WO 99/52910
`
`PCT/IB99/00503
`
`-29-
`
`x 10° /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 nl 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 ul.
`
`10
`
`All conditions were performedin triplicate. After a four hour incubation at 37°C in an humidified
`
`CO, incubator, plates were removed and centrifuged (10 minutes at approximately 250 x g) and
`
`the supernatants removed and assayed for TNFa using the R&D ELISAKit.
`
`Inhibition of Soluble TNF-a Production
`
`The ability of the compounds or the pharmaceutically acceptable salts thereof to inhibit
`
`I5
`
`the cellular release of TNF-a and, consequently, demonstrate their effectiveness for treating
`
`diseasesinvolving the disregulation of soluble TNF-a is shown bythe following in vitro assay:
`
`Methodfor the evaluation of recombinant TNF-a Converting EnzymeActivity
`Expression of recombinant TACE
`
`A DNA fragment coding for the signal sequence, preprodomain, prodomain and
`
`20
`
`catalytic domain of TACE (amino acids 1-473), can be amplified by polymerase chain reaction
`
`using a human lung cDNAlibrary 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 DH10Bacis transfected into SF9 insect cells. The
`
`virus particles is then amplified to P1, P2, P3 stages. The P3 virus is infected into both Sf9
`
`25
`
`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 quenched substrate:
`
`A model peptidic TNF-a
`
`substrate (LY-LeucineAlanineGlutamineAlanineValine-
`
`ArginineSerine-SerineLysine(CTMR)-Arginine
`
`(LY=Lucifer
`
`Yellow;
`
`30
`
`CTMR=Carboxytetramethyl-Rhodamine)) is prepared and the concentration estimated by
`
`absorbance at 560 nm (E560, 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." Bioconjugate Chem. 7, 385-391 (1995).
`
`This peptide encompasses the
`
`cleavage cite on pro-TNF which is cleaved in vivo by TACE.
`
`35
`
`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
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1401
`
`page 1401
`
`

`

`WO 99/52910
`
`PCT/1IB99/00503
`
`-30-
`
`bacmid prepared using pFastBacin E. coli DH10Bac 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 furtherpurification.
`
`Enzymereaction
`The reaction, carried out in a 96 well plate (Dynatech),
`
`is comprised of 70 ul of buffer
`
`solution (25 mM Hepes-HCl, pH7.5, plus 20 uM ZnCl,), 10 pl of 100 yM fluorescent quenched
`
`substrate, 10 pl 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
`
`Initial rates of cleavage are monitored by measuring the
`by HPLC and mass spectrometry.
`rate of increase in fluorescence at 530 nm (excitation at 409 nm) over 30 minutes. The
`
`for background fluorescence of substrate; 2) for
`is controlled as follows: 1)
`experiment
`fluorescenceof 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% vaiue. The rate of reaction in the
`
`presence of test compound was comparedto 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 ICs, value determined.
`
`All of the compounds of the invention have ICgo of less than 1 uM, preferably less than
`
`50nM. Mostpreferred compounds of the invention are at least 100 fold less potent againstr-
`
`MMP-1 than in the above TACE assay.
`
`Human Monocyte Assay
`
`10
`
`15
`
`20
`
`25
`
`Human mononuclearcelis are isolated from anti-coagulated human blood using a one-
`
`30
`
`step Ficoll-hypaque separation technique.
`
`(2) The mononuclearcells are washed three timesin
`
`Hanks balanced salt solution (HBSS) with divalent cations and resuspended to a density of 2 x
`10° /mi 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.
`
`35
`
`180m of the ceil 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 ul.
`
`All conditions were performedin triplicate. After a four hour incubation at 37°C in an humidified
`
`CO,incubator, plates were removed and centrifuged (10 minutes at approximately 250 x g) and
`the supernatants removed and assayed for TNF-a using the R&D ELISAKit.
`
`AQUESTIVE EXHIBIT 1004=page 1402
`
`AQUESTIVE EXHIBIT 1004 page 1402
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`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`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 10°cells per well into 48 well plates with 5 pCi / ml 35S (1000 Ci/mmol) sulphurin type
`| collagen coated plates. Cells are allowed to incorporate label into their proteoglycan matrix
`(approximately 1 week) at 37°C, under an atmosphere of 5% CQ).
`~
`The night beforeinitiating the assay, chondrocyte monolayers are washed two times
`
`in DMEM/ 1% PSF/G and then allowed to incubate in fresh DMEM /1% FBSovernight.
`
`The following morning chondrocytes are washed once in DMEM/1%PSF/G. The final
`
`washis allowed to sit on the plates in the incubator while making dilutions.
`
`Media and dilutions can be made as describedin the Table below.
`——orereev<GV<3V—SLLLL—g]37—ooooooe—————S—
`DMEM alone (control media)
`[
`
`Control Media
`
`
`
`10
`
`15
`
`
`
`
`
`:
`
`|
`
`|
`|
`
`IL-1 Media
`
`Drug Dilutions
`
`
`
`
`
`
`
`
`DMEM+ IL-1 (5 ng/ml)
`
`Makeall compoundsstocks at 10 mM in DMSO.
`
`Make a 100 uM stock of each compound in DMEMin 96 well plate.
`
`
`
`
`
`
`Store in freezer overnight.
`
`The next day perform serial dilutions in DMEM with IL-1 to 5 uM,
`;
`500 nM,and 50 nM.
`:
`Aspirate final wash from wells and add 50 ul of compound from |
`
`
`above dilutions to 450 ul of IL-1 media in appropriate wells of the
`48 well plates.
`
`Final compound concentrations equal 500 nM, 50 nM, and 5 nM.
`All samples completed in triplicate with Control and IL-1 alone
`
`samples on eachplate.
`
`Plates are labeled and only the interior 24 weils of the plate are used. On one of the
`
`plates, several columns are designated as !L-1 (no drug) and Control (no IL-1, no drug).
`These control columns are periodically counted to monitor 35S-proteoglycan release. Control
`
`20
`
`and IL-1 media are added to wells (450 ul) followed by compound (50 ul) so asto initiate the
`
`assay. Plates are incubated at 37°C, with a 5% CO, atmosphere.
`
`At 40-50 % release (when CPM from IL-1 media is 4-5 times control media) as
`
`assessedby 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
`
`25
`
`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/m! papain) is added to
`
`AQUESTIVE EXHIBIT 1004=page 1403
`
`AQUESTIVE EXHIBIT 1004 page 1403
`
`

`

`WO 99/52910
`
`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).
`
`10
`
`15
`
`is determined.
`The percent of released counts from the total present in each well
`Averagesofthe triplicates are made with control background subtracted from each well. The
`percent of compound inhibition is based on IL-1 samples as 0% inhibition (100% of total
`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
`
`intramuscular or
`intravenous,
`(e.g.,
`oral, parenteral
`routes may be used including
`subcutaneous), buccal, anal and topical.
`In general, the active compoundwill be administered
`at dosages between about 0.1 and 25 mg/kg body weight of the subject to be treated per day,
`preferably from about 0.3 to 5 mg/kg. Preferably the active compoundwill 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 compoundsof this invention are
`present in such dosage forms at concentration levels ranging from about 5.0% to about 70% by
`
`weight.
`
`25
`
`30
`
`35
`
`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 laury!sulfate and talc are often very useful for tabletting purposes.
`Solid compositions of a similar type may also be empioyed asfillers 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 containedin
`
`an animal feed or drinking water in a concentration of 5-5000 ppm, preferably 25 to 500 ppm.
`and
`For parenteral
`administration (intramuscular,
`intraperitoneal,
`subcutaneous
`intravenous use) a sterile injectable solution of the active ingredient
`is usually prepared.
`
`40
`
`AQUESTIVE EXHIBIT 1004=page 1404
`
`AQUESTIVE EXHIBIT 1004 page 1404
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-33-
`
`Solutions of a therapeutic compound of the present invention in either sesame or peanutoil or in
`aqueous propylene glycol may be employed. The aqueoussolutions 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 subcutaneousinjection purposes.
`The preparation of all these solutions under sterile conditions is readity accomplished by
`
`standard pharmaceutical techniques weli Known to those skilled in the art.
`
`In the case of
`
`animals, compounds can be administered intramuscularly or subcutaneously at dosagelevels 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-hydroxyethyimethacrylate and co-polymers thereof),
`
`or a hydrophilic polymer shield. The materials can also be applied as a contact lens or via a
`
`jocal reservoir or as a Subconjunctival formulation.
`
`10
`
`15
`
`20
`
`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
`
`25
`
`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 understerile 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
`
`30
`
`mg/kg/day, advantageously 0.2 to 10 mg/kg/day givenin a single dose or up to 3 divided doses.
`
`The active compoundsof the invention may also be formulated in rectal compositions
`
`such as suppositories or retention enemas, e.g., 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,
`
`trichiorofluoromethane,
`
`dichlorotetrafluorcethane,
`
`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=page 1405
`
`AQUESTIVE EXHIBIT 1004 page 1405
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-34-
`
`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 insufflator may be
`
`formulated containing a powder mix of a compound of the invention and a suitable powder
`
`base such as lactose or starch.
`
`The foliowing Preparations and Examplesillustrate the preparation of the compounds
`of the present invention. Melting points are uncorrected. NMR data are reported in partsper
`
`10
`
`million (5) 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 32-63 mm silica gel and
`
`15
`
`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.
`
`20
`
`25
`
`30
`
`35
`
`Preparation 1:
`4-(4-Fluorophenoxy)thiophenol
`
`Lithium aluminum hydride (9.95 grams, 0.26 mole) was added in portionsto 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 Celite™M 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 thetitle compound as a white solid (23 grams, 100%).
`
`Preparation 2
`
`4'-Fluorobipheny!-4-thiol
`
`Lithium aluminum hydride (0.95 grams, 25 mmole) was added in portions to a stirred
`
`solution of 4'-fluorobipheny!-4-sulfonyltchloride (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 Celite™M 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=page 1406
`
`AQUESTIVE EXHIBIT 1004 page 1406
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-35-
`
`insoluble material byfiltration and concentration ofthe filtrate provided thetitle compound as a
`yellow solid (1.4 grams, 69%).
`
`Preparation 3
`4-(4-Chlorophenoxy)thiophenol
`
`10
`
`20
`
`25
`
`Lithium aluminum hydride (6.5 grams, 0.17 mote) was addedin portions, maintaining
`gentle reflux,
`to a stirred solution of 4-(4-chlorophenoxy)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 thetitle compound
`as a white solid (15.9 grams, 99%).
`
`Example 1
`3-EX0-[4-(4-FLUOROPHENOXY)BENZENESULFONYLAMINO]-8-OXA-
`BICYCL.Of3.2.1]-OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`A)
`3-(Benzhydrylideneamino)-8-oxabicyclo[3.2.1Joctane-3-carboxylic acid
`ethyl! ester
`in N,N-
`17.1. mmole)
`grams,
`(0.41
`sodium hydride
`suspension of
`To a
`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(hydroxymethyl)-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 andthe 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 brownoil, from which
`
`30
`
`the title compound (1.42 grams, 51%, a 3:1 mixture of exo/endo diastereomers) was isolated
`
`by chromatography onsilica gel (20% ethyl acetate in hexaneas eluant).
`
`B)
`
`3-Amino-8-oxabicyclo[3.2.1Joctane-3-carboxylic
`
`acid
`
`_ethy!
`
`ester
`
`hydrochloride
`
`35
`
`of 3-(benzhydrylideneamino)-8-oxabicyclo[3.2.1]octane-3-
`A two-phase mixture
`carboxylic acid ethy! ester (1.4 grams, 3.9 mmole) in aqueous 1N hydrochioric acid solution
`(100 mL) and diethyl ether (100 mL) wasstirred 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=page 1407
`
`AQUESTIVE EXHIBIT 1004 page 1407
`
`

`

`WO 99/52910
`
`PCT/1IB99/00503
`
`-36-
`
`C)
`
`3-exo-[4-(4-Fluorophenoxy)benzenesulfonylamino]-8-
`
`oxabicyclo[3.2.1Joctane-3-carboxylic acid ethyl ester
`A solution
`of 3-amino-8-oxabicyclo[3.2.1Joctane-3-carboxylic
`acid
`ethyl
`ester
`hydrochloride (690 mg, 2.9 mmole), 4-(4-fluorophenoxy)benzenesulfonyichloride (923 mg, 3.2
`mmole) and triethylamine (0.9 mL, 6.5 mmole) in N,N-dimethylformamide (45 mL) wasstirred
`at room temperature overnight. The solvent was removed under vacuum andthe residue was
`taken up in saturated aqueous sodium bicarbonate solution. After extracting twice with
`
`10
`
`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 onsilica using 1% methanol in methylene chloride as eluant.
`
`15
`
`D)
`
`3-exo-[4-(4-Fluorophenoxy)benzenesulfonylamino]-8-
`
`oxabicyclo[3.2.1joctane-3-carboxylic acid
`Sodium hydroxide (1.5 grams, 38 mmole) was added to a solution of 3-exo-[4-(4-
`
`fluorophenoxy)benzenesulfonyiaminoj-8-oxabicyclo[3.2. 1Joctane-3-carboxylic acid ethy! ester
`
`(492 mg, 1.09 mmole) in a mixture of ethanol (10 mL) and water (10 mL). The mixture was
`
`20
`
`heated at reflux for 6 days, cooled and acidified with aqueous 1N hydrochloric acid solution.
`
`The mixture was extracted with ethy! 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-Fluorophenoxy)benzenesulfonylamino]-8-
`
`25
`
`oxabicyclo[3.2.1joctane-3-carboxylic acid benzyloxyamide
`To a
`solution of 3-exo-[4-(4-fluorophenoxy)benzenesulfonylamino)-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 ethy! 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
`
`30
`
`35
`
`acetate in hexane as eluant).
`
`AQUESTIVE EXHIBIT 1004=page 1408
`
`AQUESTIVE EXHIBIT 1004 page 1408
`
`

`

`WO 99/52910
`
`PCT/1B99/00503
`
`-37-
`
`3-exo-[4-(4-Fluorophenoxy)benzenesulfonylamino]-8-oxa-
`F)
`bicyclo[3.2.1]octane-3-carboxylic acid hydroxyamide
`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
`atmospheres pressure for 4 hours in a Parr T™ shaker. The catalyst was removed by
`passage through a 0.45 ym nylonfilter and the filtrate was concentrated to a white foam.
`Crystallization from methylene chloride provided the title compound as a white solid (62 mg,
`32%).
`A second crop (62 mg, 32%) was obtained by crystallization from ethyi
`acetate/hexane.
`
`10
`
`15
`
`M.p. 138-141°C. 1H NMR (dg-DMSC): 5 10.50 (brs, 1H), 8.56 (br s, 1H), 7.67 (d, J
`
`20
`
`25
`
`30
`
`= 8.7 Hz, 2 H), 7.66 (brs, 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 (brs, 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 m/e (M-H). Further confirmation of structure and stereochemistry was carried
`out by single crystal X-ray crystallography.
`Example 2
`3-EXO-[4-(4-FLUOROPHENOXY)BENZENESULFONYLMETHYL]-8-
`OXABICYCLO-[3.2.1]-OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`A)
`8-Oxabicyclo[3.2.1]octane-3,3-dicarboxylic acid diethyl! ester
`Sodium hydride (2.28 grams, 95 mmole) was addedin portionsto a stirred solution of
`diethy! 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 anoil.
`Distillation under vacuum affordedthetitle compound (7.8 grams, 71%) as a clearoil.
`B)
`3-exo-Hydroxymethy!-8-oxabicyclo[3.2.1]octane-3-carboxylic 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 diethy! 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=page 1409
`
`AQUESTIVE EXHIBIT 1004 page 1409
`
`

`

`WO 99/52910
`
`PCT/1IB99/00503
`
`-38-
`
`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 wasstirred at room temperature
`overnight and was quenched by dropwise addition of saturated aqueous sodium sulfate
`
`solution. Ethyl acetate was added and, afterstirring for 20 minutes, the insoluble material was
`removed byfiltration through Celite™M Thefiltrate was washed with brine, dried over
`magnesium sulfate and concentrated to afford the title compound (5.1 grams, 80%) as a clear
`oil.
`
`10
`
`C)
`
`3-exo-Hydroxymethy!-8-oxabicyclo[3.2.1Joctane-3-carboxylic acid
`
`Lithium hydroxide hydrate (2.5 grams, 59.5 mmole) was added to a solution of 3-exo-
`
`(5.1 grams, 23.8
`hydroxymethyl-8-oxabicyclo[3.2.1]octane-3-carboxylic acid ethyl ester
`mmole) in a mixture of methanol (25 mL), tetrahydrofuran (25 mL) and water (2.5 mL). The
`
`15
`
`mixture was heated at reflux overnight, cooled and quenched by addition of Amberlite IR-
`
`120™ion exchange resin. After stirring for 20 minutes, the resin was removedbyfiltration,
`washing with tetrahydrofuran. Evaporation of the solvents andtrituration of the residue with
`diethyl! ether afforded the title compound (2.35 grams, 53%) as a white solid.
`
`
`
`3',8-Dioxaspirofbicyclo[3.2.1Joctane-3,1'-cyclobutane]-2'-one
`Benzenesulfonylchloride (1.7 mL, 13.5 mmole) was added dropwiseto a solution of 3-
`
`D)
`
`20
`
`exo-hydroxymethy!-8-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 wasstirred at 0°C for 1 hour, diluted with
`
`25
`
`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%).
`
`3-exo0-[4-(4-Fluoropheno
`
`henylsulfanyimethyl}-8-oxabicyclo[3.2.1]octane-
`
`3-carboxylic acid
`A solution of 4-(4-fluorophenoxy)thiophenol (2.2 grams, 10 mmole) in tetrahydrofuran
`
`30
`
`(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[bicycio[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 andstirring was
`continued at room temperature for 2 hours after which the mixture was quenched with
`
`35
`
`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 thetitle
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 1410
`
`page 1410
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-39-
`
`compound (1.8 grams (47%) as a white solid. Concentration of the motherliquor followed by
`chromatographyonsilica gel (2% methanolin chioroform as eluant) gave moreofthe the title
`
`compound (500 mg, 13%).
`F) 3-exo-[4-(4-Fluorophenoxy)phenytsulfany!methyl]-8-oxabicyclo[3.2.1]octane-
`3-carboxylic acid benzyloxyamide
`To a solution of 3-exo-[4-(4-fluorophenoxy)benzenesulfanyilmethyl]-8-oxabicycto-
`
`10
`
`[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-yloxy)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-
`
`15
`
`benzylhydroxyiamine 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
`
`20
`
`foam (405 mg, 32%) was isolated by chromatography (30% ethyl acetate in hexane as
`
`eluant).
`
`G) 3-exo-[4-(4-Fluoropheno
`
`henyisulfony!lmethy!]-8-oxabicyclo[3.2.1]octane-
`
`3-carboxylic acid benzyloxyamide
`Solid 57-85% meta-chloroperbenzoic acid (283 mg) was added to a solution of 3-exo-
`
`25
`
`acid
`[4-(4-fluorophenoxy)phenylsulfanyimethy]}-8-oxabicyclo{3.2.1]Joctane-3-carboxylic
`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 methyiene chloride, the organic layer was separated and
`
`washed with saturated aqueous sodium bicarbonate solution, water and brine. The organic
`
`30
`
`layer was dried over magnesium sulfate and concentrated to give the tithe compound as a
`
`white foam (390 mg, 90%).
`H)
`3-exo-[4-(4-Fluorophenoxy)benzenesulfonyimethy!]-8-oxabicyclo-[3.2.1]-
`
`octane-3-carboxylic acid hydroxyamide
`A
`solution
`of
` 3-exo-[4-(4-fluorophenoxy)benzenesulfonylmethyl]-8-oxabicyclo-
`
`35
`
`[3.2.1Joctane-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 um nylonfilter 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
`
`page 1411
`
`

`

`WO 99/52910
`
`PCT/IB99/00503
`
`-40-
`
`M.p. 134-139°C. 1H NMR (dg-DMSO): 6 8.55 (brs, 1H), 7.76 (d, J = 7.5 Hz, 2H),
`7.30-7.26 (m, 2 H), 7.20-7.16 (m, 2 H), 7.09 (d, J = 7.5 Hz, 2H), 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, 2H). MS mle 434 (M—H).
`Further confirmation of structure and stereochemistry was carried out by single crystal X-ray
`crystallography.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`same
`
`procedure
`
`as
`
`Example
`
`2,
`
`using
`
`4-
`
`Example 3
`3-(4-PHENOXYBENZENESULFONYLMETHYL)-8-OXABICYCLOJ3.2.1 JOCTANE-3-
`
`CARBOXYLIC ACID HYDROXYAMIDE
`Prepared
`according
`to
`the
`phenoxyphenylthiophenolin step E.
`1H NMR (dg-DMSO): 6 8.54 (brs, 1H), 7.75 (d, J = 8.9 Hz, 2 H), 7.44-7.40 (m, 2 H),
`7.23 7.21 (m, 1H), 7.11-7.07 (m, 4 H), 4.11 (brs, 2 H), 3.38 (s, 2H), 2.22 (d, J = 14.3 Hz, 2
`H), 1.80-1.70 (m, 4 H), 1.60-1.50 (m, 2H). MS m/e 416 (M-H).
`Example 4
`3-EX0-+4'-FLUOROBIPHENYL-4-SULFONYLMETHYL)-8-OXABICYCLOJ3.2.1 ]-
`OCTANE-3-CARBOXYLIC ACID HYDROXYAMIDE
`
`Prepared according to the same procedure as Example 2 using 4'-fluorobiphenyl-4-
`thiol in step E.
`1H NMR (dg-DMSO): & 10.60 (br s, 1H), 8.58 (brs, 1H), 7.88-7.85 (m, 4 H), 7.81-
`7.78 (m, 2 H), 7.36-7.31 (m, 2 H), 4.13 (br s, 2H), 3.47 (s, 2H), 2.25 (d, J = 14.5 Hz, 2 H),
`1.80-1.76 (m, 4H), 1.60-1.55 (m, 2 H). MS m/e 418 (M-H).
`Example 5
`3-EXO-[4-(4