United States Patent [19]
`Failli et at
`
`[54] RAPAMYCIl" HYDRAZONES
`
`[75]
`
`Inventors: Amedeo A. Failli, Princeton
`Junction; Robert J. Steffan,
`Langhorne, both of N.J.
`
`[73] Assignee: American Home Products
`Corporation, New York, N.Y.
`
`[21] Appl. No.: 667,684
`
`[22] Filed:
`
`Mar. 8, 1991
`
`Int. Cl.5 ................... A61K 311395; C07D 491/06
`[51]
`[52] U.S. Cl ..................................... 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. ....................... 424/122
`3,993,74911/1976 Seghaletal. ....................... 424/122
`4.316,885 2/1982 Rakhit ................................ 424/122
`4,401,653 8/1983 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. 28,721-726 (1975).
`J. Antibiot. 28,727-732 (1975).
`J. Antibiot. 31,539-545 (1978).
`Can. J. Physiol. Pharmacol. 55,48 (1977).
`FASEB 3,3411 (1989).
`F ASEB 3,5256 (1989).
`Lancet, pp. 1183-1185 (1978).
`Med. Sci. Res., pp. 877-878 (1989), vol. 17.
`J. Am. Chern. Soc., vol. 103, pp. 3215-3217 (1981).
`
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Firm-Arnold S. Milowsky
`
`ABSTRACT
`[57]
`A compound of the structure
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`USOO5120726A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,120,726
`Jun. 9, 1992
`
`,0::,
`c0/ " ..
`
`N
`
`II
`o
`
`OMe
`
`---
`
`wherein
`Rl is hydrogen, alkyl or aralkyl; R2 is hydrogen, alkyl,
`cycloalkyl, aralkyl, -COR3, -C02R3, -S02R3,
`-CONR4R5, -CSNR4R5, -COCONR4R5, or Ar;
`R3 is alkyl, aralkyl, or Ar; R4 and R5 are each, inde(cid:173)
`pendently, hydrogen, alkyl, cycJoalkyl, allyl, aralkyl,
`or Ar;
`Ar is
`
`or
`
`R6, R7, R8 are each, independently, hydrogen, alkyl,
`alkoxy, hydroxy, cyano, halo, nitro, carbalkoxy, tri(cid:173)
`fluoromethyl, trifluoromethoxy, amino, or a carbox(cid:173)
`ylic acid; X is CH or N; Y is NH, 0, or S; or a phar(cid:173)
`maceutically acceptable salt thereof, which by virtue
`of its immunosuppressive activity is useful in treating
`transplantation rejection, host vs. graft disease, auto(cid:173)
`immune diseases and diseases of inflammation; by
`virtue of its antitumor activity is useful in treating
`solid tumors; and by virtue of its antifungal activity is
`useful in treating fungal infections.
`
`18 Claims, No Drawings
`
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`RAPAMYCIN HYDRAZONES
`
`BACKGROUND OF THE INVENTION
`
`5
`
`2
`
`OH
`
`15
`
`This invention relates to hydrazones 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, solid tumors, and 10
`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 [CO Vezina et aI., J.
`Antibiot. 28, 721 (1975); S. N. Sehgal et aI., J. Antibiot.
`28, 727 (1975); H. A. Baker et aI., J. Antibiot. 31, 539 20
`(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 25
`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; 30
`in the adjuvant arthritis model, a model for rheumatoid
`arthritis; and effectively inhibited the formation of IgE(cid:173)
`like antibodies.
`The immunosuppressive effects 'of rapamycin have 35
`been disclosed in FASEB 3, 3411 (1989) and its ability
`to prolong survival time of organ grafts in histoincom(cid:173)
`patible rodents was disclosed by R. Morris [Med. Sci.
`Res. 17: 877 (1989)]. The ability ofrapamycin to inhibit
`T-cell activation was disclosed by M. Strauch [FASEB
`3: 3411 (1989)]. Cyclosporin A and FK-506, other mac(cid:173)
`rocyclic molecules, also have been shown to be effec- 45
`tive as immunosuppressive agents, therefore useful in
`preventing transplant rejection [F ASEB 3, 3411 (1989);
`FASEB 3, 5256 (1989); and R. Y. Caine et aI., Lancet
`1183 (1978)].
`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 55
`(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- 60
`tions.
`
`40
`
`50
`
`DESCRIPTION OF THE INVENTION
`
`This invention provides derivatives of rapamycin 65
`which are useful as immunosuppressive, anti-inflamma(cid:173)
`tory, and antifungal agents having the structure
`
`N
`
`(
`o
`
`OMe
`
`wherein
`Rl is hydrogen, alkyl of 1-6 carbon atoms, or aralkyl
`of 7-10 carbon atoms;
`R 2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl
`of 3-10 carbon atoms, aralkyl of 7-10 carbon
`-COR3,
`-C02R3,
`-S02R3,
`atoms,
`-CONR4R5, -CSNR4R5, -COCONR4R5, or
`Ar;
`R3 is alkyl of 1-6 carbon atoms, aralkyl of 7-10 car(cid:173)
`bon atoms, or Ar;
`R4 and R5 are each, independently, hydrogen, alkyl of
`1-6 carbon atoms, cycloalkyl of 3-10 carbon
`atoms, allyl, aralkyl of 7-10 carbon atoms, or Ar;
`Ar is
`
`or
`
`R6, R7, R8 are each, independently, hydrogen, alkyl
`of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms,
`hydroxy, cyano, halo, nitro, carbalkoxy of 2-7
`carbon atoms, trifluoromethyl, trifluoromethoxy,
`amino, or a carboxylic acid;
`XisCHorN;
`.
`Y is NH, 0, or S;
`or a pharmaceutically acceptable salt thereof.
`The pharmaceutically acceptable salts are those de(cid:173)
`rived from such organic and inorganic acids such as:
`acetic, lactic, citric, tartaric, succinic, maleic, malonic,
`gluconic, hydrochloric, hydrobromic, phosphoric,. ni-
`
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`4
`
`3H-control thymus cells H3-rapam\,cin-treated thymus cells
`3H-controlthymus cells - H3_test compound-treated cells
`
`3
`tric, sulfuric, methanesulfonic, and similarly known
`acceptable acids.
`Of these compounds, preferred members are those in
`which RI
`is hydrogen;
`those
`in which R2
`is
`-CONR4R5; those in which RI is hydrogen and R2 is 5
`-CONR4R5; those in which RI is hydrogen, R2 is
`-CONR4R5, and R5 is Ar; those in which R2 is
`-COR3; those in which RI is hydrogen and R2 is
`-COR3; those in which R2 is Ar; those in which RI is
`hydrogen, R2 is Ar; those in which RI is hydrogen, R2 10
`is Ar, and
`
`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 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,
`irradiated spleen cells from BALB/c donors are in-
`15 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
`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 aHo(cid:173)
`genic control. Rapamycin at a dose of 6 mglkg, p.o.
`gave 86% suppression, whereas cyc1osporin A at the
`same dose gave 43% suppression. Results are expressed
`by the following ratio:
`
`Although compounds of this invention can be pre(cid:173)
`pared by conventional methods that are described in the 20
`literature, because functional groups are contained in a
`large macrocycle ring, functional groups reactivity
`cannot be readily predicted [R. B. Woodward et aI., J.
`Am. Chern. Soc. 103, 3215, (1981)].
`Rapamycin has carbonyl groups at 15, 27 and 33 25
`positions. Based on X-ray crystallographic analysis the
`33 function was predicted to be the most reactive cen(cid:173)
`ter; unexpectedly however, hydrazone formation oc(cid:173)
`curred predominantly at the 27 position.
`The compounds of this invention can be prepared by 30
`the following route from rapamycin:
`
`3H-PLN cells control C3H mouse -
`3H-PLN cells rapamycin-treated C3H mouse
`3H-PLN cells control C3H mouse -
`3H-PLN cells test compound-treated C3H mouse
`
`(or Pyridine)
`
`1/
`R
`
`~.
`II
`N
`I
`N
`"
`
`,
`R-
`
`The second in vivo test procedure is designed to
`35 determine the survival time of pinch skin 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
`40 the dorsum of the recipient as a homograft, and an
`autograft is used as control in the same region. The
`The hydrazine derivatives (hydrazines, semicarba-
`recipients are treated with either varying con centra-
`zides, semithiocarbazides, and semioxamazides) used to
`tions of cyclosporin A as test control or the test com-
`prepare the compounds of the invention are commer-
`pound, intraperitoneally. Untreated recipients serve as
`cially available or can be prepared by methods that are
`45 rejection control. The graft is monitored daily and ob-
`disclosed in the literature.
`servations are recorded until the graft becomes dry and
`Immunosuppressive activity was evaluated in an in
`forms a blackened scab. This is considered as the rejec-
`vitro standard pharmacological test procedure to mea-
`tion day. The mean graft survival time (number of
`sure lymphocyte proliferation (LAF) and in two in vivo
`days±S.D.) of the drug treatment group is compared
`standard pharmacological test procedures. The first in
`vivo procedure was a popliteal lymph node (PLN) test 50 with the control group.
`procedure which measured the effect of compounds of
`The following table summarizes the results of repre-
`sentative compounds of this invention in these three
`this invention on a mixed lymphocyte reaction and the
`second in vivo procedure evaluated the survival time of
`standard test procedures.
`TABLE I
`a pinch skin graft.
`The comitogen-induced thymocyte proliferation pro- 55 ________ ---=..:..::=.::;.::;....:.... _______ _
`LAF
`PLN
`Skin Graft
`cedure (LAF) was used as an in vitro measure of the
`(days + SO)
`Compound
`1Cso(nM)
`(ratio)"
`(ratio)"
`immunosuppressive effects of representative com(cid:173)
`pounds. Briefly, cells from the thymus of normal
`Example I
`17.3
`10.50 ± 0.6
`+
`0.50
`EXample 2
`19.2
`0.45
`7.83 ± 1.3
`+
`BALB/c mice are cultured for 72 hours with PHA and
`Example 3
`15.9
`0.55
`+
`+
`IL-l and pulsed with tritiated thymidine during the last 60
`Example 4
`0.24
`37.2
`1.07
`+
`six hours. Cells are cultured with and without various
`Rapamycin
`8.5-8.8
`I
`I
`12.0 ± 1.7
`concentrations of rapamycin, cycJosporin A, or test
`-Calculation of ratios was described supra.
`compound. Cells are harvested and incorporated radio(cid:173)
`+ Not evaluated
`activity is determined. Inhibition of lymphoprolifera(cid:173)
`tion is assessed as percent change in counts per minute 65
`from non-drug treated controls. The results are ex(cid:173)
`pressed by the following ratio, or as the percent inhibi(cid:173)
`tion of lymphoproliferation at 1 ILM.
`
`The results of these standard pharmacological test
`procedures demonstrate immunosuppressive activity
`both in vitro and in vivo for the compounds of this
`invention. Positive ratios in the LAF and PLN test
`
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`6
`Liquid pharmaceutical compositions which are sterile
`procedures indicate suppression of T cell proliferation.
`solutions or suspensions can be utilized by, for example,
`As a transplanted pinch skin grafts are typically re-
`jected within 6-7 days without the use of an immuno-
`intramuscular, intraperitoneal or subcutaneous injec-
`tion. Sterile solutions can also be administered intrave-
`suppressive agent, the increased survival time of the
`skin graft when treated with the compounds of this 5 nously. The compounds can be administered orally
`either in liquid or solid composition form. The com-
`invention further demonstrates their utility as immuno-
`suppressive agents.
`pounds also may be administered rectally in the form of
`Because the compounds of this invention are structur-
`a conventional suppository. For administration by intra-
`nasal or intrabronchial inhalation, or insufflation, the
`ally similar to rapamycin and have a similar activity
`profile to rapamycin, the compounds of this invention 10 compounds may be formulated into an aqueous or par-
`tially aqueous solution, which can be utilized in the
`also are considered to have antitumor and antifungal
`activities.
`form of an aerosal.
`Based on the results of these standard pharmacologi-
`The dosage requirements vary with the particular
`cal test procedures, the compounds are useful in the
`compositions employed, the route of administration, the
`treatment of transplantation rejection such as, heart, 15 severity of the symptoms presented and the particular
`kidney, liver, bone marrow, skin transplants and the
`subject being treated. Treatment will generally be initi-
`like; autoimmune diseases such as, lupus, rheumatoid
`ated with small dosages less than the optimum dose of
`arthritis, diabetes mellitus, myasthenia gravis, multiple
`the compound. Thereafter the dosage is increased until
`sclerosis and the like; and diseases of inflammation such
`the optimum effect under the circumstances is reached;
`as, psoriasis, dermatitis, eczema, seborrhea, inflamma- 20 precise dosages for oral, parenteral, intranasal, intra-
`tory bowel disease uveitis, and the like; diseases of pul-
`bronchial, or rectal administration will be determined
`monary inflammation, such as, asthma, chronic obstruc-
`by the administering physician based on experience
`tive pulmonary disease, emphysema, acute respiratory
`with the individual subject treated.
`Preferably, the pharmaceutical composition is in unit
`distress syndrome, bronchitis, and the like; solid tumors;
`and fungal infections.
`25 dosage form, e.g. as tablets or capsules. In such form,
`The compounds may be administered neat or with a
`the composition is sub-divided in unit dose containing
`appropriate quantities of the active ingredient; the unit
`pharmaceutical carrier to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`dosage forms can be packaged compositions, for exam-
`A solid carrier can include one or more substances
`pIe, packeted powders, vials, ampoules, prefilled syrin-
`which may also act as flavoring agents, lubricants, solu- 30 ges or sachets containing liquids. The unit dosage form
`can be, for example, a capsule or tablet itself, or it can be
`bilizers, suspending agents, fillers, glidants, compression
`aids, binders or tablet-disintegrating agents; it can also
`the appropriate number of any such compositions in
`be an encapsulating material. In powders, the carrier is
`package form. The dosage to be used in the treatment
`a finely divided solid which is in admixture with the
`must be subjectively determined by the attending physi-
`finely divided active ingredient. In tablets, the active 35 ciano
`ingredient is mixed with a carrier having the necessary
`In addition, the compounds of this invention may be
`compression properties in suitable proportions and
`employed as a solution, cream, or lotion by formulation
`compacted in the shape and size desired. The powders
`with pharmaceutically acceptable vehicles containing
`and tablets preferably contain up to 99% of the active
`0.1-0.5 percent, preferably 2%, of . active compound
`ingredient. Suitable solid carriers include, for example, 40 which may be administered to a fungally affected area.
`The following examples illustrate the preparation of
`calcium phosphate, magnesium stearate, talc, sugars,
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu-
`representative compounds of this invention.
`lose, sodium carboxymethyl cellulose, polyvinylpyr(cid:173)
`rolidine, low melting waxes and ion exchange resins.
`Liquid carriers are used in preparing solutions, sus- 45
`pensions, emulsions, syrups, elixirs and pressurized
`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 50
`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. Suitable examples of liquid car- 55
`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 60
`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(cid:173)
`pyl myristate. Sterile liquid carriers are useful in sterile
`liquid form compositions for parenteral administration. 65
`The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutially
`acceptable propellent.
`
`EXAMPLE I
`Rapamycin 27-(methylcarbonyl hydrazone)
`Under anhydrous conditions, a mixture of rapamycin
`(2.5 g, 2.7 mmole) and acethydrazide (0.3 g, 4.1 mmole)
`in 2.5 mL of pyridine were heated at 60° C. overnight.
`The reaction mixture was concentrated under reduced
`pressure and the residue triturated with water. The
`precipitate was collected, washed with water and dried
`in vacuo to yield 0.62 g of crude product which was
`further purified by flash chromatography (on silica gel
`Merck 60, eluant ethyl acetate).
`IH NMR (CDCh. 400 MHz): 0 1.599 (s, 3H,
`CH3C=C), 1.652 (s, 3H, CH3C=C),2.253 (s, 3H,
`CH3CO), 3.163 (s, 3H, CH30), 3.314 (s, 3H, CH30),
`3.40 (s, 3H, CH30), 8.482 (s, IH, NNH-).
`MS (neg. ion FAB, m/z): 969 (M-), 590,377
`Anal. Calc'd for CS3Hs3N3013: C, 65.61; H, 8.62; N,
`4.33. Found: C, 65.45; H, 8.42; N, 4.18.
`The following representative compounds can be pre(cid:173)
`pared from rapamycin and the appropriate hydrazine by
`employing the method used to prepare the title com(cid:173)
`pound in Example 1.
`Rapamycin 27-[(N-methyl N-methylcarbonyl)hydra(cid:173)
`zone]
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`Rapamycin 27-[(2-naphthylcarbonyl)hydrazone]
`Rapamycin 27-[(4-chlorophenylcarbonyl)hydrazine]
`Rapamycin
`27-[(N-ethyl N-(4-hydroxyphenylcar-
`bonyl»hydrazone]
`Rapamycin 27-[(N-butyl N-(2-pyridylcarbonyl»hydra- 5
`zone]
`Rapamycin 27-[(2-furylcarbonyl)hydrazone]
`Rapamycin
`27-[(N-methylphenyl
`methylimidazol ylcarbonyl) )hydrazone]
`Rapamycin 27-[methoxycarbonyl hydrazone]
`Rapamycin 27-[(N-(2-oxazolyl) N-«phenylmethoxy)(cid:173)
`carbonyl»hydrazone]
`Rapamycin 27-[phenylsulfonyl hydrazone]
`Rapamycin
`27-[(N-(4-chlorophenyl) N-(4-methyl-
`phenylsulfonyl»hydrazone]
`
`N-(2-(l-
`
`8
`3.152 (2s, 3H, CH30), 3.27, 3.274 and 3.296 (3s, 3H,
`CH30), 3.413 (s, 3H, CH30), 6.68-6.74 (m, 1H, ArH),
`7.19-7.24 (m, lH, ArH), 7.51-7.58 (m, 1H, ArH), 7.99
`(s, 1H, NH), 8.03-8.08 (m, 1H, ArH).
`MS (neg. ion FAB, m/z): 1004 (M-), 590,412
`Anal, Calcd. for C56Hs4N4012.0.4H20: C, 66.43; H,
`8.44; N, 5.53. Found: C, 66.19; H, 8;08; N, 5.82.
`The following representative compounds can be pre(cid:173)
`pared from rapamycin and the appropriate hydrazine by
`10 employing the method used to prepare the title com(cid:173)
`pound in Example 3.
`Rapamycin 27-(phenylhydrazone)
`Rapamycin
`27-(N-methyl N-(4(1,4-tetrahydroox-
`azinyl»hydrazone)
`15 Rapamycin 27-(N-phenylmethyl N-(4-thiazolyl)hydra(cid:173)
`zone)
`Rapamycin 27-(N-(3,4-dihydroxyphenyl) N-cyclohex(cid:173)
`ylhydrazone
`Rapamycin 27-(N-methyl N-(2-indolylhydrazone)
`Rapamycin 27-(2-thionaphthylhydrazone)
`Rapamycin 27-(N,N-dimethylhydrazone)
`
`EXAMPLE 4
`
`EXAMPLE 2
`Rapamycin 27-(4-Phenylsemicarbazone)
`A solution of rapamycin (2 g, 2.2 mmole), phenyl(cid:173)
`semi carbazide (0.33 g, 2.2 mmole) and sodium acetate 20
`(0.18 g. 2.2 mmole) in dry methanol (10 mL) was heated
`at 60° C. for 6 hrs. After stirring overnight at room
`temperature, the reaction mixture was diluted with 50
`mL of water. The precipitate was collected, washed
`with water and dried to yield 2.3 g of crude product. 25
`Rapamycin 27-semicarbazone
`Purification was achieved by MPLC (on Lobar silica
`A solution of rapamycin (1.5 g, 1.6 mmole), semicar-
`bazide hydrochloride (0.29 g, 2.45 mmole) and anhy-
`gel Merck 60, ethyl acetate-methanol 98:2, flow rate 20
`drous sodium acetate (0.39 g, 4,8 mmole) in anhydrous
`mL/min).
`IH NMR (CDCI), 400 MHz): 8 1.646 (s, 3H,
`methanol(15 mL) was heated at 50° C. for 14 hours. The
`CH)C=C), 1.805 (5, 3H. CH)C=C), 3.150 (5, 3H, 30 mixture was diluted with water, the precipitate is col-
`CH30), 3.312 (s, 3H, CH30), 3.402 (5, 3H, CH30), 7.049
`lected, washed with water and dried in vacuo. The
`(t, lH, ArH), 7.297 (t, 2H, ArH), 7.49 (d, 2H, ArH), 8.17
`crude product was purified by MPLC (on Lichrosorb
`(s, IH, NH), 8.29 (broad, 1H, NH).
`RP-8, 310-25 Merck, acetonitrile-water 55:45, flow rate
`13C NMR (CDCI3, 400 MHz): 212.7, 191.87, 168.95,
`20 mL/min) to provide the pure title compound.
`167.D1, 153.56, 150.93, 138.69,98.48
`IH NMR (CDCI3, 400 MHz): 8 1.654 (s,3H,
`MS (neg.ion FAB. m/z): 1045 (M-H-), 590,454
`CH3C=C), 1.784 (s, 3H, CH3C=C), 3.133 and 3.169
`Anal. Calcd. for C5sHs6N4013·H20; C, 65.39; H,
`(2s, 3H, CH30), 3.305 (s,3H, CH30), 3.412, 3.414 and
`8.32; N, 5.26. Found: C, 65.00; H, 8.22; N, 5.29.
`3.426 (3s, 3H, CH30), 7.93 (broad, 1H, NH), 8.47
`The following representative compounds can be pre-
`(broad, 2H, NH2)
`pared from rapamycin and the appropriate semicarba- 40
`13C NMR (CDCI3, 400 MHz): 216.35,191.19, 170.38,
`zide, semithiocarbazide, or semioxamazide by employ-
`3
`7 3 9
`3
`5
`168.56, 167.11, 167.02, 157.7 , 15 . 2, 8.97,98. 4,98.2
`.
`h
`h
`d h
`h
`. I
`d .
`mg t e met od use
`t e prepare t e tit e compoun m
`MS (neg ion FAB, m/z): 970 (M-), 590
`Example 2.
`Anal. Calcd for C52Hs2N4013.0.75H20: C, 63.62; H,
`Rapamycin 27-(4-methyl 4-phenylsemicarbazone)
`45 8.55; N, 5.60. Found: C, 63.58; H, 8.47; N, 5.79.
`Rapamycin 27-(4-(2-pyridyl) 4-methylsemicarbazone)
`Rapamycin 27-(4-(3-isoquinolinyl)semicarbazone)
`The following representative compounds can be pre-
`pared f~om rapamycin. semithiocarbazide and semithi-
`Rapamycin 27-(4-phenylsemithiocarbazone)
`Rapamycin 27-(4,4-diphenylmethylsemithiocarbazone)
`ocar?azlde by empl?ymg the method used tp prepare
`the title c?mpound. m. Example 4.
`Rapamycin 27-(5-phenylsemioxamazone)
`Rapamycin 27-(5-(phenylmethyl) 5-propylsemioxama- 50 Rapamyc~n 27-sem~thlOcarbazone
`zone)
`Rapamycm 27-semlOxamazone
`
`35
`
`EXAMPLE 5
`EXAMPLE 3
`Rapamycin 27-hydrazone
`Rapamycin 27-(2-Pyridinylhydrazone)
`Under anhydrous conditions, a solution of rapamycin 55 A solution of Rapamycin (0.1 g, 0.11 mmole) and
`(1 g, 1.1 mmole), 2-hydrazino pyridine dihydrochloride
`85% hydrazine hydrate (0.0065 g, 0.11 mmole) in anhy-
`(0.3 g, 1.65 mmole) and anhydrous sodium acetate (0.4
`drous methanol (1 mL) was heated in an oil bath at 60°
`g, 4.95 mmole) in 10 mL of anhydrous methanol was
`C. overnight. The mixture was concentrated in vacuo
`heated at reflux for 1 hour. The reaction mixture was
`and the residue was purified by preparative TLC (on
`diluted with water and the precipitate is collected, 60 20 X 20 cm Merck-6O silica gel plates, eluant: dichloro-
`washed with water and dried in vacuo. Purification of methanemethanol 9: 1) to provide the pure title com-
`the crude product by flash chromatography (on silica
`pound as a white solid.
`gel Merck 60, gradient from 19:1 dichloromethane-
`IH NMR (CDCI3, 400 MHz): 8 1.71 (m, 3H,
`CH3C=C), 1.728 (m, 3H, CH3C=C), 3.14 (s, 3H,
`ethyl acetate gradient to pure ethyl acetate) provides
`the pure title compound most likely as a mixture of E 65 CH30), 3.415 (s, 6H, CH30), 6.786 (broad, 2H, NH2).
`MS (neg ion FAB, m/z): 927 (M)-
`and Z isomers (based upon the NMR spectral data).
`IH NMR (CDCI3, 400 MHz): 8 1.639 and 1.691 (2s,
`What is claimed is:
`3H, CH3C=C), 1.788 (s, 3H, CH3C=C), 3.135 and
`1. A compound of the formula
`
`NOVARTIS EXHIBIT 2038
`Par v Novartis, IPR 2016-00084
`Page 5 of 7
`
`

`
`9
`
`OH
`
`II
`o
`
`N
`
`-'::::> o
`
`----
`
`OMe
`
`,.#
`
`5,120,726
`
`10
`4. A compound of claim 1 where R I is hydrogen and
`R2 is -CONR4R5 or a pharmaceutically acceptable salt
`thereof.
`5. A compound of claim 1 where R I is hydrogen, R2
`5 is -CONR4R5, and R5 is Ar or a pharmaceutically
`acceptable salt thereof.
`6. A compound of claim 1 where R2 is -COR3 or a
`pharmaceutically acceptable salt thereof.
`7. A compound of claim 1 where R I is hydrogen and
`10 R2 is -COR3 or a pharmaceutically acceptable salt
`thereof.
`8. A compound of claim 1 where R2 is Ar or a phar(cid:173)
`maceutically acceptable salt thereof.
`9. A compound of claim 1 where RI is hydrogen and
`15 R2 is Ar or a pharmaceutically acceptable salt thereof.
`10. A compound of claim 1 where R I is hydrogen, R2
`is Ar, and Ar is
`
`fB}R'
`
`X
`
`20
`
`wherein
`RI is hydrogen, alkyl of 1-6 carbon atoms, or phenyl(cid:173)
`alkyl of 7-10 carbon atoms;
`R2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl
`of 3-10 carbon atoms, phenylalkyl 7-10 carbon
`-COR3,
`-C02R3,
`-S02R3, 25
`atoms,
`-CONR4R5, -CSNR4R5, -COCONR4R5, or
`Ar;.
`R3 is alkyl of 1-6 carbon atoms, phenylalkyl of 7-10
`carbon atoms, or Ar;
`R4 and R5 are each, independently, hydrogen, alkyl of 30
`1-6 carbon atoms, cycloalkyl of 3-10 carbon
`atoms, allyl, phenylalkyl of 7-10 carbon atoms, or
`Ar;
`Ar is
`
`or a pharmaceutically acceptable salt thereof.
`11. A compound of claim 1 which is rapamycin 27-
`(methylcarbonylhydrazone) or a pharmaceutically ac(cid:173)
`ceptable salt thereof.
`12. A compound of claim 1 which is rapamycin 27-(4-
`phenylsemicarbazone) or a pharmaceutically accept(cid:173)
`able salt thereof.
`13. A compound of claim 1 which is rapamycin 27-(2-
`pyridinylhydrazone), or a pharmaceutically acceptable
`35 salt thereof.
`14. A compound of claim 1 which is rapamycin 27-
`semicarbazone or a pharmaceutically acceptable salt
`thereof.
`15. A compound of claim 1 which is rapamycin 27-
`hydrazone or a pharmaceutically acceptable salt
`thereof.
`16. A method of treating transplantation rejection,
`host vs. graft disease, autoimmune diseases, and diseases
`45 of inflammation in a mammal by administering an effec(cid:173)
`tive amount of a compound having the formula
`
`40
`
`or
`
`OH
`
`50
`
`55
`
`R6, R7, R8 are each, independently, hydrogen, alkyl
`of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms,
`hydroxy, cyano, halo, nitro, carbalkoxy of 2-7
`carbon atoms, trifluoromethyl, trifluoromethoxy, 60
`amino, or -C02H;
`X is CH or N;
`Y is NH, 0, or S;
`or a pharmaceutically acceptable salt thereof.
`2. A compound of claim 1 where R I is hydrogen or a 65
`pharmaceutically acceptable salt thereof.
`3. A compound of claim 1 where R2 is -CONR4R5
`or a pharmaceutically acceptable salt thereof.
`
`N
`
`I
`o
`
`wherein
`R I is hydrogen, alkyl of 1-6 carbon atoms, or phenyl(cid:173)
`alkyl of 7-10 carbon atoms;
`
`NOVARTIS EXHIBIT 2038
`Par v Novartis, IPR 2016-00084
`Page 6 of 7
`
`

`
`11
`R2 is hydrogen, alkyl of 1-6 carbon atoms, cycloalkyl
`
`5,120,726
`
`of 3-10 carbon atoms, phenylalkyl of 7-10 carbon
`
`atoms,
`
`-COR3,
`
`-S02R3, 5
`
`12
`-continued
`
`Ar;
`
`R3 is alkyl of 1-6 carbon atoms, phenyl alkyl of7-1O 10
`
`carbon atoms, or Ar;
`
`R 4 and R 5 are each, independently, hydrogen, alkyl of
`
`1-6 carbon atoms, cycloalkyl of 3-10 carbon 15
`
`or
`
`~'
`
`atoms, allyl, phenylalkyl of 7-10 carbon atoms, or
`
`Ar;
`
`Ar is
`
`20
`
`R6, R7, R8 are each, independently, hydrogen, alkyl
`of 1-6 carbon atoms, alkoxy of 1-6 carbon atoms,
`hydroxy, cyano, halo, nitro, carbalkoxy of 2-7
`carbon atoms, trifluoromethyl, trifluoromethoxy,
`amino, or -C02H;
`X is CH or N;
`Y is NH, 0, or S;
`or a pharmaceutically acceptable salt thereof.
`17. A pharmaceutical composition for use as an im-
`25 munosuppressive agent comprising an immunosuppres(cid:173)
`sive amount of a compound of claim 1.
`18. A composition as claimed in claim 17 in unit dos(cid:173)
`age form.
`
`* * * * *
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`NOVARTIS EXHIBIT 2038
`Par v Novartis, IPR 2016-00084
`Page 7 of 7