Fluoro taxols
This invention relates to a fluorinated taxol of formula I ##STR1## in which R.sup.1 is benzoyl or t-butyloxycarbonyl; R.sup.2 is acetoxy, hydrogen or hydroxy; and the wavy line indicates either the .alpha.- or the .beta.-configuration.Further provided by this invention are pharmaceutical formulations and useful intermediates for the fluorinated taxols of formula I. A method of treating mammalian tumors using a compound of formula I is also provided.
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Taxol was first isolated from the stem bark of Western Yew, Taxus brevifolia Nutt (Taxaceae) and has the following structure (with the 2'-, 7-, 10- and 13th-positions indicated) ##STR2## In ongoing clinical trials sponsored by the National Cancer Institute (NCI), taxol has shown promising results in fighting advanced cases of ovarian, breast, and other cancers.
Taxol is unique among antimitotic drugs in that it promotes the assembly of stable microtubules from tubulin even under otherwise unfavorable conditions. The drug binds to microtubules, stabilizing them from depolymerization, thus disrupting the tubulin-microtubule equilibrium and consequently inhibiting mitosis. The mechanism of action, toxicology, clinical efficacy, etc. of taxol are reviewed in a number of articles, such as in an article by Rowinsky et al. in Taxol: A Novel Investigational Antimicrotubule Agent, J. Natl. Cancer Inst., 82: p 1247 (1990).
Since the discovery of significant effectiveness in cancer treatment, many laboratories have launched to design taxol analogues in search of better pharmacological profiles. Out of such program was the discovery of taxotere of the formula ##STR3## which has been reported to be as effective as taxol at promoting the assembly of microtubules and approximately twice as cytotoxic. See, Biologically Active Taxol Analogues with Deleted A-Ring Side Chain Substitutents and Variable C-2' Configurations, J. Med. Chem., 34, p 1176 (1991); Relationships between the Structure of Taxol Analogues and Their Antimitotic Activity, J. Med. Chem., 34, p 992 (1991).
In recent years, the introduction of fluorine into pharmacologically active compounds has led to the discovery of some profound and unexpected results. (For a comprehesive review on the advances in the preparation of biologically active organofluorine compounds, see: Advances in the Preparation of Biologically Active Organofluorine Compounds, Tetrahedron, 43, No. 14, p 3123 (1987).) It is the intention of the present invention to provide fluorinated taxols and their derivatives.
SUMMARY OF INVENTION This invention relates to a fluorinated taxol derivative of formula I ##STR4## in which R.sup.1 is benzoyl or t-butyloxycarbonyl; R.sup.2 is acetoxy, hydrogen or hydroxy; and the wavy line indicates either the .alpha.- or the .beta.-configuration.Further provided by this invention are pharmaceutical formulations and useful intermediates for the fluorinated taxols of formula I. A method of treating mammalian tumors using a compound of formula I is also provided.
DETAILED DESCRIPTION OF INVENTION This invention relates to a fluorinated taxol derivative of formula I ##STR5## in which R.sup.1 is benzoyl or t-butyloxycarbonyl; R.sup.2 is hydrogen, acetoxy or hydroxy; and the wavy line indicates either the .alpha.- or the .beta.-configuration.The synthesis of a fluorinated taxol derivative of formula I can be accomplished by a wide variety of ways. In one embodiment, a process of Scheme I may be employed to make a compound of formula I. In Scheme I, 2'-hydroxy protected taxol of formula II is reacted with diethylaminosulfur trifluoride (DAST) to afford a 7-fluoro taxol of formula III (Step (a)). In formula II, R.sup.3 is a conventional hydroxy protecting group. Depending on the reaction conditions, different isomeric ratios of the 7-fluoro taxol may be obtained. For example, it has been observed that a choice of solvent was found to influence the ratio of .alpha.- and .beta.-isomers produced. In general, the use of ethereal solvent (such as THF alone or THF/diethyl ether) tends to produce higher amount of .alpha.-isomer than the use of a halogenated solvent such as methylene chloride. The mixture of isomers may be separated into an individual isomer and each used independently in subsequent steps, or the mixture may be used in subsequent steps without any separation. The separation of the isomers can be effected by conventional purification techniques normally employed by a person skilled in the art to separate isomers. The methods include chromatography, fractional crystallization, etc. A particularly suitable method for the separation of 7-.alpha.- and .beta.-fluoro isomers is HPLC (High Pressure Liquid Chromatography). The removal of a R.sup.3 protecting group from a compound of formula III in Step (b) affords a compound of formula Ia, which is within the scope of formula I compounds.
As used herein, conventional hydroxy protecting groups are moieties which can be employed to block or protect a hydroxy function, and they are well-known to those skilled in the art. Preferably, said groups are those which can be removed by methods resulting in no appreciable destruction to the remaining portion of the molecule. Examples of such readily removable hydroxy protecting groups include chloroacetyl, methoxymethyl, 2,2,2-trichloroethyoxymethyl, 2,2,2-trichloroethyloxycarbonyl (or simply trichloroethyloxycarbonyl), tetrahydropyranyl, tetrahydrofuranyl, t-butyl, benzyl, p-nitrobenzyl, p-methoxybenzyl, diphenylmethyl, triC.sub.1-6 alkylsilyl, triphenylsilyl, and the like. Other suitable protecting groups which may be used are found in Chapter 2 of "Protecting Groups in Organic Synthesis", Second Ed., by Theodora W. Greene and Peter G.M. Wuts (1991, John Wiley & Sons). A particularily advantageous protecting group for formula II compounds is benzyloxycarbonyl, which can be removed conveniently by catalytic hydrogenolysis.
In another embodiment, a compound of formula Ib may be made by a process of Scheme II. In the Scheme, (C)13-side chain is removed reductively from a compound of formula Ia by a reducing agent such as tetrabutylammonium borohydride to afford a fluoro baccatin III of formula IV (Step (a)). Azetidinone XVa is reacted with a compound of formula IV in Step (b). Holton in European Patent Application 0,400,971, published on Dec. 5, 1990, describes a related process whereby an azetidinone is reacted with C-13 hydroxy of baccatin III derivatives to afford taxol analogues with a variety of (C)13-side chains. (In Step (b), it is advantageous to convert the hydroxy group on the carbon marked with the asterisk into a metal alkoxide before the coupling. The metal cation of said metal alkoxide is preferably selected from Group Ia or IIa metals. The formation of a desired metal alkoxide may be done by reacting a compound of formula IV with a strong metal base, such as lithium diisopropylamide, C.sub.1-6 alkyllithium, lithium bis(trimethylsilyl)amide, phenyllithium, sodium hydride, potassium hydride, lithium hydride, or the like base. For example when lithium alkoxide is desired, a compound of formula IV may be reacted with n-butyllithium in an inert solvent such as tetrahydrofuran.) Triethylsilyl group is removed from a compound of formula V in Step (c) to afford a compound of formula Ib. The removal of triC.sub.1-6 alkylsilyl group, such as triethylsilyl group, can be accomplished with fluoride ion or with a mineral acid in alcohol or acetonitrile. The removal with fluoride ion is conducted in an inert solvent such as tetrahydrofuran, methylene chloride, 1,4-dioxane, DMF, chloroform, or in the like solvent; and preferably the reaction medium is buffered With a weak acid such as acetic acid.
A compound of formula Ic, which is further within the scope of formula I compounds, may be made by a process of Scheme III. In Step (a), when a compound of formula VI is treated with between one to two equivalents of a conventional hydroxy protecting reagent, preferably trichloroethyl chloroformate, a mixture of 2'- and 7-hydroxy protected (a compound of formula XIII) and 2'- and 10-hydroxy protected (a compound of formula VII) taxol derivatives may be simultaneously obtained.
A compound of formula XIII is subsequently reacted with 1,1,2-trifluoro-2-chlorotriethylamine in Step (b) to afford a dieneone of formula VIII. In Step (c), protecting groups R.sup.3 are removed. (The removal of trichloroethyloxycarbonyl group can be done by zinc dust in acetic acid.) In Step (d), the diene of a compound of formula IX is catalytically hydrogenated to afford a compound of formula X. Subsequently in Step (e), 2'-hydroxy group is once again protected, this time preferably with benzyloxycarbonyl, to afford a compound of formula XI. Treating a compound of formula XI with DAST yields a fluoro compound of formula XII. Once again, the choice of solvent used in Step (f) of Scheme III and Step (b) of Scheme IV, described hereinbelow, may influence the outcome of the .alpha.- and .beta.-isomer ratio. Removal of protecting group R.sup.3 in Step (g) affords a compound of formula Ic.
Scheme IV describes a process for making a compound of formula Id, which is further within the scope of formula I compounds. In Step (a), a compound of formula VII is reacted with DAST to afford a compound of formula XIV. Removal of R.sup.3 protecting groups affords a compound of formula Id.
In the instant application, the numbers in subscript after the symbol "C" define the number of carbon atoms a particular group may contain. For example, C.sub.1-6 alkyl refers to straight and branched chain alkyl groups with one to six carbon atoms and such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, n-hexyl, 3-methylpentyl, or the like alkyl groups. ##STR6##
DESCRIPTION OF SPECIFIC EMBODIMENTThe specific examples which follow illustrate the synthesis of representative compounds of the instant invention and are not to be construed as limiting the invention in sphere or scope. The methods may be adopted to variations in order to produce compounds embraced by this invention but not specifically disclosed. Further, variations of the methods to produce the same compounds in somewhat different fashion will also be evident to one skilled in the art.
All temperatures are understood to be in Centigrade (C) when not specified. The nuclear magnetic resonance (NMR) spectral characteristics refer to chemical shifts (.delta.) expressed in parts per million (ppm) versus tetramethylsilane (TMS) as reference standard. The relative area reported for the various shifts in the proton NMR spectral data corresponds to the number of hydrogen atoms of a particular functional type in the molecule. The nature of the shifts as to multiplicity is reported as broad singlet (bs), broad doublet (bd), broad triplet (bt), broad multiplet (bm), broad quartet (bq), singlet (s), multiplet (m), doublet (d), quartet (q), triplet (t), doublet of doublet (dd), doublet of triplet (dt), and doublet of quartet (dq). The solvents employed for taking NMR spectra are DMSO-d.sub.6 (perdeuterodimethylsulfoxide), D.sub.2 O (deuterated water), CDCl.sub.3 (deuterochloroform) and other conventional deuterated solvents. "Exch." means exchangeable with CD.sub.3 OD. (For example, "d plus exch." means a doublet plus an exchangeable signal. The total signal collapses to just a doublet after the other proton has been exchanged.) "Incl." means including.
The infrared (IR) spectral description includes only absorption wave numbers (cm.sup.-) having functional group identification value.
Celite is a registered trademark of the Johns-Manville Products Corporation for diatomaceous earth.
The abbreviations used herein are conventional abbreviations widely employed in the art. Some of which are:
______________________________________
Ac acetyl
Ar aryl
Bz benzoyl
Cbz benzyloxycarbonyl
DCI desorption chemical ionization
DMF dimethylformamide
DMSO dimethyl sulfoxide
FAB fast atom bombardment
h hour(s)
HRMS high resolution mass spectrometry
i-PrOH isopropylalcohol
min minute(s)
MS mass spectrometry
NOBA m-nitrobenzylalcohol
Ph phenyl
rt room temperature
tBu tertiarybutyl
TES triethylsilyl
THF tetrahydrofuran
tlc thin layer chromatography
v/v volume/volume
Y yield
______________________________________
The Tables 1 and 2 list the compounds Whose syntheses are described in the Examples below.
TABLE 1
__________________________________________________________________________
##STR7##
COMPOUND #
R.sup.1
R.sup.a R.sup.b
R.sup.c
__________________________________________________________________________
IIa
##STR8##
AcO
##STR9##
OH (.beta.-isomer)
IIIa'
##STR10##
AcO
##STR11##
F (1:1 .alpha./.beta.- isomers)
Ia'
##STR12##
AcO H F (1:1 .alpha./.beta.- isomers)
Ia"
##STR13##
AcO H F (.alpha.-isomer)
IIIa"
##STR14##
AcO
##STR15##
F (.alpha.-isomer)
Va'
##STR16##
AcO Et.sub.3 Si
F (3:2 .alpha./.beta.- isomers)
Ib'
##STR17##
AcO H F (3:2 .alpha./.beta.- isomers)
VIa
##STR18##
HO H OH (.beta.-isomer)
XIIIa
##STR19##
HO
##STR20##
##STR21##
VIIa
##STR22##
##STR23##
##STR24##
OH (.beta.-isomer)
Xa
##STR25##
H H OH (.beta.-isomer)
XIa
##STR26##
H
##STR27##
OH (.beta.-isomer)
XIIa"
##STR28##
H
##STR29##
F (.alpha.-isomer)
Ic"
##STR30##
H H F (.alpha.-isomer)
Id"
##STR31##
HO H F (.alpha.-isomer)
XIVa"
##STR32##
##STR33##
##STR34##
F (.alpha.-isomer)
__________________________________________________________________________
TABLE 2
______________________________________
##STR35##
COMPOUND # R.sup.1 R.sup.b R.sup.d
______________________________________
VIIIa
##STR36##
##STR37##
##STR38##
IXa
##STR39##
H H
______________________________________
EXAMPLE 1
2'-O-(Benzyloxycarbonyl)taxol (IIa)
To a stirred solution of taxol (150 mg, 0.176 mmol) and N,N-diisopropylethylamine (93 .mu.L, 0.534 mmol, 3 eq.) in anhydrous CH.sub.2 Cl.sub.2 (4 mL) at room temperature was added benzyl chloroformate (75 .mu.L, 0.525 mmol, 3 eq.) at room temperature. The reaction mixture was stirred at room temperature for 3 h. The reaction mixture was concentrated to 2 mL in volume and the product was purified on a silica gel column, using 1:1 of EtOAc/hexanes as eluant, to obtain 150 mg (0.152 mmol, Y:86%) of the title compound, IIa. as a white powder: mp, 140.degree.-150.degree. C. (decomposition); [.alpha.].sub.D.sup.20 -53.5.degree. (c =0.2, 95% EtOH); .sup.1 H-NMR (300 MHz, acetone-d.sub.6) .delta. ppm: 1.18 (3H, s, 17-H.sub.3), 1.92 (3H, s, 16-H.sub.3), 1.66 (3H, s, 19-H.sub.3), 1.96 (3H, s, 18-H.sub.3), 2.16 (3H, s, 10-OAc), 2.5 (3H, s, 4-OAc), 3.53 (1H, d, J=5.89 Hz, 7-OH, exchanged with D.sub.2 O), 3.85 (1H, d, J=7.19 Hz, 3-H), 3.9 (1H, s, 1-OH, exchanged with D.sub.2 O), 4.17 (2H, ABq, 20-H.sub.2), 4.25 (1H, m, 7-H), 4.97 (1H, d, J=9.56 Hz, 5-H), 5.19 (2H, ABq, OCH.sub.2 C.sub.6 H.sub.5), 5.54 (1H, d, J=5.5 Hz, 2'-H), 5.68 (1H, d, J=7.13 Hz, 2-H), 6.01 (1H, dd, J=5.5, 9.05 Hz, 3'-H), 6.17 (1H, bt, J=9.0 Hz, 13 -H), 6.42 (1H, s, 10-H), 7.28-7.69 (16H, m), 7.87 (2H, "d", J=8 Hz, 3'-NHCOPh), 8.14 (2H, "d", J=8 Hz, 2-CO.sub.2 Ph), 8.55 (1H, d, J=9.06 Hz, NH, exchanged with D.sub.2 O); MS (FAB-NOBA/NaI+KI): m/e 988 (M+H).sup.+, 1010 (M+Na).sup.+, 1026 (M+K).sup.+ ; IR (KBr) .nu. max: 3448, 1748 (C=O), 1726 (CONH), 1250 (C-O) cm.sup.-1 ; UV (MeOH:H.sub.2 O, 1:1) .lambda. max: 198 (.epsilon. 7.3.times.10.sup.4), 230 nm (.epsilon. 2.7.times.10.sup.4). HRMS calcd for C.sub.55 H.sub.58 NO.sub.16 (MH.sup.-): 988.3756. Found: 988.3766.
Anal. calcd for C.sub.55 H.sub.57 NO.sub.16.H.sub.2 O: C, 65.67; H, 5.92; N, 1.40.
Found: C, 65.99; H, 5.64; N, 1.33.
EXAMPLE 2 2'-O-Benzyloxycarbonyl-7-fluorotaxol (IIIa')DAST (18.7 .mu.L, 0.141 mmol) was dissolved in dry dichloromethane (0.5 mL), and this solution was cooled to 0.degree. C. A solution of compound IIa (71 mg, 0.072 mmol) in dichloromethane (1 mL) was added and the resulting solution was kept at 0.degree. C. for 30 min and at room temperature for 4 h. The water (0.15 mL) was added to the reaction mixture in order to quench the reaction and the resultant mixture was concentrated to leave a residue. The residue was chromatographed on a silica gel column (being eluted with 40% ethyl acetate in hexane) to yield 61 mg (Y: 85.7%) of compound IIIa' (a 1:1 mixture of 7.alpha.- and 7.beta.-isomers) as a white amorphous solid; H-NMR (CDCl.sub.3) .delta. 8.08 (d, J=8.7 Hz, 2H) 7.65-7.17 (m, 18H) 6.85 (exch. d, J=9.4 Hz, 1H) 6.49 (s, 1H, H-10) 6.25-6.14 (m, 1H, H-13) 5.92 (dd, J=9.4 Hz, J'=2.4 Hz, 1H, H-3') 5.68 (d, J=7.2 Hz, 1H, H-2) 5.38 (m, 1H, H-2') 5.06 (m, 2H) 4.96 (br d, 1H, H-5) 4.80-4.35 (m, 1H, H-7) 4.31-4.20 (m, 2H, H-20) 3.94 (d, H=7.2 Hz, 1H, H-3) 2.47-1.64 (m, 17H incl. s at 2.38, 3H, at 2.11, 3H, at 1.78, 3H, 1.65, 3H) 1.10 (s, 3H) 1.07 (s, 3H).
EXAMPLE 3 7-Fluorotaxol (Ia')Compound IIIa' (89 mg, 0.090 mmol, a 1:1 mixture of 7.alpha.- and 7.beta.-isomers) was dissolved in ethyl acetate (3 mL) and the mixture was stirred under slightly over one atmospheric pressure of hydrogen in the presence of palladium on charcoal (10% Pd, 29 mg, 0.027 mmol). After 12 h, the solvent was removed, and the residue was purified by silica gel chromatography (being eluted with 40% ethyl acetate in hexane) to afford 67.7 mg (Y: 88%) of the title compound as a white solid; .sup.1 H-NMR (CDCl.sub.3) .delta. 8.11 (d, J=8.7 Hz, 2H) 7.72-7.07 (m, 14H) 6.50 (s, 1H, H-10) 6.14 (bt, 1H, H-13) 5.80 (dd, J=9.0 Hz, J'=2.4 Hz, 1H, H-3') 5.74 (d, J=7.2, 1H, H-2) 4.98 (d, J=8.1 Hz, 1H, H-5) 4.77 (m, 1H, H-2') 4.70-4.40 (m, 1H, H-7) 4.40- 4.21 m, 2H, H-20) 4.02 (d, J=7.2 Hz, 1H, H-3) 2.60-1.55 (m, 17H, incl. s at 2.37, 3H, 2.20, 3H, 1.77, 3H, 1.74, 3H) 1.14 (s, 3H) 1.12 (s, 3H).
The HPLC method used to separate the .alpha.-isomer from the .beta.-isomer was:
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Equipment
Pump: PE Series 4
Column: Shandon Hypercarb (graphitized carbon), 7.mu., 100
.times. 4.6 mm, #59864750 (information on preparative
size columns may be obtained from Keystone
Scientific, Bellefonte, PA)
Injector:
PE ISS-100
Detector:
HP-1040M
Conditions
Mobile 85:15 methylene chloride:hexane
Phase: Separation not lost at 80:19:1 methylene
chloride:hexane:isopropyl alcohol
Flow Rate:
2.5 mL/min
Detector:
254 nm
Diluent: Sample dissolved in methylene chloride
______________________________________
EXAMPLE 4
7.alpha.-Fluorotaxol (Ia")
Compound IIa (258 mg, 0.26 mmol) was dissolved in THF (1.7 mL) and diethyl ether (3.4 mL), and the solution was cooled to -78.degree. C. To this solution, DAST (69 .mu.L, 0.52 mmol) was added, and the mixture stirred for 30 min at -78.degree. C., and then at room temprature overnight. Water (0.3 mL) was added to quench the reaction and the mixture was concentrated to leave a residue. The residue was purified by silica gel chromatography (being eluted with 30% ethyl acetate in hexane) to give 87 mg (Y: 33.7%) of 2'-O-benzyloxycarbonyl-7.alpha.-fluorotaxol (IIIa") as an amorphous solid. The .sup.1 H-NMR spectrum was essentially identical to that reported in Example 2; .sup.19 F-NMR (CDCl.sub.3) .phi. (vs. CF.sub.3 COOH) 90 (ddd, J.sub.F,H7 =49.6 Hz, J.sub.F,H6 =40.1 Hz, J.sub.F,H6' =21.6 Hz).
The removal of 2'-O-benzyloxycarbonyl group as in Example 3 gave the title compound in 87% yield. The .sup.1 H-NMR was consistent for the structure. HRMS calcd for MH.sup.+ : 856.3344, found: 856.3367.
EXAMPLE 5 N-debenzoyl-N-t-butoxycarbonyl-2'-O-triethylsilyl-7-fluorotaxol (Va')7-Fluorotaxol (572 mg, 0.669 mmol, a 3:2 mixture of 7.alpha.- and 7.beta.- isomers) was treated with tetrabutylammonium borohydride (286 mg, 1.111 mmol) in dry dichloromethane (7 mL) at rt overnight. The excess borohydride was quenched with acetic acid (0.4 mL); the solvent was evaporated to leave a crude product. The crude product thus obtained was purified on a silica gel column (being eluted with 50% ethyl acetate in hexane) to afford 271 mg (Y: 69%) of 7-fluoro baccatin III (IV) as a white foam. The NMR spectrum was consistent for the structure.
A solution of compound IV (130 mg, 0.220 mmol) in dry THF (1 mL) was cooled to -40.degree. C. and n-butyllithium (1.63M in hexane, 0.164 mL, 0.260 mmol) was added dropwise under argon. After 15 min, a solution of 1-t-butoxycarbonyl-(3R,4S)-cis-3 -triethylsilyoxy-4-phenylazetidinone (XVa) (203 mg, 0.530 mmol) in dry THF (0.5 mL) was added, and the mixture was warmed to 0.degree. C. The reaction was allowed to continue for 90 min at 0.degree. C. and quenched with saturated aqueous ammonium chloride. The reaction mixture was extracted with ethyl acetate. The ethyl acetate layer was dried, filtered and concentrated in vacuo to leave a crude oil. This oil was purified by silica gel chromatography (being eluted with 40% ethyl acetate in hexane) to provide 143 mg (Y: 68%) of the title compound as a white foam; .sup.1 H-NMR (300 MHz, CDCl.sub.3) .delta. 8.14 (d, 2H) 7.45-7.17 (m, 8H) 6.56 (s, 0.6H, H-10) 6.32 (s, 0.4H, H-10) 6.28 (m, 1H, H-13) 5.72 (d, 0.6H, H-2) 5.62 (d, 0.4H, H-2) 5.44 (m, 1H, H-3') 5.28 (exch. m, 1H, N-H) 5.00 (d, 1H, H-5) 4.70-4.45 (m, 1H, H-7) 4.50 (bs, 1H, H-2') 4.40-4.35 (m, 2H, H-20) 4.05 (d, 1H, H-3) 2.63-1.15 (m, 32H) 0.73 (m, 9H) 0.34 (m, 6H).
EXAMPLE 6 N-debenzoyl-N-t-butoxycarbonyl-7-fluorotaxol (Ib')To a solution of Va' (100 mg, 0.10 mmol) in acetonitrile (1 mL) at -5.degree. C. was added aqueous HCl (0.0192 mL, 0.30 mmol, 36% solution). The reaction mixture was stirred for 10 min and was diluted with ethyl acetate (1.5 mL). The organic phase was washed with water, dried, filtered, and concentrated to leave a residue. The residue was purified by silica gel chromatography (being eluted with 40% ethyl acetate in hexane) to afford 73 mg (Y: 87%) of the title product as a foam; .sup.1 H-NMR (300 MHz, CDCl.sub.3) .delta. 8.11 (m, 2H) 7.60-7.22 (m, 8H) 6.50 (s, 0.6H, H-10) 6.30 (s, 0.4H, H-10) 6.22 (m, 1H, H-13) 5.72 (d, 0.6H, H-2) 5.61 (d, 0.4H, H-2) 5.50-5.42 (m, 1H, H-3') 5.28 (exch. bd, 1H, N-H) 5.00 (d, 1H, H-5) 4.70-4.40 (m, 1H, H-7) 4.60 (bs, 1H, H-2') 4.40-4.23 (m, 2H, H-20) 4.02 (d, 1H, H-3) 3.40 (exch. bs, 1H, O-H) 2.65-1.10 (m, 32H). HRMS Calcd. for MH.sup.+ 852.3607, found 852.3604.
EXAMPLE 7 Preparation of 1-t-butoxycarbonyl-(3R,4S)-cis-3-triethylsilyoxy-4-phenylazetidinone (XVa), SCHEME V(L)-Threonine methyl ester hydrochloride (1.26 g, 7.44 mmol) in anhydrous dichloromethane (15 mL) was stirred with imidazole (1.01 g, 14.89 mmol) and t-butoxydiphenylsilyl chloride (2.274 g, 7.816 mmol) for 16 h at room temperature. The reaction mixture was partitioned between water and dichloromethane. The organic phase was washed with 5% aqueous sodium bicarbonate and water, dried and concentrated to give 2.88 g of a crude oil, which was used directly in the next step; .sup.1 H-NMR (CDCl.sub.3) .delta. 7.70-7.25 (m, 10H) 4.44 (m, 1H) 3.62 (s, 3H) 3.31 (d, J=3 Hz, 1IH) 2.12 (bs, 2H) 1.31-1.15 (m, 12H).
The foregoing oil (548 mg, 1.414 mmol) in anhydrous dichloromethane (10 mL) was treated with benzaldehyde (0.158 mL, 1.55 mmol) at rt overnight in the presence of 4.ANG. molecular sieves to afford compound of formula XVIIa in situ. Upon cooling the solution containing compound XVIIa to -40.degree. C., triethylamine (0.20 mL, 1.698 mmol) was added, followed by acetoxyacetyl chloride (XVIa) (0.182 mL, 1.698 mmol) over 10 min. The mixture was allowed to reach rt over 4 h and the product was partitioned between dichloromethane and water. The organic phase was further washed with water and brine, dried and concentrated. Silica gel chromatography (being eluted with 1:4 EtOAc/hexane) gave 411 mg of compound XVIIIa as a ca. 10:1 mixture of 3R,4S : 3S,4R diastereomers.
This mixture of diastereomers (245.1 mg, 0.414 mmol) in dry THF (2 mL) was treated with acetic acid (0.15 mL) and tetrabutylammonium fluoride (TBAF, 1M in THF, 1.20 mL). The solution was stirred for 14 h at rt, then partitioned between ethyl acetate and 5% aqueous sodium bicarbonate. The organic phase was dried and concentrated. Flash silica gel chromatography using 1:1 ethyl acetate/hexane as eluant gave 66 mg (Y: 50%) of compound XIXa (one diastereomer) as a foam; .sup.1 H-NMR (CDCl.sub.3) .delta.: 7.42-7.25 (m, 5H) 5.90 (d, J=4.8 Hz, 1H) 5.09 (d, J=4.8 Hz, 1H) 4.28 (m, 1H) 4.01 (d, J=4.8 Hz, 1H) 3.70 (s, 3H) 1.73 (s, 3H) 1.19 (d, J=6.6 Hz, 3H).
Compound of formula XIXa (9.8 g, 0.0305 mol) in dry dichloromethane (100 mL) was treated at -78.degree. C. with triethylamine (9.40 mL, 0.0671 mol) and methanesulfonyl chloride (MsCl, 3.50 mL, 0.0457 mol). The solution was allowed to reach rt overnight. The reaction mixture was partitioned between water and dichloromethane. The organic layer was washed with 5% aqueous sodium bicarbonate, dilute aqueous HCl, water and brine, and concentrated to afford compound XXa as a crude oily residue. The crude residue (10.0 g) was dissolved in dichloromethane (250 mL) and ozonized at -78.degree. C. until the color of the solution stayed as blue. Addition of methyl sulfide (11 mL) and concentration of the reaction mixture gave compound of formula XXIa (crude).
The compound of formula XXIa was dissolved in THF (150 mL) and treated at -78.degree. C. with hydrazine hydrate (10 mL). After 2 h, the mixture was poured into dilute aqueous HCl and ethyl acetate, and the two phases were separated. The organic phase was washed with more acid, water and brine and concentrated to afford a crude product, which was purified by silica gel chromatography using 1-5% methanol in methylene chloride as eluant to yield 4.40 g (Y: 71%) of compound of formula XXIIa; .sup.1 H-NMR (CDCl.sub.3) .delta. 7.38-7.24 (m, 5H) 6.31 (bs, 1H) 5.87 (bm, 1H) 5.04 (d, J=4.8 Hz, 1H) 1.67 (s, 3H).
To a cooled (-5.degree. C.) mixture of 1M aqueous KOH (140 mL) and acetonitrile (100 mL), a solution of compound XXIIa (2.39 g, 11.22 mmol) in acetonitrile (130 mL) was added dropwise. The mixture was stirred at 0.degree. C. for 1 h and diluted with ethyl acetate (300 mL), water (50 mL) and saturated aqueous bicarbonate (50 mL). The organic phase was separated, and the aqueous layer further extracted with ethyl acetate (3.times.200 mL). The organic phases were combined, dried, filtered and concentrated to give compound of formula XXIIIa (crude), which was recrystallized from hexane/acetone (mp, 1.degree.84-6.degree. C.); yield, 1.53 g (Y: 82%).
To azetidinone XXIIIa (580 mg, 3.55 mmol) in dry THF (5.0 mL) was added imidazole (265.5 mg, 3.90 mmol), followed by triethylsilyl chloride (TESCl, 0.654 mL, 3.90 mmol). The mixture was allowed to be stirred for 1 h. Ethyl acetate was added and the organic layer was washed with brine, 10% aqueous HCl and dried. Silica gel chromatography (being eluted with 25% ethyl acetate in hexane) gave 670 mg (Y: 68%) of compound XXIVa as a foam.
To a stirred solution of compound XXIVa (2.20 g, 7.92 mmol) in dry THF (25 mL) was added diisopropylethylamine (1.65 mL, 9.51 mmol) at 0.degree. C. under argon atmosphere. The solution was stirred for 5 min, then di-tert-butyl carbonate (Boc.sub.2 O, 2.08 g, 9.51 mmol) and 4-dimethylaminopyridine (193.6 mg, 1.58 mmol) were added. The reaction mixture was stirred at 0.degree. C. for 60 min. The reaction was diluted with ethyl acetate (25 mL), and the mixture was washed with brine, 10% aqueous sodium bicarbonate, 10% aqueous HCl, dried over magnesium sulfate, and concentrated to leave an oil. Silica gel flash chromatography (being eluted with 15% ethyl acetate in hexane) gave 2.40 g (Y: 83%) of compound XVa as a white solid; .sup.1 H-NMR (CDCl.sub.3) .delta. 7.28 (m, 5H) 5.03 (m, 2H) 1.38 (s, 9H) 0.76 (t, J =7.56, 9H) 0.43 (m, 6H).
EXAMPLE 8 7.alpha.-Fluoro-10-Desoxytaxol (Ic")10-Desacetyltaxol VIa (140 mg, 0.173 mmol) in dry dichloromethane (3.5 mL) was treated at 0.degree. C. with pyridine (0.028 mL, 0.346 mmol) and trichloroethyl chloroformate (0.0724 mL, 0.260 mmol). After 1 h at this temperature, the cold bath was removed and the mixture was stirred at rt overnight. The solvent was evaporated and the residue chromatographed on a silica gel column (being eluted with 30-50% ethyl acetate in hexane) to afford 92.3 mg (Y: 46%) of compound XIIIa as a foam. Continued elution also afforded compound VIIa in 16% yield as a foam.
Compound XIIIa (92.3 mg, 0.079 mmol) in dry dichloromethane (2 mL) was treated with 1,1,2-trifluoro-2-chlorotriethylamine (0.0384 mL, 0.238 mmol). The solution was stirred overnight, the solvent evaporated and the residue purified by silica gel chromatography (being eluted with 25% ethyl acetate in hexane) to yield 42.8 mg (Y: 47%) of compound VIIIa as a white solid.
Dienone VIIIa (39 mg, 0.034 mmol) was dissolved in methanol (0.5 mL) and acetic acid (0.5 mL). Zinc dust ##STR40## (66.4 mg, 1.02 mmol) was added, and temperature of the mixture was maintained at 40.degree. C. for 1 h. The insoluble matter was removed by filtration. The filtrate was concentrated and silica gel chromatography of the residue (being eluted with 60% ethyl acetate in hexane) gave 22 mg (Y: 81.5%) of compound IXa as a foam.
Dienone IXa (22 mg, 0.028 mmol) in ethyl acetate (0.7 mL) was hydrogenated at slightly over one atmospheric pressure in the presence of 10% palladium on charcoal (14.7 mg) for 5.5 h at rt. Removal of the catalyst by filtration, and purification of the product by silica gel chromatography (being eluted with 1:1 ethyl acetate/hexane) gave 15 mg (Y: 68%) of compound Xa as a foam.
Compound Xa (27 mg, 0.034 mmol) in dichloromethane (1 mL) was treated with benzyl chloroformate (0.0146 mL, 0.102 mmol), followed by diisopropylethylamine (0.0177 mL, 0.102 mmol). The reaction mixture was stirred at 0.degree. C. for 45 min, and at rt for 12 h. Evaporation of the solvent and silica gel chromatography (being eluted with 40% ethyl acetate in hexane) gave 25.5 mg (Y: 81%) of compound XIa as a foam.
Compound XIa (25.5 mg, 0.028 mmol) in dichloromethane (0.8 mL) at 0.degree. C. was treated with DAST (0.0071 mL, 0.055 mmol). After 45 min at 0.degree. C., the reaction was allowed to proceed for 5 h at rt. Evaporation of the solvent and chromatography gave XIIa" as a crude foam. This compound was dissolved in ethyl acetate (1 mL) and was stirred under slightly over one atmosphere of hydrogen in the presence of palladium on charcoal (10%, 8.9 mg) for 12 h at rt. The catalyst was removed by filtration and silica gel chromatography of the product gave 10 mg (Y: 40% over two steps) of compound Ic" as a foam; .sup.1 H-NMR (CDCl.sub.3) .delta. 8.08 (d, 2H) 7.70 (d, 2H) 7.68-7.28 (m, 11H) 7.04 (d, 1H) 6.04 (bt, 1H) 5.75 (dd, 1H) 5.69 (d, 1H) 4.92 (d, 1H), 4.72 (dd, 1H) 4.55 (dd, J.sub.H-F =47 Hz) 4.30-4.21 (m, 3H) 3.81 (dd, 1H) 3.47 (d, exch, 1H) 3.37 (bd, 1H) 2.48-1.30 (m, 13H, incl. singlets at 2.30, 1.72, 1.61) 1.07 (s, 3H) 1.02 (s, 3H); HRMS Calcd for MH.sup.+ : 798.3290, found, 798.3264.
EXAMPLE 9 7.alpha.-Fluoro-10-desacetyltaxol (Id")A solution of compound VIIa (obtained as described above, 120 mg, 0.103 mmol) in dichloromethane (2 mL) was cooled at 0.degree. C. and treated With DAST (0.0266 mL, 0.207 mmol). The solution was stirred at 0.degree. C. for 30 min and at rt for 4 h. The reaction was quenched by adding water (0.05 mL). The reaction mixture was concentrated and the residue was purified by silica gel chromatography (being eluted with 30% ethyl acetate in hexane) to afford 81 mg (Y: 68%) of compound XIVa" as a foam. This compound (63 mg, 0.054 mmol) was dissolved in methanol (0.5 mL) and acetic acid (0.5 mL) and treated with zinc dust (104 mg, 1.62 mmol) for 90 min at 45.degree. C. The reaction mixture was filtered and the filtrate was concentrated. Silica gel chromatography (being eluted with 40% hexane in 60% ethyl acetate) of the residue afforded 38 mg (Y: 86%) of compound Id" as a white solid; .sup.1 H-NMR (CDCl.sub.3) .delta. 8.17 (d, 2H) 7.78 (d, 2H) 7.66-7.26 (m, 11H) 7.15 (d, 1H) 6.20 (bt, 1H) 5.83 (dd, 1H) 5.76 (d, 1H) 5.22 (s, 1H) 5.01 (d, 1H) 4.80 (m, 1H) 4.56 (dd, J.sub.H-F =47 Hz) 4.40 (m, 2H) 4.10 (d plus exch. s, 2H) 3.55 (d, exch. 1H) 2.66-1.70 (m, 13H, incl. s at 2.41, 1.82, 1.76) 1.12 (s, 3H) 1.03 (s, 3H); HRMS calcd for MH.sup.+ : 814 3239, found 814.3214.
BIOLOGICAL DATA In vitro cytotoxicity dataThe 7-fluorotaxol derivatives of the present invention showed in vitro cytoxicity activity against human colon carcinoma cells HCT-116 and HCT-116/VM46. The HCT-116/VM46 cells are cells that have been previously selected for teniposide resistance and express the multi-drug resistance phenotype, including resistance to taxol. Cytotoxicity was assessed in HCT-116 human colon carcinoma cells by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfpphenyl) -5-[(phenylamino)carbonyl]2H-tetrazolium hydroxide) assay as reported in D.A. Scudiero, et al., "Evaluation of soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines," Cancer Res. 48:4827-4833, 1988. Cells were plated at 4000 cells/well in 96 well microtiter plates and 24 hours later drugs were added and serial diluted. The cells were incubated at 37.degree. C. for 72 hours at which time the tetrazolium dye, XTT, was added. A dehydrogenase enzyme in live cells reduces the XTT to a form that absorbs light at 450 nm which can be quantitated spectrophotometrically. The greater the absorbance, the greater the number of live cells. The results are expressed as an IC.sub.50, which is the drug concentration required to inhibit cell proliferation (i.e., absorbance at 450 nm) to 50% of that of untreated control cells. The IC.sub.50 values for compounds evaluated in this assay are given in Table I.
TABLE I
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In vitro cytotoxicity data against human colon
carcinoma cells.
IC.sub.50 (.mu.M)
Compound HCT-116 HCT-116/VM46
______________________________________
Ic" 0.301 0.979 (3.3)*
Id" 0.053 1.30 (25)
Ib' 0.004 0.008 (2.3)
Ia' 0.007 0.173 (25)
Ia" 0.011 0.158 (14)
Toxol 0.004 0.440 (124)
______________________________________
*Value is parenthesis is fold resistance relative to HCT116 cells.
Mice M109 Model
Balb/c x DBA/2 F1 hybrid mice were implanted intraperitoneally, as described by William Rose in Evaluation of Madison 109 Lung Carcinoma as a Model for Screening Antitumor Drugs, Cancer Treatment Reports, 65, No. 3-4 (1981), with 0.5 mL of a 2% (w/v) brei of M109 lung carcinoma.
Mice were treated with compound under study by receiving intraperitoneal injections of various doses on either days 1, 5 and 9 post-tumor implant or days 5 and 8 post-implant. Mice were followed daily for survival until approximately 75-90 days post-tumor implant. One group of mice per experiment remained untreated and served as the control group.
Median survival times of compound-treated (T) mice were compared to the median survial time of the control (C) mice. The ratio of the two values for each compoundtreated group of mice was multiplied by 100 and expressed as a percentage (i.e. % T/C) in Table II for a representative compound.
TABLE II
______________________________________
Maximum % T/C (dose in
Compound mg/kg/injection; schedule)
______________________________________
Ia' 239 (40; d. 1, 5 + 9) [336]*
Ib' 147 (40; d. 5 + 8) [225]
______________________________________
*% T/C for taxol on the same schedule
In summary the foregoing tests show that the compounds of the instant invention have tumor inhibiting activities in mammals. Thus, another aspect of the instant invention concerns with a method for inhibiting mammalian tumors sensitive to a compound of formula I. The present invention also provides intermediates useful for making 7-fluoro taxol derivatives of formula I.
The present invention also provides pharmaceutical compositions (formulations) containing a compound of formula I in combination with one or more pharmaceutically acceptable, inert or physiologically active, carriers, excipients, diluents or adjuvants. Examples of formulating taxol or its related derivatives (including a possible dosage) are described in numerous literatures, for example in U.S. Pat. Nos. 4,960,790 and 4,814,470, and such examples may be followed to formulate the compounds of this invention. For example, the new compounds are administrable in the form of tablets, pills, powder mixtures, capsules, injectables, solutions, suppositories, emulsions, dispersions, food premix, and in other suitable forms. The pharmaceutical preparation which contains the compound is conveniently admixed with a nontoxic pharmaceutical organic carrier or a nontoxic pharmaceutical inorganic carrier, usually about 0.01 mg up to 2500 mg, or higher per dosage unit, preferably 50-500 mg. Typical of pharmaceutically acceptable carriers are, for example, manitol, urea, dextrans, lactose, potato and maize starches, magnesium stearate, talc, vegetable oils, polyalkylene glycols, ethyl cellulose, poly(vinylpyrrolidone), calcium carbonate, ethyl oleate, isopropyl myristate, benzyl benzoate, sodium carbonate, gelatin, potassium carbonate, silicic acid, and other conventionally employed acceptable carriers. The pharmaceutical preparation may also contain nontoxic auxiliary substances such as emulsifying, preserving, wetting agents, and the like as for example, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene monostearate, glyceryl tripalmitate, dioctyl sodium sulfosuccinate, and the like.
The compounds of the invention can also be freeze dried and, if desired, combined with other pharmaceutically acceptable excipients to prepare formulations suitable for parenteral, injectable administration. For such administration, the formulation can be reconstituted in water (normal, saline), or a mixture of water and an organic solvent, such as propylene glycol, ethanol, and the like.
The mode, dosage and schedule of administration of taxol in human cancer patients have been extensively studied. See, for example Ann. Int. Med., 111, pp 273-279 (1989). For the compounds of this invention, the dose to be administered, whether a single dose, multiple dose, or a daily dose, will of course vary with the particular compound employed because of the varying potency of the compound, the chosen route of administration, the size of the recipient and the nature of the patient's condition. The dosage to be administered is not subject to definite bounds, but it will usually be an effective amount, or the equivalent on a molar basis of the pharmacologically active free form produced from a dosage formulation upon the metabolic release of the active drug to achieve its desired pharmacological and physiological effects. The dosage to be administered will be generally in the range of 0.8 to 8 mg/kg of body weight or about 50-275 mg/m.sup.2 of the patient. An oncologist skilled in the art of cancer treatment will able to ascertain, without undue experimentations, appropriate protocols for effective administration of the compounds of this present invention by referring to the earlier studies of taxol and its derivatives.
Claims
1. A compound of formula I ##STR41## in which R.sup.1 is benzoyl or t-butyloxycarbonyl; R.sup.2 is acetoxy, hydrogen or hydroxy; and the wavy line indicates either the.alpha.- or the.beta.-configuration.
2. A compound of claim 1 that is 7-fluorotaxol.
3. The compound of claim 2 that is 7.alpha.-fluorotaxol.
4. The compound of claim 2 that is 7.beta.-fluorotaxol.
5. A compound of claim 1 that is N-debenzoyl-N-t-butoxycarbonyl-7-fluorotaxol.
6. The compound of claim 5 that is N-debenzoyl-N-t-butoxycarbonyl-7.alpha.-fluorotaxol.
7. The compound of claim 5 that is N-debenzoyl-N-t-butoxycarbonyl-7.beta.-fluorotaxol.
8. A compound of claim 1 that is 7-fluoro-10-desoxytaxol.
9. The compound of claim 8 that is 7-fluoro-10-desoxytaxol.
10. The compound of claim 8 that is 7.beta.-fluoro-10-desoxytaxol.
11. A compound of claim 1 that is 7-fluoro-10-desacetyltaxol.
12. The compound of claim 11 that is 7.alpha.-fluoro-10-desacetyltaxol.
13. The compound of claim 11 that is 7.beta.-fluoro-10-desacetyltaxol.
14. A pharmaceutical formulation which comprises as an active ingredient a compound as claimed in any one of claims 1 to 13 associated with one or more pharmaceutically acceptable carriers, excipients or diluents therefor.
15. A method for treating mammalian tumors which comprises administering to a mammal a tumor sensitive amount of a compound as claimed in any one of claims 1 to 13.
16. A fluoro baccatin III of formula IV ##STR42## in which the wavy line indicates either the.alpha.- or the.beta.-configuration.
17. The fluoro baccatin III of claim 16 in which fluorine is in the.alpha.-configuration.
18. The fluoro baccatin III of claim 16 in which fluorine is in the.beta.-configuration.
| 4814470 | March 21, 1989 | Colin et al. |
| 4960790 | October 2, 1990 | Stella et al. |
| 5136060 | August 4, 1992 | Holton |
| 400971A2 | December 1990 | EPX |
- Welch, Tetrahedron, vol. 43, #14, pp. 3123-3197 (1987). Francioise Gueritte-Voegelein et al, "Relationships between the Structure of Taxol Analogues and Their Antimitotic Activity," Journal of Medicinal Chemistry, 1991, 34, pp. 992-998. William P. McGuire, M.D. et al, "Taxol: A Unique Antineoplastic Agent with Significant Activity in Advanced Ovarian Epithelial Neoplasms," Annals of Internal Medicine, Aug. 15, 1989, vol. 111, No. 4, pp. 273-279. Charles S. Swindell et al, "Biologically Active Taxol Analogues with Deleted A-Ring Side Chain Substituents and Variable C-2' Configurations," Journal of Medicinal Chemistry, 1991, 34, pp. 1176-1184.
Type: Grant
Filed: Jul 1, 1992
Date of Patent: Mar 9, 1993
Assignee: Bristol-Myers Squibb Company (New York, NY)
Inventors: Shu-Hui Chen (New Haven, CT), Vittorio Farina (West Hartford, CT)
Primary Examiner: James H. Reamer
Attorney: William T. Han
Application Number: 7/907,261
International Classification: A01N 4302;
