenched with 15% ammonium chloride solution (5 ml). The separated organic layer was washed with 15% ammonium chloride solution (10 ml), then with brine (10 ml), dried over magnesium sulfate and evaporated to dryness. The ¹H NMR spectra of the crude product showed a diastereomeric mixture ˜2.5/1. The resulting crude product (516 mg) was purified by column chromatography (cyclohexane/ethyl acetate (90:10 to 80:20), silica (15-40 μm) 25 g) to provide the minority diastereomer like a yellow oil (55 mg, 13.7%) showing the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.06 (1H, m, H-3′); 4.41 (1H, m, H-4″); 4.25 (4H, m, CH₂-5″ and O CH₂CH₃); 3.72 (1H, s, OH); 3.52 and 3.41 (2H, 2d, J_AB=17.9, CH₂-3); 2.36 (1H, m, H-6″); 2.16 (1H, m, CH₂″); 1.92 (1H, m, CH₂′); 1.75 (2H, m, CH₂′); 1.67 (3H, s, CH₃′); 1.59 (3H, s, CH₃′); 1.30 (3H, t, J=7.1, OCH₂CH₃); 0.89 (3H, d, J=7.0, CH₃″); 0.87 (3H, d, J=6.9, CH₃″).

Then the majority diastereomer like a pale yellow oil (93 mg, 23.2%) showing the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.06 (1H, m, H-3′); 4.40 (1H, m, H-4″); 4.23 (4H, m, CH₂-5″ and O CH₂CH₃); 3.68 (1H, s, OH); 3.46 (2H, s, CH₂-3); 2.33 (1H, m, H-6″); 2.16 (1H, m, CH₂′); 1.91 (1H, m, CH₂′); 1.75 (2H, m, CH₂′); 1.67 (3H, s, CH₃′); 1.59 (3H, s, CH₃′); 1.28 (3H, t, J=7.1 OCH2CH₃); 0.90 (3H, d, J=7.0, CH₃″); 0.87 (3H, d, J=6.9, CH₃″).

EXAMPLE 41

Preparation of ethyl 2-(1′R,2′S,5′R)-methoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate

##STR00144##

To a stirred commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (14 ml, 14 mmol), was added menthyl (1R,2S,5R)-(−)-acetate (2.8 g, 14.1 mmol) at −78° C. and this was allowed the reaction to proceed at −78° C. for 30 minutes. To a stirred mixture of ethyl 2-oxo-6-methylhept-5-enoate prepared above (867 mg, 4.7 mmol) in anhydrous tetrahydrofurane (12 ml) was added the lithium enolate at −78° C. over a period of 15 minutes and the resulting mixture was stirred at −78° C. for 3 minutes (CCM monitoring). The mixture was quenched with hydrochloric acid 1N (30 ml). The separated aqueous layer was extracted with tert-butyl methyl ether (2×15 ml) and the combined organic layers were washed with brine (3×15 ml), dried over magnesium sulfate and evaporated to dryness. The ¹H NMR spectra of the crude product showed a diastereomeric mixture ˜6/4. The resulting crude product was purified by column chromatography (cyclohexane/ethyl acetate (98:2), silica (15-40 μm) 70 g) to provide the expected products (1 g, 57%). The separated diastereomers thus obtained showed the following characteristics:

Majority Diastereomer:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.03 (1H, t, J=7.0, H-3′); 4.69 (1H, td, J=10.9 and 4.3, H-1_Men); 4.24 (2H, q, J=7.0, OCH₂CH₃); 3.77 (1H, s, 2-OH); 2.91 and 2.67 (2H, 2d, J_AB=16.4, CH₂CO₂); 2.13 (1H, m, CH′); 1.97 (1H, m, H-6eq_Men); 1.85 (2H, m, CH′ and H-7_Men); 1.75-1.6 (4H, m, CH₂′ and H-3eq, 4eq_Men); 1.67 (3H, s, CH₃′); 1.58 (3H, s, CH₃′); 1.45 (1H, m, H-5_Men); 1.35 (1H, m, H-2ax_Men); 1.30 (3H, t, J=6.9, OCH₂CH₃); 1.03 (1H, m, H-3ax_Men); 0.93 (1H, m, H-6ax_Men); 0.89 (6H, d, J=6.8, 2×CH_3(Men)); 0.87 (1H, m, H-4ax_Men); 0.73 (3H, d, J=6.9, CH_3(Men)).

Minority Diastereomer:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 5.05 (1H, t, J=7.0, H-3′); 4.67 (1H, td, J=10.7 and 4.7, H-1_Men); 4.25 (2H, m, OCH₂CH₃); 3.74 (1H, s, 2-OH); 2.92 and 2.65 (2H, 2d, J_AB=15.9, CH₂CO₂); 2.12 (1H, m, CH′); 1.97 (1H, m, H-6eq_Men); 1.86 (2H, m, CH′ and H-7_Men); 1.75-1.6 (4H, m, CH₂′ and H-3eq, 4eq_Men); 1.67 (3H, s, CH₃′); 1.58 (3H, s, CH₃′); 1.48 (1H, m, H-5_Men); 1.36 (1H, m, H-2ax_Men); 1.31 (3H, t, J=7.0, OCH₂CH₃); 1.15-0.8 (3H, m, H-3ax, 6ax, 4ax_Men); 0.89 (6H, d, J=6.9, 2×CH_3(Men)). 0.76 (3H, d, J=7.0, CH_3(Men)).

EXAMPLE 42

Preparation of ethyl 2-[(R)-1′,2′-triphenyl-ethan-2′-ol]ethoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate

##STR00145##

A commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane (6 ml, 6 mmol) was added to a stirred solution of R-(+)-1,1,2-triphenyl-1,2-ethanediol 2-acetate (665 mg, 2 mmol) in anhydrous tetrahydrofurane (6 ml) at −78° C. The temperature was allowed to rise to 0° C. over a period of 3 hour, then was added anhydrous heptane (10 ml). To this stirred reaction mixture at −78° C. a solution of ethyl 2-oxo-6-methylhept-5-enoate (368 mg, 2 mmol) in anhydrous tetrahydrofurane (2 ml) was added and the temperature was allowed to rise to −40° C. over a period of 1 hour. After monitoring in CCM, the freezing bath was removed and the mixture was quenched with 15% ammonium chloride solution (10 ml). The separated organic layer was washed with 15% ammonium chloride solution (10 ml) and evaporated to dryness. The aqueous layers were extracted with dichloromethane (2×10 ml). The organic layers were combined with the concentrate and washed with brine (10 ml), dried over magnesium sulfate and evaporated to dryness. The ¹H NMR spectra of the crude product showed a diastereomeric mixture ˜60/40. The resulting crude product (820 mg) was purified by column chromatography (cyclohexane/ethyl acetate (97:3 to 95:5), silica (15-40 μm) 80 g) to provide the expected products (361 mg, 35%). The separated diastereomers thus obtained like white crystalline compounds showed the following characteristics:

Minority Diastereomer:

¹H NMR 400 M (CDCl₃) (δ ppm, J Hz): 7.66 (2H, d, J=7.5, o-Ph); 7.43 (2H, t, J=7.7, m-Ph); 7.35-7.0 (11H, m, Ph); 6.72 (1H, s, H-1″); 4.95 (1H, m, H-3′); 4.41 (2H, m, OCH₂CH₃); 3.42 (1H, s, 2-OH); 2.90 and 2.67 (2H, 2d, J_AB=16.5, CH₂-3); 2.53 (1H, s, 2″-OH); 1.98 (1H, m, CH₂); 1.8-1.5 (3H, m, CH₂); 1.63 (3H, s, CH₃); 1.52 (3H, s, CH₃); 1.38 (3H, t, J=7.1, OCH₂CH₃).

Majority Diastereomer:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.52 (2H, d, J=7.5, o-Ph); 7.36 (2H, t, J=7.6, m-Ph); 7.27 (1H, t, J=7.3, p-Ph); 7.2-7.0 (10H, m, Ph); 6.59 (1H, s, H-1″); 4.98 (1H, m, H-3′); 3.90 and 3.34 (2H, 2m, OCH₂CH₃); 3.56 (1H, s, 2-OH); 3.22 (1H, s, 2″-OH); 2.88 and 2.69 (2H, 2d, J_AB=16.7, CH₂-3); 2.06 and 1.79 (2H, 2m, CH₂); 1.7-1.5 (2H, m, CH₂); 1.64 (3H, s, CH₃); 1.54 (3H, s, CH₃); 0.99 (3H, t, J=7.1, OCH₂CH₃).

EXAMPLE 43

Preparation of tert-butyl 2-[(R)-1′,2′,2′-triphenylethan-2′-ol]ethoxycarbonylmethyl-2-hydroxy-6-methylhept-5-enoate

##STR00146##

A commercial solution of lithium bis-(trimethylsilylamide) 1M in tetrahydrofurane tetrahydrofuran (3 ml, 3 mmol) was added to a stirred solution of R-(+)-1,1,2-triphenyl-1,2-ethanediol 2-acetate (330 mg, 1 mmol) in anhydrous tetrahydrofurane tetrahydrofuran (3.5 ml) at −78° C. The temperature was allowed to rise to −10° C. over a period of 3 hour hours then was added anhydrous heptane (5 ml). To this stirred reaction mixture at −78° C. a solution of tert-butyl 2-oxo-6-methylhept-5-enoate (276 mg, 1.5 mmol) in anhydrous tetrahydrofurane tetrahydrofuran (2 ml) was added and the temperature was allowed to rise to −40° C. over a period of 1 hour. After monitoring in CCM, the freezing cold bath was removed and the mixture was quenched with 15% ammonium chloride solution (5 ml). The separated organic layer was washed with 15% ammonium chloride solution (5 ml) and evaporated to dryness. The aqueous layers were extracted with dichloromethane (2×10 ml). The organic layers were combined with the concentrate and washed with brine (5 ml), dried over magnesium sulfate and evaporated to dryness. The ¹H NMR spectra of the crude product showed a diastereomeric mixture ˜75/25. The resulting crude product (550 mg) was purified by column chromatography (cyclohexane/ethyl acetate (96:4, 95/5 then 90:10), silica (15-40 □m μm) 60 g) to provide the expected products (217 mg, 40%). The majority diastereomer thus obtained like was a white crystalline compound that showed the following characteristics:

¹H NMR 400 MHz (CDCl₃) (δ ppm, J Hz): 7.53 (2H, d, J=7.4, o-Ph); 7.36 (2H, t, J=7.6, m-Ph); 7.28 (1H, t, J=7.3, p-Ph); 7.2-7.0 (10H, m, Ph); 6.66 (1H, s, H-1″); 5.00 (1H, m, H-3′); 3.50 (1H, s, 2-OH); 2.94 (1H, s, 2″-OH); 2.76 and 2.61 (2H, 2d, J_AB=16.3, CH₂—CO2); 2.06 and 1.78 (2H, 2m, CH₂); 1.65 (3H, s, CH₃); 1.55 (3H, s, CH₃ and 2H, m, CH₂); 1.23 (9H, s, tert-BuO).

EXAMPLE 44

Preparation of 2-carboxymethyl-6,6-dimethyl-2-tetrahydro-pyranecarboxylic acid or O-demethylanhydrohomoharringtonic acid

##STR00147##
(Method D)

A solution of ethylenic diacid resulting from the Example 3 (1.5 g, 6.94 mmol) in formic acid (2.6 ml) was stirred at 60° C. for 16 hours. After return at ambient temperature, formic acid was removal in vacuo and the resulting crude product was dried at 40° C. in vacuo for 20 hours (1.5 g, 100%).

EXAMPLE 45

Preparation of 2-methoxycarbonylmethyl-6,6-dimethyl-2-tetrahydro-pyranecarboxylic or anhydrohomoharringtonic acid from product resulting from Example 39

##STR00148##

A solution of tert-butyl 2-methoxycarbonylmethyl-2-hydroxy-6-methylhept-enoate resulting from Example 39 (50 mg, 0.175 mmol) in formic acid (0.5 ml) was stirred at room temperature for 9 days. After removal of formic acid in vacuo, the residue was treated with 5% sodium hydrogen carbonate solution up to pH 8. The aqueous layer was washed with ethyl acetate then, after acidification (pH 1) with hydrochloric acid 1N, was extracted with ethyl acetate. The organic layer was dried over magnesium sulfate and evaporated to dryness to provide anhydrohomoharringtonic acid (20 mg, 50%).

EXAMPLE 46

Preparation of Purified (−) Cephalotaxine from Total Alkaloidic Extract of Cephalotaxus sp

##STR00149##

Partially racemized cephalotaxine [H. Wenkui; L. Yulin; P. Xinfu, Scientia Sinica,; 23; 7; 835 (1980)]

¹H NMR of two batches of cephalotaxine (extracted in the same conditions as above) with the optically active NMR shift reagent europium(III) tris[3-(heptafluoropropylhydroxymethylene)-(+)-camphorate (1 éq) showed the following results:

Batch A: ¹H NMR 400 MHz (CDCl₃) (δ ppm): 6.06 (1H, OCH20 (+)-cephalotaxine) and 5.82 (1H, OCH20 (+)-cephalotaxine); 5.99 (1H, OCH20 (−)-cephalotaxine) and 5.76 (1H, OCH20 (−)-cephalotaxine).

Presence of 11±5% de (+)-cephalotaxine.

[α]²²=−134.0° (c=0.214; CHCl3): calculated rate 25±5%

Batch B: Slightly Racemized (1%)

[α]¹⁹=−173.3° (c=0.208; CHCl3)

Enantiomeric Enrichment of the Natural Cephalotaxine:

Crude chromatographied cephalotaxine (20 g) was dissolved at 55° C. in dry methanol (100 ml). Crystallization occurs by cooling with rotary evaporator and after filtration the product thus obtained showed 99.9% of HPLC purity,

[α]²⁰_D=−130° (Cl, CHD₃) corresponding to 10% of racemization. The crystallized product thus obtained (20 g) was dissolved again in hot methanol (100 ml).

Slowly cooling the solution allows translucent prisms composed of pure enantiomeric (−)-cephalotaxine [α]²⁰_D=−185° (Cl,CHCl₃).

After filtration, the mother liquors was allowed to slowly evaporate at room temperature and crystals in the form of macled needles exclusively composed of racemic cephalotaxine [α]^D₂₀=0.5° (Cl; CHCl₃) were obtained.

After filtration, the second mother liquors allowed prisms composed of (−)-cephalotaxine identical to this obtained at the first crystallization.

After filtration, the third mother liquors still allowed macled needles (urchins) composed of (±)-cephalotaxine.

The cycle is repeated three times. The combined prismatic crystals was recrystallized once to give enantiomerically pure (−)-cephalotaxine, while the combined macled needles treated in the same way gives 100% racemic cephalotaxine.

Chemical Evaluation of the Enantiomeric Purity of Natural Cephalotaxine:

A sample of partially racemized natural cephalotaxine was inserted in the process, which sequence is described in the Examples 1, 2, 3, 4, 5, 6, 15, 19 and 21, by using a pure (2R)-homoharrintonic acid resulting from Example 19.

The HPLC analysis of the diastereomeric mixture of anhydro-homoharrintonine thus obtained showed a significant enantio-epi-homoharringtonine rate (11%±3%) corresponding to the (+)-cephalotaxine content in the racemic mixture of origin (it has been demonstrated that the two antipodes of the homoharringtonic acid react in a stoichiometric way comparable to the pure enantiomeric cephalotaxine).

EXAMPLE 47

Preparation of Homoharringtonine, from Anhydro-Homoharringtonine

##STR00150##
1°) Method A

A commercial solution of hydrobromic acid in acetic acid (17.4 ml, 86.6 mmol, HBr 30% w/w) was added to a stirred solution of anhydrohomoharringtonine resulting from Example 21 (50.8 g, 9.63 mmol) in anhydrous dichloromethane (25.6 ml) at −10° C. After stirring at −10° C. for 3 hours was added water (240 ml) and the reaction mixture was become viscous. The temperature was allowed to rise to room temperature and after stirring for 2.5 hours was added sodium carbonate 0.76M (406 ml) to pH 8. The resulting aqueous layer was saturated with sodium chloride, then was extracted with dichloromethane (3×230 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness to afford a foam. After phase reverse chromatography below-mentioned were obtained 4.03 g of homoharringtonine (77%). The product thus obtained showed identical characteristics to this resulting from Example 25.

2°) Method B

To a stirred solution of anhydrohomoharringtonine resulting from Example 21 (214 mg, 0.406 mmol) in anhydrous dichloromethane (1.1 ml) was added at −10° C. a commercial solution of hydrobromic acid in acetic acid (0.728 ml, 3.6 mmol, HBr 30% w/w). After stirring at −10° C. for 3 hours, was added water (13 ml) and then the temperature was raised to 20° C. After stirring at 20° C. for 3 hours, was added a sodium carbonate solution (0.76M; 31.5 ml) up to pH 8. The resulting aqueous layer, after saturation with sodium chloride, was extracted with dichloromethane (3×20 ml) and the combined organic layers were dried over magnesium sulfate and evaporated to dryness. The resulting crude product was purified by phase reverse chromatography below-mentioned to provide homoharringtonine (166 mg, 75%). The product thus obtained showed identical characteristics to this resulting from Example 25.

EXAMPLE 48

Preparation of Harringtonine Drug Substance by Purification of Commercial Natural Harringtonine

A. Analytical Profile of Starting Product

By combination of HPLC analysis with UV detection (see FIG. 8) and mass spectrometry detection (see FIGS. 9 and 10) a total of 6.5% of related compound (identified as b,c: position isomer of harringtonine=3.4%; d: homoharringtonine=3%; e: 4′-demethyl harringtonine=0.01%; f: drupacine derivative: 0.05%) are found in the starting product.

B. Chromatography of Natural Harringtonine

Natural harringtonine (5 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 80 mm; length: 1000 mm) containing 1000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi). Unwanted fractions are discarded based upon in-line UV spectrophotometric detection. Kept fractions are collected in 16 separate containers which each are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase). During the development phase, a dual in-line UV-MS detection is used. After discarding of the fractions representing more than 0.5% of the total content of harringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure. Then crude concentrated solution of harringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 1.5%.

C. Crystallization of Raw Harringtonine

Under a laminar flow hood, the above raw harringtonine (4.1 grams) is dissolved in methanol (5 ml), at 30° C. The resulting alcoholic solution was filtered on a 0.25μ sterile Millipore filter to remove microparticles and germs and collected in a sterilized rotary flask. Then, deionized water (50 mL) is added and methanol is completely removed under vacuum at 30° C. in using a decontaminated rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of harringtonine is kept under vacuum and rotation is continued during appearance of white crystals of pure harringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (10 mL×2). The white translucent crystals are then dried using high vacuum at 40° C. for 24 hours. Overall yield is 76%. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 400 therapeutic units dosed at 10 mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. FIGS. 9 and 10 11 compare HPLC chromatogram before and after purification in using this process. Table II shows the comparison of the corresponding related compound content.

TABLE II
Impurity Content Decrease After Application Of This Process
		Before this	After this
Peak	Related Compound (impurities)	process	process
a	Harringtonine (HA)	93.49	99.97
b	HA isomer	1.76	0
c	HA isomer	1.67	0
d	Homoharringtonine	3.01	0
e	4′-dimethyl-HA	0.01	0.03
f	Drupacine analog	0.05	0
	Sum of Related Compounds	6.49	0.03
	Rate 6.49/0.03	216

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC) thermogravimetry, 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. FIG. 7 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline harringtonine obtained by this process. A series of sharp absorption bands are noted at 615, 654, 674, 689, 709, 722, 750, 761 805, 850, 928, 989, 1022, 1033, 1062, 1083, 1112, 1162, 1205, 1224, 1262, 1277, 1308, 1340, 1364, 1382, 1438 1486, 1508, 1625, 1656, 1725, 1745, 2883, 2936, 2972, 3079, 3353, 3552 and 3647 cm⁻¹

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG)

Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 12 mg of harringtonine drug substance were accurately weighed (12.4471 mg) into a DSC pan. The sample was heated from 25° C. to 200° C. at a rate of 10° C./min The DSC data were obtained following a standard method in the art. The DSC curve of crystalline harringtonine drug substance ((FIG. 6), exhibits a melting endotherm at 79.5° C. No subsequent decomposition occurred under the upper tested temperature 200° C. Simultaneous TG measurement, indicated a loss on drying of 1.3% which did not correspond to a lost of structural molecule of solvent or water.

EXAMPLE 49

Preparation of Homoharringtonine Drug Substance by Purification of Raw Semi-Synthetic (Hemi-Synthetic) Homoharringtonine

A. Analytical Profile of Starting Product

Crude reaction mixture of raw homoharringtonine contains a potential of 250 grams of homoharringtonine DS together with process impurities such as catalyst, unchanged starting product (anhydro-homo-harringtonine), and some related side product. HPLC analysis with UV detection (see left-side chromatogram on FIG. 12) indicated a total of 9% of related impurities.

B. Chromatography of Semi-Synthetic Homoharringtonine

Raw semi-synthetic homoharringtonine (550 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 450 mm; length: 1000 mm) containing 48,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 540 L/hour). Unwanted fractions are discarded based upon by-passed in-line UV spectrophotometric detector. Kept fractions are collected in 30 separate stainless steel containers (20 or 50 L each) which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5% of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5% (see right-side chromatogram on FIG. 12)

C. Crystallization of Homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above raw homoharringtonine DS (210 grams) is dissolved in methanol (240 mL), at 30° C. The resulting alcoholic solution is filtered on a 0.25μ sterile Millipore filter to remove microparticles and germs and collected in a sterilized pilot rotary flask. Then, desionized water (2400 mL) is added and methanol is completely removed under vacuum at 30° C. in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (450 mL×2). The white crystals are then dried using high vacuum at 60° C. for 48 hours. Overall yield is 88% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied. This clinical batch corresponds to 40,000 therapeutic units dosed at 5 mg.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. FIG. 13 shows HPLC chromatogram before and after crystallization. Total of related impurities of homoharringtonine DS is 0.03%.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC), thermogravimetry (TD), 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectrometry:

Identical IR spectra were obtained by either the KBr pellet and/or mineral oil mull preparation technique. FIG. 5 shows typical infrared spectrum (KBr) for unambiguous identification at the solid state of the crystalline homoharringtonine obtained by this process. A series of sharp absorption bands are noted at 612, 703, 771, 804, 826, 855, 879, 932, 1029, 1082, 1119, 1135, 1161, 1191, 1229, 1274, 1344, 1367, 1436, 1457, 1488, 1505, 1653, 1743. 2814, 2911, 2958, 3420, and 3552 cm⁻¹

Differential Scanning Calorimetry (DSC) And Thermogravimetry (TG)

Measurement of DSC and TG were obtained on a Mettler Toledo STAR System. Approximately 11 mg of homoharringtonine drug substance were accurately weighed (10.6251 mg) into a DSC pan. The sample was heated from 25° C. to 250° C. at a rate of 5° C./min. The DSC data were obtained following a standard method in the art. The DSC curve of crystalline homoharringtonine drug substance (FIG. 3), exhibits a melting endotherm at 145.6° C. Melting range performed by the capillary method (Bucchi Apparatus) gave 143-145° C. Literature indicated 144-146° C. [Anonymous, Acta Bot. Sin. 22, 156 (1980) cited by L. Huang and Z. Xue, Cephalotaxus Alkaloids, in “The Alkaloids”, vol. XXIII, pp 157, (1988). Crystallization medium was not published. This is the only literature reference regarding melting point of a crystalline form of HHT]

X-Ray Powder Diffraction

X-ray powder diffraction pattern was collected on a INEL microdiffractomer microdiffractometer, model DIFFRACTINEL. Powdered homoharringtonine DS was packed in a glass capillary tube and was analyzed according to a standard method in the art. The X-ray generator was opered operated at 45 kV and 40 mA, using the copper Kalpha line as the radiation source. The sample was rotated along the chi axis and data was collected between 0 and 120 deg 2-theta. A collection time of 1200 sec was used. As showed on shown in FIG. 2 4, the x-ray powder diffraction for this crystalline form of homoharringtonine shows a typical pattern including major reflection peaks at approximately 7.9, 9.2, 10.9, 14.9, 16.0, 17.7, 19.5, 19.7, 21.78, 23.1, 25.3, 25.4 and 25.7 deg 2-theta.

EXAMPLE 50

Preparation of Homoharringtonine Drug Substance by Purification of a Commercial Sample of Impure Homoharringtonine from Chinese Source

A. Analytical Profile of Starting Product

Analytical HPLC chromatogram of natural homoharringtonine (China National Pharmaceutical) is displayed on FIG. 14 (bottom left).

B. Chromatography of Natural Homoharringtonine

Natural homoharringtonine (25 grams) is injected on a preparative high-pressure liquid chromatography (HPLC) system (Prochrom stainless steel; permanent axial compression; diameter: 200 mm; length: 1000 mm) containing 12,000 grams of reverse phase octadecylsilane specially dedicated for basic compounds as stationary phase. Then elution is performed in using a gradient of pH 3 buffered methanol-water solution as mobile phase (pressure 1200 psi, flow-rate 120 L/hour). Unwanted fractions are discarded based upon by-passed in-line UV spectrophotometric detector. Kept fractions are collected in 22 separate stainless steel containers which are individually checked in using an analytical HPLC system exhibiting a different selectivity pattern (octadecylsilane as stationary phase and buffered acetonitrile-water system as mobile phase) and equipped with a diode array detector. After discarding of the fractions representing more than 0.5% of the total content of homoharringtonine, fractions which complied with pre-established specification were gathered, neutralized then evaporated under reduce pressure in using a mechanically stirred thin film evaporator. Then crude concentrated solution of homoharringtonine are alkalinized at pH 8.5 with aqueous ammonia and partitioned with dichloromethane. Resulting organic solution is concentrated under high vacuum. In-process HPLC analysis indicated a total of related compound lower than 0.5%.

C. Crystallization of Homoharringtonine DS

In a controlled clean room, under a laminar flow hood, the above chromatographied homoharringtonine DS (18 grams) is dissolved in methanol (35 mL), at 30° C. The resulting alcoholic solution is filtered on a 0.25 g sterile Millipore filter to remove microparticles and germs and collected in a sterilized pilot rotary flask. Then, desionized water (300 mL) is added and methanol is completely removed under vacuum at 30° C. in using a decontaminated pilot rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of homoharringtonine DS is kept under vacuum and rotation is continued during appearance of white crystals of pure homoharringtonine. The stirring is continued until no more crystal occurs. Under a laminar flow hood, the suspension of is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized water (50 mL×2). The white crystals are then dried using high vacuum at 60° C. for 48 hours. Overall yield is 84% from potential content of homoharringtonine in raw semi-synthetic homoharringtonine. All operations were documented prior to start the process and full current Good Manufacturing Practices were applied.

D. Analysis

Routine analytical procedure includes solvent residues, loss on drying, water determination, melting point, IR and NMR spectrum, related compound and assay by HPLC. FIG. 14 (bottom right) shows HPLC chromatogram after crystallization. Total of related impurities of homoharringtonine DS is 0.05%.

For the aim of further characterization, more advanced studies were performed including differential scanning calorimetry (DSC), thermogravimetry (TD), 2D NMR, solid NMR and X-ray powder diffractometry.

Infrared Spectra, Differential Scanning Calorimetry (DSC) and X-Ray Powder Diffraction gave patterns strictly superimposable to the one of example 2 49 obtained from semi-synthetic homoharringtonine (FIGS. 5, 3, and 4, respectively).

EXAMPLE 51

Crystallization of the (+)-Homoharringtonine, Non Natural Enantiomer of the Homoharringtonine

The mother liquors coming from the fractioned crystallization of example 3 are A solution containing (+)-homoharringtonine is concentrated then dissolved in methanol at 30° C. The resulting alcoholic solution is filtered on a 0.25μ sterile Millipore filter. Then, desionized deionized water is added and methanol is completely removed under vacuum at 30° C. in using a rotary evaporator. After removing methanol, heating is stopped and the aqueous solution of (+)-homoharringtonine is kept under vacuum and rotation is continued during appearance of white crystals of pure (+)-homoharringtonine. The stirring is continued until no more crystal crystalization occurs. The suspension of crystals is poured on a sintered glass filter with house vacuum. The resulting crystalline solid cake is washed two times with cold desionized deionized water (50 mL×2). The white crystals are then dried using high vacuum at 60° C. for 48 hours.

Partially enantiomerically enriched crystals thus obtained are dissolved in methanol at 30° C. and the above-mentioned operations (filtration, addition of desionized deionized water, removal of methanol, obtention obtaining of crystals) are repeated (mean: 3 times) until the obtention of a constant rotary power is obtained ([a] D20=−110 [α]_D²⁰=+110 (c=0.25; CHCl3 CHCl₃)). The (+)-homoharringtonine thus obtained present has all the analytical characteristics of the natural homoharringtonine (levogyrous), except its rotatory power which is equal but of opposite sign).

DESCRIPTION AND DEFINITION OF HARRINGTONINES
	EXAMPLES OF HARRINGTONIC ACIDS (R = H), 3
CEPHALOTAXANES	EXAMPLES OF HARRINGTONINES (R = CTX), 4
##STR00151##	##STR00152##
CEPHALOTAXINES	R′	R7	n	R
	EXAMPLES OF HARRINGTONIC ACIDS
	(not isolated in the natural form as such)
##STR00153##	H H H H   H	(CH3)2C(OH)— (CH3)2C(OH)— (CH3)2C(OH)— (CH3)2CH— —C (CH3)2—O— Ph—	2 3 1 2 3 1	H H H H H H	harringtonic acid homoharringtonic acid norharringtonic acid desoxyharringtonic acid anhydrohomoharringtonic acid neoharringtonic acid
Examples of cephalotaxines:	EXAMPLES OF HARRINGTONINES (natural)
cephalotaxine = CTXOH	H	(CH3)2C(OH)—	2	CTX	Harringtonine: HT
2a	H	(CH3)2C(OH)—	3	CTX	Homoharringtonine: HHT
R1 = OH, R2 = R4 = H, R3 = OMe;	H	(CH3)2C(OH)—	1	CTX	Norharringtonine
acetylcephalotaxine, 2b	H	(CH3)2CH—	2	CTX	Desoxyharringtonine
R1 = Ac, R2 = R4 = H, R3 = OMe;	H	(CH3)2CH—	1	CTX	Nordesoxyharringtonine
cephalotaxinone, 2c	H	(CH3)2CH—	3	CTX	Homodesoxyharringtonine
R1, R2 = O, R3 = OMe, R4 = H;	H	(CH3)2CH—	4	CTX	Bishomodesoxyharringtonine
demethylcephalotaxine, 2d		—C(CH3)2—O—	2	CTX	Anhydroharringtonine
R1 = OH, R2 = R4 = H, R3 = OH;	H	Ph—	1	CTX	Neoharringtonine
demethylcephalotaxinone, 2e	H	Ph—	2	CTX	Homoneoharringtonine
R1, R2 = O, R3 = OH, R4 = H;
11-hydroxycephalotaxine,
2f = 2b + R4 = OH;
ESSENCE OF PRIOR ART
##STR00154##
i) Impossible on account of the steric hindrance [K. L. Mikolajczak et coll., Tetrahedron, p. 1995, (1972];
ii) HCN, H+;
iii) MeOH, H+;
i′) Silver salt of 3c [K. L. Mikolajczak et coll. J. Med. Chem., p. 63, (1975)];
iv) 2a/pyridine-dichloromethane;
v) according to [T. R. Kelly et coll., Tetrahedron Lett., 3501 (1973)];
ORIGIN of the ANHYDROHARRINGTONINES
##STR00155##
[D. Z. Wang et coll., Yaoxue Xuebao, p. 173, (1992)]
[D. Z. Wang et coll., Yaoxue Xuebao, p. 178, (1992)]

Claims

1. A purified compound of the following formula cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

2. The purified compound of claim 1, wherein n=3.

3. The purified compound of claim 1, wherein n=2.

4. A purified crystalline compound of the formula:

5. A purified crystalline compound of the formula Crystalline cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

6. A purified crystalline compound of the formula Crystalline cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

7. A purified crystalline compound of the formula Crystalline cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

8. A purified crystalline compound of the formula:

9. A purified crystalline compound of the formula:

10. The purified compound of claim 1, wherein said purified compound is present in tautomeric forms and salts thereof.

11. The purified compound of claim 1, wherein said purified compound is an enantiomer and the total content of impurities includes the other enantiomeric forms.

12. A pharmaceutical composition comprising an effective amount of one or more purified compounds according to claim 1, together with cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

13. The pharmaceutical composition of claim 12, wherein said pharmaceutically acceptable inactive component is selected from the group consisting of carriers, excipients, adjuvants and diluents.

14. A pharmaceutical composition comprising an effective amount of one or more purified compounds according to claim 2, together with one or more pharmaceutically acceptable inactive components.

15. A pharmaceutical composition comprising an effective amount of one or more purified compounds according to claim 3, together with one or more pharmaceutically acceptable inactive components.

16. A pharmaceutical composition comprising an effective amount of one or more purified crystalline compounds according to claim 5, together with one or more pharmaceutically acceptable inactive components.

17. A pharmaceutical composition comprising an effective amount of one or more purified crystalline compounds according to claim 6, together with one or more pharmaceutically acceptable inactive components.

18. A pharmaceutical composition comprising an effective amount of one or more purified crystalline compounds according to claim 9, together with one or more pharmaceutically acceptable inactive components.

19. A method for treatment of a mammalian parasitic disease comprising administering to a patient in need of such treatment an effective amount of a purified compound as defined in claim 1 for treatment of said parasitic disease.

20. A method for immunosuppressive therapy comprising administering to a patient in need of such therapy an effective amount of a purified compound as defined in claim 1 for said immunosuppressive therapy.

21. A method for the treatment of leukemia comprising administering to a patient in recognized need of such therapy an effective amount of a purified compound as defined in claim 1 for treatment of said leukemia cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

22. The A method according to claim 21, wherein said leukemia is selected from the group consisting of for the treatment of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and, and/or myeloproliferative disorders comprising administering to a patient in recognized need of such treatment cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

23. The A method according to claim 22, wherein said myeloproliferative disorder is for the treatment of chronic myelogenous leukemia comprising administering to a patient in recognized need of such treatment cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

24. The method according to claim 21, wherein said purified compound is administered as an adjuvent therapy of resistance to other chemotherapeutic agents.

25. The method according to claim 21 23, wherein said purified compound cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester), and/or pharmaceutically acceptable salt thereof, is administered by a parenteral mode of administration.

26. The method according to claim 21, wherein said purified compound is administered by an oral mode of administration.

27. The method according to claim 21, wherein said purified compound is administered by an anal administration.

28. The method according to claim 21, wherein said purified compound is administered by a topical mode of administration.

29. The method according to claim 26, wherein said parenteral mode of administration is subcutaneous.

30. The cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

31. A pharmaceutical composition comprising semi-synthetic and/or synthetic cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

32. The cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester) according to claim 5 wherein said cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester) is semi-synthetic.

33. A salt of cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

34. A pharmaceutical composition comprising semi-synthetic and/or synthetic cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):

35. A pharmaceutical composition comprising cephalotaxine, 4-methyl (2′R)-2-hydroxy-2-(4-hydroxy-4-methylpentyl) butanedioate (ester):