PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PC1)
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Oassification 5 :
`C07D 498/18, C07F 7 /18
`A61K 31/435 // C07D 498/18
`C07D 311 :00, 273 :00, 221 :00
`
`(11) International Publication Number:
`
`WO 94/09010
`
`Al
`
`(43) International Publication Date:
`
`28 April 1994 (28.04.94)
`
`(21) International Application Number:
`
`PCT /EP93/02604
`
`(22) International Filing Date:
`
`24 September 1993 (24.09.93)
`
`(72) Inventors; and
`(75) Inventors/ Applicants (for US only) : CO TIE NS, Sylvain
`[CH/CH]; In den Reben 12, CH-4108 Witterswil (CH).
`SEDRANI, Richard [LU/CH]; Herrengrabenweg 15,
`CH-4054 Basie (CH).
`
`(30) Priority data:
`9221220.8
`
`9 October 1992 (09.10.92)
`
`GB
`
`(74) Common Representative: SANDOZ LTD.; Patents &
`Trademarks Div., Lichtstrasse 35, CH-4002 Basie (CH).
`
`(71) Applicant (for AT only): SANDOZ-ERFINDUNGEN
`VERWALTUNGSGESELLSCHAFT M.B.H. [AT/A1];
`Brunner Strasse 59, A-1230 Vienna (A1).
`
`(71)Applicant (for DE only): SANDOZ-PATENT-GMBH [DE/
`DE]; Humboldstrasse 3, D-79539 Li:irrach (DE).
`
`(81) Designated States: AU, CA, CZ, FI, HU, JP, KR, NO, NZ,
`PL, RO, RU, SK, US, European patent (AT, BE, CH,
`DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT,
`SE).
`
`(71) Applicant (for all designated States except AT DE US): SAN-
`DOZ LTD. [CH/CH]; Lichtstrasse 35, CH-4002 Basie
`(CH).
`
`Published
`With international search report.
`
`(54)Title: 0-ALKYLATED RAPAMYCIN DERIVATIVES AND THEIR USE, PARTICULARLY AS IMMUNOSUPPRES(cid:173)
`SANTS
`
`(57) Abstract
`
`Novel 0-alkylated derivatives of rapamycin of for(cid:173)
`mula (I), especially 40-0-alkylated derivatives, are found
`to have pharmaceutical utility, particularly as immunosup(cid:173)
`pressants.
`
`41
`'0
`· 1
`.
`RO,,,.,~ 42
`I :;a I 31
`
`so,,,, .... · i 34
`~~a
`
`2
`
`-
`
`(I)
`
`11
`
`0
`
`0......-
`
`13
`
`15
`
`18
`
`20
`
`?'
`19
`
`West-Ward Exhibit 1026
`Cottens WO '010
`Page 001
`
`

`

`,/
`
`...
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`Cl
`CM
`CN
`cs
`CZ
`DE
`DK
`ES
`Fl
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Den mar Ir.
`Spain
`Finland
`
`FR
`GA
`GB
`GN
`GR
`HU
`IE
`IT
`JP
`KP
`
`KR
`KZ
`LI
`LK
`LU
`LV
`MC
`MC
`ML
`MN
`
`France
`Gabon
`United Kingdom
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Democratic People's Republic
`or Korea
`Republic or Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Madagascar
`Mali
`Mongolia
`
`MR
`MW
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SI
`SK
`SN
`TD
`TG
`UA
`us
`uz
`VN
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovak Republic
`Senegal
`Chad
`Togo
`Ukraine
`United States or America
`Uzbekistan
`Viet Nam
`
`West-Ward Exhibit 1026
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`Page 002
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`W094/090JO
`
`PCT/EP93/02604
`
`0-ALKYLATED RAPAMYCIN DERIVATIVES AND THEIR USE, PARTICULARLY AS IMMUNO(cid:173)
`SUPPRESSANTS
`
`This invention comprises novel alkylated derivatives of rapamycin having
`
`pharmaceutical utility, especially as immunosuppressants.
`
`Rapamycin is a known macrolide antibiotic produced by Srreptomvces
`
`hvtrroscopicus, having the structure depicted in Formula A:
`
`41
`
`42
`
`36
`
`35
`
`33
`
`4
`
`,,, ...
`,,,
`
`3
`
`30
`
`0
`
`0
`
`OH
`
`(A)
`
`0
`
`24
`
`'o
`
`5
`
`6
`
`11
`
`12
`
`See, e.g., McAlpine, J.B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S.L., et al., J. Arn.
`
`Chem. Soc. (1991) 113: 7433; US Patent No. 3 929 992. Rapamycin is an exrremely
`
`..
`
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`W094/09010
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`PCT /EP93/02604
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`- 2 -
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`potent immunosuppressant and has also been shown to have anti.tumor and anti.fungal
`
`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 cenain 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 Compounds are alkylated derivatives of rapamycin having the
`
`structure of Formula I:
`
`41
`
`42
`
`37
`
`36
`
`3.J
`
`30
`
`0
`
`OR.2
`
`y
`
`(I)
`
`0..........-
`
`18
`
`20
`
`24
`
`wherein
`
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`WO 94/09010
`
`PCT /EP93/02604
`
`-3-
`
`X is (H,H) or 0;
`
`Y is (H.OH) or O;
`
`R 1 and R2 are independently selected from
`H, alkyl, tbioalky4 arylalk:yl, hydroxyalkyl, dihydroxyalk:yl.
`hydroxyalkylarylalkyl. dihydroxyalkylazylalkyl, alkoxyalkyl, acyloxyalkyl,
`
`aminoalk:yl. alkylaminoalkyl, a!koxycarbonylaminoalkyl, acylaminoalkyl,
`
`arylsuifonamidoalkyl, ally!, dihydroxyalkylallyl, dioxolanylallyl,
`) 3Si where each R3 is independently selected from a
`carbalkoxyalkyl. and (R3
`methyl, ethyl, isopropyl, I-butyl. aild phenyl; wherein "alk-" or "alkyl" refers
`to Ci.6 alkyl, branched or linear preferably C1•3 alkyl. in which the carbon
`chain may be optionally imemlpted by an ether (-0-) linkage; and
`
`R' is methyl, or R4 and R1 together form Cu alkylenc;
`
`provided that R1 and R1 are not both H; and
`provided that where R1 is (R:o)3Si or carbalkoxyalkyl. X and Y are not both 0.
`
`Preferred Novel Compounds include the following:
`
`40-0-Benzyl-rapamycin
`
`40-0-(4'-Hydroxymethyl)benzyl-rapamycin
`
`40-0-[ 4' -(1.2-Dihydroxyethyl)]benzyl-rapamycin
`
`40-0-Allyl-rapamycin
`
`40-0-[3' -(2,2-Dimethyl-l,3-dioxolan-4(S)-yl)-prop-2'-en-l '-yl]-rapamycin
`
`(2'E, 4'S)-40-0-(4' ,5'-Dihydroxypent-2'-en-l' -yl)-rapamycin
`
`40-0-(2-Hydroxy)ethoxycarbonyl.methyl-rapa.mycin
`
`40-0-(2-Hydroxy)erhyl-rapamycin
`
`1.
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
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`- 4 -
`
`40-0-(3-H ydroxy )propyl-rapamycin
`
`40-0-(6-Hydroxy)hexyl-rapamycin
`
`40-0-[2-(2-Hydroxy)ethoxy]ethyl-rapamycin
`
`40-0-[(3S)-2,2-Dimethyldioxolan-3-yl]methyl-rapamycin
`
`40-0-[(2S)-2,3-Dihydroxyprop-1-yl]-rapamycin
`
`40-0-(2-Acetoxy )ethy 1-rapamycin
`
`40-0-(2-Nicotinoyloxy )ethyl-rapamycin
`
`40-0-[2-(N-Morpholino )acetoxy ]ethyl-rapamycin
`
`40-0-(2-N-Imidazolylacetoxy)ethyl-rapamycin
`
`40-0-[2-(N-Methyl-N' -piperazinyl)acetoxy ]ethyl-rapamycin
`
`39-0-Desmethyl-39 ,40-0,0-ethylene-rapamycin
`
`(26R)-26-Dihydro-40-0-(2-hydroxy)ethyl-rapamycin
`
`28-0-Methyl-rapamycin
`40-0-(2-Aminoethyl)-rapamycin
`
`40-0-(2-Acetaminoethyl)-rapamycin
`
`40-0-(2-Nicotinamidoethyl)-rapamycin ·
`
`40-0-(2-(N-Methyl-imidazo-2' -ylcarbethoxamido )ethyl)-rapamycin
`
`40-0-(2-Ethoxycarbonylaminoethyl)-rapamycin
`
`40-0-(2-Tolylsulfonamidoethyl)-rapamycin
`
`40-0-[2-( 4' ,5 '-Dicarboethoxy-1 ',2' ,3 '-triazol-1 '-yl)-ethyl]-rapamycin
`
`9.
`
`10.
`
`11.
`
`12.
`
`13.
`
`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`27.
`
`28.
`
`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 R1 is
`other than H; most preferably where R 1 is selected from hydroxyalkyl, hydroxyalkoxyalkyl,
`acylaminoalkyl, and aminoalkyl; especially 40-0-(2-hydroxy)ethyl-rapamycin, 40-0-(3-
`
`hydroxy)propyl-rapamycin, 40-0-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, and
`
`40-0-(2-acetaminoethyl)-rapamycin).
`
`Preferably, 0-substitution at C40 or 0,0-disubstitution at C28 and C40 is performed
`
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`- 5 -
`
`according to the following general process: Rapamycin (or dihyd.ro or deoxorapamycin) is
`
`reacted with an organic radical attached to a leaving group (e.g., RX where R is the organic
`
`radical, e.g., an alkyl, allyl, or benzyl moiety, which is desired- as the 0-substiruent, and X is
`the leaving group, e.g., CC13C(NH)O or CF3S03) under suitable reaction conditions,
`preferably acidic or neutral conditions, e.g., in the presence of an acid like
`
`trifluoromethanesulfonic acid, camphorsulfonic acid, p-toluenesulfonic acid or their
`
`respective pyridinium or substituted pyridinium salts when X is CC13C(NH)O or in the
`presence of a base like pyridine, a substituted pyridine, diisopropylethylamine or
`
`pentamethylpiperidine when X is CF3S03•
`the same manner, but with prior protection at C40. Further modifications are possible. For
`
`0-substirutions at C28 only are accomplished in
`
`example, where the substiruent is allyl, the isolated, monosubstituted double bond of the
`
`allyl moiety is highly amenable to further modification.
`
`The 9-deoxorapamycin compounds are preferably produced by reducing a rapamycin
`
`using hydrogen sulfide, by reacting rapamycin with diphenyldiselenide and tributylphosphine
`
`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 transplantation.
`
`b)
`
`Treatment and prevention of autoimmune disease and of inflammatory
`
`conditions, in particular inflammatory conditions with an etiology including an autoimmune
`
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`
`component such as anhritis (for example rheumatoid arthritis, anhritis chronica progrediente
`
`and arthritis deformans) and rheumatic diseases. Specific autoimmune diseases for which
`
`the compounds of the invention may be employed include, autoimmune hematological
`
`disorders (including e.g. hemolytic anaemia, aplasric anaemia, pure red cell anaemia and
`
`idiopathic thrombocytopenia), systemic lupus erythematosus, polychondritis, sclerod.oma,
`
`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 idiopathic nephrotic syndrome or
`
`minimal change nephropathy) and juvenile dermatomyositis.
`
`c)
`
`d)
`
`Treannent and prevention of asthma.
`
`Treannent of multi-drug resistance (MDR). The Novel Compounds suppress
`
`P-glycoproteins (Pgp), which are the membrane transpon 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 treannent and control of multidrug resistant conditions such
`
`as multidrug resistant cancer or mulridrug resistant AIDS.
`
`e)
`
`Treannent of proliferative disorders, e.g. tumors, hyperproliferative skin
`
`disorder and the like.
`
`f)
`
`g)
`
`of steroids.
`
`Treannent of fungal infections.
`
`Treannent and prevention of inflammation, especially in potentiating the action
`
`h)
`
`Treannent and prevention of infection, especially infection by pathogens
`
`having Mip or Mip-like factors.
`
`i)
`
`Treannent of overdoses of FK-506, rapamycin, immunosuppressive Novel
`
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`W094/09010
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`PCT /EP93/02604
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`- 7 -
`
`Compounds, and other macrophilin 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 amount of a Novel Compound to a patient in need
`
`thereof, use of a Novel Compound in the manufacture of a medicament for treaonent or
`
`prevention of the above-described disorders, and pharmaceutical compositions comprising a
`
`Novel Compound in combination or association with a pharmaceutically acceptable diluent
`
`or carrier.
`
`Most of the Novel Compounds described herein are highly im.munosuppressive,
`
`especially those Novel Compounds which are 0-substituted at C40, and these Novel
`
`Compounds are particularly useful in indications a and b, but not in indication i. Those of
`
`the Novel Compounds which are less im.munosuppressive, especially those which are 0-
`
`substituted at C28 only, are particularly useful in indications h and i, but are less preferred
`
`in indications a or b.
`
`The Novel Compounds are utilized by administration of a pharmaceutically effective
`
`dose in pharmaceutically acceptable fonn to a subject in need of treaonent. 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 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 lOmg/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 doses 2 to 4x per day, or on
`
`administration parenterally, e.g. intravenously, for example by i.v. drip or infusion, at
`
`dosages on the order of from 0.01 to 2.5 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 500
`
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`- 8 -
`
`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 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 lOOOng/ml, i.e. analogously to
`
`methods of dosaging currently employed for 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 autoimmune disease, the Novel
`
`Compounds may be used in combination with Ciclosporin, FK-506, or their
`
`immunosuppressive derivatives; corticosteroids; azathioprene; immunosuppressive
`
`monoclonal antibodies, e.g., monoclonal antibodies to CD3, CD4, CD25, CD28, or CD45;
`
`and7or other immunomodulatory compounds. For anti-inflammatory applications, the Novel
`
`Compounds can be used together with anti-inflammatory agents, e.g., corticosteroids. For
`
`anti-infective applications, the Novel Compounds can be used in combination with other
`
`anti-infective agents, e.g., anti-viral drugs or antibiotics.
`
`The Novel Compounds are administered by any conventional 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 administration comprise, e.g. from 1 to 50 mg of a compound of the
`
`invention, usually 1 to 10 mg. Pharmaceutical compositions 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.
`
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`The pharmacological activity of the Novel Compounds are demonstrated in, e.g., the
`
`following tests:
`
`1.
`
`~1ixed lvmphocvte reaction Gvll .. R)
`
`The Mixed Lymphocyte Reaction was originally developed 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
`
`immunosuppressive effect of the Novel Compounds. Spleen cells (0.5 x 106
`) from Balb/c
`mice (female, 8-10 weeks) are co-incubated for 5 days with 0.5 x 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
`
`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
`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
`
`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 recombinant 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
`
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`another 6 hours, harvested and counted by liquid scintillation. (3-H)-thymidine incorporation
`
`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 concentration
`
`resulting in 50% inhibition of cell proliferation (IC50). 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).
`
`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 protein or FKBP-12), and
`
`this binding is thought to be related to the immunosuppressive activity of these compounds.
`
`The ~ovel Compounds also bind strongly to macrophilin-12, as is demonstrated in a
`
`competitive binding assay.
`
`In this assay, FK-506 coup1ea IO o:,ri. iS useu LO coat IIllcromer we11s.
`recombinant human macrophilin-12 (biot-MAP) is allowed to bind in the presence or
`
`isiotinylated
`
`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, followed by washing and subsequent addition of
`
`p-nitrophenyl phosphate as a substrate. The read-out is the OD at 405nm. 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 (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., ICs0-test sample/
`IC50-free FK-506).
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`4. Localized Graft-Versus-Host (GvH) Reaction
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`In vivo efficacy of the Novel Compounds is proved in a suitable animal mod.el, as
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`described, e.g., in Ford et al, TRANSPLANTATION 10 (1970) 258. Spleen cells (1 x 107
`
`)
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`from 6 week old female Wistar/Funh (WF) rats are injected subcutaneously on day 0 into
`
`the left hind-paw of female (F344 x WF)F1 rats weighing about lOOg. Animals are treated
`for 4 consecutive days and the popliteal lymph nodes are removed and weighed on day 7.
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`The difference in weight between the two lymph nodes is taken as the parameter. for
`
`evaluating the reaction.
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`5. Kidnev Allograft Reaction in Rat
`
`One kidney from a female fisher 344 rat is transplanted onto the renal vessel of a
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`unilaterally (left side) nephrectomized WF recipient rat using an end-to-end anastomosis.
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`Ureteric anastomosis is also end-to-end. Treatment commences on the day of transplantation
`
`and is continued for 14 days. A contralatera.l nephrectomy is done seven days after
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`transplantation, leaving the recipient relying on the performance of the donor kidney.
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`Survival of the graft recipient is taken as the parameter for a functional graft
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`6. Experimentally Induced Allergic Encephalomvelitis (EAE) in Rats
`
`Efficacy of the Novel Compounds in EAE is measured, e.g., by the procedure
`
`described in Levine & Wenk, AMER J PATii 47 (1965) 61; Mcfarlin et al, J IMMUNOL
`113 (1974) 712; Borel, TRANSPLANT. & CLIN. IMMUNOL ll (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
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`disease (paralysis of the tail and both hind legs) usually develop within 16 days. The
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`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, ARTIIRITIS & RHEUMATISM .2. (1966) 394;
`Billingham & Davies, HANDBOOK OF EXPERIMENTAL PHARMACOL (Vane &
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`Ferreira Eds, Springer-Verlag, Berlin) .2Q/11 (1979) 108-144. OFA and Wistar rats (male or
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`female, 150g 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 smegmatis.
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`In the developing arthritis model, treatment is staned immedlately after the injection of the
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`adjuvant (days 1 - 18); in the established arthritis model treatment is staned on day 14,
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`when the secondary inflammation is well developed (days 14-20). At the end of the experi(cid:173)
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`ment, 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 appropriate drug resistant cell line
`
`and control (parental) cell line, generated, e.g. as described by Ling et al., J. Cell. Physiol.
`fil, 103-116 (1974) and Bech-Hansen et al. J. Cell. Physiol. ll., 23-32 (1976). Particular clones
`
`chosen are the multi-drug resistant (e.g. colchicine resistant) line CHR (subclone C5S3.2)
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`and the parental, sensitive line AUX Bl (subclone ABl Sil).
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`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
`
`resistant to drug substance DR, VC, AM, ET, 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, 1Q, 1131 (1982).
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`Equivalent results may be obtained employing the Novel Compounds test models of
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`comparable design, e.g. in vitro, or employing test animals infected with drug-resistant and
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`drug sensitive viral strains, antibiotic (e.g. penicillin) resistant 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 occurring sub-strains of Plasmodium
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`falciparum exhibiting acquired chemotherapeutic, anti-malarial drug resistance.
`
`9. FKBP binding
`
`Cenain of the Novel Compounds are not immunosuppressive, panicularly those
`
`which are 0-substituted at C28 only, such as 28-0-methyl-rapamycin. This can be shown in
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`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 reponer
`
`gene assay. Rapamycin, although not active in the IL-2 reponer gene assay, strongly
`
`inhibits IL-6 dependent T-cell proliferation. Both compounds are very potent inhibitors of
`
`the mixed lymphocyte reaction. Nonimmunosuppressivity 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 confers cenain 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(cid:173)
`
`binding immunosuppressants, such as FK506, rapamycin, and the immunosuppressive Novel
`
`Compounds.
`
`10. Steroid potentiation
`
`The macrophilin binding activity of the Novel Compounds also makes them useful in
`
`enhancing or potentiating the action of corticosteroids. Combined treatment with the
`
`compounds of the invention and a corticosteroid, such as dexamethasone, results in greatly
`
`enhanced steroidal activity. This can be shown, e.g., in the murine mammary tumor virus-
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`chloramphenicol acetyltransferase (MMTV-CAT) reporter gene assay, e.g., as described in
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`Ning, et al., J. Biol. 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 variety of Mip (macrophage
`
`infectivity potentiator) and Mip-li.ke factors, which are structurally similar to macrophilin.
`
`Mip and Mip-like factors are virulence factors produced by a wide variety of pathogens,
`
`including those of the genera Chlamidia. e.g., Chlamidia trachomatis: Neisseria, e.g.,
`
`Neisseria meningitidis: and Legionella, e.g., Legionella pneumophilia; and also by the
`
`obligately parasitic members of the order Rickensiales. These factors play a critical role in
`
`the establishment of intracellular infection. The efficacy of the Novel Compounds in
`
`reducing the infectivity of 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. Microbiol. (1993)
`
`7: 777. The nonimmunosuppressive compounds of the invention are preferred for use in
`
`this indication 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 the presence or amount of
`
`macrophilin-binding compounds, e.g., in competitive assays for diagnostic or screening
`
`purposes. 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
`
`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 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., fluoro-, enzymatically- or
`
`radio-labelled, e.g., a Novel Compound which has been 0-substiruted at C40 and/or C28
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`with a labelling group. The plates are washed and the amount of bound labelled compound
`
`is measured. The amount of macrophilin-bind.ing 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 concentrations of macrophilin bind
`
`compound.
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`EXAMPLES:
`
`In the following examples, characteristic spectrescopic data is given to facilitate
`
`identification. Peaks which do not differ significantly from rapamycin are not included.
`
`Biological 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,
`
`an~ to FK-506 in the macrophilin binding assay (MBA). A higher IC50 correlates with lower
`binding affinity.
`
`Example 1: 40-0-Benzvl-rapamvcin
`To a stirred solution of 183 mg (0.200 mmol) of rapamycin in 2.1 mL of 2:1 cyclo(cid:173)
`hexane-methylene chloride is added 75 µL (0.402 mmol) of benzyl-trichloroaceri.midate,
`
`followed by 2.6 µL (29 µmol 15 mol%) of trifluoromethanesulfonic acid whereupon the
`
`mixture turned immediately yellow. After 3h the mixture is diluted with ethyl acetate and
`
`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 afford 40-0-benzyl-rapamycin as a white
`amorphous solid: 1H NMR (CDC13) o 0.73 (lH, dd), 1.65 (3H, s), 1.73 (3H, s), 3.12 (4H, s
`and m), 3.33 (3H, s), 3.49 (3H, s), 4.15 (IH, bd), 4.65 (lH, d), 4.71 (IH, d), 7.22-7.38 (5H,
`
`m); MS (FAB) m/z 1026 ([M+Nat), 972 ([M-OCH3)t), 954 .([M-(OCH3+H20)r>.
`MBA (rel. IC50)
`1.8
`
`IL-6 dep. prol. (rel. IC50)
`
`MLR (rel. IC50)
`
`10
`
`110
`
`Example 2: 40-0-( 4 '-Hydroxvmethvl)benzvl-rapamvcin
`
`a) 40-0-[ 4' -(t-Butyldimethylsilyl)oxymethyl]benzyl-rapamycin
`
`To a stirred, cooled (-78°C) solution of 345 µL (2.0 mmol) of triflic anhydride in 5
`mL of methylene chloride is added a solution of 504 mg (2.0 mmol) of 4-(t-
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`butyldimethylsilyl)oxymethyl-benzyl alcohol and 820 mg (4.0 mmol) of 2,6-di+butyl-4-
`
`methyl-pyridine in 5 mL of methylene chloride. The resulting mixture is warmed to -20°C
`
`and stirring is continued at this temperature for 0.5h. The mixture is then cooled back to -
`78°C and a solution of 914 mg (1.0 mmol) of rapamycin in 5 mL of methyle