WO 99/52910
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`PCT/IB99/00503
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`-2 9-
`
`x 106 /ml in HBSS containing 1% BSA. Differential counts determined using the Abbott Cell Dyn
`
`3500 analyzer indicated that monocytes ranged from 17 to 24% of the total cells in these
`
`preparations.
`
`180 pl of the cell suspension was aliquoted into flat bottom 96 well plates (Costar).
`
`Additions of compounds and LPS (100 ng/ml final concentration) gave a final volume of 200 pl.
`
`All conditions were performed in triplicate. After a four hour incubation at 37°C in an humidified
`
`002 incubator, plates were removed and centrifuged (10 minutes at approximately 250 x g) and
`
`the supematants removed and assayed for TNFa using the R&D ELISA Kit.
`
`Inhibition of Soluble TNF-g_ Production
`
`The ability of the compounds or the pharmaceutically acceptable salts thereof to inhibit
`
`the cellular release of TNF-cx and, consequently, demonstrate their effectiveness for treating
`
`diseases involving the disregulation of soluble TNF-a is shown by the following ip yitfl assay:
`
`Method for the evaluation of recombinant TNF—g Converting Enzyme Activity
`
`Expression of recombinant TACE
`
`A DNA fragment coding for the signal sequence, preprodomain. prodomain and
`
`catalytic domain of TACE (amino acids 1-473), can be amplified by polymerase chain reaction
`
`using a human lung cDNA library as a template. The amplified fragment is then cloned into
`
`pFastBac vector. The DNA sequence of the insert is confirmed for both the strands. A
`
`bacmid prepared using pFastBac in E. coli DH1OBac is transfected into SF9 insect cells. The
`
`virus particles is then amplified to P1. P2. P3 stages. The P3 virus is infected into both Sf9
`
`and High Five insect cells and grown at 27°C for 48 hours. The medium is collected and used
`
`for assays and further purification.
`
`Preparation of fluorescent guenched substrate:
`
`A model peptidic TNF-a
`
`substrate (LY-LeucineAlanineGlutamineAlanineValine-
`
`ArginineSen‘ne-SerineLysine(CTMR)-Arginine
`
`(LY=Lucifer
`
`Yellow;
`
`CTMR=Carboxytetramethyl~Rhodamine)) is prepared and the concentration estimated by
`
`absorbance at 560 nm (E550. 60,000 M-1CM-1) according to the method of Geoghegan, KF,
`
`"Improved method for converting an unmodified peptide to an energy-transfer substrate for a
`
`proteinase." Bioconiugate Chem. 7, 385-391 (1995).
`
`This peptide encompasses the
`
`cleavage cite on pro-TNF which is cleaved in vivo by TACE.
`
`Expression of recombinant TACE
`
`A DNA fragment coding for the signal sequence, preprodomain, prodomain and
`
`catalytic domain of TACE (amino acids 1-473),
`
`is amplified by polymerase chain reaction
`
`using a human lung cDNA library as a template. The amplified fragment
`
`is cloned into
`
`pFastBac vector. The DNA sequence of the insert is confirmed for both the strands. A
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1401
`
`page 1401
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`

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`WO 99/52910
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`PCT/IB99/00503
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`-30-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`bacmid prepared using pFastBac in E. coli DH1OBac is transfected into SF9 insect cells. The
`
`virus particles were amplified to P1. P2, P3 stages. The P3 virus is infected into both Sf9 and
`
`High Five insect cells and grown at 27°C for 48 hours. The medium is collected and used for
`
`assays and further purification.
`
`Enzyme reaction
`
`The reaction, carried out in a 96 well plate (Dynatech),
`
`is comprised of 70 pl of Differ
`
`solution (25 mM Hepes-HCI, pH7.5, plus 20 uM ZnClz), 10 ul of 100 pM fluorescent quenched
`
`substrate, 10 ul of a DMSO (5%) solution of test compound. and an amount of r-TACE
`
`enzyme which will cause 50% cleavage in 60 minutes - in a total volume of 100 pl. The
`
`specificity of the enzyme cleavage at the amide bond between alanine and valine is verified
`
`by HPLC and mass spectrometry.
`
`Initial rates of cleavage are monitored by measuring the
`
`rate of increase in fluorescence at 530 nm (excitation at 409 nm) over 30 minutes. The
`
`experiment
`
`is controlled as follows: 1)
`
`for background fluorescence of substrate; 2) for
`
`fluorescence of fully cleaved substrate; 3) for fluorescence quenching or augmentation from
`
`solutions containing test compound.
`
`Data is analyzed as follows. The rates from the non-test compound containing
`
`“control" reactions were averaged to establish the 100% value. The rate of reaction in the
`
`presence of test compound was compared to that in the absence of compound, and tabulated
`
`as “percent of non-test compound containing control. The results are plotted as “% of control“
`
`vs. the log of compound concentration and a half-maximal point or leo value determined.
`
`Alt of the compounds of the invention have leo of less than 1 pM, preferably less than
`
`50nM. Most preferred compounds of the invention are at least 100 fold less potent against r-
`
`MMP-1 than in the above TACE assay.
`
`Human Monocfle Assay
`
`Human mononuclear cells are isolated from anti—coagulated human blood using a one-
`
`step Ficoll-hypaque separation technique.
`
`(2) The mononuclear cells are washed three times in
`
`Hanks balanced salt solution (HBSS) with divalent cations and resuspended to a density of 2 x
`
`106 /ml in HBSS containing 1% BSA. Differential counts determined using the Abbott Cell Dyn
`
`3500 analyzer indicated that monocytes ranged from 17 to 24% of the total cells in these
`
`preparations.
`
`180m of the cell suspension was aliquoted into flat bottom 96 well plates (Costar).
`
`Additions of compounds and LPS (100 ng/ml final concentration) gave a final volume of 200 pl.
`
`All conditions were performed in triplicate. After a four hour incubation at 37°C in an humidified
`
`C02 incubator, plates were removed and centrifuged (10 minutes at approximately 250 x g) and
`
`the supematants removed and assayed for TNF-o: using the R&D ELISA Kit.
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1402
`
`page 1402
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`WO 99/52910
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`PCT/IB99/00503
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`-31-
`
`Aggrecanase Assay
`
`Primary porcine chondrocytes from articular joint cartilage are isolated by sequential
`
`trypsin and collagenase digestion followed by collagenase digestion overnight and are plated
`
`at 2 x 105 cells per well into 48 well plates with 5 pCi I ml 358 (1000 Ci/mmol) sulphur in type
`
`l collagen coated plates. Cells are allowed to incorporate label into their proteoglycan matrix
`
`(approximately 1 week) at 37°C, under an atmosphere of 5% 002.
`
`The night before initiating the assay. chondrocyte monolayers are washed two times
`
`in DMEM/ 1% PSF/G and then allowed to incubate in fresh DMEM /1% FBS overnight.
`
`The following morning chondrocytes are washed once in DMEM/1%PSF/G. The final
`
`wash is allowed to sit on the plates in the incubator while making dilutions.
`
`Media and dilutions can be made as described in the Table below.
`
`Control Media
`
`DMEM alone (ctOn———————e—j
`
`
`
`
`
`;
`
`I
`V
`
`
`
`
`
`2
`
`
`IO
`
`15
`
`20
`
`25
`
`lL-1 Media
`
`Drug Dilutions
`
`
`
`
`
`
`
`
`
`
`
`DMEM + lL—1 (5 ng/ml)
`
`Make all compounds stocks at 10 mM in DMSO.
`
`Make a 100 uM stock of each compound in DMEM in 96 well plate.
`Store in freezer overnight.
`The next day perform serial dilutions in DMEM with lL-1 to 5 uM.
`above dilutions to 450 ul of lL-1 media in appropriate wells of the
`
`500 nM, and 50 nM.
`Aspirate final wash from wells and add 50 ul of compound from
`
`48 well plates.
`Final compound concentrations equal 500 nM. 50 nM. and 5 nM.
`
`i
`"
`I
`t
`
`I
`‘
`
`l
`
`All samples completed in triplicate with Control and lL-1 alone
`
`
`
`samples on each plate.
`
`.
`
`Plates are labeled and only the interior 24 wells of the plate are used. On one of the
`
`plates, several columns are designated as lL-1 (no drug) and Control (no lL—1, no drug).
`
`These control columns are periodically counted to monitor SSS-proteoglycan release. Control
`
`and IL-1 media are added to wells (450 ul) followed by compound (50 ul) so as to initiate the
`
`assay. Plates are incubated at 37°C, with a 5% CO2 atmosphere.
`
`At 40-50 °/o release (when CPM from lL-‘l media is 4-5 times control media) as
`
`assessed by liquid scintillation counting (LSC) of media samples, the assay is terminated (9-
`
`12 hours). Media is removed from all wells and placed in scintillation tubes. Scintillate is
`
`added and radioactive counts are acquired (LSC). To solubilize cell layers, 500 ul of papain
`
`digestion buffer (0.2 M Tris. pH 7.0, 5 mM EDTA, 5 mM DTT, and 1 mg/ml papain) is added to
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1403
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`page 1403
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`WO 99/52910
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`PCT/IB99/00503
`
`-32-
`
`each well. Plates with digestion solution are incubated at 60°C overnight The cell layer is
`
`removed from the plates the next day and placed in scintillation tubes. Scintillate is then
`
`added, and samples counted (LSC).
`
`The percent of released counts from the total present in each well
`
`is determined.
`
`Averages of the triplicates are made with control background subtracted from each well. The
`
`10
`
`percent of compound inhibition is based on IL-1 samples as 0% inhibition (100% of Fatal
`
`counts).
`
`For administration to mammals,
`
`including humans,
`
`for
`
`the inhibition of matrix
`
`metalloproteinases or the production of tumor necrosis factor (TNF), a variety of conventional
`
`routes may be used including
`
`oral. parenteral
`
`($1.,
`
`intravenous,
`
`intramuscular or
`
`15
`
`subcutaneous), buccal, anal and topical.
`
`In general, the active compound will be administered
`
`at dosages between about 0.1 and 25 mglkg body weight of the subject to be treated per day,
`
`preferably from about 0.3 to 5 mglkg. Preferably the active compound will be administered orally
`
`or parenterally. However. some variation in dosage will necessarily occur depending on the
`
`condition of the subject being treated. The person responsible for administration will,
`
`in any
`
`20
`
`event, determine the appropriate dose for the individual subject.
`
`The compounds of the present invention can be administered in a wide variety of
`
`different dosage forms, in general, the therapeutically effective compounds of this invention are
`
`present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by
`
`weight.
`
`25
`
`For oral administration, tablets containing various excipients such as microcrystalline
`
`cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed
`
`along with various disintegrants such as starch (and preferably corn, potato or tapioca starch),
`
`alginic
`
`acid
`
`and
`
`certain
`
`complex
`
`silicates,
`
`together with
`
`granulation
`
`binders
`
`like
`
`polyvinylpyrrolidone. sucrose, gelation and acacia. Additionally,
`
`lubricating agents such as
`
`magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes.
`
`Solid compositions of a similar type may also be employed as fillers in gelatin capsules;
`
`preferred materials in this connection also include lactose or milk sugar as well as high
`
`molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired
`
`for oral administration, the active ingredient may be combined with various sweetening or
`
`flavoring agents, coloring matter or dyes, and,
`
`if so desired, emulsifying and/or suspending
`
`agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and
`
`various like combinations thereof.
`
`In the case of animals, they are advantageously contained in
`
`an animal feed or drinking water in a concentration of 5-5000 ppm, preferably 25 to 500 ppm.
`
`For parenteral
`
`administration (intramuscular,
`
`intraperitoneal,
`
`subcutaneous
`
`and
`
`intravenous use) a sterile iniectable solution of the active ingredient
`
`is usually prepared.
`
`30
`
`35
`
`40
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1404
`
`page 1404
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`

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`WO 99/52910
`
`PCT/IB99/00503
`
`-33-
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`Solutions of a therapeutic compound of the present invention in either sesame or peanut oil or in
`
`aqueous propylene glycol may be employed. The aqueous solutions should be suitably adjusted
`
`and buffered, preferably at a pH of greater than 8,
`
`if necessary and the liquid diluent first
`
`rendered isotonic. These aqueous solutions are suitable intravenous injection purposes. The
`
`' oily solutions are suitable for intraarticular, intramuscular and subcutaneous injection purposes.
`
`The preparation of all these solutions under sterile conditions is readily accomplished by
`
`standard pharmaceutical techniques well known to those skilled in the art.
`
`In the case of
`
`animals, compounds can be administered intramuscularly or subcutaneously at dosage levels of
`
`about 0.1 to 50 mg/kg/day. advantageously 0.2 to 10 mg/kg/day given in a single dose or up to 3
`
`divided doses.
`
`For topical ocular administration. direct application to the affected eye may be
`
`employed in the form of a formulation as eyedrops, aerosol. gels or ointments, or can be
`
`incorporated into collagen (such as poly-2-hydroxyethylmethacrylate and co—polymers thereof).
`
`or a hydrophilic polymer shield. The materials can also be applied as a contact lens or via a
`
`local reservoir or as a subconjunctival formulation.
`
`For intraorbital administration a sterile injectable solution of the active ingredient is
`
`usually prepared. Solutions of a therapeutic compound of the present invention in an aqueous
`
`solution or suspension (particle size less than 10 micron) may be employed. The aqueous
`
`solutions should be suitably adjusted and buffered, preferably at a pH between 5 and 8,
`
`if
`
`necessary and the liquid diluent first rendered isotonic. Small amounts of polymers can be
`
`added to increase viscosity or for sustained release (such as cellulosic polymers. Dextran,
`
`polyethylene glycol. or alginic acid). These solutions are suitable for intraorbital
`
`injection
`
`purposes. The preparation of all these solutions under sterile conditions is readily accomplished
`
`by standard pharmaceutical techniques well known to those skilled in the art.
`
`In the case of
`
`animals, compounds can be administered intraorbitally at dosage levels of about 0.1 to 50
`
`mg/kg/day, advantageously 0.2 to 10 mg/kg/day given in a single dose or up to 3 divided doses.
`
`The active compounds of the invention may also be formulated in rectal compositions
`
`such as suppositories or retention enemas. 93., containing conventional suppository bases
`
`such as cocoa butter or other glycerides.
`
`For intranasal administration or administration by inhalation, the active compounds of
`
`the invention are conveniently delivered in the form of a solution or suspension from a pump
`
`spray container that
`
`is squeezed or pumped by the patient or as an aerosol spray
`
`presentation from a pressurized container or a nebulizer, with the use of a suitable propellant,
`
`_e_.g_.,
`
`dichlorodifluoromethane,
`
`trichlorofluoromethane,
`
`dichlorotetrafluoroethane,
`
`carbon
`
`dioxide or other suitable gas.
`
`In the case of a pressurized aerosol, the dosage unit may be
`
`40
`
`determined by providing a valve to deliver a metered amount. The pressurized container or
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1405
`
`page 1405
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`

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`WO 99/52910
`
`PCT/IB99/00503
`
`-34-
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`nebulizer may contain a solution or suspension of the active compound. Capsules and
`
`cartridges (made, for example,
`
`from gelatin) for use in an inhaler or insuffiator may be
`
`formulated containing a powder mix of a compound of the invention and a suitable powder
`
`base such as lactose or starch.
`
`The following Preparations and Examples illustrate the preparation of the compounds
`
`of the present invention. Melting points are uncorrected. NMR data are reported in parts‘per
`
`million (8) and are referenced to the deuterium lock signal
`
`from the sample solvent
`
`(deuteriochloroform unless otherwise specified). Commercial reagents were utilized without
`
`further purification. THF refers to tetrahydrofuran. DMF refers to N,N-dimethylformamide.
`
`Chromatography refers to column chromatography performed using 3263 mm silica gel and
`
`executed under nitrogen pressure (flash chromatography) conditions. Room or ambient
`
`temperature refers to 20—25°C.
`
`All non-aqueous reactions were run under a nitrogen
`
`atmosphere for convenience and to maximize yields. Concentration at reduced pressure
`
`means that a rotary evaporator was used.
`
`Preparation 1:
`4- 4-Fluoro heno
`thio henol
`
`Lithium aluminum hydride (9.95 grams, 0.26 mole) was added in portions to a stirred solution
`
`of 4-(4—fluorophenoxy)benzenesulfonylchloride (30 grams, 0.105 mole) in tetrahydrofuran (700
`
`mL). The resulting mixture was heated at reflux for 1.5 hours, cooled in an ice bath and
`
`quenched by addition of 10% aqueous sulfuric acid solution (100 mL). After stirring for 30
`
`minutes, the mixture was filtered through CeliteTM and the tetrahydrofuran was removed
`
`under vacuum. The residue was diluted with water and extracted with diethyl ether. The
`
`organic layer was washed with water and brine, dried over magnesium sulfate and
`
`concentrated under vacuum to provide the title compound as a white solid (23 grams, 100%).
`
`Preparation 2
`
`4'-Fluorobi9henyl-4-thiol
`
`Lithium aluminum hydride (0.95 grams, 25 mmole) was added in portions to a stirred
`
`solution of 4'-fluorobiphenyl-4-sulfonylchloride (2.7 grams, 10 mmole) in tetrahydrofuran (75
`
`mL). The resulting mixture was heated at reflux for 4 hours, cooled in an ice bath and
`
`quenched by addition of 10% aqueous sulfuric acid solution (100 mL). After stirring for 30
`
`minutes, the mixture was filtered through CeliteTM and the tetrahydrofuran was removed
`
`under vacuum. The residue was diluted with water and extracted with diethyl ether. The
`
`organic layer was washed with water and brine, dried over magnesium sulfate and
`
`concentrated under vacuum to a solid. Trituration of the solid with diethyl ether, removal of
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1406
`
`page 1406
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-35-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`insoluble material by filtration and concentration of the filtrate provided the title compound as a
`
`yellow solid (1.4 grams, 69%).
`
`Preparation 3
`4- 4-Chloro henox thio henol
`
`Lithium aluminum hydride (6.5 grams, 0.17 mote) was added in portions, maintaining
`
`gentle reflux,
`
`to a stirred solution of 4-(4-chIorophenoxy)benzenesulfonyl-chloride (20.5
`
`grams, 68 mmole) in tetrahydrofuran (400 mL). The resulting mixture was stirred at room
`
`temperature for 2 hours, cooled in an ice bath and quenched by addition of 10% aqueous
`
`sulfuric acid solution (100 mL). After stirring for 30 minutes, the mixture was diluted with
`
`water and extracted with diethyl ether. The organic layer was washed with water and brine,
`
`dried over magnesium sulfate and concentrated under vacuum to provide the title compound
`
`as a white solid (15.9 grams, 99%).
`
`Example 1
`
`3-EXO-[4-(4-FLUOROPHENOXY)BENZENESULFONYLAMIN01-8-OXA—
`
`BICYCLO[3.2.1]-OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`
`A)
`
`3-(Benzhydrylideneaminol-s-oxabicyclopz.1loctane-3-carboxylic
`
`acid
`
`ethyl ester
`
`To a
`
`suspension of
`
`sodium hydride
`
`(0.41
`
`grams,
`
`17.1 mmole)
`
`in N,N-
`
`dimethylformamide (50 mL) at 0°C was added dropwise a solution of N-diphenylmethylene
`
`glycine ethyl ester (2.07 grams, 7.8 mmole) in N,N-dimethylformamide (50 mL). After stirring
`
`for 30 minutes at room temperature, a solution of cis-2,5-bis(hydroxymethyI)-tetrahydrofuran
`
`ditosylate (4.1 grams, 9.3 mmole) in N,N—dimethylformamide (50 mL) was added dropwise.
`
`The reaction mixture was gradually heated to 100°C in an oil bath and stirred at this
`
`temperature overnight. The solvent was evaporated under vacuum and the residue was taken
`
`up in water and extracted twice with diethyl ether. The combined organic extracts were
`
`washed with brine, dried over magnesium sulfate and concentrated to a brown oil, from which
`
`the title compound (1.42 grams, 51%, a 3:1 mixture of exo/endo diastereomers) was isolated
`
`by chromatography on silica gel (20% ethyl acetate in hexane as eluant).
`
`B)
`
`3-Amino-8-oxabic clo 3.2.1 octane-S—carbox lic
`
`acid
`
`eth I
`
`ester
`
`hydrochloride
`
`A two—phase mixture
`
`of
`
`3-(benzhydrylideneamino)—8-oxabicyclo[3.2.1]octane-3-
`
`carboxylic acid ethyl ester (1.4 grams, 3.9 mmole) in aqueous 1N hydrochloric acid solution
`(100 mL) and diethyl ether (100 mL) was stirred at room temperature overnight. The aqueous
`
`layer was concentrated to provide the title compound (0.70 grams, 78%, a 3:1 mixture of
`
`exo/endo diastereomers) as a pale yellow solid.
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1407
`
`page 1407
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-36-
`
`C)
`
`3-exo-[4-(4-Fluorophenoxy)benzenesulfonylaminol-8-
`
`oxabicyclol3.2.1[octane-3-carboxylic acid ethyl ester
`
`A solution
`
`of 3-amino-8-oxabicyclo[3.2.1]octane-3-carboxylic
`
`acid
`
`ethyl
`
`ester
`
`hydrochloride (690 mg, 2.9 mmole), 4-(4-fluorophenoxy)benzenesulfonylchloride (923 mg, 3.2
`
`mmole) and triethylamine (0.9 mL, 6.5 mmole) in N,N-dimethylformamide (45 mL) was stirred
`
`at room temperature overnight. The solvent was removed under vacuum and the residue was
`
`taken up in saturated aqueous sodium bicarbonate solution. After extracting twice with
`
`methylene chloride,
`
`the combined organic layers were washed with brine, dried over
`
`magnesium sulfate and concentrated to a brown oil. The title compound (492 mg, 38%) was
`
`isolated by chromatography on silica using 1% methanol in methylene chloride as eluant.
`
`D)
`
`3-exo- 4- 4-Fluoro heno
`
`benzenesulfon Iamino -8-
`
`oxabicyclo[3.2.11octane-3—carboxylic acid
`
`Sodium hydroxide (1.5 grams, 38 mmole) was added to a solution of 3-exo-[4—(4-
`
`fluorophenoxy)benzenesulfonylamino]~8—oxabicyclo[3.2.1]octane-3-carboxylic acid ethyl ester
`
`(492 mg, 1.09 mmole) in a mixture of ethanol (10 mL) and water (10 mL). The mixture was
`
`heated at reflux for 6 days, cooled and acidified with aqueous 1N hydrochloric acid solution.
`
`The mixture was extracted with ethyl acetate and the organic layer was washed with brine,
`
`dried over magnesium sulfate and concentrated to provide the title compound (411 mg, 89%)
`
`as a tan foam.
`
`E)
`
`3-exo- 4- 4-Fluoro heno
`
`benzenesulfon lamino -8-
`
`oxabicyclo[3.2.1]octane-3-carboxyiic acid bengyloxyamide
`
`To a
`
`solution of 3—exo-[4-(4-fluorophenoxy)benzenesulfony|amino]-8—oxabicyclo-
`
`[3.2.1]octane-3—carboxylic acid (411 mg, 0.98 mmole) and triethylamine (0.19 mL, 1.36
`
`mmole)
`
`in N,N-dimethylformamide
`
`(30 mL) was
`
`added
`
`(benzotriazol—1-yloxy)tris-
`
`(dimethylamino)phoshonium hexafluoroborate (474 mg, 1.07 mmole). After stirring at room
`
`temperature
`
`for
`
`1
`
`hour,
`
`additional
`
`triethylamine
`
`(0.22 mL,
`
`1.58 mmole)
`
`and O-
`
`benzylhydroxylamine hydrochloride (187 mg, 1.17 mmole) were added. The reaction mixture
`
`was stirred for 1 day at room temperature and then for 1 day at 50°C. After concentration
`
`under vacuum,
`
`the residue was dissolved in ethyl acetate and washed sequentially with
`
`aqueous 1N hydrochloric acid solution, saturated aqueous sodium bicarbonate solution, and
`
`brine. The solution was dried over magnesium sulfate and concentrated to an oil from which
`
`the title compound, a white solid (237 mg, 46%) was isolated by chromatography (50% ethyl
`
`acetate in hexane as eluant).
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
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`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1408
`
`page 1408
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`

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`WO 99/52910
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`PCT/IB99/00503
`
`-37-
`
`F)
`
`3—exo-[4j4-Fluoroghenoxy)benzenesulfonylamino]-8-oxa-
`
`bicyclo|3.2.1]octane-3-carboxylic acid hydrogamide
`
`A
`
`solution
`
`of
`
`3-exo-{4-(4-fluorophenoxy)benzenesulfonylamino]-8—oxabicyclo-
`
`[3.2.1]octane-3-carboxylic acid benzyloxyamide (237 mg, 0.45 mmole) in methanol (25 mL)
`
`was treated with 5% palladium on barium sulfate (150 mg) and hydrogenated at 3
`
`10
`
`atmospheres pressure for 4 hours in a Parr TM shaker. The catalyst was removed by
`
`passage through a 0.45 pm nylon filter and the filtrate was concentrated to a white foam.
`
`Crystallization from methylene chloride provided the title compound as a white solid (62 mg,
`
`A second crop (62 mg, 32%) was obtained by crystallization from ethyl
`32%).
`acetate/hexane.
`
`15
`
`20
`
`25
`
`30
`
`M.p. 138-141°C. 1H NMR (dB-DMSO): 6 10.50 (br s, 1 H), 8.56 (br s, 1 H), 7.67 (d, J
`
`= 8.7 Hz, 2 H), 7.66 (br s, 1 H, overlapped), 7.26-7.22 (m, 2 H), 7.16—7.12 (m, 2 H), 7.01 (d, J
`
`= 8.5 Hz, 2 H), 4.09 (br s, 2 H), 2.32 (d, J = 14.1 Hz, 2 H), 1.68-1.63 (m, 4 H), 1.51-1.48 (m, 2
`
`H). MS: 435 We (M—H). Further confirmation of structure and stereochemistry was carried
`
`out by single crystal X‘ray crystallography.
`
`Example 2
`
`3-EXO-[4j4-FLUOROPHENOXY)BENZENESULFONYLMETHYL1-8-
`
`OXABICYCLO-[3.2.1]-OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`
`A)
`
`8-Oxabicyclo[3.2.1]octane-3,3-dicarbo§ylic acid diethyl ester
`
`Sodium hydride (2.28 grams, 95 mmole) was added in portions to a stirred solution of
`
`diethyl malonate (15 mL, 99 mmole) in N,N-dimethylformamide (400 mL). The mixture was
`
`stirred for 45 minutes at which time evolution of hydrogen was complete. A solution of cis-2,5-
`
`bis(hydroxymethyl)tetrahydrofuran
`
`ditosylate
`
`(19.0
`
`grams,
`
`43 mmole)
`
`in
`
`N,N-
`
`dimethylformamide (400 mL) was then added dropwise. The mixture was heated in an oil
`
`bath at 140°C overnight. After cooling to room temperature, the mixture was quenched by
`
`addition of saturated aqueous ammonium chloride solution and concentrated under vacuum.
`
`The residual oil was taken up in water and extracted with diethyl ether. The organic extract
`
`was washed with water and brine, dried over magnesium sulfate and concentrated to an oil.
`
`Distillation under vacuum afforded the title compound (7.8 grams, 71%) as a clear oil.
`
`B)
`
`3-exo-Hydroxymethyl-8-oxabicyclo[3.2.1]octane-3—carbo:_(ylic acid ethyl
`
`35
`
`ester
`
`A 1.2 M solution of diisobutylaluminum hydride in toluene (62.5 mL, 75 mmole) was
`
`added dropwise to a solution of 8-oxabicyclo[3.2.1]octane—3,3-dicarboxylic acid diethyl ester
`
`(7.8 grams, 30 mmole) in toluene (80 mL) at 40°C. The mixture was allowed to warm to 0°C
`
`while stirring for a period of 3 hours.
`
`It was then cooled to -15°C and ethanol (8 mL) was
`
`AQUESTIVE EXHIBIT 1004
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`AQUESTIVE EXHIBIT 1004 page 1409
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`W0 99/5291 0
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`PCT/IB99/00503
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`-33-
`
`added slowly while maintaining this temperature. After stirring at ~15°C for 1 hour, sodium
`
`borohydride (1.1 grams. 30 mmole) was added. The mixture was stirred at room temperature
`
`overnight and was quenched by dropwise addition of saturated aqueous sodium sulfate
`
`solution. Ethyl acetate was added and, after stirring for 20 minutes. the insoluble material was
`
`removed by filtration through CeliteTM. The filtrate was washed with brine. dried over
`
`magnesium sulfate and concentrated to afford the title compound (5.1 grams, 80%) as a cEar
`oil.
`
`C)
`
`3-exo-H dro meth l-8-oxabic clo 3.2.1 octane-S-carbo
`
`Iic acid
`
`Lithium hydroxide hydrate (2.5 grams, 59.5 mmole) was added to a solution of 3-exo-
`
`hydroxymethyl-8—oxabicyclo[3.2.1]octane-3-carboxy|ic acid ethyl ester
`
`(5.1 grams, 23.8
`
`mmole) in a mixture of methanol (25 mL), tetrahydrofuran (25 mL) and water (2.5 mL). The
`
`mixture was heated at reflux overnight. cooled and quenched by addition of Amberlite iR-
`
`120TM ion exchange resin. After stirring for 20 minutes, the resin was removed by filtration,
`
`washing with tetrahydrofuran. Evaporation of the solvents and trituration of the residue with
`
`diethyl ether afforded the title compound (2.35 grams, 53%) as a white solid.
`
`D)
`
`3',8-Dioxas iro bic clo 3.2.1 octane-3,1'-c clobutane -2'-one
`
`
`
`
`Benzenesulfonylchloride (1.7 mL, 13.5 mmole) was added dropwise to a solution of 3-
`
`exo-hydroxymethyl-B-oxabicyclo[3.2.1]octane-3-carboxylic acid (2.3 grams, 12.3 mmole),
`
`triethylamine (3.4 mL, 24.7 mmole) and 4-dimethylaminopyridine (300 mg, 2.5 mmole) in
`
`methylene chloride (50 mL) at 0°C. The mixture was stirred at 0°C for 1 hour, diluted with
`
`methylene chloride and washed with aqueous 1N hydrochloric acid solution. saturated
`
`aqueous sodium bicarbonate solution and brine. After drying over magnesium sulfate, the
`
`solvent was evaporated to provide the title compound as a white solid (1.8 grams, 90%).
`
`E) 3-exo-4 4-Fluoro heno
`
`hen lsulfan lmeth l-8—oxabic clo 3.2.1 octane-
`
`3-carboglic acid
`
`A solution of 4-(4-fiuorophenoxy)thiophenol (2.2 grams, 1O mmole) in tetrahydrofuran
`
`(10 mL) was added dropwise to a slurry of sodium hydride (270 mg, 11.3 mmole)
`
`in
`
`tetrahydrofuran (20 mL) at -10°C. The mixture was allowed to warm to room temperature
`
`while
`
`stirring for 30 minutes.
`
`After cooling again to -10°C.
`
`a solution of 3',8-
`
`dioxaspiro[bicyclo[3.2.1]octane-3,1'cyclobutane]-2'-one
`
`(1.8
`
`grams,
`
`10 mmole)
`
`in
`
`tetrahydrofuran (20 mL) was added dropwise. The cooling bath was removed and stirring was
`
`continued at room temperature for 2 hours after which the mixture was quenched with
`
`aqueous 1N hydrochloric acid solution and extracted twice with methylene chloride. The
`
`combined organic extracts were washed with water and brine. dried over magnesium sulfate
`
`and concentrated to a solid. Recrystallization from diethyl ether/hexane afforded the title
`
`10
`
`15
`
`20
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`25
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`30
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`35
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`AQUESTIVE EXHIBIT 1004
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`AQUESTIVE EXHIBIT 1004 page 1410
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`WO 99/52910
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`PCT/IB99/00503
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`-39-
`
`IO
`
`15
`
`20
`
`25
`
`3O
`
`35
`
`compound (1.8 grams (47%) as a white solid. Concentration of the mother liquor followed by
`
`chromatography on silica gel (2% methanol in chloroform as eluant) gave more of the the title
`
`compound (500 mg. 13%).
`
`F) 3-exo-[4-(4-Fluoroghenoxymhenylsulfanylmethyl]-8-oxabicyclo[3.2.1]octane-
`
`3-carboxylic acid bengloxyamide
`
`To a solution of 3-exo-[4-(4-fluorophenoxy)benzenesulfanylmethyl]-8-oxabicyclo—
`
`[3.2.1]octane-3-carboxylic acid (1.0 grams, 2.6 mmole) and diisopropylethylamine (0.5 mL. 2.9
`
`mmole)
`
`in N,N-dimethylformamide
`
`(20 mL) was
`
`added
`
`(benzotriazol-1-y|oxy)tris-
`
`(dimethylamino)phoshonium hexafluoroborate (1.2 grams. 2.7 mmole). After stirring at room
`
`temperature for 2.5 hours, additional diisopropylethylamine (0.86 mL, 4.9 mmole) and O-
`
`benzylhydroxylamine hydrochloride (525 mg, 3.3 mmole) were added. The reaction mixture
`
`was stirred for 16 hours at 50°C. After concentration under vacuum,
`
`the residue was
`
`dissolved in ethyl acetate and washed sequentially with aqueous 1N hydrochloric acid
`
`solution, saturated aqueous sodium bicarbonate solution, and brine. The solution was dried
`
`over magnesium sulfate and concentrated to an oil from which the title compound, a white
`
`foam (405 mg, 32%) was isolated by chromatography (30% ethyl acetate in hexane as
`
`eluant).
`
`G) 3-exo— 4- 4-Fluoro heno
`
`hen lsulfon lmeth l-8-oxabic clo 3.2.1 octane-
`
`3—carboxylic acid benzyloxyamide
`
`Solid 57-85% meta-chloroperbenzoic acid (283 mg) was added to a solution of 3-exo-
`
`[4-(4-fluorophenoxy)phenylsulfanyImethyl]-8—oxabicyclo[3.2.1]octane-3-carboxylic
`
`acid
`
`benzyloxyamide in methylene chloride (15 mL). The resulting mixture was stirred at room
`
`temperature overnight. and was then quenched by addition of saturated aqueous sodium
`
`bisulfite solution. After dilution with methylene chloride, the organic layer was separated and
`
`washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic
`
`layer was dried over magnesium sulfate and concentrated to give the title compound as a
`
`white foam (390 mg, 90%).
`
`H)
`
`3-exo- 4- 4-Fluoro heno
`
`benzenesulfon lmeth l.8-oxabic clo- 3.2.1 -
`
`octane-3-carboxylic acid hydroxyamide
`
`A
`
`solution
`
`of
`
`3~exo~[4-(4-fluorophenoxy)benzenesulfonylmethyI]-8-oxabicyclo-
`
`[3.2.1]octane-3-carboxylic acid benzyloxyamide (390 mg, 0.74 mmole) in methanol (20 mL)
`
`was treated with 5% palladium on barium sulfate (195 mg) and hydrogenated at 3
`
`atmospheres pressure for 3.5 hours in a Parr TM shaker. The catalyst was removed by
`
`passage through a 0.45 pm nylon filter and the filtrate was concentrated to a white foam.
`
`Crystallization from a mixture of ethyl acetate and hexane provided the title compound as a
`
`40
`
`white solid (230 mg, 71%).
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1411
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`

`WO 99/52910
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`PCT/IB99/00503
`
`.40-
`
`Mp. 134-139°C. 1H NMR (de-DMSO): 6 8.55 (br s. 1 H), 7.76 (d, J = 7.5 Hz, 2 H).
`
`7.30-7.26 (m, 2 H), 7.20-7.16 (m. 2 H), 7.09 (d, J = 7.5 Hz. 2 H). 4.13 (br s, 2 H), 3.40 (s, 2 H),
`2.24 (d, J = 14.3 Hz. 2 H), 1.78-1.73 (m. 4 H), 1.57-1.55 (m, 2 H). MS m/e 434 (M—H).
`Further confirmation of structure and stereochemistry was carried out by single crystal X-ray
`crystallography.
`
`Example 3
`
`3-(4-PHENOXYBENZENESULFONYLMETHYL[-8-OXABICYCLOI3.2.1[OCTANE-3-
`CARBOXYLIC ACID HYDROXYAMIDE
`
`to
`according
`Prepared
`phenoxyphenylthiophenol in step E.
`
`the
`
`same
`
`procedure
`
`as
`
`Example
`
`2,
`
`using
`
`4-
`
`1H NMR (de-DMSO): 5 8.54 (br s, 1 H), 7.75 (d, J = 8.9 Hz. 2 H), 7.44-7.40 (m, 2 H).
`7.23 7.21 (m. 1 H), 7.11-7.07 (m. 4 H), 4.11 (br s. 2 H). 3.38 (s. 2 H), 2.22 (d, J = 14.3 Hz. 2
`H), 1.80-1.70 (m, 4 H), 1.60—1.50 (m, 2 H). MS m/e 416 (M—H).
`
`10
`
`15
`
`Example 4
`
`20
`
`3-EXOj4‘-FLUOROBlPHENYL-46ULFONYLMETHYL)-8-OXAB|CYCLO|3.2.1l-
`OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`
`Prepared according to the same procedure as Example 2 using 4'-fluorobiphenyl-4-
`thiol