NZ750153B2 - Condensed thiophene derivatives useful as napi-iib inhibitors - Google Patents

Condensed thiophene derivatives useful as napi-iib inhibitors Download PDF

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NZ750153B2
NZ750153B2 NZ750153A NZ75015317A NZ750153B2 NZ 750153 B2 NZ750153 B2 NZ 750153B2 NZ 750153 A NZ750153 A NZ 750153A NZ 75015317 A NZ75015317 A NZ 75015317A NZ 750153 B2 NZ750153 B2 NZ 750153B2
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New Zealand
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amino
methyl
phenyl
ethyl
difluoro
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NZ750153A
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NZ750153A (en
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David Andrew Coates
Kevin Robert Fales
Jeffrey Alan Peterson
Jeffrey Michael Schkeryantz
Quanrong Shen
Matthew John Valli
Ii John Rowley Wetterau
Dariusz Stanislaw Wodka
Yangping Xu
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Eli Lilly And Company
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Priority claimed from PCT/US2017/045843 external-priority patent/WO2018034883A1/en
Publication of NZ750153A publication Critical patent/NZ750153A/en
Publication of NZ750153B2 publication Critical patent/NZ750153B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4995—Pyrazines or piperazines forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/50—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D333/52—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes
    • C07D333/62—Benzo[b]thiophenes; Hydrogenated benzo[b]thiophenes with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
    • C07D333/66—Nitrogen atoms not forming part of a nitro radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/08—Bridged systems

Abstract

The invention provides compounds of the formula: (A), pharmaceutically acceptable salts, pharmaceutical compositions thereof, and methods of using these compounds, salts, or compositions to treat hyperphosphatemia, chronic kidney disease, and/or the cardiovascular disease associated with chronic kidney disease. ney disease.

Description

CONDENSED THIOPHENE DERIVATIVES USEFUL AS NAPI-IIB INHIBITORS The invention provides compounds to treat the phosphate excess or hyperphosphatemia associated with chronic kidney disease (CKD), dialysis ts with end stage renal disease (ESRD), and related cardiovascular e.
In patients with impaired renal function, such as chronic kidney e and dialysis patients with end stage renal disease, phosphorus accumulates in the body resulting in a rise in phosphorus concentration in the blood and a phosphate excess.
In some patients this phosphate burden reaches a state referred to as hyperphosphatemia. The elevated phosphate burden in CKD and ESRD in turn brings about the hypersecretion of parathyroid hormones, i.e., secondary hyperparathyroidism, and causes bone s. hosphatemia has been linked with calcification of the coronary arteries and aorta, as well as cardiovascular and all-cause mortality. Vascular ication is considered to promote dysfunction of the heart leading to death. Dysfunction of phosphate regulation has serious clinical consequences, and studies show that even small increases in serum phosphate levels, within the normal or near-normal range, may correlate with increased ity and mortality. Phosphate excess in CKD stage three and four patients, and the body’s compensating response to the the phosphate excess, has been implicated in the associated cardiovascular morbidity and mortality. Decreasing phosphate absorption in CKD stage three and four patients may mitigate or prevent these responses and preserve cardiovascular health. Controlling phosphate load early in CKD may te or prevent morbidity and mortality in ed patients (C. S. Ritter and E. Slatopolsky, Phosphate ty in CKD: The Killer Among Us, Clin. J. Am. Soc. Nephrol. 11:1088–1100, 2016).
Three isoforms of NaPi-II have been identified. NaPi-IIa (type IIa, also referred to as SLC34A1) is mainly expressed in the kidney, while NaPi-IIb (type IIb, also referred to as SLC34A2) is expressed in the small intestine and can be regulated by vitamin D. NaPi-IIc (type IIc, also referred to as SLC34A3) is also expressed in the kidney. Phosphate absorption in the intestinal tract is performed in large part by NaPi-IIb, whereas phosphate in the blood is filtered by renal glomeruli and reabsorbed in necessary s mainly by NaPi-IIa and Ic in the renal tubule oto, et al. Sodium-Dependent Phosphate Cotransporters: s from Gene Knockout and Mutation Studies, J. Pharm. Science, 100(9):3719-30, 2011).
In spite of progress made for treatment of ate excess, and/or hyperphosphatemia, there remains a significant unmet need for safe and effective therapies to treat these conditions. Current ents employ ate adsorbents to suppress phosphate absorption in the gastrointestinal tract. These include for example nonmetallic polymer adsorbents, for example sevelamer carbonate and mer hloride, calcium salt preparations, for example precipitated calcium ate, and metallic adsorbents, for example num carbonate. However, these agents have each been reported to have adverse effects such as constipation, diarrhea, hypercalcemia, and metal accumulation. In on, treatment with adsorbents requires daily intake on the order of a few grams of adsorbent, and noncompliance with therapy is a common problem. Accordingly, there remains an unmet need for treatment of phosphate excess, and/or hyperphosphatemia, which provide ed safety, efficacy, and convenience.
Inhibition of NaPi-IIb may suppress phosphate absorption in the gastrointestinal tract resulting in decreased phosphate concentration in blood as an approach to treat hyperphosphatemia gh et al, inal Phosphate Transport, Adv. c Kidney Dis., 18(2):85–90, 2011). Suppression of phosphate absorption by NaPi-IIb inhibition employs a different mechanism of action, as compared to current phosphate adsorbents, and may provide clinically useful advantages for prevention and or treatment of phosphate excess and/or hyperphosphatemia. Further, NaPi-IIb transporter tors may provide additional benefits for secondary hyperparathyroidism, chronic kidney disease, and/or cardiovascular disease associated with chronic kidney disease more generally, by decreasing the absorption of dietary phosphate.
The compounds of the present invention are inhibitors of NaPi-IIb transporter and demonstrate potent inhibition of NaPi-IIb. As such, compounds of the present invention are believed to be useful for the treatment of conditions in which NaPi-IIb mediated phosphate absorption plays a role, such as chronic kidney disease and hyperphosphatemia.
United States Application Publication US 2013/0053369 discloses n tetrahydrobenzothiophene compounds as inhibitors of Ib, and recites the compounds as useful in treating a number of diseases including hyperphosphatemia.
The need for a safe and convenient treatment of phosphate excess, hyperphosphatemia, chronic kidney disease, and/or cardiovascular disease associated with chronic kidney disease, without the disadvantages possessed by adsorbents, or other agents known in the field, continues to be a concern for treatment of patients renal disease. The present invention provides ative compounds which are useful in treatment of phosphate excess, hyperphosphatemia, c kidney e, and/or cardiovascular disease associated with c kidney disease. In addition, the compounds provided address the need for treatments of conditions associated with NaPi-IIb activity with ed efficacy and/or advantageous side effect and tolerability profiles.
The present invention provides a compound of the formula: Formula II wherein Y is a fused cyclohexane ring or a fused phenyl ring, wherein A is or , wherein the crossed lines indicate bonds for the point of attachment to the core of Formula II, and the dashed lines indicate bonds for the point of attachment to R2, wherein R2 is selected from the group consisting of -CH3, -(CH2)3OH, -(CH2)3OCH3, -(CH2)3CO2H, 3, , -CO(CH2)3CH3, -COCH(CH3)2, -CO(CH2)2CO2H, NH2, N(CH3)2, -SO2N[(CH2)2OCH3]2, -SO2NHCH3, -SO2(CH2)2OCH3, -CONH(CH2)4OH, -CONH(CH2)4OCH3, -CONHCH3, -CONH(CH2)2CO2H, -CONH(CH2)2OCH3, -CON(CH2CH2OCH3)2, -CSNHCH3, , , and , wherein the dashed lines represent the point of attachment, wherein R’ is -CO2H or -CONH2, or a pharmaceutically acceptable salt thereof.
The present invention further provides a compound of Formula II as described above wherein Y is a fused cyclohexane ring , A is , and R’ is -CO2H, or a pharmaceutically able salt thereof.
The present invention further provides a compound of Formula II as described above wherein Y is a fused cyclohexane ring, A is , and R’ is -CO2H, or a pharmaceutically able salt thereof.
Further, the present invention es a compound of the formula: R' wherein R is selected from the group consisting of -(CH2)3OH, -(CH2)3OCH3, -(CH2)3CO2H, -CONH(CH2)4OH, -COCH2NH2, -SO2N[(CH2)2OCH3]2, -CONH(CH2)4OCH3, and 2)2CO2H, wherein R’ is -CO2H or , or a pharmaceutically acceptable salt thereof.
The present invention further provides a compound of Formula I as described above wherein R’ is -CO2H, or a pharmaceutically acceptable salt thereof.
The following particular embodiments are compounds and/or salts of Formula I and/ or II.
The present invention provides a compound which is 4-[2-[2,6-difluoro[[2-[[3-[[4- (4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof.
The present invention provides a compound which is 4-[2-[2,6-difluoro[[2-[[3-[[4- (3-hydroxypropyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically able salt thereof.
The present invention provides a compound which is 4-[2-[4-[[2-[[3-[[4-[bis(2- methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof.
The t invention provides a compound which is 4-[2-[4-[[2-[[3-[[4-(3- carboxypropyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof.
The present invention provides a compound which is 2,6-difluoro[[2-[[3-[[4- (3-methoxypropyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- ydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt f.
The present invention provides a compound which is 4-[2-[4-[[2-[[3-[[4-(2- aminoacetyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof.
The present invention provides a compound which is 4-[2-[2,6-difluoro[[2-[[3-[[4- (4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a ceutically able salt thereof.
The present invention provides a compound which is 4-[4-[[3-[[3-[[4-[2-(4- carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinyl]oxo-butanoic acid, or a ceutically acceptable salt thereof.
The present invention provides a salt which is 2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, um.
The present invention provides a solid dispersion formulation of 4-[2-[2,6-difluoro [[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium, wherein the formulation comprises 30% 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium, and 70% polyvinylpyrrolidone-vinyl acetate.
The present invention further provides a compound, or a ceutically acceptable salt thereof, selected from the group consisting of: 4-[2-[2,6-difluoro[[2-[[3-[(2,2,4-trimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid formic acid salt; 4-[[2-[[3-[[2,2-dimethyl(methylcarbamoyl)piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[[2,2-dimethyl(methylcarbamothioyl)piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[[4-(2-carboxyethylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, formic acid salt; 4-[2-[2,6-difluoro[[2-[[3-[(4-methoxycarbonyl-2,2-dimethyl-piperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid; 4-(2,6-difluoro(2-(3-(((1R,5S)pentanoyl-3,8-diazabicyclo[3.2.1]octan yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene carboxamido)phenethyl)benzoic acid; 4-[2-[4-[[2-[[3-[[2,2-dimethyl(methylsulfamoyl)piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro[[2-[[3-[[8-(tetrahydropyranylcarbamoyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octanyl)methyl]benzoyl]amino]-4,5,6,7- ydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate; 2,6-difluoro[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate; 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate; 4-[2-[4-[[2-[[3-[[8-[2-(dimethylamino)acetyl]-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid; 2,6-difluoro[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro[[2-[[3-[[8-[2-(4-methylpiperazinyl)acetyl]-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid; 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid; and 4-[2-[4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazinyl)methyl]benzoyl]amino]-4,5,6,7- ydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid.
Further, the present invention provides a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient. Further, the present invention provides a pharmaceutical composition comprising a compound of formula II, or a pharmaceutically able salt thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
Further, the present invention provides a compound of formula I or II, or a pharmaceutically acceptable salt thereof, for use in therapy. The present invention provides a salt which is 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl- piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, um, for use in therapy.
Further, the t invention provides a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of hyperphosphatemia.
Further, the present ion es a compound of formula II, or a pharmaceutically acceptable salt thereof, for use in the treatment of hyperphosphatemia. Further, the present invention provides a compound of formula I or II, or a pharmaceutically acceptable salt thereof, for use in the ent of chronic kidney disease. The present invention es a salt which is 2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl- piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, for use in the treatment of hyperphosphatemia. The present invention provides a solid dispersion formulation of 4-[2- [2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, n the formulation comprises % 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, and 70% polyvinylpyrrolidone-vinyl acetate for use in the ent of hyperphosphatemia.
The present invention provides a salt which is 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium, for use in the treatment of chronic kidney disease. The present invention provides a solid dispersion formulation of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl- piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, wherein the formulation comprises % 2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, and 70% polyvinylpyrrolidone-vinyl acetate for use in the treatment of chronic kidney disease.
The present invention provides a compound or salt which is 4-[2-[2,6-difluoro[[2- [[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically able salt thereof, for use in the treatment of cardiovascular disease associtated with c kidney disease. The t invention provides a salt which is 4-[2- [2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, for use in the treatment of cardiovascular disease associtated with chronic kidney disease. Described herein is a method of treating cardiovascular e associtated with chronic kidney disease comprising administrating to a patient in need thereof an effective amount of a compound or salt which is 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof.
Further, the present invention provides the use of a compound of a I or II, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating hyperphosphatemia, and/or chronic kidney disease. r, described herein is a method of treating hyperphosphatemia, sing administering to a t in need thereof an effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof. Further, described herein is a method of treating hyperphosphatemia, comprising administering to a t in need thereof an effective amount of a compound of formula II, or a pharmaceutically acceptable salt thereof.
Further, described herein is a method of treating hyperphosphatemia comprising administrating to a patient in need thereof an effective amount of 4-[2-[2,6-difluoro[[2- [[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, or a pharmaceutically acceptable salt thereof. r, described herein is a method of treating hyperphosphatemia comprising administrating to a patient in need thereof an effective amount of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethylpiperazinyl ]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, um. Further, described herein is a method of treating hyperphosphatemia comprising administrating to a t in need thereof an effective amount of a solid dispersion formulation of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium, wherein the formulation comprises 30% 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)- 2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, and 70% polyvinylpyrrolidone-vinyl acetate.
The term “pharmaceutically acceptable salt” includes an acid addition salt that exists in conjunction with the basic portion of a compound of formula I or II, or basic addition salt that exists in conjunction with the acidic n of a compound of formula I or II. Such salts include the pharmaceutically acceptable salts, for example those listed in Handbook of Pharmaceutical Salts: ties, Selection and Use, P. H. Stahl and C. G. Wermuth (Eds.), Wiley-VCH, New York, 2002 which are known to the skilled artisan.
In addition to pharmaceutically able salts, other salts are contemplated in the invention. They may serve as intermediates in the purification of compounds or in the preparation of other pharmaceutically-acceptable salts, or are useful for identification, terization or purification of compounds of the invention.
As used herein, the term “patient” refers to a warm blooded animal such as a mammal and includes a human. A human is a red patient.
Cardiovascular disease associated with chronic kidney e may include sudden cardiac death, arrhythmia, angina, myocardial tion, and heart e (Kestenbaum et al., Serum Phosphate Levels and Mortality Risk Among People with Chronic Kidney Disease, J. Am. Soc. Nephrol. 16: 8, 2005).
One skilled in the art may treat hyperphosphatemia and/or chronic kidney disease by administering to a patient presently displaying symptoms an effective amount of the nd of formula I. Thus, the terms “treatment” and “treating” are intended to refer to all processes wherein there may be a slowing, interrupting, arresting, controlling, or stopping of the progression of an existing disorder and/or symptoms thereof, but does not necessarily indicate a total elimination of all symptoms.
One skilled in the art may treat hyperphosphatemia and/or chronic kidney disease by administering to a patient having recognized risk factors for hyperphosphatemia and/or chronic kidney disease an effective amount of the compound of formula I. For instance, Patients having ate levels in the high end of the normal range, in eration with other factors such as ension and/or diabetes, may be considered as having recognized risk for hyperphosphatemia and/or chronic kidney disease, and cardiovascular disease associated with chronic kidney disease.
As used herein, the term “effective amount” of a compound of a I or II refers to an amount which is effective in treating a disorder, such as hyperphosphatemia and/or chronic kidney e described herein. One skilled in the art can determine an effective amount by the use of conventional techniques and by ing results obtained under circumstances considered to be informative to the current patient. In determining an effective amount or dose of a compound of formula I or II, a number of factors are considered, including, which compound of formula I or II is administered; whether co- administration of other agents exists; the species of mammal; its size, age, and general health; the degree of involvement or the severity of the disorder, such as hyperphosphatemia and/or chronic kidney disease; the se of the individual patient; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen ed; and other relevant circumstances.
The compounds of the present ion can be administered alone or in the form of a pharmaceutical composition combined with pharmaceutically acceptable carriers or excipients, the proportion, and nature of which are determined by the solubility and chemical properties, including stability, of the compound selected, the chosen route of administration, and standard pharmaceutical practice. The compounds of the present invention, while effective themselves, may also be formulated and administered in the form of their pharmaceutically acceptable salts.
One d in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular teristics of the nd selected, the er or condition to be treated, the stage of the disorder or condition, and other relevant circumstances (See, e.g., ton: The Science and Practice of cy, L.V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012).
Certain abbreviations are defined as follows: “AcOH” refers to acetic acid; “ACN” refers to acetonitrile; “BOP” refers to benzotriazoleyl-oxy-tris-(dimethylamino)- phosphonium hexafluorophosphate; “DCM” refers to dichloromethane or methylene chloride; “DIPEA” refers to N,N-diisopropylethylamine; “DMF” refers to N,N- dimethylformamide; “DMSO” refers to dimethylsulfoxide; “EDCI” refers to 3- (ethyliminomethyleneamino)-N,N-dimethylpropanamine; “EtOAc” refers to ethyl acetate; “EtOH” refers to ethanol; “FBS” refers to fetal bovine serum; “HATU” refers to 1- [bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate; “HEPES” refers to 2-[4-(2-hydroxyethyl)piperazinyl]ethanesulfonic acid’ “HOBT” refers to hydroxybenzotriazole; ‘hr” refers to hour or hours; “IC50” refers to the concentration of an agent that produces 50% of the maximal tory response possible for that agent; “LC-ES/MS” refers to Liquid tography Electrospray Mass ometry; “min” refers to minute or minutes; “MeOH” refers to methanol or methyl alcohol; “MTBE” refers to -tert-butyl ether; “33P” refers to phosphorus-33; “psi” refers to pounds per square inch; “PyBOP” refers to benzotriazolyloxytripyrrolidinophosphonium hexafluorophosphate; “RT” refers to room temperature; “TEA” refers to triethylamine; “TFA” refers to trifluoroacetic acid; “THF” refers to tetrahydrofuran; “Tris” refers to 2-aminohydroxymethyl-propane-1,3-diol ; “U/mL” refers to units per milliliter; “PVP-VA” refers to polyvinylpyrrolidone-vinyl acetate.
Scheme 1 S O H N N BOC O NH Cl II N O H H N BOC III N O H H N H HCl HCl O IV O Va R = (CH2)3OCH3 Vb R = (CH2)3CO2CH3 Vc R = CONH(CH2)4OH Vd R = COCH2NHBOC Ve R = SO2N[(CH2)2OCH3]2 Vf R = CONH(CH2)4OCH3 Ia R = (CH2)3OCH3 Ib R = (CH2)3CO2H Ic R = H2)4OH Id R = COCH2NH2 Ie R = SO2N[(CH2)2OCH3]2 If R = CONH(CH2)4OCH3 Ig R = (CH2)3OH Formula Ix Scheme 1 depicts the synthetic route to compounds of Formula Ix. Generally, the alkyl halide compound II may be aminated under various conditions well appreciated in the art, for e, using an amine and an appropriate non-nucleophilic base such as TEA, DIPEA, or in a suitable organic t such as THF, ACN, or DMF. More specifically, about 2 equivalents of tert-butyl 3,3-dimethylpiperazinecarb oxylate may be heated in a microwave reaction vessel at 110 oC in the presence of about 1 lent of alkyl halide II and about 12 equivalents of DIPEA in ACN to obtain the alkyl amine III. The Boc protecting group of Compound III may be removed under acidic conditions well described in the art. More specifically, compound II may be treated with an excess of HCl in 1,4-dioxane in DCM to yield the hydrochloride salt IV.
Subsequent tion of compound IV may be carried out under a wide array of conditions well known in the art, such as treatment with an alkyl halide under alkylation conditions, for example, with an appropriately substituted alkyl halide and the amine IV in the presence of a non-nucleophilic base, such as TEA, DIPEA, pyridine, or 1,8- diazabicycloundecene, in an appropriate organic solvent such as DCM, ACN or DMF. onally, alkylation of nd IV may be performed under reductive amination conditions, such as with an riately substituted aldehyde in the presence of a reducing agent such as sodium borohydride, sodium toxyborohydride, or sodium cyanoborohydride, and a catalytic amount of an organic acid, such as AcOH or TFA, in an appropriate organic solvent, such as MeOH, EtOH, ACN, DCM, THF, or DMF. More specifically, compound IV may be treated with about 2 equivalents DIPEA and subsequently treated with 3-methoxyproprionaldehyde or 4-oxobutanoic acid methyl ester in the presence of about 2 equivalents of sodium triacetoxyborohydride and catalytic AcOH in DCM, to obtain compounds Va and Vb, respectively.
Acylation or sulfonylation of compound IV may be accomplished under conditions well known in the art, for example, using an acyl halide or sulfonyl halide under basic conditions using an excess of an appropriate non-nucleophilic base such as TEA or DIPEA in a suitable organic solvent such as DCM, THF, ACN, or DMF. More specifically, compound IV may be treated with about 4 lents of DIPEA and about 2 lents of N-(4- hydroxybutyl)-3,3-dimethyl-piperazinecarboxamide hydrochloride in a mixture of about 1:10 MeOH:ACN and heated in a microwave reactor at 100 oC, to obtain the compound Vc.
Acylation of the amine IV may also be performed under standard amide coupling conditions well known in the art, for example, using EDCI and HOBT, HATU, BOP, or PyBOP, in the presence of a non-nucleophilic base such as TEA or DIPEA, and in a le organic t such as MeOH, ACN, THF, DCM, or DMF, or a combination thereof. More specifically, about 1.2 equivalents of compound IV may be treated with about 0.9 equivalents of 1,8- diazabicyclo [5.4.0]undecene and subsequently treated with about 1 equivalent N -(tert- butoxycarbonyl)glycine, 0.5 equivalents HOBT, and 1.2 equivalents EDCI in DMF to obtain compound Vd. Sulfonylation of compound IV may be achieved in the presence of about 1.5 equivalents bis(2-methoxyethyl)sulfamoyl chloride and about 5 e quivalents TEA in DMF with heating, to obtain nd Ve.
Moreover, acylation of amines to obtain ureas, by nucleophilic addition of an amine to an isocyanate under basic conditions, are also well described in the art. Specifically, about 1 equivalent of compound IV may be treated with about 2 equivalents of 1-isocyanato methoxy-butane in the presence of about 4 equivalents TEA in DCM to obtain compound Vf.
The compounds of a Ix may be prepared by saponification of the methyl ester moiety of compounds V under either acidic or basic conditions as well known in the art.
More specifically, compounds Va-Vf may be treated with about 1-5 equivalents of LiOH in THF, MeOH, H2O, or an riate e thereof, to obtain compounds of Formula Ia-f.
These conditions may simultaneously saponify the additional ester moiety in compound Vb.
Additional protecting groups may be removed by well-known methods, for example, treatment of compound Ve with an excess of 4 M HCl in dioxane, after the ester saponification is completed. Moreover, additional heteroatom-protected alkyl aldehydes, specifically 3-[(tert-butyldimethylsilyl)oxy]propanal, may b e treated with compound IV under reductive ion conditions as described above, ected in situ, and ultimately saponified as bed above. More specifically, 3 -[(tert-butyldimethylsilyl)oxy] propanal and compound IV may be treated to reductive amination conditions, using sodium toxyborohydride and catalytic AcOH as described over, with subsequent removal of the silyl group in situ, using excess HCl in oxane. Final saponification with LiOH, as described above, may be performed to obtain compound of Formula Ig.
Scheme 2 VIII N O H N H O X O a Ic An alternative synthesis to compound of Formula Ic is depicted in Scheme 2. A mixture of about 1 equivalent methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethyl]benzoate XII and 1.1 equivalents 3-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene carboxylic acid (Aurum Pharmatech) in DCM may be treated with about 1.25 equivalents bis-(2-oxooxazolidinyl)phosphinic chloride in the presence of about 5 equivalents DIPEA to give the product compound VII, which may be ected under standard conditions well known in the art, specifically with excess 4 N HCl in 1,4-dioxane and DCM as solvent, to obtain amine compound VIII. Amine VIII may be subjected to acylation conditions as described above, specifically with about 1.1 equivalents 3-(chloromethyl)benzoyl chloride in the ce of pyridine with DCM as solvent, to obtain compound IX. The alkyl chloride IX may be aminated under a wide array of ion conditions well known in the art, more specifically with about 2 equivalents N-(4-hydroxybutyl)-3,3-dimethyl-piperazine carboxamide hydrochloride in the presence of about 4 equivalents DIPEA in a e of ACN/MeOH as described above to obtain compound X, and uent saponification as described above may yield the compound of Formula Ic.
Scheme 3 The sis of aniline compound XII is depicted in Scheme 3. Generally, palladium-copper mediated Sonogashira cross-coupling between an aryl halide and a substituted acetylene are well known in the art. Specifically, about 1 equivalent 3,5-difluoro- 4-iodoaniline (AstaTech) and about 1 equivalent methyl nylbenzoate (Alfa Aesar) may be heated in the presence of about 0.4 equivalents iphenylphosphine)-palladium(II) dichloride and 0.07 equivalents CuI with about 10 lents DIPEA in THF to give the diaryl acetylene compound XI, which may be reduced under standard conditions well described in the art, ically catalytic hydrogenation at about 60 psi in the presence of palladium black in a solvent mixture of THF/H2O, to obtain the requisite aniline compound XII.
Scheme 4 XIII Scheme 4 illustrates the synthesis of amine XV. Nucleophilic addition of 1 equivalent t-butyl 2,2-dimethylpiperazinecarboxylate to about 1.1 equivalent of methyl 4- isocyanatobutanoate in the presence of 3 equivalents DIPEA with DCM as reaction solvent may yield the urea compound XIII. Reduction of the ester moiety may be accomplished using a wide array of conditions well described in the ture, including BH3, lithium borohydride, and diisobutyl aluminum hydride. More specifically, 1 equivalent of ester XII may be treated with about 3 equivalents of lithium borohydride in THF to give d product XIV; subsequent removal of the BOC protecting group as described above gives the requisite compound XV.
Scheme 5 NH3/dioxane XVII XVIII XIX F NH2 S H NH F Formula Iy Scheme 5 depicts the synthesis of compound of Formula Iy. Hydrolysis of the ester moiety in compound XVI may be achieved under conditions well known in the art, such as with an alkaline base such as NaOH, KOH, or LiOH in aqueous, organic, or ic solvent mixtures. More specifically, the ester XVI may be treated with 5 lents LiOH in a mixture of aqueous THF; mild acidification of the saponified acid may yield compound XVII. Subsequent direct amidation via the acid chloride may be accomplished under mild conditions (E. Valeur and M. Bradley, Chem. Soc. Rev., 2009, 38, 606–631). More specifically, the acid chloride of acid compound XVII may be generated in situ with 1.2-2.5 equivalents of bis(2-oxooxazolidinyl)phosphinic chloride at room temperature followed by ent with excess ammonia to obtain the desired primary amide XVIII. Removal of the BOC protecting group as bed above gives the requisite compound XIX as described above. Acylation of the unmasked piperizine nitrogen may be achieved under conditions well known in the art, specifically treatment of compound XIX with 5 equivalents of a suitable non-nucleophilic base such as DIPEA in the presence of an acylating agent such as succinic anhydride to obtain the nd of Formula Iy. Other compounds of Formula I and/or II may be ed by the skilled artisan by procedures analogous to those described herein using appropriate starting materials and cations as needed.
Scheme 6 O O F O O S N F NH + F O NH H2N F O O O O F F O O N F N F NH S Cl NH2 H Cl O O O O OH F F O O N F N F H H S S NH NH O O N N N H N H N N O O OH OH D X The synthesis of nd X is depicted in Scheme 6. A BOPCl mediated amide coupling yields compound A. Removal of the BOC protecting group as described above gives the requisite compound B. Acylation of the amino group maybe achieved under conditions well known in the art, specifically treatment of B with 3-(chloromethyl)-benzoyl chloride in presence of pyridine to obtain compound C. Nucleophilic substitution of benzylic chloride with substituted piperazine yields methyl benzoate D. Subsequent saponification as described above affords the nd X.
Preparations and Examples The following Preparations and Examples further illustrate the invention and represent typical synthesis of the compound of the invention. The reagents and starting materials are readily available or may be y synthesized by one of ordinary skill in the art. It should be understood that the Preparations and Examples are set forth by way of illustration and not limitation, and that various modifications may be made by one of ordinary skill in the art.
LC-ES/MS is performed on an AGILENT® HP1100 liquid chromatography system.
Electrospray mass spectrometry measurements (acquired in positive and/or negative mode) are performed on a Mass ive or quadrupole mass spectrometer interfaced to the HP1100 HPLC. LC-MS conditions (low pH): column: PHENOMENEX® GEMINI® NX C18 2.1 × 50 mm 3.5 μm; gradient: 5-100% B in 3 min, then 100% B for 0.75 min, or 5- 95% B in 1.5 min, then 95% B for 0.25 min; column temperature: 50 °C +/-10 °C; flow rate: 1.2 mL/min; Solvent A: deionized water with 0.1% HCOOH; Solvent B: ACN with 0.1% formic acid; ngth 214 nm. Alternate LC-MS conditions (high pH): : XTERRA® MS C18 columns 2.1×50 mm, 3.5 μm; nt: 5% of t A for 0.25 min, gradient from 5% to 100% of solvent B in 3 min and 100% of solvent B for 0.5 min or 10% to 100% of t B in 3 min and at 100% of solvent B for 0.75 min or 5-95% B in 1.5 min, then 95% B for 0.25 min; column temperature: 50 °C +/-10 °C; flow rate: 1.2 mL/min; Solvent A: 10 mM NH4HCO3 pH 9; Solvent B: ACN ; wavelength: 214 nm.
NMR spectra are performed on a Bruker AVIII HD 400 MHz NMR Spectrometer or a Varian VNMRS 300 or 400 MHz NMR Spectrometer, obtained as CDCl3 or DMSO-d6 solutions reported in ppm, using residual t [CDCl3, 7.26 ppm; DMSO-d6, 2.50 ppm] as reference standard. When peak multiplicities are reported, the following abbreviations may be used: s et), d (doublet), t (triplet), q (quartet), m (multiplet), br s (broad singlet), dd (doublet of doublets), dt (doublet of triplets). Coupling constants (J), when reported, are reported in hertz (Hz).
Preparation 1 methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethynyl]benzoate A suspension of 3,5-difluoroiodoaniline (14.7 g, 55.9 mmol), CuI (0.745 g, 3.91 mmol), iphenylphosphine)palladium(II) dichloride (1.59 g, 2.24 mmol), methyl 4- ethynylbenzoate (9.05 g, 55.9 mmol), TEA (114 mL) and THF (44.1 mL) is stirred at 60 ºC for 3 hr. The mixture is cooled to RT and the t evaporated to dryness under reduced pressure. EtOAc (100 mL) and H2O (100 mL) are added, and the resulting solid is filtered over diatomaceous earth. The organic layer from the filtrate is separated, dried over MgSO4, and ated to dryness under reduced pressure. A 1:1 mixture of DCM:heptane (400 mL) is added to the resulting residue and the mixture is stirred at RT ght. The resulting solid is collected by filtration and dried under vacuum to obtain the title compound (8.0 g, 45.8% yield) as a brown solid. 1H NMR (300 MHz, DMSO-d 6) δ 3.86 (s, 3H), 6.26-6.37 (m, 4H), 7.59 (d, J = 8.2 Hz, 2H), 7.96 (d, J = 8.5 Hz, 2H).
Preparation 2 methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethyl]benzoate A 500 mL Parr shaker is charged with Pd black (0.53 g, 5.0 mmol) under N2. A degassed 4:1 solution of MeOH/THF (25 mL) is added followed by a degassed solution of methyl 4-[2-(4-amino-2,6-difluoro-phenyl)ethynyl]benzoate (1.17 g, 3.38 mmol) in a 4:1 mixture of MeOH/THF (25 mL) under N2. The resulting mixture is purged with N2 and pressurized with H2 to 60 psi. The sealed vessel is heated at 40 oC for 14 hr. The resulting suspension is filtered h a pad of diatomaceous earth under N2 and evaporated to dryness in vacuo. The resulting residue is ed by chromatography over silica, g with a gradient of 25-35% hexanes/THF, to afford the title compound as a white solid (404 mg, 40% yield) after solvent evaporation and drying under vacuum. 1H NMR (400.13 MHz, DMSO-d6) δ, 2.71-2.83 (m, 4H), 3.84 (s, 3H), 5.52 (s, 2H), 6.10-6.15 (m, 2H), 7.28 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.2 Hz, 2H). MS (m/z) 292 [M+1].
Preparation 3 tert-butyl 4-[(4-methoxyoxo-butyl)carbamoyl]-2,2-dimethyl-piperazinecarboxylate A 30 mL scintillation vial is charged with t-butyl methylpiperazine carboxylate (500 mg, 2.28 mmol) and DCM (12 mL) . The resulting solution is cooled in an ice/water bath and DIPEA (1.20 mL, 6.85 mmol) is added in one p ortion. A solution of methyl 4-isocyanatobutanoate (447 mg, 2.97 mmol) in DCM (3 mL) is added drop wise over min. The reaction e is slowly warmed to RT and stirred for a further 15 min. The mixture is partitioned between 5% aqueous citric acid (100 mL) and DCM (20mL). The organic layer is separated, and the s layer is extracted twice more with DCM (20 mL each). The combined organic extracts are washed tially with saturated aqueous NaHCO3 (30 mL) and saturated aqueous NaCl (30 mL), dried over anhydrous Na2SO4, filtered, and evaporated under d pressure. The resulting residue is ed by chromatography over silica, eluting with a gradient of 30-50% hexanes/acetone, to obtain the title compound as colorless viscous oil (848mg, 95% yield) after solvent removal and drying under vacuum. 1H NMR (399.8 MHz, CDCl 3) δ 1.34 (s, 6H), 1.45 (s, 9H), 1.83 (t, J = 6.9 Hz, 2H), 2.37 (t, J = 7.1 Hz, 2H), 3.25-3.30 (m, 2H), 3.37 (t, J = 5.7 Hz, 2H), 3.47 (s, 2H), 3.65 (s, 3H), 3.71 (t, J = 5.7 Hz, 2H), 4.65-4.68 (m, 1H). LC-ES/MS (m/z) 358 [M+1].
Preparation 4 tert-butyl 4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinecarboxylate A 2 M solution of LiBH4 in THF (3.02 mL, 6.04 mmol) is added drop wise to a 100 mL round bottom flask containing tert-butyl 4-[(4-methoxyoxo-butyl)carbamoyl]-2,2- dimethyl-piperazinecarboxylate (783 mg, 2.01 mmol) and THF (2 mL) at RT. The resulting mixture is stirred for 12 hr at RT. The reaction mixture is quenched with 0.5 mL of MeOH, stirred at RT for 20 min, and partitioned between 5% aqueous NaHCO3 (150 mL) and DCM (50 mL). The organic layer is separated, and the s layer is extracted twice more with DCM (50 mL each). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous , filtered, and evaporated to dryness under reduced pressure to afford the title compound as a white solid (634mg, 96% yield). 1H NMR (399.8 MHz, CDCl3) δ 1.36 (s, 6H), 1.46 (s, 9H), 1.58-1.63 (m, 4H), 3.31-3.28 (m, 2H), 3.49 (s, 2H), 3.66-3.69 (m, 2H), 3.71-3.74 (m, 2H), .39 (m, 2H). LC-ES/MS (m/z) 330 [M+1].
Preparation 5 N-(4-hydroxybutyl)-3,3-dimethyl-piperazinecarboxamide hydrochloride A 30 mL scintillation vial is charged with a solution of utyl 4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinecarboxylate (631 mg, 1.92 mmol) in DCM (20 mL). A 4 N solution of HCl in dioxane (2.4 mL, 9.58 mmol) is added drop wise over 5 min and the resulting solution is stirred at RT for 2 hr. The volatiles are removed in vacuo and the residue is dried under vacuum to afford the title compound as a hygroscopic white oily solid (100% yield, quantitative), le for use in the next step without further purification. LC-ES/MS (m/z) 230 [M+1].
Preparation 6 methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate N F A 100mL round bottom flask is charged with methyl 4-[2-(4-amino-2,6-difluorophenyl )ethyl]benzoate (2.11 g, 7.24 mmol), 3-(tert-butoxycarbonylamino)-4,5,6,7- tetrahydrobenzothiophenecarboxylic acid (2.37 g, 7.97mmol), and DCM (60 mL). The resulting suspension is cooled in an ice/water bath, and DIPEA (5.05 mL, 29.0 mmol) is added drop wise to afford a yellow-brown turbid solution. Solid bis-(2-oxo oxazolidinyl)phosphinic chloride (2.30 g, 9.05 mmol) is added in small portions over 30 min at 0 oC. The reaction mixture is then warmed to RT and stirred for 24 hr. Additional solid 3- (tert-butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophenecarboxylic acid (0.6 g, 2.0 mmol) is added followed by additional DIPEA (1.26 mL, 7.25 mmol) and solid -oxo oxazolidinyl)phosphinic chloride (0.58 g, 2.26 mmol) in small portions over 5 min. The resulting turbid brown reaction mixture is stirred at RT for 12 hr. The reaction mixture is partitioned n 5% s citric acid (150 mL) and DCM (25 mL), the organic layer is separated, and the aqueous layer is extracted twice more with DCM (50 mL each). The combined organic extracts are washed sequentially with 10% aqueous NaHCO3 (50 mL), saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under d pressure. The resulting brown oily solid is purified by chromatography over silica, eluting with a gradient of 15-40% hexanes/(10% MTBE in DCM). The collected ons containing desired product are combined and concentrated under reduced pressure, and the ing residue is triturated with MTBE (15 mL). The resulting solid is collected by filtration and dried under vacuum to afford the title nd (2.75 g, 66% yield). 1H NMR (400.1 MHz, DMSO-d6) δ 1.44 (s, 9H), 1.69-1.77 (m, 4H), 2.53-2.65 (m, 4H), 2.91 (br s, 4H), 3.84 (s, 3H), 7.30-7.34 (m, 4H), 7.86 (d, J = 8.3 Hz, 2H), 9.80 (br s, 1H), 9.94 (s, 1H).
MS (m/z) 569 [M-1].
Preparation 7 methyl 4-[2-[4-[(2-amino-4,5,6,7-tetrahydrobenzothiophenecarbonyl)amino]-2,6-difluorophenyl ]ethyl]benzoate hydrochloride A 30 mL scintillation vial is charged with methyl 4-[2-[4-[[2-(tert- butoxycarbonylamino)-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluorophenyl ]ethyl]benzoate (351 mg, 0.61 mmol) and DCM (6 mL). The resulting light yellow solution is degased by g through a gentle stream of N2. A solution of 4 N HCl in e (155 mL, 6.1 mmol) is added drop wise over 5 min and the resulting solution is stirred at RT for 12 hr. The reaction mixture is trated in vacuo and the resulting pale yellow residue is triturated with a minimal amount of DCM. The resulting solid is collected by filtration and dried under vacuum to afford the title compound as an off-white powder (337 mg, 99% yield). 1H NMR (399.8 MHz, DMSO-d6) δ 1.73-1.75 (m, 4H), 2.40-2.44 (m, 2H), 2.56-2.59 (m, 2H), 2.85 (s, 4H), 3.80 (s, 3H), 7.26-7.28 (m, 4H),7.81-7.83 (m, 2H), 9.21 (s, 1H). LC-ES/MS (m/z) 571 [M+1].
Preparation 8 methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate A suspension of methyl 4-[2-[4-[(2-amino-4,5,6,7-tetrahydrobenzothiophene yl)amino]-2,6-difluoro-phenyl]ethyl]benzoate hydrochloride (2.43 g, 4.55 mmol) in DCM (80 mL) in a 250mL round bottom flask is cooled to 0 oC with an ice/water bath.
Pyridine (0.92 mL, 11 mmol) is added drop wise with stirring over 5 min. The resulting pale yellowish solution is stirred for an additional 5 min at 0 oC, and a solution of 3- (chloromethyl)benzoyl chloride (0.71 mL, 5.0 mmol) in DCM (20 mL) is added drop wise over 5 min. The reaction mixture is stirred for additional 30 min at 0 oC. The reaction mixture is diluted with 150 mL of 10% aqueous citric acid and stirred at RT for 1 hr. The organic layer is separated and the aqueous layer is extracted twice more with DCM (50 mL each). The combined c extracts are washed sequentially with 5% aqueous NaHCO3 (2 x 50 mL) and saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and ated to dryness under reduced pressure. The resulting residue is triturated with EtOH (30 mL), and the resulting solid is ted by filtration, washed with EtOH (15 mL), and dried under vacuum to yield the title compound as a tan solid (2.61 g, 92% yield). 1H NMR (400.1 MHz, DMSO-d6) δ), 1.73-1.81 (m, 4H), 2.68 (m, 4H), 2.92 (s, 4H), 3.84 (s, 3H), 4.83 (s, 2H), 7.32 (d, J = 8.3 Hz, 2H) 7.38-7.344 (m, 2H), 7.56 (t, J = 7.7 Hz, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.82 (d, J = 7.9 Hz, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.96 (br s, 1H), 10.10 (s, 1H), 11.34 (s, 1H). MS (m/z-) 621 [M-1].
Preparation 9 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin- 1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate N O H H N N F O O A 2 mL microwave vial is charged with methyl 4-[2-[4-[[2-[[3- (chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate (50 mg, 0.08 mmol), ydroxybutyl)-3,3-dimethylpiperazinecarboxamide hydrochloride (42.7 mg, 0.16 mmol) and DIPEA (0.056 mL, 0.32 mmol) in a mixture of ACN (1.5 mL) and MeOH (50 μL). The resulting yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 4 hr. The reaction mixture is concentrated in vacuo and the e is ioned between 5% aqueous NaHCO3 (75 mL) and DCM (25 mL). The organic layer is separated, the aqueous layer is extracted with twice more with DCM (25 mL each), and the combined organic extracts are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and evaporated to dryness under reduced pressure. The ing residue is purified by reverse phase chromatography over C-18 silica, eluting with a gradient of 0-100% of a mixture of 5% HCOOH in H2O/ACN, to afford the title compound as a light yellow foamy solid (30.2 mg, 46% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.05 (s, 6H), 1.37- 1.44 (m, 4H), .85 (m, 4H), 2.25-2.27 (m, 2H), 2.65-2.677 (m, 4H), 2.91 (br s, 4H), 2.96-3.05 (m, 2H), 3.12 (s, 2H), 3.17-3.26 (m, 2H), 3.35-3.41 (m, 2H), 3.51 (s, 2H), 3.84 (s, 3H), 4.36 (t, J = 5.1 Hz, 1H), .39 (m, 1H), 7.32 (d, J = 8.3 Hz, 2H), 7.51-7.56 (m, 4H), 7.70-7.80 (m, 1H), 7.87 (m, 3H), 10.01 (br s, 1H), 11.45 (br s, 1H). LC-ES/MS (m/z) 816 [M+1].
Preparation 10 tert-butyl 4-[[3-[[3-[[3,5-difluoro[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]- 4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine carboxylate S NH O N N O A 20mL microwave reaction vessel is charged with methyl 4-[2-[4 -[[2-[[3- (chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- ro-phenyl]ethyl]benzoate (1.85 g, 2.97 mmol), tert-butyl methylpiperazine carboxylate (0.91 g, 4.16 mmol), and DIPEA (2.07 mL, 11.9 mmol) in 15 mL of ACN. The resulting yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 4 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned n 5% aqueous NaHCO3 (150 mL) and DCM (50 mL). The organic layer is separated, the aqueous layer is extracted with DCM (2 x 25 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness under d pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-100% of a mixture of 9:1 DCM/acetone in hexane, to afford the title compound as a yellow solid (1.63 g, 69% yield) after t evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.04 (s, 6H), 1.38 (s, 9H), 1.81-1.74 (m, 4H), 2.28 (t, J= 5.0 Hz, 2H), 2.70 (br s, 4H), 2.91 (s, 4H), 3.13 (s, 2H), 3.26 (s, 2H), 3.52 (s, 2H), 3.84 (s, 3H), 7.31 (d, J= 8.3 Hz, 2H), 7.44-7.39 (m, 2H), 7.55-7.46 (m, 2H), 7.75 (d, J= 7.6 Hz, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 801 [M+1].
Preparation 11 methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride Cl H O Cl H S NH A solution of tert-butyl 4-[[3-[[3-[[3,5-difluoro[2-(4- methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (1.13 g, 1.41 mmol) in DCM (14.1 mL) is stirred at RT and 4 N HCl in dioxane (3.5 mL, 14.1 mmol) is added via syringe. Upon complete addition, the on is stirred at RT overnight and concentrated to dryness under d pressure. The solid is triturated with DCM/Et2O, the ing precipitate is collected via vacuum filtration, and the filter cake is dried in a vacuum oven at 50°C to afford the title compound as a white solid (0.93 g, 1.20 mmol, 85% yield). LCES /MS (m/z) 701 [M+1].
Preparation 12 methyl 4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- ro-phenyl]ethyl]benzoate O O F O O N S N NH N O O To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (190.8 mg, 0.24 mmol) and TEA (0.165 mL, 1.18 mmol) in 4 mL of THF, bis(2-methoxyethyl)sulfamoyl chlorid e (87 mg, 0.36 mmol) in 4 mL of THF is added drop wise. The resulting mixture is heate d to 50 °C for 18 hr. After cooling to RT, the reaction mixture is diluted with a mixture of 5% aqueous NaHCO3 (75 mL) and DCM (25 mL). The layers are separated, and the aqueous layer is washed with additional DCM (2 x 25 mL). The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over ous Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue is purified by tography over silica, eluting with a gradient of 10-30% of a mixture of acetone in hexanes, to afford the desired product as a light yellow solid (150.1 mg, 70.8% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d6) δ 1.13 (s, 6H), .70 (m, 4H), 2.43-2.36 (m, 2H), 2.75-2.65 (m, 4H), 3.03-2.86 (m, 8H), 3.24 (s, 6H), .32 (m, 4H), 3.44 (t, J= 5.8 Hz, 4H), 3.54 (s, 2H), 3.84 (s, 3H), 7.32 (d, J= 8.3 Hz, 2H), 7.52-7.38 (m, 3H), 7.56 (d, J= 7.6 Hz, 1H), 7.74 (d, J= 7.6 Hz, 1H), 7.91-7.84 (m, 3H), 10.00 (s, 1H), 11.47 (s, 1H). LC-ES/MS (m/z) 896 [M+1].
Preparation 13 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-methoxyoxo-butyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate O O F O F OH O NH N To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl] amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl] benzoate dihydrochloride (148.4 mg, 0.1688 mmol), DIPEA (60 μ L, 0.34 mmol) and 4- oxobutanoic acid methyl ester (40 mg, 0.34 mmol) in 4 mL of DCM is added AcOH (0.01 mL), followed by sodium triacetoxyborohydride (0.073 g, 0.34 mm ol). The resulting reaction e is allowed to stir at RT for 12 hr. The reaction mixture is diluted with a mixture of 5% aqueous NaHCO3 (75 mL ) and DCM (25 mL). The layers are separated, and the aqueous layer is washed with additional DCM ( 2 x 25 mL). The c extracts are ed, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue is ed by chromatography over silica, using a gradient of 35-40% of a mixture of 9:1 EtOH/DCM in hexane, to afford the desired product as a light yellow solid (132.1 mg, 91.8% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.09 (s, 6H), 1.70-1.58 (m, 2H), 1.87-1.70 (m, 4H), 2.20-2.09 (m, 2H), 2.36-2.27 (m, 4H), 2.63-2.54 (m, 2H), 2.75-2.64 (m, 4H), 2.92 (s, 4H), 3.30-3.28 (m, 2H), 3.65-3.53 (m, 5H), 3.84 (s, 3H), 7.32 (d, J= 8.3 Hz, 2H), 7.58-7.37 (m, 4H), .69 (m, 1H), 7.91-7.83 (m, 3H), 9.99 (s, 1H), 11.45 (s, 1H).
LC-ES/MS (m/z) 801 [M+1].
Preparation 14 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate O O O NH N To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl] -4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl] benzoate dihydrochloride (0.108 g, 0.13 mmol), 3-methoxypropion aldehyde (0.023 g, 0.25 mmol), and DIPEA (0.043 mL, 0.25 mmol) in 4 mL of DCM, AcOH (0. 01 mL) is added, followed by sodium triacetoxyborohydride (0.053 g, 0.25 mmol). The resulting on mixture is stirred at RT for 12 hr. The reaction e is diluted with of 5% aqueous NaHCO3 (75 mL)and of DCM (25 mL). The layers are separated, and the aqueous layer is washed with onal DCM (2 x 25 mL). The c extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, using a gradient of 25-80% acetone in hexane, to afford the desired product as a light yellow solid (26.2 mg, 27.6% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 11.44 (m, 1H), 9.98 (m, 1H), 7.92-7.82 (m, 3H), 7.76-7.70 (m, 1H), 7.57-7.38 (m, 4H), 7.36-7.27 (m, 2H), 3.84 (s, 4H), 3.27-3.16 (m, 4H), 2.97-2.87 (m, 4H), 2.76-2.64 (m, 4H), 2.37-2.28 (m, 2H), 2.25-2.15 (m, 2H), 1.90-1.71 (m, 4H), 1.67-1.54 (m, 2H), 1.09 (s, 6H).
LC-ES/MS (m/z) 773 [M+1].
Preparation 15 methyl 4-[2-[4-[[2-[[3-[[4-[2-(tert-butoxycarbonylamino)acetyl] -2,2-dimethyl-piperazin hyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate N F N O To a round bottom flask is added methyl 4-[2-[4-[[2 -[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (500 mg, 1.4 mmol), DMF (25 mL), and 1,8- diazabicyclo ]undecene (0.19 mL, 1.27 mmol) . The mixture is stirred briefly and treated with HOBT (103 mg, 0.67 mmol), EDCI (250 mg, 1.6 mmol), and N-(tertbutoxycarbonyl )glycine (132 mg, 0.75 mmol). The mixture is sti rred at RT for 18 hours.
The mixture is diluted with water (25 mL) and the resulting slurry is d at RT for 3 hr.
The resulting light yellow solid is collected by filtration, washed with water, and air-dried.
The resulting powder is ved in DCM, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue is purified by chromatography over silica, eluting with 15% EtOAc in DCM for 20 min then 25% EtOAc in DCM, to afford the title compound (451 mg, 38% yield) after solvent ation. 1H NMR (400.13 MHz, DMSO) δ 11.52-11.46 (m, 1H), 9.99 (s, 1H), 7.90-7.86 (m, 3H), .74 (m, 1H), 7.57-7.55 (m, 1H), 7.52-7.47 (m, 1H), 7.44-7.39 (m, 2H), 7.33 (d, J= 7.6 Hz, 2H), 6.76-6.71 (m, 1H), 3.84 (s, 3H), 3.81-3.76 (m, 2H), 3.57-3.54 (m, 2H), 3.43-3.38 (m, 2H), 3.32 (s, 12H, water), 3.28- 3.22 (m, 2H), 2.91 (s, 4H), 2.70 (s, 4H), 2.51-2.50 (m, 16H, DMSO), 2.39-2.35 (m, 2H), .81 (m, 4H), .35 (m, 10H), 1.29-1.25 (m, 1H), 1.07 (d, J= 21.1 Hz, 6H). LCES /MS (m/z) 858 [M+1].
Preparation 16 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin- 1-yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate N N O S N O NH In a 30 mL scintillation vial, a solution of methyl 4-[2-[4-[[2-[[3-[(2,2- dimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride (120 mg, 0.155 mmol) and TEA (63 mg, 0.62 mmol) in DCM (3 mL) is stirred at RT as a solution of 1-isocyanato- oxy-butane (30 mg, 0.23 mmol) in DCM (1 mL) is added via syringe. The resulting reaction mixture is allowed to stir at RT for 4 hours. The reaction mixture is concentrated under reduced pressure and purified by chromatography over silica, eluting with a gradient of 0-100% EtOAc/hexanes, to afford the title compound as a tan foam (102 mg, 79% yield) after t evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.03 (s, 6H), 1.35-1.45 (m, 4H), 1.68-1.82 (m, 4H), 2.21-2.25 (m, 2H), 2.63-2.70 (m, 4H), 2.89 (s, 4H), 2.95-3.02 (m, 2H), 3.095 (s, 2H), 3.18 (s, 3H); 3.15-3.22 (m, 2H), 3.25-3.30 (m, 2H), 3.48 (s, 2H), 3.815 (s, 3H), 6.334 (t, J = 5.5 Hz, 1H), 7.30 (d, J = 7.9 Hz, 2H), 7.35-7.42 (m, 2H), 7.42-7.50 (m, 1H), ), 7.50-7.55 (m, 1H), 7.71 (d, J= 7.6 Hz, 1H), 7.83-7.89 (m, 3H), 9.981 (s, 1H), 11.44 (s, 1H). LC-ES/MS (m/z) 830 [M+1].
Preparation 17 4-[2-[4-[[2-[[3-[(4-tert-butoxycarbonyl-2,2-dimethyl-piperazinyl)methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid O F S NH O N N O A 60 mL llation vial is charged with tert-butyl 4-[[3-[[3-[[3,5-difluoro[2-(4- methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (3.92 g, 4.8 mmol), lithium hydroxide (570 mg, 24 mmol), THF (20 mL), MeOH (10 mL) and H2O (10 mL) and the resulting suspension is stirred at RT for 12 hr. The reaction e is diluted with H2O (40 mL) and concentrated in vacuo to ~ ½ volume. The pH of the resulting mixture is adjusted to ~ 5-6 with 10 % aqueous citric acid and the resulting colorless suspension is partitioned n 150 mL of water and 50 mL of 4:1 chloroform/isopropanol. The organic layer is separated, the pH of the aqueous layer is adjusted again to pH ~5 with 10 % s citric acid, and the e is extracted twice with additional 4:1 chloroform/isopropanol (2 x 50 mL). The organic layers are combined, washed with saturated aqueous NaCl, dried over anhydrous , filtered, and the filtrate is concentrated under reduced pressure to give the title compound (3.7 g, >99% yield) as an off-white solid that may be used in the subsequent step without additional purification. LC-ES/MS (m/z) 787 [M+1].
Preparation 18 tert-butyl 4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]- 4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine carboxylate O F S NH O N N O A 500mL round bottom flask is charged with a on of 4-[2-[4-[[2-[[3-[(4-tertbutoxycarbonyl-2 ,2-dimethyl-piperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid (3.50 g, 4.45 mmol) and DIPEA (3.1 mL, 17.8 mmol) in DCM (75 mL). To this solution solid bis(2- oxooxazolidinyl)phosphinic chloride (1.4 g, 5.3 mmol) is added in small portions over 10 min and the resulting suspension is stirred at RT for 10 min. A 0.5 M solution of NH3 in 1,4- dioxane (40 mL, 22.25 mmol) is added in one portion and the resulting suspension is stirred for 2 h at RT. Additional bis(2-oxooxazolidinyl)phosphinic chloride (0.7g, 2.7 mmol) is added in small portions over 5 min and the resulting suspension is left to stir for 12 h at RT. onal bis(2-oxooxazolidinyl)phosphinic chloride (0.7g, 2.7 mmol) is again added in small portions over 5 min and the resulting suspension is left to stir for 12 h at RT. The resulting on mixture is partitioned between 300 mL of 5% aqueous NaHCO3 and 50 mL of DCM. The layers are separated, and the aqueous layer is extracted twice with additional DCM (2 x 100 mL). The combined c layers are washed with saturated aqueous NaCl, dried over anhydrous Na2SO4, ed, and concentrated to dryness in vacuo. The resulting yellow foamy residue is ed by chromatography over silica, eluting with a gradient of 10-12% acetone/DCM ; the mobile phase is then switched to 35% MeOH / DCM. The collected fractions containing desired t are combined and concentrated under reduced pressure and the residue is triturated with DCM. The resulting solid is collected by filtration and dried under vacuum to afford the title compound. The trituration filtrate is recovered and evaporated to dryness in vacuo. The resulting residue is purified by chromatography over silica, eluting with a gradient of 5-10% MeOH in DCM and the ted fractions containing desired product are combined and evaporated and added to the material obtained from the first purification to give the title nd as a light yellow solid (2.43 g, 70% yield). 1H NMR (400.1 MHz, DMSO-d6) δ 1.05 (s, 6H), 1.39 (s, 9H), 1.68-1.90 (m, 4H), 2.29 (m, 2H), 2.70 (m, 4H), 2.89 (m, 4H), 3.14 (s, 2H), 3.28 (s, 2H), 3.51 (s, 2H), 7.16-7.34 (m, 3H), 7.36- 7.58 (m, 4H), 7.68-7.73 (m, 3H), 7.90 (m, 2H), 10.00 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 786 [M+1].
Preparation 19 N-[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl][[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarboxamide dihydrochloride N F H Cl H NH Cl H A solution of tert-butyl 4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluorophenyl ]carbamoyl]-4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3- dimethyl-piperazinecarboxylate ( 2.43 g, 3.1 mmol) in a mixture of DCM (80 mL) and MeOH (8 mL) in a 250 mL bottom flask is thoroughly ed under nitrogen, and a 4 N solution of HCl in 1,4-dioxane (16 mL, 62 mmol) is added drop wise over 10 min. The resulting solution is stirred for 12 h at RT. Volatiles are removed in vacuo to give the title compound as a yellow, hygroscopic solid (2.45 g, >99%) which may be used in the next step without further purification.
Preparation 20 methyl 4-[2-[2,6-difluoro[[2-[[3-[(2,2,4-trimethylpiperazinyl)methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoate F O S H F A 5mL microwave reaction vessel is charged with methyl 4-[2-[4 -[[2-[[3- omethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate (225 mg, 361 mmol) and a solution of 1,3,3- trimethylpiperazine (65 mg, 0.488 mmol) and DIPEA (0.25 mL, 1.4 mmol) in 3 mL of ACN.
The resulting yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 3 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned n 5% aqueous NaHCO3 (75 mL) and DCM (25 mL). The c layer is separated, the aqueous layer is extracted with DCM (2 x 25 mL). The combined organic layers are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The resulting e is purified by chromatography over silica, eluting with a gradient of 20-50% of a mixture of 10% 7N NH3/MeOH in dcm in MTBE, to afford the title compound as a light yellow solid (195 mg, 76% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6): δ 1.11 (two s, 6H).1.90-1.64 (m, 4H), 2.25-1.94 (m, 4H), 2.40-2.26 (m, 2H), 2.50 (under residual dmso resonance, 3H), 2.79-2.60 (br s, 4H), 2.92 (s, 4H), .76 (br, 2H), 3.84 (s, 3H), 7.32 (m, 2H), 7.60-7.36 (m, 4H), 7.79-7.67 (m, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.46 (s, 1H). LCES /MS (m/z) 715 [M+1]. ation 21 tert-butyl 4-[(3-methoxyoxo-propyl)carbamoyl]-2,2-dimethyl-piperazinecarboxylate mL scintillation vial is charged with a solution of tert-butyl 2,2-dimethylpiperazine carboxylate (500 mg, 2.29 mmol) in DCM (12 mL) and cooled down in ice bath. While cooling DIPEA (1.20 mL, 6.86 mmol) is added followed by a solut ion of methyl 3- isocyanatopropanoate (404 mg, 2.97 mmol) in DCM (3 mL) added dr opwise over 5min. The resulting mixture is allowed to warm up to rt and stirred at rt for 15min. The rxn mixture is diluted with 5% aqueous citric acid (100 mL) and DCM (25 mL), and the layers are separated. The aqueous layer is extracted with DCM (2 x 25 mL) . The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous , filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 30-53% of e in s, to afford the title com pound as a colorless thick oil (726 mg, material contains ~10% of residual DCM, 83% yield) after solvent ation. 1H NMR (399.8 MHz, CDCl 3): δ 1.34 (s, 6H), 1.45 (s, 9H), 2.58-2.48 (m, 2H), 3.37 (t, J= 5.7 Hz, 2H), 3.54-3.43 (m, 4H), 3.75-3.62 (m, 5H), .99 (m, 1H). LC-ES/MS (m/z) 344 [M+1].
Preparation 22 methyl 3-[(3,3-dimethylpiperazinecarbonyl)amino]propanoate hydrochloride To a 20 mL scintillation vial containing a solution of tert-but yl 4-[(3-methoxyoxopropyl moyl]-2,2-dimethyl-piperazinecarboxylate (250 mg , 0.65 mmol) in DCM (7 mL), 4 N hydrochloric acid in dioxane (1.63 mL, 6.54 mmol) is a dded dropwise with stirring.
The resulting suspension is stirred at rt for 1 hr, concentrated in vacuo and the residue is dried under v accum to to obtain the title compound as white hygroscopic solid which is used in the next step without as it is. LC-ES/MS (m/z) 244 [M+1].
Preparation 23 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-[(3-methoxyoxo-propyl)carbamoyl]-2,2-dimethylpiperazinyl l]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate A 5mL microwave reaction vessel is charged with methyl 4-[2-[4 [3- (chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate (180 mg, 0.29 mmol), a solution of methyl 3-[(3,3- dimethylpiperazinecarbonyl)amino]propanoate hydrochloride (1 62 mg, 0.58 mmol) and N,N-DIPEA (0.202 mL, 1.16 mmol) in a mixture of 3 mL of ACN and 0.5 mL of MeOH.
The ing yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 3 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% s NaHCO3 (75 mL) and DCM (25 mL). The organic layer is separated; the aqueous layer is extracted with DCM (2 x 25 mL). The combined organic layers are washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over C-18 s ilica, eluting with a gradient of 15-80% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2 O for 15 minutes, to obtain the title compound as a pale orange solid (81 mg, 34% yield) after solvent evaporation. LC-ES/MS (m/z) 830 [M+1].
Preparation 24 tert-butyl 3-[[3-[[3-[[3,5-difluoro[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]- 4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,8- icyclo[3.2.1]octanecarboxylate O F O S N O NH H O F A 20mL microwave on vessel is charged with methyl 4-[2-[ 4-[[2-[[3- (chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate (1.80 g, 2.89 mmol) and a solution of tert-butyl 3,8- diazabicyclo ]octanecarboxylate (760 mg, 3.47 mmol) and DIPEA (1.01 mL, 5.78 mmol) in ACN (12 mL). The resu lting yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 1 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% s NaHCO3 (150 mL) and DCM (50 mL). The organic layer is ted, the aqueous layer is extracted with DCM (2 x 50 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to s under reduced re. The resulting residue is purified by chromatography over silica, eluting with a gradient of 15-45% of EtOAc in hexanes to afford the title compound as a light yellow solid (2.23 g, 97% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6): δ 1.38 (s, 9H), 1.95-1.60 (m, 8H), 2.16 (d, J= 10.0 Hz, 2H), 2.57 (d, J= 10.0 Hz, 1H), 2.70 (br s, 4H), 2.92 (s, 4H), 3.51 (s, 2H), 3.84 (s, 3H), 4.08-3.96 (m, 2H), 7.35-7.26 (m, 2H), 7.45-7.35 (m, 2H), 7.58-7.45 (m, 2H), .72 (m, 1H), 7.92-7.79 (m, 3H), 10.00 (s, 1H), 11.41 (s, 1H),. LC-ES/MS (m/z) 799 [M+1].
Preparation 25 methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octanylmethyl)benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate ochloride S F O NH H O F O N HCl Under N2, to a 250 mL RBF containing a solution tert-butyl [[3-[[ 3,5-difluoro[2- (4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,8-diazabicyclo[3.2.1]octanecarboxylate (2.23 g, 2.79 mmol) in DCM (45 mL), 4 N hydrochloric acid in dioxane (7.0 mL, 28 mmol) is added dropwise with stirring. The resulting suspension is stirred at rt for 12 hr, concentrated in vacuo and the residue is dried under vacuum to afford the title compound as light yellowish solid which is used in the next step without further purificati on. LC-ES/MS (m/z) 699 [M+1].
Preparation 26 methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoate O F S N mL scintillation vial is charged with methyl 4-[[2-[[3 -(3,8- diazabicyclo[3.2.1]octanylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate ochloride (1.04 g, 1.27 mmol), DCM (8 mL) and TEA (0.89 mL, 6.4 mmol). The resu lting suspension is agitated at rt until all solids dissolved. A solution of methylaminoformyl chlorid e (144 mg, 1.46 mmol) in 2 mL of DCM is added dropwise with stirring and stirring is conti nued for additional 15 min.
The rxn mixture is diluted with 5% aqueous NaHCO3 (150 mL) and DCM (50 mL), and the layers are separated. The aqueous layer is extracted with DCM (2 x 50 mL). The c extracts are combined, washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resu lting residue is purified by chromatography over C-18 silica, eluting with a gradient of 10-20% of a mixture of 5% MeOH in 10 mM ammonium bicarbonate in ACN over 5 min and 20-80% of a e of % MeOH in 10 mM ammonium bicarbonate in ACN over 15 minutes, to obtain the title nd as an off-white solid (53 mg, 68% yield) after solvent evaporation. The title compound was isolated as a minor component of the mixture as a yellowish green solid (177 mg, 17% yield). The material is used in the next step t further purification. LCES /MS (m/z) 752 [M+1]. ation 27 methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate S NH O NH A solution of tert-butyl 4-[[3-[[3-[[3,5-difluoro[2-(4- methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (480 mg, 0.599 mmol) in DCM (5 mL) is stirred at RT and 4 N HCl in e (4 mL, 16 mmol) is added via syringe. Upon complete addition, the reaction is stirred at RT for 2 hours, during which time a precipitate formed. The resulting mixture is concentrated in vacuo to a light yellow solid then further dried under high vacuum at ambient temperature for 16 h to yield 464 mg of the intermediate hydrochloride salt as a light yellow solid. This solid is partitioned n saturated sodium bicarbonate and ethyl acetate. The layers are separated and the aqueous portion washed with an additional portion of ethyl e. The combined organic extracts are dried over sodium sulfate, decanted, then concentrated in vacuo and dried under vacuum at 55 °C for 16 h to yield the title product as a yellow-brown solid (323 mg, 0.461 mmol, 77% yield). LC-ES/MS (m/z) 701 [M+1].
Preparation 28 methyl 4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate S NH N O A solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- ro-phenyl]ethyl]benzoate (226 mg, 0.322 mmol) in dichloromethane (10 mL) is stirred at RT and treated with acetyl chloride (46 μL, 0.645 mmol). To the resulting mixture is slowly added saturated aqueous sodium bicarbonate solution (5 mL). The mixture is stirred rapidly at r.t. for 1h, and then diluted with saturated aqueous sodium bicarbonate solution (10 mL) and dichloromethane (15 mL). The layers are separated and the organic layer dried with magnesium sulfate. The mixture is filtered and the filter cake with dichloromethane. The resulting filtrate is dried in vacuo. The crude product is purified via normal phase flash chromatography, eluting with 3:1 ethyl e/hexanes under tic conditions, collecting ons at 240 nM. Product containing fractions are pooled and concentrated in vacuo to afford the title product as light yellow solids (169 mg, 227 mmol, 71% yield). LC-ES/MS (m/z) 741 [M-1].
Preparation 29 methyl 4-(2,6-difluoro(2-(3-(((1R,5S)pentanoyl-3,8-diazabicyclo[3.2.1]octan yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene carboxamido)phenethyl)benzoate S NH O N N O To a solution methyl 2-(3-(((1R,5S)-3,8-diazabicyclo[3.2.1]octan yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophenecarboxamido)-2,6- difluorophenethyl)benzoate dihydrochloride (200 mg, 0.24 mmol) and TEA (0.145 mL, 4.0 eq., 1.04 mmol) in 2.60 mL of DCM, is added penta noyl chloride dissolved in 0.5 mL DCM (87 mg, 1.5 eq., 0.39 mmol) via syringe. The resulting mixture is stirred at rt for 6 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between water (15 mL) and EtOAc (10 mL). The organic layer is separated, the aqueous layer is extracted with EtOAc (2 x 10 mL). The ed organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure to afford the desired t as a light yellow solid (214 mg, 100% yield). LCES /MS (m/z) 783 [M+1].
Preparation 30 methyl 4-(4-(2-(3-(((1R,5S)(dimethylglycyl)-3,8-diazabicyclo[3.2.1]octan yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophenecarboxamido)-2,6- difluorophenethyl)benzoate S NH O N N O A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan yl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluorophenyl ]benzoate;dihydrochloride (200 mg, 0.2592 mmol), 2-(dimethylamino)acetic acid (40 mg, 0.39 mmol), triethylamine (4 equiv., 1.037 mmol), EDCI (74 mg, 1.5 equiv., 0.39 mmol) and 1-hydroxyazobenzotriazole (53 mg, 1.5 equiv., 0.39 mmol) in DCM (2.6 mL) was stirred at rt overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water (15 mL) and EtOAc (10 mL). The organic layer is separated, the aqueous layer is ted with EtOAc (2 x 10 mL). The ed organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (0-5% MeOH/DCM) to afford the desired product as a pale yellow solid (135 mg, 66% yield). LC-ES/MS (m/z) 785 [M+1].
Preparation 31 methyl 4-[[3-[[3-[[3,5-difluoro[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]- 4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine carboxylate S NH O N N O To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (200 mg, 0.26 mm ol) and TEA (0.146 mL, 1.03 mmol) in 3 mL of DCM, methyl chloroformate (36 mg, 0.39 mm ol) in 1 mL of DCM is added drop wise. The ing mixture was stirred at room temperature overnight. The reaction mixture is diluted with a diluted with EtOAc and partitioned with water. The product was extracted with EtOAc. All organics were ed, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue is purified by chromatography over silica, g with a gradient of 0-30% of a mixture of EtOAc in hexanes, to afford the d product as a tan foam (157 mg, 80% yield) after solvent evaporation. LC-ES/MS (m/z) 757 [M-1].
Preparation 32 methyl 4-[2-[4-[[2-[[3-[[2,2-dimethyl(methylsulfamoyl)piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- ro-phenyl]ethyl]benzoate S NH O N To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (201 mg, 0.26 mm ol) and TEA (0.146 mL, 1.03 mmol) in 3 mL of DCM, N-methylsulfamoyl chloride (50 mg, 0 .39 mmol) in 1 mL of DCM is added drop wise. The resulting mixture was stirred at r oom temperature overnight.
The on mixture is diluted with a d with EtOAc and partitioned with water. The product was extracted with EtOAc. All organics were combined, washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting e is purified by chromatography over silica, eluting with a gradient of 0-50% of a mixture of EtOAc in hexanes, to afford the desired product as a tan foam (55 mg, 27% yield) after solvent evaporation. LC-ES/MS (m/z) 792 [M-1].
Preparation 33 methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]- 3,8-diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene- onyl]amino]phenyl]ethyl]benzoate S NH O N N O A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluorophenyl ]ethyl]benzoate;dihydrochloride (150 mg, 0.194 mmol), 1- (methoxymethyl)cyclopropanecarboxylic acid (30 mg, 0.233 mmol), triethylamine (4 equiv., 0.972 mmol), EDCI (56 mg, 1.5 equiv., 0.29 mmol) and 1-hydroxyazobenzotriazole (39 mg, 1.5 equiv., 0.29 mmol) in DCM (2.6 mL) was d at rt overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water and EtOAc.
The organic layer is separated, the aqueous layer is extracted with EtOAc. The combined organic layers are washed with ted aqueous NaCl, dried over anhydrous Na2SO4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by column chromatography (0-100% EtOAc/hexanes) to afford the desired product as a white foam (128 mg, 81% . LC-ES/MS (m/z) 810 [M-1]. ation 34 methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-[2-(4-methylpiperazinyl)acetyl]-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate S NH O N N O A solution of methyl 4-[2-[4-[[2-[[3-(3,8-diazabicyclo[3.2.1]octan ylmethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluorophenyl ]ethyl]benzoate;dihydrochloride (200 mg, 0.26 mmol), 2-(4-methylpiperazin yl)acetic acid (62 mg, 0.39 mmol), Hunig’s Base (0.17 g, 1.30 mmol) and HATU (127 mg, 0.32 mmol) in DMF (2.6 mL) was stirred at rt overnight. The reaction mixture is concentrated in vacuo and the residue is partitioned between water and EtOAc. The organic layer is separated, the aqueous layer is extracted with EtOAc. The combined organic layers are washed with saturated aqueous NaCl, dried over anhydrous Na2SO4, ed, and concentrated to s under reduced pressure. The e was purified by column chromatography (0-10% MeOH/DCM) to afford the desired product as a white foam (220 mg, 100% yield). LC-ES/MS (m/z) 840 [M+1].
Preparations 35 - 41 below are prepared in a manner substantially similar to Preparation 29.
Preparation 35 N F N O methyl 2,6-difluoro[[2-[[3-[[8-(tetrahydropyranylcarbamoyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate Prepared using 4-isocyantotetrahydropyran, 52% yield, MS (m/z) 824 [M-1] Preparation 36 N F N O methyl 4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octanyl)methyl]benzoyl]amino]- 7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate Prepared using acetyl chloride, 83% yield, MS (m/z) 741 [M+1] Preparation 37 N F methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate ed using 2-methoxyethanesulfonyl chloride, 64% yield, MS (m/z) 820 [M-1] Preparation 38 methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8- icyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate Prepared using 1-isocyanatomethoxy-ethane, 95% yield, MS (m/z) 800 [M+1] Preparation 39 methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate Prepared using 1-isocyanatomethoxy-butane, 90% yield, MS (m/z) 827 [M-1] Preparation 40 methyl 4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate Prepared using s(2-methoxyethyl)carbamoyl chloride, 54% yield, MS (m/z) 857 [M-1] Preparation 41 N F N O methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate Prepared using 2-methylpropanoyl chloride, 100 % yield, MS (m/z) 767 [M-1] Preparation 42 tert-butyl 4-[[3-[[3-[[3,5-difluoro[2-(4-methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]- 4,5,6,7-tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazine carboxylate A 20mL microwave reaction vessel is d with methyl 4-[2-[4 -[[2-[[3- (chloromethyl)benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate (1.85 g, 2.97 mmol), tert-butyl 3,3-dimethylpiperazine carboxylate (0.91 g, 4.16 mmol), and DIPEA (2.07 mL, 11.9 mmol) in 15 mL of ACN. The ing yellow suspension is heated in a BIOTAGE® Initiator microwave synthesizer at 110 °C for 4 hr. The reaction mixture is concentrated in vacuo and the residue is partitioned between 5% aqueous NaHCO3 (150 mL) and DCM (50 mL). The organic layer is ted, the aqueous layer is extracted with DCM (2 x 25 mL). The combined organic layers are washed with saturated aqueous NaCl (50 mL), dried over anhydrous Na2SO4, ed, and concentrated to dryness under reduced pressure. The resulting residue is purified by chromatography over silica, eluting with a gradient of 0-100% of a mixture of 9:1 DCM/acetone in hexane, to afford the title compound as a yellow solid (1.63 g, 69% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.04 (s, 6H), 1.38 (s, 9H), .74 (m, 4H), 2.28 (t, J= 5.0 Hz, 2H), 2.70 (br s, 4H), 2.91 (s, 4H), 3.13 (s, 2H), 3.26 (s, 2H), 3.52 (s, 2H), 3.84 (s, 3H), 7.31 (d, J= 8.3 Hz, 2H), 7.44-7.39 (m, 2H), 7.55-7.46 (m, 2H), 7.75 (d, J= 7.6 Hz, 1H), 7.87 (m, 3H), 9.99 (s, 1H), 11.49 (s, 1H). LC-ES/MS (m/z) 801 [M+1].
Preparation 43 methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate dihydrochloride A solution of tert-butyl [[3-[[3,5-difluoro[2-(4- methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (1.13 g, 1.41 mmol) in DCM (14.1 mL) is stirred at RT and 4 N HCl in e (3.5 mL, 14.1 mmol) is added via syringe. Upon complete addition, the reaction is stirred at RT ght and concentrated to dryness under reduced pressure. The solid is triturated with DCM/Et2O, the resulting precipitate is collected via vacuum filtration, and the filter cake is dried in a vacuum oven at 50°C to afford the title compound as a white solid (0.93 g, 1.20 mmol, 85% yield). LCES /MS (m/z) 701 [M+1].
Preparation 44 methyl 4-[2-[4-[[2-(tert-butoxycarbonylamino)benzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate N F O O A round bottom flask is d with 2-(tert-butoxycarbonylamin o)benzothiophene- 3-carboxylic acid (2.60 g, 8.87 mmol), methyl 4-amino-2,6 -difluoro-phenyl)ethyl]- benzoate (2.35 g, 8.07 mmol) and 30 mL of CH 2Cl2. The resulting suspension is cooled in an ice/water bath, and DIPEA (5.63 mL, 32.3 mmol) is added drop wise to afford a yellowbrown turbid solution. Solid bis-(2-oxooxazolidinyl)phosphinic de (2.57 g, 10.1 mmol) is added in small portions over 30 min at 0 oC. The reaction mixture is then warmed to RT and stirred for 48 hr. Additional solid 2-(tert-butoxycarbonylamino)benzothiophene carboxylic acid (1.3 g, 4.43 mmol) is added followed by additional DIPEA (2.8 mL) and solid bis-(2-oxooxazolidinyl)phosphinic chloride (1.3 g, 5.1 mmol) in small portions over min. The resulting turbid brown reaction mixture is stirred at RT for 12 hr. The reaction e is diluted with 40 mL of methylene chloride and then washed with 5% aqueous citric acid (150 mL), brine (2 x 25 mL). The organic layer is separated and dried over anhydrous Na2SO4, filtered, and trated under reduced pressure. The resulting brown foamy solid is purified by flash chromatography to yield the title compound (2.2 g, 48% yield). LCES /MS (m/z) 565 [M-1].
Preparation 45 methyl 4-[2-[4-[(2-aminobenzothiophenecarbonyl)amino]-2,6-difluoro- phenyl]ethyl]benzoate hydrochloride N F NH2 H Cl A round bottom flask is charged with methyl 4-[2-[4-[[2-(tertbutoxycarbonylamino )-benzothiophenecarbonyl]amino]-2,6-difluorophenyl ]ethyl]benzoate (2.2 g, 3.9 mmol) in 30 mL of CH 2Cl2. 4N HCl in dioxane (9.7 mL, 39 mmol) was added dropwise. The resulting mixture is allowed to stand at r.t. for 12 h. The light yellow suspension was concentrated to dryness under vacuum to yield 2.0 g (100%) of the title nd. LC-ES/MS (m/z) 467 [M+1].
Preparation 46 methyl 4-[2-[4-[[2-[[3-(chloromethyl)benzoyl]amino]benzothiophenecarbonyl]amino]- 2,6-difluoro-phenyl]ethyl]benzoate N F A round bottom flask is charged with methyl 4-[2-[4-[(2- enzothiophenecarbonyl)amino]-2,6-difluoro-phenyl]ethyl]benzoate;hydrochloride (2 g, 3.976 mmol) in 24 mL of CH oC in an ice 2Cl2. The resulting suspension is cooled to 0 bath. Pyridine (0.804 mL, 9.940 mmol) was added dropwise. To the resulting yellow suspension, a solution of 3-(chloromethyl)-benzoyl chloride (0. 622 mL, 4.374 mmol) in 6 mL of CH 2Cl2 was added dropwise over 5min to yield a dark yellow solution which is allowed to warm up to r.t. for 1 h. The on mixture is diluted with 75mL of 10% aq NaHCO3. The aqueous layer is washed with CH2Cl2 (2 x 25mL). The combined organic layer is washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product is triturated using 20 mL of EtOH. The resulting slurry is stirred at r.t. for 30 min and then ed to yield 2.2 g (89%) of the title compound as light yellow solid. LC-ES/MS (m/z) 617 [M-1]. ation 47 methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin- 1-yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoate N F N H A microwave flask is charged with methyl 4-[2-[4-[[2-[[3- (chloromethyl)benzoyl]-amino]benzothiophenecarbonyl]amino]-2,6-difluorophenyl ]ethyl]benzoate (0.98 g, 1.6 mmol), N-(4-hydroxybutyl)-3, 3-dimethyl-piperazine carboxamide;hydrochloride (0.59 g, 2.2 mmol) and a solution of DIPEA (1.1 mL, 6.3 mmol) in 12 mL of CH oC in microwave for 4 h. The 3CN. The reaction mixture is heated to 110 resulting yellow solution is cooled down to r.t. and concentrated to dryness under a vacuum.
The residue is partitioned n 25 mL of CH2Cl2 and 75 mL of 5% aqueous NaHCO3.
The organic layer is washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. 1.3 g (100%) of the title compound is yielded as a yellow foamy solid. LC-ES/MS (m/z) 812 [M+1].
Example 1 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid N O H H N N F O O A 30 mL scintillation vial is d with methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoate (196 mg, 0.24 mmol), lithium hydroxide monohydride (17.2 mg, 0.72 mmol), THF (4 mL), MeOH (2 mL) and H2O (2 mL). The ing suspension is stirred at RT for 12 hr. The reaction mixture is diluted with 4 mL of H2O and concentrated under reduced pressure to approximately ½ of the volume. An aqueous solution of 1 N HCl is added drop wise to provide a thick off-white suspension which is evaporated to dryness in vacuo. The resulting residue is purified by reverse phase chromatography over C-18 silica, eluting with a gradient of 0-100% of a mixture of 5% NH4HCO3 in H2O/ACN, to afford the title compound as a pale yellow solid (82.5mg, 41% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.05 (s, 6H) 1.37-1.42 (m, 4H), 1.74-1.85 (m, 4H), 2.25-2.30 (m, 2H), .76 (m, 4H), 2.91 (s, 4H), 2.96-3.02 (m, 2H), 3.12-3.15 (m, 2H), 3.17-3.25 (m, 2H), 3.35-3.40 (m, 2H), 3.51 (s, 2H), 4.36 (t, J = 5.1 Hz, 1H), 6.36 (t, J = 5.4 Hz, 1H), 7.30 (d, J = 8.3 Hz, 2H), 7.45-7.55 (m, 4H), 7.74-7.79 (m, 1H), 7.84-7.87 (m, 4H), 10.01 (s, 1H), 11.45 (s, 1H), 12.82 (br s, 1H).
LC-ES/MS (m/z) 802 [M+1].
A spray-dried powder solid dispersion of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid is prepared as an amorphous product containing 30% 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- ybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium / 70% PVP-VA (Polyvinylpyrrolidone-vinyl e). All materials are tested by ion exchange tography and are shown to be consistent with the ed stoichiometry. Cation exchange chromatography with evaporative light scattering detection (ELSD) is used to tate the levels of sodium in the active pharmaceutical ingredient (API) 4-[2-[2,6- difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid, and the solid dispersion formulation of this active pharmaceutical ingredient. Cation ge chromatography (HPLC) is performed under conditions as follows: ELSD: 60°C, Pump: 2.0mL/minute, Nitrogen:1.4L/min, Column Temp: 30°C, Column: PHENOMENEX® LUNA® 5μ SCX 100A (15cmx4.6mm, 5um), Injection Volume: 50uL, Mobile Phase A: 0.1M Ammonium Formate Buffer, pH 4.5, Mobile Phase B: 100% ACN, Run time: 4 minutes.
Scale-up of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2- dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid, disodium, is performed by placing 126 mg of 4- [2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, free base, in 5 mL of acetone at 60°C while stirring at 1000 rpm, resulting in a slurry of white solid. 18 µL of sodium hydroxide (2.17 equivalents) is added. The sample turns yellow, and polarized light microscopy shows a semi-amorphous solid. The yellow solid is isolated by vacuum filtration, giving a cake of canary yellow material. 102 mg is recovered. X-ray powder diffraction (XRD) shows a poorly crystalline solid.
The solid dispersion of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)- 2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, disodium, is formulated as 30% 4-[2-[2,6- difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid, disodium / 70% PVP-VA. The scale-up of a spray dried solid dispersion containing a 30% drug load of 2,6-difluoro[[2-[[3-[[4- (4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid is performed by placing 1040.5 mg of 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2- dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, free base, and 2426.7 mg of PVP-VA in 50 mL methanol. The al is stirred resulting in a white slurry. 0.519 mL of 5N sodium hydroxide (2.0 mole lents) is added to the slurry and bath sonicated until a clear yellow solution is formed. The solution is slowly pumped into a spray dryer with a stream of hot nitrogen ing in a solid powder that is collected and further dried in a vacuum oven at 50°C under vacuum overnight to dry.
Conditions for spray drying are as follows: Equipment Water Bath Oil Bath Nitrogen ng Temp. Final Temp.
Setting 60°C 200°C 60 psi 45°C 50°C The recovered spray dried material is observed to be microscopically non-birefringent particles of approximately 2.5μm in diameter.
Observed levels of sodium in the active pharmaceutical ingredient, and the solid dispersion formulation of the active pharmaceutical ingredient are shown below: Theoretical Observed Material % Sodium % Sodium (n=3) Example 1, um .43 5.59 salt e 1, disodium 1.63 1.73 salt, solid dispersion Example 2 4-[2-[2,6-difluoro[[2-[[3-[[4-(3-hydroxypropyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid HCO2H H N N To a solution of methyl 4-[2-[4-[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (214.6 mg, 0.28 mmol), DIPEA (0.097 mL, 0.55 mmol) in DCM (4 mL) in DCM (4 mL) are added 3-[(tert-butyl dimethylsilyl)oxy] propanal (55 mg, 0.28 mmol) and AcOH (25 μL). The resulting so lution is d to stir at RT for 30 min. Sodium triacet oxyborohydride (0.12 g, 0.55 mmol) is added in one portion.
The mixture is stirred at RT for 12 h, diluted with 10% aqueous NaHCO3 (75mL) and DCM (25mL), and the layers are separated. The s layer is extracted with DCM (2 x 25 mL).
The organic extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, ed, and concentrated under reduced pressure. The resulting residue is dissolved in DCM (3 mL) and 4 M HCl in 1,4-dioxane is added. The resulting solution is stirred at RT for 24 hr. The organic solvent is removed under reduced pressure to afford a yellow solid. The material is dissolved in MeOH (2 mL) and LiOH (0.36 g, 1.4 mmol) is added. The resulting mixture is stirred at RT for 16 h, diluted with H2O (4 mL), and concentrated under reduced pressure to ca. 50% of the volume. An aqueous solution of 1 N HCl is added (5 mL), and the mixture is concentrated under reduced pressure. The resulting residue is ed by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/CH3CN in 0.1% formic acid/H2O for 5 minutes, then a gradient of 20-50% of a mixture of 0.1% formic acid/CH3CN in 0.1% formic acid/H2O over 20 minutes, to obtain the title compound as mono-formic acid salt (73 mg, 33% yield) as a yellow solid after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6): δ 1.10 (s, 6H), 1.55 et, J= 6.7 Hz, 2H), .69 (m, 4H), 2.41-2.09 (m, 8H), 2.81-2.61 (m, 4H), 2.91 (s, 4H), 3.65-3.36 (m, 4H), 7.29 (d, J= 8.2 Hz, 2H), 7.57-7.36 (m, 4H), 7.75-7.72 (m, 1H), 7.94-7.80 (m, 3H), 8.15 (s, 1H), .1 (br, 3H). LC-ES/MS (m/z) 745 [M+1].
Example 3 4-[2-[4-[[2-[[3-[[4-[bis(2-methoxyethyl)sulfamoyl]-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid O F S NH N O O N S N O O A solution of methyl 4-[2-[4-[[2-[[3-[[4-[bis(2-met hoxyethyl)sulfamoyl]-2,2- dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (148.4 mg, 0 .17 mmol) and LiOH (0.016 g, 0.66 mmol) in a 2:1:1 mixture of OH:H 2 O (6 mL) is stirred at RT for 12hr.
The reaction mixture is diluted with H 2O (2 mL), concentrated under reduced pressure to ~ 1/3 volume, and 1 N HCl (4 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, g with a gradient of 15-20% of a e of 0.1% formic acid/CH3CN in 0.1% formic acid/H2O for 5 minutes, then a nt of 20-50% of a mixture of 0.1% formic acid/CH3CN in 0.1% formic acid/H2O over 20 minutes, to obtain the title compound as a yellow solid (99.0 mg, 67.8% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d6) δ 1.13 (s, 6H), 1.87-1.70 (m, 4H), 2.44-2.36 (m, 2H), 2.76-2.64 (m, 4H), 3.02- 2.91 (m, 8H), 3.24 (s, 6H), 3.36-3.31 (m, 4H), 3.44 (t, J= 5.8 Hz, 4H), 3.54-3.49 (br s, 2H), 7.30 (d, J= 8.3 Hz, 2H), 7.59-7.39 (m, 4H), 7.75 (d, J= 7.6 Hz, 1H), 7.90-7.81 (m, 3H), 10.01 (s, 1H), 11.47 (s, 1H), 12.83 (s, 1H). LC-ES/MS (m/z) 883 [M+1].
Example 4 4-[2-[4-[[2-[[3-[[4-(3-carboxypropyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid A mixture of methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-met -oxo-butyl)-2,2- dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzo- thiophene carbonyl]amino]phenyl]ethyl]benzoate (120.8 mg, 0.14 mmol) and LiOH (0.017 g, 0.71 mmol) in 2:1:1 mixture of THF:MeOH:H 2 O (4 mL ) is stirred at RT for 12 hr. The resulting solution is diluted with H2O (2 mL) and then concentrated to ~1/3 of the volume under reduced pressure. An aqueous solution of 1 N HCl (4 mL) is added, and the solvent is removed under reduced pressure. The resulting residue is purified by chromatography over C18 silica, eluting with a gradient of 15-20% of a e of 0.1% formic acid/CH3CN in 0.1% formic acid/H2O for 5 minutes, then a gradient of 20-50% of a mixture of 0.1% formic H3CN in 0.1% formic acid/H2O over 20 minutes, to obtain the title compound as a yellow solid (72.5 mg, 62.3% yield) after solvent ation. 1H NMR (400.1 MHz, DMSO-d6) δ 1.11 (s, 6H), 1.68-1.55 (m, 2H), 1.88-1.69 (m, 4H), 2.40-2.03 (m, 10H), 2.79- 2.61 (m, 4H), 2.91 (s, 4H), 3.70-3.40 (m, 2H), 7.29 (d, J= 8.3 Hz, 2H), 7.58-7.36 (m, 4H), 7.79-7.69 (m, 1H), 7.92-7.79 (m, 3H), 10.00 (br s, 1H), 11.45 (br s, 1H), 12.54 (br, 2H). LC- ES/MS (m/z) 773 [M+1].
Example 5 4-[2-[2,6-difluoro[[2-[[3-[[4-(3-methoxypropyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid, formic acid salt O F S NH O N O A e of methyl 2,6-difluoro[[2-[[3-[[4-(3-met hoxypropyl)-2,2- dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate (22.7 mg, 0.029 mmol) and LiOH (7 mg, 0.3 mmol) in a mixture of 2:1:1 THF:MeOH:H 2 O (4 mL) is stirred at RT for 16 hr. The resulting mixture is diluted with H2O (3 mL) and concentrated to ~1/2 volume under reduced pressure.
An aqueous solution of 1 N HCl (5 mL) is added, and the solvent is removed under reduced pressure. The resulting e is purified by chromatography over C-18 silica, eluting with a gradient of 15-70% over 10 minutes, to obtain the title compound as a yellow solid (16.2 mg, 68.3% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.11 (s, 6H), 1.68-1.55 (m, 2H), 1.88-1.69 (m, 4H), 2.43-2.07 (m, 6H), 2.79-2.62 (m, 4H), 2.91 (s, 4H), 3.20 (s, 3H), 3.62-3.23 (m,6H), 7.29 (d, J= 8.3 Hz, 2H), .36 (m, 4H), 7.79-7.68 (m, 1H), 7.85 (d, J= 8.2 Hz, 3H), 8.14 (s, 1H), 10.00 (br s, 1H), 11.45 (br s, 1H), 13.14- 12.45(br, 1H). LC-ES/MS (m/z) 759 [M+1].
Example 6 4-[2-[4-[[2-[[3-[[4-(2-aminoacetyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid To a 25 mL microwave reaction vial is charged methyl 4-[2-[4-[[ 2-[[3-[[4-[2-(tertbutoxycarbonylamino )acetyl]-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (0.451 g, 0.53 mmol), THF (12 mL), MeOH (8 mL), and 1 N LiOH in THF (2.5 mL, 2.5 mmol). The mixture is heated via ave at 100 °C for 30 min. To this e is added 1 N aqueous HCl (2.5 mL), and the resulting mixture is stirred under a nitrogen stream to evaporate volatiles. The resulting oily residue is dissolved in EtOAc (25 mL), diluted with saturated s NaCl (10 mL), and the layers ted. The organic layer is dried over MgSO4, filtered, and the filter cake washed with solvent. The filtrate is concentrated under d pressure to afford the crude title com pound (0.431 g) as a light yellow powder. To a round- bottomed flask is added the crude title com pound (0.422 g, 0.56 mmol), DCM (25 mL), and 4 N HCl in dioxane (1.25 mL, 5 mmol). The mixture is diluted with THF (10 mL) and stirred at RT under nitrogen for 24 hr, then for 4 h at 50 °C. At this point, an additional portion of 4 N HCl in dioxane (1.25 mL, 5 mmol) is added. Heat ing is continued for 2 hr, and the mixture is cooled to RT. The resu lting slurry is diluted with hexanes (50 mL) and filtered to collect crude product (364 mg). The crude material is dissolved in MeOH/DMSO and purified by reverse phase HPLC on a Waters E® 30 x 75 mm 5 μm C-18 OBD column, eluting with a gradient of 27-50% of a mixture of 10 mM aqueous um bicarbonate/ACN in MeOH at 85 mL/min over 6 min while monitoring at 205 and 237 nm. riate fractions are concentrated to s under reduced pressure and dried for 18 hr at 40 °C to obtain the title com pound (0.16 g, 43% yield). 1H NMR (400.13 MHz, DMSO) δ 7.93 (s, 1H), 7.88-7.79 (m, 3H), 7.46 (t, J= 6.3 Hz, 1H), 7.42-7.36 (m, 3H), 7.31-7.28 (m, 2H), 4.00-3.97 (m, 30H, broad, exchangeable protons), 2.89-2.86 (m, 4H), 2.86-2.78 (m, 3H), 2.58-2.54 (m, 2H), 2.51-2.50 (m, 18H, DMSO), 2.44-2.41 (m, 3H), 1.76-1.72 (m, 4H), 1.06 (d, J = 12.3 Hz, 6H). LC-ES/MS (m/z) 744 [M+1].
Example 7 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-methoxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid O F N O S NH O N NH A solution of methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-methoxybutylcarbamoyl)- 2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate (93 mg, 0.11 mmol) in a 3:2:1 mixture of THF:MeOH:water (3 mL) is stirred at RT as LiOH (14 mg, 0.56 mmol) is added in one portion. The ing reaction mixture is stirred at RT for 6 hours and concentrated in vacuo. The resulting residue is purified by reverse phase chromatography on a ENEX® GEMINI-NX® C-18 column, eluting with a nt of 23-57% of a mixture of 5% MeOH in 10mM aqueous ammonium bicarbonate (pH ~ 10) and ACN over 7 min, to afford the title compound as a light yellow foamy solid (83 mg, 91%) after t evaporation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.03 (s, 6H), 1.38-1.43 (m, 4H), 1.72-1.76 (m, 4H), 2.22-2.28 (m, 2H), 2.60-2.67 (m, 2H), 2.67-2.75 (m, 2H), 2.88 (s, 4H), 2.95-3.02 (m, 2H), 3.095 (s, 2H), 3.18 (s, 3H); 3.15-3.22 (m, 2H), 3.23-3.28 (m, 2H), 3.49 (s, 2H), 6.34 (t, J = 4.8 Hz, 1H), 7.27 (d, J = 7.9 Hz, 2H), 7.35-7.42 (m, 2H), 7.40-7.48 (m, 1H), ), 7.47- 7.52 (m, 1H), 7.72-7.77 (m, 1H), 7.81-7.88 (m, 3H), 10.01 (br s, 1H), 11.49 (br s, 1H), 12.75 (br s, 1H). LC-ES/MS (m/z) 816 [M+1].
Example 8 4-[4-[[3-[[3-[[4-[2-(4-carbamoylphenyl)ethyl]-3,5-difluoro-phenyl]carbamoyl]-4,5,6,7- tetrahydrobenzothiophenyl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinyl] oxo-butanoic acid N F DIPEA (0.1 mL, 0.55 mmol) is added to a solution of N-[4-[2-(4- carbamoylphenyl)ethyl]-3,5-difluoro-phenyl][[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarboxamide dihydrochloride (92.0 mg, 0.11 mmol) in DCM (4mL) followed by succinic anhydride (160 mg, 1.65 mmol) all at once. The resulting solution is stirred at RT for 20 min, onal succinic anhydride (53.4 mg, 0.05 mmol) is added, and the resulting mixture is stirred for an additional 20 min.
The reaction mixture is quenched by the addition of MeOH (0.5 mL), volatiles are removed in vacuo, and the resulting yellow oily residue is purified by reverse phase chromatography on a PHENOMENEX® GEMINI-NX® C-18 , eluting with a gradient of 20-70% of a mixture of 5% MeOH in 10mM aqueous ammonium onate (pH ~ 10) and ACN over 6 min, to afford the title compound as a light yellow solid (50.9 mg, 61% yield) after solvent ation. 1H NMR (400.1 MHz, DMSO-d 6) δ 1.03 (s, 3H), 1.09 (s, 3H), 1.62-1.89 (m, 4H), 2.28 (m, 1H), 2.30 (m, 1H), 2.40-2.47 (m, 2H), 2.58-2.81 (br m, 4H), 2.88 (s, 4H), 3.27 (s, 2H), 3.30 (m, 2H, partial overlap with residual water peak), 3.38 (br s, 2H), 3.54 (s, 2H), 7.19-7.31 (m, 3H), 7.31-7.59 (m, 3H), 7.78 (d, J= 8.1 Hz, 3H), 7.92-7.85 (m, 2H), 8.36 (br s, 1H), 8.56 (br s, 1H), 9.99 (br s, 1H), 11.51 (br s, 1H), 11.93 (br s, 1H). LC-ES/MS (m/z) 786 [M+1].
Example 9 4-[2-[2,6-difluoro[[2-[[3-[(2,2,4-trimethylpiperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid formic acid salt A solution of methyl 4-[2-[2,6-difluoro[[2-[[3-[(2,2,4-trimethylpiperazin hyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate (167 mg, 0.233 mmol) and l ithium hydroxide (22 mg, 0.93 mmol) in a 2:1:1 mixture of THF:MeOH:H 2 O (6 mL) is stirred at RT for 12 hr. The reaction mixture is diluted with water (2 mL), concentrated und er reduced pressure to ~ 1/3 volume, and 1 N HCl (3 mL) is added. The mixture is subsequently concentrated to s under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O for 5 minutes, then a nt of 20-50% of a mixture of 0.1% formic CN in 0.1% formic acid/H2O over 20 minutes, to afford the title compound as a yellow solid (122 mg, 68% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6): δ 1.11 (s, 6H), 1.89-1.67 (m, 4H), 2.15 (s, 3H), 2.41-2.30 (m, 4H), 2.70 (m, 4H), 2.91 (s, 4H), 4.10-3.10 (br, 4H), 7.29 (m, 2H), 7.57-7.36 (m, 4H), 7.75 (m, 1H), 7.90-7.81 (m, 3H), 8.15 (s, 1H), 10.8-9.7 (br, 1H), 12.9-11.1(br, 1H). LC-ES/MS (m/z) 701 [M+1].
Example 10 4-[[2-[[3-[[2,2-dimethyl(methylcarbamoyl)piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid S NH N O mL scintillation vial is charged methyl 4 -[[2-[[3-[(2,2-dimethylpiperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (145 mg, 0.187 m mol) and a solution of TEA (0.13 mL, 0.94 mmol) in DCM (2 mL). The resu lting suspension is stirred until it is clear. A solution of N-methylcarbamoyl chloride (21 mg, 0.22 m mol) in 1 mL of DCM is added dropwise. The ing solution is allowed to stir at rt for 15 min. The rxn mixture is diluted with 5% aqueous NaHCO3 (75mL) and DCM (25mL), and the layers are separated.
The aqueous layer is extracted with DCM (2 x 25 mL). The organi c extracts are combined, washed with saturated aqueous NaCl (25 mL), dried over anhydrous Na2SO4, ed, and concentrated under d pressure. The ing crude material and lithium hydroxide (23 mg, 0.94 mmol) are suspended in a 2:1:1 mixture of THF:MeOH:H 2 O (4 mL) and stirred at rt for 12 hr. The on mixture is diluted with water (3 mL), concentrated under reduced pressure to ~ 1/3 volume, and 1 N HCl (4 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting e is purified by chromatography over C-18 silica, eluting with a gradient of 10-15% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O for 5 minutes, then a gradient of 15-50% of a mixture of 0.1% formic CN in 0.1% formic acid/H2O over 20 minutes, to afford the title compound as a pale yellow solid (108 mg, 77% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d6): δ 1.06 (s, 6H), 1.88-1.65 (m, 4H), 2.27 (s, 2H), 2.55 (d, J= 4.3 Hz, 3H), 2.70 (m, 4H), 2.91 (s, 4H), 3.10 (s, 2H), 3.25-3.15 (m, 2H), 3.51 (s, 2H), 6.33 (m, 1H), 7.34-7.24 (m, 2H), 7.59-7.37 (m, 4H), 7.83-7.79 (m, 1H), 7.93-7.80 (m, 3H), 10.02 (br s, 1H), 11.46 (br s, 1H), 12.79 (br s, 1H). LC-ES/MS (m/z) 744 [M+1].
Example 11 4-[2-[4-[[2-[[3-[[2,2-dimethyl(methylcarbamothioyl)piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid S N S mL scintillation vial is charged with methyl 4-[2-[4-[[2-[[ 3-[(2,2-dimethylpiperazin hyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate dihydrochloride (165 mg, 0.212 m mol) and a solution of TEA (0.15 mL, 1.1 mmol) in DCM (3 mL). The resu lting suspension is stirred until it is clear. A solution of methyl isotiocyanate (18 mg, 0.24 mmol) i n 1 mL of DCM is added dropwise. The resulting solution is allowed to stir at rt for 15 min. The rxn mixture is diluted with 5% s NaHCO3 (75mL) and DCM (25mL), and the layers are separated.
The aqueous layer is extracted with DCM (2 x 25 mL). The organi c extracts are combined, washed with saturated s NaCl (25 mL), dried over anhydrous Na2SO4, filtered, and trated under d pressure. The resulting crude material and lithium hydroxide (26 mg, 1.1 mmol) are suspended in a 2:1:1 e of THF:MeOH:H 2 O (4 mL) and stirred at rt for 12 hr. The reaction mixture is diluted with water (3 mL), concentrated under reduced pressure to ~ 1/3 volume, and 1 N HCl (4 mL) is added. The mixture is subsequently trated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with a gradient of 15-20% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O for 5 minutes, then a gradient of 20-60% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O over 20 minutes, to afford the title compound as an off-white solid (73 mg, 45% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d6): δ 1.06 (s, 6H), 1.89-1.68 (m, 4H), 2.34 (m, 2H), 2.77-2.61 (m, 4H), 2.99-2.82 (m, 7H), 3.52 (s, 2H), 3.60 (s, 2H), 3.71 (br s, 2H), .22 (m, 2H), 7.53-7.35 (m, 3H), 7.68-7.53 (m, 2H), 7.80-7.68 (m, 1H), 7.94-7.80 (m, 3H), 10.02 (br s, 1H), 11.47 (br s, 1H), 12.79 (br s, 1H),. LC-ES/MS (m/z) 760 [M+1].
Example 12 4-[2-[4-[[2-[[3-[[4-(2-carboxyethylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoic acid A solution of methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-[(3-methoxyoxopropyl )carbamoyl]-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoate (80 mg, 0.097 mmol) and lithium hydroxide (12 mg, 0.49 mmol) in a 2:1:1 mixture of THF:MeOH:H 2 O (6 mL) is stirred at rt for 12 hr. The reaction mixture is diluted with H 2O (3 mL), concentrated under reduced pressure to ~ 1/3 volume, and 1 N HCl (2 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is ed by chromatography over C-18 , eluting with a gradient of 10-15% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O for 1 minutes, then a gradient of 15-50% of a mixture of 0.1% formic CN in 0.1% formic acid/H2O over 20 minutes, to obtain the title nd as an off-white solid (53 mg, 68% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d6): δ 1.05 (s, 6H), 1.88-1.66 (m, 4H), 2.31- 2.20 (m, 2H), 2.42- 2.31 (m, 2H), 2.79-2.60 (m, 4H), 2.91 (s, 4H), 3.11 (m, 2H), 3.27-3.15 (m, 4H), 3.51 (s, 2H), 6.48 (m, 1H), 7.35-7.23 (m, 2H), 7.60-7.36 (m, 4H), 7.80-7.68 (m, 1H), 7.94-7.80 (m, 3H), 10.02 (br s, 1H), 11.46 (br s, 1H), 12.78-12.15 (br, 1H). LC-ES/MS (m/z) 802 [M+1].
Example 13 4-[2-[2,6-difluoro[[2-[[3-[[8-(methylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, formic acid salt F OH S H F O O A solution of methyl 4-[2-[2,6-difluoro[[2-[[3-[[8-(methylcarbamoyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]benzothiophene carbonyl]amino]phenyl]ethyl]benzoate (100 mg, 0.13 mmol) and li thium hydroxide (15 mg, 0.63 mmol) in a 2:1:1 mixture of THF:MeOH:H 2 O (4 mL) is stirred at rt for 12 hr. The reaction mixture is diluted with water (3 mL), concentrated und er reduced pressure to ~ 1/3 volume, and 1 N HCl (3 mL) is added. The mixture is subsequently concentrated to dryness under reduced pressure, and the resulting residue is purified by chromatography over C-18 silica, eluting with 10% of a e of 0.1% formic CN in 0.1% formic acid/H2O for 5 minutes, then a nt of 10-65% of a mixture of 0.1% formic acid/ACN in 0.1% formic acid/H2O over 15 minutes, to afford the title compound as a yellowish tan solid (67 mg, 67% yield) after solvent evaporation. 1H NMR (400.1 MHz, DMSO-d 6): δ .57 (m, 2H), 1.97-1.79 (m, 2H), 2.19 (d, J= 10.2 Hz, 2H), 2.52 (overlaps with residual dmso nce, 2H), 2.56 (m, 3H), 2.94 (s, 4H), 3.53 (s, 2H), 4.13 (br s, 2H), 6.37 (m, 1H), 7.65-7.25 (m, 8H), 8.08-7.78 (m, 6H), 8.14 (s, 1H), 10.63 (s, 1H), 11.93 (s, 1H), 12.79 (br s, 1H). LCES /MS (m/z) 738 [M+1].
Example 14 4-[2-[2,6-difluoro[[2-[[3-[(4-methoxycarbonyl-2,2-dimethyl-piperazin yl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid S NH O N N O A 25 mL scintillation vial is charged with methyl 4-[[3-[[3-[[3,5-difluoro[2-(4- methoxycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (152 mg, 0.20 mmol), lithium hydroxide (25 mg, 5 mmol), THF (1.5 mL), MeOH (1.0 mL) and H2O (0.5 mL) and the resulting suspension is stirred at RT for 8 hr. The reaction mixture is evaporated, reconstituted in DMSO (2 mL) and purified on Phenomenex Kinetex EVO C18 column ing a gradient elution. (32-67% Acetonitrile/Aqueous 10mM um bicarbonate pH10/5% MeOH). Eluent was trated under reduced pressure to give the title compound (124 mg, 83% yield) as an off-white solid. LC-ES/MS (m/z) 746 [M+1].
Example 15 4-[2-[4-[[2-[[3-[[2,2-dimethyl(methylsulfamoyl)piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid S NH N NH O O A 25 mL llation vial is charged with methyl 4-[[3-[[3-[[3,5-difluoro[2-(4- ycarbonylphenyl)ethyl]phenyl]carbamoyl]-4,5,6,7-tetrahydrobenzothiophen yl]carbamoyl]phenyl]methyl]-3,3-dimethyl-piperazinecarboxylate (152 mg, 0.20 mmol), lithium hydroxide (25 mg, 5 mmol), THF (1.5 mL), MeOH (1.0 mL) and H2O (0.5 mL) and the resulting suspension is stirred at RT for 8 hr. The reaction mixture is evaporated, reconstituted in DMSO (2 mL) and purified on Phenomenex Kinetex C18 column utilizing a gradient elution. % Acetonitrile/Aqueous 10mM Ammonium bicarbonate pH10/5% MeOH). Eluent was concentrated under reduced pressure to give the title compound (124 mg, 83% yield) as an off-white solid. LC-ES/MS (m/z) 746 [M+1].
Example 16 4-(2,6-difluoro(2-(3-(((1R,5S)pentanoyl-3,8-diazabicyclo[3.2.1]octan yl)methyl)benzamido)-4,5,6,7-tetrahydrobenzo[b]thiophene carboxamido)phenethyl)benzoic acid S NH O N N O A 60 mL scintillation vial is charged with methyl 4-(2,6-difluoro(2-(3-(((1R,5S) pentanoyl-3,8-diazabicyclo[3.2.1]octanyl)methyl)benzamido)-4,5,6,7- tetrahydrobenzo[b]thiophenecarboxamido)phenethyl)benzoate (214 mg, 0.27 mmol), lithium hydroxide (33 mg, 6.0 eq., 1.37 mmol), THF (1.5 mL), MeOH (1.0 mL) and H2O (0.5 mL) and the resulting suspension is stirred at RT for 12 hr. The reaction mixture is diluted with H2O (5 mL) and concentrated in vacuo to ~ ½ volume. The pH of the ing mixture is adjusted to ~ 5-6 with 10 % aqueous citric acid and the resulting colorless suspension is ioned between 15 mL of water and 5 mL of 4:1 chloroform/isopropanol. The organic layer is separated, the pH of the aqueous layer is adjusted again to pH ~5 with 10 % aqueous citric acid, and the mixture is ted twice with additional 4:1 chloroform/isopropanol (2 x 15 mL). The organic layers are combined, washed with saturated aqueous NaCl, dried over anhydrous Na2SO4, filtered, and the filtrate is concentrated under reduced pressure to give the title compound (114 mg, 54% yield) as an ite solid. LC-ES/MS (m/z) 769 [M+1].
Examples 17 - 27 below are prepared in a manner ntially similar to Example 16.
Example 17 4-[2-[2,6-difluoro[[2-[[3-[[8-(tetrahydropyranylcarbamoyl)-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid 17% Yield, MS m/z 812 [M+1] Example 18 S NH O N 4-[2-[4-[[2-[[3-[(8-acetyl-3,8-diazabicyclo[3.2.1]octanyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate 85% yield, MS m/z 727 [M+1] Example 19 N F N O 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methoxyethylsulfonyl)-3,8-diazabicyclo[3.2.1]octan hyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate 70% yield, MS m/z 807 [M+1] Example 20 S NH H O N 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methoxyethylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate 28% yield, MS m/z 786 [M+1] e 21 4-[2-[2,6-difluoro[[2-[[3-[[8-(4-methoxybutylcarbamoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate 84% yield, MS m/z 815 [M+1] e 22 S NH O O O N 4-[2-[4-[[2-[[3-[[8-[bis(2-methoxyethyl)carbamoyl]-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]-2,6- difluoro-phenyl]ethyl]benzoate 23% yield, MS m/z 844 [M+1] Example 23 S NH O N 4-[2-[2,6-difluoro[[2-[[3-[[8-(2-methylpropanoyl)-3,8-diazabicyclo[3.2.1]octan yl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoate 62% yield, MS m/z 755 [M+1] Example 24 S NH O N 4-[2-[4-[[2-[[3-[[8-[2-(dime ino)acetyl]-3,8-diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid 73% yield, MS m/z 770 [M+1] Example 25 4-[2-[2,6-difluoro[[2-[[3-[[8-[1-(methoxymethyl)cyclopropanecarbonyl]-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene yl]amino]phenyl]ethyl]benzoic acid 98% yield MS m/z 797 [M+1] Example 26 N F O O N N N 4-[2-[2,6-difluoro[[2-[[3-[[8-[2-(4-methylpiperazinyl)acetyl]-3,8- diazabicyclo[3.2.1]octanyl]methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]phenyl]ethyl]benzoic acid 59% yield, MS m/z 825 [M+1] Example 27 4-[2-[4-[[2-[[3-[(4-acetyl-2,2-dimethyl-piperazinyl)methyl]benzoyl]amino]-4,5,6,7- tetrahydrobenzothiophenecarbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoic acid O F S NH O N O To a 25 mL microwave reaction vial is charged methyl methyl 4-[2-[4-[[2-[[3-[(4-acetyl- 2,2-dimethyl-piperazinyl)methyl]benzoyl]amino]-4,5,6,7-tetrahydrobenzothiophene carbonyl]amino]-2,6-difluoro-phenyl]ethyl]benzoate (169 mg, 0.227 mmol), tetrahydrofuran (12 mL, 148 mmol), methanol (8 mL, 197 mmol), and 1M aqueous lithium ide solution (1.14 mL, 1.14 mmol). The vial is capped and the resulting mixture is stirred and heated via microwave irradiation at 100 °C for 30 minutes. The reaction mixture is then carefully treated with 1M aqueous hydrochloric acid solution (1.14 mL, 1.14 mmol), resulting in a on mixture pH of ~4. The resulting e is concentrated to s in vacuo and the product is purified via low pH, reversed phase chromatography. 1H NMR (400.1 MHz, DMSO-d6) δ 1.04 (s, 3H), 1.09 (s, 3H), 1.84-1.72 (m, 4H), 1.97 (bs, 3H), 2.29-2.27 (m, 1H), 2.37-2.33 (m, 1H), 2.75-2.65 (m, 4H), 2.91 (s, 4H), 3.25-3.3.24 (m, 2H), 3.38-3.34 (m, 2H), 3.54 (s, 2H), 7.29 (d, J= 7.6 Hz, 2H), 7.45-7.37 (m, 2H), 7.51-7.46 (m, 1H), 7.55 (d, J= 7.5 Hz, 1H), 7.76 (d, J= 7.5 Hz, 1H), 7.85 (d, J= 8.0 Hz, 2H), 7.91 (s, 1H), 9.99 (s, 1H), 11.49 (s, 1H), 12.84 (s, 1H). LC-ES/MS (m/z) 729 [M+1].
Example 28 4-[2-[2,6-difluoro[[2-[[3-[[4-(4-hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin yl]methyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid To a solution of methyl 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazin hyl]benzoyl]amino]benzothiophenecarbonyl]amino]phenyl]-ethyl]benzoate (0.20 g, 0.25 mmol) in 8 mL of a mixed solvent (THF/CH3OH/H2 O in 2:1:1 ratio), LiOH (18 mg, 0.75 mmol) is added. The resulting mixture is allowed to stand at r.t. for 12 h. Additional LiOH (18 mg, 0.75 mmol) is added. And the mixture is allowed to stand at r.t. for 48 h and diluted with 1mL of 4N HCl in dioxane. The solvent is removed under a vacuum. The crude product is purified by flash chromatography to yield 120 mg (57%) of the title compound as a white solid. 1H NMR (399.80 MHz, DMSO-d 6): d 1.06 (s, 6H), 1.31-1.48 (m, 4H), 2.34 (s, 2H), 2.87-2.97 (m, 4H), 3.07-3.09 (m, 2H), 3.09-3.18 (s, 2H), .28 (m, 2H), 3.38 (partially overlaps with res water peak, 1H), 3.54 (s, 2H), 4.38 (m, 2H), 6.33 (m, 2H), 7.25- 7.71 (m, 8H), 7.73-8.08 (m, 6H), 10.64 (s, 1H), 11.99 (s, 1H), 12.8 (br s, 1H). LC-ES/MS (m/z) 798 [M+1].
Inhibition of NaPi-IIb In Vitro Inhibition of 33P uptake is measured in human and mouse NaPi-IIb T-REXTM-CHO stable cell lines. cDNA for NaPi-IIb is subcloned in plasmid SLC34A2 pcDNA5/TO (human) and SLC34A2 pcDNA5/TO (mouse) and stable cell lines are generated from clonal isolation for both human and mouse respectively. Mouse and human stable lines are maintained in uous culture in growth media (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (3:1), 10% Heat Inactivated FBS , 1% penicillin/streptomycin/FUNGIEZONE® (HYCLONETM), 20 mM HEPES, 250 μg/mL hygromycin, 5 μg/mL blasticidin). Cells are harvested from T225 cell culture flasks NG®) using 0.25% Trypsin, and plated in 96 well CYTOSTAR-TTM scintillating microplates (Amersham Systems) at 40,000 cells/well in 100 μL of growth media plus 100 ng/mL of tetracycline. Cell plates are incubated overnight at 37 °C and 5% CO2. The next day, compounds are ly d using one to three ons in 100% DMSO. Cell plates may remain in the tor until ready to be assayed. A cell plate is removed from the incubator and media removed. Cells are washed 3 times with 200 μL assay buffer (137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2, 1.2 mM MgSO4, and 14 mM Cl buffer, pH ~ 7.5), ng buffer in between washes. Serially diluted compounds in DMSO are further diluted 50 fold in assay buffer and 50 μL added to the CYTOSTAR-TTM assay plate, immediately followed by the addition of 50 μL of 33P solution (PERKIN-ELMER®, Walton, MA; 0.05 μCi/50 μL). CYTOSTAR-TTM assay plates are covered with foil to protect from light and incubated for 60 min at RT. After the 60 min incubation, 100 μL of a stop solution (assay buffer + 400 μM Phloretin) is added to the assay plate to stop 33P uptake. The plate is immediately read on a Wallac MICROBETA® Trilux liquid scintillation r and luminometer after stop solution is added with 1 minute count per well. Each plate may be processed separately and staggered in time so there may be no delay in counting after stop solution is added. Percent inhibition at all concentrations tested (final assay concentrations 100-0.005 μM) are calculated relative to 1% DMSO (minimum effect), and the effect of 100 μM of a fully efficacious NaPi-IIb inhibitor (maximum effect). IC50 values were calculated using a 4 parameter logistic curve fitting equation. The numbers ted are the geometric means with standard deviation (SD) calculated where n is the number of runs. Thus, Table 1 describes the relative IC50 values for Examples 1-28 t human NaPi-IIb and murine NaPi-IIb, respectively.
Table 1. Relative IC50 (rel IC50) values for Examples 1-28 against human and murine NaPi-IIb in vitro data in T-REXTM Chinese Hamster Ovarian-stable cell lines. h NaPi-IIb rel IC50 m Ib rel IC50 Example n n (nM), (SD) (nM), (SD) 1 32.4, (23.0) 3 43.9, (24.2) 3 2 13.6, (18.2) 4 17.5, (9.05) 3 3 51.1, (32.3) 2 40.9 1 4 18.6, (9.8) 3 26.0, (37.3) 3 76.3, (4.2) 2 23.6, (8.2) 2 6 8.7, (6.7) 6 9.9, (4.2) 3 7 16.2, (2.6) 2 8.56 1 8 32.6, (19.5) 3 35, (7.8) 2 9 197, (162) 5 314, (526) 5 3.4, (4.1) 5 5.9, (5.0) 5 11 10.8, (5.6) 4 12.6, (2.2) 4 12 156, (73.6) 3 217, (122) 3 13 4.3, (9.4) 2 9.0 1 14 5.3, (2.5) 3 11.3, (0.8) 4 6.5, (4.4) 4 16.8, (8.5) 4 16 60.0, (50.1) 3 31.2, (6.7) 3 17 3.5, (4.6) 3 8.8, (6.8) 3 18 5.1, (9.2) 3 20.3, (37.4) 3 19 60.3, (36.3) 2 20.0, (31.6) 3 9.5, (23.1) 3 9.1, (15.5) 3 21 19.6, (8.7) 2 5.3, (11.7) 2 22 9.4, (14.4) 3 27.3, (16.6) 2 23 46.5, (47.8) 3 47.1, (6.5) 3 24 16.6, (12.7) 3 30.2, (9.6) 3 22.5, (3.2) 2 8.3, (11.0) 3 26 2.4 1 15.3 1 27 25.9, (1.9) 3 22.6, (6.4) 3 28 6.2, (1.2) 3 7.6, (1.6) 3 Inhibition of NaPi-IIb In Vivo For test article and vehicle control preparation, add vehicle, 20% hydroxypropyl-betacyclodextrin (HPBCD) in water, to the test article. Sonicate to reduce particle size in an ultrasonic water bath as needed. If necessary, use a polytron to break down any visible particles in test e solution. Add 1 N NaOH as ted in Table 2 below. The pH of the vehicle control is adjusted to 8.0 to 8.5 with 1N NaOH.
Table 2. Amount of 1N NaOH to add to indicated testing compound.
Example 1 N NaOH per No. mg of compound (μL) 1 2.5 2 3.8 3 2.3 4 3.9 3.7 6 2.7 7 2.5 8 2.5 Radioactive phosphate preparation: Prepare a 16.25 mM Na2HPO4 solution using 0.9% saline as the vehicle. Mix until a clear solution is formed. Adjust pH to approximately 7.4.
Filter using a sterile 0.2 μm polyethersulfone membrane. For the final preparation of the ctive phosphate dosing solution, add 0.5 µl of stock H333PO4 per 1 mL of Na2HPO4.
Mix thoroughly then sterile filter the H333PO4 + Na2HPO4 solution using a 0.2 µm polyethersulfone membrane prior to dosing. Measure the radioactivity of each sample with the scintillation counter. If the DPMs are between 0 and 150,000 proceed with dosing.
In-vivo ol: Male C57Bl6 male mice at the age of about 8-9wks old are fasted for 16hrs the day before the study. They are assigned to treatment groups based on body weight on the day of study. Mice are dosed orally with either the test article, prepared as described above, or vehicle control at a 10 mls/kg dose volume. Fifteen s later, the radioactive phosphate dosing solution is given by oral gavage. Fifteen minutes later, blood is collected by orbital bleed. Plasma is ed and 50 µl of EDTA plasma from each mouse is mixed with 10ml of scintillation fluid and the counts determined by scintillation counting.
The effect of the test article is determined by comparing the counts in the plasma from the test article treated s to counts in the plasma of the vehicle control d animals [Percent Inhibition = s in the plasma of test article treated animals / counts in the plasma of vehicle treated animals) X 100%]. Percent inhibition is measured at 30 s post administration of the compound or vehicle, and at 15 minutes post administration of the labelled phosphate. Percent Inhibition for indicated Examples are illustrated in Table 3 below.
Table 3: In vivo data for Example compounds 4, 6, and 8 Percent Example Treatment n SEM Inhibition 4 5 mg/kg 6 52 5.4 6 5 mg/kg 6 62 2.5 8 5 mg/kg 6 60 5.2 Alternative vehicles, for example polyvinylpyrrolidone-co-vinyl acetate (PVPVA ), at varying concentrations, may be used. Example 1 can be assayed in PVP-VA, and for this study the compound is formulated as described in the solid dispersion preparation of Example 1, followed by dissolving in water then dosing by oral gavage. The e control is water with varying concentrations of PVP-VA, matching the concentration found in the t dose of Example 1, the lowest dose of Example 1, and an ediate dose of of Example 1. No effect of varying PVP-VA on the uptake of radiolabeled phosphate is observed over several studies, and thus all the vehicles are averaged to calculate percent inhibition.
Table 4: In vivo data for Example 1 in PVP-VA formulation Percent Group Treatment n SEM Inhibition 1 Vehicle 23 0 5.12 2 30 mg/kg Example 1 7 75.83 3.54 3 10 mg/kg Example 1 7 75.64 4.02 4 3 mg/kg Example 1 7 68.71 4.30 1 mg/kg Example 1 7 66.90 3.10 6 0.3 mg/kg Example 1 7 51.21 7.71 7 0.1 mg/kg Example 1 7 30.61 7.57 8 0.03 mg/kg Example 1 7 0.75 14.45 The effect of a test compound on gastric emptying can be assessed by dosing the compound and radiolabeled ate in a manner similar to that used to assess a compound’s y to inhibit NaPi-IIb in vivo. After bleeding the mice, they were sacrificed and their stomachs harvested. The ted stomachs are digested in 10 ml of 1N NaOH overnight, and the recovered abeled phosphate DPMs ined by scintillation counting. The compound mediated effect on the rate of gastric emptying is determined by comparing the dpms in the stomach of animals treated with compound, to that of animals treated with vehicle.
Compounds of the ion, for instance Example 1, show advantageous pharmacological properties, such as potency, in vivo distribution, in vivo efficacy, and favorable lack of toxicity in preclinical testing. For instance Example 1 shows surprisingly advantageous margin of Ib inhibition with respect to inhibition of in vivo gastric ng. In addition, Example 1 is generally well tolerated when administered in vivo to normal rats for a period of four days, and shows an advantageous lack of toxicity in this in vivo experiment.

Claims (15)

We claim:
1. A compound of the formula: Formula II 5 wherein Y is a fused cyclohexane ring or a fused phenyl ring, wherein A is or , wherein the crossed lines indicate bonds for the point of attachment to the core of Formula II, and the dashed lines indicate bonds for the point of attachment to R2, wherein R2 is selected from the group consisting of 10 -CH3, -(CH2)3OH, -(CH2)3OCH3, -(CH2)3CO2H, -COOCH3, , - CO(CH2)3CH3, -COCH(CH3)2, -CO(CH2)2CO2H, -COCH2NH2, -COCH2N(CH3)2, -SO2N[(CH2)2OCH3]2, -SO2NHCH3, -SO2(CH2)2OCH3, -CONH(CH2)4OH, -CONH(CH2)4OCH3, H3, -CONH(CH2)2CO2H, -CONH(CH2)2OCH3, -CON(CH2CH2OCH3)2, -CSNHCH3, , , and , wherein the dashed lines represent the point of attachment, wherein R’ is -CO2H or -CONH2, or a ceutically acceptable salt thereof. 5
2. The compound of claim 1, wherein Y is a fused cyclohexane ring , A is , and R’ is -CO2H, or a pharmaceutically acceptable salt thereof.
3. The compound of claim 1 which is of the formula: wherein R2 is selected from the group consisting of -(CH2)3OH, -(CH2)3OCH3, -(CH2)3CO2H, CH2)4OH, -COCH2NH2, -SO2N[(CH2)2OCH3]2, -CONH(CH2)4OCH3, and -CO(CH2)2CO2H, wherein R’is -CO2H or , or a pharmaceutically acceptable salt thereof. 5
4. The compound of claim 3 wherein R’ is -CO2H, or a pharmaceutically acceptable salt thereof.
5. The compound of claim 1 or claim 3 which is 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- ybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, 10 which can be structurally represented as: N O H H N N F O O or a pharmaceutically acceptable salt thereof.
6. The compound of claim 5 which is 4-[2-[2,6-difluoro[[2-[[3-[[4-(4- hydroxybutylcarbamoyl)-2,2-dimethyl-piperazinyl]methyl]benzoyl]amino]- 15 4,5,6,7-tetrahydrobenzothiophenecarbonyl]amino]phenyl]ethyl]benzoic acid, disodium.
7. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 6, and a pharmaceutically acceptable carrier, diluent or excipient.
8. A solid dispersion ation comprising 30% of a compound of claim 6, and 70% polyvinylpyrrolidone-vinyl acetate. 5
9. Use of a compound or salt according to any one of claims 1 to 6 for the manufacture of a medicament for treating hosphatemia.
10. Use of a compound or salt according to any one of claims 1 to 6 for the manufacture of a medicament for ng chronic kidney disease.
11. Use of a compound or salt according to any one of claims 1 to 6 for the cture 10 of a medicament for treating cardiovascular disease associated with chronic kidney disease.
12. A compound or salt as claimed in any one of claims 1 to 6 substantially as herein described with reference to any example thereof.
13. A pharmaceutical composition as claimed in claim 7 substantially as herein described 15 with reference to any e thereof.
14. A solid dispersion formulation as claimed in claim 8 substantially as herein described with reference to any example thereof.
15. Use as claimed in any one of claims 9 to 11 substantially as herein described with reference to any example thereof.
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