United States Patent c191
`Cottens et al.
`
`11•11111111111111
`
`US005665772A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,665,772
`Sep.9, 1997
`
`[54] 0-ALKYLATED RAPAMYCIN DERIVATIVES
`AND THEIR USE, PARTICULARLY AS
`IMMUNOSUPPRESSANTS
`
`Int. CI.6
`•.••••••••..•.••••••••• A61K 31/395; C07D 498/16
`[51]
`[52] U.S. CI .............................................. 514/514; 540/456
`[58] Field of Search .............................. 540/456; 514/514
`
`[75]
`
`Inventors: Sylvain Cottens, Witterswil; Richard
`Sedrani. Basel, both of Switzerland
`
`[56]
`
`References Cited
`U.S. PA1ENT 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, 1995
`
`[87] PCT Pub. No.: W094/09010
`
`PCT Pub. Date: Apr. 28, 1994
`
`[30]
`
`Foreign Application Priority Data
`
`6/1992 Failli et al .....••..•.•..•.........•..... 540/542
`5,120,842
`9/1992 Caufield et al .....•.••••.•............•. 514/63
`5,151,413
`5,258,389 1111993 Goulet et al ............................ 514/291
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Finn-Robert S. Honor; Melvyn M.
`Kassenoff; Thomas 0. 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
`immunosuppressants.
`
`Oct. 9, 1992
`
`[GB] United Kingdom ................... 9221220
`
`10 Claims, No Drawings
`
`Roxane Labs., Inc.
`Exhibit 1001
`Page 001
`
`

`
`5,665,772
`
`2
`
`1
`0-ALKYLATED RAPAMYCIN DERIVATIVES
`AND THEIR USE, PARTICULARLY AS
`IMMUNOSUPPRESSANTS
`
`This application is a 371 of PCT/EP93/02604, filed Sep. 5
`24, 1993.
`This invention comprises novel alkylated derivatives of
`rapamycin having pharmaceutical utility, especially as
`immunosuppressants.
`Rapamycin is a known macrolide antibiotic produced by 10
`Streptomyces hygroscopicus, having the structure depicted
`in Formula A:
`
`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-butyl, and phenyl;
`wherein "alk-" or "alkyl" refers to cl-6 alkyl, branched
`or linear preferably cl-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 R 1 is (R3 hSi or carbalkoxyalkyl, X and Y are not both
`0.
`Preferred Novel Compounds include the following:
`1. 40-0-Benzyl-rapamycin
`2. 40-0-( 4'-Hydroxymethyl)benzyl-rapamycin
`3. 40-0-[ 4'-( 1,2-Dihydroxyethyl) ]benzyl-rapamycin
`4. 40-0-Allyl-rapamycin
`25 5. 40-0-[3'-(2,2-Dimethyl-l,3-dioxolan-4(S)-yl)-prop-2'(cid:173)
`en-1'-yl]-rapamycin
`6. (2'E,4'S)-40-0-( 4' ,5' -Dihydroxypent-2' -en-1 '-yl)(cid:173)
`rapamycin
`7. 40-0-(2-Hydroxy)ethoxycarbonylmethyl-rapamycin
`30 8. 40-0-(2-Hydroxy)ethyl-rapamycin
`9. 40-0-(3-Hydroxy )propyl-rapamycin
`10. 40-0-(6-Hydroxy)hexyl-rapamycin
`11. 40-0-[2-(2-Hydroxy)ethoxy]ethyl-rapamycin
`12. 40-0-[(3S)-2,2-Dimethyldioxolan-3-yl]methyl-
`rapamycin
`13. 40-0-[(2S)-2,3-Dihydroxyprop-1-yl]-rapamycin
`14. 40-0-(2-Acetoxy)ethyl-rapamycin
`15. 40-0-(2-Nicotinoyloxy)ethyl-rapamycin
`16. 40-0-[2-(N-Morpholino)acetoxy]ethyl-rapamycin
`40 17. 40-0-(2-N-Imidazolylacetoxy)ethyl-rapamycin
`18. 40-0-[2-(N-Methyl-N'-piperazinyl)acetoxy]ethyl-
`rapamycin
`19. 39-0-Desmethyl-39 ,40-0,0-ethylene-rapamycin
`20. (26R)-26-Dihydro-40-0-(2-hydroxy)ethyl-rapamycin
`21. 28-0-Methyl-rapamycin
`22. 40-0-(2-Aminoethyl)-rapamycin
`23. 40-0-(2-Acetaminoethyl)-rapamycin
`24. 40-0-(2-Nicotinamidoethyl)-rapamycin
`25. 40-0-(2-(N-Methyl-imidazo-2'-ylcarbethoxamido)
`ethyl)-rapamycin
`26. 40-0-(2-Ethoxycarbonylaminoethyl)-rapamycin
`27. 40-0-(2-Tolylsulfonamidoethyl)-rapamycin
`28. 40-0-[2-( 4' ,5'-Dicarboethoxy-1' ,2' ,3'-triazol-1 '-yl)(cid:173)
`ethyl ]-rapamycin
`The Novel Compounds for immunosuppressive use are
`preferably the 40-0-substituted rapamycins where X and Y
`are both 0, R2 is H, R4 is methyl, and R 1 is other than H;
`most preferably where R 1 is selected from hydroxyalkyl,
`hydroxyalkoxyalkyl, acylaminoalkyl, and aminoalkyl; espe-
`60 cially 40-0-(2-hydroxy)ethyl-rapamycin, 40-0-(3-hydroxy)
`propyl-rapamycin, 40-0-[2-(2-hydroxy)ethoxy]ethyl(cid:173)
`rapamycin, and 40-0-(2-acetaminoethyl)-rapamycin).
`Preferably 0-substitution at C40 or 0,0-disubstitution at
`C28 and C40 is performed according to the following
`65 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,
`
`15
`
`20
`
`36
`
`(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 35
`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- 45
`pounds are alkylated derivatives of rapamycin having the
`structure of Formula I:
`
`50
`
`55
`
`(I)
`
`24
`
`wherein
`
`Roxane Labs., Inc.
`Exhibit 1001
`Page 002
`
`

`
`5,665,772
`
`10
`
`3
`e.g., an alkyl, allyl, or benzyl moiety, which is desired as the
`0-substituent, and X is the leaving group, e.g., CC13C
`(NH)O or CF3S03) under suitable reaction conditions, pref(cid:173)
`erably acidic or neutral conditions, e.g., in the presence of an
`acid like trifiuoromethanesulfonic acid, camphorsulfonic 5
`acid, p-toluenesulfonic acid or their respective pyridinium or
`substituted pyridinium salts when Xis CC13(NH)O or in the
`presence of a base like pyridine, a substituted pyridine,
`diisopropylethylamine or pentamethylpiperidine when X is
`CF3S03• 0-substituents at C28 only are accomplished in the
`same manner, but with prior protection at C40. Further
`modifications are possible. For example, where the substitu(cid:173)
`ent is allyl, the isolated, monosubstituted double bond of the
`allyl moiety is highly amenable to further modification.
`The 9-deoxorapamycin compounds are preferably pro(cid:173)
`duced by reducing a rapamycin using hydrogen sulfide, by
`reacting rapamycin with diphenyldiselenide and tribu(cid:173)
`tylphosphine or by other suitable reduction reaction.
`The 26-dihydro-rapamycins are preferably produced by
`reducing rapamycins or 9-deoxorapamycins from keto to
`hydroxy at C26 by a mild reduction reaction, such as a
`borohydride reduction reaction.
`The Novel Compounds are particularly useful for the
`following conditions:
`a) Treatment and prevention of organ or tissue transplant
`rejection, e.g. for the treatment of recipients of e.g.
`heart, lung, combined heart-lung, liver, kidney,
`pancreatic, skin or corneal transplants. They are also
`indicated for the prevention of graft-versus-host
`disease, such as following bone marrow transplanta(cid:173)
`tion.
`b) Treatment and prevention of autoimmune disease and
`of inflammatory conditions, in particular inflammatory
`conditions with an etiology including an autoimmune
`component such as arthritis (for example rheumatoid
`arthritis, arthritis chronica progrediente and arthritis
`deformans) and rheumatic diseases. Specific autoim(cid:173)
`mune diseases for which the compounds of the inven(cid:173)
`tion may be employed include, autoimmune hemato(cid:173)
`logical disorders (including e.g. hemolytic anaemia,
`aplastic anaemia, pure red cell anaemia and idiopathic
`throm.bocytopenia), systemic lupus erythematosus,
`polychondritis, sclerodoma, Wegener granulamatosis,
`dermatomyositis, chronic active hepatitis, myasthenia
`gravis, psoriasis, Steven-Johnson syndrome, idiopathic
`sprue, autoimmune inflammatory bowel disease
`(including e.g. ulcerative colitis and Crohn's disease)
`endocrine ophthalmopathy, Graves disease,
`sarcoidosis, multiple sclerosis, primary billiary
`cirrhosis, juvenile diabetes (diabetes mellitus type I),
`uveitis (anterior and posterior), keratoconjunctivitis
`sicca and vernal keratoconjunctivitis, interstitial lung
`fibrosis, psoriatic arthritis, glomerulonephritis (with
`and without nephrotic syndrome, e.g. including idio(cid:173)
`pathic nephrotic syndrome or minimal change
`nephropathy) and juvenile dermatomyositis.
`c) Treatment and prevention of asthma.
`d) Treatment of multi-drug resistance (MDR). The Novel
`Compounds suppress P-glycoproteins (Pgp ), which are
`the membrane transport molecules associated with
`MDR. MDR is particularly problematic in cancer
`patients and AIDS patients who will not respond to
`conventional chemotherapy because the medication is
`pumped out of the cells by Pgp. The Novel Compounds
`are therefore useful for enhancing the efficacy of other
`chemotherapeutic agents in the treatment and control of
`multidrug resistant conditions such as multidrug resis(cid:173)
`tant cancer or multidrug resistant AIDS.
`
`4
`e) Treatment of proliferative disorders, e.g. tumors, hyper(cid:173)
`proliferative skin disorder and the like.
`f) Treatment of fungal infections.
`g) Treatment and prevention of inflammation. especially
`in potentiating the action of steroids.
`h) Treatment and prevention of infection, especially infec(cid:173)
`tion by pathogens having Mip or Mip-like factors.
`i) Treatment of overdoses of FK-506, rapamycin, immu(cid:173)
`nosuppressive Novel Compounds, and other macrophi(cid:173)
`lin binding immunosuppressants.
`The invention thus provides the Novel Compounds
`described herein, for use as novel intermediates or as
`pharmaceuticals, methods of treating or preventing the
`above-described disorders by administering an effective
`15 amount of Novel Compound to a patient in need thereof, use
`of a Novel Compound in the manufacture of a medicament
`for treatment or prevention of the above-described disorders,
`and pharmaceutical compositions comprising a Novel Com(cid:173)
`pound in combination or association with a pharmaceuti-
`20 cally acceptable diluent or carrier.
`Most of the Novel Compounds described herein are
`highly immunosuppressive, especially those Novel Com(cid:173)
`pounds which are 0-substituted at C40, and these Novel
`Compounds are particularly useful in indications a and b, but
`25 not in indication i. Those of the Novel Compounds which are
`less immunosuppressive, especially those which are
`0-substituted at C28 only, are particularly useful in indica(cid:173)
`tions h and i, but are less preferred in indications a or b.
`The Novel Compounds are utilized by administration of a
`30 pharmaceutically effective dose in pharmaceutically accept(cid:173)
`able form to a subject in need of treatment. Appropriate
`dosages of the Novel Compounds will of course vary, e.g.
`depending on the condition to be treated (for example the
`disease type or the nature of resistance), the effect desired
`35 and the mode of administration.
`In general however satisfactory results are obtained on
`administration orally at dosages on the order of from 0.05 to
`5 or up to 10 mg/kg/day, e.g. on the order of from 0.1 to 2
`or up to 7.5 mg/kg/day administered once or, in divided
`40 doses 2 to 4x per day, or on administration parenterally, e.g.
`intravenously, for example by i.v. drip or infusion, at dos(cid:173)
`ages on the order of from 0.01to2.5 up to 5 mg/kg/day, e.g.
`on the order of from 0.05 or 0.1 up to 1.0 mg/kg/day.
`Suitable daily dosages for patients are thus on the order of
`45 500 mg p.o., e.g. on the order of from 5 to 100 mg p.o., or
`on the order of from 0.5 to 125 up to 250 mg i.v., e.g. on the
`order of from 2.5 to 50 mg i.v.
`Alternatively and even preferably, dosaging is arranged in
`patient specific manner to provide pre-determined trough
`50 blood levels, e.g. as determined by RIA technique. Thus
`patient dosaging may be adjusted so as to achieve regular
`on-going trough blood levels as measured by RIA on the
`order of from 50 or 150 up to 500 or 1000 ng/rnl, i.e.
`analogously to methods of dosaging currently employed for
`55 Ciclosporin immunosuppressive therapy.
`The Novel Compounds may be administered as the sole
`active ingredient or together with other drugs. For example,
`in immunosuppressive applications such as prevention and
`treatment of graft vs. host disease, transplant rejection, or
`60 autoimmune disease, the Novel Compounds may be used in
`combination with Ciclosporin, FK-506, or their immuno(cid:173)
`suppressive derivatives; corticosteroids; azathioprene;
`immunosuppressive monoclonal antibodies, e.g., mono(cid:173)
`clonal antibodies to CD3, CD4, CD25, CD28, or CD45; and
`65 7 or other immunomodulatory compounds. For anti(cid:173)
`infiammatory applications, the Novel Compounds can be
`used together with anti-inflammatory agents, e.g., corticos-
`
`Roxane Labs., Inc.
`Exhibit 1001
`Page 003
`
`

`
`5,665,772
`
`5
`teroids. For anti-infective applications, the Novel Com(cid:173)
`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(cid:173)
`tional route, in particular enterally, e.g. orally, for example 5
`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(cid:173)
`istration comprise, e.g. from 1 to 50 mg of a compound of
`the invention, usually 1 to 10 mg. Pharmaceutical compo- 10
`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 15
`are demonstrated in, e.g., the following tests:
`1. Mixed lymphocyte reaction (MLR)
`The Mixed Lymphocyte Reaction was originally devel(cid:173)
`oped in connection with allografts, to assess the tissue
`compatibility between potential organ donors and recipients, 20
`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 immunosuppressive effect of the Novel 25
`Compounds. Spleen cells (0.5xl06
`) from Balb/c mice
`(female, 8-10 weeks) are co-incubated for 5 days with
`0.5x106 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 30
`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 35
`Compounds on the Balb/c cells is measured at various
`dilutions and the concentration resulting in 50% inhibition
`of cell proliferation (IC50) 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 40
`rapamycin).
`2. IL-6 mediated proliferation
`The capacity of the Novel Compounds to interfere with
`growth factor associated signalling pathways is assessed
`using an interleukin-6 (IL-6)-dependent mouse hybridoma 45
`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, L Lefkovits and B. Pernis, eds., Academic Press
`1981, Vol. Il, pp. 263-275), supplemented with 1 ng recom- 50
`binant IL-6/ml. Following a 66 hour incubation in the
`absence or presence of a test sample, cells are pulsed with
`1 µCi (3-H)-thymidine/well for another 6 hours, harvested
`and counted by liquid scintillation. (3-H)-thymidine incor(cid:173)
`poration into DNA correlates with the increase in cell 55
`number and is thus a measure of cell proliferation. A dilution
`series of the test sample allows the calculation of the
`concentration resulting in 50% inhibition of cell prolifera(cid:173)
`tion (IC50). The inhibitory capacity of the test sample may
`be compared to rapamycin and expressed as a relative IC50 60
`(i.e. IC50 test sample/IC50 rapamycin).
`3. Macrophilin binding assay
`Rapamycin and
`the structurally related
`immunosuppressant, FK-506, are both known to bind in
`vivo to macrophilin-12 (also known as FK-506 binding 65
`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(cid:173)
`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(cid:173)
`lowed by washing and subsequent addition of p-nitrophenyl
`phosphate as a substrate. The read-out is the OD at 405 run.
`Binding of a test sample to biot-MAP results in a decrease
`in the amount ofbiot-MAPbound 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 (IC50). 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/IC50-free
`FK-506).
`4. Localized Graft-Versus-Host (GvH) Reaction
`In vivo efficacy 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 (lx107
`)
`from 6 week old female Wistar/Furth (WF) rats are injected
`subcutaneously on day 0 into the left hind-paw of female
`(F344xWF)F 1 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(cid:173)
`mized WF recipient rat using an end-to-end anastomosis.
`Ureteric anastomosis is also end-to-end. Treatment com(cid:173)
`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(cid:173)
`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
`Efficacy 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,
`ARI'HRII1S & RHEUMATISM 9 (1966) 394; Billingham
`& Davies, HANDBOOK OF EXPERIMENTALPHARMA(cid:173)
`COL (Vane & Ferreira Eds, Springer-Verlag, Berlin) 50/Il
`(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
`
`Roxane Labs., Inc.
`Exhibit 1001
`Page 004
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`

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`5,665,772
`
`7
`established arthritis model treatment is started on day 14,
`when the secondary inflammation is well developed (days
`14-20). At the end of the experiment, the swelling of the
`joints is measured by means of a micro-caliper. ED50 is the
`oral dose in mg/kg which reduces the swelling (primary or
`secondary) to half of that of the controls.
`8. Antitumor and MDR activity
`The antitumor activity of the Novel Compounds and their
`ability to enhance the performance of antitumor agents by
`alleviating multidrug resistance is demonstrated, e.g., by
`administration of an anticancer agent, e.g., colchicine or
`etoposide, to multidrug resistant cells and drug sensitive
`cells in vitro or to animals having multidrug resistant or drug
`sensitive tumors or infections, with and without
`co-administration of the Novel Compounds to be tested, and
`by administration of the Novel Compound alone.
`Such in vitro testing is performed employing any appro(cid:173)
`priate drug resistant cell line and control (parental) cell line,
`generated, e.g. as described by Llng et al., J. Cell. Physiol.
`83, 103-116 (1974) and Bech-Hansen et al. J. Cell. Physiol.
`88, 23-32 (1976). Particular clones chosen are the multi(cid:173)
`drug resistant (e.g. colchicine resistant) line CHR (subclone
`C5S3.2) and the parental, sensitive line AUX Bl (subclone
`ABl Sll).
`In vivo anti-tumor and anti-MDR activity is shown, e.g.,
`in mice injected with multidrug resistant and drug sensitive
`cancer cells. Ehrlich ascites carcinoma (EA) sub-lines resis(cid:173)
`tant to drug substance DR, VC, AM, Er, TE or CC are
`developed by sequential transfer of EA cells to subsequent
`generations of BALB/c host mice in accordance with the
`methods described by Slater et al., J. Clin. Invest, 70, 1131
`(1982).
`Equivalent results may be obtained employing the Novel
`Compounds test models of comparable design, e.g. in vitro,
`or employing test auimals infected with drug-resistant and
`drug sensitive viral strains, antibiotic (e.g. penicillin) resis(cid:173)
`tant and sensitive bacterial strains, anti-mycotic resistant and
`sensitive fungal strains as well as drug resistant protozoa!
`strains, e.g. Plasmodial strains, for example naturally occur(cid:173)
`ring sub-strains of Plasmodium falciparum exhibiting
`acquired chemotherapeutic, anti-malarial drug resistance.
`9. FKBP binding
`Certain of the Novel Compounds are not
`immunosuppressive, particularly those which are
`0-substituted at C28 only, such as 28-0-methyl-rapamycin.
`This can be shown in standard in vitro assays in comparison
`to FK506 and rapamycin. FK506, for example, is known to
`be a potent inhibitor of IL-2 transcription, as can be shown
`in an IL-2 reporter gene assay. Rapamycin, although not
`active in the IL-2 reporter gene assay, strongly inhibits IL-6
`dependent T-cell proliferation. Both compounds are very
`potent inhibitors of the mixed lymphocyte reaction. Nonim(cid:173)
`munosuppressivity can also be shown in the in vivo models
`1-7 above. Even those Novel Compounds which are not
`immunosuppressive, however, bind to macrophilin, which 55
`confers certain utilities in which nonimmunosuppressivity is
`an advantage.
`Those of the Novel Compounds which bind strongly to
`macrophilin and are not themselves immunosuppressive can
`be used in the treatment of overdoses of macrophilin- 60
`binding immunosuppressants, such as FK506, rapamycin,
`and the immunosuppressive Novel Compounds.
`10. Steroid potentiation
`The macrophilin binding activity of the Novel Com(cid:173)
`pounds also makes them useful in enhancing or potentiating 65
`the action of corticosteroids. Combined treatment with the
`compounds of the invention and a corticosteroid, such as
`
`8
`dexarnethasone, results in greatly enhanced steroidal activ(cid:173)
`ity. This can be shown, e.g., in the murine mammary tumor
`virus-chloramphenicol acetyltransferase (MMTV-CXI')
`reporter gene assay, e.g., as described in Ning, et al., J. Biol.
`5 Chem. (1993) 268: 6073. This synergistic effect allows
`reduced doses of corticosteroids, thereby reducing the risk of
`side effects in some cases.
`11. Mip and Mip-like factor inhibition
`Additionally, the Novel Compounds bind to and block a
`10 variety of Mip (macrophage infectivity potentiator) and
`Mip-like factors, which are structurally similar to macro(cid:173)
`philin. Mip and Mip-like factors are virulence factors pro(cid:173)
`duced by a wide variety of pathogens, including those of the
`genera Chlamidia, e.g., Chlamidia trachomatis; Neisseria,
`15 e.g., Neisseria meningitidis; and Legionella, e.g., Legionella
`pneumophilia; and also by the obligately parasitic members
`of the order Rickettsiales. These factors play a critical role
`in the establishment of intracellular infection. The efficacy
`of the Novel Compounds in reducing the infectivity of
`20 pathogens which produce Mip or Mip-like factors can be
`shown by comparing infectivity of the pathogens in cells
`culture in the presence and absence of the macrolides, e.g.,
`using the methods described in Lundemose, et al., Mol.
`Microbial. (1993) 7: 777. The nonimmunosuppressive com-
`25 pounds of the invention are preferred for use in this indica(cid:173)
`tion for the reason that they are not immunosuppressive, thus
`they do not compromise the body's natural immune defenses
`against the pathogens.
`The Novel Compounds are also useful in assays to detect
`30 the presence or amount of macrophilin-binding compounds,
`e.g., in competitive assays for diagnostic or screening pur(cid:173)
`poses. Thus, in another embodiment, the invention provides
`for use of the Novel Compounds as a screening tool to
`determine the presence of macrophilin-binding compounds
`35 in a test solution, e.g., blood, blood serum, or test broth to
`be screened. Preferably, a Novel Compound is immobilized
`in microtiter wells and then allowed to bind in the presence
`and absence of a test solution to labelled macrophilin-12
`(FKBP-12). Alternatively, the FKBP-12 immobilized in
`40 microtiter wells and allowed to bind in the presence and
`absence of a test solution to a Novel Compound which has
`been labelled, e.g., :fiuoro-, enzymatically- or radio-labelled,
`e.g., a Novel Compound which has been 0-substituted at
`C40 and/or C28 with a labelling group. The plates are
`45 washed and the amount of bound labelled compound is
`measured. The amount of macrophilin-binding substance in
`the test solution is roughly inversely proportional to the
`amount of bound labelled compound. For quantitative
`analysis, a standard binding curve is made using known
`50 concentrations of macrophilin bind compound. ·
`
`EXAMPLES:
`In the following examples, characteristic spectroscopic
`data is given to facilitate identification. Peaks which do not
`differ significantly from rapamycin are not included. Bio(cid:173)
`logical data is expressed as a relative IC50, compared to
`rapamycin in the case of the mixed lymphocyte reaction
`(MLR) and IL-6 dependent proliferation (IL-6 dep. prol.)
`assays, and to FK-506 in the macrophilin binding assay
`(MBA). A higher IC50 correlates with lower binding affinity.
`Example 1: 40-0-Benzyl-rapamycin
`To a stirred solution of 183 mg (0.200 mmol) of rapamy(cid:173)
`cin in 2.1 mL of 2:1 cyclo-hexane-methylene chloride is
`added 75 µL (0.402 mmol) of benzyl-trichloroacetimidate,
`followed by 2.6 µL (29 µmol 15 mol % ) of tri:fiuoromethane(cid:173)
`sulfonic acid whereupon the mixture turned immediately
`yellow. After 3 h the mixture is diluted with ethyl acetate and
`
`Roxane Labs., Inc.
`Exhibit 1001
`Page 005
`
`

`
`5,665,772
`
`9
`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 5
`pressure. The residue is purified by column chromatography
`on silica gel (50:50 hexane-ethyl acetate) to afford 40-0-
`benzyl-rapamycin as a white amorphous solid: 1H NMR
`(CDC13) 80.73 (lH, dd), 1.65 (3H, s), 1.73 (3H, s), 3.12 (4H,
`sand m), 3.33 (3H, s), 3.49 (3H, s), 4.15 (lH, bd), 4.65 (lH,
`d), 4.71 (lH, d), 7.22-7.38 (SH, m); MS (FAB) rn/z 1026
`([M+Na]+), 972 ([M-OCH3)]+), 954 ([M-(OCH3+H20)]+).
`
`MBA (rel. IC50)
`IL-0 dep. pro!. (rel. IC50)
`MLR (rel. IC50)
`
`1.8
`IO
`I10
`
`•
`
`•
`
`10
`4-yl)benzyl trichloroacetimidate, followed by 0.14 mL (0.64
`mmol) of 2,6-di-t-butylpyridine 56 µL (0.64 rnrnol) of
`trifiuoromethanesulfonic acid. To this mixture is ·added a
`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(cid:173)
`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-
`IO drous sodium sulfate, filtered and concentrated. The residue
`is purified by column chromatography on silica gel (50:50
`hexane-ethyl acetate) to give 40-0-[4'-(2,2-Dimethyl-l,3-
`dioxolan-4-yl)]benzyl-rapamycin as a white, amorphous
`15 solid: 1H NMR (CDC13) 00.73 (lH, dd), 1.48 (3H, s), 1.55
`(3H, s), 1.65 (3H, s), 1.74 (3H, s), 3.67 (3H, m), 4.28 (lH,
`dd), 4.62 (lH, d), 4.69 (lH, d), 5.06 (lH, dd), 7.33 (4H, m),
`MS (FAB) rn/z 1126 ([M+Na]+), 1072 ([M-OCH3]+), 1054
`([M-OCH3+H20W), 1014 ([M-(OCH3+cH3COCH3W),
`Example 2: 40-0-(4'-Hydroxymethyl)benzyl-rapamycin
`a) 40-0-[4'-(t-Butyldimethylsilyl)oxymethyl]benzyl- 20 996 ([M-(OCH3+H2o+cH3COCH3)]+:J, 978 ([M-(OCH3+
`2H2o+cH3COCH3W).
`rapamycin
`To a stirred, cooled (-78° C.) solution of 345 µL (2.0
`b) 40-0-[4'-(l,2-Dihydroxyethyl)]benzyl-rapamycin
`To a solution of 90.7 mg (0.08 rnrnol) of 40-0-[ 4'-(2,2-
`rnrnol) of triflic anhydride in 5 mL of methylene chloride is
`added a solution of 504 mg (2.0 mmol) of 4-(t-
`Dimethyl-1,3-dioxolan-4-yl)]benzyl-rapamycin in 4 mL of
`butyldimethylsilyl)oxymethyl-benzyl alcohol and 820 mg 25 methanol is added 1 mL of lN aqueous HCl. After 2 h the
`(4.0 rnrnol) 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
`20 0 c
`d tirrin'
`d t thi t
`tur ,, 0 5
`extracted twice with ethyl acetate. The organic solution is
`ti'
`s empera e ior .
`g is con nue a
`.
`.
`.
`.
`-
`. an s
`h. The mixture is then cooled back to_ 7go c. and a solution
`washed with brine, dried over anhydrous sodium sulfate and
`of 914 mg (1.0 rnrnol) of rapamycin in 5 mL of methylene 30 concentrat~~· The residue is purified by col~mn chromato~-
`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 (CDC13) 00.73 (lH, dd),
`temperature overnight and is then quenched with 10%
`1.65 (3H, s), 1.74 (3H, s), 3.70 (4H, m), 4.63 (lH, d), 4.69
`aqueous sodium bicarbonate. The layers are separated and
`the aqueous layer is extracted with ethyl acetate. The com-
`(lH, d), 4.80 (lH, dd), 7.33 (4H, m); MS (FAB) rn/z 1086
`bined organic solution is washed with saturated brine, dried 35 ([M+Nat), 1032 ([M-OCH3t), 1014 ([M-OCH3+H20)]+:J,
`over sodium sulfate, filtered under reduced pressure and
`996 ([M-(OCH3+2H20)t).
`concentrated. The residue is purified by column chromatog-
`raphy on silica gel (50:50 hexane-ethyl acetate) to afford
`40-0-[ 4' -( t-butyldimethy lsilyl)oxymethy l]benzyl(cid:173)
`rapamycin a white foam: MS (FAB) rn/z 1170 ([M+Na]+),