1 SYNTHETIC PROCESS FOR THE MANUFACTURE OF ECTEINASCIDIN COMPOUNDS FIELD OF THE INVENTION [0001] The present invention relates to the field of organic chemical synthesis, in particular to synthetic processes for the manufacture of ecteinascidin compounds. 5 BACKGROUND OF THE INVENTION [0002] Ecteinascidins are a group of naturally occurring marine compounds and analogs thereof, which are well identified and structurally characterized, and are disclosed to have antibacterial and cytotoxic properties. See for example, EP 309477; WO 03/66638; WO 03/08423; WO 01/77115; WO 03/014127; R. Sakai et al., 1992, Proc. Natl. Acad. Sci. USA 89, pages 11456-11460; R. Menchaca et al., 2003, J. Org. 10 Chem.68(23), pages 8859-8866; and I. Manzanares et al., 2001, Curr. Med. Chem. Anti-Cancer Agents, 1, pages 257-276; and references therein. Examples of ecteinascidins are provided by ET-743, also named trabectedin, ET-729, ET-745, ET-759A, ET-759B, ET-759C, ET-770, ET-815, ET-731, ET-745B, ET-722, ET-736, ET-738, ET-808, ET-752, ET-594, ET-552, ET-637, ET-652, ET-583, ET-597, ET-596, ET-639, ET-641, lurbinectedin, ecubectedin, and derivatives thereof, such as acetylated forms, formylated forms, 15 methylated forms, and oxide forms. [0003] International patent application publication WO 01/87895 described several syntheses of trabectedin, including a route that transforms compound 1 into compound 4 as shown is Scheme I:
Scheme I 20 [0004] International patent application publication WO 2020/155613 described that the first step of Scheme I had poor selectivity. They stated that during the esterification of the primary alcohol group of compound 1 with a cysteine derivative, the phenolic group in the structure also easily undergoes an esterification reaction to give compound 5.
2
[0005] In addition, they isolated compounds 2 and 3 as oils, and reported that these compounds are difficult to purify and store. [0006] WO 2020/155613 inverts the order of the reactions according to Scheme II: 5
Scheme II [0007] Impurity 5 is difficult to remove, and it is carried over during the synthetic route even after compound 4 is synthetized. This results in the formation of impurity 6 during the manufacture of compound 4.
3
. [0008] There remains a need to develop more efficient processes, in particular hemisynthetic routes, to manufacture ecteinascidin compounds and related compounds. The present invention addresses this need. SUMMARY OF THE INVENTION 5 [0009] In a first aspect of the present invention there is provided a process for the synthesis of an ecteinascidin compound or a pharmaceutically acceptable salt thereof, the process comprising a step of coupling compound 1 with a compound of Formula A, or a stereoisomer thereof, to give a compound of Formula I:
10 wherein R6 is ProtNH, wherein ProtNH is a protecting group for amino; R7 is ProtSH, wherein ProtSH is a protecting group for SH; and the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a salt thereof, such as its hydrochloride (EDC·HCl), in presence of 4-dimethylaminopyridine (DMAP) and water, wherein the total 15 amount of water (w/w) in the reaction mixture versus the amount of compound 1 initially present is from about 2.5% to about 6%. [0010] In a second aspect, there is provided a synthetic process for the synthesis of an ecteinascidin compound or a pharmaceutically acceptable salt thereof, the process comprising a step of protecting the
4 phenolic group in a compound of formula I by reacting it with a compound of formula R1X in presence of a base MOH and water in tetrahydrofuran to give a compound of formula II:
wherein 5 R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; R7 is ProtSH; X is selected from Cl, Br, and I; and MOH is selected from sodium hydroxide, potassium hydroxide and lithium hydroxide. 10 [0011] In a third aspect, there is provided a synthetic process for the synthesis of an ecteinascidin compound or a pharmaceutically acceptable salt thereof, the process comprising a step of de-allylating a compound of Formula II to provide a compound of Formula III:
with a palladium catalyst in presence of a secondary amine as reducing agent; 15 wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; and R7 is ProtSH. BRIEF DESCRIPTION OF THE FIGURES
5 Figure 1. Effect of water addition on the kinetics of the synthesis of two batches of compound 3. Figure 2. Effect of water content in the suspension of NaOH and MEMCl in the synthesis of compound 3. DETAILED DESCRIPTION OF THE INVENTION [0012] The following apply to all aspects of the present invention. 5 General chemical definitions [0013] In the compounds of the present invention, the alkyl groups may be branched or unbranched, and preferably have from 1 to about 12 carbon atoms. One more preferred class of alkyl groups has from 1 to about 6 carbon atoms. Even more preferred are alkyl groups having 1, 2, 3 or 4 carbon atoms. Methyl, ethyl, n-propyl, isopropyl, and butyl, including n-butyl, isobutyl, sec-butyl and tert-butyl are particularly preferred 10 alkyl groups in the compounds of the present invention. [0014] In the compounds of the present invention, the alkenyl groups may be branched or unbranched, have one or more double bonds and from 2 to about 12 carbon atoms. One more preferred class of alkenyl groups has from 2 to about 6 carbon atoms. Even more preferred are alkenyl groups having 2, 3 or 4 carbon atoms. Ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, and 3-butenyl are 15 particularly preferred alkenyl groups in the compounds of the present invention. [0015] In the compounds of the present invention, the alkynyl groups may be branched or unbranched, have one or more triple bonds and from 2 to about 12 carbon atoms. One more preferred class of alkynyl groups has from 2 to about 6 carbon atoms. Even more preferred are alkynyl groups having 2, 3 or 4 carbon atoms. 20 [0016] In the compounds of the present invention, the alkoxy groups may be branched or unbranched, have one or more oxygen linkages and from 1 to about 12 carbon atoms, more preferably 1 to about 6 carbon atoms, and most preferably 1, 2, 3, or 4 carbon atoms. [0017] Suitable aryl groups in the compounds of the present invention include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups. Typical aryl 25 groups contain from 1 to 3 separated and/or fused rings and from 6 to about 18 carbon ring atoms. Preferably aryl groups contain from 6 to about 10 carbon ring atoms. Specially preferred aryl groups included substituted or unsubstituted phenyl, substituted or unsubstituted naphthyl, substituted or unsubstituted biphenyl, substituted or unsubstituted phenanthryl and substituted or unsubstituted anthryl. [0018] Suitable heterocyclic groups include heteroaromatic and heteroalicyclic groups containing from 1 30 to 3 separated and/or fused rings and from 5 to about 18 ring atoms. Preferably heteroaromatic and heteroalicyclic groups contain from 5 to about 10 ring atoms, most preferably 5, 6, or 7 ring atoms. Suitable heteroaromatic groups in the compounds of the present invention contain one, two or three heteroatoms selected from N, O or S atoms and include, e.g., coumarinyl including 8-coumarinyl, quinolyl including 8- quinolyl, isoquinolyl, pyridyl, pyrazinyl, pyrazolyl, pyrimidinyl, furyl, pyrrolyl, thienyl, thiazolyl,
6 isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridyl. Suitable heteroalicyclic groups in the 5 compounds of the present invention contain one, two or three heteroatoms selected from N, O or S and include, e.g., pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridyl, 2-pirrolinyl, 3- pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl,10 dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3- azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl, and quinolizinyl. [0019] The groups above mentioned may be substituted at one or more available positions by one or more suitable groups such as OR’, =O, SR’, SOR’, SO2R’, NO2, NHR’, NR’R’, =N-R’, NHCOR’, N(COR’)2, NHSO2R’, NR’C(=NR’)NR’R’, CN, halogen, COR’, COOR’, OCOR’, OCONHR’, OCONR’R’, 15 CONHR’, CONR’R’, protected OH, protected amino, protected SH, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group, where each of the R’ groups is independently selected from the group consisting of hydrogen, OH, NO2, NH2, SH, CN, halogen, COH, COalkyl, CO2H, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, 20 substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group. Where such groups are themselves substituted, the substituents may be chosen from the foregoing list. In addition, where there are more than one R’ groups on a substituent, each R’ may be the same or different. [0020] In the compounds for the present invention, the halogen substituents include F, Cl, Br, and I. 25 [0021] The term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts can be carried out by methods known in the art. 30 [0022] For instance, pharmaceutically acceptable salts of the compounds provided herein are synthesized from the parent compounds, which contain a basic or acidic moiety, by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of both. Generally, nonaqueous media like ether, ethyl acetate, ethanol, 2-propanol or acetonitrile are 35 preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of the alkali addition salts include
7 inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, triethanolamine and basic aminoacids salts. [0023] The compounds of the invention may be in crystalline or amorphous form either as free compounds 5 or as solvates (e.g. hydrates) and it is intended that all forms are within the scope of the present invention. Methods of solvation are generally known within the art. [0024] Stereoisomerism about the asymmetric carbons with unspecified stereochemistry is possible, therefore in such cases the asymmetric carbons can have (R) or (S) configuration. All diastereomers generated by a specific configuration of such asymmetric carbons in conjunction with the other asymmetric 10 carbons present in the molecule, and mixtures thereof, are considered within the scope of the present invention. Stereoisomerism about the double bond (geometric isomerism) is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer. If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same or different than the stereoisomerism of the other double bonds of the molecule. Furthermore, compounds referred to herein 15 may exist as atropoisomers. The single stereoisomers including diastereoisomers, geometric isomers and atropoisomers of the compounds referred to herein, and mixtures thereof fall within the scope of the present invention. [0025] In addition, compounds referred to herein may exist in isotopically labelled forms. All pharmaceutically acceptable salts, esters and isotopically labelled forms of the compounds referred to 20 herein, and mixtures thereof, are considered within the scope of the present invention. [0026] Protected forms of the compounds disclosed herein are considered within the scope of the present invention. Suitable protecting groups are well known for the skilled person in the art. A general review of protecting groups in organic chemistry is provided by Wuts, P.G.M. in Greene’s Protective Groups in Organic Synthesis, 5th Ed. Wiley, and by Kocienski P.J. in Protecting Groups, 3rd Ed. Georg Thieme Verlag. 25 These references provide sections on protecting groups for OH, SH and amino groups. All these references are incorporated by reference in their entirety. [0027] Within the scope of the present invention, a protecting group for OH is defined to be the O-bonded moiety resulting from the protection of the OH group through the formation of a suitable protected OH group. Examples of such protected OH groups include ethers, silyl ethers, esters, sulfonates, sulfenates and 30 sulfinates, carbonates, and carbamates. In the case of ethers, the protecting group for the OH can be selected from methyl, methoxymethyl, 1H,1H,2H,2H,3H,3H-perfluorooctyloxymethyl, 1H,1H,2H,2H,3H,3H- perfluoroundecyloxymethyl, methylthiomethyl, (phenyldimethylsilyl)methoxymethyl, benzyloxymethyl, p-methoxybenzyloxymethyl, [(3,4-dimethoxybenzyl)oxy]methyl, p-nitrobenzyloxymethyl, o- nitrobenzyloxymethyl, [(R)-1-(2-nitrophenyl)ethoxy]methyl, (4-methoxyphenoxy)methyl, guaiacolmethyl, 35 [(p-phenylphenyl)oxy]methyl, t-butoxymethyl, 4-pentenyloxymethyl, siloxymethyl, acyloxymethyl, phthalimidomethyl, 2-methoxyethoxymethyl, 2-cyanoethoxymethyl, methylsulfonylethoxymethyl, 2-(4- tolylsulfonyl)ethoxymethyl, bis(2-chloroethoxy)methyl, 2,2,2-trichloroethoxymethyl, 2-(trimethylsilyl)-
8 ethoxymethyl, menthoxymethyl, 2-cyano-2,2-dimethylethanimine-N-oxymethyl, 2’-O-{[2,2-dimethyl-2- (2-nitrophenyl)acetyl]oxy}methyl, O-bis(2-acetoxyethoxy)methyl, tetrahydropyranyl, fluorous tetrahydropyranyl, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S- 5 dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl, 1-(2-fluorophenyl)-4-methoxypiperidin- 4-yl, 1-(4-chlorophenyl)-4-methoxypiperidin-4-yl, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1- ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-hydroxyethyl, 2-bromoethyl, 1-[2-(trimethylsilyl)ethoxy]ethyl, 1- methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl, 1-methyl-1-10 phenoxyethyl, 2,2,2-trichloroethyl, 1,1-dianisyl-2,2,2-trichloroethyl, 1,1,1,3,3,3-hexafluoro-2- phenylisopropyl, 1-(2-cyanoethoxy)ethyl, 2-trimethylsilylethyl, 2-(benzylthio)ethyl, 2- phenylselenyl)ethyl, 4-hydroxyphenacyl, 4-methoxyphenacyl, t-butyl, cyclohexyl, 1-methyl-1’- cyclopropylmethyl, allyl, prenyl, cinnamyl, 2-phenallyl, propargyl, 1-naphthylpropargyl, 4- trifluoromethylphenylpropargyl, p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, 2,4-dinitrophenyl, 15 2,3,5,6-tetrafluoro-4-(trifluoromethyl)phenyl, 2,6-diphenylphenyl, benzyl, p-methoxybenzyl, perfluoroalkoxybenzyl, 3,4-dimethoxybenzyl, 2,6-dimethoxybenzyl, 4-(3,4-dimethoxyphenyl)benzyl, o- nitrobenzyl, p-nitrobenzyl, pentadienylnitrobenzyl, pentadienyl-nitropiperonyl, halobenzyl, 2,6- dichlorobenzyl, 2,4-dichlorobenzyl, 2,6-difluorobenzyl, p-cyanobenzyl, fluorous benzyl, 4- fluorousalkoxybenzyl, trimethylsilylxylyl, p-phenylbenzyl, 2-phenyl-2-propyl, p-acylaminobenzyl, p-20 azidobenzyl, 4-azido-3-chlorobenzyl, 2-trifluoromethylbenzyl, 4-trifluoromethylbenzyl, 2,6- bis(trifluoromethyl)benzyl, p-(methylsulfinyl)benzyl, p-siletanylbenzyl, 4-acetoxybenzyl, 4-(2- trimethylsilyl)ethoxymethoxybenzyl, 2-naphthylmethyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido, 2-quinolinylmethyl, 6-methoxy-2-(4-methylphenyl-4-quinolinemethyl, 1-pyrenylmethyl, diphenylmethyl, 4-methoxydiphenylmethyl, bis(4-methoxyphenyl)methyl, 4-phenyldiphenylmethyl, 9-fluorenyl, (2,6- 25 dichloro-4-alkoxyphenyl)-(2,4-dichlorophenyl)methyl, p,p’-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, tris(4-t-butylphenyl)methyl, α-naphthyldiphenylmethyl, p- methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4,4’- dimethoxy-4’’-methanesulfinyltrityl, 4-(4’-bromophenacyloxy)phenyldiphenylmethyl, 4,4’,4’’-tris(4,5- dichlorophthalimidophenyl)methyl, 4,4’,4’’-tris(levulinoyloxyphenyl)methyl, 4,4’,4’’-tris(benzoyloxy-30 phenyl)methyl, 4,4’-dimethoxy-3’’-[N-(imidazolylmethyl)]trityl, 4,4’-dimethoxy-3’’-[N- (imidazolylethyl)carbamoyl]trityl, diphenyl-(2-pyridyl)methyl, bis(4-methoxyphenyl)-1’-pyrenylmethyl, 4 (17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4,4’’-dimethoxytrityl, 3-dimethylaminophenyldiphenylmethyl, 9- anthryl, 9-(9-phenyl)xanthenyl, 9-phenylthioxanthyl, 9-(9-phenyl-10-oxo)anthryl, 1,3-benzodithiolan-2-yl, 4,5-bis(ethoxycarbonyl)-[1,3]-dioxolan-2-yl, and benzisothiazolyl-S,S-dioxido. In the case of silyl ethers, 35 the protecting group for the OH can be selected from trimethylsilyl, triethylsilyl, triisopropylsilyl, dimethylisopropylsilyl, diethylisopropylsilyl, dimethylthexylsilyl, 2-norbornyldimethylsilyl, t- butyldimethylsilyl, di-t-butylisobutylsilyl, t-butyldiphenylsilyl, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl, di-t-butylmethylsilyl, bis(t-butyl)-1-pyrenylmethoxysilyl, allyl-t- butylmethylsilyl, tris(trimethylsilyl)silyl, (2-hydroxystyryl)dimethylsilyl, (2-40 hydroxystyryl)diisopropylsilyl, t-butylmethoxyphenylsilyl, t-butoxydiphenylsilyl, 1,1,3,3-tetraisopropyl-
9 3-[2-(triphenylmethoxy)ethoxy]disiloxane-1-yl, bis(trimethylsiloxy)cyclododecyloxysilyl, (-)-(R)-(1- methoxy-2,2,2-triphenylethyl)dimethylsilyl, (+)-(S)-(1-methoxy-2,2,2-triphenylethyl)dimethylsilyl, and fluorous silyl. In the case of esters, the protecting group for the OH together with the oxygen atom of the unprotected OH group to which it is attached form an ester that can be selected from formate, 5 benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trichloroacetamidate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate, phenylacetate, p-P-phenylacetate, diphenylacetate, azulen-1-yl-oxoacetate, 2-chloroisobutyrate, 3- phenylpropionate, bisfluorous chain type propanoate, 4-pentenoate, 4-oxopentanoate, 4,4- (ethylenedithio)pentanoate, 5-[3-bis(4-methoxyphenyl)hydroxymethylphenoxy]levulinate, pivaloate, 1- 10 adamantoate, crotonate, 4-methoxycrotonate, (E)-3-(4-diethoxycarbonylmethylamino-2-hydroxyphenyl) acrylate, benzoate, 2-(trimethylsilyl)benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate, 4- bromobenzoate, 2,5-difluorobenzoate, pentafluorobenzoate, p-nitrobenzoate, 2-(2- methoxyphenyl)alkynylbenzoate, picolinate, nicotinate, 2-(azidomethyl)benzoate, 4-azidobutyrate, (2- azidomethyl)phenylacetate, 2-{[(tritylthio)oxy]methyl}benzoate, 2-{[(4-methoxytritylthio)oxy]methyl}-15 benzoate, 2-{[methyl(tritylthio)amino]methyl}benzoate, 2-{{[(4-methoxytrityl)thio]methylamino}- methyl}benzoate, 2-(allyloxy)phenylacetate, 2-(prenyloxymethyl)benzoate, 6-(levulinyloxymethyl)-3- methoxy-2-nitrobenzoate, 6-(levulinyloxymethyl)-3-methoxy-4-nitrobenzoate, benzyloxybutyrate, 4- trialkylsilyloxybutyrate, 4-acetoxy-2,2-dimethylbutyrate, 2,2-dimethyl-4-(4-methoxyphenoxy)butyrate, 2,2-dimethyl-4-azidobutyrate, 2,2-dimethyl-4-pentenoate, 2-iodobenzoate, 4-nitro-4-methylpentanoate, o-20 (dibromomethyl)benzoate, 2-formylbenzenesulfonate, 4-(methylthiomethoxy)butyrate, 2-(methylthio- methoxymethyl)benzoate, 2-(chloroacetoxymethyl)benzoate, 2-[(2-chloroacetoxy)ethyl]benzoate, 2-[2- (benzyloxy)ethyl]benzoate, 2-[2-(4-methoxybenzyloxy)ethyl]benzoate, 3-(2’-benzoyloxy-4’,6’- dimethylphenyl-3,3-dimethylpropanoate, (2-nitrophenyl)acetate, 4-nitrophthalimidobutyrate, 2,6-dichloro- 4-methylphenoxyacetate, 2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate, 2,4-bis(1,1-25 dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuccinate, (E)-2-methyl-2- butenoate, o-(methoxycarbonyl)benzoate, p-P-benzoate, α-naphthoate, nitrate, alkyl N,N,N’,N’- tetramethylphosphorodiamidate, and 2-chlorobenzoate. In the case of sulfonates, sulfenates and sulfinates the protecting group for the OH together with the oxygen atom of the unprotected OH group to which it is attached form a group that can be selected from sulfate, allylsulfonate, methanesulfonate, benzylsulfonate,30 tosylate, 2-[(4-nitrophenyl)ethyl]sulfonate, 2-trifluoromethylbenzenesulfonate, 4-monomethoxy- tritylsulfenate, 2,4-dinitrophenylsulfenate, and 2,2,5,5-tetramethylpyrrolidin-3-one-1-sulfinate. In the case of carbonates, the protecting group for the OH together with the oxygen atom of the unprotected OH group to which it is attached from a carbonate group that can be selected from methyl carbonate, methoxymethyl carbonate, azidomethyl carbonate, 9-fluorenylmethyl carbonate, acridin-9-ylmethyl carbonate, ethyl 35 carbonate, bromoethyl carbonate, 2-(methylthiomethoxy)ethyl carbonate, 2-(methylsulfonyl)ethyl carbonate, [2-(4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecyl)sulfonyl]ethyl carbonate, 2,2,2-trichloroethyl carbonate, 1,1-dimethyl-2,2,2-trichloroethyl carbonate, 2-(trimethylsilyl)ethyl carbonate, 2-[dimethyl(2-naphthylmethyl)silyl]ethyl carbonate, 2-(phenylsulfonyl)ethyl carbonate, 2- (triphenylphosphonio)ethyl carbonate, cis-[4-[[(4-methoxytrityl)sulfenyl]oxy]tetrahydrofuran-3-yl] 40 carbonate, isobutyl carbonate, t-butyl carbonate, vinyl carbonate, allyl carbonate, cinnamyl carbonate,
10 propargyl carbonate, p-chlorophenyl carbonate, p-nitrophenyl carbonate, 4-ethoxy-1-naphthyl carbonate, 6-bromo-7-hydroxycoumarin-4-ylmethyl carbonate, (4-oxo-3-phenyl-1,1-dioxo-4H-thiochromen-2- yl)methyl carbonate, 7-[bis-[2-[[2-(dimethylamino)ethyl]-2-oxoethyl]amino]coumarin-4-ylmethyl carbonate, benzyl carbonate, o-nitrobenzyl carbonate, p-nitrobenzyl carbonate, p-methoxybenzyl 5 carbonate, 3,4-dimethoxybenzyl carbonate, anthraquinon-2-ylmethyl carbonate, 2-dansylethyl carbonate, 2-(4-nitrophenyl)ethyl carbonate, 2-(2,4-dinitrophenyl)ethyl carbonate, 2-(2-nitrophenyl)propyl carbonate, 2-(3,4-methylenedioxy-6-nitrophenyl)propyl carbonate, 2-cyano-1-phenylethyl carbonate, 2-(2- pyridyl)amino-1-phenylethyl carbonate, 2-[N-methyl-N-(2-pyridyl)]amino-1-phenylethyl carbonate, phenacyl carbonate, 3’,5’-dimethoxybenzoin carbonate, methyl dithiocarbonate, S-benzyl thiocarbonate 10 and S-phenyl thiocarbonate. And in the case of carbamates the protecting group for the OH together with the oxygen atom of the unprotected OH group to which it is attached form a carbamate that can be selected from dimethylthio carbamate, N,N-bis(perfluoroalkyl)thiocarbamate, 1,1-dioxothio- morpholinethionocarbamate, N-phenyl carbamate, N-methyl-N-(o-nitrophenyl) carbamate, 3-pyrroline carbamate, p-toluenesulfonyl carbamate. 15 [0028] Within the scope of the present invention a protecting group for amino is defined to be the N- bonded moiety resulting from the protection of the amino group through the formation of a suitable protected amino group. Examples of such protected amino groups include carbamates, ureas, amides, N- alkyl amines, N-alkenyl amines, N-alkynyl amines, N-aryl amines, enamines, quaternary ammonium salts, N-metal derivatives, N-N derivatives, N-P derivatives, N-Si derivatives, N-S derivatives, heterocyclic 20 systems and imines. In the case of carbamates, the protecting group for the amino group together with the nitrogen atom of the unprotected amino group to which it is attached form a carbamate that can be selected from carbamate derived from the unprotected amine and CO2, methyl carbamate, ethyl carbamate, 9- fluorenylmethyl carbamate, 2,6-di-t-butyl-9-fluorenylmethyl carbamate, 2,7-bis(trimethylsilyl)fluorenyl- methyl carbamate, 2-(2-ethylhexyl)-9-fluorenylmethyl carbamate, 2.7-bis-(2-ethylhexyl)-9- 25 fluorenylmethyl carbamate, 9-(2-sulfo)-fluorenylmethyl carbamate, 9-(2,7-dibromo)fluorenylmethyl carbamate, 17-tetrabenzo[a,c,g,i]fluorenylmethyl carbamate, 2-chloro-3-indenylmethyl carbamate, benz[f]inden-3-ylmethyl carbamate, 1,1-dioxobenzo[b]-thiophene-2-ylmethyl carbamate, azidomethyl carbamate, 2-(hydroxymethyl)-3-phenyl-4H-1-benzothiopyran-4-one 1,1-dioxide carbamate, 2- methylsulfonyl-3-phenyl-1-prop-2-enyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10- 30 tetrahydrothioxanthyl)]methyl carbamate, 2,2,2-trichloroethyl carbamate, 2-trimethylsilylethyl carbamate, (2-phenyl-2-trimethylsilyl)ethyl carbamate, 2-(triphenylsilyl)ethyl carbamate, 2-phenylethyl carbamate, 2- chloroethyl carbamate, 2-bromo-3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluoro-1-decyl carbamate, 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate, 1,1-dimethyl-2,2,2- trichloroethyl carbamate, 2-(2’-pyridyl)ethyl carbamate, 2-(4’-pyridyl)ethyl carbamate, 2,2-bis(4’-35 nitrophenyl)ethyl carbamate, 2-(t-butyldisulfanyl)ethyl carbamate, phenyldithioethyl carbamate, 2- pyridyldithioethyl carbamate, 2-[(2-nitrophenyl)dithio]-1-phenylethyl carbamate, 2-(N,N- dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate, C8F19CH2CH2C(CH3)2-carbamate, (1- methyl)cyclopropyl carbamate, 1-adamantyl carbamate, 2-adamantyl carbamate, 1-(1-adamantyl)-1- methylethyl carbamate, 1-methyl-1-(4-biphenylyl)ethyl carbamate, 1-(3,5-di-t-butylphenyl)-1-methylethyl 40 carbamate, N-(2-pivaloylamino)-1,1-dimethylethyl carbamate, triisopropylsilyl carbamate, vinyl
11 carbamate, allyl carbamate, prenyl carbamate, 1-isopropylallyl carbamate, cinnamyl carbamate, 4- nitrocinnamyl carbamate, 3-(3’-pyridyl)prop-2-enyl carbamate, hexadienyl carbamate, propargyl carbamate, but-2-ynylbisoxy carbamate, 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate, 3,5-di-t-butylbenzyl carbamate, p-methoxybenzyl carbamate, p- 5 nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl carbamate, 4-trifluoromethylbenzyl carbamate, 3,5- bis(trifluoromethyl)benzyl carbamate, C8F17CH2CH2-C6H4-CH2- carbamate, (C8F17CH2CH2)3Si-C6H4- CH2- carbamate, 2-naphthylmethyl carbamate, 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 4- phenylacetoxybenzyl carbamate, 4-azidobenzyl carbamate, 4-azidomethoxybenzyl carbamate, m-chloro-p-10 acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate, 2- (trifluoromethyl)-6-chromonylmethyl carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p-toluenesulfonyl)ethyl carbamate, 2-[(4-fluorophenyl)sulfonyl)ethyl carbamate, 2-(4- nitrophenylsulfonyl)ethyl carbamate, 2-(2,4-dinitrophenylsulfonyl)ethyl carbamate, 2-(4- trifluoromethylphenylsulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methyl carbamate, 2-phosphonioethyl 15 carbamate, 2-[phenyl(methyl)sulfonio]ethyl carbamate, 1-methyl-2-(triphenylphosphonio)ethyl carbamate, 1,1-dimethyl-2-cyanoethyl carbamate, 2-dansylethyl carbamate, 2-(4-nitrophenyl)ethyl carbamate, 4- methylthiophenyl carbamate, 2,4-dimethylthiophenyl carbamate, m-nitrophenyl carbamate, 3,5- dimethoxybenzyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, α-methylnitropiperonyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate, 3,4-dimethoxy 6-nitrobenzyl 20 carbamates, phenyl(o-nitrophenyl)methyl carbamate, 2-nitrophenylethyl carbamate, 6-nitroveratryl carbamate, 2,5-dimethylphenacyl carbamate, 4-methoxyphenacyl carbamate, 3’,5’-dimethoxybenzoin carbamate, 9-xanthenylmethyl carbamate, N-methyl-N-(o-nitrophenyl) carbamate, methyl 3-hydroxy-2- methyl-2-(9-oxo-9H-xanthen-2-yl) carbamate, 6-bromo-7-hydroxycoumarin-4-ylmethyl carbamate, 6- bromo-7-methoxycoumarin-4-ylmethyl carbamate, N-methylpicoliniummethyl carbamate, dinitroindolinyl25 carbamate, 1-naphthaldehyde oxime carbamate, t-amyl carbamate, 1-methylcyclobutyl carbamate, 1- methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate, cyclobutyl carbamate, cyclopentyl carbamate, cyclohexyl carbamate, isobutyl carbamate, isobornyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, diisopropylmethyl carbamate, 2,2- dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, 1,1-dimethyl-3-30 (N,N-dimethylcarboxamido)propyl carbamate, butynyl carbamate, 1,1-dimethylpropynyl carbamate, 2- iodoethyl carbamate, 1-methyl-1-(4’-pyridyl)ethyl carbamate, 1-methyl-1-(p-phenylazophenyl)ethyl carbamate, p-(p’-methoxyphenylazo)benzyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6- trimethylbenzyl carbamate, isonicotinyl carbamate, 4-(trimethylammonium)benzyl carbamate, p- cyanobenzyl carbamate, di(2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, phenyl carbamate, 35 2,4,6 tri-t-butylphenyl carbamate, 1-methyl-1-phenylethyl carbamate, and S-benzyl thiocarbamate. In the case of ureas the protecting groups for the amino group can be selected from aminocarbonyl, phenothiazinyl-(10)-carbonyl, N’-p-toluenesulfonylaminocarbonyl, 4-hydroxyphenylaminocarbonyl, 3- hydroxytryptaminocarbonyl, and N’-phenyl-aminothiocarbonyl. In the case of amides the protecting group for the amino group together with the nitrogen atom of the unprotected amino group to which it is attached 40 form an amide group that can be selected from formamide, acetamide, chloroacetamide, trichloroacetamide,
12 trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, pent-4-enamide, picolinamide, 3- pyridylcarboxamide, N-benzoylphenylalaninamide, benzamide, p-phenylbenzamide, o- nitrophenylacetamide, 2,2-dimethyl-2-(o-nitrophenyl)acetamide, o-nitrophenoxyacetamide, 3-(o- nitrophenyl)propanamide, 2-methyl-2-(o-nitrophenoxy)propanamide, 3-methyl-3-nitrobutanamide, o- 5 nitrocinnamide, o-nitrobenzamide, 3-(4-t-butyl-2,6-dinitrophenyl)-2,2-dimethylpropanamide, o- (benzoyloxymethyl)benzamide, 2-(acetoxymethyl)benzamide, 2-[(t-butyldiphenylsiloxy)methyl]benz- amide, 3-(3',6'-dioxo-2',4',5'-trimethylcyclohexa-1',4'-diene)-3,3-dimethylpropionamide, o-hydroxy-trans- cinnamide, 2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide, acetoacetamide, 3-(p- hydroxyphenyl)propanamide, (N'-dithiobenzyloxycarbonylamino)acetamide, and N- 10 acetylmethioninamide. In the case of N-alkyl, N-alkenyl, N-alkynyl or N-aryl amines the protecting group for the amino group can be selected from N-methyl, N-t-butyl, N-allyl, N-prenyl, N-cinnamyl, N-2- phenallyl, N-propargyl, N-methoxymethyl, N-(triisopropylsilyloxy)methyl, N-[2- (trimethylsilyl)ethoxy]methyl, N-3-acetoxypropyl, N-cyanomethyl, N-2-azanorbornene, N-2,4- dinitrophenyl, N-o-methoxyphenyl, N-p-methoxyphenyl, 2,5-dimethyl-4-methoxyphenyl, N-benzyl, N-4-15 methoxybenzyl, N-2,4-dimethoxybenzyl, N-2-hydroxybenzyl, N-9-phenylfluorenyl, N-9-(4-bromophenyl)- 9-fluorenyl, N-9-fluorenyl, N-ferrocenylmethyl, N-2-picolylamine N'-oxide, N-7-methoxycoumar-4- ylmethyl, N-diphenylmethyl, N-bis(4-methoxyphenyl)methyl, N-bis(3,5-dimethyl-4- methoxyphenyl)methyl, N-5-dibenzosuberyl, N-triphenylmethyl, N-(4-methylphenyl)diphenylmethyl, and N-(4-methoxyphenyl)diphenylmethyl. In the case of enamines the protecting group for the amino group20 can be selected from N-(5,5-dimethyl-3-oxo-1-cyclohexenyl), N-2,7-dichloro-9-fluorenylmethylene, N-1- (4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, N-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3- methylbutyl, N-(1,3-dimethyl-2,4,6-(1H,3H,5H)-trioxopyrimidine-5-ylidene)methyl, N-4,4,4-trifluoro-3- oxo-1-butenyl, N-(1-isopropyl-4-nitro-2-oxo-3-pyrrolin-3-yl, and 2,2-bis(ethoxycarbonyl)vinyl). In the case of N-metal derivatives the protecting group for the amino group together with the nitrogen atom of the25 unprotected amino group form a derivative that can be selected from N-borane derivative, N- diphenylborinic acid derivative, N-diethylborinic acid derivative, N-9-borabicyclononane derivative, N- difluoroborinic acid derivative, and N-3,5-bis(trifluoromethyl)phenylboronic acid derivative; and also including N-[phenyl(pentacarbonylchromium)carbenyl]amine, N-[phenyl(pentacarbonyltungsten)- carbenyl]amine, N-copper chelate, N-zinc chelate, and a 18-crown-6-derivative. In the case of N-N 30 derivatives the protecting group for the amino group together with the nitrogen atom of the unprotected amino group to which it is attached form a derivative that can be selected from N-nitro derivative, N-nitroso derivative, N-oxide derivative, azide derivative, triazene derivative, and N-trimethylsilylmethyl-N- benzylhydrazine. In the case of N-P derivatives the protecting group for the amino group together with the nitrogen group of the unprotected amino group to which it is attached form an N-P derivative that can be 35 selected from dimethylphosphinamide, diphenylphosphinamide, dimethylthiophosphinamide, diphenylthiophosphinamide, dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, and iminotriphenylphosphorane. In the case of N-Si derivatives the protecting group for the amino group can be selected from t-butyldiphenylsilyl and triphenylsilyl. In the case of N-S derivatives the protecting group for the amino group together with the nitrogen atom of the unprotected amino group 40 to which it is attached form an N-S derivative that can be selected from N-sulfenyl or N-sulfonyl derivatives.
13 The N-sulfenyl derivatives can be selected from benzenesulfenamide, 2-nitrobenzenesulfenamide, 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide, 1-(2,2,2)-trifluoro-1,1-diphenylethylsulfenamide, and N-3-nitro-2- pyridinesulfenamide. The N-sulfonyl derivatives can be selected from methanesulfonamide, (9H-fluoren- 5 9-yl)methanesulfonamide, trifluoromethanesulfonamide, t-butylsulfonamide, benzylsulfonamide, 2- (trimethylsilyl)ethanesulfonamide, 2-(1,3-dioxan-2-yl)ethylsulfonamide, p-toluenesulfonamide, benzenesulfonamide, o-anisylsulfonamide, 2-nitrobenzenesulfonamide, 4-nitrobenzenesulfonamide, 2,4- dinitrobenzene-sulfonamide, 2-naphthalenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzene- sulfonamide, 2-(4-methylphenyl)-6-methoxy-4-methylsulfonamide, 9-anthracenesulfonamide, pyridine-2- 10 sulfonamide, 8-quinolylsulfonamide, benzothiazole-2-sulfonamide, phenacylsulfonamide, trichloroethoxysulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide, 2,4,6-trimethoxybenz- enesulfonamide, 2,6-dimethyl-4-methoxybenzenesulfonamide, pentamethylbenzenesulfonamide, 2,3,5,6- tetramethyl-4-methoxybenzenesulfonamide, 4-methoxybenzenesulfonamide, 2,4,6- trimethylbenzenesulfonamide, 2,6-dimethoxy-4-methylbenzenesulfonamide, 3-methoxy-4-t-15 butylbenzenesulfonamide, 2,2,5,7,8-pentamethylchroman-6-sulfonamide, 2,2,4,6,7- pentamethyldihydrobenzofuranylsulfonamide, 1,2-dimethylindole-3-sulfonamide, 2-thienylsulfonamide, 1,2,5-thiadizoline-1,1-dioxide, N,N-dimethylsulfamide. In the case of heterocyclic systems the protecting group for the amino group together with the nitrogen atom of the unprotected amino group to which it is attached form a heterocyclic system that can be selected from 4,5-diphenyl-3-oxazolin-2-one, N-20 tetramethylsuccinimide, N-2,3-dicyclohexylsuccinimide, N-phthalimide, N-dichlorophthalimide, N- tetrachlorophthalimide, N-4-nitrophthalimide, N-thiodiglycoloyl amine, N-diglycoloyl amine, N- dithiasuccinimide, N-2,3-diphenylmaleimide, N-2,3-dimethylmaleimide, N-2,5-dimethylpyrrole, N-2,5- bis(triisopropylsiloxy)-pyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct, N-1,1,3,3 tetramethyl- 1,3-disilaisoindoline, N-diphenylsilyldiethylene amine, N-5-substituted-1,3-dimethyl-1,3,5-25 triazacyclohexan-2-one, N-5-substituted-1,3-benzyl-1,3,5-triazacyclohexan-2-one, 1-substituted 3,5- dinitro-4-pyridone, 1,3,5-dioxazine, 1,3,5-dithiazane, 1.2-dimethoxy-4,5-dimethylenebenzene, and 4- piperidinone. In the case of imines the protecting group for the amino group can be selected from N-1,1- dimethylthiomethylene, N-isobutylmethylmethylene, N-benzylidene, N-p-methoxybenzylidene, N-2- trifluoromethylbenzylidene, N-3-nitrobenzylidene, N-diphenylmethylene, N-9-fluorenylidene, N-30 xanthonylidene, N-[2-pyridyl)mesityl]methylene, N-(N',N'-dimethylaminomethylene), N-(N',N'- dibenzylaminomethylene), N-(N'-t-butylaminomethylene), N-(N’,N’-dibutylaminomethylene), N-(N’,N’- diisopropylaminomethylene), N-1-pyrroline-2-yl, N,N'-isopropylidene, N-p-nitrobenzylidene, N- salicylidene, N-5-chlorosalicylidene, N-(5-chloro-2-hydroxyphenyl)phenylmethylene, N-cyclohexylidene, N-t-butylidene, N-4,5-dihydrothiazoline, and N-2-pyrimidyl. 35 [0029] Within the scope of the present invention a protecting group for SH is defined to be the S-bonded moiety resulting from the protection of the SH group through the formation of a suitable protected SH group. Examples of such protected SH groups include thioethers, disulfides, silyl thioethers, thioesters, thiocarbonates, thiocarbamates, sulfenyl derivatives, and S-P derivatives. In the case of thioethers, the protecting group for the SH can be selected from S-alkyl, S-benzyl, S-p-methoxybenzyl, S-o-40 hydroxybenzyl, S-p-hydroxybenzyl, S-o-acetoxybenzyl, S-p-acetoxybenzyl, S-p-nitrobenzyl, S-o-
14 nitrobenzyl, S-2,4,6-trimethylbenzyl, S-2,4,6-trimethoxybenzyl, 2,2,4,6,7-pentamethyl-2,3- dihydrobenzofuran-5-methyl, S-4-pycolyl, S-2-picolyl N-oxide, S-quinolinylmethyl, S-9-antrylmethyl, S-9- fluorenylmethyl, S-xanthenyl, S-ferrocenylmethyl, S-diphenylmethyl, S-bis(4-methoxyphenyl)methyl, S-5- dibenzosuberyl, S-triphenylmethyl, 4-methoxytrityl, S-diphenyl-4-pyridylmethyl, S-phenyl, S-2,4- 5 dinitrophenyl, S-2-quinolyl, S-t-butyl, S-1-adamantyl, S-methoxymethyl, S-isobutoxymethyl, S- benzyloxymethyl, S-4-methoxybenzyloxymethyl, S-t-butyldimethylsilyloxymethyl, S-1-ethoxyethyl, S-2- tetrahydropyranyl, S-benzylthiomethyl, S-phenylthiomethyl, S-acetamidomethyl (Acm), S- trimethylacetamidomethyl, S-benzamidomethyl, S-allyloxycarbonylaminomethyl, S-[[2-[8-[[(1,1- dimethylethyl)dimethylsilyl]oxy]octahydro-1(2H)-quinolinyl]-acetyl]amino]methyl, S-N-methylphenacyl- 10 oxycarbamidomethyl, S-N-[2,3,5,6-tetrafluoro-4-(N’-piperidino)-phenyl-N-allyloxycarbonylaminomethyl, S-phthalimidomethyl, S-phenylacetamidomethyl, S-acetylmethyl, S-carboxymethyl, S-cyanomethyl, S-(2- nitro-1-phenyl)ethyl, S-2-(2,4-dinitrophenyl)ethyl, S-2-(4’-pyridyl)ethyl, S-2-cyanoethyl, S-2- (trimethylsilyl)ethyl, S-2,2-bis(carboethoxy)ethyl, S-(1-m-nitrophenyl-2-benzoyl)ethyl, S-2- phenylsulfonylethyl, S-1-(4-methylphenylsulfonyl)-2-methylprop-2-yl, S-p-hydroxyphenacyl, S-phenacyl, 15 and S-tosylvinyl. In the case of disulfides, the protected SH group can be selected from S-ethyl disulfide, S-t-butyl disulfide, S-2-nitrophenyl disulfide, S-2,4-dinitrophenyl disulfide, S-2-phenylazophenyl disulfide, S-2-carboxyphenyl disulfide S-3-nitro-2-pyridyl disulfide and S-(N-methyl-N-phenylthiocarbamate. In the case of silyl thioethers the protecting group for the SH can be selected from the list of groups that was listed above for the protection of OH groups with silyl ethers. In the case of thioesters, the protecting group for20 the SH can be selected from S-acetyl, S-benzoyl, S-2-methoxyisobutyryl, S-trifluoroacetyl, S-[N-[(p- biphenylyl)isopropyloxy]carbonyl]-N-methyl-γ-aminothiobutyrate, and S-N-(t-butoxycarbonyl)-N-methyl- γ-aminothiobutyrate. In the case of thiocarbonate the protecting group for the SH can be selected from S- 2,2,2-trichloroethoxycarbonyl, S-t-butoxycarbonyl, S-benzyloxycarbonyl, S-p- methoxybenzyloxycarbonyl, and S-fluorenylmethyloxycarbonyl. In the case of thiocarbamate the protected 25 SH group can be selected from S-(N-ethyl) carbamate and S-(N-methoxymethyl) carbamate. In the case of sulfenyl derivatives the protected SH group can be selected from S-sulfonate derivative, S-thiosulfonate derivative, S-sulfenylthiocarbonate, S-3-nitro-2-pyridinesulfenyl sulfide, S-[tricarbonyl[1,2,3,4,5-η]-2,4- cyclohexadien-1-yl]-iron(1+) derivative, and oxathiolones. In the case of S-P derivatives the protecting group for the SH group is selected from S-(dimethylphosphino)thioyl and S-(diphenylphosphino)thioyl. 30 [0030] The mention of these groups should not be interpreted as a limitation of the scope of the invention, since they have been mentioned as a mere illustration of protection groups for OH, amino and SH groups, but further groups having said function may be known by the skilled person in the art, and they are to be understood to be also encompassed by the present invention. [0031] The term “ecteinascidin compound” may refer to compounds of Formula E:
15
wherein: R101 and R104 are independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, C(=O)R10c, 5 C(=O)OR10b, C(=O)NR10dR10e, and a protecting group for OH; R102 is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2- C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, C(=O)R10c, C(=O)OR10b, C(=O)NR10dR10e, and a protecting group for amino; R103 is CN or OH; 10 R105 and R106 together to the carbon to which they are attached form a group: (a) C(=O); (b) CH(OR107) or CH(NR108R109) wherein R107 is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a 15 protecting group for OH; and R108 and R109 are independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for amino; (c) a group of formula: 20
wherein X101 and X102 are independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; R110 and R111 are independently selected from hydrogen, C(=O)R10c, C(=O)OR10b, 25 C(=O)NR10dR10e, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; or (d) a group of formula:
16
wherein X is -NR112- or -O-; Y101 is selected from hydrogen, OR10b, OC(=O)R10c, OC(=O)OR10b, OC(=O)NR10dR10e, SR10f, 5 SOR10c, SO2R10c, C(=O)R10c, C(=O)OR10b, C(=O)NR10dR10e, NO2, NR10dR10e, N(R10d)C(=O)R10c, N(R10d)-OR10b, C(R10c)=NOR10b, N(R10d)C(=O)OR10b, N(R10d)C(=O)NR10dR10e, CN, halogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; 10 Y102 and Y103 are independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; R112 and R113 are independently selected from hydrogen, C(=O)R10c, C(=O)OR10b, C(=O)NR10dR10e, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; and 15 each R10b is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for OH; each R10c is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, 20 substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each R10d and R10e is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting 25 group for amino; each R10f is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for SH; 30 or a pharmaceutically acceptable salt thereof. [0032] Preferably, R101 may be hydrogen or the same as R1 as described herein. [0033] Preferably, R102 may be methyl. [0034] Preferably, R104 may be the same as R8 as described herein. [0035] Preferably, X101 may be the same as X1 as described herein.
17 [0036] Preferably, X102 may be the same as X2 as described herein. [0037] Preferably, R110 may be hydrogen. [0038] Preferably, R111 may be hydrogen. [0039] Preferably, Y101 may be the same as Y1 as described herein. 5 [0040] Preferably, Y102 may be the same as Y2 as described herein. [0041] Preferably, Y103 may be the same as Y3 as described herein. [0042] Preferably, R112 may be hydrogen. [0043] Preferably, R113 may be hydrogen. [0044] In the first and second aspect of the present invention and in their preferred embodiments, water 10 content is calculated as % w/w regarding compound 1 or compound of formula I, respectively. [0045] In the present invention, reference is made to the total amount of water (w/w) present in the reaction mixture with respect to a compound, for example compound 1, compound 2 or compound I. Since the amount of compound decreases over time as the reaction proceeds, the total amount of water is assessed versus the amount of compound present at the start of the reaction. 15 [0046] To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term “about”. It is understood that, whether the term “about” is used explicitly or not, every quantity given herein is meant to refer to the actual given value, and it also meant to refer to the approximation of such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to experimental and/or measurement conditions for such 20 given value. Selective acylation of compound 1 [0047] In a first aspect of the present invention, there is provided a process for the synthesis of an ecteinascidin compound, or a pharmaceutically acceptable salt thereof, the process comprising a step of coupling compound 1 with a compound of Formula A, or a stereoisomer thereof, to give a compound of 25 Formula I:
18
wherein: R6 is ProtNH, wherein ProtNH is a protecting group for amino; R7 is ProtSH, wherein ProtSH is a protecting group for SH; and 5 the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a salt thereof, such as its hydrochloride (EDC·HCl), in presence of 4-dimethylaminopyridine (DMAP) and water wherein the total amount of water (w/w) in the reaction mixture with respect to the amount of the compound 1 initially present is from about 2.5% to about 6%. Preferred embodiments for the first aspect of the present invention: 10 [0048] In some preferred embodiments, R6 together with the NH group to which it is attached form a carbamate group. [0049] In a preferred embodiment, R6 together with the NH group to which it is attached form a carbamate group selected from allylcarbamate, 2,2,2-trichloroethylcarbamate, benzylcarbamate, 9-fluorenylmethyl- carbamate and t-butylcarbamate, being more preferably t-butylcarbamate. 15 [0050] In some preferred embodiments R7 together with the S atom to which it is attached form a thioether group, being S-9-fluorenylmethyl (Fm) the most preferred R7 group. [0051] Preferably, the compound of formula A is:
, or a stereoisomer thereof, and more preferably is . [0052] In a preferred embodiment, the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)- 20 carbodiimide hydrochloride (EDC·HCl). [0053] In a preferred embodiment, the total amount of water (% w/w) with respect to compound 1 in the reaction mixture is from about 2.5% to about 5%, more preferably from about 2.5% to about 4%, even more preferably about 2.5% to about 3.5%, most preferably about 3%.
19 [0054] In a preferred embodiment, a number of equivalents of the compound of Formula A with respect to compound 1 is from about 2 to about 3, preferably from about 2 to about 2.5, more preferably from about 2 to about 2.1, even more preferably about 2.02. [0055] In a preferred embodiment, a number of equivalents of the coupling agent with respect to 5 compound 1 is from about 1.0 to about 1.8, preferably from about 1.5 to about 1.8, more preferably from about 1.6 to about 1.8, even more preferably about 1.7. [0056] In a preferred embodiment, a number of equivalents of 4-dimethylaminopyridine (DMAP) with respect to compound 1 in the reaction mixture is from about 0.65 to about 0.75, preferably about 0.7. [0057] In a preferred embodiment, the coupling step is carried out at from about -10 ºC to about -6 ºC, 10 preferably from about -9 ºC to about -7 ºC, more preferably at about -8 ºC. [0058] In a preferred embodiment, the concentration of compound 1 is from about 0.04 g/mL to about 0.14 g/mL, preferably from about 0.04 g/mL to about 0.10 g/mL, more preferably from about 0.07 g/mL to about 0.09 g/mL, most preferably about 0.08 g/mL. [0059] In a preferred embodiment, the reaction time of the coupling step is from about 30 min to about 15 120 min, preferably from about 45 min to about 90 min, more preferably from about 55 min to about 65 min, even more preferably about 60 min. [0060] In a preferred embodiment, the coupling step is carried out in a halogenated solvent, most preferably dichloromethane. [0061] In a preferred embodiment, the coupling step is quenched with water and the resulting mixture is 20 washed sequentially with saturated aqueous NH4Cl and saturated aqueous NaCl, more preferably wherein the pH of the resulting aqueous phase is between about 5 and about 7. Preferably, the organic layer is dried, evaporated, dissolved in methanol and re-evaporated to give a foam. [0062] In a preferred embodiment, the foam is dissolved in methanol and treated with water to precipitate the compound of formula I. 25 [0063] In a preferred embodiment, the amount of water added to the methanol solution (v/v) is from about 25% to about 30%, more preferably about 27%. [0064] In a preferred embodiment, the concentration of the compound of formula I in the mixture methanol/water is from about 20 g/L to about 40 g/L, preferably from about 25 g/L to about 35 g/L, more preferably about 30 g/L. 30 [0065] In a preferred embodiment, the precipitation of the compound of formula I is carried out at from about 0 ºC to about 25 ºC, more preferably at from about 0 º C to about 10 ºC, most preferably at about 0 ºC.
20 [0066] In a preferred embodiment, the precipitated compound of formula I is dried at 30 ºC for three days under vacuum. [0067] In a preferred embodiment, the water content of the compound of formula I after drying is about 1% w/w. 5 [0068] In a preferred embodiment, the amount of impurity Boc-L-Cys(Fm)-OH relative to the compound of formula I is below 6% w/w. Protection of the phenolic group [0069] In a second aspect of the present invention there is provided a process for the synthesis of an ecteinascidin compound, or a pharmaceutically acceptable salt thereof, comprising a step of protecting the 10 phenolic group in a compound of formula I by reacting it with a compound of formula R1X in presence of a base MOH and water in tetrahydrofuran to give a compound of formula II:
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; 15 R6 is ProtNH; R7 is ProtSH; X is selected from Cl, Br and I; and MOH is selected from sodium hydroxide, potassium hydroxide and lithium hydroxide. Preferred embodiments for the second aspect of the present invention: 20 [0070] In a preferred embodiment, the compound of formula I used for the manufacture of the compound of formula II has been obtained as described in the first aspect of the present invention, optionally including one or more of its preferred embodiments. [0071] In a preferred embodiment, R1 is selected from methoxymethyl (MOM) and methoxyethoxymethyl (MEM), more preferably methoxyethoxymethyl (MEM). 25 [0072] Preferably, R6 and R7 are as described herein.
21 [0073] In a preferred embodiment, X is Cl or Br, more preferably Cl. [0074] In a preferred embodiment, MOH is powdered MOH. [0075] In a preferred embodiment, the powdered MOH has a mean particle size from about 50 µm to about 500 µm, preferably from about 60 µm to about 300 µm, more preferably from about 75 µm to about 5 250 µm, even more preferably from about 100 µm to about 200 µm. [0076] In another preferred embodiment, about 60% to about 90% of the particles of powdered MOH have a particle size from about 100 μm to about 200 μm. [0077] In a preferred embodiment, MOH is NaOH, preferably powdered NaOH. [0078] In a preferred embodiment, a number of equivalents of MOH with respect to the compound of 10 formula I is from about 2 to about 3, preferably from about 2.3 to about 2.7, more preferably from about 2.4 to about 2.6, even more preferably about 2.55. [0079] In a preferred embodiment, a number of equivalents of R1X with respect to the compound of formula I in the reaction mixture is from about 1.9 to about 3.3, preferably from about 2.2 to about 3.0, more preferably from about 2.5 to about 2.9, even more preferably about 2.8. 15 [0080] In a preferred embodiment, the total amount of water in the reaction mixture when the reaction is completed is from about 7% w/w to about 12% w/w of the amount of the compound of formula I initially present in the reaction, preferably the total amount of water in the reaction mixture when the reaction is completed is from about 7% w/w to about 11% w/w, more preferably from about 7% w/w to about 10% w/w, even more preferably from about 7% to about 9% w/w and most preferably from about 7% w/w to 20 about 8% w/w versus the amount of the compound of formula I initially present. The reaction is completed when the amount of the compound of formula I present in the reaction mixture is about 1% or lower than 1% of its initial amount. More preferably the amount of the compound of formula I in the reaction mixture is determined by semiquantitative TLC or by HPLC-DAD chromatography. [0081] In a more preferred embodiment, water is added in one or more portions during the reaction until 25 it is completed. [0082] In a preferred embodiment, the protection step is carried out by adding a solution of the compound of formula I to a suspension of MOH and R1X; preferably wherein the temperature of the reaction mixture during the addition is maintained below -10 ºC. In embodiments the temperature is about -15ºC [0083] In a preferred embodiment, after the addition of the compound of formula I, the protection step is 30 carried out at from about -7 ºC to about -3 ºC, preferably from about -6 ºC to about -4 ºC, more preferably at about -5 ºC. [0084] In a preferred embodiment, the water content in the solution of the compound of formula I is below 1% w/w.
22 [0085] In a preferred embodiment, the suspension of MOH and R1X in tetrahydrofuran has a water content, (w/w) from about 2% to about 4.5%, preferably about 2.5% to about 4%, more preferably from about 2.5% to about 3.5% and most preferably about 3% versus the compound of formula I. More preferably, once the addition of the solution of the compound of formula I to such suspension is finished, 5 one or more portions of water is/are added until the total amount of water in the reaction mixture is from about 7% w/w to about 12% w/w, preferably from about 7% w/w to about 11% w/w, more preferably from about 7% w/w to about 10% w/w, even more preferably from about 7% to about 9% w/w and most preferably from about 7% w/w to about 8% w/w versus the compound of formula I. [0086] In another embodiment, if the reaction is not completed in 30 min after the addition of the 10 compound of formula I, then water is added in an amount of from about 4% to about 8% (% w/w), being more preferred to add about 4% w/w of water. [0087] In a preferred embodiment, the reaction time after the addition of the compound of formula I is from about 0.5 h to about 2 h, more preferably from about 30 to 50 minutes, even more preferably about 40 min. 15 De-allylation reaction [0088] In a third aspect of the present invention, there is provided a process for the synthesis of an ecteinascidin compound, or a pharmaceutically acceptable salt thereof, the process comprising a step of de- allylating a compound of Formula II to provide a compound of Formula III:
20 with a palladium catalyst in presence of a secondary amine as reducing agent; wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; and R7 is ProtSH. 25 Preferred embodiments for the third aspect of the present invention:
23 [0089] In a preferred embodiment, the compound of formula II used for the manufacture of the compound of formula III is obtained following the procedure described in the second aspect of the present invention, optionally including one of more of its/their preferred embodiments. [0090] Preferably, R1, R6 and R7 are as described herein. 5 [0091] In a preferred embodiment, the deprotection of the compound of Formula II to give the compound of Formula III is typically conducted using palladium (0) or palladium (II) catalysts in the presence of a secondary amine as reducing agent. [0092] Most preferred palladium (0) catalyst is Pd(PPh3)4 and most preferred palladium (II) catalyst is PdCl2(PPh3)2. Preferred secondary amine is diethylamine. 10 [0093] In a preferred embodiment, the number of equivalents of the palladium catalyst is from about 0.03 to about 0.1, more preferably from about 0.04 to about 0.08, most preferably about 0.06 equivalents. [0094] In a preferred embodiment, the deprotection is carried out at from about 30 ºC to about 50 ºC. Most preferably, the deprotection is carried out at 40 ºC. [0095] In a preferred embodiment, the reaction mixture, after the reaction is completed, is cooled at about 15 0 ºC before quenching it with aqueous HCl 1M. [0096] Preferably, the deprotection reaction is carried out in dichloromethane. Further synthetic steps [0097] The coupling reaction of compound 1 with compounds of formula A and/or the protection of the compound of formula I to give the compound of formula II, and/or the de-allylation reaction of a compound 20 of formula II to give a compound of formula III as described herein may be utilized in the early stages of ecteinascidin compound synthesis. For example, the processes described herein may be used in the generation of starting materials and intermediate compounds for preparing ecteinascidin compound syntheses. [0098] The process may comprise further synthetic steps as disclosed in Cragg et al. Anticancer Agents 25 from Natural Products (2012), 2nd Edition – Chapter 12: “Ecteinascidin-743 (Yondelis®), Aplidin®, and Irvalec® (pages 291-316); WO 00/69862 A2 (and references contained therein); WO 01/87895 A1 (and references contained therein); WO 03/014127 A1 (and references contained therein); WO 2011/147828 A1 (and references contained therein); WO 2018/197663 A1 (and references contained therein); WO2025/077996 A1 (and references contained therein); all of which (and the references contained therein) 30 are incorporated by reference. [0099] A preferred set of further synthetic steps is provided below.
24 [0100] Preferably, the process further comprises a step of oxidizing the compound of formula III to give a compound of formula IV:
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; 5 R6 is ProtNH; R7 is ProtSH; and the wavy line represents a mixture of epimers. [0101] Preferably, R1, R6 and R7 are as described herein. [0102] The oxidation reaction of a compound of Formula III to form a compound of Formula IV is 10 typically effected by reaction with a suitable oxidant, for example with hydrogen peroxide, an organic peroxide, a perbenzoic acid, a periodate, lead tetraacetate, lead oxide, selenium dioxide, hypervalent iodine oxidants such as 2-iodoxybenzoic acid (IBX), or with an organic seleninic anhydride such as (PhSeO)2O. [0103] Preferably, the process further comprises a step of conducting a cyclisation reaction on a compound of Formula IV to provide a compound of Formula V:
15 wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; R7 is ProtSH;
25 R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; and the wavy line represents a mixture of epimers. [0104] Preferably, R1, R6 and R7 are as described herein. 5 [0105] Preferably, R8 is acetyl. [0106] The cyclisation reaction of a compound according to Formula IV to provide a compound of Formula V is typically effected by forming an exendo quinone methide at the 4-position of ring B, allowing the methide to react with the sulphur atom of the cysteine residue and capturing the resulting phenoxide with an acylating reagent. Typically, the methide is formed by reaction of the compound of Formula IV 10 with an in situ-generated Swern reagent (such as by reaction of Tf2O with DMSO) followed by treatment with a base (such as tert-butyltetramethylguanidine). Typically, the cyclization is carried out by removing the protecting group for SH under conditions that allow the formation of a thiolate ion, followed by nucleophile addition of sulphur to the quinone methide to generate the 10-membered lactone bridge, and the resulting phenoxide is captured using an acylating agent (such as acetic anhydride, a mixed acetyl 15 anhydride, or acetyl chloride) to give the acylated compound of Formula V. [0107] Preferably, the process further comprises a step of conducting a deprotection reaction on a compound of Formula V to provide a compound of Formula VI:
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; 20 R6 is ProtNH; and R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. [0108] Preferably, R1, R6 and R8 are as described herein. [0109] The conversion of the compound of Formula V to the compound of Formula VI typically involves 25 deprotection of the amino group on R6. Suitable conditions and reagents for the deprotection of these protecting groups are known to the skilled person (see Wuts, PGM and Greene TW in Protecting Groups in Organic Synthesis, 5th Ed. Wiley-Interscience, and Kocienski PJ in Protecting Groups, 3rd Ed. Georg Thieme Verlag). For example, in the case of t-butyl carbamate, the deprotection is typically conducted
26 using acidic conditions. A preferred reagent is p-toluenesulfonic acid or p-toluenesulfonic acid monohydrate. [0110] The conversion of the compound of Formula V to the compound of Formula VI typically involves deprotection of the phenol group on R1. The deprotection is typically conducted using acidic conditions. 5 Particular reagents include p-toluenesulfonic acid or p-toluenesulfonic acid monohydrate or HCl (e.g. in EtOAc). [0111] Preferably, the deprotection of the amino group on R6 and the deprotection of the phenol group on R1 is conducted in one-pot manner. [0112] Preferably, the process may comprise a step of conducting a deprotection reaction on a compound 10 of Formula V to provide a compound of Formula VI-Int-a:
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; and R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, 15 substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. [0113] Preferably, R1, R6 and R8 are as described herein. [0114] The conversion of the compound of Formula V to the compound of Formula VI-Int-a typically involves deprotection of the phenol group on R1. The deprotection is typically conducted using acidic conditions. Particular reagents include p-toluenesulfonic acid or p-toluenesulfonic acid monohydrate or 20 HCl (e.g. in EtOAc). [0115] Preferably, the process further comprises a step of conducting a deprotection reaction on a compound of Formula VI-Int-a to provide a compound of Formula VI:
27
wherein R6 is ProtNH; and R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. 5 [0116] The conversion of the compound of Formula VI-Int-a to the compound of Formula VI typically involves deprotection of the amino group on R6. Suitable conditions and reagents for the deprotection of these protecting groups are known to the skilled person (see Wuts, PGM and Greene TW in Protecting Groups in Organic Synthesis, 5th Ed. Wiley-Interscience, and Kocienski PJ in Protecting Groups, 3rd Ed. Georg Thieme Verlag). 10 [0117] Preferably, the process further comprises a step of conducting a deprotection reaction on a compound of Formula V to provide a compound of Formula VI-Int-b:
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; and 15 R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. [0118] Preferably, R1, R6 and R8 are as described herein. [0119] The conversion of the compound of Formula V to the compound of Formula VI-Int-b typically involves deprotection of the amino group on R6. Suitable conditions and reagents for the deprotection of 20 these protecting groups are known to the skilled person (see Wuts, PGM and Greene TW in Protecting
28 Groups in Organic Synthesis, 5th Ed. Wiley-Interscience, and Kocienski PJ in Protecting Groups, 3rd Ed. Georg Thieme Verlag). [0120] Preferably, the process further comprises a step of conducting a deprotection reaction on a compound of Formula VI-Int-b to provide a compound of Formula VI:
5 wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; and R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. [0121] Preferably, R1 and R8 are as described herein. 10 [0122] The conversion of the compound of Formula VI-Int-b to the compound of Formula VI typically involves deprotection of the phenol group on R1. The deprotection is typically conducted using acidic conditions. Particular reagents include p-toluenesulfonic acid or p-toluenesulfonic acid monohydrate or HCl (e.g. in EtOAc). [0123] Preferably, the process further comprises a step of conducting a transamination reaction on a 15 compound of Formula VI to provide a compound of Formula VII:
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl. [0124] Preferably, R8 is as described herein.
29 [0125] The transamination of the compound of Formula VI to provide a compound of Formula VII is typically conducted using a suitable carbonyl reagent such as a hindered 1,2-benzoquinone or a pyridine- or pyridinium carboxaldehyde. More preferred carbonyl reagents are the methiodide of pyridine-4- carboxaldehyde and the methylbenzenesulfonate of pyridine-4-carboxaldehyde. 5 [0126] Preferably, the process further comprises a step of reacting a compound of Formula VII with a compound of Formula B to provide a compound of Formula VIIIa:
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; and 10 X1 and X2 are independently selected from hydrogen and unsubstituted or unsubstituted C1-C12 alkyl. [0127] Preferably, R8 is as described herein. [0128] Preferably, X1 is hydrogen. [0129] Preferably, X2 is hydrogen. [0130] The conversion of the compound of Formula VII to provide the compound of Formula VIIIa is 15 typically conducted using a Pictet-Spengler reaction. Typically, the reaction is conducted under acid catalysis. A preferred catalyst is silica gel. [0131] Preferably, the compound of Formula B is:
. [0132] Preferably, the process further comprises a step of replacing a cyano group with a hydroxy group 20 in a compound of Formula VIIIa to provide a compound of Formula IXa:
30
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; and X1 and X2 are independently selected from hydrogen and unsubstituted or unsubstituted C1-C12 alkyl. 5 [0133] Preferably, R8, X1 and X2 are as described herein. [0134] The conversion of the compound of Formula VIIIa to provide the compound of Formula IXa is typically carried out by reaction with a nitrile-coordinating transition metal salt. More preferred salts are salts of Ag(I) or Cu(I). The most preferred salts are AgNO3 and CuCl. [0135] Preferably, the compound of Formula IXa is trabectedin: 10
. [0136] Preferably, the process further comprises a step of reacting a compound of Formula VII with a compound of Formula C to provide a compound of Formula VIIIb:
31
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; X is -NH- or -O-; 5 Y1 is selected from hydrogen, ORb, OC(=O)Rc, OC(=O)ORb, OC(=O)NRdRe, SRf, SORc, SO2Rc, C(=O)Rc, C(=O)ORb, C(=O)NRdRe, NO2, NRdRe, N(Rd)C(=O)Rc, N(Rd)-ORb, C(Rc)=NORb, N(Rd)C(=O)ORb, N(Rd)C(=O)NRdRe, CN, halogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2- C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; 10 Y2 and Y3 are independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; each Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for OH; each Rc is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or 15 unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each Rd and Re is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for amino; and 20 each Rf is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for SH. [0137] Preferably, R8 is as described herein. [0138] Preferably, Y1 is selected from hydrogen and ORb. In some more preferred embodiments, Y1 is 25 hydrogen. In other more preferred embodiments, Y1 is ORb, preferably methoxy. [0139] Preferably, Y2 is hydrogen. [0140] Preferably, Y3 is selected from hydrogen, -CH2OH, -CH2OC(=O)Rg, -CH2NH2 and -CH2NHRh, wherein Rg is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl, and Rh is ProtNH.
32 [0141] The conversion of the compound of Formula VII to provide the compound of Formula VIIIb is typically conducted using a Pictet-Spengler reaction. Typically, the reaction is conducted under acid catalysis. Preferred catalysts are acetic acid and trifluoroacetic acid. [0142] Preferably, the step of reacting the compound of Formula VII with the compound of Formula C 5 to provide the compound of Formula VIIIb comprises reacting the compound of Formula VII with a compound of Formula C-1 to provide a compound of Formula VIIIb-1:
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; 10 X is -NH- or -O-; Y1 is hydrogen or a -ORb group, wherein Rb is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; Y3 is selected from hydrogen, -CH2OH, -CH2OC(=O)Rg, -CH2NH2 and -CH2NHRh, wherein Rg is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted 15 or unsubstituted C2-C12 alkynyl, and Rh is ProtNH. [0143] Preferably, R8 and Y1 are as described herein. [0144] Preferably, Y3 is selected from -CH2NH2 and -CH2NHRh, more preferably -CH2NHRh. [0145] Preferably, Rh is Alloc. [0146] Preferably, Y3 is -CH2NHRh, and the process further comprises a step of conducting a deprotection 20 reaction to convert -CH2NHRh to -CH2NH2, after the step of reacting the compound of Formula VII with the compound of Formula C to provide the compound of Formula VIIIb. Suitable conditions and reagents for the deprotection of this protecting groups are known to the skilled person (see Wuts, PGM and Greene TW in Protecting Groups in Organic Synthesis, 5th Ed. Wiley-Interscience, and Kocienski PJ in Protecting Groups, 3rd Ed. Georg Thieme Verlag). For example, in the case of Alloc, the deprotection is typically 25 conducted using metal catalysis. Preferred catalysts include palladium(0) catalysts and palladium (II) catalysts in the presence of a reducing reagent, such as a trialkyltin hydride or secondary amines.
33 [0147] Preferably, the step of reacting the compound of Formula VII with the compound of Formula C to provide the compound of Formula VIIIb comprises reacting the compound of Formula VII with a compound of Formula C-1A to provide a compound of Formula VIIIb-1A:
5 wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; Y1 is hydrogen or a -ORb group, wherein Rb is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; Y3 is selected from hydrogen, -CH2OH and -CH2OC(=O)Rg, wherein Rg is selected from substituted or10 unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2- C12 alkynyl. [0148] Preferably, R8 and Y1 are as described herein. [0149] Preferably, Y3 is selected from hydrogen and -CH2OH. In some more preferred embodiments, Y3 is hydrogen. In other more preferred embodiments, Y3 is -CH2OH. 15 [0150] Preferably, the compound of Formula C is:
[0151] Preferably, the compound of Formula C is:
[0152] Preferably, the compound of Formula C is: 20
[0153] Preferably, the compound of Formula C is:
34
[0154] Preferably, the step of reacting the compound of Formula VII with the compound of Formula C to provide the compound of Formula VIIIb comprises reacting the compound of Formula VII with a compound of Formula C-1B to provide a compound of Formula VIIIb-1B: 5
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; Y1 is hydrogen or a -ORb group, wherein Rb is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; 10 Y3 is selected from hydrogen, -CH2OH, -CH2OC(=O)Rg, wherein Rg is selected from substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2- C12 alkynyl. [0155] Preferably, R8 and Y1 are as described herein. [0156] Preferably, Y3 is selected from hydrogen and -CH2OH. In some more preferred embodiments, Y3 15 is hydrogen. In other more preferred embodiments, Y3 is -CH2OH. [0157] Preferably, the process further comprises a step of replacing a cyano group with a hydroxy group in a compound of Formula VIIIb to provide a compound of Formula IXb:
35
wherein R8 is a -C(=O)Ra group, wherein Ra is selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, and substituted or unsubstituted C2-C12 alkynyl; X is -NH- or -O-; 5 Y1 is selected from hydrogen, ORb, OC(=O)Rc, OC(=O)ORb, OC(=O)NRdRe, SRf, SORc, SO2Rc, C(=O)Rc, C(=O)ORb, C(=O)NRdRe, NO2, NRdRe, N(Rd)C(=O)Rc, N(Rd)-ORb, C(Rc)=NORb, N(Rd)C(=O)ORb, N(Rd)C(=O)NRdRe, CN, halogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2- C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; 10 Y2 and Y3 are independently selected from hydrogen and substituted or unsubstituted C1-C12 alkyl; each Rb is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for OH; each Rc is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or 15 unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, and substituted or unsubstituted heterocyclic group; each Rd and Re is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for amino; and 20 each Rf is independently selected from hydrogen, substituted or unsubstituted C1-C12 alkyl, substituted or unsubstituted C2-C12 alkenyl, substituted or unsubstituted C2-C12 alkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group, and a protecting group for SH. [0158] The conversion of the compound of Formula VIIIb to provide the compound of Formula IXb is typically carried out by reaction with a nitrile-coordinating transition metal salt. More preferred salts are 25 salts of Ag(I) or Cu(I). The most preferred salts are AgNO3 and CuCl. [0159] Preferably, the compound of Formula IXb is lurbinectedin:
36
. [0160] Preferably, the compound of Formula IXb is ecubectedin:
. [0161] Preferably, the compound of Formula IXb is: 5
. [0162] Preferably, the compound of Formula IXb is:
.
37 [0163] Preferred stereochemistry of the compounds of formula A is:
wherein R6 and R7 are as defined above for formula A. [0164] Preferred stereochemistry of the compounds of formula I is: 5
wherein R6 and R7 are as defined above for formula I. [0165] Preferred stereochemistry of the compounds of formula II is:
wherein R1, R6 and R7 are as defined above for formula II. 10 [0166] Preferred stereochemistry of the compounds of formula III is:
wherein R1, R6 and R7 are as defined above for formula III.
38 [0167] Preferred stereochemistry of the compounds of formula IV is:
wherein the wavy line, R1, R6 and R7 are as defined above for formula IV. [0168] Preferred stereochemistry of the compounds of formula V is: 5
wherein R1, R6 and R8 are as defined above for formula V. [0169] Preferred stereochemistry of the compounds of formula VI is:
wherein R8 is as defined above for formula VI. 10 [0170] Preferred stereochemistry of the compounds of formula VI-Int-a is:
39 wherein R6 and R8 are as defined above for formula VI-Int-a. [0171] Preferred stereochemistry of the compounds of formula VI-Int-b is:
wherein R1 and R8 are as defined above for formula VI-Int-b. 5 [0172] Preferred stereochemistry of the compounds of formula VI is:
wherein R8 is as defined above for formula VI. Abbreviations Alloc Allyloxycarbonyl 10 Boc t-Butyloxycarbonyl Cbz Benzyloxycarbonyl Cys Cysteine EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide DMAP 4-dimethylaminopyidine 15 DMSO Dimethylsulfoxide Fm 9-Fluorenylmethyl Fmoc 9-Fluorenylmethyloxycarbonyl HPLC-DAD High Performance Liquid Chromatography Diode Array Detector MEM Methoxyethoxymethyl 20 MOM Methoxymethyl Relative retention time THF Tetrahydrofuran TLC Thin Layer Chromatography TROC 2,2,2-trichloroethyloxycarbonyl
40 [0173] The present invention is further described in the following non-limiting examples. EXAMPLES Example 1. Synthesis of compound 2 and effect of water addition on the amount of impurity 5. [0174] a) A solution of compound 1 in dichloromethane (12 mL/g) was treated at -15 º C with EDC (1.8 5 equiv.), DMAP (0.7 equiv.) and Boc-L-Cys(Fm)-OH (2.6 equiv., with a 0.2% humidity determined by Karl- Fisher). The reaction mixture was warmed up to – 5 ºC and stirred at that temperature for 1 hour. The reaction was quenched by addition of water (10 mL). The separated organic phase was washed sequentially with saturated aqueous NH4Cl (10 mL) and brine (10 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was evaporated. The crude was analysed by HPLC detecting a 12.1% area 10 of compound 5. [0175] b) A solution of compound 1 in dichloromethane (12 mL/g) was treated at -15 ºC with EDC (1.8 equiv.), DMAP (0.7 equiv.) and Boc-L-Cys(Fm)-OH (2.6 equiv., with a 2% humidity determined by Karl- Fisher). The reaction mixture was warmed up to – 5 ºC and stirred at that temperature for 1 hour. The reaction was quenched by addition of water (10 mL). The separated organic phase was washed sequentially 15 with saturated aqueous NH4Cl (10 mL) and brine (10 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was evaporated. The crude was analysed by HPLC detecting a 0.27% area of compound 5. [0176] c) A solution of compound 1 in dichloromethane (12 mL/g) was treated at -15 ºC with EDC (1.8 equiv.), DMAP (0.7 equiv.) and Boc-L-Cys(Fm)-OH (2.6 equiv., same batch used in Example 1(a) (spiked 20 with water, 2% w/w respect to Boc-L-Cys(Fm)-OH), The reaction mixture was warmed to – 5 ºC and stirred at that temperature for 1 hour. The reaction was quenched by addition of water (10 mL). The separated organic phase was washed sequentially with saturated aqueous NH4Cl (10 mL) and brine (10 mL). The organic phase was dried over anhydrous Na2SO4, filtered and the solvent was evaporated. The crude was analysed by HPLC detecting a 0.25% area of compound 5. 25 [0177] These results demonstrate that the formation of compound 5 can be controlled by modulating the presence of water in the reaction mixture. Example 2 Scale-up of the synthesis of compound 2 [0178] A solution of compound 1 (220.6 g) in dichloromethane (1.76 L) in a 10 L reactor under Argon atmosphere was treated with a mixture of EDC·HCl (139.2 g) and water (7 mL). The reaction mixture was30 cooled to -15 ºC and DMAP (36.5 g) was added followed by the addition of a solution of Boc-L-Cys(Fm)- OH (342.9 g) in dichloromethane (650 mL) for 30 min while maintaining the reaction temperature below - 10 ºC. Once the addition has been finished, the reaction mixture was agitated for 60 – 80 min at -8 ºC. The reaction was quenched by addition of water (2150 mL). The organic phase was decanted and washed with aqueous NH4Cl (2.1 L; prepared by dissolving 748 g of NH4Cl in 2.2 L of water), aqueous NaCl (2.1 L; 35 prepared by dissolving 616 g of NaCl in 2.2 L of water). If the pH of the aqueous phase is lower than 5, the
41 washing with aqueous NaCl is repeated. If the pH of the aqueous phase is between 5 to 7, the organic layer was decanted and evaporated under vacuum to dryness. The resulting foam was dissolved in methanol (2660 mL) and evaporated to dryness to remove traces of dichloromethane and re-dissolved in methanol (9.28 L). The resulting methanolic solution was treated at 0 ºC with water (3.4 L) for 30 min. The resulting 5 suspension was filtered over a number 3 or 4 filter plate, washed with water (12.0 L) to give a solid that was dried under vacuum at 35 ºC for at least 36 h to give 366.1 g of solid compound 2 that was employed in the next synthetic step without further purification. Conclusions [0179] The formation of impurity 5 has been significantly reduced in a reproducible way by adding a 10 controlled amount of water to the reaction mixture. This avoids the carry-over of impurity 5 through the synthetic route and the formation of more impurities derived from it such as impurity 6. [0180] A precipitation process has been developed to obtain compound 2 in solid form. This process significantly reduces the amount of the impurity Boc-L-Cys(Fm)-OH with respect to compound 2 to a level below 6% w/w. 15 Example 3 Stability of compound 2 a) in dichloromethane solution [0181] Two solutions of compound 2 in dichloromethane were stored, respectively, at 5 º C and 22 ºC for 14 days. The solutions were analysed at day 0, 5 and 14. The stability results are summarised in Table 1. Table 1
20 [0182] These results demonstrate that a solution of compound 2 in dichloromethane is stable at 5 ºC – 22 ºC for at least 14 days. b) In solid state At -20 ºC 25 [0183] Three batches of compound 2 stored at -20 ºC were analysed after 4, 5 and 6 months, respectively, and no degradation was observed during this time period. Therefore, solid compound 2 is stable at -20 ºC for at least 6 months. At 30 ºC
42 [0184] A sample of compound 2 was dried at 30 ºC under vacuum for up to 30 h and no degradation was observed during this period. The results are summarised in Table 2. Table 2 Stability of solid compound 2 at 30 ºC
5 At 60 ºC [0185] A sample of compound 2, with a 6.8% of Boc-L-Cys(Fm)-OH, was stored at 60 ºC for 3 days. At the third day a 6% of degradation was observed. The results of this analysis are summarised in Table 3. Table 3
10 Conclusions: [0186] A dichloromethane solution of compound 2 obtained as described in Example 2 is stable at 5 ºC - 22 ºC for at least 14 days. [0187] Solid compound 2 obtained as described in Example 2 is stable at 30 ºC for at least 72 h and at - 20 ºC for at least six months. 15 [0188] The process of the present invention allows the isolation of solid compound 2 that is stable both in solid state and in dichloromethane solution. Example 4 Development of a new process for the manufacture of compound 3. [0189] Although the synthesis of compound 3 disclosed in WO 01/87895 has a good yield (87%), it requires the use of a large excess of MEMCl (15 equiv.) to complete the reaction. This excess results in the 20 formation of polyether subproducts with 1H-NMR signals in the range 4.7-3.8 ppm and not detectable by conventional HPLC-DAD chromatography. These subproducts must be removed at that stage because otherwise they would be carried over during next synthetic steps. In addition, MEMCl is a toxic alkylating agent that may cause cancer. For both reasons, it is highly desirable to reduce the amount of MEMCl that
43 is required to complete the synthesis of compound 3 while the reaction yield is maintained or improved and the impurity profile of compound 3 is kept under control. [0190] Attempts to reduce the number of equivalents of MEMCl in presence of DIPEA and DMAP resulted in low conversions and/or degradation. 5 [0191] Several experimental conditions were tested to achieve the synthesis of compound 3 using a lower amount of MEMCl. They are summarized in Table 4: Table 4
[0192] wherein compounds 7 and 8 have the following structures: 10
[0193] The best conversion for this set of experiments was achieved with 1.5 equivalents of MEMCl and NaOH (1 equiv.) as base in THF, however significant degradation was observed.
44 [0194] The effect of the order of addition was studied in the following experiments. a) Addition of NaOH to a mixture of compound 2 and MEMCl. [0195] To a solution of compound 2 (3 g, 3.33 mmol) in THF (90 mL) at -10 ºC was added MEMCl (0.57 mL, 4.99 mmol, 1.5 equiv.) and NaOH (0.40 g, 10.0 mmol, 3 equiv.) as a solid in portions. After stirring 5 the reaction mixture for 6 h, the reaction was quenched with saturated aqueous NH4Cl (90 mL) and dichloromethane (90 mL). The organic layer was separated, washed with brine (90 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. b) Addition of compound 2 to a mixture of NaOH and MEMCl [0196] To a suspension of NaOH (270 mg, 6.7 mmol, 3 equiv.) in THF (36 mL) at -15 ºC was added 10 MEMCl (0.76 mL, 6.7 mmol, 3 equiv.) and a solution of compound 2 (2 g, 2.22 mmol) in THF (4 mL) that was added dropwise. After the addition was finished, the reaction mixture was stirred at -10 ºC for 30 minutes. The reaction was quenched with saturated aqueous NH4Cl (30 mL) and dichloromethane (40 mL). The organic phase was separated, washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. 15 c) Addition of MEMCl to a mixture of compound 2 and NaOH [0197] To a suspension of NaOH (270 mg, 6.7 mmol, 3 equiv.) in THF (36 mL) at -15 ºC was added dropwise a solution of compound 2 (2 g, 2.22 mmol) in THF (4 mL) and MEMCl (0.76 mL, 6.7 mmol, 3 equiv.). After the addition was finished, the reaction mixture was stirred at -10 ºC for 30 minutes. The reaction was quenched with saturated aqueous NH4Cl (30 mL) and dichloromethane (40 mL). The organic 20 phase was separated, washed with brine (30 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. [0198] Analytical results of experiments a) – c) are summarized in Table 5 Table 5
25 [0199] wherein impurity 1-MEM has the following formula:
45
d) Effect of the number of equivalents of MEMCl, NaOH, concentration and temperature over the synthesis of compound 3. [0200] A solution of compound 2 (1.3 – 4.0 g) obtained as described in Example 2, in THF was added 5 dropwise at -15 ºC to a mixture of NaOH (equiv. as indicated below) and MEMCl (equiv. as indicated below). After the addition was finished, the reaction mixture was stirred at the indicated temperature during at least 1.5 h. Analytical data, expressed as% of area, from the reaction crudes at times 0.5 h and 1.5 h is provided in Table 6. Table 6
10 e) Effect of water in the synthesis of compound 3. e1) with dry NaOH. [0201] During the development of the synthesis of compound 3, it was observed that the reaction kinetics was significantly influenced by the presence of water in the reaction media.
46 [0202] NaOH powdered under nitrogen atmosphere was used as base in the synthesis of two batches of compound 3 by reacting compound 2, with a water content of less than 1% w/w, with MEMCl (Batches 1 and 2). Since the reaction did not progress significantly after the first 40-60 min, water was added in several portions to the reaction mixture. The reaction kinetics is showed in Figure 1. A clear acceleration of the 5 formation of compound 3 was observed when water was first-time added. Further additions of water allowed the completion of the reaction. e2) with controlled water content in the NaOH, MEMCl suspension [0203] The effect of the amount of water in the NaOH, MEMCl suspension in THF was tested in three experiments, with 2.5%, 4% and 5%. of water (w/w relative to compound 2), respectively. 10 [0204] Micro pearls of NaOH (42 g) were triturated in a dry box under N2 atmosphere. This triturated NaOH (780 mg, 61-89% particles between 100 μm and 200 μm) was added to a 250 ml jacketed reactor under N2 atmosphere and suspended in a solution of water in tetrahydrofuran (123 mL) wherein the amount of water in each experiment was, respectively, 2.5% w/w, 4% w/w and 5% w/w regarding the amount of compound 2 initially present. The resulting suspension was cooled to -15 ºC and treated sequentially with 15 MEMCl (2.4 mL) and a solution of compound 2 (6.86 g) in tetrahydrofuran (14 mL) while maintaining the reaction mixture at less than -10 ºC during the additions. Once the addition was finished, the temperature of the jacket coolant was raised to – 7 ºC. Water (275 μL, 4% w/w versus the amount of compound 2 initially present) was added 40 minutes after the addition of compound 2 started and the reaction was quenched with aqueous saturated NH4Cl 90 min after the addition of compound 2 started and extracted with 20 dichloromethane (140 mL). The organic layer was decanted and washed with diluted brine (96 mL) and brine (x2, 100 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum. Aliquots of the reaction mixture were taken 40 min and 60 min after the addition of compound 2 started and analyzed by HPLC-DAD chromatography. Reaction kinetics are showed in Figure 2. [0205] The poor performance observed in the experiment with 5% w/w water in the NaOH suspension 25 was due to NaOH aggregation. Example 5 Scale-up of the synthesis of compound 3 [0206] A solution of compound 2 (366.1 g, 406 mmol, with less than 1% w/w water content) in THF (740 mL) under Argon atmosphere was added during 15 – 25 min to a suspension of pulverized NaOH (41.5 g, 1.035 mol, with about 3% w/w water content regarding compound 2) and MEMCl (128 mL, 1.121 mol) in 30 THF (6.6 L) at -15 ºC. During the addition the reaction temperature must not be higher than -10 ºC. After the addition the reaction mixture is allowed to warm to -7 ºC and agitated for 40-45 min. The reaction mixture was treated with water (15 mL, 4% w/w regarding compound 2) and agitated for 20 min. The reaction mixture was quenched by adding cold aqueous NH4Cl (5L, prepared by dissolving NH4Cl (1875 g) in water (5.52 L)) and extracted with dichloromethane (7.36 L). The organic layer was decanted and 35 washed sequentially with water (3.50 L), aqueous NaCl (1.7 L, prepared from NaCl (1282 g) and water (5.5 L) and twice with aqueous NaCl (2 x 5 L). The pH of the aqueous phase was adjusted between 5 and 7. The
47 organic layer was decanted and dried over Na2SO4 (739 g) and evaporated under vacuum to dryness, dissolved in toluene (1L) and evaporated to give 437.3 g of crude compound 3 that was used in next step without further purification. Conclusions 5 [0207] A significant reduction in the number of equivalents of MEMCl required to complete the reaction has been achieved. This avoids the formation of polyether impurities and simplifies the working up of the reaction. The reaction kinetics and the formation of new impurities is controlled by adjusting the number of equivalents of MEMCl, NaOH and water. Example 6 Stability of crude compound 3. 10 [0208] Two dichloromethane solutions of crude compound 3, obtained before co-evaporation in toluene, were stored at 5 ºC and 22 ºC, respectively for six days. Their chromatographic profiles at day 0 and day 6 are summarised in Table 7. The toluene signal has been omitted. Table 7
15 [0209] Two concentrated toluene solutions of crude compound 3, obtained after co-evaporation in toluene, were stored at 5 ºC and 22 ºC, respectively, for six days. Their chromatographic profiles at day 0 and at day 6 are summarised in Table 8. The toluene signal has been omitted. Table 8
20 Conclusions [0210] A solution of crude compound 3, obtained as described in Example 5, in dichloromethane was stable 6 days at 5 ºC and at 22 ºC.
48 [0211] A concentrated solution of crude compound 3, obtained as described in Example 5, after co- evaporation in toluene was stable 5 days at 5 º C and at 22 ºC. No isolation of compound 3 is required and its dichloromethane solution is employed in the next step. Example 7 Development of a new process for the manufacture of compound 4. 5 [0212] The de-allylation process employed for the synthesis of compound 4 described in WO01/87895 has a very good yield (95%), but it employs a toxic reagent (Bu3SnH) that generates sub-products such as Bu3SnSnBu3 that are difficult to remove. [0213] Bu3SnH was replaced with a secondary amine such as diethylamine with good results when the reaction was carried out in dichloromethane at about 40 ºC. These results are summarized in Table 9. 10 Table 9
Example 8 Scale-up of the synthesis of compound 4 [0214] Crude compound 3, obtained in example 5, was dissolved in dichloromethane under Argon atmosphere (6L). Pd(PPh3)4 (28.6 g) and diethylamine (425 mL) were added sequentially. The reaction 15 mixture was agitated at 39 ºC for 60 min. The reaction mixture was cooled to 0 ºC and treated with cold 1M HCl (6.58 L) maintaining the temperature during the addition below 10 ºC. The organic layer was decanted and washed sequentially with water (6.0 L) and aqueous NaCl (6.0 L). If the pH of the aqueous phase is lower than 5, the washing step with aqueous NaCl is repeated and the organic layer is decanted and filtered through a nº 4 filter plate. The resulting filtrate was utilized in the next reaction step without 20 further purification. Conclusions [0215] A toxic and difficult to remove reagent has been successfully replaced. The acidic washing allowed the removal of diethylamine salts. The decrease in chromatographic purity of compound 4 after the acidic treatment described in the previous example was controlled by keeping the temperature below 10 ºC during 25 the acidic treatment. Example 9 Stability of crude compound 4.
49 [0216] A dichloromethane solution of compound 4 stored at -20 ºC for 14 days to evaluate its stability in solution. Its chromatographic profile at days 0, 9 and 14 is summarised in Table 10. The toluene signal has been omitted. Table 10
5 nd. Not detected [0217] Impurities 9,10 and 11 have the following structures:
. [0218] Two solutions of crude compound 4 in dichloromethane were stored at 5 ºC and 22 ºC, respectively, for 5 days to evaluate its stability in solution. Their chromatographic profiles at days 0 and 5 10 are summarized in Table 11. The toluene signal has been omitted. Table 11
50 References EP309477 WO 03/66638 WO 03/08423 5 WO 01/77115 WO 03/014127 Sakai, R. et al., 1992, Proc. Natl. Acad. Sci. USA 89, pages 11456-11460 Menchaca, R. et al., 2003, J. Org. Chem.68(23), pages 8859-8866 Manzanares, I; et al., 2001, Curr. Med. Chem. Anti-Cancer Agents, 1, pages 257-276 10 WO 01/87895 WO 2020/155613 Wuts, P.G.M. in Greene’s Protective Groups in Organic Synthesis, 5th Ed. Wiley Kocienski P.J. in Protecting Groups, 3rd Ed. Georg Thieme Verlag Cragg et al. Anticancer Agents from Natural Products (2012), 2nd Edition – Chapter 12: “Ecteinascidin-743 15 (Yondelis®), Aplidin®, and Irvalec® (pages 291-316) WO 00/69862 WO 2011/147828 WO 2018/197663 WO2025/077996
51 CLAUSES 1. A process for the synthesis of an ecteinascidin compound or a pharmaceutically acceptable salt thereof, the process comprising a step of coupling compound 1 with a compound of Formula A, or a stereoisomer thereof, to give a compound of Formula I: 5
wherein: R6 is ProtNH, wherein ProtNH is a protecting group for amino; R7 is ProtSH, wherein ProtSH is a protecting group for SH; and the coupling agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or a salt thereof, such as its 10 hydrochloride (EDC·HCl), in presence of 4-dimethylaminopyridine (DMAP) and water, where the total amount of water (w/w) in the reaction mixture versus the amount of compound 1 initially present is from about 2.5% to about 6%. 2. The process according to clause 1, wherein R6 together with the NH group to which it is attached form a carbamate group. 15 3. The process according to clause 2, wherein R6 together with the NH group to which it is attached form a carbamate group selected from allylcarbamate, 2,2,2-trichloroethylcarbamate, benzylcarbamate, 9- fluorenylmethylcarbamate and t-butylcarbamate. 4. The process according to clause 3, wherein R6 together with the NH group to which it is attached form a t-butylcarbamate. 20 5. The process according to any preceding clause, wherein R7 together with the S atom to which it is attached form a thioether group. 6. The process according to clause 5, wherein R7 is S-9-fluorenylmethyl (Fm). 7. The process according to any preceding clause, wherein the compound for formula A is:
52
, or a stereoisomer thereof. 8. The process according to clause 7, wherein the compound of formula A is:
. 9. The process according to any preceding clause, wherein the coupling agent is 1-ethyl-3-(3- 5 dimethylaminopropyl)-carbodiimide hydrochloride (EDC·HCl). 10. The process according to any preceding clause, wherein the total amount of water (% w/w) to compound 1 in the reaction mixture is from about 2.5% to about 5%. 11. The process according to clause 10, wherein the total amount of water (% w/w) to compound 1 in the reaction mixture is from about 2.5% to about 4%. 10 12. The process according to clause 11, wherein the total amount of water (% w/w) to compound 1 in the reaction mixture is from about 2.5% to about 3.5%. 13. The process according to clause 12, wherein the total amount of water (% w/w) to compound 1 in the reaction mixture is about 3%. 14. The process according to any preceding clause, wherein the number of equivalents of the 15 compound of formula A with respect to compound 1 is from about 2 to about 3. 15. The process according to clause 14, wherein the number of equivalents of the compound of formula A with respect to compound 1 is from about 2 to about 2.5. 16. The process according to clause 15, wherein the number of equivalents of the compound of formula A with respect to compound 1 is from about 2 to about 2.1. 20 17. The process according to clause 16, wherein the number of equivalents of the compound of formula A with respect to compound 1 is about 2.02. 18. The process according to any preceding clause, wherein the number of equivalents of the coupling agent with respect to compound 1 is from about 1.0 to about 1.8. 19. The process according to clause 18, wherein the number of equivalents of the coupling agent with 25 respect to compound 1 is from about 1.5 to about 1.8. 20. The process according to clause 19, wherein the number of equivalents of the coupling agent with respect to compound 1 is about 1.7.
53 21. The process according to any preceding clause, wherein the number of equivalents of 4- dimethylaminopyridine (DMAP) with respect to compound 1 is from about 0.65 to about 0.75. 22. The process according to clause 21, wherein the number of equivalents of 4- dimethylaminopyridine (DMAP) is about 0.7. 5 23. The process according to any preceding clause, wherein the coupling step is carried out at from about -10 ºC to about -6 ºC. 24. The process according to clause 23, wherein the coupling step is carried out at from about -9 ºC to about -7 ºC. 25. The process according to clause 24, wherein the coupling step is carried out at -8 ºC. 10 26. The process according to any preceding clause, wherein the concentration of compound 1 is from about 0.04 g/mL to about 0.14 g/mL. 27. The process according to clause 26, wherein the concentration of compound 1 is from about 0.04 to about 0.10 g/mL. 28. The process according to clause 27, wherein the concentration of compound 1 is from about 0.07 15 to about 0.09 g/mL. 29. The process according to clause 28, wherein the concentration of compound 1 is about 0.08 g/mL. 30. The process according to any preceding clause, wherein the reaction time is from about 30 to about 120 min. 31. The process according to clause 30, wherein the reaction time is from about 55 to about 65 min. 20 32. The process according to clause 31, wherein the reaction time is about 60 min. 33. The process according to any preceding clause wherein the coupling step is carried out in a halogenated solvent. 34. The process according to clause 33, wherein the coupling step is carried out in dichloromethane. 35. The process according to any preceding clause, wherein the coupling step is quenched with water 25 and the resulting mixture is washed sequentially with saturated aqueous NH4Cl and saturated aqueous NaCl. 36. The process according to clause 35, wherein the pH of the resulting aqueous phase is from about 5 to about 7. 37. The process according to any preceding clause, wherein the resulting organic layer is dried, evaporated, dissolved in methanol and re-evaporated to give a foam.
54 38. The process according to clause 37, wherein the foam is dissolved in methanol and treated with water to precipitate the compound of formula I. 39. The process according to clause 38, wherein the amount of water added to the methanol solution (v/v) is from about 25 to 30% v/v. 5 40. The process according to clause 39, wherein the amount of water added to the methanol solution is about 27% v/v. 41. The process according to any one of clauses 38 to 40, wherein the concentration of the compound of formula I in the mixture methanol/water is from about 20 g/L to about 40 g/L. 42. The process according to clause 41, wherein the concentration of the compound of formula I is 10 from about 25 g/L to about 35 g/L. 43. The process according to clause 42, wherein the concentration of the compound of formula I is about 30 g/L. 44. The process according to any one of clauses 38 to 43, wherein the precipitation is carried out at from about 0 ºC to about 25 ºC. 15 45. The process according to clause 44, wherein the precipitation is carried out at from about 0 ºC to about 10 ºC. 46. The process according to clause 45, wherein the precipitation is carried out at about 0 ºC. 47. The process according to any one of clauses 38 to 46, wherein the precipitated compound of formula I is dried at 30 ºC for three days under vacuum. 20 48. The process according to clause 47, wherein the water content of the precipitated compound of formula I after drying is about 1% w/w. 49. The process according to any one of clauses 38 to 48, when dependent on clause 8, wherein the amount of impurity Boc-L-Cys(Fm)-OH relative to the precipitated compound of formula I is below 6% w/w. 25 50. A process for the synthesis of an ecteinascidin compound, or a pharmaceutically acceptable salt thereof, the process comprising a step of protecting the phenolic group in the compound of formula I by reacting it with a compound of formula R1X in presence of a base MOH and water in tetrahydrofuran to give a compound of formula II:
55
wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; R6 is ProtNH; 5 R7 is ProtSH; X is selected from Cl, Br and I; and MOH is selected from sodium hydroxide, potassium hydroxide and lithium hydroxide. 51. The process according to clause 50, wherein the compound of formula I used in the manufacture of the compound of formula II has been obtained as described in any one of clauses 1 to 49. 10 52. The process according to clause 50 or 51, wherein R1 is selected from methoxymethyl (MOM) and methoxyethoxymethyl (MEM). 53. The process according to clause 52, wherein R1 is methoxyethoxymethyl (MEM). 54. The process according to any one of clauses 50 to 53, wherein X is Cl or Br. 55. The process according to clause 54, wherein X is Cl. 15 56. The process according to any one of clauses 50 to 55, wherein MOH is powdered MOH. 57. The process according to clause 56, wherein the mean particle size of powdered MOH is from about 50 µm to about 500 µm. 58. The process according to clause 57, wherein the mean particle size of powdered MOH is from about 60 µm to about 300 µm. 20 59. The process according to clause 58, wherein the mean particle size of powdered MOH is from about 75 µm to about 250 µm. 60. The process according to clause 59, wherein the mean particle size of powdered MOH is from about 100 µm to about 200 µm.
56 61. The process according to clause 56, wherein about 60% to about 90% of the particles of powdered MOH have a particle size from about 100 μm to about 200 μm. 62. The process according to any one of clauses 50 to 61, wherein MOH is NaOH. 63. The process according to any one of clauses 50 to 62, wherein the number of equivalents of MOH 5 with respect to the compound of formula I is from about 2 to about 3. 64. The process according to clause 63, wherein the number of equivalents of MOH is from about 2.3 to about 2.7. 65. The process according to clause 64, wherein the number of equivalents of MOH is from about 2.4 to about 2.6. 10 66. The process according to clause 65, wherein the number of equivalents of MOH is about 2.55. 67. The process according to any one of clauses 50 to 66, wherein the number of equivalents of R1X with respect to the compound of formula I is from about 1.9 to about 3.3. 68. The process according to clause 67, wherein the number of equivalents of R1X is from about 2.2 to about 3.0. 15 69. The process according to clause 68, wherein the number of equivalents of R1X is from about 2.5 to about 2.9. 70. The process according to clause 69, wherein the number of equivalents of R1X is about 2.8. 71. The process according to any one of clauses 50 to 70, wherein a solution of the compound of formula I is added to a suspension of MOH and R1X. 20 72. The process according to clause 71, wherein the temperature of the reaction mixture during the addition is maintained below -10 ºC. 73. The process according to any one of clauses 50 to 72, wherein the protection step is carried out at a temperature from about -7 ºC to about -3 ºC once the addition of the compound of formula I has finished. 74. The process according to clause 73, wherein the protection step is carried out at a temperature 25 from about -6 ºC to about -4 ºC. 75. The process according to clause 74, wherein the protection step is carried out at about -5 ºC. 76. The process according to any one of clauses 50 to 75, wherein the total amount of water in the reaction mixture when the reaction is completed is from about 7% w/w to about 12% w/w versus the amount of the compound of formula I initially present.
57 77. The process according to clause 76, wherein the total amount of water in the reaction mixture when the reaction is completed is from about 7% w/w to about 11 % w/w regarding compound of formula I. 78. The process according to clause 77, wherein the total amount of water in the reaction mixture 5 when the reaction is completed is from about 7% w/w to about 10% w/w regarding compound of formula I. 79. The process according to clause 78, wherein the total amount of water in the reaction mixture when the reaction is completed is from about 7% w/w to about 9% w/w regarding compound of formula I. 80. The process according to clause 79, wherein the total amount of water in the reaction mixture 10 when the reaction is completed is from about 7% w/w to about 8% w/w regarding compound of formula I. 81. The process according to any one of clauses 71 to 80, wherein the water content in the solution of the compound of formula I is below 1% w/w regarding compound of formula I. 82. The process according to any one of clauses 71 to 81, wherein the suspension of MOH and R1X in tetrahydrofuran has a water content from about 2% w/w to about 4.5% w/w regarding compound of 15 formula I. 83. The process according to any one of clauses 71 to 82, wherein the suspension of MOH and R1X in tetrahydrofuran has a water content from about 2.5% w/w to about 4 % w/w regarding compound of formula I. 84. The process according to any one of clauses 71 to 83, wherein the water content of the suspension 20 of MOH and R1X in tetrahydrofuran has a water content from about 2.5% w/w to about 3.5% w/w regarding compound of formula I. 85. The process according to any one of clauses 71 to 84, wherein the water content of the suspension of MOH and R1X in tetrahydrofuran has a water content of about 3% w/w regarding compound of formula I. 25 86. The process according to any one of clauses 50 to 85, wherein water is added in one of more portions to the reaction mixture until it is completed. 87. The process according to any one of clauses 76 to 80 or 81 to 86, when dependent on any one of clauses 76 to 80, wherein the reaction is completed when the amount of the compound of formula I present in the reaction mixture is about 1% or lower than 1% of its initial amount determined by semiquantitative 30 TLC or by HPLC-DAD chromatography. 88. The process according to any one of clauses 50 to 75, wherein if the reaction is not completed in 30 min after the addition of the compound of formula I, then water is added in an amount of from about 4% to about 8% (w/w) regarding the initial amount of the compound of formula I.
58 89. The process according to clause 88, wherein the amount of water added is about 4% w/w regarding compound of formula I. 90. A process for the synthesis of an ecteinascidin compound, or a pharmaceutically acceptable salt thereof, comprising a step of de-allylating the compound of formula II to provide a compound of Formula 5 III:
with a palladium catalyst in presence of a secondary amine as reducing agent; wherein R1 is selected from alkoxymethyl and alkoxyalkoxymethyl; 10 R6 is ProtNH; and R7 is ProtSH. 91. The process according to clause 90, wherein the compound of formula II used in the manufacture of the compound of formula III has been obtained as defined in any one of clauses 50 to 89. 92. The process according to clause 90 or 91, wherein the palladium catalyst is a palladium (0) 15 catalyst. 93. The process according to clause 92, wherein the palladium catalyst is Pd(PPh3)4. 94. The process according to clause 90 or 91, wherein the palladium catalyst is a palladium (II) catalyst. 95. The process according to clause 94, wherein the palladium catalyst is PdCl2(PPh3)2. 20 96. The process according to any one of clauses 90 to 95, wherein the number of equivalents of the palladium catalyst is from about 0.03 to about 0.1. 97. The process according to clause 96, wherein the number of equivalents of the palladium catalyst is from about 0.04 to about 0.08.
59 98. The process according to clause 97, wherein the number of equivalents of the palladium catalyst is about 0.06. 99. The process according to any one of clauses 90 to 98, wherein the secondary amine is diethylamine. 5 100. The process according to any one of clauses 90 to 99, wherein the deprotection is carried out at from about 30 ºC to about 50 ºC. 101. The process according to clause 100, wherein the deprotection is carried out at 40 ºC. 102. The process according to any one of clauses 90 to 101, wherein the reaction mixture, after the reaction is completed, is cooled at about 0 ºC before quenching it with aqueous HCl 1M. 10 103. The process according to any one of clauses 90 to 102, wherein the deprotection reaction is carried out in dichloromethane. 104. The process according to any one of clauses 1 to 103, wherein the ecteinascidin compound is selected from: 15