Lipase-catalyzed regioselective esterification of rapamycin: Synthesis of temsirolimus (CCI-779)

Article abstract of DOI:10.1021/ol0514395

(Chemical Equation Presented) A lipase-catalyzed acylation of the immunosuppressant rapamycin with complete regioselectivity is described. The method was successfully applied to the synthesis of 42-hemiesters and temsirolimus (CCI-779), an investigational oncology drug.

Full text of DOI:10.1021/ol0514395

ORGANIC
LETTERS
2005
Vol. 7, No. 18
3945-3948
Lipase-Catalyzed Regioselective
Esterification of Rapamycin: Synthesis
of Temsirolimus (CCI-779)
Jianxin Gu,* Mark E. Ruppen, and Ping Cai
Department of Bioprocess DeVelopment, Wyeth Research,
401 North Middletown Road, Pearl RiVer, New York 10965
guj2@wyeth.com
Received June 21, 2005
ABSTRACT
A lipase-catalyzed acylation of the immunosuppressant rapamycin with complete regioselectivity is described. The method was successfully
applied to the synthesis of 42-hemiesters and temsirolimus (CCI-779), an investigational oncology drug.
Rapamycin 1 is a 31-member macrocyclic polyketide first
isolated from Streptomyces hygroscopicus NRRL5491 as an
antifungal agent1 and later shown to be a immunosuppressive
and antiproliferative agent2,3 with a mechanism of action
different from that of cyclosporine A and FK-506.4 The
compound is currently approved for use in renal transplanta-
tion (sirolimus, Rapamune) and shows promise as a coating
for coronary stents to prevent restenosis following angio-
plasty.5
The biological activities of rapamycin are dependent on
the binding of the left-hand portion of the molecule, from
C8 to C31 (so-called “binding domain”), to FKBP12 (FK-
506 binding protein) and the subsequent formation of a
tertiary complex with mTor (mammalian target-of-rapamy-
cin) protein6 through the remaining portion of the macrolide
(so-called “effector domain”). This unique mode of action,
along with its recognized relationship to FK-506, has led to
a number of research programs focused on the identification
of rapamycin analogues with improved activity. A number
of different approaches have been taken, including biological
modifications such as precursor-directed biosyntheses7 and
biotransformations,8 semisynthesis, including four total syn-
theses,9 as well as synthetic modifications of different regions
through selective manipulation of functional groups.10 Ma-
nipulations of the binding domain such as selective reduction
of the C15 ketone of the “tricarbonyl” unit and the C27
ketone,11 switching the stereochemistry of C31-C33 hy-
droxyl ketone through a two-step retroaldol/aldol sequence,12
(7) Graziani, E. I.; Ritacco, F. V.; Summers, M. Y.; Zabriskie, T. M.;
Yu, K.; Bernan, V. S.; Greenstein, M.; Carter, G. T. Org. Lett. 2003, 5,
2385-2388.
(1) (a) Sehgal, S. N.; Baker, H.; Vezina, C. J. Antibiot. 1975, 28 (10),
721-726. (b) Singh, K.; Sun, S.; Vezina, C. J. Antibiot. 1979, 32 (6), 630-
645.
(2) Sehgal, S. N. Clin. Biochem. 1998, 31 (5), 335-340.
(3) Abraham, R. T. Cell 2002, 111, 9-12.
(4) Li, Y.-T.; Hsieh, Y.-L.; Henion, J. D.; Ocain, T. D.; Schiehser, G.
A.; Ganem, B. J. Am. Chem. Soc. 1994, 116, 7487-7493.
(5) Marx, S. O.; Marks, A. R. Circulation 2001, 104 (8), 852-855.
(6) Abraham, R. T. Curr. Opin. Immunol. 1998, 10 (3), 330-336.
(8) Kuhnt, M.; Bitsch, F.; Ponelle, M.; Fehr, T.; Sanglier, J. J. Enzyme
Microb. Technol. 1997, 21, 405-412.
(9) For a review, see: Farooq, A.; Anjum, S.; Ur-Rahman, A. Curr. Org.
Chem. 1998, 2, 281-328.
(10) For a review, see: Caufield, C. E. Curr. Pharm. Des. 1995, 1 (2),
145-160.
(11) Luengo, J. I.; Rozamus, L. W.; Holt, D. A. Tetrahedron Lett.1994,
35, 6469-6472.
10.1021/ol0514395 CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/12/2005

Scheme 1. Lipase-Catalyzed Esterification of Rapamycin
Scheme 2. Synthesis of Rapamycin 42-Hemisuccinate
42-OH with Enol Esters
the presence of 25-100% (w/w) enzyme, and nearly
quantitative yields of the corresponding 42-esters were
obtained after the removal of enzyme. HPLC analysis of the
reaction demonstrated that the enzymatic acylation proceeded
in an extremely regioselective fashion toward the 42-
hydroxyl moiety of rapamycin; no 31-acylation or 31,42-
diacylation byproducts were detected. In addition to vinyl
esters, anhydrides such as acetic anhydride, propionic
anhydride, and isobutyric anhydride also gave exclusively
42-acylated product under the conditions described above,
but the reaction proceeded at slower rate.
Rapamycin 42-hemiesters of dicarboxylic acid such as
succinic acid, glutaric acid, or adipic acid can serve as
precursors for the synthesis of rapamycin-immunogenic
protein conjugates that are useful as immunogens for the
generation of antibodies for rapamycin as well as for isolating
rapamycin binding proteins for immunoassays.15 These 42-
hemiesters are generally obtained by direct esterification of
rapamycin with the corresponding anhydrides in the presence
of a weak base. This procedure usually gives poor yields of
desired 42-hemiesters due to the poor regioselectivity and
the instability of rapamycin in the presence of a base. For
example, with succinic anhydride and (dimethylamino)-
pyridine (DMAP), rapamycin 42-hemisuccinate 3 was ob-
tained in 18% yield after RP-HPLC purification.16 A group
at Abbott reported a two-step chemoenzymatic process17 in
which corresponding benzyl and methyl ester of rapamycin
42-hemisuccinate were hydrolyzed using a lipase from
Pseudomonas sp. and gave 50% yield of 3 from the methyl
ester and 29% from the benzyl ester after HPLC purification.
The benzyl and methyl ester of rapamycin 42-hemisuccinate,
in turn, were made by reaction of rapamycin with methyl
succinyl chloride or benzyl succinyl chloride in the presence
of a base.
modification of its effector domain through selective func-
tionalization of the C1-C6 triene subunit via Sharpless’s
AD reaction,13 and nucleophilic substitution of C7 methoxy
group with different nucleophiles such as alcohols, thiols,
and electron-rich aromatic systems14 often resulted in the
loss of its binding affinity and/or immunosuppressive activity.
Modification of the cyclohexyl region, particularly in the 42-
OH position, however, resulted in the discovery of new
derivatives with good activity. One of them, Temsirolimus
(CCI-779) (7), a 42-ester derivative with 2,2-bis(hydroxy-
methyl) propionic acid, is in clinical trials as an oncology
agent.
Regioselective acylation of rapamycin at the 42-OH has
proven to be difficult, as there is another secondary hydroxyl
group at C31 surrounded by a number of sensitive function-
alities. Here we report a lipase-catalyzed synthesis of 42-
ester derivatives with various acylating agents with complete
regioselectivity and high yields under mild conditions. This
discovery provided a practical and efficient method for the
synthesis of rapamycin 42-hemisuccinate, 42-hemiadipate,
and CCI-779.
The starting point of the present work was to identify an
appropriate enzyme catalyst suitable for acylation of rapa-
mycin at the sterically less hindered 42-OH position. Among
the wide range of lipases/solvents tested using vinyl acetate
as an acyl donor, lipase Novozym 435 (Candida antarctica
B) and lipase PS “Amano” (Burkholderia cepacia), particu-
larly its immobilized form, lipase PS-C “Amano” II (im-
mobilized on ceramic particles), suspended in anhydrous tert-
butyl methyl ether (TBME) were identified as effective
catalysts for rapamycin 42-OH acylation.
The reaction, depicted in Scheme 1, can proceed either at
room temperature or, when less reactive enol esters such as
vinyl crotonate, vinyl benzoate, and vinyl decanoate are used,
at 45 °C. The reaction was completed within 12-48 h in
The lipase-mediated direct acylation using succinic an-
hydride as an acylating reagent (Scheme 2) offered a simple
and efficient route to access rapamycin 42-hemisuccinate 3.
A systematic screening of commercially available lipases
showed that only Novozym 435 was able to catalyze the
reaction and furnish 3 in good yield (91% isolated yield).
Solvent screening revealed that toluene is the most suitable
(12) Yang, W.; Digits, C. A.; Hatada, M.; Narula, S.; Rozamus, L. W.;
Huestis, C. M.; Wong, J.; Dalgarno, D.; Holt, D. A. Org. Lett. 1999, 1,
2033-2035.
(15) (a) Gonzalez, E., Russell, J. C.; Molnar-Kimber, K. L. WO 94/
25022. (b) Yatscoff, R. W.; Malcolm, A. J.; Naicker, S. WO 98/45333.
(16) Molnar-Kimber, K. L.; Caufield, C. E.; Ocain, T. O.; Failli, A. A
U.S. Patent 2001/0010920A1, 2001.
(17) Adamczyk, M.; Gebler, J. C.; Mattingly, P. G. Tetrahedron Lett.
1994, 35, 1019-1022.
(13) Sedrani, R.; Thai, B.; France, J.; Cottens, S. J. Org. Chem. 1998,
63, 10069-10073.
(14) Luengo, J. I.; Konialian-Beck, A.; Rozamus, L. W.; Holt, D. A. J.
Org. Chem. 1994, 59, 6512-6513.
3946
Org. Lett., Vol. 7, No. 18, 2005

Scheme 3. Synthesis of Rapamycin 42-Hemiadipate
Scheme 4. Synthesis of CCI-779 with Ketal-Protected Vinyl
Ester
medium for this reaction in regard to the rate of conversion.
Once again, the lipase demonstrated excellent regioselectivity
toward 42-hydroxyl group.
When similar reactions were carried out with other
dicarboxylic anhydrides such as glutaric anhydride, diglycolic
anhydride, and 3-methylglutaric anhydride, the conversion
was low; the yields of the corresponding 42-hemiesters were
10-20% even with prolonged reaction times. The results
indicated that these six-membered cyclic anhydrides are poor
substrates for Novozym 435 lipase.
Divinyl adipate, a bifunctional acylating reagent that had
been used previously to prepare a paclitaxel derivative via
an enzyme-catalyzed two-step strategy,18 was found to be a
good acyl donor to synthesize 42-hemiadipate 5. The
synthesis of 5 via enzymatic transesterification/hydrolysis
catalyzed by the same lipase was performed in a one-pot,
two-step procedure as illustrated in Scheme 3. In the first
step, rapamycin was reacted with divinyl adipate in anhy-
drous TBME in the presence of Novozym 435 to give
intermediate 4. Subsequent hydrolysis of the terminal vinyl
ester group by adding wet acetonitrile (containing 5% H2O)
to the reaction mixture furnished rapamycin 42-adipic acid
with 89% isolated yield. In this step, the lipase-catalyzed
hydrolysis proceeded quickly, and the cleavage of the vinyl
ester was completed within a few minutes.
a chemoenzymatic approach, as shown in Scheme 4, we were
able to derive CCI-779 from a lipase-catalyzed acylation with
ketal-protected vinyl ester 10 and subsequent acid-catalyzed
deprotection of resulting intermediate 6 with excellent yield.
The procedure offers a simple and efficient method to
synthesize CCI-779 without the need of extra steps of
protection-deprotection.
Enzyme-solvent screening showed that the most effective
lipase to accomplish this transformation was lipase PS-C
“Amano” II suspended in anhydrous TBME. Surprisingly,
Novozym 435, a lipase well-known for its ability to
accommodate substrates with varying sizes and complexities,
and which had shown good activity in acylating rapamycin
42-OH as described above, showed no activity toward ester
10. Under the optimized conditions, the enzymatic step was
carried out with a 1.5 molar excess of enol ester 10 at 42-
43 °C in the presence of molecular sieves, and >98%
conversion of rapamycin was achieved with 100 wt % lipase
after 48 h. Maintaining a low amount of moisture by adding
molecular sieves to the reaction mixture was found to be
important for achieving high product yield, as it can minimize
the formation of seco-rapamycin, a lactone ring-opened
product. Experiments showed that 5 Å molecular sieves were
particularly suitable for this reaction.
A remarkable application of this enzymatic acylation
approach was further demonstrated through the synthesis of
temsirolimus (CCI-779) 7, a compound currently under
development as an mTor inhibitor for a number of tumor
types. In a previously reported regioselective synthesis of
CCI-779, both 31- and 42-OH in rapamycin were silylated,
and the 42-OH was then selectively unmasked by mild acid
hydrolysis. An isopropylidene ketal-protected 2,2-bis(hy-
droxymethyl) propionic acid side chain was then introduced
via a 2,4,6-trichlorobenzyl mixed anhydride.19 By employing
To investigate whether different protecting groups other
than the acetonide in ester 10 could be accepted by the lipase
and thus eliminate the need for acid-catalyzed deprotection,
the cyclic methylboronate 12 was prepared and tested as a
substrate to lipase. Enzyme screening revealed that both
lipase PS-D “Amano” I and lipase PS-C “Amano” II could
effectively catalyze the synthesis of boronate-protected CCI-
779 8 under the conditions shown in Scheme 5. However,
the acylation process stopped after reaching about 80%
conversion; apparently, the enzyme lost activity at the later
(18) Khmelnitsky, Y. L.; Budde, C.; Arnold, J. M.; Usyatinsky, A.; Clark,
D. S.; Dordick, J. S. J. Am. Chem. Soc. 1997, 119, 11554-11555.
(19) Shaw, C. C.; Sellstedt, J.; Noureldin, R.; Cheal, G. K.; Fortier, G.
U.S. Patent 6277983, 2001.
Org. Lett., Vol. 7, No. 18, 2005
3947

Scheme 5. Synthesis of CCI-779 with
Scheme 6. Synthesis of Vinyl Ester 10 and 12
Methylboronate-Protected Vinyl Ester
straightforward. Ketal-protected 10 was prepared from 922
through a vinyl-exchange reaction with vinyl acetate and
PdCl2-LiCl as a catalyst.23 Deprotection with 0.25 N H2-
SO4 and subsequent treatment with trimethylboroxine gave
O-methylboranediyl-protected 12 in good overall yield.
In summary, we established for the first time that rapa-
mycin can be enzymatically derivatized with various acy-
lating agents at the 42-hydroxyl position with complete
regioselectivity. The synthetic utility of this reaction was
demonstrated by the synthesis of 42-hemiesters, intermediates
for rapamycin conjugates, and the synthesis of CCI-779, an
investigational oncology drug, with excellent yields.
stages of the reaction. The exact cause of this deactivation
was not clear, as this was the first example in which a cyclic
boronate derivative had been used as a substrate. Changing
the temperature and solvent media and increasing the amount
of lipase or ester 12 failed to drive the reaction to completion.
Nevertheless, by employing the cyclic methylboronate as the
protecting group, CCI-779 could be isolated in about 80%
yield together with 16-20% recovered rapamycin, simply
by adding alcoholic solvents20 such as methanol, ethanol, or
2-methylpentane-2, 5-diol21 to the reaction mixture.
Acknowledgment. We thank Dr. Raveendranath Panolil,
Dr. Chia-Cheng Shaw for valuable suggestions, and Dr.
Xidong Feng for FT-ICRMS analysis.
Further evaluation showed that other cyclic boronate-
protected vinyl esters such as butyl boronate gave low
conversion, as the reaction went rather sluggish; a phenyl
boronate-protected vinyl ester gave no reaction under similar
conditions. Interestingly, the enzymatic acylation did not take
place with the unprotected vinyl ester 11.
Supporting Information Available: Experimental pro-
cedures for 3, 5-8, and 10-12; NMR (1H, 13C) spectra for
3 and 5-7. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0514395
The synthesis of vinyl esters 10 and 12 (Scheme 6) is
(22) Bruice, T. C.; Piszkiewicz, D. J. Am. Chem. Soc. 1967, 89, 3568-
3576.
(20) Dahlhoff, W. V.; Koster, R. J. Org. Chem. 1976, 41, 2316-2320.
(21) Bertounesque, E.; Florent, J.-C.; Monneret, C. Synthesis 1991, 270.
(23) Bjorkquist, D. W.; Bush, R. D.; Ezra, F. S.; Keough, T. J. Org.
Chem. 1986, 51, 3192-3196.
3948
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