IIIII
`US005233036A
`[ii] Patent Number:
`[45] Date of Patent:
`
`ABSTRACT
`[57]
`A compxiund of the structure
`
`.5,233,036
`Aug. 3, 1993
`
`United States Patent m
`Hughes
`
`[54] RAPAMYCIN ALKOXYESTERS
`
`[75]
`
`Inventor: Philip F. Hughes, Hopewell, N.J.
`
`[73] Assignee: American Home Products
`
`Corporation, New York, N.Y.
`
`[21] Appl. No.: 839,653
`
`[22] Filed:
`
`Feb. 20,1992
`
`Related U.S. Application Data
`[63] Continuation of Ser. No. 598.270, Oct. 16, 1990, aban(cid:173)
`doned.
`
`[51] Int. Cl.5
`[52] U.S. Cl
`[58] Field of Search
`
`C07D 269/00; A61K 31/33
`540/455
`540/455; 514/183
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975
`Sehga! et al
`3,993.749 11/1976
`Sehgal et al
`4,316.885 2/1982
`Rakhit et al
`4,401,653 4/1983
`Maruyama et al
`4,650,803 3/1987
`Stella et al
`4.885.171 12/1989
`Sehgal et al
`4,929,611 5/1990 Okuhava
`OTHER PUBLICATIONS
`Can. J. Physiol. Pharmacol. 55, 48 (1977).
`FASEB 3, 3411 (1989).
`FASEB 3, 5256 (1989).
`Lancet, 1183,(1978).
`J. Am. Chem. Soc. 103, 3215 (1981).
`J. Am. Chem. Soc. 104, 6787 (1982).
`J. Antibiot. 28, 721-726 (1975).
`J. Antibiot. 28, 727-732 (1975).
`J. Antibiot. 31, 539-545 (1978).
`
`Primary Examiner—C. Warren Ivy
`Assistant Examiner—Celia Chang
`Attorney, Agent, or Firm—Arnold S. Milowsky
`
`wherein R1 is
`
`424/122
`424/122
`424/122
`424/122
`546/90
`424/122
`514/183
`
`o
`II
`—CH2COR-:
`
`R2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of
`3-8 carbon atoms which is optionally unsaturated,
`aralkyl of 7-10 carbon atoms, or phenyl which is
`optionally mono-, di-, or tri-substituted with a substit(cid:173)
`uent selected from alkyl of 1-6 carbon atoms, alkoxy
`of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,
`carbalkoxy of 2-7 carbon atoms, trifluoromethyl,
`amino, or a carboxylic acid;
`or a pharmaceutically acceptable salt thereof when R2is
`hydrogen, which are useful in the treatment of trans(cid:173)
`plantation rejection, host vs. graft disease, autoimmune
`diseases, diseases of inflammation, or fungal infections.
`
`4 Oaims, No Drawings
`
`West-Ward Pharm.
`Exhibit 1045
`Page 001
`
`

`

`5,233,036
`
`RAPAMYON ALKOXYESTERS
`
`OR1
`
`This is a continuation of application Ser. No. 5
`07/598,270 filed Oct. 16, 1990 now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`This invention relates to novel ethers of rapamycin
`and a method for using them in the treatment of trans(cid:173)
`plantation rejection, host vs. graft disease, autoimmune
`diseases, diseases of inflammation or fungal infections.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`albicans, both in vitro and in vivo [C. Vezina et al., J.
`Antibiot. 28, 721 (1975); S. N. Seghal et al., J. Antibiot.
`28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539
`(1978); U.S. Pat. No. 3,929,992; and U.S Pat. No.
`3,993,749].
`
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com(cid:173)
`bination with picibanil (U.S. Pat. No. 4,401,653) has
`been shown to have antitumor activity. R. Martel et al.
`[Can. J. Physiol. Pharmacol. 55. 48 (1977)] disclosed
`that rapamycin is effective in the experimental allergic
`encephalomyelitis model, a model for multiple sclerosis;
`in the adjuvant arthritis model, a model for rheumatoid
`arthritis; and effectively inhibited the formation of IgE-
`like antibodies.
`
`The immunosuppressive effects of rapamycin have
`been disclosed in FASEB 3, 3411 (1989). Cyclosporin A
`and FK-506, other macrocyclic molecules, also have
`been shown to be effective as immunosuppressive
`agents, therefore useful in preventing transplant rejec(cid:173)
`tion [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); and
`R. Y. Calne et al., Lancet 1183 (1978)].
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`Mono- and diacylated derivatives of rapamycin (es(cid:173)
`terified at the 28 and 43 positions) have been shown to
`be useful as antifungal agents (U.S. Pat. No. 4,316,885)
`and used to make water soluble prodrugs of rapamycin
`(U.S. Pat. No. 4,650,803). Recently, the numbering
`convention for rapamycin has been changed; therefore
`according to Chemical Abstracts nomenclature, the
`esters described above would be at the 31- and 42- posi(cid:173)
`tions.
`
`50
`
`55
`
`60
`
`DESCRIPTION OF THE INVENTION
`
`This invention provides derivatives of rapamycin 65
`which are useful as immunosuppressive, anti-inflamma(cid:173)
`tory, or antifungal agents having the structure
`
`wherein R' is
`
`O
`II
`—CH2COR2;
`
`R2is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of
`3-8 carbon atoms which is optionally unsaturated,
`aralkyl of 7-10 carbon atoms, or phenyl which is
`optionally mono-, di-, or tri-substituted with a substit(cid:173)
`uent selected from alkyl of 1-6 carbon atoms, alkoxy
`of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,
`carbalkoxy of 2-7 carbon atoms, trifluoromethyl,
`amino, or a carboxylic acid;
`or a pharmaceutically acceptable salt thereof when R2is
`hydrogen.
`Of the compounds, preferred members are those in
`which R2 is alkyl Of 1-6 carbon atoms.
`The pharmaceutically acceptable salts may be formed
`from inorganic cations such as sodium, potassium, cal(cid:173)
`cium, magnesium and the like or may be in the form of
`a quaternary ammonium salt.
`The compounds of this invention can be prepared by
`reacting rapamycin with the appropriately substituted
`ester of diazoacetic acid in the presence of a divalent
`cation salt, such as rhodium (II) diacetate dimer or
`copper (II) triflate, as shown below.
`
`HN2CCO2R2 ^
`OH M(I1)
`> ">
`
`•>
`
`OCH2CO2R2
`
`This method of preparing alkoxyesters has been de(cid:173)
`scribed by B. Ganem et al., J. Am. Chem. Soc. 104,6787
`(1982). The starting materials utilized are either com(cid:173)
`mercially available or can be prepared by methods dis(cid:173)
`closed in the literature.
`The compounds of this invention, rapamycin-42-
`ethers, provide stability against hydrolysis ofthe 42-side
`chain by virtue of the ether moiety connecting the side
`chain to rapamycin at the 42-position.
`Immunosuppressive activity was evaluated in an in
`vitro sUndard pharmacological test procedure to mea(cid:173)
`sure lymphocyte proliferation (LAF) and in two in vivo
`standard pharmacological test procedures. The first in
`vivo procedure was a popliteal lymph node (PLN) test
`procedure which measured the effect of compounds of
`this invention on a mixed lymphocyte reaction and the
`
`West-Ward Pharm.
`Exhibit 1045
`Page 002
`
`

`

`• 4
`3
`The following table summarizes the results of repre-
`second in vivo procedure evaluated the survival time of
`sentative compounds of this invention in these three
`a pinch skin graft.
`standard test procedures,
`The comitogen-induced thymocyte proliferation pro-
`cedure (LAF) was used as an in vitro measure of the __
`TABLE 1
`immunosuppressive effects of representative com(cid:173)
`PLN*
`L A F*
`pounds. Briefly, cells from
`the thymus of normal
`(ratio)
`(ratio)
`BALB/c mice are cultured for 72 hours with PHA and
`0.74**
`IL-1 and pulsed with tritiated thymidine during the last
`+
`1.0
`six hours. Cells are cultured with and without various
`concentrations of rapamycin, cyclosporin A, or test
`compound. Cells are harvested and incorporated; radio(cid:173)
`activity is determined. Inhibition of lymphoprolifera-
`tion is assessed in percent change in counts per minute
`from non-drug treated controls. The results are ex- iS
`pressed by the following ratio, or as the percent inhibi-
`tion of lymphoproliferation of 1 pM.
`
`5,233,036
`
`Compound
`Example 1
`Exampie 2
`Rapamycin
`
`0.46
`0.27
`1.0
`
`•Calculation of ratios was
`. described supra.
`••A result of -0.14 also
`was obtained for Example 1.
`-rNot evaluated
`
`10
`
`Skin Graft
`(days + SD)
`7.5 ± 1.2
`+
`12.0 ± 1.7
`
`The results of the LAF standard pharmacological
`test procedure demonstrates that the compound of Ex(cid:173)
`ample 2 suppressed T-cell proliferation, and is therefore
`useful as an immunosuppressive agent. Based on the
`results of the in vitro and in vivo standard pharmaco(cid:173)
`logical test procedures, it was unclear whether the com-
`p o u nd 0f Example 1 had immunosuppressive activity. A
`r a t io of QM ^
`t he L AF a nd Q 74 in t he pLN
`t e st p r o c e.
`dures indicates that the compound of Example 1 sup-
`preSSed T-cell proliferation; however, the -0.14 ratio
`A mixed lymphocyte reaction (MLR) occurs when
`t h at w as ajso obtained in the PLN test procedure could
`lymphoid cells from genetically distinct animals are
`combined in tissue culture. Each stimulates the other to 25 be indicative of immunostimulation.
`undergo blast transformation which results in increased
`Antifungal activity of the compounds of this inven-
`DNA synthesis that can be quantified by the incorpora-
`tion was measured against 5 strains of Candida albicans
`tion of tritiated thymidine. Since stimulating a MLR is a
`using a plate test procedure for measurement of inhibi-
`function of disparity at Major Histocompatibility anti-
`tion. The following represents the typical procedure
`gens, as in vivo popliteal lymph node (PLN) test proce- 3Q used. Compound to be tested was placed on sterile dried
`dure closely correlates to host vs. graft disease. Briefly,
`J" plate disks, and allowed to dry. Agar plates were
`irradiated spleen cells from BALB/c donors are in-
`seeded with fungi and allowed to solidify. The impreg-
`jected into the right hind foot pad of recipient C3H
`nated disks were placed on the seeded Agar surface and
`mice The drug is given daily, p.o. from Day 0 to Day
`incubated for the time required for the particular cul-
`4 On Day 3 and Day 4, tritiated thymidine is given i.p., 35 t u r e- R e s u l ts a re expressed in MIC (pg/ml)
`to inhibit
`b i.d.. On Day 5, the hind popliteal lymph nodes are
`growth. The results of this test procedure showed that
`?he compounds of this invention have antifungal activ-
`removed and dissolved, and radioactivity counted. The
`corresponding left PLN serves as the control for the
`"y-
`PLN from the injected hind foot. Percent suppression is
`calculated using the non-drug treated animals as allo(cid:173)
`genic control. Rapamycin at a dose of 6 mgAg, p.o.
`gave 86% suppression, whereas cyclosporin A at the
`same dose gave 43% suppression. Results are expressed
`by the following ratio:
`
`^H-controi thymus cells - H^-rapamYcin-ireated thymus cells
`-'H-control thymus cells - H'-test compound-treated cells
`
`40
`
`45
`
`Compound
`Example 1
`Example 2
`Rapamycin
`
`TABLE 2*
`Strain of Candida albicans
`ATCC
`ATCC
`ATCC
`10231
`38246
`38247
`0.5
`0.1
`0.2
`0.05
`0.2
`0.05
`0.003
`0.025
`0.003
`
`ATCC
`38248
`0.1
`0.1 •
`0.006
`
`3669
`
`0.05
`0.2
`0.025
`
`•expressed as MIC (fig/ml)
`
`-'H-PLN cells control C3H mouse -
`3H-PLN cells ranamvcin-treated C3H mouse—
`3U D,3^"PnN c e"s c o m r o li:,-, H,m^^?lr „,...
`jH-PLN cells test compound-treated C3H mouse
`
`Based on the results of these standard pharmacologi-
`cal test procedures, the compounds are useful in the
`r
`.
`K .
`'
`.
`50 treatment of transplantation rejection such as, heart,
`k i d n ey> "^ ^nt marrow and skin transplants; auto-
`The second in vivo test procedure is designed to
`" ™e dlsf,a
`ses s u ch «*• ^
`rheumatoid arthntis,
`determine the survival time of pinch skin graft from
`f
`diabetes mellitus, myasthenia gravis, and multiple scle-
`, X^TW /-. J
`-
`1
`.
`_ I T > A I D /„
`.A
`.
`male DBA/2 donors transplanted to male BALB/c
`, • „
`.•
`x.
`• •
`rosis; and diseases of inflammation such as, psoriasis,
`. •
`j
`, c
`n-ii-
`t.
`r>
`L,
`.
`. ^ i^
`recipients. The method is adapted from BdhnghamR. 55 d e n n a t i ti
`e c z e m ai
`inf la m m a t o r£
`^wel
`E. and Medawar P. B., J Exp. Biol. 28:385-402, (1951).
`d i s e a s e; or f
`i n f e c t i o ns
`Briefly, a pinch skin graft from the donor is grafted on
`T he c o m p o u n ds m ay ^
`a d ministered neat or with a
`the dorsum of the recipient as a homograft, and an
`pharmaceutical carner to a mammal in need thereof,
`autograft is used as control in the same region. The
`^g pharmaceutical carrier may be solid or liquid,
`recipients are treated with either varymg concentra- ^
`substances
`A s o l id c a r r i er c an jn ciu de o ne or m0Tt
`tions of cyclosporin A as test control or the test com-
`whjch may also act as flavoring agents, lubricants, solu-
`pound, intraperitoneally. Untreated recipients serve as
`bilizers, suspending agents, flllers, glidants, compression
`rejection control. The graft is monitored daily and ob-
`ajds, binders or tablet-disintegrating agents; it can also
`servations are recorded until the graft becomes dry and
`be an encapsulating material. In powders, the carrier is
`forms a blackened scab. This is considered as the rejec- 65 a finely divided solid which is in admixture with the
`tion day. The mean graft survival time (number of
`finely divided active ingredient. In tablets, the active
`days±S.D.) of the drug treatment group is compared
`ingredients is mixed with a carrier having the necessary
`with the control group.
`compression properties in suitable proportions and
`
`West-Ward Pharm.
`Exhibit 1045
`Page 003
`
`

`

`5,233,036
`
`10
`
`15
`
`30
`
`35
`
`compacted in the shape and size desired. The powders
`chloride for 3 d to yield a white powder which was
`and tablets preferably contain up to 99% of the active
`filtered
`to give 42-deoxy-42-(2-ethoxy-2-oxoethoxy)-
`ingredient. Suitable solid carriers include, for example,
`rapamycin (496 mg, 23%). The product was isolated as
`calcium phosphate, magnesium stearate, talc, sugars,
`the hemihydrate. IR (KBr) 1680, 1730, 2920, 3430
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu(cid:173)
`c m - '; IH-NMR (CDCL3)81.28 (3H, t, J=7.14 Hz),
`lose, sodium carboxymethyl cellulose, polyvinylpyr-
`1.65 (3H, s), 1.74 (3H, s) 3.14 (3H, s), 3.34 (3H, s), 3. 41
`rolidine, low melting waxes and ion exchange resins.
`(3H, s), 4.20 (2H, q, J = 7.14 Hz), 4.30 (2H, dd); Mass
`Liquid carriers are used in preparing solutions, sus(cid:173)
`Spect (neg. ion FAB) m/z 999 (94%), 590 (15%), 407
`pensions, emulsions, syrups, elixirs and pressurized
`(16%), 379 (4%), 253 (6%), 167 (100%).
`compositions. The active ingredient can be dissolved or
`Analysis Calcd for C55 Hgs N Ou .0.5 H20:C, 65.45;
`suspended in a pharmaceutically acceptable liquid car(cid:173)
`H, 8.59; N, 1.39 Found: C, 65.29; H, 8.64; N, 1.60.
`rier such as water, an organic solvent, a mixture of both
`The following representative compounds can be pre(cid:173)
`or pharmaceutically acceptable oils or fats. The liquid
`pared from rapamycin and the appropriately substituted
`carrier can contain other suitable pharmaceutical addi(cid:173)
`ester of diazoacetic acid by employing the method used
`tives such as solubilizers, emulsifiers, buffers, preserva(cid:173)
`to prepare the title compound in Example 1. 42-Deoxy-
`tives, sweeteners, flavoring agents, suspending agents,
`42-(2-phenoxy-2-oxoethoxy)rapamycin
`42-Deoxy-42-
`thickening agents, colors, viscosity regulators, stabiliz(cid:173)
`ers or osmo-regulators. Suitable examples of liquid car(cid:173)
`[2-(4-chlorophenoxy)-2-oxoethoxy]rapamycin
`42-
`riers for oral and parenteral administration include
`Deoxy-42-(2-phenylmethoxy)-2-oxoethoxy)rapamycin
`water (partially containing additives as above, e.g. cel- 20 42-Deoxy-42-(2-cyclobotoxy)-2-oxoethoxy)rapamycin
`lulose derivatives, preferably sodium carboxymethyl
`42-Deoxy-42-[2-(cyclohex-2-enyloxy)-2-oxoethoxy]-
`cellulose solution), alcohols (including monohydric
`rapamycin
`alcohols and polyhydric alcohols, e.g. glycols) and their
`derivatives, and oils (e.g. fractionated coconut oil and
`arachis oil). For parenteral administration, the carrier 25
`can also be an oily ester such as ethyl oleate and isopro(cid:173)
`pyl myristate. Sterile liquid carriers are useful in sterile
`liquid form compositions for parenteral administration.
`The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically
`acceptable propellent.
`Liquid pharmaceutical compositions which are sterile
`solutions or suspensions can be utilized by, for example,
`intramuscular, intraperitoneal or subcutaneous injec(cid:173)
`tion. Sterile solutions can also be administered intrave(cid:173)
`nously. The compound can also be administered orally
`either in liquid or solid composition form.
`Preferably, the pharmaceutical composition is in unit
`dosage form, e.g. as tablets or capsules. In such form,
`the composition is sub-divided in unit dose containing ^
`appropriate quantities of the active ingredient; the unit
`dosage forms can be packaged compositions, for exam(cid:173)
`ple, packeted powders, vials, ampoules, prefilled syrin(cid:173)
`ges or sachets containing liquids. The unit dosage form
`can be, for example, a capsule or tablet itself, or it can be
`the appropriate number of any such compositions in
`package form. The dosage to be used in the treatment
`must be subjectively determined by the attending physi(cid:173)
`cian.
`In addition, the compounds of this invention may be
`employed as a solution, cream, or lotion by formulation
`with pharmaceutically acceptable vehicles containing
`0.1-0.5 percent, preferably 2%, of active compound
`which may be administered to a fungally affected area.
`The following examples illustrate the preparation of 55
`representative compounds of this invention.
`
`EXAMPLE 2
`42-Deoxy-42-[2-(l,l-dimethylethoxy)-2-oxoethoxy]-
`rapamycin
`To solution of rapamycin (2.0 g, 2.187 mmol) and
`rhodium (H) diacetate (20 mg, 0.04 mmol) in methylen
`chloride (50 mL) was added with t-butyl diazoacetate
`(750 mg, 690 pL, 6.56 mmol) in methylene chloride (20
`mL) over 1.5 h and the reaction mixture was allowed to
`stir overnight. The mixture was concentrated and puri(cid:173)
`fied by chromatography (silica gel, ethyl acetate-hex(cid:173)
`ane, 4:6) to give the product as a glass. The product was
`dissolved with methylene chloride and concentrated to
`give the product as a while solid (588 mg, 26%). The
`product was then dried in vacua at 68° C. overnight and
`isolated as the hemihydrate. IR (KBr) 1650, 1725, 1750,
`2940, 3440 c m - '; 'H-NMR (CDCL3)81.47 (9H, s), 1.65
`(3H, s), 1.74 (3H, s), 3.14 (3H, s) 3.34 (3H, s) 3.34 (3H, s),
`4.20 (2H, dd); Mass spect (neg. ion FAB) m/z 1027
`(32%), 590 (13%), 435 (9%), 167 (100%).
`Analysis Calcd for C57H89N Oi5«0.5 H20:C, 66.00;
`H, 8.74; N, 1.35 Found: C, 65.83; H, 8.60; N, 1.29.
`What is claimed is:
`1. A compound of the structure
`
`.,
`
`50
`
`EXAMPLE "l
`42-Deoxy-42-(2-ethoxy-2-oxoethoxy)rapamycin
`A solution of rapamycin (2.0 g, 2.187 mmol) and
`rhodium (II) diacetate (37 mg, 0.08 mmol) in benzene
`(50 mL) was heated to reflux and ethyl diazoacetate
`(750 mg, 690 ^iL, 6.56 mmol) in benzene (10 mL) was
`added over 10 min. The mixture was concentrated and
`purified by chromatography (silica gel, ethyl acetate-
`hexane, 1:1) to give 700 mg of product (32%) as a glass.
`The product was stirred with hexane with the addition
`of small amounts of ethyl acetate, ether and methylene
`
`60
`
`65
`
`wherein R1 is
`
`West-Ward Pharm.
`Exhibit 1045
`Page 004
`
`

`

`5,233,036
`
`—CH:COR-:
`
`R2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of
`3-8 carbon atoms which is optionally unsaturated
`phenylalkyl of 7-10 carbon atoms, or phenyl which is
`optionally mono-, di-, or tri-substituted with a substit(cid:173)
`uent selected from alkyl of 1-6 carbon atoms, alkoxy
`of 1-6 carbon atoms, hydroxy, cyano, halo, nitro,
`
`• 8
`carbalkoxy of 2-7 carbon atoms, trifluoromethyl,
`amino, or a carboxylic acid;
`or a pharmaceutically acceptable salt thereof when R2is
`hydrogen.
`2. A compound of claim 1 where R2 is alkyl of 1-6
`carbon atoms.
`3. A compound of claim 1 which is 42-deoxy-42-(2-
`ethoxy-2-oxoethoxy)rapamycin.
`4. A compound of claim 1 which is 42-deoxy-42-[2-
`10 (1,1 -dimethylethoxy)-2-oxoethoxy]-rapamycin.
`
`5
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`West-Ward Pharm.
`Exhibit 1045
`Page 005
`
`