United States Patent [191
`Caufield
`
`[54] RAPAMYCIN ESTERS
`Inventor: Craig E. Caufield, Plainsboro, N.J.
`[75]
`[73] Assignee: American Home Products
`Corporation, New York, N.Y.
`[21] Appl. No.: 777,983
`Oct. 17, 1991
`[22] Filed:
`
`Related U.S. Application Data
`[63] Continuation-in-part of Ser. No. 584,833, Sep. 19, 1990,
`abandoned.
`Int. CI.s .................... C07D 487/06; A61K 31/33
`[51]
`[52] U.S. Cl ...................................... 514/183; 540/455
`[58] Field of Search ......................... 540/455; 514/183
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 12/1975 Sehgal et al ........................ 424/122
`(List continued on next page.) ·
`
`OTHER PUBLICATIONS
`Rosen et al "Dictionary of Immunology" pp. 18,88
`Mar. 1991.
`Huffer et al "Introduction to Human Immunology" p.
`(List continued on next page.)
`
`Primary Examiner-C. Warren Ivy
`Assistant Examiner-Celia Chang
`Attorney, Agent, or Firm-Arnold S. Milowsky
`
`[57]
`ABSTRACT
`A compound of the structure
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll lllll lllll lllll llllll Ill lllll llll
`US005221670A
`S,221,670
`[11] Patent Number:
`Jun.22, 1993
`[45] Date of Patent:
`
`where
`RI, R2, and R3 are each, independently, hydrogen or
`
`0
`II
`-CR4
`
`with the proviso that RI, R2, and R3 are not all
`hydrogen;
`R4 is -(CH2)mX(CH2)nC02RS or
`
`RS and R6 are each, independently, alkyl, aralkyl, or
`phenyl which is optionally mono-, di-, or tri-sub(cid:173)
`stituted with a substituent selected from alkyl, alk(cid:173)
`oxy, hydroxy, cyano, halo, nitro, carbalkoxy, tri(cid:173)
`fluoromethyl, amino, or a carboxylic acid;
`
`Xis
`
`R7
`I
`-c-,,0, or S;
`is
`
`R7 and R8 are each, independently, hydrogen or al-
`kyl;
`Yis CH or N;
`m is 04; n is 0-4;
`with the proviso that m and n are not both 0 when X
`is 0 or S;
`or a pharmaceutically acceptable salt thereof, which
`is by virtue of its immunosuppresive activity is
`useful in treating transplantation rejection, host vs.
`graft diSea.se, autoimmune diseases, and diseases of
`inflammation, by virtue of its antitumor activity
`useful in treating tumors, and by virtue of its anti-
`. fungal activity is useful in treating fungal infec(cid:173)
`tions.
`
`19 Claims, No Drawinp
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 001
`
`

`

`5,221,670
`
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`3,993,749 11/1976 Sehgal et al. ....................... 424/122
`4,316,885 2/1982 Rakhit ................................. 424/122
`4,401,653 4/1983 Eng ..................................... 424/122
`4,650,803 3/1987 Stella et al. ........................... 546/90
`4,885,171 12/1989 Surendra et al. ................... 424/122
`5,078,999 1/1992 Warner ............................... 514/291
`S,080,899 1/1992 Sturm et al ......................... 514/291
`5,091,389 2/1992 Ondeyka et al. .................... 514/291
`5,100,883 3/1992 Schiehser ............................ 514/183
`S,100,899 3/1992 Caine ................................... 514/291
`
`OTHER PUBLICATIONS
`161, Jan. 1987.
`J. Antibiot. 28, 721-726 (1975).
`J. Antibiot. 28, 727-732 (1975).
`J. Antibiot. 31, 539-545 (1978).
`Can. J. Physiol. Pharmacol. 55, 48 (1977).
`FASEB 3, 3411 (1989).
`FASEB 3, 5256 (1989).
`Lancet, 1183, (1978).
`Immunology, C. V. Moseby Co., pp. 12.8-12.11 (1989).
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 002
`
`

`

`1
`
`RAPAMYCIN ESTERS
`
`5,221,670
`
`2
`
`CROSS REFERENCE TO RELATED
`APPLICATION
`
`This application is a continuation in part of Ser. No.
`584,833, filed Sep. 19, 1990 now abandoned.
`
`BACKGROUND OF THE INVENTION
`
`5
`
`10
`
`CJ N
`
`wherein
`RI, R2, and R3 are each, independently, hydrogen or
`
`This invention relates to novel esters of rapamycin
`and a method for using them in the treatment of trans(cid:173)
`plantation rejection, host vs. graft disease, autoimmune 15
`diseases, diseases of inflammation, tumors, and fungal
`infections.
`Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found 20
`to have antigugal activity, particularly against Candida
`a/bicans, both in vitro and in vivo [C. Vezina et al., J.
`Antibiot. 28, 721 (1975); S. N. Seghal et al., J. Antibiot.
`28, 727 (1975); H. A. Baker et al., J. Antibiot. 31,539 25
`(1978); U.S. Pat. No. 3,929,992; and U.S. Pat. No.
`3993,749].
`Rapamycin alone (U.S. Pat. No. 4,885,171) or in com(cid:173)
`bination with picibanil (U.S. Pat. No. 4,401,653) has 30
`been shown to have antitumor activity. R. Martel et al.
`[Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed
`that rapamycin is effective in the experimental allergic
`encephalomyelitis model, a model for multiple sclerosis; 35
`in the adjuvant arthritis model, a model for rheumatoid
`arthritis; and effectively inhibited the formation of lgE(cid:173)
`like antibodies.
`The immunosuppressive effects of rapamycin have 40
`been disclosed in FASEB 3, 3411 (1989), rapamycin has
`been shown to be effective in inhibiting transplant rejec(cid:173)
`tion (U.S. Pat. application Ser. No. 362,354 filed Jun. 6,
`1989). Cyclosporin A and FK-506, other macrocyclic 45
`molecules, also have been shown to be effective as im(cid:173)
`R7
`munosuppressive agents, therefore useful in preventing
`I
`transplant rejection [FASEB 3, 3411 (1989); FASEB 3,
`-C-,0,orS;
`!s
`5256 (1989); and R. Y. Caine et al., Lancet 1183 (1978). 50
`Mono- and diacylated derivatives of rapamycin (es-
`R7 and R8 are each, independently, hydrogen or alkyl
`terified at the 28 and 43 positions) have been shown to
`of 1-6 carbon atoms;
`be useful as antifungal agents (U.S. Pat. No. 4,316,885)
`Y is CH or N;
`and used to make water soluble prodrugs of rapamycin 55 m is 0-4;
`n is 0-4;
`(U.S. Pat. No. 4,650,803). Recently, the numbering
`convention for rapamycin has been changed; therefore
`with the proviso that m and n are not both 0 when X
`. bl sa1 h
`f
`according to Chemical Abstracts nomenclature, the
`is O or S; "call
`.
`.
`or a pharmaceut1
`y accepta e
`t t ereo .
`esters descnbed above would be at the 31- and 42- pos1- 60 Of the compounds, preferred members are those in
`which R4 is -(CH2)mX(CH2)11C02R5•
`tions.
`Aryl is defined as an organic radical derived from an
`aromatic hydrocarbon by the removal of one atom; e.g.,
`phenyl from benzene. Aralkyl is defined as an arylated
`65 .alkyl radical; a radical in which an alkyl H atom is
`substituted by an aryl group. The definition of aryl and
`aralkyl are also intended to encompass compounds in
`which the phenyl groups of such moieties are optionally
`
`0
`II
`-cR.4
`
`with the proviso that RI, R2, and R3 are not all
`hydrogen;
`R4 is -(CH2)mX(CH2)nC02R5 or
`
`R5 and R6 are each, independently, alkyl of 1-6 car(cid:173)
`bon atoms, aralkyl of7-10 carbon atoms, or phenyl
`which is optionally mono-, di-, or tri-substituted
`with a substituent selected from alkyl of 1-6 carbon
`atoms, alkoxy of 1-6 carbon atoms, hydroxy, cy(cid:173)
`ano, halo, nitro, carbalkoxy of 2-7 carbon atoms,
`trifluoromethyl, amino, or a carboxylic acid;
`Xis
`
`DESCRIPTION OF THE INVENTION
`This invention provides derivatives of rapamycin
`
`which are useful as immunosuppressive, anti-inflamma-
`tory, antiti:unor, and antifungal agents having the struc-
`ture
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 003
`
`

`

`5,221,670
`
`3
`mono-, di-, or tri-substituted with a substituent selected
`from alkyl of 1-6 carbon atoms, alkoxy of 1-6 carbon
`atoms, hydroxy, cyano, halo, nitro, carbalkoxy of 2-7
`carbon atoms, trifluoromethyl, amino, a carboxylic
`acid, or the like.
`The pharmaceutically acceptable salts may be formed
`from inorganic cations such as sodium, potassium, and
`the like; mono-, di-, and trialkyl amines of 1-6 carbon
`atoms, per alkyl group and mono-, di-, and trihydroxy(cid:173)
`alkyl amines of 1-6 carbon atoms per alkyl group. Pre(cid:173)
`ferred salts are formed from sodium cations and tris(hy(cid:173)
`droxymethyl)methylamine.
`The compounds of this invention can be prepared by
`acylating rapamycin with an acylating agent having the
`general structure
`
`4
`On Day 5, the hind popliteal lymph nodes are removed
`and dissolved, and radioactivity counted. The corre(cid:173)
`sponding left PLN serves as the control for the PLN
`from the injected hind foot. Percent suppression is cal-
`5 culated using the non-drug treated animals as allogenic
`control. Rapamycin at a dose of 6 mg/kg, p.o. gave
`86% suppression, whereas cyclosporin A at the same
`dose gave 43% suppression. Compounds evaluated in
`the PLN test procedure were administered orally, un-
`10 less otherwise indicated, as being administered intraper(cid:173)
`itoneally. Carboxymethyl cellulose was used as the
`vehicle for administration, unless otherwise indicated.
`Results are expressed by the following ratio, unless
`otherwise indicated:
`
`15
`
`3ff.PLN cells control
`C3H mouse
`3ff.PLN cells control
`C3H mouse
`
`3ff-PLN cells rapamycin-treated
`C3H mouse
`3tt-PLN cells test compound-treated
`C3H mouse
`
`0
`II
`XCR4
`
`20
`
`f
`l"
`. h
`where X is OH, m t e presence o a coup mg reagent,
`such as dicyclohexylcarbodimide. The compounds of
`this invention also can be prepared using an anhydride
`of the above described carboxylic acid as the acylating
`species. Alternatively, the acylating species can be an 25
`acid halide, where X can be Cl, Br, or I. The acylating
`groups used to prepare the compounds of this invention
`are commercially available or can be prepared by meth(cid:173)
`ods that are disclosed in the literature.
`Immunosuppressive activity was evaluated in an in 30
`vitro standard pharmacological test procedure to mea(cid:173)
`sure lymphocyte proliferation (LAF) and in two in vivo
`standard pharmacologiCal test procedures. The first in
`vivo procedure was a popliteal lymph node (PLN) test
`procedure which measured the effect of compounds of 35
`this invention on a mixed lymphocyte reaction and the
`second in vivo procedure evaluated the survival time of
`a pinch skin graft.
`The comitogen-induced thymocyte proliferation pro(cid:173)
`cedure (LAF) was used as an in vitro measure of the 40
`immunosuppressive effects of representative com(cid:173)
`pounds. Briefly, cells from the thymus of normal
`BALB/c mice are cultured for 72 hours with PHA and
`IL-1 and pulsed with tritiated thymidine during the last
`six hours. Cells are cultured with and without various 45
`concentrations of rapamycin, cyclosporin A, or test
`compound. Cells are harvested and incorporated; radio(cid:173)
`activity is determined. Inhibition of lymphoprolifera(cid:173)
`tion is assessed in percent change in counts per minute
`from non-drug treated controls. The results are ex- 50
`pressed by the following ratio:
`
`ff3.rapamycin-treated thymus cells
`3ff-control thymus cells -
`3ff-control thymus cells -
`ff3·test compound-treated cells
`
`SS
`
`A mixed lymphocyte reaction (MLR) occurs when
`lymphoid cells from genetically distinct animals are
`combined in tissue culture. Each stimulates the other to
`undergo blast transformation which results in increased
`DNA synthesis that can be quantified l:iY the incorpora- 60
`tion of tritiated thymidine. Since stimlllating a MLR is a
`function of disparity at Major Histocompatibility anti-
`gens, an in vivo popliteal lymph node (PLN) test proce-
`dure closely correlates to host vs. graft disease. Briefly,
`irradiated spleen cells from BALB/c donors are in- 65
`jected into the right hind foot pad of recipient C3H
`mice. The drug is given daily, p.o. from Day 0 to 4. On
`Day 3 and Day 4, tritiated thymidine is given i.p., b.i.d.
`
`The second in vivo test procedure is designated to
`determine the survival time of pinch skin graft from
`male DBA/2 donors transplanted to male BALB/c
`recipients. The method is adapted from Billingham R.
`E. and Medawar P. B., J. Exp. Biol. 28:385-402, (1951).
`Briefly, a pinch skin graft from the donor is grafted on
`the dorsum of the recipient as a homograft, and an
`autograft is used as control in the same region. The
`recipients are treated with either varying concentra(cid:173)
`tions of cyclosporin A as test control or the test com(cid:173)
`pound, intraperitoneally. Untreated recipients serve as
`rejection control. The graft is monitored daily and ob(cid:173)
`servations are recorded until the graft becomes dry and
`forms a blackened scab. This is considered as the rejec(cid:173)
`tion day. The mean graft survival time (number of
`days±S.D.) of the drug treatment group is compared
`with the control group.
`The following table summarizes the results of repre(cid:173)
`sentative compounds of this invention in these three
`standard test procedures.
`TABLE 1
`PLN*
`(ratio)
`++
`0.69**
`1.04••
`0.20
`2.08
`1.68
`0.42
`J.l I
`0.48
`0.10••
`-1.93
`0.37••.+
`0.41
`0.39
`0.09
`1.04
`1.02
`0.40
`12.0 ± 1.7
`1.0
`1.0
`Rapamycin
`-.Calc--ula-110n-. _o_r_ra_tios_wu_desc __ ribed-su-pra-.----------
`**Administered using crcmophore/ethanol IS the vehicle.
`:~~~=!trapcritoncally.
`+++Result exprcoaed .. percent inhibition at 100 nM.
`
`Compound
`
`Example I
`Example 2
`Example 3
`
`Example 4
`
`Example 5
`Example 6
`Example 7
`Example 8
`
`Example 9
`Example 10
`Example 11
`Example 12
`Example 13
`
`LAF*
`(ratio)
`
`0.37
`0.9
`3.27
`
`O.S6
`
`O.Q2
`0.01
`0.97
`0.22
`
`0.22
`0.18
`0.00
`97%+++
`2.11
`
`Skin Graft
`(days+ SD)
`8.2 ± 1.2
`10.7 ± 1.2
`12.7 ± 0.9
`
`10.2 ± 1.7
`
`8.0 ± 1.7
`8.0 ± 0.9
`12.0 ± 1.0
`9.3 ± 1.6
`
`10.2 ± 1.2
`10.8 ± 0.8
`7.8 ± 1.7
`10.8 ± 0.4
`10.6 ± 0.9
`
`The results of these standard pharmacological test
`procedures demonstrate immunosuppressive activity
`both in vitro and in vivo for the compounds of this
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 004
`
`

`

`5,221,670
`
`6
`TABLE 2*-continued
`Strain of Candida albicans
`ATCC
`ATCC
`ATCC
`38247
`38246
`10231
`Compound
`Rapamycin
`0.003
`•expressed as MIC (µg/ml)
`
`ATCC
`38248
`0.006
`
`3669
`0.025
`
`O.Q25
`
`0.003
`
`5
`invention. Positive ratios in the LAF and PLN test
`procedures indicate suppression of T cell proliferation.
`As a transplanted pinch skin grafts are typically re(cid:173)
`jected within 6-7 days without the use of an immuno(cid:173)
`suppressive agent, the increased survival time of the 5
`skin graft when treated with the compounds of this
`invention further demonstrates their utility as immuno(cid:173)
`suppressive agents. While it appears that the compound
`disclosed by Example 8 may cause T cell proliferation
`in the PLN test procedures because of the -1.93 ratio 10
`obtained, it is believed that this result is merely an
`anomaly in light of the other data obtained. Spurious
`results have been obtained in the PLN test procedure
`using compounds that have low bioavailability. Low
`bioavailability can be due to the compound itself, the 15
`dose used, the vehicle, the route of administration, or a
`combination of any of the above factors. When the
`negative ratio was obtained for the compound of Exam(cid:173)
`ple 8, it was administered orally in carboxymethylcellu(cid:173)
`lose. A negative ratio in the PLN test procedure was 20
`not observed for the compounds of Examples 9 and 10,
`which are pharmaceutical salts of the compound of
`Example 8. When the compound of Example 8 was
`administered i.p. in a mixture of cremophore and etha(cid:173)
`nol as the vehicle, a positive ratio was obtained indicat- 25
`ing the compound had immunosuppressive activity.
`The positive ratio obtained in the LAF test procedure
`coupled with the increased survival time observed in
`the skin graft test procedure confirm the immunosup(cid:173)
`pressive activity of the compound of Example 8. The 30
`negative ratio obtained when the compound of Exam(cid:173)
`ple 8 was administered orally in carboxymethyl cellu(cid:173)
`lose is therefore beleived to be attributed to low bi(cid:173)
`oavailability, and not a function of its immunosuppres-
`sive activity.
`Antifungal activity of the compounds of this inven(cid:173)
`tion was measured against 5 strains of Candida albicans
`using a plate test procedure for measurement of inhibi(cid:173)
`tion. The following represents the typical procedure
`used. Compound to be tested was placed on sterile dried 40
`! " plate disks, and allowed to dry. Agar plates were
`seeded with fungi and allowed to solidify. The impreg(cid:173)
`nated disks were placed on the seeded Agar surface and
`incubated for the time required for the particular cul(cid:173)
`ture. Results are expressed in MIC (µg/ml) to inhibit 45
`growth. The results of this test procedure showed that
`the compounds of this invention have antifungal activ(cid:173)
`ity; however, it was surprising that the compounds of
`this invention were less active that the parent com(cid:173)
`pound, rapamycin. The compounds of Examples 12 and 50
`13 were not evaluated for antifungal activity, but be(cid:173)
`cause of the structural similarity to the ones that were
`evaluated, they too are considered to have antifungal
`activity.
`
`35
`
`TABLE 2*
`Strain of Candida albicans
`ATCC
`ATCC
`ATCC
`10231
`38246
`38247
`
`>0.4
`>0.4
`0.2
`>0.4
`0.4
`0.4
`0.1
`0.4
`0.2
`0.1
`>0.4
`
`>0.4
`0.4
`0.1
`0.2
`>0.4
`>0.4
`0.4
`>0.4
`>0.4
`>0.4
`>0.4
`
`>0.4
`>0.4
`0.4
`>0.4
`>0.4
`0.4
`0.1
`0.4
`0.2
`0.2
`>0.4
`
`ATCC
`38248
`
`>0.4
`0.4
`0.1
`0.2
`>0.4
`>0.4
`0.1
`>0.4
`0.4
`0.4
`>0.4
`
`3669
`
`>0.4
`0.4
`0.1
`0.4
`>0.4
`>0.4
`0.2
`>0.4
`>0.4
`>0.4
`>0.4
`
`Compound
`Example I
`Example 2
`Example 3
`Example 4
`Example 5
`Example 6
`Example 7
`Example 8
`Example9
`Example JO
`Example II
`
`65
`
`Based on the results of these standard pharmacologi(cid:173)
`cal test procedures, the compounds are useful in the
`treatment of transplantation rejection such as, heart,
`kidney, liver, bone marrow, and skin transplant; autoim(cid:173)
`mune diseases such as, lupus, rheumatoid arthritis, dia(cid:173)
`betes mellitus, myasthenia gravis, and multiple sclerosis;
`diseases of inflammation such as, psoriasis, dermatitis,
`eczema, seborrhea, and inflammatory bowel disease;
`and fungal infections. As the compounds of this inven(cid:173)
`tion are structurally related to rapamycin, which has
`antitumor activity, they too are considered to be useful
`as antitumor agents.
`The compounds may be administered neat or with a
`pharmaceutical carrier to a mammal in need thereof.
`The pharmaceutical carrier may be solid or liquid.
`A solid carrier can include one or more substances
`which may also act as flavoring agents, lubricants, solu(cid:173)
`bilizers, suspending agents, fillers, glidants, compression
`aids, binders or tablet-disintegrating agents; it can also
`be an encapsulating material. In powders, the carrier is
`a finely divided solid which is in admixture with the
`finely divided active ingredient. In tablets, the active
`ingredient is mixed with a.carrier having the necessary
`compression properties in suitable proportions and
`compacted in the shape and size desired. The powders
`and tablets preferably contain up to 99% of the active
`ingredient. Suitable solid carriers include, for example,
`calcium phosphate, magnesium stearate, talc, sugars,
`lactose, dextrin, starch, gelatin, cellulose, methyl cellu(cid:173)
`lose, sodium carboxymethyl cellulose, polyvinylpyr(cid:173)
`rolidine, low melting waxes and ion exchange resins.
`Liquid carriers are used in preparing solutions, sus(cid:173)
`pensions, emulsions, syrups, elixirs and pressurized
`compositions. The active ingredient can be dissolved or
`suspended in a pharmaceutically acceptable liquid car(cid:173)
`rier such as water, an organic solvent, a mixture of both
`or pharmaceutically acceptable oils or fats. The liquid
`carrier can contain other suitable pharmaceutical addi-
`tives such as solubilizers, emulsifiers, buffers, preserva(cid:173)
`tives, sweeteners, flavoring agents, suspending agents,
`thickening agents, colors, viscosity regulators, stabiliz(cid:173)
`ers or osmo-regulators. Suitable examples of liquid car(cid:173)
`riers for oral and parenteral administration include
`water (partially containing additives as above, e.g. cel(cid:173)
`lulose derivatives, preferably sodium carboxymethyl
`55 cellulose solution), alcohols (including monohydric
`alcohols and polyhydric alcohols, e.g. glycols) and their
`derivatives, and oils (e.g. fractionated coconut oil and
`arachis oil). For parenteral administration, the carrier
`can also be an oily ester such as ethyl oleate and isopro-
`60 pyl myristate. Sterile liquid carriers are useful in sterile
`liquid form compositions for parenteral administration.
`The liquid carrier for pressurized compositions can be
`halogenated hydrocarbon or other pharmaceutically
`acceptable propellent.
`Liquid pharmaceutical compositions which are sterile
`solutions or suspensions can be utilized by, for example,
`intramuscular, intraperitoneal or subcutaneotis injec(cid:173)
`tion. Sterile solutions can also be administered intrave-
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 005
`
`

`

`5,221,670
`
`7
`nously. The compound can also be administered orally
`either in liquid or solid composition form.
`Preferably, the pharmaceutical composition is in unit
`dosage form, e.g. as tablets or capsules. In such form,
`the composition is sub-divided in unit dose containing 5
`appropriate quantities of the active ingredient; the unit
`dosage forms can be packaged compositions, for exam(cid:173)
`ple, packeted powders, vials, ampoules, prefilled syrin(cid:173)
`ges or sachets containing liquids. The unit dosage form
`can be, for example, a capsule or tablet itself, or it can be 10
`the appropriate number of any such compositions in
`package form. The dosage to be used in the treatment
`must be subjectively determined by the attending physi-
`be 15
`·
`f h" ·
`d
`.
`h
`cianln. dd" ·
`a 1t10n, t e compoun s o t 1s invention may
`employed as a solution, cream, or lotion by formulation
`with pharmaceutically acceptable vehicles containing
`0.1-5 percent, preferably 2%, of active compound
`which may be administered topically.
`The following examples illustrate the preparation of
`representative compounds of this invention.
`
`20
`
`8
`EXAMPLE2
`Rapamycin-31,42-bis(monobenzylsuccinate)
`Fraction #2, obtained from the procedure employed
`in Example 1, gave 1.25 g (17.7%) of pure rapamycin-
`31,42-bis(monobenzylsuccinate) upon concentration.
`IH NMR (CDCIJ, 400 MHz) 8 7.351 (bs, lOH, arom),
`5.168 (d, J=2.0 Hz, lH, CH-02C), 5.125 (m, 4H,
`CH2Ph), 4.680 (m, lH, C02CH-CHOMe), 3.356 (s,
`3H, CH30-), 3.329 (s, 3H, CH30-), 3.146 (s, 3H,
`CH30-), 2.639 (m, SH, 02CCH2CH2C02CH2Ph),
`1.748 (s, 3H, CH3C=C), 1.654 (s, 3H, CH3C=C); IR
`(KBr) 3450 (OH), 2940 (CH), 1740 (C=O), 1650, 1455,
`1380, 1355, 1160, 1105, 995 cm-1; MS (neg. ion FAB)
`1294 (M-), 1202, 1103, 1012, 590, 511, 475, 297, 207,
`167, 148, 99 (100); High Res. MS (neg. ion FAB) Calcd
`for C73H99N019 1293.68108, found 1293.6811.
`Analysis Calcd for C73H99N019.H20: C 66.82; H
`7.70; N 1.07; Found: C 67.17; H 7.67; N 1.23.
`The following representative compounds can be pre(cid:173)
`pared from rapamycin and the appropriate half acid(cid:173)
`ester by employing the method used to prepare the title
`25 compound in Example 2.
`Rapamycin-31,42-bis (monomethylsuccinate)
`Rapamycin-31,42-bis
`(monophenyl-3' ,3' -dimethyl-
`glutarate)
`Rapamycin-31,42-bis (mono t-butyl-3' -methylgluta(cid:173)
`rate)
`Rapamycin-31,42-bis (monobenzylthiodiglycolate)
`Rapamycin-31,42-bis (monohexyldiglycolate)
`Rapamycin-31,42-bis (monopropylphthalate)
`Rapamycin-31,42-bis (monoethyl-2' ,6' -pyridinedicar(cid:173)
`boxylate)
`
`EXAMPLE 1
`Rapamycin-14,31,42-tris(monobenzylsuccinate)
`To a solution of 5.0 g (5.47 mmol) of rapamycin, 3.41
`g (16.41 mmol) of monobenzylsuccinate, and 3.15 g
`(16.41 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcar(cid:173)
`bodiimide hydrochloride in 20 mL of dry dichlorometh- 30
`ane was added 200 mg of 4-dimethylaminopyridine.
`The solution was stirred at room temperature for 3
`days. The reaction mixture was poured into 2N HCI and
`extracted three times with ethyl acetate. The organic
`layers were combined, washed with brine, dried over 35
`anhydrous sodium sulfate, decanted, and concentrated
`in vacuo to give a light yellow foam. Flash chromatog(cid:173)
`raphy on a 60mmX150 mm silica gel column eluting
`EXAMPLE 3·
`with 20% ethyl acetate/hexane to 75% ethyl acetate/(cid:173)
`Rapamycin-42-(monobenzylsuccinate)
`hexane gave three fractions. Fraction #1, upon concen- 40
`Fraction #3, obtained from the procedure employed
`tration, gave 330 mg (4.1%) of pure rapamycin-
`in Example 1, gave 930 mg (15.4%) of pure rapamycin-
`14,31,42-tris(monobenzylsuccinate).
`42-monobenzylsuccinate upon concentration.
`1H NMR (CDCl3, 400 MHz) 87.353 (bs, 15H, arom),
`IH NMR (CDCI3, 400 MHz) 8 7.355 (bs, SH, arom),
`5.168 (d, J=2.0 Hz, lH, CH-02C), 5.148 (m, 6H,
`CH2Ph), 4.672 (m, lH, C02CH-CHOMe), 3.355 (s, 45 5.141 (m, 2H, CH2Ph), 4.680 (m,
`lH, C02CH--
`3H, CH30-), 3.337 (s, 3H, CH30-), 3.327 (s, 3H,
`CHOMe), 3.364 (s, 3H, CH30-), 3.333 (s, 3H,
`CH30-), 2.697 (m, 12H, 02CCH2CH2C02CH2Ph),
`CHJO-), 3.141 (s, 3H, CH30-), 2.698 (m, 4H,
`o2CCHiCH2C02CH2Ph), 1.751 (s, 3H, CH3C=C),
`1.745 (s, 3H, CH3C=C), 1.655 (s, 3H, CH3C=C); IR
`(KBr) 3450 (OH), 2950 (CH), 1745 (C=O), 1650, 1460,
`1.655 (s, 3H, cH3C=C); IR (KBr) 3450 (OH), 2940
`1385, 13~, 1160, 1105, 995 cm-I.
`(CH), 1740 (C=O), 1645, 1455, 1380, 1165, 1105, 990
`AnalyS1s Calcd for C84ff109N021.3H20: C 66.27; H
`cm-1; MS (neg. ion FAB) 1103 (M-), 1045, 1012, 624,
`7.56; N 0.92; ~ound: C 65.9~; H 7.24; N 1.00.
`590, l67, 99 (JOO); High Res. MS (neg. ion FAB) Calcd
`The follow1Dg repr.esentat1ve compo~ds can be p~e-
`for 42H89N016 1103.61Sl, found 1103.6048.
`pared from rap~yclD and the appropriate half ac:id- 5S Analysis Calcd for 42Hs9N016-H20: C 66.36; H
`ester by employmg the method used to prepare the title
`8 02. N 1 24. F
`d· C 66 02. H 7 69. N
`1 26
`compound ID Example 1.
`· •
`· • . oun ·
`· .'
`·
`,
`·
`·
`The followmg repr.esentat1ve compo~ds can be p~e-
`Rapamycin-14,31,42-tris (monomethylsuccinate)
`Rapamycin-14 3142-tris
`(monophenyl-3' 3'-dime-
`pared from rapamyclD and the appropnate half ac1d-
`thylglutarat;) '
`'
`ester by employing the method used to prepare the title
`compound ID Example 3.
`Rapamycin-14,31,42-tris
`t-butyl-3'-methyl-
`glutarate)
`Rapamycin-42-monophenyl-3',3'-dimethylglutarate)
`Rapamycin-14,31,42-tris
`Rapamycin-42-(mono t-butyl-3'-methylglutarate)
`late)
`Rapamycin-42-(monobenzylthiodiglycolate)
`Rapamycin-14,31,42-tris (monohexyldiglycolate)
`Rapamycin-42-(monohexyldiglycolate)
`Rapamycin-14,31,42-tris (monopropylphthalate)
`Rapamycin-42-(monopropylphthalate)
`Rapamycin-14,31,42-tris
`(monoethyl-2' ,6' -
`Rapamycin-42-(monoethyl-2' ,6' -pyridinedicarboxy-
`pyridinedicarboxylate)
`late)
`
`(monobenzylthiodiglyco-
`
`50
`
`60
`
`65
`
`(mono
`
`West-Ward Exhibit 1066
`Caufield USP '670
`Page 006
`
`

`

`5,221,670
`
`10
`99 (100); High Res. MS (neg. ion FAB) Calcd for
`C61Hs9019NNa (M-Na) 1162.5926, Found 1162.5899.
`Analysis Calcd for C61Hs9019NNa2. H20: C 60.85; H
`7.56; N 1.16; Found: C 60.67; H 7.36; N 1.ss.
`
`9
`EXAMPLE4
`Rapamycin-31,42-bishemiglutarate
`To a solution of 2.0 g (2.2 mmol) of rapamycin in 10
`mL of dry dichloromethane was added 1.24 g (10.9 5
`EXAMPLE6
`mmol) of glutaric anhydride followed by 881 uL (861
`Rapamycin-31,42-bishemiglutarate bistromethamine
`mg, 10.9 mmol) of pyridine. To this was added 200 mg
`salt
`of 4-dimethylaminopyridine and the reaction mixture
`Purified bis-31,42 hemiglutarate of rapamycin (950
`was allowed to reflux for 8 h. The solution was cooled 10
`mg, 833 umol), prepared as described in Example 4, was
`to room temperature, poured into 2N HCl, and ex-
`dissolved in 5 mL of 95% ethanol and 197 mg (1.63
`tracted three times with dichloromethane. The com-
`mmol) of tris(hydroxymethyl)methylamine was added.
`bined organic extracts were washed with brine, dried
`over anhydrous sodium sulfate, decanted, and concen- Water (1 mL) was added to completely dissolve the
`trated in vacuo to give a yellow foam. The crude prod- 15 amine. Once dissolved, the yellow solution was concen-
`trated in vacuo to give a foamy yellow solid. The very
`uct was purified via reverse phase HPLC on a c 18 col-
`hygroscopic foam was dried in a drying pistol for 24 h,
`umn eluting starting with 60% acetonitrile/water. Col-
`lected, after, concentration, 586 mg (24%) of rapamy-
`refluxing over acetone at reduced pressure to give 900
`cin-31,42-bishemiglutarate.
`mg (78%) of the bistromethamine salt.
`1H NMR (d6·DMSO, 400 MHz) 8 5.253 (m, lH,
`lH, 20
`IH NMR (CDCl3, 400 MHz) 8 5.398 (m,
`-C02CHCHOMe), 4.683 (m, lH, -C02CHCHOMe),
`-CH02C), 4.523 (m, lH, MeOCHCH02C-), 3.347 (s,
`3.364 (s, 3H, CH30-), 3.362 (s, 3H, CH30-), 3.106 (s,
`6H, 2 CH30-), 3.276 (s, 3H, CH30-), 2.289 (m, 8H,
`3H, CH30-), 2.407 (m, 8H, -02CCH2CH2CH-
`02CCH2CH2CH2C02-), 1.681 (s, 3H, CH3C=C),
`2C02H), 1.960 (m, 4H, -o2ccH2cH2cH2co2H),
`1.595 (s, 3H, CH3C=C); IR (KBr) 3400 (OH), 2920
`1.770 (s, 3H, CH3C=C), 1.653 (s, 3H, cH3C=C); !3C 25 (CH), 1730 (C==O), 1620, 1_555, 1450, 1400, 1370, 1185,
`l~, 980 cm- 1; MS (neg. ion FAB) 1140 (M-H, free
`NMR (CDC13, MHz) 211.45 (C==O), 206.84 (C==O),
`200.44 (C==O), 177.83 (C==O), 177.04 (C==O), 172.43
`~c1d), 1028, 167, 148, 131(100),113; High Res. MS (neg.
`(C==O), 171.20 (C==O), 165.27 (C==O), 159.08 (C==O);
`ion FAB) Calcd for C61H9Q019N (M-H, free acid)
`IR (KBr) 3430 (OH) 2940 (CH) 2880 (CH) 1745
`1140.6107, Found 1140.6069.
`(C==O), 1685, 1625, 15180, 1450, 138,5, 1330, 12oo: 1140, 30 Analysis Calcd for C69H10302sN3.2 H20: C 58.77; H
`1100, 990 cm-1; MS (neg. ion FAB) 1140 (M-H),
`7.58; N 2.98; Found: C 58.47; H 7.94; N 3.58.
`1122, 1026, 990, 946, 913, 590, 475, 435, 321, 167, 148,
`EXAMPLE 7
`131 (100), 113; High Res. MS (neg. ion FAB) Calcd for
`C61H90019N (M-H) 1140.6107, Found 1140.6106.
`Analysis Calcd for C61H91019N.H20: C 63.15; H 35
`8.02; N 1.20; Found: C 63.35; H 7.88; N 1.40.
`The following representative compounds can be pre-
`pared from rapamycin and the appropriate anhydride
`by employing the method used to prepare the title com-
`pound in Example 4.
`Rapamycin-31,42-bishemi-3'-methylglutarate
`Rapamycin-31,42-bishemi-3',3'-dimethylglutarate
`Rapamycin-31,42-bishemi-3'-oxoglutarate
`Rapamycin-31,42-bishemi-3'-thioglutarate
`Rapamycin-31,42-bishemi-phthalate
`Rapamycin-31 42-bishemi-2' 3'-pyridine dicarboxyl-
`ate
`'
`'
`
`Rapamycin-42-hemi-3'-oxoglutarate
`To a solution of 3.0 g (3.3 mmol) of rapamycin in 20
`mL of dry dichloromethane was added 1.90 g (16.4
`mmol) of diglycolic anhydride followed by 1.32 mL
`(l.29 g, 16.4 mmol) of pyridine. To this was added 200
`mg of 4-dimethylaminopyridine and the reaction mix-
`40 ture was allowed to stir at room temperature for 2 days.
`~e solution was cooled to room t~mpera~ure,.J?Oured
`mto 2N HCl, and e~tracted t~ee trmes with dtchloro-
`m~than~. The ~ombmed orgamc extra~ts were washed
`with bnne, dned over an~ydrous sod1u°!' sulfate, de-
`45 canted, and concentrated m va~uo to. give a yellow
`foam. The crude product was punfied via reverse phase
`HPLC on a C1s column eluting starting with 60%
`acetonitrile/water. After concentration, 870 mg (26%)
`of rapamycin-42-hemi-3'-oxoglutarate and 500 mg
`EXAMPLES
`so (13 % ) of rapamycin-31,42-bishemi-3 '-oxoglutarate were
`Rapamycin-31,42-hemiglutarate bissodium salt
`isolated.
`IH NMR (CDC13, 400 MHz) 8 4.768 (m, lH, C02.
`Purified bis-31,42-hemiglutarate of rapamycin (740
`CH-CHOMe), 4.250 (m, 4H, o2ccH20CH2C02),
`mg, 649 umol), prepared as described in Example 4, was
`3.356 (s, 3H, CH30-), 3.331 (s, 3H, CH30-), 3.139 (s,
`dissolved in 5 mL of 95% ethanol and 107 mg (127
`mmol) of sodium bicarbonate was added. Water (1 mL) 55 3H, CH30-), 1.759 (s, 3H, CH3C=C), 1.653 (s, 3H,
`CH3C=C); IR (KBr) 3420 (OH), 2920 (CH), 2875
`was added to completely dissolve the salt. Once dis-
`solved, the light yellow solution was concentrated in
`(CH), 1740 (C==O), 1720 (C==O), 1640, 1625, 1445,
`vacuo to give a foamy yellow solid. The foam was dried
`1370, 1320, 1200, 1135, 1095, 980 cm-1; MS (neg. ion
`in a drying pistol for 24 h, refluxing over acetone at
`FAB) 1028 (M-H), 327, 167 (100), 148, 133, 115; High
`reduced pressure to give 520 mg of the bissodium salt. 60 Res. MS (neg. ion FAB) Calcd for CssHs2017N
`lff NMR (d6-DMSO, 400 MHz) 8 5.235 (m, lH,
`(M-H) 1028.5597, Found 1028.5599.
`-CH02C), 4.498 (m, lH, MeOCHCH02C-), 3.287 (s,
`Analysis Calcd for CssHs3017N.3 H20: C 60.97; H
`6H, 2 CH30-), 3.236 (s, 3H, CH30-), 2.245 (m, 8H,
`8.22; N 1.29; Found: C 61.33; H 7.74; N 1.69.
`02CCH2CH2CH2C02-), 1.712 (s, 3H, CH3C=C),
`The following representative compounds can be pre-
`1.593 (s, 3H, CH3C=C); IR (KBr) 3420 (OH), 2920 65 pared from rapamycin and the appropriate half acid-
`(CH), 1725 (C==O), 1675, 1620, 1560, 1450, 1400, 1375,
`ester by employing the method used to prepare the title
`1230, 1195, 1130, 1090, 980 cm-1; MS (neg. ion