United States Patent [19]
`Caufield
`
`[54] REDUCTION PRODUCTS OF RAPAMYCIN
`
`[75]
`
`Inventor: Craig E. Caufield, Plainsboro, N.J.
`
`[73] Assignee: American Home Products
`Corporation, New York, N.Y.
`[21] Appl. No.: 696,692
`[22] Filed:
`May 7, 1991
`Int. a.s ..........•........ A61K 31/395; COlD 491/00
`[51]
`[52] U.S. a ..................................... 514/183; 514/321;
`540/456
`[58] Field of Search ................. 540/456; 514/183, 321
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Seghal et al. ....................... 122/122
`3,993,749 11/1976 Seghal et al. ....................... 424/122
`4,316,885 2/1982 Rakhit ................................. 424/122
`4,401,653 4/1983 Eng ..................................... 424/114
`4,650,803 3/1987 Stella et al. ........................... 546/90
`4,885,171 12/1989 Surendra et al. ................... 424/122
`
`OTHER PUBLICATIONS
`J. Antibiot. 28, 721-732, (1975).
`J. Antibiot. 31, 539-545 (1978).
`FASEB 3, 3411, 5256 (1989).
`Lancet, pp. 1183-1185 (1978).
`J. Am. Chern. Soc. 103, pp. 3215-3217 (1981).
`Immunology, C. V. Moseby Co., pp. 12.8-12.11 (1989).
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Firm-Robert F. Boswell, Jr.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`US005102876A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,102,876
`Apr. 7, 1992
`
`ABSTRACT
`[57]
`Reduction of rapamycin with DIBAL furnishes either
`the 15-hydroxyrapamycin or 15,27-bis(hydroxy)rapa(cid:173)
`mycin, depending upon the reaction conditions, having
`the general structure
`
`O OH
`os--' y' ~'~I
`
`N
`
`(
`o
`
`OMe
`
`MeO'
`
`where Y is -CO- -CH(OH)-.
`The compounds of this invention are useful in treating
`transplant rejection, host vs. graft disease, autoimmune
`diseases, diseases of inflammation, and fungal infections.
`
`7 Claims, No Drawings
`
`NOVARTIS EXHIBIT 2039
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`Page 1 of 6
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`

`
`1
`
`5,102,876
`
`2
`
`REDUCIION PRODUCIS OF RAP AMYCIN
`
`BACKGROUND OF THE INVENTION
`
`5
`
`10
`
`This invention relates to compounds of formula I or
`pharmaceutically acceptable salts thereof, which pos-
`sess immunosuppressive and/or antifungal and/or anti-
`tumor and/or antiinflammatory activity in vivo and/or
`inhibit thymocyte proliferation in vitro and are there(cid:173)
`fore useful in the treatment of transplantation rejection,
`autoimmune diseases (i.e. lupus, rheumatoid arthritis, 15
`diabetes mellitus, mUltiple sclerosis), Candida albicans
`infections, and diseases of inflammation.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found 20
`to have antifungal activity, particularly against Candida
`albicans, both in vitro and in vivo [CO Vezina et aI., J.
`
`0 0
`(j _\ ____ , Y -- ~M'I
`
`"
`
`Formula I
`
`N
`
`(
`o
`
`OMe
`
`..----
`
`Under Formula I Y is -CO- or -CH(OH)-. For-
`
`Antibiot. 28, 721 (1975); S. N. Seghal et a!., J. Antibiot. 25 mula I also encompasses the pharmaceutically accept-
`28, 727 (1975); H. A. Baker et a!., J. Antibiot. 31, 539
`(1978); U.S. Pat. Nos. 3,929,992; and 3,993,749].
`
`able salts which may be formed from inorganic cations
`
`such as sodium, potassium and the like.
`
`DETAILED DESCRIPTION OF THE
`
`INVENTION
`
`35
`
`40
`
`The Formula I compounds of the present invention
`
`are
`
`prepared
`
`by
`
`reacting
`
`rapamycin with
`
`diisobutylaluminum hydride (DIBAL) or a similar rea-
`
`gent as outlined in schemes A and B below when only
`
`the pertinent portion of the molecular structure are
`
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com-
`bination with picibanil (U.S. Pat. No. 4,401,653) has 30
`been shown to have antitumor activity. R. Martel et a!.
`[Can. J. Physio!. 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). Rapamycin
`has been shown to be effective in inhibiting transplant
`rejection (U.S. patent application Ser. No. 362,544 filed 45 shown.
`June 6, 1989). Cyclosporin A and FK-506, other macro(cid:173)
`cyclic molecules, also have been shown to be effective
`as immunosuppressive agents, therefore useful in pre(cid:173)
`venting transplant rejection [FASEB 3, 3411 (1989); 50
`FASEB 3, 5256 (1989); and R. Y. CaIne et a!., Lancet
`1183 (1978)]'
`
`Scheme A. Reduction of rapamycin at position 15
`
`O OH
`~~----yZ
`
`l ) 0
`(
`o
`
`N
`
`0
`
`DIBAL>
`-78· C.
`
`SUMMARY OF THE INVENTION
`
`55
`
`This invention provides derivatives of rapamycin
`which are useful as immunosuppressive, antiinflll;ffima(cid:173)
`tory, antifungal, and antitumor agents. The invention 60
`compounds thus are useful in the treatment of transplant
`rejection, autoimmune diseases (i.e., lupus, rheumatoid
`arthritis, diabetes mellitus, multiple sclerosis), Candida
`albicans infections, and diseases of inflammation.
`The compounds of this invention are represented by
`Formula I below:
`
`65
`
`NOVARTIS EXHIBIT 2039
`Par v Novartis, IPR 2016-00084
`Page 2 of 6
`
`

`
`5,102,876
`
`O OH
`~~/~
`
`-continued
`Scheme A. Reduction of rapamycin at position 15
`
`N
`
`(
`
`l ) °
`°
`
`°
`
`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
`5 second in vivo procedure evaluated the survival time of
`a pinch skin graft.
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LA F) was used as an in vitro measure of the
`immunosuppressive effects of representative com-
`10 pounds. Briefly, cells from the thymus of normal
`BALB/c mice are cultured for 72 hours with PHA and
`IL-l and pulsed with tritiated thymidine during the last
`six hours. Cells are cultured with and without various
`concentrations of rapamycin, cyclosporin A, or test
`15 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-
`pressed by the following ratio, or as the percent inhibi(cid:173)
`tion of lymphoproliferation of 1 p.M.
`
`3H-control thymus cells - H3-rapamvcin-treated th"mus cells
`3H-control thymus cells - H3_test compound-treated cells
`
`Reaction of rapamycin with DIBAL at - 78° C. in 20
`anhydrous tetrahydrofuran (THF) results in the keto
`group at position 15 being reduced.
`When the reaction mixture is allowed to proceed at
`_20° C. following addition of DIBAL at _78° c.,
`reduction of the keto groups at positions 15 and 27 25
`occurs giving the 15, 27 diol as shown in Scheme B.
`
`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
`30 DNA synthesis that can be quantified by the incorpora(cid:173)
`tion of tritiated thymidine. Since stimulating a MLR is a
`function of disparity at Major Histocompatibility anti(cid:173)
`gens, an in vivo popliteal lymph node (PLN) test proce(cid:173)
`dure closely correlates to host vs. graft disease. Briefly,
`35 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.,
`b.i.d. On Day 5, the hind popliteal lymph nodes are
`40 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(cid:173)
`genic control. Rapamycin at a dose of 6 mg/kg, p.o.
`45 gave 86% suppression, whereas cyclosporin A at the
`same dose gave 43% suppression. Results are expressed
`by the following ratio:
`
`Scheme B. Reduction of Rapamycin at positions 15 and 27
`
`O OH
`~~/-yZ~
`°
`l ) °
`./'
`II
`°
`
`N
`
`""" °
`
`DIBAL :::,...
`-20' C. 7'
`
`O OH
`~~/~50
`l ) °
`°
`
`OH
`
`N
`
`(
`
`Diastereomers obtained in Scheme B are separated by
`chromatographic procedures, i.e., preparative high
`pressure liquid chromatography.
`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
`
`3H-PLN cells control C3H mouse -
`3H_PLN cells raoamycin-tre<lted C3H mouse
`JH-PLN cells control C3H mouse -
`3H-PLN cells test compound-treated C3H mouse
`
`The second in viv.o test procedure is designed to
`55 determine the survival time of pinch sy..in graft from
`male DBA/2 donors transplanted to male BALB/c
`recipients. The method is adapted from Billingham R.
`E. and Medawar P. B., J. Exp. BioI. 28: 385-402, (1951).
`Briefly, a pinch skin graft from the donor is grafted on
`60 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(cid:173)
`tions of cyclosporin A as test control or the test com(cid:173)
`pound, intraperitoneally. Untreated recipients serve as
`65 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(cid:173)
`tion day. The mean graft survival time (number of
`
`NOVARTIS EXHIBIT 2039
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`Page 3 of 6
`
`

`
`5,102,876
`
`5
`days±S.D.) of the drug treatment group is compared
`with the control group.
`The following table summarizes the results of the
`compounds of this invention in these three standard test
`procedures.
`
`TABLE I
`
`Compound
`Example I
`Example 2
`0.92 ip
`Example 3
`• Activity of analog at 100 nM as compared with rapamycin
`
`LAP
`
`0.58
`0.67
`0.84
`
`PLN*
`-2.47 po, -0.07 ip
`
`Skin Graft
`(days + SD)
`9.8 ± 1.0
`9.2 ± 0.41
`9.5 ± 5.5
`
`6
`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,
`5 lactose, dextrin, starch, gelatin, cellulose, methyl cellu(cid:173)
`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
`10 compositions. The active ingredient can be dissolved or
`suspended in a pharmaceutically acceptable liquid car(cid:173)
`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(cid:173)
`tives, sweeteners, flavoring agents, suspending agents,
`thickening agents, colors, viscosity regulators, stabiliz(cid:173)
`ers or osmo-regulators. Suhable examples of liquid car(cid:173)
`riers for oral and parenteral administration include
`water (partially containing additives as above, e.g. cel(cid:173)
`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
`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.
`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
`the compounds of this invention.
`
`The results of these standard pharmacological test
`procedures demonstrate immunosuppressive activity 15
`both in vitro and in vivo for the compounds of this
`invention. Positive ratios in the LAF and PLN test
`procedures indicate suppression of T cell proliferation.
`As transplanted pinch skin grafts are typically rejected
`with 6-7 days without the use of an immunosuppressive 20
`agent, the increased survival time of the skin graft when
`treated with the compounds of this invention further
`demonstrates their utility as immunosuppressive agents.
`Antifungal activity of the compounds of this inven(cid:173)
`tion was measured against 5 strains of Candida albicans 25
`using a plate test procedure for measurement of inhibi(cid:173)
`tion. The following represents the typical procedure
`used. The compound to be tested was placed on sterile
`dried !" plate disks, and allowed to dry. Agar plates
`were seeded with fungi and allowed to solidify. The 30
`impregnated disks were placed on the seeded Agar
`surface and incubated for the time required for the par:
`ticular culture. Results are expressed in MIC (f-Lg/ml) to
`inhibit growth. The results of this test procedure
`showed that the compounds of this invention have anti- 35
`fungal activity.
`The compound of Example 1 had the following mini(cid:173)
`mal inhibitory concentrations (50%) against 5 strains of
`Candida albicans.
`
`40
`
`Compound
`Example I
`Rapamycin
`
`ATCC
`10231
`
`0.Q25
`0.003
`
`Strains of Candida albicans
`ATCC
`ATCC
`ATCC
`38246
`38247
`38248
`
`0.2
`0.025
`
`0.025
`0.003
`
`0.05
`0.006
`
`3669
`
`0.2
`0.Q25
`
`45
`
`Based on the results of these standard pharmacologi(cid:173)
`cal test procedures, the compounds are useful in the
`treatment of transplantation rejection such as, heart, 50
`kidney, liver, bone marrow, and skin transplants; auto(cid:173)
`immune diseases such as, lupus, rheumatoid arthritis,
`diabetes mellitus, myasthenia gravis, and multiple scle(cid:173)
`rosis; and diseases of inflammation such as, psoriasis,
`dermatitis, eczema, seborrhea, inflammatory bowel 55
`disease; and fungal infections.
`The compounds may be administered neat or with a
`pharmaceutical carrier to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`A solid carrier can include one or more substances 60
`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 65
`finely divided active ingredient. In tablets, the active
`ingredient is mixed with a carrier having the necessary
`compression properties in suitable proportions and
`
`EXAMPLE 1
`15-Deoxo-15-hydroxyrapamycin
`To a solution of 750 mg (821 f-Lmol) of rapamycin in
`15 mL of dry THF was added dropwise at _78° c., 3.6
`mL (3.6 mmol) of a l.OM solution of DIBAL in hex(cid:173)
`anes. After 1 h, the reaction was worked up by adding
`1.0N HCl to dissolve the aluminum salts. The aqueous
`solution was extracted three times with ethyl acetate,
`the combined organics washed with brine, dried over
`sodium sulfate, and concentrated in vacuo to give a
`
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`

`
`5,102,876
`
`7
`colorless transparent solid. The residue was recrystal(cid:173)
`lized from diisopropyl etherlhexane to give after filtra(cid:173)
`tion, 275 mg (37%) of 15-deoxo-15-hydroxyrapamycin,
`mp 118'_122' C.
`The spectral data follow: IH NMR (CDCI3, 400
`MHz) 04.42 (d, 1H, -OH), 4.17 (bs, 1H, anomeric OH),
`3.40 (s, 3H, OCH3), 3.39 (s, 3H, OCH3), 3.14 (s, 3H,
`OCH3), 1.75 (s, 3H, CH3C=C), 1.60 (s, 3H, CH3C=C); 10
`J3C NMR (CDCb, 100 MHz) 8216.0, 209.0, 174.5,
`169.9; IR (KBr) 3440, 2940, 2860, 1740, 1720, 1630,
`1450, 1380, 1195, 1090 cm- I; MS (neg. ion FAB) 914
`(M-H), 592, 339, 184, 168 (100).
`Analysis Calcd. for C5IHsIN013: C, 66.86; FI, 8.91;
`N, 1.53. Found: C, 66.46; H, 9.03; N, 1.36.
`
`5
`
`15
`
`EXAMPLE 2
`
`15,27 -Bis(deoxo )-15,27 -bis(hydroxy )rapamycin
`
`20
`
`where Y is -CO- or -CH(OH)-, or a pharmaceuti(cid:173)
`
`cally acceptable salt thereof.
`
`2. A compound according to claim II which is 15-
`
`deoxo-15-hydroxyrapamycin.
`
`4. A method of treating transplantation rejection,
`
`host vs. graft 'disease, autoimmune diseases, and diseases
`
`of inflammation in a mammal by administering thereto
`
`an effective amount of a compound having the formula
`
`To a solution of 1.43 g (1.56 mmo1) ofrapamycin in 20
`mL of dry THF at _78' C. was added dropwise 6.87
`mL (6.87 mmol) of a 1.0M solution of DIBAL in tolu(cid:173)
`ene. The reaction was slowly warmed to '_20' over a 4 25
`h period and then quenched by stirring over LON HCI
`for 20 min. The reaction mixture was extracted three
`3. A compound according to claim II which is 15,27-
`times with ethyl acetate, the organic. layers combined,
`washed with brine, dried over sodium sulfate, filtered, 30 bis(deoxo)-15,27-bis(hydroxy)rapamycin.
`and concentrated in vacuo to give a foamy solid. The
`residue was purified via HPLC chromatography (2 in
`Dynamax silica column, 100% ethyl acetate, 35
`mLimin) gave 90 mg (6%) of 15,27-bis(deoxy)-15,27- 35
`bis(hydroxy)rapamycin.
`The spectral data follow: IH NMR (CDCI3, 400
`MHz) 04.35 (m, 1H, -OH), 4.17 (bs, 1H, anomeric
`OH), 3.39 (s, 3H, OCH3), 3.33 (s, 3H, OCH3), 3.12 (s, 40
`3H, OCH3), 1.71 (s, 3H, CH3C=C), 1.58 (s, 3H,
`CH3C=C); IR (KBr) 3435, 2930, 2870, 1740, 1720,
`1640,1450,1380,1195,1090 cm- I; MS(neg. ion FAB)
`917 (M-).
`Analysis Calcd. for C51HS3N013.2H20: C, 64.19; H,
`9.19; N, 1.47. Found: C, 64.07; H, 8.28; N, 1.62.
`
`45
`
`EXAMPLE 3
`
`15,27 -Bis(deoxo )-15,27-bis(hydroxy)rapamycin
`
`50
`
`55
`
`Using the above procedure, also collected as a second
`fraction, 60 mg (4%) of 15,27-bis(deoxo)-15,27-bis(hy-
`droxy)rapamycin.
`The spectral data follow: IH NMR (CDCI3, 400
`MHz) 04.39 (m, 1H, -OH), 4.21 (m, 1H, anomeric
`OH), 3.41 (s, 3H, OCH3), 3.40 (s, 3H, OCH3), 3.15 (s,
`3H, OCH3), 1.73 (s, 3H, CH3C=C), 1.60 (s, 3H, 60
`CH3C=C); IR (KBr) 3440, 2930, 2880, 1740, 1720,
`1640,1450, 1380, 1190, 1090 cm- I; MS (neg. ion FA B)
`917 (M-), 359, 168 (100).
`Analysis Calcd. for C5IHs3N013.1.5H20: C, 64.80; 65
`H, 9.17; N, 1.48. Found: C, 64.69; H, 8.86; N, 1.59.
`What is claimed is:
`1. A compound of the formula
`
`'OOH
`(j - ........
`__ ~_/
`-- ~,Me
`
`----
`
`y
`
`°
`
`(
`
`°
`
`I
`
`N
`
`HO
`
`OMe
`
`-----
`
`where Y is -CO- or -CH(OH)-, or a pharmaceuti(cid:173)
`
`cally acceptable salt thereof.
`
`5. A method of treating fungal infections in mammals
`
`by administering thereto an effective amount of a com(cid:173)
`
`pound having the formula
`
`NOVARTIS EXHIBIT 2039
`Par v Novartis, IPR 2016-00084
`Page 5 of 6
`
`

`
`9
`
`5,102,876
`
`5
`
`10
`
`15
`
`10
`
`O OH
`()S/~Y~~'I
`
`N
`
`(
`o
`
`OMe
`,
`
`25
`
`where Y is -CO- or -CH(OH)-, or a pharmaceuti-
`20 cally acceptable salt thereof.
`6. A pharmaceutical composition for treating trans(cid:173)
`plantation rejection, host vs graft disease, autoimmune
`diseases, diseases of inflammation, and fungal infections
`comprising:
`a. a therapeutically effective amount of a compound
`according to claim 1 or a pharmaceutically accept(cid:173)
`able salt thereof and
`b. a pharmaceutical carrier.
`7. A pharmaceutical composition according to claim
`30 6 in unit dosage form.
`• • • • •
`
`35
`
`40
`
`45
`
`·50
`
`. 55
`
`60
`
`65
`
`NOVARTIS EXHIBIT 2039
`Par v Novartis, IPR 2016-00084
`Page 6 of 6