US005665772A
`5,665,772
`[11] Patent Number:
`Umted States Patent
`Cottens et al.
`[45] Date of Patent:
`Sep. 9, 1997
`
`
`[19]
`
`[54] O-ALKYLATED RAPAMYCIN DERIVATIVES
`AND THEIR USE: PARTICULARLY AS
`IMMUNOSUPPRESSANTS
`
`[51]
`Int. Cl.‘5 ....................... A61K 31/395; C07D 498/16
`[52] US. Cl. ......................................... 514/514; 540/456
`
`[53] Field of Search .............................. 540/456; 514/514
`
`[75]
`
`Inventors: Sylvain Cottens, Witterswil; Richard
`Sedrani. Basel, both of Switzerland
`
`[56]
`
`References Cited
`US. PATENT DOCUMENTS
`
`[73] Assignee: Sandoz Ltd., Basel, Switzerland
`[21] Appl. No.:
`416,673
`[22] PCT Filed:
`Sep. 24, 1993
`_
`[86] PCT No"
`PCT/EP93/02604
`§ 371 Date:
`Apr. 7, 1995
`§102(e) Date: Apr‘ 7’199s
`[37] Pcrpub, No_; W094/09010
`
`PCT P“b- D3t°5AP1'- 23: 1994
`Foreign Application Priority Data
`
`[30]
`
`............................. 540/542
`6/1992 Failli et al.
`5,120,342
`al.
`..
`9/1992 Ca field
`5,151,413
`
`........................... 514/291
`11/1993 Goltlllet eteztll.
`5,253,339
`Primary Examiner—Robe1't T. Bond
`Attomey, Agent, or Firm—Robe1t S. Honor; Melvyn M.
`Kassenoff; Thomas O. McGovern
`[57]
`ABSTRACT
`Novel derivatives of rapamycin, particularly
`9-deoxorapamycins. 26-dihydro-rapamycins. and 40-0-
`substituted and 28.40-0,0-di—substituted rapamycins, are
`found to have pharmaceutical utility, particularly as an
`““m““°S“PPI€S5a“*S-
`
`Oct. 9, 1992
`
`[GB]
`
`United Kingdom ................... 9221220
`
`10 Claims, N0 Drawings
`
`Roxane Labs., Inc.
`Exhibit 1009
`
`Page 001
`
`Roxane Labs., Inc.
`Exhibit 1009
`Page 001
`
`

`
`5 ,665,772
`
`1
`O-ALKYLATED RAPAMYCIN DERIVATIVES
`AND THEIR USE, PARTICULARLY AS
`IMMUNOSUPPRESSANTS
`
`This application is a 371 of PCT/EP93/02604. filed Sep.
`24. 1993.
`This invention comprises novel alkylated derivatives of
`rapamycin having pharmaceutical utility, especially as
`immunosnppressants.
`Rapamycin is a known macrolide antibiotic produced by
`Streptomyces hygroscopicus, having the structure depicted
`in Formula A:
`
`(A)
`
`24
`
`See. e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44:
`688; Schreiber, S. L., et al.. J. Am. Chem. Soc. (1991) 113:
`7433; U.S. Pat. No. 3,929,992. Rapamycin is an extremely
`potent immunosuppressant and has also been shown to have
`antitumor and antifungal activity.
`Its utility as a
`pharmaceutical, however, is restricted by its Very low and
`variable bioavailability as well as its high toxicity.
`Moreover, rapamycin is highly insoluble, making it difficult
`to formulate stable galenic compositions.
`It has now surprisingly been discovered that certain novel
`derivatives of rapamycin (the Novel Compounds) have an
`improved pharmacologic profile over rapamycin, exhibit
`greater stability and bioavailability, and allow for greater
`ease in producing galenic formulations. The Novel Com-
`pounds are alkylated derivatives of rapamycin having the
`structure of Formula I:
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`
`
`(1)
`
`50
`
`55
`
`65
`
`X is (H,H) or O;
`Y is (H,OH) or O;
`R1 and R2 are independently selected from H. alkyl,
`thioalkyl, arylalkyl, hydroxyalkyl, dihydroxyalkyl.
`hydroxyalkylarylalkyl, dihydroxyalkylarylalkyl,
`alkoxyalkyl, acyloxyalkyl, aminoalkyl,
`alkylaminoalkyl, alkoxycarbonylaminoalkyl,
`acylaminoalkyl. arylsulfonamidoalkyl, allyl,
`dihydroxyalkylallyl. dioxolanylallyl, carbalkoxyalkyl,
`and (R3)3Si where each R3 is independently selected
`from H. methyl, ethyl, isopropyl. t—buty1, and phenyl;
`wherein “all:-” or “alkyl” refers to C1_5 alkyl, branched
`or linear preferably C1_3 alkyl, in which the carbon
`chain may be optionally interrupted by an ether
`(—-0-) linkage; and
`R4 is methyl, or R4 and R1 together form C2_5 alkylene;
`provided that R1 and R2 are not both H; and provided that
`where R1 is (R3)3Si or carbalkoxyalkyl, X and Y are not both
`0.
`
`6.
`
`Preferred Novel Compounds include the following:
`1. 40-0-Benzyl-rapamycin
`2. 40-O-(4'—Hydroxymethyl)benzyl-rapamycin
`3. 40-O-[4'-(1,2-Dihydroxyethyl)]benzyl-rapamycin
`4. 40-O-Allyl-rapamycin
`5. 40-O-[3'-(2,2-Dimethyl-1,3-dioxolan-4(S)-yl)-prop-2'-
`en-1'-yl]-rapamycin
`(2'E,4'S)—40—O-(4',5'-Dihydroxypent-2'-en-1’-yl)-
`rapamycin
`7. 40—O-(2-Hydroxy)ethoxycarbonylmethyl-rapamycin
`8. 40-O-(2-Hydroxy)ethyl-rapamycin
`9. 40-O-(3-Hydroxy)propyl—rapamycin
`10. 40-0-(6-Hydroxy)hexyl-rapamycin
`11. 40-O-[2-(2-Hydroxy)ethoxy]ethyl-rapamycin
`12. 40-O-[(3S)-2,2-Dimethyldioxolan-3-y1]methyl-
`rapamycin
`13. 40-O-[(2S)—2,3-Dihydroxyprop-1-yl]-rapamycin
`14. 40-O-(2-Acetoxy)ethy1-rapamycin
`15. 40—O—(2-Nicotinoyloxy)ethy1-rapamycin
`16. 40-O-[2-(N-Morpho1ino)acetoxy]ethyl-raparnycin
`17. 40-O-(2-N-Imidazoly1acetoxy)ethyl-rapamycin
`18. 40-O-[2-(N-Methyl-N’-piperazinyl)acetoxy]ethyl-
`rapamycin
`19. 39-O-Desmethyl-39,40—0,0-ethylene-rapamycin
`20. (26R)—26-Dihydro—40—O-(2—hydroxy)ethy1—rapamycin
`21. 28-O-Methyl-rapamycin
`22. 40-O-(2-Aminoethyl)-rapamycin
`23. 40-O-(2-Acetaminoethyl)-rapamycin
`24. 40-O-(2-Nicotinamidoethyl)-rapamycin
`25. 40-O—(2-(N-Methyl-imidazo-2'-ylcarbethoxamido)
`ethyl)-rapamycin
`26. 40-O—(2—Ethoxycarbonylaminoethyl)-rapamycin
`27. 40-O-(2-Tolylsulfonamidoethyl)-rapamycin
`28. 40-O-[2-(4',5'-Dicarboethoxy-1',2',3'-triazol-1'-yl)-
`ethyl]-rapamycin
`The Novel Compounds for immunosuppressive use are
`preferably the 40-O-substituted rapamycins where X and Y
`are both 0, R2 is H. R4 is methyl, and R’ is other than H;
`most preferably where R1 is selected from hydroxyalkyl,
`hydroxyalkoxyalkyl, acylaminoalkyl. and aminoalkyl; espe-
`cially 40-O-(2-hydroxy)ethy1-rapamycin, 40-0-(3-hydroxy)
`propyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-
`rapamycin. and 40-O-(2-acetaminoethyl)-raparnycin).
`Preferably O-substitution at C40 or 0,0-disubstitution at
`C28 and C40 is performed according to the following
`general process: Rapamycin (or dihydro or
`deoxorapamycin) is reacted with an organic radical attached
`to a leaving group (e.g.. RX where R is the organic radical.
`Roxane Labs., Inc.
`Exhibit 1009
`
`Page 002
`
`Roxane Labs., Inc.
`Exhibit 1009
`Page 002
`
`

`
`3
`
`4
`
`5,665,772
`
`e) Treatment of proliferative disorders, e.g. tumors, hyper-
`e.g., an alkyl, allyl, or benzyl moiety, which is desired as _the
`proliferative skin disorder and the like.
`O-substituent, and X is the leaving group, e.g., CCl3C
`(NH)O or CF3SO3) under suitable reaction conditions, pref-
`f) Treatment of fungal infections.
`erably acidic or neutral conditions, e.g., in the presence of an
`g) Treatment and prevention of inflammation. especially
`acid like trifluoromethanesulfonic acid, camphorsulfonic
`in potentiating the action of steroids.
`acid, p-toluenesulfonic acid or their respective pyridinium or
`h) Treatment and prevention of infection, especially infec-
`substituted pyridinium salts when X is CCl3(NH)O or in the
`tion by pathogens having Mip or Mip-like factors.
`presence of a base like pyridine, a substituted pyridine,
`i) Treatment of overdoses of FK-506, rapamycin, immu-
`diisopropylethylarnine or pentamethylpiperidine when X is
`nosuppressive Novel Compounds, and other macrophi-
`-CF3SO3. O-substituents at C28 only are accomplished in the
`1m.bmdm.g 1mmun°SupessamS'
`same manner, but with prior protection at C40. Further 10
`d Th?b;‘1‘V1‘:““‘?“ glue Pro‘/Ides thle .Nt°Vel C?-"Pounds
`modifications are possible. For example, where the substitu-
`25°11
`figtlim’ 0;! us: a; govt; In erme a C; or $5
`ent is allyl, the isolated, monosubstituted double bond of the
`P ammceu _
`S’
`‘Fe O
`0
`ea _
`°r_ Preven "3 _ C
`allyl moiety is highly amenable to further modification.
`above-described disorders by admrnrstenng an effective
`The 9_dcoX01.apamycin Compounds are Preferably pm_
`duced by reducing a rapamycin using hydrogen sulfide’ by 15 amount of Novel Comppund to a patient 1n need thereof, use
`reacting rapamycin with diphenyldiselenide and tribu-
`Of a NoVel Compound in the manufacture of a medicament
`tylphosphing 01'
`other suitablg reduction ]_'eacfion_
`fOl' lIC3.lIl1CIlt 0I'pI'CVCIlti0Il Of fl'lC above-described dl.SOl'd€l' S,
`The 26-djhyd;o.1apamycjns are preferably produced by
`and pharmaceutical compositions comprising a Novel Com-
`reducing rapamycins or 9-deoxorapamycins from keto to
`pound in combination or association with a pharmaceuti-
`hydroxy at C26 by a mild reduction reaction, such as a 20 cally acceptable diluent or carrier.
`borohydride reduction reaction.
`Most of the Novel Compounds described herein are
`The Novel Compounds are particularly useful for the
`highly irnmunosuppressive, especially those Novel Com-
`following conditions?
`pounds which are O-substituted at C40, and these Novel
`a) Treatment and prevention of organ or tissue transplant
`Compounds are particularly useful in indications a and b, but
`rejection: e-g~ for the treatment of reciPients of e-g- 25 notinindication i. Those of the Novel Compounds which are
`heart,
`lung, combined heart'lung.~ liVer, kidneys
`less immunosuppressive, especially those which are
`Peuereeee Ski“ °r eemeel t”an5P1a“t5- They are 315°
`O-substituted at C28 only, are particularly useful in indica-
`indicated fer the Prevention °f g1'afi'Ver5“5‘h°5t
`tions h and i, but are less preferred in indications a or b.
`disease’ Such as f°u°Wing bone marrow t1'a“5P1a“ta'
`The Novel Compounds are utilized by administration of a
`tie“
`30 pharrnaceutically effective dose in pharrnaceutically accept-
`b) Treatment and prevention of autoimmune disease and
`able form to a subject in need of treatment_ Appropriate
`of inflammatory conditions, in Particular inilammatory
`dosages of the Novel Compounds will of course vary, e.g.
`conditions With an etiology including an autoimmune
`depending on the condition to be treated (for example the
`component such as arthritis (tor eXamPle rheumatoid
`disease type or the nature of resistance), the effect desired
`arthritis, arthritis chronica progrediente and arthritis 35 and the mode of adm1m's1Iati0n_
`deformahs) and rheumatic diseases Specific autoi-tn‘
`In general however satisfactory results are obtained on
`mune diseases for Which the comPounds of the inVen‘
`administration orally at dosages on the order of from 0.05 to
`tion may he emPloyed include, autoimmune hemato'
`5 or up to 10 mg/kg/day, e.g. on the order of from 0.1 to 2
`logical disorders (including e-g- hemolytic anaemia:
`or up to 7.5 mg/kg/day administered once or, in divided
`aPlastic anaemia: Pure red cell anaemia and idiOPathic 40 doses 2 to 4x per day, or on administration parenterally, e.g.
`thromhocytOPenia)a systemic luPus erythematosnsa
`intravenously, for example by i.v. drip or infusion, at dos-
`Polychondrltis, sCler0d0Ina.~ Wegener granularnatosisa
`ages on the order of fiom 0.01 to 2.5 up to 5 mg/kg/day, e.g.
`dermatomyosilis. chronic active hepatitis. myasthenia
`on the order of from 0.05 or 0.1 up to 1.0 mg/kg/day.
`graVisa Psoriaslsa SteVen'JohnsOn syndromes idiopathic
`Suitable daily dosages for patients are thus on the order of
`sPruea autoimmune inflammatory ho“/el disease 45 500 mg p.o., e.g. on the order of from 5 to 100 mg p.o., or
`(including e-g- ulceratiVe colitis and Crohn’s disease)
`on the order of from 0.5 to 125 up to 250 mg i.v., e.g. on the
`endocrine ophthalmopathy, Graves disease,
`order of from 25 to 50 mg i_V_
`-
`sa-1'Coidosisa multiPle sclerosis, Primary hilliary
`Alternatively and even preferably, dosaging is arranged in
`cl1‘1'hosis,J'uVenile diabetes (diabetes mellitus tyPe Du
`patient specific manner to provide pre-determined trough
`nVeitis (anterior and Posterior), keratoconlunctiVitis 50 blood levels, e.g. as determined by RIA technique. Thus
`sicca and vernal keratoconjunctivitis, interstitial lung
`patient dosaging may be adjusted so as to achieve regular
`fibrosis, Psoriatic arthritis, glomerulonephritis (With
`on-going trough blood levels as measured by RIA on the
`‘and without nephrofic syndrome, e-g- including idi<>
`order of from 50 or 150 up to 500 or 1000 ng/ml, i.e.
`Pathic nePhrotic syndrome or minimal change
`analogously to methods of dosaging currently employed for
`nePh1'oPathy) and luVenile dermatomyosiiis
`55 Ciclosporin irnmunosuppressive therapy.
`C)TICatII1€I1t and p1'CV€I1ti0Il Of aSthma-
`The Novel Compounds may be administered as the sole
`d) Treatment of multi-drug resistance (MDR). The Novel
`active ingredient or together with other drugs. For example,
`Compounds suppress P-glycoproteins (Pgp), which are
`in irnmunosuppressive applications such as prevention and
`the membrane transport molecules associated with
`treatment of graft vs. host disease, transplant rejection. or
`MDR. MDR is particularly problematic in cancer 60 autoimmune disease, the Novel Compounds may be used in
`patients and AIDS patients who will not respond to
`combination with Ciclosporin, FK-506, or their immuno-
`conventional chemotherapy because the medication is
`suppressive derivatives; corticosteroids; azathioprene;
`pumped out of the cells by Pgp. The Novel Compounds
`immunosuppressive monoclonal antibodies, e.g., mono-
`clonal antibodies to CD3, CD4, CD25, CD28, or CD45; and
`are therefore useful for enhancing the eflicacy of other
`chemotherapeutic agents in the treatment and control of
`7 or other immunomodulatory compounds. For anti-
`multidrug resistant conditions such as multidrug resis-
`inflammatory applications, the Novel Compounds can be
`tant cancer or multidrug resistant AIDS.
`used together with anti-inflammatory agents, e.g., corticos-
`
`65
`
`Roxane Labs., Inc.
`Exhibit 1009
`
`Page 003
`
`Roxane Labs., Inc.
`Exhibit 1009
`Page 003
`
`

`
`5,665,772
`
`5
`
`teroids. For anti-infective applications, the Novel Com-
`pounds can be used in combination with other anti-infective
`agents, e.g., anti-viral drugs or antibiotics.
`The Novel Compounds are administered by any conven-
`tional route, in particular enterally, e.g. orally, for example
`in the form of solutions for drinking, tablets or capsules or
`parenterally, for example in the form of injectable solutions
`or suspensions. Suitable unit dosage forms for oral admin-
`istration comprise, e.g. from 1 to 50 mg of a compound of
`the invention, usually 1 to 10 mg. Pharmaceutical compo-
`sitions comprising the novel compounds may be prepared
`analogously to pharmaceutical compositions comprising
`rapamycin, e.g., as described in EPA 0 041 795, which
`would be evident to one skilled in the art.
`
`The pharmacological activity of the Novel Compounds
`are demonstrated in, e.g., the following tests:
`1. Mixed lymphocyte reaction (MLR)
`The Mixed Lymphocyte Reaction was originally devel-
`oped in connection with allografts.
`to assess the tissue
`compatibility between potential organ donors and recipients,
`and is one of the best established models of immune reaction
`
`in vitro. A murine model MLR, e.g., as described by T. Meo
`in “Immunological Methods”, L. Lefkovits and B. Peris,
`Eds., Academic Press, N.Y. pp. 227-239 (1979), is used to
`demonstrate the irnmunosuppressive effect of the Novel
`Compounds. Spleen cells (0.5x10‘) from Balb/c mice
`(female, 8-10 weeks) are co-incubated for 5 days with
`0.5><106 irradiated (2000 rads) or mitomycin C treated
`spleen cells from CBA mice (female, 8-10 weeks). The
`irradiated allogeneic cells induce a proliferative response in
`the Balb/c spleen cells which can be measured by labeled
`precursor incorporation into the DNA. Since the stimulator
`cells are irradiated (or mitomycin C treated) they do not
`respond to the Balb/c cells with proliferation but do retain
`their antigenicity. The antiproliferative effect of the Novel
`Compounds on the Balblc cells is measured at various
`dilutions and the concentration resulting in 50% inhibition
`of cell proliferation (IC5o) is calculated. The inhibitory
`capacity of the test sample may be compared to rapamycin
`and expressed as a relative IC50 (i.e. IC50 test sample/IC50
`rapamycin).
`2. IL-6 mediated proliferation
`The capacity of the Novel Compounds to interfere with
`gowth factor associated signalling pathways is assessed
`using an interleukin-6 (IL-6)-dependent mouse hybridoma
`cell line. The assay is performed in 96-well microtiter plates.
`5000 cells/well are cultivated in serum-free medium (as
`described by M. H. Schreier and R. Tees in Immunological
`Methods, I. Lefkovits and B. Pernis, eds., Academic Press
`1981, Vol. II, pp. 263-275), supplemented with 1 ng recom-
`binant IL-6/ml. Following a 66 hour incubation in the
`absence or presence of a test sample, cells are pulsed with
`1 uCi (3-H)-thymidindwell for‘ another 6 hours, harvested
`and counted by liquid scintillation. (3-H)-thyrnidine incor-
`poration into DNA correlates with the increase in cell
`number and is thus a measure of cell proliferation. A dilution
`series of the test sample allows the calculation of the
`1 concentration resulting in 50% inhibition of cell prolifera-
`tion (IC50). The inhibitory capacity of the test sample may
`be compared to raparnycin and expressed as a relative IC5o
`(i.e. IC50 test sample/[C50 rapamycin).
`3. Macrophilin binding assay
`structurally related
`Rapamycin and the
`irnmunosuppressant, FK-506, are both known to bind in
`vivo to macrophilin-12 (also known as FK-506 binding
`protein or FKBP-12). and this binding is thought to be
`related to the immunosuppressive activity of these com-
`
`6
`pounds. The Novel Compounds also bind strongly to
`macrophilin- 12, as is demonstrated in a competitive binding
`assay.
`In this assay, FK-506 coupled to BSA is used to coat
`microtiter wells. Biotinylated recombinant human
`macrophilin- 12 (biot-MAP) is allowed to bind in the pres-
`ence or absence of a test sample to the immobilized FK—506.
`After washing (to remove non-specifically bound
`macrophilin), bound biot-MAP is assessed by incubation
`with a streptavidin-alkaline phosphatase conjugate, fol-
`lowed by washing and subsequent addition of p-nitrophenyl
`phosphate as a substrate. The read-out is the OD at 405 nm.
`Binding of a test sample to biot-MAP results in a decrease
`in the amount of biot-MAP bound to the FK-506 and thus in
`
`a decrease in the OD405. A dilution series of the test sample
`allows determination of the concentration resulting in 50%
`inhibition of the biot-MAP binding to the immobilized
`FK-506 (ICSO). The inhibitory capacity of a test sample is
`compared to the IC50 of free FK-506 as a standard and
`expressed as a relative IC50 (i.e., IC50-test sample/IC5O-free
`FK-506).
`,
`4. Localized Graft-Versus-Host (GVH) Reaction
`In vivo efiicacy of the Novel Compounds is proved in a
`suitable animal model, as described, e.g., in Ford et al,
`TRANSPLANTATION 10 (1970) 258. Spleen cells (1><107)
`from 6 week old female Wistar/Furth (WF) rats are injected
`subcutaneously on day 0 into the left hind-paw of female
`(F344><WF)F1 rats weighing about 100 g. Animals are
`treated for 4 consecutive days and the popliteal lymph nodes
`are removed and weighed on day 7. The difference in weight
`between the two lymph nodes is taken as the parameter for
`evaluating the reaction.
`5. Kidney Allograft Reaction in Rat
`One kidney from a female fisher 344 rat is transplanted
`onto the renal vessel of a unilaterally (left side) nephrecto-
`mized WF recipient rat using an end-to-end anastomosis.
`Ureteric anastomosis is also end-to-end. Treatment com-
`mences on the day of transplantation and is continued for 14
`days. A contralateral nephrectomy is done seven days after
`transplantation, leaving the recipient relying on the perfor-
`mance of the donor kidney. Survival of the graft recipient is
`taken as the parameter for a functional graft.
`6. Experimentally Induced Allergic Encephalomyelitis
`(EAE) in Rats
`Eflicacy of the Novel Compounds in EAE is measured,
`e.g., by the procedure described in Levine & Wenk, AMER
`J PATH 47 (1965) 61; McFarlin et al, J IMMUNOL 113
`(1974) 712; Borel, TRANSPLANT. & CLIN. IMMUNOL
`13 (1981) 3. EAE is a widely accepted model for multiple
`sclerosis. Male Wistar rats are injected in the hind paws with
`a mixture of bovine spinal cord and complete Freund’s
`adjuvant. Symptoms of the disease (paralysis of the tail and
`both hind legs) usually develop within 16 days. The number
`of diseased animals as well as the time of onset of the disease
`are recorded.
`7. Freund’s Adjuvant Arthritis
`Efficacy against experimentally induced arthritis is shown
`using the procedure described, e.g.,
`in Winter & Nuss,
`ARTHRITIS & RHEUMATISM 9 (1966) 394; Billingham
`& Davies, HANDBOOK OF EXPERIMENTAL PHARMA-
`COL (Vane & Ferreira Eds, Springer-Verlag. Berlin) 50/11
`(1979) 108-144. OFA and Wistar rats (male or female, 150
`g body weight) are injected i.e. at the base of the tail or in
`the hind paw with 0.1 ml of mineral oil containing 0.6 mg
`of lyophilized heat-killed Mycobacterium smegrnatis. In the
`developing arthritis model. treatment is started immediately
`after the injection of the adjuvant (days 1-18);
`in the
`
`-
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`Roxane Labs., Inc.
`Exhibit 1009
`
`Page 004
`
`Roxane Labs., Inc.
`Exhibit 1009
`Page 004
`
`

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`5,665,772
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`7
`
`8
`established arthritis model treatment is started on day 14,
`dexamethasone, results in greatly enhanced steroidal activ-
`when the secondary inflammation is well developed (days
`ity. This can be shown, e.g.. in the murine mammary tumor .
`virus-chloramphenicol acetyltransferase (MMTV-CA1")
`14-20). At the end of the experiment, the swelling of the
`joints is measured by means of a micro-caliper. ED50 is the
`reporter gene assay, e.g., as described in Ning, et al., J. Biol.
`5 Chem. (1993) 268: 6073. This synergistic effect allows
`oral dose in mg/kg which reduces the swelling (primary or
`reduced doses of corticosteroids, thereby reducing flre risk of
`secondary) to half of that of the controls.
`side efl°ects in some cases.
`8. Antiurmor and MDR activity
`11. Mip and Mip-like factor inhibition
`The antitumor activity of the Novel Compounds and their
`Additionally. the Novel Compounds bind to and block a
`ability to enhance the performance of antitumor agents by
`alleviating multidrug resistance is demonstrated, e.g., by 10 variety of Mip (macrophage infectivity potentiator) and
`administration of an anticancer agent, e.g., colchicine or
`Mip-like factors, which are structurally similar to macro-
`etoposide, to multidrug resistant cells and drug sensitive
`phflin. Mip and Mip-like factors are virulence factors pro-
`cells in vitro or to animals having multidrug resistant or drug
`duced by a wide variety of pathogens, including those of the
`sensitive tumors or
`infections, with and without
`genera Chlamidia, e.g., Chlamidia trachomatis; Neisseria,
`co-administration of the Novel Compounds to be tested, and 15 e.g., Neisseria meningiiidis; and Legionella, e.g., Legionella
`by administration of the Novel Compound alone.
`pneumophilia; and also by the obligately parasitic members
`Such in vitro testing is performed employing any appro-
`of the order Rickettsiales. These factors play a critical role
`priate drug resistant cell line and control (parental) cell line,
`in the establishment of intracellular infection. The efficacy
`generated, e.g. as described by Ling et al., J. Cell. Physiol.
`of the Novel Compounds in reducing the infectivity of
`83, 103-116 (1974) and Bech-Hansen et al. J. Cell. Physiol. 20 pathogens which produce Mip or Mip-like factors can be
`88, 23-32 (1976). Particular clones chosen are the multi-
`shown by comparing infectivity of the pathogens in cells
`drug resistant (e.g. colchicine resistant) line CHR (subclone
`culture in the presence and absence of the macrolides, e.g.,
`C5S3.2) and the parental, sensitive line AUX B1 (subclone
`using the methods described in Lundemose, et al., Mal.
`ABl S11).
`Microbiol. (1993) 7: 777. The nonirnrnunosuppressive com-
`In vivo anti-tumor and anti-MDR activity is shown, e.g., 25 pounds of the invention are preferred for use in this indica-
`in mice injected with multidrug resistant and drug sensitive
`tion for the reason that they are not immunosuppressive, thus
`cancer cells. Ehrlich ascites carcinoma (EA) sub-lines resis-
`they do not compromise the body’ s natural immune defenses
`tant to drug substance DR, VC, AM, ET, TE or CC are
`against the pathogens.
`developed by sequential transfer of EA cells to subsequent
`The Novel Compounds are also useful in assays to detect
`generations of BALB/c host mice in accordance with the 30 the presence or amount of macrophilin-binding compounds,
`methods described by Slater et al., J. Clin. Invest, 70, 1131
`e.g., in competitive assays for diagnostic or screening pur-
`(1982).
`poses. Thus, in another embodiment, the invention provides
`Equivalent results may be obtained employing the Novel
`for use of the Novel Compounds as a screening tool to
`Compounds test models of comparable design, e.g. in vitro,
`determine the presence of macrophflin-binding compounds
`or employing test animals infected with drug-resistant and 35 in a test solution, e.g., blood, blood serum, or test broth to
`drug sensitive viral strains, antibiotic (e.g. penicillin) resis-
`be screened. Preferably, a Novel Compound is immobilized
`tant and sensitive bacterial strains, anti-mycotic resistant and
`in microtiter wells and then allowed to bind in the presence
`sensitive fungal strains as well as drug resistant protozoal
`and absence of a test solution to labelled macrophilin-12
`strains, e.g. Plasmodial strains, for example naturally occur-
`(FI$P- 12). Alternatively,
`the FIG3P- 12 immobilized in
`ring sub-strains of Plasmodium falciparum exhibiting 40 microtiter wells and allowed to bind in the presence and
`acquired chemotherapeutic, anti-malarial drug resistance.
`absence of a test solution to a Novel Compound which has
`9. FKBP binding
`been labelled, e.g., fluoro-, enzymatically- or radio-labelled,
`the Novel Compounds are not
`Certain of
`e.g., a Novel Compound which has been O-substituted at
`immunosuppressive, particularly those which are
`C40 and/or C28 with a labelling group. The plates are
`O-substituted at C28 only, such as 28-O-methyl-rapamycin. 45 washed and the amount of bound labelled compound is
`This can be shown in standard in vitro assays in comparison
`measured. The amount of macrophilin-binding substance in
`to F1606 and rapamycin. FK506, for example, is known to
`the test solution is roughly inversely proportional to the
`be a potent inhibitor of IL-2 transcription, as can be shown
`amount of bound labelled compound. For quantitative
`in an IL-2 reporter gene assay. Raparnycin, although not
`analysis, a standard binding curve is made using known
`active in the IL-2 reporter gene assay, strongly inhibits IL-6 50 concentrations of macrophilin bind compound. ‘
`dependent T-cell proliferation. Both compounds are very
`EXAMPLES :
`potent inhibitors of the mixed lymphocyte reaction. Nonim-
`In the following examples, characteristic spectroscopic
`munosuppressivity can also be shown in the in vivo models
`data is given to facilitate identification. Peaks which do not
`1-7 above. Even those Novel Compounds which are not
`immunosuppressive, however, bind to macrophilin, which 55 difier significantly from rapamycin are not included. Bio-
`confers certain utilities in which nonirmnunosuppressivity is
`logical data is expressed as a relative IC5o, compared to
`an advantage.
`rapamycin in the case of the mixed lymphocyte reaction
`Those of the Novel Compounds which bind strongly to
`(MLR) and IL-6 dependent proliferation (IL-6 dep. prol.)
`macrophilin and are not themselves immunosuppressive can
`assays, and to FK-506 in the macrophilin binding assay
`be used in the treatment of overdoses of macrophilin- 60 (MBA). Ahigher IC5o correlates with lower binding aflfinity.
`binding irnmunosuppressants, such as FK506, rapamycin,
`Example 1: 40-O-Benzyl-rapamycin
`and the immunosuppressive Novel Compounds.
`To a stirred solution of 183 mg (0.200 mmol) of rapamy-
`l0. Steroid potentiation
`cin in 2.1 mL of 2:1 cyclo-hexane-methylene chloride is
`The macrophilin binding activity of the Novel Com-
`added 75 pL (0.402 mmol) of benzyl-trichloroacetimidate,
`pounds also makes them useful in enhancing or potentiating 65 followed by 2.6 11L (29 umol 15 mol %) of trifluoromethane-
`the action of corticosteroids. Combined treatment with the
`sulfonic acid whereupon the mixture turned immediately
`compounds of the invention and a corticosteroid,_ such as
`yellow. After 3 h the mixture is diluted with ethyl acetate and
`
`Roxane Labs., Inc.
`Exhibit 1009
`
`Page 005
`
`Roxane Labs., Inc.
`Exhibit 1009
`Page 005
`
`

`
`9
`
`10
`
`5,665,772
`
`4-yl)benzyl tiichloroacetimidate, followed by 0.14 mL (0.64
`mmol) of 2,6—di-t-butylpyridine 56 11L (0.64 mmol) of
`trifluoromethanesulfonic acid. To this mixture is added a
`
`quenched with 10% aqueous sodium bicarbonate. The layers
`are separated and the aqueous layer is extracted twice with
`ethyl acetate. The combined organic solution is washed with
`10% aqueous sodium bicarbonate, dried over anhydrous
`sodium sulfate. filtered and concentrated under reduced
`
`pressure. The residue is purified by column chromatography
`on silica gel (50:50 hexane-ethyl acetate) to atford 40-O-
`benzyl-rapamycin as a white amorphous solid:
`lH NMR
`(CDCI3) 60.73 (1H, dd), 1.65 (3H, s), 1.73 (3H, s). 3.12 (4H,
`s and In), 3.33 (3H, s). 3.49 (3H, s). 4.15 (1H, bd), 4.65 (1H,
`d), 4.71 (1H, d). 7.22—-7.38 (SH, In); MS (FAB) m/z 1026
`([M+Na]*), 972 ([M-OCH3)]+), 954 ([M—(OCH3+H2O)]+).
`
`
`10
`
`MBA(1-cl. Icso)
`1.8
`IL6dc1>~ vr°1« (rot I050)
`10
`
`ML“ ‘"1’ 1°”)
`11°
`
`25
`
`30
`
`35
`
`°-92
`fiAd<f1~ 10150)
`10.5
`.
`.( 1.1c5o
`MLR (,§1F’f§50)"’
`22
`)
`
`
`solution of 587 mg (0.64 mmol) of rapamycin in 2 mL of
`methylene chloride. The reaction is stirred overnight at room
`temperature and quenched with aqueous sodium bicarbon-
`ate. The layers are separated and the aqueous layer is
`extracted twice with ethyl acetate. The combined organic
`solution is washed with saturated brine, dried over anhy-
`drous sodium sulfate. filtered and concentrated. The residue
`is purified by column chromatography on silica gel (50:50
`hexane-ethyl acetate) to give 40-O-[4'-(2.2-Dimethyl-1.3-
`dioxolan-4-yl)]benzyl-rapamycin as a white, amorphous
`15 solid: in NMR (CDCl3) 50.73 (1H, dd), 1.48 (3H, s). 1.55
`(3H, s). 1.65 (3H, s), 1.74 (3H, s), 3.67 (3H. m), 4.23 (111,
`dd), 4.62 (1H, d), 4.69 (1H, d), 5.06 (1H, dd), 7.33 (4H, in),
`MS (FAB) mlz 1126 ([M+Na]*), 1072 ([M-OCH3]*), 1054
`([M—OCH3+H2O)]+), 1014 ([M-(OCH3+CH3COCH3)]*).
`Example 2: 40-0-(4'-Hydroxymethy1)benzy1—rapamycin
`a) 40-O-[4'-(t-Butyldimethylsilyl)oxymethyl]benzyl- 20 996 ([M-(OCH3+H._,0+CH3COCH3)]*), 978 ([M-(OCH3+
`rapamycin
`2H2O+CH3COCH3)]+).
`To a stirred, cooled (—78° C.) solution of 345 uL (2.0
`b) 40-O-[4'-(1,2-Dihydroxyethyl)]benzyl-rapamycin
`mmol) of triflic anhydride in 5 mL of methylene chloride is
`To a solution of 90.7 mg (0.08 mmol) of 40-O-[4'-(2,2-
`added a solution of 504 mg (2.0 mmol) of 4-(t-
`Dimethyl-1,3-dioxolan-4-y1)]benzyl-rapamycin in 4 mL of
`butyldimethylsilyl)oxymethyl-benzyl alcohol and 820 mg
`methanol is added 1 mL of 1N aqueous HC1. After 2 h the
`(4.0 mmol) of 2.6-di-t-butyl-4—methyl-pyridine in 5 mL of
`mixture is quenched with aqueous sodium bicarbonate and
`methylene chloride. The resulting mixture is warmed to
`extracted twice with ethyl acetate. The organic solution is
`—20° C. and stirring is continued at this temperature for 0.5
`washed with brine. dried over anhydrous sodium sulfate and
`h. The mixture is then cooled back to -78° C. and a solution
`concentrated. The residue is purified by column chromatog-
`of 914 mg (1.0 mmol) of rapamycin in 5 mL of methylene
`chloride is added. This mixture is allowed to warm to room
`raphy on silica gel (ethyl acetate) and the title compound is
`obtained as a white foam: 1H NMR (CDCI3) 80.73 (1H, dd),
`temperature overnight and is then quenched with 10%
`1.65 (3H, s), 1.74 (3H, s), 3.70 (4H, m), 4.63 (1H, d). 4.69
`aqueous sodium bicarbonate. The layers are separated and
`(1H, d), 4.80 (1H, dd), 7.33 (4H, m); MS (FAB) m/z 1086
`the aqueous layer is extracted with ethyl acetate. The com-
`bined organic solution is washed with saturated brine, dried
`([M+Na]+). 1032 ([M-OCH3]+), 1014 ([M-OCH3+H2O)]"),
`over sodium sulfate, filtered under reduced pressure and
`996 ([M-(OCH3+2H2O)]+).
`concentrated. The residue is puri.fied by column chromatog-
`raphy on silica gel (50:50 hexane-ethyl acetate) to afiord
`40—O-[4'-(t-butyldimethylsilyl)oxymethy1]benzyl-
`.
`.
`rapamycin a wlnte foam: MS (FAB) m/z 1170 ([M+Na]+), 40
`1098 ([M-(OCH3+H2O)]+).
`b) 40-O{4'-Hydroxymethyl)benzyl-raparnycin
`To a stirred, cooled (0° C.) solution of 98 mg (0.093
`mmol) of the compound obtained in example 2 in 2 mL of
`acetonitrile is added 0.2 mL of HF-pyridine. The resulting
`mixture is stirred for 2 h and quenched with aqueous sodium
`bicarbonate, then extracted with ethyl acetate. The organic
`solution is washed with brine, dried over sodium sulfate,
`filtered and concentrated. The residue is purified by column
`chromatography on silica gel (20:80 hexane-ethyl acetate) to
`atford the title compound as a white foam: ‘H NMR (CDCI3)
`50.73 (1H, dd), 1.65 (3H, s), 1.74 (3H, s), 3.22 (1H, m), 4.67
`(4H, m), 7.35 (4H, m); MS (FAB) m/z 1056 ([M+Na]“‘),
`1002 ([M-OCH3]*). 984 ([M-(OCH3+H2O)]+), 966 ([M-
`(OCH3+2H2O)]"), 934 ([M—(OCH3+CH3OH+2H2O)]*').
`
`
`$2». (:61. tcfo) I IC50
`M