Biowganic & aeon CMw Letkm, Vol. 4, No. 15, pp. 1861-1864, 1994 Copydght a3 1994 EIsevitr Science Ud Printed in Great Britain. All rights reserved 096&894x/94 $7.oot0.00
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`0960-894X(94)00248-7
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`SYNTHESIS AND ~T~MOR EVALUATION OF 2’-OXYCARBONYLPACLITAXELS (PACLITAXEL-2WARBONATES) Yasutsugu UC&*. Henry Wang, John I), MatiskclIa, acne B. ~~~~, Vittorio Fatina$, Craig Fairchildt, W* C. Rose?, Stephen W. Mamber, Bpn I-l Longt, Edward H. Kerns, Anna Maria Casazzif, and Dolatrai M. Vyas
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`Bristol-Myers Squibb Pharnvacetuical Research imitute
`5 Research Parkway, P.O. B~.K 5100, W~lin~ord~ Cmnecticw 06492-7660
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`Ab&ract: A number of ~~yc~yl~~~els betel-2~~~) 3 have been prepared and evaluated for their cytotoxicity and Zn vhro antitumor activity. Most of these paclitaxel-r-ca&onates were found to cxhiiit in
`vivo antitumar
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`under
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`activity in the ip. Ml09 mutinc tumor model system, comparable to that of the parent drug. Taxol@ (1) &aclitaxel)l is regarded as one of the most promising antitumor agents to emerge in the past decade. It has broad specmun of antitumor activity against several human tumors and a unique mechanism of action, promoting tubulin ~l~~~tion and ~bi~g the ~s~s~bly process.2 Paclitaxel was recently approved by the FDA for the treatment of ovarian cancer and has also been shown to exhibit promising clinical effkxy against other foams such as v&5 antitumor activity, indicating the 2’-acetate 2a was readily hydrolyzed back to pa&axe11
`a cell culture bioassay and in viva conditions.5 These observations lead to the design of a number of prodrugs of pa&axe1 by introduction of a variety of acyl moieties at the 2+osition. While many of these 2’-acylpaclitaxels @aclitaxcl-Z- esters) 2 proved to behave as prodrugs, some suffered f&m poorchemical stability, limiting ~~~~.6 some of 2’~yl~~~e~ ~~~~-~~ ) such as ~-~c~~yl~a~ 3 (R = -CO2CH$Cl3) have been used as 2’-protected paclitaxcls for further chemical manipulation,7a however their antitumor activity has not been repot%& The carbonates, in gene&, ax considered to be more stable than the along esters to chemical or enzymatic hydrolysis. It is our a~umption that on this basis, the 2’- carbonates of pa&axe1 3 were never invcsdgatcd for their cytotoxicity or in viw, antitumor activity.7b In our mgram of paclitaxel modification, we have prepared a number of T-carbo~tcs of paclitaxel3a - 31 and investigated their cytomxEty against HCl’ cell line and
`antitumor activity against the intrapexitoneal (i.p.) Ml09 mm& ttmxx model using i.p. administration of each compound8 H&n, we describe the synthesis ~~ti~~~~d~~~~~ allalogues of paclitaxcL 1861
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`in vivo
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`1862 Y. t&DA ef ai. a
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`1
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`COPh CC4R’ 4 (2-4 eq.) I-Q,NEt I CH&I2 3 &OPh OAc The cytotoxkity and in viva antitumor activity are summarized in Table 2. When tested in cytotoxicity assay, paclitaxel-2’-carbonates 3 were 2-10 times less cytotoxic than paclitaxel against human colon cancer cell line (HCT 116). All carbonates were inactive in tubulin polymerization assay. However, after incubation in rat plasma at 37YJ for 18 hrs, some of these carbonate derivatives, ~~1~1~ 3a and 3b were found to promote mi~~bule assembly, indicating the g~n~don of the parent compound, paclitaxef in rat plasma. In the murine
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`2’-Oxycarbonylpaclitaxels 1863 lung carcinoma model M109,* all wbonates were efktive (%T/C > 125%). The methylcarbonate 3a was less active than paclitaxel, but all the other carbonates 3b
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`l
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`31 exhibited comparable in vivo antitumar activity relative to paclitaxel. Apparent superiority of the 2’-ethylwbonate 3b (%T/C > 475%) and T-vinylcarbonate 3f (%T/C > 475%) compared to pa&t&xc1 (%T/C = 275%) and most of the other carbonates evaluated was a function of the early initiation of thempy used in that pakular expeknent and did not represent a signiflcaut advantage. In all but one other experiment peffoima treatment lnhiatlon was delayed until the fifth day post-tumor implant in order to inuease the stringency of the antiaunor assay.11 Table 2: Cytotoxidty and In Viva Antitumor Activity (Lp. Ml09 Mice Tumor Model) of Paclitaxel-2’.carbonates, 3r - 3i I 31 I 0.016 1 226%(8O)C t 262% (75)c a Cytotoxiclty is expressed as IC50 against human colon caucer cell line HcTll6. IC50: Drug concentration required to inhibit cell proliferation to 50% vs. untreated cells, incubated at 37OC for 72 h. Paclitaxel, IC50 (HCT 116) 0.004 @f. b The Madison 109 murine lun Drugs administered i.p. in 1 Fk caknoma (M109) i.p. (lntrape&meal) implant model, Twecn 80 in salii (paclitaxel), in 10% DMSO in saline (3b - 3f’), or ln 10% DMSO in H20 plus a few drops of Twecn 80 (I, 3g, 3h, and 3i). %T/C nfers to the pacentage of the median survival time of drug-tmated mice (six I(!? dose) to saline-treated control. %T/c at the maximum tolerated dose arc listed in the table. %T >125% is defined as active in this tumormodel. C Dose adminlstend i.p. on days 5, and 8. d Dose administered i.p. on days 1.5. and 9. These in vitro and in vivo results indicate that the paclitaxel-2’carbonates 3 must have been converted to the parent paclltaxel under the in viva conditbns and act like prodrugs of paclitaxel. The in vitro drug uptake study using liquid chromatography coupled to tandem mass spec@oscopy indicated that incubation of cells with paclitaxel-2’-carbonaks, such as 3b and 3e resulted in rapid accumulation of paclitaxel and the carbonates in Ha 116 human colon catzlnom a cells. This result clearly demonstrates metabolii conversion of 2’-carbonates 3 to the parent paclitaxel. ‘Ike favorable in vivo effkacy observed with most of the paclitaxel-2’-carbonates may
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`1864 Y. t&DA et al. be a partial reflection of their unique distribution and pharmacology in vivo system. Such cases are well documented in medicinal chemistry of antitumor agents (cg., CC-1065, ~amptothecin).*~
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`Acknowiedgementsz
`and our
`We would like to thank Dr. A.R. Crosswell for preliminary in v&o evaluation, Dr. T.W. Doyle for constructive guidance,
`Analytical Research staff members for spectroscopic and analytical meas~ents.
`
`References and Notes:
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`Drugs Future, E&6,1
`J. Natl. Cancer
`1990,82,1247.
`1, and references cited themin. ’ Rowinsky, E.K; Casenave, L.A.; Donehower, R.C.
`Prod. f990,_53,1;
`Pharmac. Ther. 1991,52,1.
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`Natl.
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`~iophys. Res. Comm. 1984,124, 329.
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`Prod. 1988,5I, 298.
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`J. Med. Chem. 1989,32,7&k
`(b) Dentsch, H.M.; Glinski, J.A.; Hemandez, M.; Haugwitx, R.D.; Narayanana, V.L.; Suffness, M.; Zalkow, L.H.
`(c) Zhao. 2.;
`Prod.,
`Med. Chem.
`1991, 54, 1607. (d) Mathew, A.E.; Mejillano, M.R.; Nath, J.P.; Hmes, RH; Stella, V.J. J.
`1992.35, 145. (e) Ueda, Y; Mikkilineni, A.B.; Knipe, J.G.; Rose, W.C.; Cassaza. A.M.; Vyas, D.M.
`Lett. 1993.3,
`Org. Chem. 1986,5Z,
`1761. (a) Magri. N.F.; Kingston, D.G.I. J.
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`BioMed. Chem.
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`5.
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`6
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`7.
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`8.
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`9.
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`10. 11. 12. present Address: Bristol-Myers Squibb pharmaceutical Research Institute, Princeton, NJ 08543 present Address: Department of Medicinal Chemistry, Boehringer-Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877. Taxol@ is a registered trademark of Bristol-Myers Squibb Company. Zee-Chen, RX-Y.; Cheng C.C.
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`797. (b) Recently, appropriately activated carbonates, 2’-aryhhioethyl- and T-arylsulfonylethyl carbonates 3 (R = -Co2CH2CH2SGt&, n = 0.2) have been reported as prodrugs of paclitaxek Nicolaou, ICC.; Riemer, C.; Kerr, M.A.; Rideout, D.; Wrasidlo, W.
`
`Nature, 1993,364,464.
`Caruer Treat. Ream, 1981,65,299.
`Anti-Cancer Drugs, 1992,3,311.
`
`@)
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`All new compounds gave satisfactory analytical and spectroscopic results in accord with the assigned StlUChue. Formation of oxazohme 5, presumably uced other cases. Isolation of oxazolone s”d by intramolecular cyclixation of 31, was not apparant in as a major product has been reported from the reaction of nichloroethylcarbonate of paclitaxel with DBU.4a The evaluation of compounds involved two different schedules of treatment, either i.p. injections ‘ven on daysL5and9 X t-tumor implant or i.p. injections given on days 5 and 8 post-tumor implant. # elatter schedule was pted in order to increase the strin ency of the assay. %T/C values for pa&axe1 varied depending on each cxpcriment and the treatment con&. tions. The in
`
`vivo activity ~paclitaxcl-2’-carbonates
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`3 was evaluated using paclitaxel as a standard in each experiment. All compounds were evaluated at several dosesdes@edtoencom F 8 a) Li, L.H.; DeKoning, their likely maximum tolerated levels. .F.; Kelly, R.C.; Krueger, W.C.; McGovern, J.P.; padbury, GE., Fetxold, G.L.; Wallace, T.L; Guding, R.J.; prairie, M.D.; Gebhard, I.
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`Cancer Research, 1992.52.4904.
`Cancer Research, 1991,5Z, 4187.
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`(Received in USA 9 May
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`accepted 22 Jwze 1994)
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`NEPTUNE GENERICS EX. 1012 00004
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`Inst.
`For a review on the chemistry of paclitaxel, see (a) Kingston, D.G.L; Samaranayake, G.; Ivey, CA. 1.
`(b) Kingston, D.G. L
`Mellado, W. ; Magri, N.F.; Kmgston, D.G.I.; G~ia-Arena, R, Grr, G.A.; Horwitz, S.G. &o&em.
`(a) Magri, N-F.; Kingston, D.G.I. J. Nati.
`Kingston, D.G.I.; Croswell, A.R. J. Natl.
`(a) Rose, W.C.
`Rose, W.C.
`b) Kawato, Y.; Aonuma, M.; Hirota, Y.; Kuga, H.; Sato, K.
`1994;
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