United States Patent [19J
`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.
`
`Int. Cl.5 .................... C07D 269/00; A61K 31/33
`[51]
`[52] U.S. Cl. .................................................... 540/455
`[58] Field of Search ......................... 540/455; 514/183
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Sehgal et al. ....................... 424/122
`3,993.749 11/1976 Sehgal et al. ....................... 424/122
`4,316.885 2/1982 Rakhit et al. ....................... 424/122
`4,401,653 4/1983 Maruyama et al. ................. 424/122
`4,650,803 3/1987 Stella et al. ........................... 546/90
`4,885.171 12/1989 Sehgal et al. ....................... 424/122
`4,929,611 5/1990 Okuhava ............................. 514/183
`
`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
`
`I lllll llllllll Ill lllll lllll lllll llllH~ll 111111111111111111111111111111111
`US005233036A
`.5,233,036
`[11) Patent Number:
`[45) Date of Patent:
`Aug. 3, 1993
`
`ABSTRACT
`[57]
`A compound of the structure
`
`wherein RI is
`
`R2is hydrogen, alkyl of 1-6 carbon atoms, cy.cloalkyl 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 Claims, No Drawings
`
`Breckenridge Exhibit 1009
`Breckenridge v. Novartis AG
`
`

`
`1
`
`5,233,036
`
`RAPAMYCIN ALKOXYESTERS
`
`This is a continuation of application Ser. No. 5
`07/598,270 filed Oct. 16, 1990 now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`2
`
`OR 1
`
`0
`
`0
`
`N
`
`I
`0
`
`~O
`
`OMe
`
`MeO __ _
`
`wherein RI is
`
`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.
`
`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 of the 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 standard 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
`
`15
`
`25
`
`35
`
`45
`
`This invention relates to novel ethers of rapamycin JO
`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-
`duced by Streptomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`a/bicans, both in vitro and in vivo [C. Vezina et al., J. 20
`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. 30
`[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 lgE(cid:173)
`like antibodies.
`The immunosuppressive effects of rapamycin have 40
`been disclosed in F ASEB 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-
`tion [F ASEB 3, 3411 ( 1989); F ASEB 3, 5256 ( 1989); and
`R. Y. Caine et al., Lancet 1183 (1978)].
`Mono- and diacylated derivatives of rapamycin (es- so
`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, t e num nng
`be
`h
`convention for rapamycin has been changed; therefore
`according to Chemical Abstracts nomenclature, the
`esters described above would be at the 31- and 42- posi- 60
`tions.
`
`55
`
`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
`
`

`
`5,233,036
`
`4
`The following table summarizes the results of repre(cid:173)
`sentative compounds of this invention in these three
`standard test procedures.
`TABLE 1
`Skin Graft
`LAF*
`PLN*
`(days+ SD)
`(ratio)
`(ratio)
`Compound
`7.5 ± 1.2
`0.46
`0.74**
`Example I
`0.27
`Example 2
`+
`+
`12.0 ± 1.7
`1.0
`Rapamycin
`1.0
`--...:....~------------------
`•Calculation of ratio!t wa~ described supra.
`.. A result of -0.14 also was obtained for Example 1.
`+Not evaluated
`
`3
`second in vivo procedure evaluated the survival time of
`a pinch skin graft.
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LAF) was used as an in vitro measure of the
`immunosuppressive effects of representative com- 5
`pounds. Briefly, cells from the thymus of normal
`BALB/c mice are cultured for 72 hours with PHA and
`IL-1 and pulsed with tritiated thymidine during the last
`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(cid:173)
`tion is assessed in percent change in counts per minute
`from non-drug treated controls. The results are ex- 15
`pressed by the following ratio, or as th~ percent inhibi(cid:173)
`tion of lymphoproliferation of I µM.
`
`10
`
`3H-control thymus cells - H'-rapamycin-treated thvmus celb
`'H-control thymus celb - H'-test compound-treated cells
`
`25
`
`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-
`20 pound of Example 1 had immunosuppressive activity. A
`ratio of0.46 in the LAF and 0. 74 in the PLN test proce(cid:173)
`dures indicates that the compound of Example 1 sup(cid:173)
`pressed T-cell proliferation; however, the -0.14 ratio
`that was also obtained in the PLN test procedure could
`be indicative of immunostimulation.
`Antifungal activity of the compounds of this inven(cid:173)
`tion was measured against 5 strains of Candida albicans
`using a plate test procedure for measurement of inhibi(cid:173)
`tion. The following represents the typical procedure
`used. Compound to be tested was placed on sterile dried
`l" plate disks, and allowed to dry. Agar plates were
`seeded with fungi and allowed to solidify. The impreg(cid:173)
`nated disks were placed on the seeded Agar surface and
`incubated for the time required for the particular cul(cid:173)
`ture. Results are expressed in MIC (µg/ml) to inhibit
`growth. The results of this test procedure showed that
`the compounds of this invention have antifungal activ(cid:173)
`ity.
`
`A mixed lymphocyte reaction (MLR) occurs when
`lymphoid cells from genetically distinct animals are
`combined in tissue culture. Each stimulates the other to
`undergo blast transformation which results in increased
`DNA synthesis that can be quantified by the incorpora(cid:173)
`tion oftritiated thymidine. Since stimulating a MLR is a
`function of disparity at Major Histocompatibility anti(cid:173)
`gens, as in vivo popliteal lymph node (PLN) test proce- 30
`dure closely correlates to host vs. graft disease. Briefly,
`irradiated spleen cells from BALB/c donors are in(cid:173)
`jected into the right hind foot pad of recipient C3H
`mice. The drug is given daily, p.o. from Day 0 to Day
`4. On Day 3 and Day 4, tritiated thymidine is given i.p., 35
`b.i.d .. On Day 5, the hind popliteal lymph nodes are
`removed and dissolved, and radioactivity counted. The
`corresponding left PLN serves as the control for the
`PLN from the injected hind foot. Percent suppression is
`calculated using the non-drug treated animals as allo- 40
`genie control. Rapamycin at a dose of 6 mg/kg, p.o.
`gave 86% suppression, whereas cyclosporin A at the
`same dose gave 43% suppression. Results are expressed
`by the following ratio:
`
`TABLE 2*
`Strain of Candida a/bicans
`ATCC
`ATCC
`ATCC
`!0231
`38246
`38247
`
`0.5
`0.05
`0.003
`
`0.1
`0.2
`0.025
`
`0.2
`0.05
`0.003
`
`ATCC
`38248
`
`0.1
`0.1.
`0.006
`
`3669
`
`0.05
`0.2
`0,025
`
`Compound
`Example I
`Example 2
`Rapamycin
`
`•expressed a. MIC (µg/mlJ
`
`45
`
`'H-PLN cells control C3H mouse -
`'H-PLN cells rapamycin-treated C3H mouse
`JH-PLN celb control C3H mouse -
`3H-PLN cells test compound-treated C3H mouse
`
`The second in vivo test procedure is designed to
`determine the survival time of pinch skin graft from
`male DBA/2 donors transplanted to male BALB/c
`recipients. The method is adapt~d from Billingham R. 55
`E. and Medawar P. B., J. Exp. Btol. 28:385-402, (1951).
`Briefly, a pinch skin graft from the donor is grafted on
`the dorsum of the recipient as a homograft, and an
`autograft is used as control in the same region. The
`recipients are treated with either varying concentra- 60
`tions of cyclosporin A as test control or the test com(cid:173)
`pound, intraperitoneally. Untreated recipients serve as
`rejection control. The graft is monitored daily and ob(cid:173)
`servations are recorded until the graft becomes dry and
`forms a blackened scab. This is considered as the rejec- 65
`tion day. The mean graft survival time (number of
`days±S.D.) of the drug treatment group is compared
`with the control group.
`
`Based on the results of these standard pharmacologi(cid:173)
`cal test procedures, the compounds are useful in the
`50 treatment of transplantation rejection such as, heart,
`kidney, liver, bone marrow, and skin transplants; auto(cid:173)
`immune diseases such as, lupus, rheumatoid arthritis,
`diabetes mellitus, myasthenia gravis, and multiple scle-
`rosis; and diseases of inflammation such as, psoriasis,
`dermatitis, eczema, seborrhea, inflammatory bowel
`disease; or fungal infections.
`The compounds may be administered neat or with a
`pharmaceutical earner to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`A solid carrier can include one or more substances
`which may also act as flavoring agents, lubricants, solu(cid:173)
`bilizers, suspending agents, fillers, glidants, compression
`aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrier is
`a finely divided solid which is in admixture with the
`finely divided active ingredient. In tablets, the active
`ingredients is miAed with a carrier having the necessary
`compression properties in suitable proportions and
`
`

`
`5,233,036
`
`6
`chloride for 3 d to yield a white powder which was
`filtered to give 42-deoxy-42-(2-ethoxy-2-oxoethoxy)(cid:173)
`rapamycin (496 mg, 23%). The product was isolated as
`the hemihydrate. IR (KBr) 1680, 1730, 2920, 3430
`cm-1; IH-NMR (CDCL3)01.28 (3H, t, J=7.14 Hz),
`1.65 (3H, s), 1.74 (3H, s) 3.14 (3H, s), 3.34 (3H, s), 3. 41
`(3H, s), 4.20 (2H, q, J=7.14 Hz), 4.30 (2H, dd); Mass
`Spect (neg. ion F AB) m/z 999 (94% ), 590 (15% ), 407
`(16%), 379 (4%), 253 (6%), 167 (100%).
`Analysis Calcd for Css Hss N 01s e0.5 H20:C, 65.45;
`H, 8.59; N, 1.39 Found: C, 65.29; H, 8.64; N, 1.60.
`The following representative compounds can be pre(cid:173)
`pared from rapamycin and the appropriately substituted
`ester of diazoacetic acid by employing the method used
`to prepare the title compound in Example 1. 42-Deoxy-
`42-(2-phenoxy-2-oxoethoxy)rapamycin 42-Deoxy-42-
`[2-( 4-chlorophenoxy )-2-oxoethoxy ]rapamycin
`42-
`Deoxy-42-(2-phenylmethoxy )-2-oxoethoxy )rapamycin
`42-Deoxy-42-(2-cyclobotoxy)-2-oxoethoxy)rapamycin
`42-Deoxy-42-[2-(cyclohex-2~nyloxy)-2-oxoethoxy]-
`rapamycin
`
`5
`compacted in the shape and size desired. The powders
`and tablets preferably contain up to 99% of the active
`ingredient. Suitable solid carriers include, for example,
`calcium phosphate, magnesium stearate, talc, sugars,
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu- 5
`lose, sodium carboxymethyl cellulose, polyvinylpyr(cid:173)
`rolidine, low melting waxes and ion exchange resins.
`Liquid carriers are used in preparing solutions, sus(cid:173)
`pensions, emulsions, syrups, elixirs and pressurized
`compositions. The active ingredient can be dissolved or JO
`suspended in a pharmaceutically acceptable liquid car~
`rier such as water, an organic solvent, a mixture of both
`or pharmaceutically acceptable oils or fats. The liquid
`carrier can contain other suitable pharmaceutical addi(cid:173)
`tives such as solubilizers, emulsifiers, buffers, preserva- J5
`tives, sweeteners, flavoring agents, suspending agents,
`thickening agents, colors, viscosity regulators, stabiliz(cid:173)
`ers or osmo-regulators. Suitable examples of liquid car(cid:173)
`riers for oral and parenteral administration include
`water (partially containing additives as above, e.g. eel- 20
`lulose derivatives, preferably sodium carboxymethyl
`cellulose solution), alcohols (including monohydric
`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-
`pyl myristate. Sterile liquid carriers are useful in sterile
`liquid form compositions for parenteral administration.
`To solution of rapamycin (2.0 g, 2.187 mmol) and
`rhodium (II) diacetate (20 mg, 0.04 mmol) in methylen
`The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically 30 chloride (50 mL) was added with t-butyl diazoacetate
`(750 mg, 690 µL, 6.56 mmol) in methylene chloride (20
`acceptable propellent.
`Liquid pharmaceutical compositions which are sterile
`mL) over 1.5 hand the reaction mixture was allowed to
`solutions or suspensions can be utilized by, for example,
`stir overnight. The mixture was concentrated and puri-
`intramuscular, intraperitoneal or subcutaneous injec-
`fied by chromatography (silica gel, ethyl acetate-hex-
`tion. Sterile solutions can also be administered intrave- 35 ane, 4:6) to give the product as a glass. The product was
`dissolved with methylene chloride and concentrated to
`nously. The compound can also be administered orally
`give the product as a while solid (588 mg, 26%). The
`either in liquid or solid composition form.
`Preferably, the pharmaceutical composition is in unit
`product was then dried in vacua at 68° C. overnight and
`dosage form, e.g. as tablets or capsules. In such form,
`isolated as the hemihydrate. IR (KBr) 1650 1725 1750
`the com_position i~ ~uh-divided i.n u?it dos_e containin~ 40 2940, 3440 cm-1; IH-NMR (CDCL3)ol.47 '(9H, ~), l.6S
`appropnate quantities of the active mg:c:dient; the umt
`(3H, s), 1. 74 (3H, s), 3.14 (3H, s) 3.34 (3H, s) 3.34 (3H, s),
`dosage forms can be pac~aged compositions, for exa~-
`4.20 (2H, dd); Mass spect (neg. ion F AB) m/z 1027
`pie, packeted powdc:r~, v1~Js, _ampoules, !?refilled syrm-
`(32%), 590 (13%), 435 (9%), l67 (100%).
`.
`ges or sachets contammg hqu1ds. The umt dosage form
`A 1 · C 1 d ,. C H N 0
`0 5 H O·C
`s1 89
`66 00. ,
`
`,.
`bl
`·
`If
`·
`b
`na ys1s
`a c 1or
`159 .
`2 . ,
`
`1
`1
`can be, ior e~amp e, a capsu e or ta et itse , or~·- can . e 45 H 8. 74· N 1.35 Found: C 65.83· H 8.60· N 1.29.
`'wh ' . '
`. d .
`'
`'
`'
`'
`'
`the appropnate number of any such compos1t1ons m
`1
`· h
`at 1s c a1me 1s:
`package form. The dosage to be used m t e treatment
`must be subjectively determined by the attending physi-
`1. A compound of the structure
`cian.
`In addition, the compounds of this invention may be 50
`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
`
`EXAMPLEi
`
`42-Deoxy-42-(2~thoxy-2-oxoethoxy)rapamycin
`A solution of rapamycin (2.0 g, 2.187 mmol) and 60
`rhodium (II) diacetate (37 mg, 0.08 mmol) in benzene
`(50 mL) was heated to reflux and ethyl diazoacetate
`(750 mg, 690 µL, 6.56 mmol) in benzene (10 mL) was
`added over 10 min. The mixture was concentrated and
`purified by chromatography (silica gel, ethyl acetate- 65
`hexane, 1:1) to give 700 mg of product (32%) as a glas.s.
`The product was stirred with hexane with the addition
`of small amounts of ethyl acetate, ether and methylene
`
`wherein RI is
`
`

`
`7
`
`5,233,036
`
`R2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl of
`3-8 carbon atoms which is optionally unsaturated
`phenylalkyl of7-10 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,
`
`5
`
`8
`carbalkoxy of 2-7 carbon atoms, trifluoromethyl,
`amino, or a carboxylic acid;
`or a pharmaceutically acceptable salt thereof when R 2 is
`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.
`• • • • •
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65