GOVERNMENT SUPPORT

Certain of the inventors were supported by National Institutes of Health Grant GM29028.

FIELD OF THE INVENTION

This invention relates to methods for the preparation of lactam- and lactone-containing macrocycles εuch as rapamycin and demethoxyrapamycin, and to intermediates useful in their preparation.

BACKGROUND OF THE INVENTION

Rapamycin and demethoxyrapamycin are two members of a growing class of acrolidε natural products possessing marked immunosuppressive properties . R-e-c-srtly, several groups have focused on the preparation of both modest and advanced fragments of the polyketide skeleton, culminating in three total syntheses of rapamycin. In addition, several groups have prepared semi-synthetic analogs of rapamycin to improve upon its impressive therapeutic profile as well as to gain insight into the as-yet-unresolved mechanism of action. To date, four research groups have reported the discovery and isolation of a 220 k_Da protein which is thought to be the direct intracellular target of the rapam.ycir.-FK3? complex. This protein shares structural homolo y with a number of known liσid kinases although its soecific role in siσnal

transduction and immunosuppression remains unclear. It has, however, been established that rapamycin interferes with a Ca ² '- independent signaling pathway emanating from the IL-2 receptor, thus prohibiting the progression of activated T cells from the Gl to the S phase of the cell cycle, perhaps via indirect inhibition of a cyclin dependent kinase specifically required for this transition.

There is a need for improved synthetic methods for the preparation of rapamycins. This invention is directed to this important end.

OBJECTS OF THE INVENTION

It is one object of the present invention to provide lactam- and/or lactone-containing macrocycles;

It is a further object to provide processes for the preparation cf rapamycin, demethoxyrapamycin, and C-27 epirapamycin.

It is another object of this invention to provide intermediates usef l in the processes.

SUMMARY OF THE INVENTION These and other objects are satisfied by the present invention, which provides synthetic methods for the preparation of macrocycles, and novel compounds useful in the syntheses.

In certain embodiments, methods are provided for the Dreoaration of a comDOund havinσ formula (VI-I)

vπ comprising the steps of: providing a first compound of formula IV:

and contacting said compound with a compound of formula (VI) :

VI for a time and under reaction conditions effective to form said compound of formula VII; wherein:

R _1# R ₄ , and R, are, independently, hydrogen or a hydroxyl protecting group;

R, is alkyl having one to about six carbons;

R ₇ is hydrogen or alkoxy having one to about six carbons; is H, OH, or alkoxy having one to about six carbons;

R _2X is alkyl having one to about six carbons;

M is a metal atom; and X is halogen.

In preferred embodiments R ₇ is H or methoxy, and M is Sn.

Preferably the hydroxyl protecting groups are selected from the group consisting of TIPS, FMBO, TESO and TBS. More preferably, R, _, is TIPS; R, is K; R ₄ is TBS; and R, is TES.

Preferred embodiments further comprise treating said compound of formula VII for a time and under reaction conditions effective to form a compound of formula XV:

XV

Preferably, the protecting groups are then removed. Also provided is a method for the preparation of a comoound of formula IV:

comprising the steps of: providing a compound of formula II

II MeO

treating said compound of formula II for a time and under conditions effective to form a terminal acetylene of formula III:

m and treating said terminal acetylene for a time and ^• under conditions effective to form said compound of formula IV.

In some preferred embodiments said compound of formula II is formed by the reaction of an epoxide of formula V:

and a reagent cf formula XVI

XVI

In other preferred embodiments said compound of formula II is formed by the reaction of an aldehyde of formula XIII:

and a reaσent of formula XVI

XVI

Also provided is a method for the preparation of a diviπyl halide of formula VI:

VI

comprising the steps of : providing an orthoester of formula IX;

I X

treating said orthoester for a time and unds conditions effective to form an aldehyde cf formula X;

contacting said aldehyde with 2-carboxy-N- acetoylpiperidine for a time and under conditions effective to form a comoound of formula XII;

XII

and treating said compound cf formula XII for a time and under conditions effective to form said divinyl halide of formula VI.

Also provided according to the invention are novel intermediates, useful in the methods of the invention, having formula I:

wnerein:

_t , R _j and R _« are independently H or a hydroxyl protecting group;

R, is alkyl having from one to six carbons; R ₇ is H or alkoxy; R. _j is carbonyl group or protected derivative thereof;

R has the formula -CK(OCKj) ₂ , -CβC-R ₁₂ or cis-CH-CK- M(πC ₄ H,),; wherein:

R _l2 is H or trimethylsilyl; and M is a metal atom.

In certain preferred embodiments R ₇ is H, and in other preferred embodiments R ₇ is methoxy.

Preferably, R _X1 has the formula -CK(0CKj) ₂ , -C_BC-R, ₂ or cis-CH«CH-M(πC ₄ H,),; and M is Sn. The compound of claim 6 wherein Rj is H.

In certain preferred embodiments R, _l is -CH(OCHj) ₂ or -CβC-R _X2 , and said protected derivative of said carbonyl group of said group R _l3 is a 1,3-dithiane.

Preferably, R ₂ is methyl; and said hydroxyl protecting groups are selected from the group consisting of TIPS, PMBO, TΞS and TBS. In particularly preferred embodiments R, is TIPS, R ₃ is PMB and R is TBS.

Also provided are compounds of formula V:

wherein R _{l f} R ₂ , R ₃ and are as defined above . Compound are also provided having formula VI

96/17816

- 12

wherein:

R, is H or a hydroxyl protecting group; R ₁₀ is H, OH or alkoxy having one to about six carbon atoms; and

X is halogen, preferably iodine.

In certain embodiments R, is H, and R ₁₀ is methoxy.

Also provided are compounds having formula VII:

vπ

wherein R _{l f} R ₂ , R , R ₇ , R, , R ₁₀ , R _2l and M are as defined above .

BRIEF DESCRIPTION OF THE DRAWINGS

The numerous objects ana advantages of the present invention may be better understood by those skilled in the art by reference to the accompanying figures, in which: Figure 1 shows a overview cf a synthesis cf the invention as applied to rapamycin and demethoxyrapamycin.

Figure 2 shows the preparation of compounds XV and VII from compounds IV and VI .

Figure 3 shows the preparation of fragment IV from precursor compound II.

Figure 4 shows exemplary synthetic routes for the derivation of compound II where R ₇ is H cr alkoxy.

Figure 5 shows the preparation of compound VI from fragment D. Figure 6 shows the formation cf compound II in the synthesis of demethoxyrapamycin.

Figure 7 shows the formation of compound II in the synthesis of rapamycin.

Figure 8 shows the synthesis of compound VI (fragment DE) from fragment D.

Figure 9 shows the completion of the synthetic routes to rapamycin and demethoxyrapamycin from compounds Ila and lib.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It has been found in accordance with the invention that the synthesis of macrocycles such as rapamycin and/or derivatives can be achieved by highly convergent synthetic procedures wherein fully functionalized fragments corresponding to carbons 21-34 of the rapamycin skeleton are coupled with pinecolinate-tricarbonyl fragments corresponding to carbons 1- 20, each fragment being available from building blocks A through E as outlined in Figure 1.

In preferred embodiments rapamycin or demethoxyrapamycin are prepared from compound VII, which is in turn prepared from precursor fragments IV and VI according to

Figure 2. Preferably, compound IV is first coupled to compound

VI by an inter olecular acylaticn to form compound VII. A

preferred condensation agent for the acylaticn is ethyl-3-(3- dimethylamino) -propyl carbcdiimide ^• KC1 (EDAC-KC1 _. , cimethylamiπo pyridine (DMA?) . Closure cf the ring in compound VII is preferably achieved by a Pd (0) -catalyzed Stille coupling. A preferred reagent for the coupling is (2- furylj?) ₂ PdCl,, diisopropylethylamine (DIPΞA) in DMF/T-. . After ring closure, the protecting groups are preferably removed by any cf several reagents known in the art to be suitable, for example tetrabutylammonium fluoride/acetic acid (TΞAF/AcOK) followed by KF-pyridine, pyridine in TKF.

The methods of the present invention allow flexibility in choice of substituents en the rapamycin skeleton. For example, R ₇ can be varied to provide different rapamycin derivatives. In one preferred embodiment R ₇ is hydrogen, and the resulting product is demethoxyrapamycin. Other similar substitutions may be mace at other positions in the rapamycin skeleton, for example, at C-16.

The preparation of fragment IV from common precursor compound II is shown in Figure 3. Compound II is first treated to unmask the aldehyde group by reaction with, for example, TsOH in acetone, and then subjected to Corey-Fuchs homologation to yield the terminal acetylene compound III. See, Corey, E.J. and Fuchs, P.L., Tetrahedron Lett. 1972 13 , 3769. Compound III is, in turn, treated to remove the protecting group at C-34. In preferred embodiments the protecting group at C-34 is FM30 (p-mεthoxybenzyloxy) , which is preferably removed oxidatively by, for example, DDQ (2, 3-dichloro-5, 6-dicyano-l, 4- benzocuinone) in CH ₂ C1 ₂ . This is followed by hydrolysis cf the dithiane group with, for example, Mel, CaC0 ₃ , and palladium(0) - mediated hydrostannylation. See Zhang, H.X. et al . , J. O ?. Cher, . 1990 55, 1857.

Compound II derives from two separate synthetic routes depending upon the nature of R ₇ . These synthetic routes are shewn in Figure 4. In the synthetic route leading, for example, to demethoxyrapamycin, fragment B from Figure 1 is coupled with the b tyliithiu derivative cf fragment A of Figure 1 tc produce precursor acetcnide VIII (fragment AS) . Fragments A

and B can be prepared according to the procedure of Smith, A.Ξ. et al., Te trahedron Le t ters 1994, 35 (28) , 4907-4910. Epoxide V is produced from compound VIII by standard techniques, such as, for example, unmasking with ca phorsulfcnic acid/methanol 5 (C≤A/MeOK) , tosylation, and epcxidaticn with K,C0 ₃ /MeOK. Compound II (R ₇ = K) is formed from the coupling of terminal epoxide V and compound XVI. Freferably, the coupling is performed at low temperature (i.e., -78°C) using an alkyllithium reagent, for example t-EuLi, in an appropriate solvent such as, for example, 10V (v/v) K ?A (hexamethylphosphoramide) /TKF. In the synthetic route leading to, for example, rapamycin, aldehyde XIII is first produced by coupling fragment A frcm Figure 1 with the lithium derivative cf dithiane compound XVII. Fragment C from Figure 1, prepared according to the procedure of Smith, A.B. et al . , Te trahedron Le t ters 1994, 35 (28) , 4911-14 is metallated with t-butyllithium and added to aldehyde XIII, and the product is methylated to give compound II (R = OCH ₃ ) . The preparation of compound VI is shown in Figure 5. Fragment D from Figure 1, prepared according to the procedure in Smith, A.B. et al. , Te trahedron Le t ters 1994, 35 (28) , 4911-14, is hydrolyzed with, for example, AcOH/TKF, and silated with, for example, 2 equivalents cf TESC1 ( t-butyldimethylsilyl chloride) , imidazole, DMF. The ester is reduced by, for example DIBAL (diisobutylaluminu hydride) reduction, and aldehyde species X is formed by subsequent oxidation, for example with DMSO and oxalyl chloride by the method of Swerπ. See, March, J. , Advanced Organic Chemistry Fourth Ed . Wiley & Sons New York, 1992 p. 1194.

Compound X is then condensed with the dianion cf (L) - N-acetylpipecolinic acid in the presence of a condensing agent such as, for example, lithium hexamethyldisilazide (LKMDS) . The products are treated with diazomethane followed by Des- Martin oxidation (5 equiv) to give the tricarbonyl species in accordance with the procedure developed by Golec, e t al . See, Batchelor, M.J. et al . , Te trahedron Le t t . , 1993, 34 , 167 ; Dees, D.B et al., J. Orσ. Che .. 1983 48 4155. Removal of the TBS σrouD at C-14 vields the hemiketal comoound XII, which is then

protected at the free hydroxyl groups by, for example, reaction with triethylsilyltrifluoro ethane sulfonate (TESOTf) . Compound VI (fragment DΞ) is then formed from compound XII by free radical hydrostannyla icn according to the procedure of Nicolaou, et al. , Syn thesis 1986, 453, using, for example BuSnK, 2,2' -azobisisccutrylcnitrile (AIBN) , tin-iodide exchange according to the procedure of Crisp, e t al . , Te trahedron Le t ters 1992, 33 (32) , 4S49, and subsequent conversion of the ester to the carboxylic acid by, for example, Lil in pyridine.

Compounds of the invention contain protecting groups. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Some representative protecting groups useful for protecting the for carbonyl functionality are 1,3-dithiane groups and dimethoxyacetal groups. Some representative protecting groups useful for protecting the for hydroxyl functionality are TIPS (triisopropylsilyl) , PM3 (p- ethoxybenzyl) , TBS (t-butyldimethylsilyl) , TΞS (triethylsilyl) and lower alkyl groups such as methyl. Other representative groups may be found in Greene, T.W. and Wuts, P.G.M., "Protective Groups in Organic Synthesis " 2d. Ed., Wiley & Sons, ^■ 1991. In preferred embodiments R _: is TIPS, R ₃ is PM3, R. is TBS, and R, is TES.

R ₂ is preferably hydrogen or alkyl having from one to about six carbons. Alkyl groups according to the invention include straight chain, branched, and cyclic hydrocarbons such as methyl, isopropyl, and cyclchexyl groups. Alkoxy groups are oxygen atoms having an alkyl group appended thereto. It will be recognized that a wide variety of compounds according to the invention can readily be prepared according to the methods of the invention.

Those skilled in the art will appreciate that numerous changes and modifications may be made to the preferred

embodiments of the invention and that such changes and modifications may be made without departing from the spirit cf the invention. It is therefore intended that the appended claims cover all equivalent variations as fall within the true soirit and scooe of the invention.