...
`
`--(3L//
`
`PCT
`
`(51) International Patent ClasdBcation 6:
`C07D 401/04
`
`WORLD INTELLECT1JAL PROPERTY ORGANIZATION
`lntcmalional Bureau
`INTERNATIONAL APPLICATION PUBLISHED UNDER TIIE PATENT COOPERATION TREATY {PCT)
`WO 97/41111
`
`(11) International Publication Number:
`
`Al
`
`(43) lnternatJonal Publication Date:
`
`6'Noveml>cr 1997(06.11.97)
`
`(21) International Application Nwnber: -
`
`PCT/IB97/00254
`
`(22) International Filing Date:
`
`13 March 1997 (13.03.97)
`
`(30) Priority Data:
`60/016,495
`
`30 April 1996 (30.04.96)
`
`us
`
`(71) Applicant (for all designated Stales ucep1 US): PAZER INC.
`(US/US); 235 East 42nd Street, New York, NY 10017 (US).
`
`(81) Designated States: AL, AM, AT, Ali; AZ. BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ. DE, DK, EE. ES_, Fl; G8. GE,
`HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC;LK, LR, LS, LT,
`LU, LV, MD, MG, MK, MN, MW, MX, NO. NZ. PL. PT.
`RO, RU, SD, SE. SG, SI,-SK, TJ, TM, TR, Tr. UA, UG,
`US, UZ, VN, YU, ARIPO patenl (GH, KE, LS, MW, SD,
`SZ. UG), Eurasian patent (AM. AZ. BY, KG, KZ, MD, RU,
`TJ, TM), European patent (AT, BE, CH, DE, DK, ES, Fl,
`FR, GB, GR, IE, IT, LU; MC, NL,. PT, SE); OAPI patent
`(BF, BJ, CF, CG, Cl, CM, GA, GN, ML, MR; NE, SN, m:
`
`TG).
`
`.
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US 011/y): URBAN. Frank, John Published
`[US/US]; 12 Twin Lakes Drive, Waterford, CT06385 (US).
`Wi1h i111erna1ional search report.
`
`(74) Agents: SPIEGa, Allen, J. et al.; Pfizer Inc., 235 East 42nd
`Sneet, New York, NY 10017 (US).
`
`(54) Title: PROCESSES AND IN"IERMEDIATES FOR PREPARING 4'-TRIFLUOROMlITHYLBIPHENYL·2-CARBOXYLIC ACJD
`(2-{2H-[ l,2,4}TR IAZOL-3-YLME'IHYL)-1,2,3,4-TETRAHYDRO-ISOQUINOLIN-6-YL]-AMIDE.
`.
`
`(57) Abstract
`
`A process for preparing the compound of formula (I) which comprises
`trea1ing the compound of fonnula (Ill) wherein R is H or R2 and R2 is selected
`from the group comprising allyl or a substituted methyl group wherein lhe
`subsdtuents comprise one to three (C6-C1o)aryl groups wherein the eryl groups
`are further optionally substituted with one or more substiruents selected from
`nitro and (C1-4)alkoxy; a) with a compound of formula (X) wherein J is a
`leaving group such as a halogen atom, an !Wdo group, a (C1-C6)acyloxy group
`or a CC6-C1o)aroyloxy group, preferably a chlorine or bromine atom; and b)
`when R is R2 further treating the product of step a) with an acid. A compound
`of fonnula (II) wherein R is R2 and R2 is as defined above and is, preferably,
`CHJOC6H4CH2-.
`
`01
`
`R
`
`J:X:JC·
`....
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`Codes used lO identify Slates pany to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`FOR THE PURPOSES OF INFORMATION ONLY
`
`Al.
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`cc
`CH
`C1
`CM
`CN
`cu
`CZ.
`DE
`DK
`EE
`
`Albania
`Aimcnia
`Auslria
`Ausnalia
`Azerbaijan
`llOlllia and Hcrugovina
`Bazbadoo
`Belgiwn
`Bwti..a Fuo
`Bulgaria
`Benin
`Brazil
`BelllUS
`Canada
`C.ennal African Republic
`Coogo
`Swil7crland
`C«e d'lvoirc
`Came<ODCI
`China
`Cuba
`Cuch Republic
`Germany
`De nm art
`EslDl'li•
`
`£S
`Fl
`FR
`CA
`CB
`CE
`CH
`CN
`CR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KC
`KP
`
`KR
`K7.
`LC
`u
`LK
`LR
`
`Spain
`Finlwl
`France
`Oabon
`Uniud Kingdom
`Ctmgia
`Ghana
`Ouinu
`Gruce
`Hung1ty
`Ireland
`Israel
`Iceland
`Italy
`Jl!'ll'
`Kel'lya
`Kytgyutan
`Dcmocnlic People'•
`Republic or Kcfta
`Rcpufllic of Kocca
`Ku.USWI
`Saini Lucia
`Liechlcnstcin
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`1.-.ho
`Lkhiwlia
`LuumbomB
`Lal via
`Moa.co
`Repubfic of Moldova
`MAd,.gucar
`1llt fonner Yugmlav
`Republic of Ma<cdonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Maico
`Niger
`Nelhertandl
`Norway
`New Ztaland
`!'bland
`l'onugal
`Romania
`Russian Fcdcralion
`SudUI
`Sweden
`Singapo"'
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UC
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slowtia
`Senepl
`Swaziland
`Oied
`Togo
`Tajikistan
`Turtmenisian
`Tudtey
`Trinidad and Tobago
`Uknint
`Uganda
`United Slllel of America
`Uzbctisun
`v;.i Nam
`Yugoslavia
`Zimbabwe
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`PCT/IB97/00254
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`-1-
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`5
`
`PROCESSES ANO INTERMEPIATES FOB PREPARING
`
`4'-TRIFLUOBOMETHYLBIPHENYL-2-CARBOXYLIC ACIP 12-l2H-1] .2.41 ·
`.
`.
`TRIAZOL-3-YLMETHYU-1 .2.3.4-TETRAHYQRO-!SOOU!NOLIN-6-YlJ-AMIDE
`
`Field Of The lnventjon
`
`This invention relates to 4'-trifluoromethylbiphenyl-2-carboxylic acid 12-(2H-
`
`10
`
`( 1 ,2,4itriazol-3-ylmettiyl)-1 ,2,3,4-tetrahydro-isoquinolin-6-yl)-amide ot fo;mula
`
`I
`
`below. More particularly it relates to an improved method, and intermediates, for the :
`
`preparation of compound I. Compound I is an inhibitor of microsomal triglyceride·
`
`transfer protein C and/or apolipoprotein B (Apo B) secretion and is, accordingly, ·
`
`useful for the prevention and treatment of atherosclerosis and its clinical sequelae,
`
`15
`
`for lowering serum lipids, and related diseases.
`Backgroynd Of The lnventjon
`
`Microsomal triglyceride transfer protein CMTP) catalyzes the transport of
`
`triglyceride, cholesteryl ester, and phospholipids.
`
`It has been implicated as a ·
`
`probable agent in the assembly of Apo B-containing lipoproteins, biomolecules which
`
`20
`
`contribute to the formation of atherosclerotic lesions. See European Patent
`
`application publication no. 0 643057A1, European Patent application publication
`
`no. 0 584 446 A2, and Wetterau et al.; Science, 258, 999-1001, 11992).
`
`Compounds which inhibit MTP and/or otherwise inhibit Apo B secretion are,
`
`therefore, useful in the treatment of atherosclerosis. Such compounds. are els~
`
`25
`
`useful in the treatment of other diseases or conditions in which, by inhibiting MTP
`
`end/or Apo B secretion, serum cholesterol and triglyceride levels can pe reduced.
`
`Such conditions include hypercholesterolemia, hypertriglyceridemia, pancreatitis, and
`
`obesity; end hypercholesterolemia, hypertriglyceridemia, and hyper!ipidemia
`
`associated with pancreetitis, obesity, and diabetes.
`
`Summary Of The Invention
`
`This invention provides a method for preparing the compound of formula
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`30
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`35
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`-2-
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`5
`
`10
`
`15
`
`which comprises treating the compound of the formula
`
`I I I
`
`wherein R is H or R2 and R2 is selected from allyl and a substituted methyl
`group wherein the substituents comprise one to three (C9-C10)aryl groups wherein the
`20 aryl groups are optionally substituted with one or more substituents selected from
`nitro and (C 1-C9)alkoxv:
`a)
`with a compound of the formula
`COJ
`
`25
`
`x
`
`30
`
`is a leaving group such as a halogen atom, an azido group, a
`wherein J
`(C 1-C8)acyloxy group or a (C11-C10)aroyloxy group, preferably a chlorine or bromine
`atom; and
`
`b)
`
`when R is R2 further treating the product of step a) with an acid
`
`such as trifluoroacetic acid (TFA), p-toluenesulfonic acid <PTSA_), methane- or
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`trifluoromethanesulfonic acid and HBr in acetic acid, preferably trifluoroacetic acid:·
`
`Reference to a moiety as "heterocyclic" means any single ring or fused ring ·
`
`system containing at least one ring heteroatom independently selected from 0, N,
`
`5
`
`and S. Thus a polycyclic fused ring system containing one or more carb~cyclic fused·
`
`saturated, partially unsaturated, or aromatic rings (usually benzene ringsl is within
`the definition of heterocyclyl so long as the system also contains at le~st Orie fused(cid:173)
`
`ring which contains at least one of the aforementioned heteroatoms. As a
`
`substituent, such heterocyclic rings may be attached to the remainder of the
`10 molecule from either a carbocyclic Ce.g., benzene) ring or from a heterocyclic ring.
`
`Reference to a moiety containing "one or more rings" is intended to mean that
`
`said moiety contains a single or fused cyclic moiety or moieties. The rings may be
`
`carbocyclic or heterocyclic, saturated or partially unsaturated. and aromatic or non(cid:173)
`
`aromatic.
`Reference to a fused polycyclic ring system or radical means that all rings in
`
`1 5
`
`the system are fused.
`
`Reference to "halo" in this specification is inclusive of fluoro, chloro, bromo,
`
`and iodo unless noted otherwise.
`Reference to an "aryl" substitutent (e.g. CC9-C 10)aryll means th~ ring qr
`substitutent is carbocyclic. Aromatic moieties which contain one or more
`
`20
`
`heteroatoms are included as a subset of the term "heterocyclic", as discussed above.
`
`Reference to an •acyl" substituent refers to an aliphatic or cyclic hydrocarbon
`
`moiety attached to a carbonyl group through which the substituent bonds.
`
`Reference to "alkyl" and "alkoxy" include both straight and, when the moiety
`
`25
`
`contains more than two carbon atoms, branched of cyclic chain radicals, but it is to
`
`be understood that references to individual radicals such as "propyl" or "propoxy"
`
`embrace only the straight chain ("normal") radical, branched chain isomers such as
`
`"isopropyl" or "isopropoxy" being referred to specifically.
`
`Certain intermediates of the formula
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`-4-
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`5
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`y
`
`.
`.
`and theirtautomers, wherein Y is NH, or NO,, ere additionally provided: as a further_
`feature of the invention. One ski11ed in the art will understand that. the above
`
`1 0
`
`compounds may-exist in several teutomeric forms all of which are included in the
`invention~ Another intermediate provided by the invention is the compound of the
`formula
`
`15
`
`20
`
`0
`II c,
`N
`H
`
`I I
`
`wherein R is R2
`
`, preferably CH 30C.,H4CH 2-.
`Qetejled Descrjotjon of the !nyentjon
`In the reaction Schemes and description which follow R, R 2~ J, K end Y as
`
`well as structural formulae I through X are as defined above.
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`-5-
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`SCHEME 1
`
`-Base
`
`R2
`I
`N
`
`fl' ;N
`
`N..._j'
`
`IX
`
`.
`
`R2
`I
`HOCHiy--N
`'N
`II
`N~;
`
`·
`
`V 111,
`
`R2
`I
`
`H2~N'\N
`N~
`
`VI
`
`7
`
`5
`
`10
`
`20
`
`25
`
`0
`
`R2
`
`~~N
`
`15
`
`VII
`
`W~)N
`
`R
`
`N--J'
`
`02N
`
`IV CR=R2>
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`SCHEME 2
`
`IV C R=Rz>
`
`Ill <R=H>
`
`I
`
`5
`
`10
`
`15
`
`20
`
`25
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`30
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`-8-
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`As shown in Scheme 1 compound IX is prepared by reacting 1 .2·,4-triazole
`with a compound of the formula R2K wherein K, is halo as defined above,: in ·the
`presence of a base. Preferably R2K is 4-~ethoxybenzyl chloride. The ·reaction is
`carried out at about room temperature in a polar solvent such as a N,N,-di(C,-
`5 C8)alkylcarboxamide, e.g •• dimethylacetamide {DMAC) end dimethylformamide
`!DMF); a ketone, e.g., acetone and methylethylketone; a {C,·C11lalkanol, e.g .• ethanol.
`methanol and isopropanol; end mixtures thereof. Bases useful in the.practice of this
`aspect of the invention include alkali metal hydroxides, carbonates and hydrogen
`carbonates. Preferably the solvent is DMF and the base is NaOH.
`Compound ~is prepared by heating compound 1.K with formaldehyde in hot
`water. The reaction is affected at reflux temperature using an external heat source
`
`10
`
`at from about 100 to about 135°C. The formaldehyde, which is used in excess, can
`
`be supplied in the form of its 37% aqueous solution (also known as formalin) or its
`
`linear polymeric (paraformaldehyde) and trimeric (trioxane) forms. Paraformaldehyde
`
`15 decomposes in hot water, and trioxane in aqueous solutions containing strong acids.
`
`to yield formaldehyde. The preferred source of formaldehyde is formalin.
`Compound VII is formed by treating compound Yfil, in a Mitsunobu reaction,
`with phthalimide in the presence of triphenylphosphine and a di(C,~C8)alkyl or
`dipiperidinyl azodicarboxylate. The preferred azodicarboxylate is the diisopropyl ester.
`
`20 The reaction may be effected at a temperature of from about 0 to about 65 °C in an
`
`aprotic solvent such as tetrahydrofuran (THF), isopropyl ether and dioxane.
`
`Preferably the reaction is carried out at about 15 °C in THF.
`
`Compound ~ is converted to compound VI by suspensio~ in an aprotic
`suspension medium such as a (C 1-C11lalkanol, e.g., methanol, ethanol or isopropanol,
`and treatment with hydrazine. The hydrazine is preferably provided in the form of its
`
`25
`
`hydrate and the preferred suspension medium is methanol. The reaction can be
`
`effected at a temperature of about room temperature to about 65 °C, preferably room
`
`temperature.
`Compound IV, {R = R2
`
`30
`
`), is formed by treatment of compound VI with
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`y
`
`v
`
`5
`
`solvent.
`
`wherein Y is selected from halo and optionally substituted (C 1-C8)alkyl-. or (C,(cid:173)
`C8)arysulfonoxy groups, in the presence of a base and an aprotic solvent. Prefer~bly
`CH3S03 (mesyloxy). The reaction is effected under an inert atmosphere, such as
`1 O nitrogen or argon, at a temperature of about ambient to the reflux temperature of the
`The bases which may be used are organic bases such as tri(C 1-
`C9)alkylamines, pyridine and N-methylmorpholine. Aprotic solvents include :THF,
`CH 2Cl 2 and DMF. The reaction is preferably effected in THF, under nitrogen, at the
`reflux temperature of the solvent. The preferred base is triethylamine. Compound ':f.,
`15 wherein Y is mesyloxy~ may be prepared by treating a suspension of compound Y
`wherein Y is OH in a (C 1-C11)haloalkane. at a low temperature under an inert
`atmosphere, with mesyl chloride in the presence of a base. Bases useful in the
`
`practice of this aspect can be selected from those described above~ The inert
`
`atmospheres may be selected from those described above. The temperature is from
`
`20
`
`about -40 to about 0°C. The reaction is preferably carried out under nitrogen, at
`
`about -30° C in the presence of triethylamine.
`As shown in Scheme 3 compound J.Y. (R = R2
`(R =HJ by treatment with an acid such as trifluoroacetic acid (TFA), p-toluenesulfonic
`
`) is converted to compound IV
`
`acid, methane or trifluoromethanesulfonic acid and HBr in acetic a_cid, preferably
`
`25
`
`trifluoroacetic acid. The reaction may be carried out at a temperature from about
`
`room temperature to about 60°C preferably at room temperature. The_ TFA may be
`used neat or dissolved in CH 2Cl 2•
`Compound n£, wherein R is hydrogen , is converted to compol.!nd l!! ', wherein
`A is hydrogen, by treatment with hydrogen, at a pressure of from about 1 to about
`
`30
`
`3 Atom in the presence of a hydrogenation catalyst and an organic , solvent.
`
`Hydrogenation catalysts include Pd, Pt and Raney Ni. The metals may be used in the
`form of salts, e.g .. Pd(0H) 2 , or on carriers, e.g., carbon. The hvdrogenation is
`effected at a temperature from about room temperature to about 50°C, preferably
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`at room temperature. The preferred hydrogenation catalyst is 10% Pd/C and :the
`
`preferred solvent is methanol.
`Compound ill wherein R is hydrogen, is converted to compound I by treatment
`with a source of the 4'-trifluoromethylbiphenyl-2-carbonyl group selected fr:om'the
`
`5
`
`halides, ezides and mixed enhydrides, in the presence of a solvent and a base. The
`
`reacti~n.is effected at a temperature from about room temperature t~ about_ 50°C
`
`preferably et room temperature. Solvents useful in aspect of the invention Jnclude
`
`aprotic sol:vents, as described above, haloalkanes and mixtures thereof. A preferred
`
`solvent is the mixture of THF and methylene chloride which is formed wh8n a
`
`10
`
`15
`
`solution of the carbonyl compound, in methylene chloride, is added to a suspension
`of compound Ill (R = H) in THF.
`Alternatively, as shown in Scheme 3, compound l can be prepared·by treating
`compound Ill wherein R is R2 with a 4'-trifluoromethylbiphenyl-2-carbonyl source, as
`described above, to form the compound of formula !land treating compound 1! with
`an acid such es trifluoroacetic acid CTFA), p-toluenesulfonic acid, methane or
`
`trifluoromethanesulfonic acid and HBr in acetic acid, preferably tri~lu.oroacetic acid
`as described above. Preferably, R2
`is p-CH 30C0H4CH 2-
`trifluoromethylbiphenyl-2-carbonyl source is the chloride.
`
`the 4'(cid:173)
`
`and
`
`The compound of formula! and its tautomers form acid addition salts and the
`
`20 expression "pharmaceutfoally-acceptable salts" is intended to include _but not.is to
`
`be limited to such salts as the hydrochloride, hydrobromide, sul~at~. hydrogen
`
`sulfate, phosphate, hydrogen phosphate, dihydrogenphosphate, acetate, succirate,
`
`citrate, methenesulfonate and p-toluenesulfonate salts. It can also.for:m polyaddition
`
`salts.
`
`25
`
`The acid addition salts of the compound of formula I, and its tautomer~. are
`
`readily prepared by reacting the base form with the appropriate acid. When the salt
`
`is of a monobasic acid (e.g., the hydrochloride, the hydrobromide,. the p(cid:173)
`
`toluenesulfonate, the acetate), the hydrogen form of a dibasic _acid (e.g., the
`
`hydrogen sulfate, the succinate) or the dihydrogen form of a tribasic acid _(e.g., the
`
`30 dihydrogen phosphate, the citrate), at least one molar equivalent end usually a _molar
`
`excess of the acid is employed. However when such salts as ~he. sulfate~ the
`hemisuccinate, the hydrogen phosphate or the phosphate are desired, the appropriate
`'
`.
`and exact chemical equivalents of acid will generally be used. The free base and the
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`acid are usually combined in a co-solvent from which the desired salt pre~lpitates,
`or can be otherwise isolated by concentration and/or addition of a non~solvent~
`The compound of formula I, its tautomers, and their pharmaceutically
`.
`.
`acceptable acid salts, (hereafter 'the active compounds") are orally administrable and··
`
`.
`
`5
`
`are accordingly used in combination with a pharmaceutically acceptable carrier or
`
`diluent suitable to oral dosage forms. Suitable pharmaceutically-acceptable carriers
`
`include inert solid fillers or diluents and sterile aqueous or organic sol'utions. • The
`.
`.
`active compounds will be present in such pharmaceutical compositions.in amounts .
`
`sufficient to provide the desired dosage amount in the range described below. Thus,
`
`1 O
`
`for oral administration the compounds can be combined with a suitable solid or liquid
`
`carrier or diluent to form capsules, tablets, powders, syrups, solutions, suspensions
`
`and the like. The pharmaceutical compositions may, if desired, contain additional
`
`components such as flavorants, sweeteners, excipients and the like. Pharmaceutical
`
`compositions comprising the active compounds are suitable for the treatment of
`
`15
`
`conditions including atherosclerosis, pancreatitis, obesity, hypercholesterolemia,
`hypertriglyceridemia, hyperlipidemia, and diabetes, comprising a com~ound of
`
`formula I as hereinbefore defined, and a pharmaceutically acceptable carrier.
`
`The active compounds inhibit or decrease apo B secretion, likely by the
`
`inhibition of MTP, although it may be possible that other mechanisrrys are involved
`
`20 as well. The compounds are useful in any of the diseases or conditions in w.hich apo
`
`B, serum cholesterol, and/or triglyceride levels are elevated. Accordingly, the
`.
`.
`invention further provides a method of treating a condition ~elected from
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`:
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`atherosclerosis, pancreatitis, obesity, hypercholesteremia, hypertriglyceridemia,
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`hyperlipidemia, and diabetes, comprising administering to a mammal, especially a
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`25
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`human, in need of such treatment an amount of a compound of formula, I as defi.ned
`
`above sufficient to decrease the secretion of apolipoprotein B. A subgroup of the
`.
`: .
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`preceding conditions includes atherosclerosis. obesity. pancreatitis, a.nd diabetes.
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`A more particular subgroup includes atherosclerosis.
`
`The term •treating" as used herein with respect to the active compounds
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`30
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`includes preventative as well as disease remitative treatment.
`
`The tablets, pills, capsules, and the like may also contain a binder such as
`
`gum tragacanth, acacia, corn starch or gelatin; excipients such a~ dicalcium
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`phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a
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`lubricant such as magnesium stearate; and a sweetening agent such as sucrose, ·
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`lactose or saccharin. When a dosage unit form is a capsule, it may ?ontain, in
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`addition to materials of the above type, a liquid carrier such as a fatty oil.
`Various other materials may be present as coatings o~ to ~odify the physical
`form of the dosage unit. For instance, tablets may be coated with shellac, sugar or
`both. A syrup or elixir may contain, in addition to the active ingred!ent, sucrose as
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`5
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`a sweetening agent, methyl and propylparabens as preservatives, :a dye and a '
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`flavoring such as cherry or orange flavor. ·
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`The active compounds may also be administered parenterally. For parenteral
`
`1 O administration the compounds can be combined with sterile aqueous or organic
`media to form injectable solutions or suspensions. Solutions or suspensions of these
`
`active compounds can be prepared in water suitably mixed with 8 surfaCtant such
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`as hydroxypropylcellulose. Dispersions can also be prepared in seS&me or peanut oil,
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`ethanol, water, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol),
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`15
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`suitable mixtures thereof, vegetable oils. N-methyl glucamine, polyvinylpyrrolidone
`and mixtures thereof in oils as well as aqueous solutions of water-soluble
`pharmaceutically acceptable salts of the compounds. Under ordinary ~~nditio~~ ~f
`storage and use, these preparations contain a preservative to prevent th~ growth of
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`microorganisms. The injectable solutions prepared in this manner can .then be
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`20 administered intravenously, intraperitoneally. subcutaneously, or intramuscularly.
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`The pharmaceutical forms suitable for injectable use include sterile aqueous
`
`solutions or dispersions and sterile powders for the extemporaneous preparation of
`
`sterile injectable solutions or dispersions. In all cases, the form must be sterile and
`
`must be fluid to the extent that easy syringability exists. It must be. stable ~nder the
`conditions of manufacture and storage and must be preserved . against the
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`25
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`contaminating action of microorganisms such as bacteria and fungi ..
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`The dose of an active compound which is administered will generally be varied
`
`according to principles well known in the art taking into account the severity of the
`
`condition being treated and the route of administration.
`
`In .general, the active
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`30
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`compound will be administered to a warm blooded animal (such as a humall) so that
`
`an effective dose, usually a daily dose administered in unitary or divided portions, is
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`received, for example a dose in the range of about 0. 1 to about 15 mg/kg body
`
`weight, preferably about 1 to about 5 mg/kg body weight. The to~al daily dose
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`received will generally be between 1 and 1000 mg, preferably between 5 and 350
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`mg.
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`The active compound may be used in conjunction with other pharmaceutical
`
`agents, including other lipid lowering agents. Such agents include cholesterol
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`5
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`biosynthesis inhibitors, especially HMG CoA reductase inhibitors . and squalene
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`synthetase inhibitors; bile acid sequestrants; fibrates; cholesterol . absorption'
`
`inhibitors; and niacin.
`
`A test compound is considered to be active if it is active 'in any of the
`
`following screens.
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`1 O
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`The activity of an active compound can be assessed by measuring inhibition
`
`of apo B secretion in HepG2 cells.
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`HepG2 cells are grown in Dulbecco's Modified Eagles Medium plus 10% fetal
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`bovine serum (growth medium; Gibco) in 96-well culture plates in e humidified
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`atmosphere containing 5% carbon dioxide until they are approximately 70%
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`confluent. Test compounds are dissolved at 10-20 mM in dimethyl sulfoxide which
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`is then diluted to 1 µMin growth medium. Serial 1 :1 dilutions of this stock are made
`
`in growth medium and 100 pl of each are added. to separate wells of a 96-well
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`culture plates containing HepG2 cells. Twenty four hours later, growth medium is
`
`collected and assayed by specific ELISA for apoB and, as a control, apoAI
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`20
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`concentrations. Inhibitors are identified as compounds that decrease apoB se.cretion
`
`into the medium without affecting the secretion of apoAI. The ELISA for apoB is
`
`performed as follows. Monoclonal antibody against human apoB (Chemicon) is
`diluted to 5 pg/ml in phosphate buffered saline/azide (PBS + 0.02% Na azide) and
`100 µL are added to each well of a 96-well plate INUNC Maxisorb). After an
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`25
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`overnight incubation at room temperature, the antibody solution is removed and wells
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`are washed 3 times with PBS/azide. Non-specific sites on the plastic are blocked by
`
`incubating wells for 1-3 hours in a solution of 1 % (w/v) bovine serum albumin (BSA)
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`made in PBS/azide. 100 µl of various dilutions of growth medium from the HepG2
`
`cells or apoB standards (made in 0.004% Tween 2011 % BSA in PBS/a~ide) are added
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`30
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`to each well and incubated for 18 hours. Wells are aspirated and washed. 3 times
`
`(0. 1 % Tween 20 in PBS) prior to adding 100 µl of a 1/1000 dilution of the
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`secondary antibody, goat anti-human apoB (Chemicon). After a 3 hpur incubation
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`at room temperature, this solution is aspirated and the wells are again washed 3
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`times as above~ 100 pl ofa 1 :1600 dilution (in PBS/1 % BSA/2mM MgCl~I of rabbit
`anti-goat lgG conjugated to alkaline phosphatase (Sigma I are then added to each well
`and incubated for 1 hour at. room tempe~ature. After aspirating, ~he: wells :are
`washed 4 times as above and 1 00 pl of 1 mg/ml p-nitrophenylphos'phate : (.pNPP;
`5 Sigma) in 25 mM sodium bicarbonate 2 mM MgCl2, pH 9.5, are added t~ each well
`and incubated for 20.-30 minutes and then the reaction is terminated by the addition
`.
`.
`of 50 µl of 0.2N NaOH. Absorbance of each well is read at 405 nm and: the
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`.
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`background at 650 nm is subtracted. ApoB concentration is calculated . from ~
`
`standard curve constructed from purified lDl standards that are run in parallel in the
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`sarrie assay. ApoAI is measured in ·an analogous manner except that antibodies for
`
`apoAI (Chemiconl are used in place of the antibodies for apoB and antigen incubation
`
`is at 37 ° instead of room temperature.
`
`Activity can also be confirmed if a test compound inhibits MTP activity
`
`directly.
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`1 5
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`Inhibition of MTP activity by a compound can be measured by observing the
`
`inhibition of transfer of radiolabeled triglyceride from donor Vflsicl~s to acceptor
`
`vesicles in. the presence of soluble human MTP. The procedure for preparing MTP
`
`is based on the method of Wetterau and Zilversmit (Biochem. Biophys. Acta ,<1986)
`.
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`875:6101. Briefly, human liver chunks, frozen at -80°C, are th.awed on ice, minced,
`
`20
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`and rinsed several times with ice cold 0.25 M sucrose. All subseq~ent steps are
`
`performed on ice. A 50% homogenate in 0.25 M sucrose is prepared using a Potter(cid:173)
`
`Elvehjem Teflon pestle. The homogenate is diluted 1 :1 with 0.25 M .. sucrose and
`
`centrifuged at 10,000 x g for 20.minutes at 4°C. The pellet is resuspended in
`
`sucrose and recentrifuged at 10,000 x g for 20 minutes. The supema~ants are
`
`25
`
`combined and the microsomes pelleted by centrifugation at 105,000 x g for 75
`
`minutes. The supernatant is discarded and the microsomal pellet is ~uspended. in a
`
`minimal volume of 0.25 M sucrose, diluted to 3 ml per gm starting liver weight with
`
`0.15 M Tris-HCI pH 8.0. This suspension is divided into 12 fr,actions, and
`
`centrifuged at 105,000 x g for 75 minutes. The supernatants are discarded and the
`
`· 30 microsomal.pellets are stored.frozen at -80°C until needed. For preparation of MTP
`: .
`prior to performing the assay, a thawed pellet is suspended in 12 ml of col.d 50.mM
`Tris-HCI, 50 mM KCI, 5 mM MgCl 2J, pH 7 .4 and 1 .2 ml of a 0.54% d,eoxycholate
`(pH 7.4) solution is added slowly with mixing to disrupt the microsomal m~mbrane.
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`After a 30 minutes incubation on. ice with gentle mixing, the suspension is·
`centrifuged et 105,000 x g for 75 minutes. The supernatant. co·ntairiing the soluble .
`MTP protein, is dialyzed for 2-3 days with 4 changes of assay buffer H50 rnM Tris(cid:173)
`HCI, 40 mM NaCl, 1 mM EDTA. 0.02% NaN3 , pH 7.4); The human.liver ·MTP is
`stored at 4°C and diluted 1:5 with assay buffer just before use. MTP preparations
`
`5
`
`show no notable toss of transfer activity with storage up to 30 days •.
`Liposomes are prepared under nitrogen by room temperat~re, ~th sonic_ation ;
`of a dispersion of 400 µM egg phosphatidytcholine (PC), 75 µM bovine heart·
`
`cardiolipin, and 0.82 µM 14C-triolein (110 Ci/moO in assay buffer. The lipids in
`
`1 O
`
`chloroform are added in the proper amounts and dried under a nitrogen stream before
`
`hydrating with assay buffer. Acceptor liposomes are prepared under nitrogen by
`
`room temperature bath sonication of a dispersion of 1.2 mM PC, 2.3 j.JM triolein and
`
`30 pM 3H-PC (50 Ci/mol) in essay buffer. The donor and acceptor liposomes are
`centrifuged at 160,000 x g for 2 hours at 7°C. The top 80% of the supernatant,
`containing small unilamellar liposomes, are carefully removed and stored at 4°C until
`
`15
`
`used for transfer assays.
`
`MTP activity is measured using a transfer assay which is initiated by mixing
`
`donor and acceptor vesicles together with the soluble MTP and test compound. To
`
`100 µL of either .a 5% BSA (control) or 5% BSA containing the test comp~und, are
`
`20 added 500 µL assay buffer, 100 µL donor liposomes, 200 µL acceptor liposomes and
`100 µL of diluted MTP protein. After incubation at 37°C for 45 minutes.,
`triglyceride transfer is terminated by adding 500 µL of a 50% (wlv) DEAE cellulose
`
`suspension in assay buffer. Following 4 minutes of agitation, the do_nc;>r lip_osomes,
`
`bound to the DEAE cellulose. are selectively sedimented by low speed centrifugation.
`
`25 An aliquot of the supernatant containing the acceptor liposomes is c~unted and. the
`3H and 14C counts are used to calculate the percent recovery of acceptor li~osomes
`and the percent triglyceride transfer using first order kinetics.
`lnhibitiQn of
`
`triglyceride transfer by test compound is manifested
`
`as a decrease in 14C
`
`radioactivity compared to controls where no test compound is present.
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`30
`
`Activity of test compounds as MTP inhibitors can also be measured. in vivo
`according to the following test.
`
`Male mice 120-30g.; various strains) are dosed by oral gavage .10.25 ml/25
`
`g. body weight) with test compound suspended in en aqueous O.!?% methyl cellulose
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`solution. Compound solutions are dosed either multiple times over several days or,
`
`alternatively, once 90 minutes before mice are euthanized and blood i~. collected for
`
`preparation of serum. The serum is assayed for triglyceride c()Ocentration by a
`
`commercial enzymatic assay.(Triglyceride G: Wako Fine Chemicals). MTP inhibitors
`
`5
`
`are identified by their ability to lower serum triglycerides as compared to contiol mice
`
`dosed w.ith vehicle.
`
`The present invention is illustrated by the following Examples.' However, it
`should be understood that the invention is not limited to the specific d~tails of these.
`examples.
`
`10
`
`Conventional methods andfor techniques of purification and separation known
`to those skilled in the art can be used to isolate the compounds of this invention.
`
`Such techniques include all types of chromatography CH PLC, column chromatography
`
`using common adsorbents such as silica gel, and thin layer chromatography).
`
`15
`
`recrystallization, and differential Ci.e.~ liquid-liquid) extractio