United States Patent [19]
`United States Patent [19]
`Caufield
`Caufield
`
`[54] Al\UDE ESTERS OF RAPAMYCI~
`[54] AMIDE ESTERS OF RAPAMYCII\~
`[75]
`Inventor: Craig E. Caufield, Plainsboro, N.J.
`[75] Inventor:
`Craig E. Cau?eld, Plainsboro, NJ.
`[73] Assignee: American Home Products
`[73] Assignee: American Home Products
`Corporation, New York, N.Y.
`Corporation, New York, NY.
`[21] App!. No.: 703,240
`[21] Appl. No.: 703,240
`[22] Filed:
`May 20, 1991
`[22] Filed:
`May 20, 1991
`[51]
`Int. CI.5 ................... A61K 311395; C07D 491/06
`[51] Int. Cl.5 ................. .. A61K 31/395; CO7D 491/06
`[52] U.S. Cl. .................................... 514/183; 514/321;
`[52] U.S. Cl. .................................. .. 514/183; 514/321;
`540/456
`540/456
`[58] Field of Search ................. 540/456: 514/183, 321
`[58] Field of Search ............... .. 540/456: 514/183, 321
`[56]
`References Cited
`[56]
`References Cited
`U.S. PATENT DOCUMENTS
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Seghah et al. ...................... 424/122
`3,929,992 12/1975 Seghah et al. .................... .. 424/122
`3.993.749 11/1976 SeghaJ et al. ....................... 424/122
`3.993.749 11/1976 Seghal et a]. .... ..
`. 424/122
`4.316.885 2/1982 Rakhit ................................. 424/122
`4.316.885 2/1982 Rakhit .............. ..
`.. 424/122
`4.401.653 8/1983 Eng ..................................... 424/114
`..
`4,401.653 8/1983 Eng .................. ..
`4,885.171 12/1989 Surendra et al. ................... 424/122
`4,885.171 12/1989 Surendra et al. ................. .. 424/122
`
`OTHER PUBLICATIONS
`OTHER PUBLICATIONS
`J. Antibiot. 28. 721-726 (1975).
`J. Antibiot. 28. 721-726 (1975).
`J. Antibiot. 28, 727-732 (1975).
`J. Antibiot. 28, 727-732 (1975).
`J. Antibiot. 31, 539-545 (1978).
`J. Antibiot. 31, 539-545 (1978).
`Can. J. Physio!. Pharmacol. 55,48(1977).
`Can. J. Physiol. Pharmacol. 55, 48(1977).
`.
`F ASEB 3.3411 (1989).
`FASEB 3.3411 (1989).
`F ASEB 3,5256 (1989).
`FASEB 3,5256 (1989).
`Lancet 1183 (1978).
`Lancet 1183 (1978).
`Med. Sci Res. 17:877 (1989).
`Med. Sci Res. 17:877 (1989).
`Primary Examiner-Robert T. Bond
`Primary Examiner—Robert T. Bond
`Allorney. Agelll, or Firm-Arnold S. Milowsky
`Attorney. Agent, or F1'rm—-Arnold S. Milowsky
`[57]
`ABSTRACT
`[57]
`ABSTRACT
`A compound of the structure
`A compound of the structure
`
`O
`
`HO
`
`lllllllllllllllllllllllllllIlllllllllllllllllllIlllllllllllllllllllllllllll
`111111111111111111111111111111111111111111111111111111111111111111111111111
`usoos 1 1 8677A
`L'SOO5118677A
`5,118,677
`5,118,677
`[11] Patent Number:
`[II] Patent Number:
`Jun. 2, 1992
`Jun. 2, 1992
`[45] Date of Patent:
`[45] Date of Patent:
`
`wherein R1 and R2 are each independently, hydrogen or
`wherein RI and R 2 are each independently, hydrogen or
`
`0
`O
`1|
`1|
`—C-—X—C—-NR3R“;
`
`X is -(CH2)m- or -Ar-; R3 and R4 are each, inde(cid:173)
`X is —(CHz)m—- 0r —Ar—; R3 and R4 are each, inde
`pendently, hydrogen, alkyl, -(CH1),,—Ar, —(CHg.
`pendently, hydrogen, alkyl, -(CH}h-Ar, -(CH2_
`)p-—NR5R6, or -(CH3)p-—N+R5R6R7Y"; R5 and R6
`)p-NR5R6, or -(CH2)p-N+R5R6R7y-; R5 and R6
`are each, independently, hydrogen, alkyl, or ---(CH;.
`are each, independently, hydrogen, alkyl, or -(CH2_
`),,—Ar; Ar is an optionally substituted group selected
`)n-Ar; Ar is an optionally substituted group selected
`from
`from
`
`(X)._()
`
`)..;
`I
`H
`
`N
`.
`
`in which the optional substituents are selected from the
`in which the optional substituents are selected from the
`group consisting of alkyl, aralkyl, alkoxy, cyano, halo,
`group consisting of alkyl, aralkyl, alkoxy, cyano, halo,
`nitro, carbalkoxy, or perfluoroalkyl; R 7 is alkyl: Y is a
`nitro, carbalkoxy, or peri'luoroalkyl; R7 is alkyl: Y is a
`halide, sulfate, phosphate, or p-toluenesulfonate anion;
`halide, sulfate, phosphate, or p-toluenesulfonate anion;
`m = 1-6; n = 1-6; P = I -6; with the proviso that R 1 and
`m=l-6; n= 1-6; p=l-6; with the proviso that R1 and
`R2 are not both hydrogen; or a pharmaceutically ac(cid:173)
`R2 are not both hydrogen; or a pharmaceutically ac
`ceptable salt thereof, which by virtue of its immunosup
`ceptable salt thereof, which by virtue of its immunosup(cid:173)
`pressive activity is useful in treating transplantation
`pressive activity is useful in treating transplantation
`rejection, host vs. graft disease, autoimmune diseases
`rejection, host vs. graft disease, autoimmune diseases
`and diseases of inflammation; by virtue of its antitumor
`and diseases of in?ammation; by virtue of its antitumor
`activity is useful in treating solid tumors; and by virtue
`activity is useful in treating-solid tumors; and by virtue
`of its antifun gal activity is useful in treating fungal infec
`of its antifungal activity is useful in treating fungal infec(cid:173)
`tions.
`tions.
`
`10 Claims, No Drawings
`10 Claims, No Drawings
`
`NOVARTIS EXHIBIT 2046
`Par v Novartis, IPR 2016-00084
`Page 1 of 7
`
`

`
`1
`1
`
`AMIDE ESTERS OF RAPAMYCI!\"
`AMIDE ESTERS or RAPAMYCIN
`
`5,118,677
`5,118,677
`
`wherein
`wherein
`R1 and R2 are each independently. hydrogen or
`RI and Rc are each independently. hydrogen or
`
`2
`2
`
`o
`0
`.
`II
`II
`-C-X-C-NR"R 4:
`
`X is -(CH2)m- or -Ar-:
`R3 and R4 are each, independently, hydrogen. alkyl of
`R3 and R4 are each, independently, hydrogen. alkyl of
`1-12 carbon atoms, —(CH;r),,—Ar, —(CH2.
`1-12 carbon atoms,
`-(CH;:)n-Ar, -(CH2.
`)p—NR5R6, or —(CH3),,—N+R5R‘>R7Y—;
`)p-NR5R6, or -(CH2)p-N+R5R6R7y-;
`RS and R6are each, independently, hydrogen, alkyl of
`R5 and R6 are each, independently, hydrogen, alkyl of
`1-12 carbon atoms, or -(CH2)n-Ar;
`1-12 carbon atoms, or —(CI-l2),,—-Ar;
`Ar is an optionally mono- or di- substituted group
`Ar is an optionally mono- or di- substituted group
`selected from
`selected from
`
`@oo
`
`00"""::::
`
`#
`
`N"'"
`
`I
`
`. or
`
`.
`
`.
`
`.
`
`.
`
`.
`
`s
`
`10
`10
`
`25
`
`BACKGROUND OF THE INVENTION
`BACKGROUND OF THE INVENTION
`5
`This invention relates to amide esters of rapamycin
`This invention relates to amide esters of rapamycin '
`and a method for using them in the treatment of trans(cid:173)
`and a method for using them in the treatment of trans
`plantation rejection, host vs. graft disease, autoimmune
`plantation rejection, host vs. graft disease, autoimmune
`diseases, diseases of inflammation, solid tumors, and
`diseases, diseases of in?ammation, solid tumors, and
`fungal infections.
`fungal infections.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`Rapamycin is a macrocyclic triene antibiotic pro
`duced by Streptomyces hygroscopicus, which was found
`duced by Srreplomyces hygroscopicus, which was found
`to have antifungal activity, particularly against Candida
`to have antifungal activity, particularly against Candida
`albicans, both in vitro and in vivo [c. Vezina et aI., J.
`albicans, both in vitro and in vivo [C. Vezina et al., J.
`Antibiot. 28. 721 (1975); S. N. Sehgal et aI., J. Antibiot. 15
`Antibiot. 28. 721 (1975); S. N. Sehgal et al., J. Antibiot.
`28, 727 (1975); H. A. Baker et aI., J. Antibiot. 31, 539
`28, 727 (1975); H. A. Baker et al., J. Antibiot. 31, 539
`(1978): U.S. Pat. No. 3,929,992; and U.S. Pat. No.
`(1978): US. Pat. No. 3,929,992; and US. Pat. No.
`3,993,749].
`3,993,749].
`'
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com(cid:173)
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com
`bination with picibanil (U.S. Pat. No. 4,401,653) has 20
`bination with picibanil (U.S. Pat. No. 4,401,653) has
`been shown to have antitumor activity. R. Martel et al.
`been shown to have antitumor activity. R. Martel et a1.
`[Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed
`[Can J. Physiol. Pharmacol. 55, 48 (1977)] disclosed
`that rapamycin is effective in the experimental allergic
`that rapamycin is effective in the experimental allergic
`encephalomyelitis model. a model for multiple sclerosis;
`encephalomyelitis model. a model for multiple sclerosis;
`in the adJ'uvant arthritis model, a model for rheumatoid
`in the adjuvant arthritis model, a model for rheumatoid
`arthritis: and effectively inhibited the formation of IgE-
`arthritis; and effectively inhibited the formation of IgE
`like antibodies.
`like antibodies.
`The immunosuppressive effects of rapamycin have
`The immunosuppressive effects of rapamycin have
`been disclosed in FASEB 3. 3411 (1989). Cyclosporin A
`been disclosed in FASEB 3. 3411 (1989). Cyclosporin A
`and FK-506, other macrocvclic molecules. also have
`and FK-506, other macrocyclic molecules, also have
`been shown to be effecti~e as immuno~uppressive 30
`been shown to be effective as immunosuppressive
`agents. therefore useful in preventing transplant rejec(cid:173)
`agents. therefore useful in preventing transplant rejec
`tion [F ASEB 3, 3411 (1989); F ASEB 3, 5256 (1989): and
`tion [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989): and
`R. Y. CaIne et aI., Lancet 1183 (1978)].
`R. Y. Calne et al., Lancet 1183 (1978)].
`Mono- and diacylated derivatives of rapamycin (es
`Mono- and diacylated derivatives of rapamycin (es- 35
`35
`terified at the 28 and 43 positions) have been shown to
`teri?ed at the 28 and 43 positions) have been shown to
`be useful as antifungal agents (U.S. Pat. No. 4.316.885)
`be useful as antifungal agents (U.S. Pat. No. 4.316.885)
`and used to make water soluble prod rugs of rapamycin
`and used to make water soluble prodrugs of rapamycin
`(U.S. Pat. No. 4.650,803). Recently, the numbering
`(U.S. Pat. No. 4.650,803). Recently, the numbering
`convention for rapamycin has been changed: therefore 40
`convention for rapamycin has been changed; therefore
`according to Chemical Abstracts nomenclature, the
`according to Chemical Abstracts nomenclature, the
`esters described above would be at the 31- and 42- posi(cid:173)
`esters described above would be at the 31- and 42- posi
`tions.
`trons.
`
`DESCRIPTION OF THE INVENTION
`DESCRIPTION OF THE INVENTION
`45
`This invention provides derivatives of rapamycin 45
`This invention provides derivatives of rapamycin
`which are useful as immunosuppressive, anti-inflamma(cid:173)
`which are useful as immunosuppressive, anti-in?amma
`tory, antifungal, and antitumor agents having the struc(cid:173)
`tory, antifungal, and antitumor agents having the struc
`ture
`
`ture
`
`-
`
`50
`50
`
`0
`
`§
`o
`
`in which the optional substituents are selected from
`in which the optional substituents are selected from
`the group consisting of alkyl of 1-6 carbon atoms,
`the group consisting of alkyl of 1-6 carbon atoms,
`aralkyl of 7-l0 carbon atoms, alkoxy of l-6 carbon
`aralkyl of 7-10 carbon atoms, alkoxy of 1-6 carbon
`atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon
`atoms, cyano, halo, nitro, carbalkoxy of 2-7 carbon
`atoms, or perfluoroalkyl of 1-6 carbon atoms;
`atoms, or per?uoroalkyl of l—6 carbon atoms;
`R7 is alkyl of 1-6 carbon atoms;
`R 7 is alkyl of 1-6 carbon atoms;
`Y is a halide, sulfate, phosphate. or p-toluenesulfonate
`Y is a halide, sulfate, phosphate, or p-toluenesulfonate
`anion;
`anion;
`m=I-6;
`n= 1-6;
`p=I-6;
`with the proviso that R1 and R2 are not both hydro
`with the proviso that R I and R 2 are not both hydro(cid:173)
`gen;
`gen;
`or a pharmaceutically acceptable salt thereof.
`55 or a pharmaceutically acceptable salt thereof.
`55
`Pharmaceutically acceptable salts may be formed
`Pharmaceutically acceptable salts may be formed
`when R3 or R4 is -(CH2)p-NR5R6 or when Ar is an
`when R3 or R4 is —(CI-I2)p—NR5R6 or when Ar is an
`optionally mono- or di- substituted pyridyl or quinolyl
`optionally mono- or di- substituted pyridyl or quinolyl
`group. The pharmaceutically acceptable salts are de
`group. The pharmaceutically acceptable salts are de-
`rived from such organic and inorganic acids such as,
`60 rived from such organic and inorganic acids such as,
`acetic, lactic, citric, tartaric, succinic, maleic, malonic,
`acetic, lactic, citric, tartaric, succinic, maleic, malonic,
`gluconic, hydrochloric, hydrobromic, phosphoric, ni
`gluconic, hydrochloric, hydrobromic, phosphoric, ni(cid:173)
`tric, sulfuric, methanesulfonic, and the like.
`tric, sulfuric, methanesulfonic, and the like.
`Of these compounds, preferred members are those in
`Of these compounds, preferred members are those in
`which X is —(CI-I2)m——; those in which X is —(CI-l2.
`65 which X is -(CH2)m-; those in which X is -(CH2.
`65
`)m- and R3 and R4 are alkyl of 1-6 carbon atoms; and
`)m— and R3 and R4 are alkyl of l-6 carbon atoms; and
`those in which X is —(CH;),,,-—, R3 is hydrogen, and
`those in which X is -(CH2)m-, R3 is hydrogen, and
`R4 is and Ar is -(CH2)n-Ar.
`R4 is and Ar is —(CI-Iz)n——Ar.
`
`NOVARTIS EXHIBIT 2046
`Par v Novartis, IPR 2016-00084
`Page 2 of 7
`
`

`
`5,118,677
`5,118,677
`4
`4
`3
`3
`activity is determined. Inhibition of lymphoprolifera
`activity is determined. Inhibition of Iymphoprolifera(cid:173)
`The compounds of this invention acylated at the
`The compounds of this invention acylated at the
`42-position can be prepared by acylating rapamycin
`tion is assessed as percent change in counts per minute
`42-position can be prepared by acylating rapamycin
`tion is assessed as percent change in counts per minute
`with an amido-acid acylating agent having the general
`with an amido-acid acylating agent having the general
`from non-drug treated controls. The results are ex(cid:173)
`from non-drug treated controls. The results are ex
`pressed by the following ratio.
`structure
`pressed by the following ratio.
`structure
`
`5
`
`o
`0
`II
`II
`AA '4
`HO
`x
`NRF‘R4
`HO
`X
`NR"R
`
`3H·control thymus celb H"-rapamycin-treated th"mus celb
`JH-control thvmus cells — H3-rapamvcin-treated thvmus cells
`3H-control thymu~ cells - H3_test compound-treated cells
`3l-l-control thymus cells — H3-tesl compound-treated cells
`
`in the presence of a coupling reagent, such as a suitably
`in the presence of a coupling reagent, such as a suitably
`substituted carbodiimide coupling reagent.
`substituted carbodiimide coupling reagent. .
`The compounds of this invention acylated at both the
`The compounds of this invention acylated at both the
`31- and 42-positions can be prepared by the method
`31- and 42-positions can be prepared by the method
`described above by increasing variables such as reaction
`described above by increasing variables such as reaction
`time, temperature, and quantity of acylating agent.
`time, temperature, and quantity of acylating agent.
`The 31 -acylated compounds of this invention can be
`The 31 -acylated compounds of this invention can be
`prepared by protecting the 42-alcohol of rapamycin
`prepared by protecting the 42-alcohol of rapamycin
`with a protecting group, such as with a tert-butyl dime(cid:173)
`with a protecting group, such as with a tert-butyl dime
`thylsilyl group in the presence of a base, such as imidaz(cid:173)
`thylsilyl group in the presence ofa base. such as imidaz
`ole, followed by acylation of the 31-position with an
`ole, followed by acylation of the Ell-position with an
`acylating agent having the general structure shown
`acylating agent having the general structure shown
`above. Removal of the protecting group provides the
`above. Removal of the protecting group provides the
`3l-acylated compounds. In the case of the tert-butyl
`31-acylated compounds. In the case of the tert-butyl
`dimethylsilyl protecting group. deprotection can be
`dimethylsilyl protecting group, deprotection can be
`accomplished under mildly acidic conditions, such as
`accomplished under mildly acidic conditions. such as
`with a mixture of aqueous acetic acid and THF.
`with a mixture of aqueous acetic acid and THF.
`Having the 3lsposition acylated and the 42-position
`Having the 31-position acylated and the 42-position
`deprotected, the 42-position can be reacted with a dif(cid:173)
`deprotected. the 42-position can be reacted with a dif
`ferent amido-acid of the generic structure described
`ferent amido-acid of the generic structure described
`above than was reacted \vith the 3 I-alcohol, to give
`above than was reacted with the 31-alcohol. to give
`compounds having different acyl moieties at the 31- and
`compounds having different acyl moieties at the 3]- and
`42-positions. Alternatively. the 42-acyl compounds.
`42-positions. Alternatively, the 42-acyl compounds,
`prepared as described above, can be reacted with an
`prepared as described above. can be reacted with an
`acylating agent having a different structure to provide
`acylating agent having a different structure to provide
`compounds having different acyl moieties at the 3l-and
`compounds having different acyl moieties at the 31-and 35
`42-positions.
`42-positions.
`The acylating groups used to prepare the compounds
`The acylating groups used to prepare the compounds
`of the invention are can be prepared by the method
`of the invention are can be prepared by the method
`outlined below from anhydrides that are either com(cid:173)
`outlined below from anhydrides that are either com
`mercially available or by methods that are that are dis- 40
`mercially available or by methods that are that are dis
`closed in the literature.
`.
`closed in the literature.
`'
`
`10 A mixed lymphocyte reaction (MLR) occurs when
`A mixed lymphocyte reaction (MLR) occurs when
`10
`lymphoid cells from genetically distinct animals are
`lymphoid cells from genetically distinct animals are
`combined in tissue culture. Each stimulates the other to
`combined in tissue culture. Each stimulates the other to
`undergo blast transformation which results in increased
`undergo blast transformation which results in increased
`DNA synthesis that can be quanti?ed by the incorpora
`DNA synthesis that can be quantified by the incorpora-
`15 tion of tritiated thymidine. Since stimulating a MLR is a
`tion oftritiated thymidine. Since stimulating a MLR is a
`function of disparity at Major Histocompatibility anti
`function of disparity at Major Histocompatibility anti(cid:173)
`gens, an in vivo popliteal lymph node (PLN) test proce(cid:173)
`gens, an in vivo popliteal lymph node (PLN) test proce
`dure closely correlates to host vs. graft disease. Briefly,
`dure closely correlates to host vs. graft disease. Briefly.
`irradiated spleen cells from BALB/c donors are in-
`irradiated spleen cells from BALB/c donors are in
`jected into the right hind foot pad of recipient C3H
`20 jected into the right hind foot pad of recipient C3H
`mice. The drug is given daily, p.o. from Day 0 to Day
`mice. The drug is given daily, p.o. from Day 0 to Day
`3 and Day 4. tritiated thymidine is given i.p.. b.i.d. On
`3 and Day 4, tritiated thymidine is given i.p .. b.i.d. On
`Day 5, the hind popliteal lymph nodes are removed and
`Day 5. the hind popliteal lymph nodes are removed and
`dissolved, and radioactivity counted. The correspond
`dissolved, and radioactivity counted. The correspond-
`25 ing left PLN serves as the control for the PLN from the
`ing left PLN serves as the control for the PLN from the
`25
`injected hind foot. Percent suppression is calculated
`injected hind foot. Percent suppression is calculated
`using the non-drug treated animals as allogenic control.
`using the non-drug treated animals as allogenic control.
`Rapamycin at a dose of 6 mg/kg, p.o. gave 86% sup(cid:173)
`Rapamycin at a dose of 6 mg/kg. po gave 86% sup
`pression, whereas cyclosporin A at the same dose gave
`pression. whereas cyclosporin A at the same dose gave
`30 43% suppression. Results are expressed by the follow(cid:173)
`43% suppression. Results are expressed by the follow
`ing ratio:
`ing ratio:
`
`"H-PLl" cells control C3H mouse - 3H·PLN cells
`3l-l-PLN cells control C3H mouse -— 3l-l'PLN cells
`rapamvcin-treated C3H mouse
`ranam\'cin-treated C3H mouse
`"H·PL;\; cell> contrc>1 C3H mou~e - 3H-PLN cells
`:‘l-l-PLN cells control C3l-l mouse — 3H-PLN cells
`test compound-treated cell,
`test compound-treated cells
`
`The second in vivo test procedure is designed to
`The second in vivo test procedure is designed to
`determine the survival time of pinch skin graft from
`determine the survival time of pinch skin graft from
`male DBA/2 donors transplanted to male BALB/c
`male DEA/2 donors transplanted to male BALB/c
`recipients. The method is adapted from Billingham R.
`recipients. The method is adapted from Billingham R.
`E. and Medawar P. B., J. Exp. BioI. 28:385-402, (1951).
`E. and Medawar P. B., J. Exp. Biol. 28:385-402, (1951).
`Briefly, a pinch skin graft from the donor is grafted 0n
`Briefly, a pinch skin graft from the donor is grafted on
`45 the dorsum of the recipient as a homograft. and an
`the dorsum of the recipient as a homograft. and an
`45
`autograft is used as control in the same region. The
`autograft is used as control in the same region. The
`recipients are treated with either varying concentra(cid:173)
`recipients are treated with either varying concentra
`tions of cyclosporin A as test control or the test com(cid:173)
`tions of cyclosporin A as test control or the test com
`pound, intraperitoneally. Untreated recipients serve as
`pound, intraperitoneally. Untreated recipients serve as
`rejection control. The graft is monitored daily and ob
`50 rejection control. The graft is monitored daily and ob(cid:173)
`servations are recorded until the graft becomes dry and
`servations are recorded until the graft becomes dry and
`Immunosuppressive activity was evaluated in an in
`Immunosuppressive activity was evaluated in an in
`forms a blackened scab. This is considered as the rejec(cid:173)
`forms a blackened scab. This is considered as the rejec
`vitro standard pharmacological test procedure to mea(cid:173)
`vitro standard pharmacological test procedure to mea
`tion day. The mean graft survival time (number of
`tion day. The mean graft survival time (number of
`sure lymphocyte proliferation (LAF) and in two in vivo
`sure lymphocyte proliferation (LAP) and in two in vivo
`daysiS. D.) of the drug treatment group is compared
`days±S. D.) of the drug treatment group is compared
`standard pharmacological test procedures. The first in
`standard pharmacological test procedures. The ?rst in
`55 with the control group.
`with the control group.
`vivo procedure was a popliteal lymph node (PLN) test
`vivo procedure was a popliteal lymph node (PLN) test
`55
`The follo'Wing table summarizes the results of repre(cid:173)
`The following table summarizes the results of repre
`procedure which measured the effect of compounds of
`procedure which measured the effect of compounds of
`sentative compounds of this invention in these three
`sentative compounds of this invention in these three
`this invention on a mixed lymphocyte reaction and the
`this invention on a mixed lymphocyte reaction and the
`standard test procedures.
`standard test procedures.
`second in vivo procedure evaluated the survival time of
`second in vivo procedure evaluated the survival time of
`a pinch skin graft.
`a pinch skin graft.
`TABLE I
`TABLE 1
`The comitogen-induced thymocyte proliferation pro
`The comitogen-induced thymocyte proliferation pro(cid:173)
`LAF
`PLN
`LAF
`PLN
`cedure (LAF) was used as an in vitro measure of the
`cedure (LAF) was used as an in vitro measure of the
`(ratio)
`(ratio)
`(ratio)
`(ratio)
`immunosuppressive effects of representative com
`immunosuppressive effects of representative com(cid:173)
`1.94
`0.62
`1.94
`0.62
`pounds. Brie?y, cells from the thymus of normal
`pounds. Briefly, cells from the thymus of normal
`+
`0.14
`0.14
`+
`BALB/c mice are cultured for 72 hours with PHA and
`BALB/c mice are cultured for 72 hours with FHA and
`0.19
`0.76
`0.19
`0.76
`IL-1 and pulsed with tritiated thymidine during the last
`IL-I and pulsed with tritiated thymidine during the last
`0.91
`0.59
`0.91
`0.59
`six hours. Cells are cultured with and without various
`1.00
`1.00
`six hours. Cells are cultured with and without various
`1.00
`1.00
`concentrations of rapamycin, cyclosporin A, or test
`concentrations of rapamycin, cyclosporin A. or test
`compound. Cells are harvested and incorporated radio-
`compound. Cells are harvested and incorporated radio
`
`o
`II
`A
`o
`V
`II
`o
`
`60
`
`65
`65
`
`Compound
`Compound
`Example 1
`Example 1
`Example 2
`Example 2
`Example 3
`Example 3
`Example 4
`Example 4
`Rapamycin
`' Rapamycin
`+ Not evaluated.
`+ Not evaluated.
`
`Skin Graft
`Skin Graft
`(days -'- SD)
`(days J» SD)
`
`-
`.;.
`-;- '
`7.5 ± 1.5
`7.5 : 1.5
`9.5 ± 0.8
`9.5 1 0.8
`12.0 = 1.7
`l2.0 : 1.7
`
`NOVARTIS EXHIBIT 2046
`Par v Novartis, IPR 2016-00084
`Page 3 of 7
`
`

`
`25
`
`5,118,677
`5,118,677
`6
`5
`6
`5
`halogenated hydrocarbon or other pharmaceutically
`halogenated hydrocarbon or other pharmaceutically
`The results of these standard pharmacological test
`The results of these standard pharmacological test
`acceptable propellent.
`procedures demonstrate immunosuppressive activity
`acceptable propellent.
`procedures demonstrate immunosuppressive activity
`Liquid pharmaceutical compositions which are sterile
`Liquid pharmaceutical compositions which are sterile
`both in vitro and in vivo for the compounds of this
`both in vitro and in vivo for the compounds of this
`solutions or suspensions can be utilized by, for example,
`solutions or suspensions can be utilized by, for example,
`invention. Positive ratios in the LAF and PLN test
`invention. Positive ratios in the LAF and PLN test
`intramuscular. intraperitoneal or subcutaneous injec
`5 intramuscular. intraperitoneal or subcutaneous injec(cid:173)
`procedures indicate suppression of T cell proliferation.
`procedures indicate suppression of T cell proliferation.
`tion. Sterile solutions can also be administered intrave(cid:173)
`tion. Sterile solutions can also be administered intrave
`As a transplanted pinch skin grafts are typically re(cid:173)
`As a transplanted pinch skin grafts are typically re
`nously. The compound can also be administered orally
`nously. The compound can also be administered orally
`jected within 6-7 days without the use of an immuno(cid:173)
`jected within 6~7 days without the use of an immuno
`either in liquid or solid composition form.
`either in liquid or solid composition form.
`suppressive agent, the increased survival time of the
`suppressive agent, the increased survival time of the
`Preferably, the pharmaceutical composition is in unit
`Preferably, the pharmaceutical composition is in unit
`skin graft when treated with the compounds of this
`skin graft when treated with the compounds of this
`dosage form, e.g. as tablets or capsules. In such form.
`10 dosage form, e.g. as tablets or capsules. In such form,
`invention further demonstrates their utility as immuno(cid:173)
`invention further demonstrates their utility as immuno
`the composition is sub-divided in unit dose containing
`the composition is sub-divided in unit dose containing
`suppressive agents.
`suppressive agents.
`appropriate quantities of the active ingredient; the unit
`appropriate quantities of the active ingredient; the unit
`Because the compounds of this invention are structur(cid:173)
`Because the compounds of this invention are structur
`dosage forms can be packaged compositions, for exam
`dosage forms can be packaged compositions, for exam(cid:173)
`ally similar to rapamycin and have a similar activity
`ally similar to rapamycin and have a similar activity
`ple, packeted powders, vials, ampoules, pre?lled syrin
`ple, packeted powders, vials, ampoules, prefilled syrin-
`profile to rapamycin, the compounds of this invention
`pro?le to rapamycin, the compounds of this invention
`ges or sachets containing liquids. The unit dosage form
`15 ges or sachets containing liquids. The unit dosage form
`also are considered to have antitumor and antifungal
`also are considered to have antitumor and antifungal
`can be, for example, a capsule or tablet itself, or it can be
`can be, for example, a capsule or tablet itself, or it can be
`activities.
`activities.
`the appropriate number of any such compositions in
`the appropriate number of any such compositions in
`Based on the results of these standard pharmacologi(cid:173)
`Based on the results of these standard pharmacologi
`package form. The dosage to be used in the treatment
`package form. The dosage to be used in the treatment
`cal test procedures, the compounds are useful in the
`cal test procedures, the compounds are useful in the
`must be subjectively determined by the attending physi
`must be subjectively determined by the attending physi(cid:173)
`treatment of transplantation rejection such as, heart,
`treatment of transplantation rejection such as, heart,
`cian.
`cian.
`kidney, liver, bone marrow, and skin transplants; auto- 20
`kidney, liver, bone marrow, and skin transplants; auto
`In addition, the compounds of this invention may be
`In addition, the compounds of this invention may be
`immune diseases such as, lupus, rheumatoid arthritis.
`immune diseases such as, lupus, rheumatoid arthritis,
`employed as a solution. cream. or lotion by formulation
`employed as a solution. cream. or lotion by formulation
`diabetes mellitus, myasthenia gravis, and multiple scle
`diabetes mellitus, myasthenia gravis, and multiple scle(cid:173)
`with pharmaceutically acceptable vehicles containing
`with pharmaceutically acceptable vehicles containing
`rosis: and diseases of inflammation such as, psoriasis,
`rosis: and diseases of in?ammation such as, psoriasis,
`0.1-5 percent. preferably 2%, of active compound
`0.1-5 percent. preferably 2%, of active compound
`dermatitis, eczema, seborrhea, inflammatory bowel
`dermatitis, eczema, seborrhea, in?ammatory bowel
`which may be administered to a fungally affected area.
`which may be administered to a fungall)~ affected area.
`disease, and eye uveitis; solid tumors; and fungal infec-
`disease, and eye uveitis; solid tumors; and fungal infec
`The following examples illustrate the preparation of
`The following examples illustrate the preparation of
`tions.
`tions.
`representative compounds of this invention.
`representative compounds of this invention.
`The compounds may be administered neat or with a
`The compounds may be administered neat or with a
`pharmaceutical carrier to a mammal in need thereof.
`pharmaceutical carrier to a mammal in need thereof.
`EXAMPLE 1
`EXAMPLE 1
`The pharmaceutical carrier may be solid or liquid.
`The pharmaceutical carrier may be solid or liquid.
`Rapamycin 42-ester with
`Rapamycin 42-ester with
`A solid carrier can include one or more substa'nces
`A solid carrier can include one or more substances
`4-(dimethylamino)-4-oxobutanoic acid
`4-( dimethyl amino )-4-oxobutanoic acid
`which may also act as flavoring agents, lubricants, solu(cid:173)
`which may also act as ?avoring agents, lubricants, solu
`bilizers, suspending agents, ?llers, glidants, compression
`bilizers, suspending agents, fillers, glidants, compression
`To a solution of 1.00 g (1.09 mmol) ofrapamycin in 20
`To a solution of 1.00 g (1.09 mmol) of rapamycin in 20
`aids, binders or tablet-disintegrating agents; it can also
`mL of dry dichloromethane was added 316 mg (2.18
`mL of dry dichloromethane was added 316 mg (2.18
`aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrier is 35
`mmol) of N.N-dimethylsuccinamic acid, 15 mg of 4~
`mmo!) of KN-dimethylsuccinamic acid, 15 mg of 4-
`be an encapsulating material. In powders, the carrier is
`N,N-dimethylaminopyridine followed by 476 mg
`a finely divided solid which is in admixture with the
`N,N-dimethylaminopyridine
`followed by 476 mg
`a ?nely divided solid which is in admixture with the
`(mmol) of l-(3-dimethylaminopropyl)-3-ethylcarbodii
`(mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodii(cid:173)
`finely divided active ingredient. In tablets. the active
`?nely divided active ingredient. In tablets, the active
`ingredient is mixed with a carrier having the necessary
`mide hydrochloride. The solution was stirred overnight
`mide hydrochloride. The solution was stirred overnight
`ingredient is mixed with a carrier having the necessary
`compression properties in suitable proportions and
`and then poured into IN HCl and extracted three times
`compression properties in suitable proportions and
`and then poured into 1N HC] and extracted three times
`with ethyl acetate. The organic layers were combined,
`with ethyl acetate. The organic layers were combined,
`compacted in the shape and size desired. The powders 40
`compacted in the shape and size desired. The powders
`and tablets preferably contain up to 99% of the active
`washed with brine, dried over anhydrous sodium sul-
`washed with brine, dried over anhydrous sodium sul
`and tablets preferably contain up to 99% of the active
`ingredient. Suitable solid carriers include, for example,
`fate, filtered, and concentrated in vacuo to give a pale
`ingredient. Suitable solid carriers include, for exam