Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • 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
  • A61K31/16—Amides, e.g. hydroxamic acids
  • A61K31/18—Sulfonamides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to methods for improving the pharmacokinetics of drugs which are metabolized by cytochrome P450 monooxygenase using PPL-100 and related pharmaceutical compositions.
• the present invention relates to methods for improving the pharmacokinetics of HIV protease inhibitors by co-administering PPL-100 with such protease inhibitors.
• proteases inhibitors of the HIV viral protease have been developed relatively recently and their use began only in 1996. Currently, they are considered the most effective drugs against HIV infection. Unfortunately, most current proteases inhibitors are relatively large hydrophobic molecules that possess rather low bioavailability. A high pill burden is therefore required to attain the therapeutic dose in a patient. This is a deterrent, which too often results in patient non-compliance and inadequate treatment results. This situation leads to sub- optimal therapeutic drug concentration that in turns leads to the development of HIV resistant strains. Consequently, there is an urgent need to improve the solubility and bioavailability of proteases inhibitors.
• Lysine-based compounds with increased solubility and improved oral bioavailability are described herein and have been described in United States patent application No. 10/902,935 filed on August 2, 2004 and published on February 2, 2006 under No. 2006/0025592A1 , the entire content of which is incorporated herein by reference. These compounds may readily be cleaved in vivo to release an active ingredient which has an affinity for aspartyl proteases and which may act as a protease inhibitor. More particularly, the active ingredient may bind, for example, to an HIV aspartyl protease (U.S. patent no. 6,632,816) and may inhibit this enzyme.
• the Lysine-based compounds are also referred herein as a protease inhibitor precursor.
• the Lysine-based compounds Upon in vivo physiological conditions (e.g., metabolic, enteric and/or gastrointestinal conditions, etc.) the Lysine-based compounds, allow for the release of a protease inhibitor (e.g., aspartyl protease inhibitor).
• a protease inhibitor e.g., aspartyl protease inhibitor
• the Lysine-based compounds may thus serve as means for improving the solubility and/or bioavailability of protease inhibitors and therefore may reduce the pill burden and/or reduce dosages needed for inhibition. Improved treatment of HIV-infected patients and favourable patient's compliance may consequently occur.
• the compounds described herein may be used to inhibit the activity of cytochrome P450 monooxygenase (CYP450). More particularly, these compounds may be used to inhibit the activity of CYP3A4, including for example, CYP3A4/5.
• CYP450 cytochrome P450 monooxygenase
• the compounds described herein may thus be used with drugs which are metabolized by cytochrome P450 monooxygenase in order to improve their pharmacokinetics. More particularly, these compounds may be used to improve the pharmacokinetics of drugs which are metabolized by CYP3A4, including CYP3A4/5.
• drugs which may benefit from inhibition of CYP450 may include, for example, the immunosupressants cyclosporine, FK-506 and rapamycin, the chemotherapeutic agents taxol and taxotere, the antibiotic clarithromycin and the HIV protease inhibitors, A-77003, A-80987, MK-639.
• Exemplary embodiments of compounds encompassed by the present invention and which may be used for inhibiting CYP450 may include, for example, a compound of formula I:
• pharmaceutically acceptable salts and derivatives thereof e.g., for example, when the compound of the present invention comprises an amino group, the pharmaceutically acceptable salt may be an ammonium salt,
• n may be, for example, 3 or 4,
• X and Y may be selected, for example, from the group consisting of H 1 a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -OCF 3 , - CN, -NO 2 , -NR 4 R 5 , -NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 , and -CH 2 OH or X and Y together define an alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH 2 O- and an ethylenedioxy group of formula - OCH 2 CH 2 O-,
• R 6 may be selected, for example, from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
• R 3 may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, and a group of formula R 3A -CO-, wherein R 3A may be selected, for example, from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms (e.g.
• a cycloalkyl group having 3 to 6 carbon atoms e.g. cyclopropyl-, cyclohexyl- etc.
• a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof e.g.
• a picolyl group selected from the group consisting of
• a picolyloxy group selected from the group consisting of
• a substituted pyridyl group selected from the group consisting of
• X' and Y' may be selected, for example, from the group consisting of H 1 a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -NO 2 , - NR 4 R 5 , -NHCOR 4 . -OR 4 , -SR 4 , -COOR 4 , -COR 4 and -CH 2 OH,
• R 4 and R 5 may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, and a cycloalkyl group of 3 to 6 carbon atoms, wherein R 2 may be selected, for example, from the group consisting of a diphenylmethyl group of formula IV a naphthyl-1 -CH 2 - group of formula V
• R 1 may be H or a physiologically cleavable unit, whereby upon ⁇ in vivo) physiological conditions the compound may be converted into an active protease inhibitor.
• the compound upon cleavage of the physiologically cleavable unit, the compound may be able to release a protease inhibitor.
• Ri may be selected, for example, from the group consisting of (HO) 2 P(O) and (MO) 2 P(O), wherein M is an alkali metal (e.g. Na, K, Cs 1 etc) or alkaline earth metal (Ca, Mg, etc.).
• M is an alkali metal (e.g. Na, K, Cs 1 etc) or alkaline earth metal (Ca, Mg, etc.).
• R 1 may be a group of formula R 1A -CO-, wherein R 1A may be selected, for example, from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms (e.g. methyl, ethyl, propyl, /so-propyl, butyl, /so-butyl, terf-butyl, tert-butyl-CH 2 -, etc.), a cycloalkyl group having 3 to 6 carbon atoms (e.g.
• cyclopropyl-, cyclohexyl- etc. a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, (e.g. cyclopropyl-CH 2 -, cyclohexyl-CH 2 -, etc.), an alkyloxy group of 1 to 6 carbon atoms (e.g.
• HI a picolyl group selected from the group consisting of
• a picolyloxy group selected from the group consisting of
• a substituted pyridyl group selected from the group consisting of
• a pharmaceutical composition which may comprise: a) a compound of formula I as described herein or a pharmaceutically acceptable salt thereof, b) a drug which may be metabolized by a cytochrome P450 monooxigenase (e.g., CYP3A4), and; c) a pharmaceutically acceptable carrier; where the compound of formula I is represented by;
• n may be 3 or 4,
• X and Y may be selected, for example, from the group consisting of H 1 a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -OCF 3 , - CN, -NO 2 , -NR 4 R 5 .
• -NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 , and -CH 2 OH or X and Y together may define an alkylenedioxy group which may be selected, for example, from the group consisting of a methylenedioxy group of formula -OCH 2 O- and an ethylenedioxy group of formula -OCH 2 CH 2 O-,
• R 8 may be selected from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
• R 3 may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, and a group of formula R 3A -CO-, where R 3A may be selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, an alkyloxy group of 1 to 6 carbon atoms, tetrahydro-3-furanyloxy, -CH 2 OH 1 -CF 3 , -CH 2 CF 3 , -CH 2 CH 2 CF 3 .
• i a picolyloxy group which may be selected from the group consisting of
• a substituted pyridyl group which may be selected from the group consisting of
• X 1 and Y may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -NO 2 , - NR 4 R 5 , -NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 and -CH 2 OH 1
• R 4 and R 5 may be selected from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, and a cycloalkyl group of 3 to 6 carbon atoms,
• R 2 may be selected from the group consisting of a diphenylmethyl group of formula IV
• R 1 may be H or a physiologically cleavable unit, whereby upon (in vivo) physiological conditions the compound may be converted into an active protease inhibitor. For example, upon cleavage of the physiologically cleavable unit, the compound may be able to release a protease inhibitor.
• Ri may be selected, for example, from the group consisting of H 1 (HO) 2 P(O) and (MO) 2 P(O) (wherein M may be, for example, an alkali metal or alkaline earth metal) and a group of formula Ri A -CO-, where Ri A rnay be selected, for example, from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, an alkyloxy group of 1 to 6 carbon atom, -CH 2 OH, CH 3 O 2 C-, CH 3 O 2 CCH 2 -, Acetyl-OCH 2 CH 2 -, HO 2 CCH 2 -, 2-hydroxyphenyl, 3-hydroxyphenyl, 4- hydroxy
• a picolyl group selected from the group consisting of
• a picolyloxy group selected from the group consisting of
• a substituted pyridyl group selected from the group consisting of
• a pharmaceutical composition which may comprise; a) a compound of formula Il and pharmaceutically acceptable salts thereof, b) a drug which may be metabolized by a cytochrome P450 monooxigenase (e.g., CYP3A4), and; c) a pharmaceutically acceptable carrier; where the compound of formula Il is represented by;
• n may be 3 or 4,
• X and Y may be selected from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F 1 Cl, Br, I, -CF 3 , -OCF 3 , -CN, -NO 2 , -NR 4 Rs, - NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 , and -CH 2 OH or X and Y together may define, for example, an alkylenedioxy group which may be selected from the group consisting of a methylenedioxy group of formula -OCH 2 O- and an ethylenedioxy group of formula - OCH 2 CH 2 O-,
• R 6 may be selected, for example, from the group consisting of a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof,
• R 3 may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, and a group of formula R 3 A-CO-, where R 3 A rnay be selected, for example, from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, an alkyloxy group of 1 to 6 carbon atoms, tetrahydro-3-furanyloxy, -
• a picolyl group which may be selected from the group consisting of
• a picolyloxy group which may be selected from the group consisting of
• a substituted pyridyl group which may be selected from the group consisting of
• X' and Y' may be selected from the group consisting of H, a straight a Iky I group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F 1 Cl, Br, I, -CF 3 , -NO 2 , -NR 4 Rs, - NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 and -CH 2 OH,
• R 4 and R 5 may be selected from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, and a cycloalkyl group of 3 to 6 carbon atoms,
• R 2 may be selected from the group consisting of a diphenylmethyl group of formula IV a naphthyl-1-CH 2 - group of formula V
• R 1 may be H or a physiologically cleavable unit, whereby upon physiological conditions (in vivo) the compound may be converted into an active protease inhibitor.
• R 1 may be selected, for example, from the group consisting of H, (HO) 2 P(O) and (MO) 2 P(O) (wherein M may be an alkali metal or alkaline earth metal), and a group of formula R 1A -CO-, where R 1A may be selected, for example, from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, an alkyloxy group of 1 to 6 carbon atoms, -CH 2 OH, CH 3 O 2 C-, CH 3 O 2 CCH 2 -, Acetyl- OCH 2 CH 2 -, HO 2 CCH 2 -, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxy phenyl,
• a picolyl group which may be selected from the group consisting of
• a picolyloxy group which may be selected from the group consisting of
• a substituted pyridyl group which may be selected from the group consisting of
• compositions which comprise compounds of formula Il wherein R 6 may be, for example, /so-butyl and n may be 3 are encompassed herewith.
• pharmaceutical compositions which comprise compounds of formula Il wherein R 8 may be, for example, /so-butyl and n may be 4 are also encompassed herewith.
• Ri may be selected, for example, from the group consisting of H, (HO) 2 P(O) and (NaO) 2 P(O).
• Ri may be selected, for example, from the group consisting of CH 3 CO, 3-pyridyl-CO, (CH 3 ) 2 NCH 2 CO and
• compositions which comprise compounds of formula Il wherein R 3 may be selected, for example, from the group consisting of CH 3 CO, CH 3 O-CO, (CH 3 ) 2 N-CO, 3- pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO are encompassed by the present invention.
• X may be 4-NH 2 and Y may be H or F.
• X' and Y' may both be H.
• compositions which comprise compounds of formula Il wherein R 2 may be selected, for example, from the group consisting of a diphenylmethyl group of formula IV 1 a naphthyl-1- CH 2 - group of formula V, a naphthyl-2-CH 2 - group of formula Vl, a biphenylmethyl group of formula VII and an anthryl-9-CH 2 - group of formula VIII are encompassed herewith.
• R 2 may, more particularly, be selected from the group consisting of a diphenylmethyl group of formula IV, a naphthyl-1-CH 2 - group of formula V, and a naphthyl- 2-CH 2 - group of formula Vl.
• the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R 6 is /so-butyl, n is 4 and R 2 is a diphenylmethyl group of formula IV.
• R 1 may be selected from the group consisting of H, (HO) 2 P(O) and (NaO) 2 P(O).
• R 1 may be selected from the group consisting of CH 3 CO, 3-pyridyl-CO, (CH 3 J 2 NCH 2 CO and (CH 3 ) 2 CHCH(NH 2 )CO.
• R 3 may be selected, for example, from the group consisting of CH 3 CO, CH 3 O-CO 1 (CH 3 ) 2 N-CO, 3- pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO.
• X may be 4-NH 2 and Y may be H or F.
• the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X may be, for example, 4-NH 2 , Y may be H, X' may be H, Y' may be H and R 3 may be CH 3 O-CO.
• Ri may be (HO) 2 P(O).
• R 1 may be (NaO) 2 P(O).
• R 1 may be H.
• the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X may be 4-NH 2 , Y may be 3-F, X' may be H, Y' may be H and R 3 may be CH 3 O- CO.
• R 1 may be (HO) 2 P(O). In accordance with another particular embodiment of the present invention, R 1 may be (NaO) 2 P(O).
• Ri may be H.
• the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X is 4-NH 2 , Y is H or 3-F, X 1 is H, Y' is H and R 3 is CH 3 CO.
• Ri may be (HO) 2 P(O).
• Ri may be (NaO) 2 P(O).
• R 1 may be H.
• the present invention further provides a pharmaceutical composition which may comprise a compound of formula II, X is 4-NH 2 , Y is H or 3-F, X * is H 1 Y' is H and R 3 is 4-morphoHne-CO.
• X may be 4-NH 2
• Y may be H
• X 1 may be H
• Y * may be H
• R 3 may be CH 3 O-CO.
• R 1 may be 3-pyridyl- CO.
• R 1 may be (CH 3 ) 2 NCH 2 CO.
• R 1 may be (CHa) 2 CHCH(NH 2 )CO.
• R 1 may be CH 3 CO.
• the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R 8 is /sobutyl, n is 4, X' and Y" are both H, R 2 is Naphtyl-1-CH 2 -, X is 4-NH 2 , Y is H, R 3 is 4-morpholine-CO and R 1 may be selected, for example, from the group consisting of H, (HO) 2 P(O) and (NaO) 2 P(O).
• the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R 6 is /so-butyl, n is 4, X 1 and Y' are both H, R 2 is Naphtyl-2-CH 2 -, X is 4-NH 2 , Y is H 1 R 3 is CH 3 O-CO and Ri may be selected, for example, from the group consisting of H, (HO) 2 P(O) and (NaO) 2 P(O).
• the present invention provides a pharmaceutical composition which may comprise; a) a compound of formula Ha
• X and Y may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -OCF 3 , - CN, -NO 2 , -NR 4 R 5 , -NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 .
• alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH 2 O- and an ethylenedioxy group of formula - OCH 2 CH 2 O-,
• X' and Y' may be selected, for example, from the group consisting of H, a straight alkyl group of 1 to 6 carbon atoms, a branched alkyl group of 3 to 6 carbon atoms, a cycloalkyl group of 3 to 6 carbon atoms, F, Cl, Br, I, -CF 3 , -NO 2 , - NR 4 R 5 , -NHCOR 4 , -OR 4 , -SR 4 , -COOR 4 , -COR 4 and -CH 2 OH 1 wherein n, R 1 , R 3 , R 4 , R 5 and R ⁇ are as defined herein;
• R 1 may be selected from the group consisting of H, (HO) 2 P(O) and (MO) 2 P(O), wherein M may be an alkali metal or alkaline earth metal and a group of formula R 1A -CO-, R 1A which may be selected from the group consisting of a straight or branched alkyl group of 1 to 6 carbon atoms, a cycloalkyl group having 3 to 6 carbon atoms, a cycloalkylalkyl group having 3 to 6 carbon atoms in the cycloalkyl part thereof and 1 to 3 carbon atoms in the alkyl part thereof, an alkyloxy group of 1 to 6 carbon atom, -CH 2 OH, CH 3 O 2 C-, CH 3 O 2 CCH 2 -, ACeIyI-OCH 2 CH 2 -, HO 2 CCH 2 -, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, (CH 3 ) 2 NCH
• a picolyl group selected from the group consisting of
• a picolyloxy group selected from the group consisting of
• R 6 may be feo-b ⁇ tyl.
• n may be 4.
• R 1 may be selected from the group consisting of H, (HO) 2 P(O) and (NaO) 2 P(O).
• Ri may be selected, for example, from the group consisting of CH 3 CO, 3-pyridyl-CO, (CH 3 J 2 NCH 2 CO and (CH 3 ) 2 CHCH(NH 2 )CO.
• R 3 may be selected from the group consisting of CH 3 CO, CH 3 O-CO 1 (CH 3 ) 2 N-CO, 3-pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO.
• R 3 may be selected from the group consisting of CH 3 CO, CH 3 O-CO, (CH 3 ) 2 N-CO, 3-pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO.
• X may be 4-NH 2 and Y may be H or F.
• X may be 4-NH 2 and Y may be H or F.
• X may be 4-NH 2
• Y may be H or 3-F
• X 1 may be H
• Y' may be H
• R 3 may be CH 3 CO.
• X may be 4-NH 2
• Y may be H or 3-F
• X' may be H
• Y' may be H
• R 3 may be 4-morpholine-CO.
• X may be 4-NH 2
• Y may be H 1
• X' may be H
• Y" may be H
• R 3 may be CH 3 O-CO and R 1 may be (HO) 2 P(O), (NaO) 2 P(O) or H.
• X may be 4- NH 2
• Y may be 3-F
• X' may be H
• Y' may be H 1
• R 3 may be CH 3 O-CO
• Ri may be (HO) 2 P(O). (NaO) 2 P(O) or H.
• X may be 4- NH 2
• Y may be H or 3-F
• X * may be H
• Y' may be H 1
• R 3 may be CH 3 CO
• Ri may be (HO) 2 P(O) 1 (NaO) 2 P(O) or H.
• X may be 4- NH 2
• Y may be H or 3-F
• X 1 may be H
• Y' may be H
• R 3 may be 4-morpholine-CO.
• X may be 4-NH 2 , Y may be H 1 X" may be H, Y' may be H 1 R 3 may be CH 3 O-CO and R 1 may be 3-pyridyl-CO, (CH 3 ) 2 NCH 2 CO, (CH 3 ) 2 CHCH(NH 2 )CO or CH 3 CO.
• X may be 4-NH 2 , Y may be 3-F, X' may be H, Y' may be H, R 3 may be CH 3 O-CO and Ri may be 3-pyridyl-CO, (CHg) 2 NCH 2 CO or (CH 3 ) 2 CHCH(NH 2 )CO.
• R 1 may be, for example, H 1 (HO) 2 P(O) or (NaO) 2 P(O). Further in accordance with the present invention, n may be 4. Y may be, for example, H. R 3 may be, for example. CH 3 O-CO. R 2 may be, for example, a diphenylmethyl group of formula IV, where X' and Y 1 may be, for example H 1
• compositions, methods, uses and kits encompassed by the present invention may comprise one or more of the following compounds and combination thereof; I O
• n 4, R 1 is (CHa) 2 NCH 2 CO 1 X is 4-NH 2 , Y is H, X' is 30 H 1 Y * is H 1 R 6 is /so-butyl and R 3 is CH 3 O-CO 1 - a compound of formula Ha wherein n is 4, R 1 is (CHa) 2 CHCH(NH 2 )CO, X is 4-NH 2 , Y is H, X' is H, Y" is H, R 6 is /so-butyl and R 3 is CH 3 O-CO,
• Any compound which is a precursor of an active ingredient described herein may be used to carry out the present invention and is also encompassed by the present invention.
• the above identified active ingredient is identified herein as PL-100 and it is to be understood herein that any precursor able to release or generate the above mentioned exemplary compound either in vivo or in vitro is encompassed by the present invention and may be used to carry out methods, pharmaceutical compositions, kits and uses described herein.
• the above identified active ingredient is identified herein as PL-337 and it is to be understood herein that any precursor able to release or generate the above mentioned exemplary compound either in vivo or in vitro may be used to carry out the present invention and is encompassed by the present invention.
• the present invention thus relates to the use of a compound described herein for improving the pharmacokinetics of a drug which may affected by CYP450 and more particularly, CYP3A4 (e.g., CYP3A4/5 etc.).
• CYP3A4 e.g., CYP3A4/5 etc.
• the present invention also relates to methods of treatment which may comprise admininistering to an individual in need, a compound described herein with one or more drugs which may be affected (metabolized) by CYP450 and more particularly, CYP3A4 (e.g., CYP3A4/5 etc.). As indicated herein, administration may be done at the same time or at different time intervals.
• the compound(s) and drug(s) may be mixed together or not.
• the present invention further relates to the use of a compound described herein in the manufacture of a medicament for improving the pharmacokinetics of a drug which may affected by CYP450 and more particularly, CYP3A4 (e.g., CYP3A4/5 etc.).
• CYP3A4 e.g., CYP3A4/5 etc.
• the present invention further relates to the use of a compound described herein and a drug which may affected by CYP450 in the manufacture of a medicament for treating a patient in need.
• pharmaceutically effective amount refers to an amount effective in treating, preventing or reducing the risk or probability of HlV infection or of reducing HIV burden.
• pharmaceutically effective amount also refers to an amount effective in treating, preventing or reducing the risk or probability of developing acquired immunodeficiency syndrome (AIDS), for delaying the apparition of AIDS, or reducing AIDS symptoms.
• AIDS acquired immunodeficiency syndrome
• a "pharmaceutically effective amount” may be construed as an amount giving a desired therapeutic effect, either taken into a single or multiple doses or in any dosage or route or taken alone or in combination with other therapeutic agents.
• a "pharmaceutically effective amount” may be understood as an amount having an inhibitory effect (partial or complete) on HIV (HIV-1 and HIV-2 as well as related viruses (e.g., HTLV-I and HTLV-II, and simian immunodeficiency virus (SIV))) infection cycle (e.g., inhibition of replication, reinfection, maturation, budding etc.) and on any organism which rely on aspartyl proteases for its life cycle.
• An inhibitory effect is to be understood herein as an effect such as a reduction in the capacity of an organism (e.g. HIV) to reproduce itself (replicate), to re-infect surrounding cells, etc, or even a complete inhibition (or elimination) of an organism.
• HIV protease and “HIV aspartyl protease” are used interchangeably and includes, for example, the aspartyl protease encoded by the human immunodeficiency virus type 1 or 2.
• pharmaceutically acceptable carrier refers to a non-toxic carrier or adjuvant that may be administered to a patient, together with one or more compounds of the present invention, and which does not destroy the pharmacological activity thereof.
• the term "precursor” refers to a compound, such as a Lysine-based compound which is able to be converted into an active ingredient in vitro or in vivo.
• the compound PL-461 is a precursor of compound PL-100 as when administered to an individual, PL-461 is converted into PL-100 in vivo (e.g., under physiological conditions).
• derivative refers to a compound which has been chemically synthesized from an original compound.
• PL-461 is a derivative of PL-100.
• composition includes the specified materials and may include other material that does not materially affect the basic characteristics of the pharmaceutical composition.
• compositions of formula I such as compounds of formulae I, II, Ha, lib, Uc, MA and HA 1
• pharmaceutically acceptable salts thereof such as, for example, ammonium salts are described herein.
• a "pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, or salt of such ester, of a compound of this invention or any other compound which, upon administration to a recipient (a mammal), is capable of providing
• a "straight alkyl group of 1 to 6 carbon atoms” includes for example, methyl, ethyl, propyl, butyl, pentyl, hexyl.
• branched alkyl group of 3 to 6 carbon atoms includes for example, without limitation, /so-butyl, tert-butyl, 2-pentyl, 3-pentyl, etc.
• a "cycloalkyl group having 3 to 6 carbon” includes for example, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclocyclohexyl (i.e., C 6 H 11 ).
• Salts derived from appropriate bases include alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N - (Ci -4 alkyl)/ salts.
• the compounds described herein contain one or more asymmetric carbon atoms and thus may occur as racemates and racemic mixtures, single enantiomer, diastereomeric mixtures and individual diastereoisomers. All such isomeric forms of these compounds are expressly included in the present invention.
• Each stereogenic carbon may be of the R or S configuration.
• Pharmaceutically acceptable salts of the compounds described herein include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
• acid salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylhydrogensulfate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycollate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2- naphthylsulfonate, nicotinate, nit
• any basic nitrogen containing groups of the compounds disclosed herein may be quaternized with any agents known to those of ordinary skill in the art including, for example, lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, and aralkyl halides including benzyl and phenethyl bromides. Water or oil-soluble or dispersible products may be obtained by such quaternization.
• lower alkyl halides such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides
• dialkyl sulfates including dimethyl, diethyl, dibutyl and di
• any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
• the mention of the range of 1 to 6 carbon atoms is to be understood herein as incorporating each and every individual number of carbon atoms as well as sub-ranges such as, for example, 1 carbon atoms, 3 carbon atoms, 4 to 6 carbon atoms, etc. with respect to reaction time, a time of 1 minute or more is to be understood as specifically incorporating herein each and every individual time, as well as sub-range, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc.;
• the compound formulae each include each and every individual compound described thereby as well as each and every possible class or sub-group or sub-class of compounds whether such class or sub-class is defined as positively including particular compounds, as excluding particular compounds or a combination thereof; for example an exclusionary definition for the formula (e.g. I) may read as follows: "provided that when one of A and B is -COOH and the other is H, -COOH may not occupy the 4' position".
• Scheme 1 illustrates a generic example for the preparation of the phosphate monoester /// derived from a primary alcohol (see I), a compound of HIV protease inhibitors (see example 1 (step G and H) in the experimental portion of this document for a specific example of this synthesis).
• R 2 and R 3 are as defined herein.
• phosphate monoester /// may use a HIV aspartyl protease inhibitor (/, see U.S. patent no. 6,632,816) as the starting material.
• the diethyl phosphotriester // was obtained in good yield upon treatment with diethyl chlorophosphate and sodium hydride in a mixture of tetrahydrofuran and triethylphosphate. Then, addition of trimethysilyl bromide in dichloromethane (DCM) gave compound /// in good to excellent yields.
• DCM dichloromethane
• Scheme 1A represents another generic example for the preparation of the phosphate monoester IHA derived from a primary alcohol (see IA), a compound of HIV protease inhibitors.
• n X, Y 1 R 2 , R 3 and Re are as defined herein.
• Scheme 2 illustrates a generic example for the preparation of the phosphate monoester ///, a compound of HIV protease inhibitors, with a different approach starting from (3S)-3- isobutylamino-azepan-2-one (IV).
• R 2 and R 3 are as defined herein.
• the phosphate monoester derivative /// was obtained from (3S)-3- isobutylamino-azepan-2-one (/V) in a seven-step reaction sequence.
• (2S)-3- isobutylamino-azepan-2-one (/V) was sulfonated with 4-acetamidobenzenesulfonyl chloride in the presence of triethylamine in dichloromethane to give compound V in excellent yields.
• the derivative Vl was obtained quantitatively upon treatment of V with di- tert-butyl pyrocarbonate and DMAP in acetonitrile.
• the reductive ring opening with sodium borohydride in ethanol lead to key intermediates VII in good yield.
• the diethyl phosphotriester VIII was obtained in good yield upon treatment with diethyl chlorophosphate and sodium hydride in a mixture of tetrahydrofuran and triethylphosphate.
• the Boc protective groups were removed upon treatment with HCI in ethanol to give compound IX quantitatively (T. W. Greene and P. G. M. Wuts, Protective groups in Organic Synthesis, 3 rd Edition, John Wiley & Sons, Inc. 1999).
• Scheme 3 presents the transformation of a diphenylmethyl derivative; (1S,5S)-(1- ⁇ 5-[(4- am ino-benzenesulf onyl)-isobutyl-am ino]-6-hyd roxy-hexy lcarbam oyl ⁇ -2 , 2-diphenyl-ethy I)- carbamic acid methyl ester (PL -100) into its fluorinated phosphate mo ⁇ oester sodium salt analog Xl.
• This reaction sequence may be used to produce any other similar compounds (compounds) made of unsubstituted (or substituted) diphenylmethyl, 1- naphthyl, 2-naphthyl, biphenyl and 9-anthryl groups described herein.
• Scheme 4 illustrates a generic example for the transformation of a phosphotriester // into its fluorinated analog XIII in a two-step reaction sequence.
• This generic example represents a second approach for the synthesis of fluorinated compounds described herein.
• the fluorine atom is added to the phosphotriester // instead of the primary alcohol derivative of general formula / or, more specifically, PL-100 as shown on scheme 3.
• This alternate reaction sequence may be used to produce any other similar compounds made of unsubstituted (or substituted) diphenylmethyl, 1-naphthyl, 2-naphthyl, biphenyl and 9-anthryl groups described herein.
• R 2 and R 3 are as defined herein.
• Scheme 5 illustrates exemplary synthesis of various ester compounds XVI described herein.
• the ester compounds are known to be easily cleaved in vivo by esterase enzymes and, as a result, may release the active ingredient.
• R 2 is set as a diphenylmethyl group.
• this reaction sequence may be used to produce any other similar compounds made of unsubstituted (or substituted) diphenylmethyl, 1- naphthyl, 2-naphthyl, biphenyl and 9-anthryl groups described herein.
• R 1A represents the "residue" of the acid molecule that is linked to the free primary alcohol group present on intermediate XV and is as defined herein.
• the compounds XVI are generally obtained in a three-step reaction sequence in high yields.
• HABt 1-hydroxybenzotriazole
• EDAC 1-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride
• acetyl ester was obtained quantitatively using acetic anhydride in the presence of ⁇ /, ⁇ /-dimethylaminopyridine (DMAP) in dichloromethane (DCM). Cleavage of the Boc protective group was achieved quantitatively upon treatment with trifluoroacetic acid (TFA) in DCM.
• TFA trifluoroacetic acid
• a second coupling with (2S)-2-methoxycarbonylamino-3,3-diphenyl-propionic acid is performed on the primary amino group of intermediate XV with HOBt and EDAC to give the desired compounds XVI in good to excellent yields.
• catalytic hydrogenation of a benzyloxycarbonyl group is performed using 10% palladium on carbon to give the final compound XVII.
• the compounds described herein may be modified by appending appropriate functionalities to enhance selective biological properties.
• modifications may include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
• compositions of this invention comprise any of the compounds of the present invention, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle.
• Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethyleneglycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
• compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles.
• parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
• the compounds described herein When the compounds described herein are administered in combination with a drug which may be metabolized by CYP450, they may be administered sequentially or concurrently to the patient. Administration of the compound and drug may also be separated by a suitable time interval.
• HIV-1 HIV-1 , -2 Human immunodeficiency virus type 1 , type 2
• analytical plates may be treated with a 0.3% ninhydrin solution in ethanol containing 3% acetic acid and/or a CAM solution made of 20 g (NH ⁇ 6 Mo 7 O 24 and 8.3 g Ce(SO 4 J 2 polyhydrate in water (750 mL) containing concentrated sulfuric acid (90 mL).
• Preparative HPLC were performed on a Gilson apparatus equipped with a C18 column, a 215 liquid handler module and 25 mL/min capacity head pumps. The HPLC is operated with a Gilson UniPoint System Software.
• HPLC system 2 Gilson #305-25 mL pumps, Gilson #215 liquid handler for injection and collection and a Gilson #155 UV-Vis absorbance detector, all controlled from a Gilson Unipoint V1.91 software
• Melting points (mp) were determined on a B ⁇ chi 530 melting point apparatus in capillary tubes and were uncorrected.
• Mass spectra were recorded on a Hewlett Packard LC/MSD 1100 system using APCI or electrospray sources either in negative mode or positive mode.
• ⁇ /o-isobutyl-L- ⁇ -amino-caprolactam (IV) (4.1 g free base) was dissolved in DCM (200 mL) and treated with 4.0 g triethylamine, followed by 4-acetamidobenzenesulfonyl chloride (5.2 g). A 0.1 g portion of dimethylaminopyridine was added and the mixture was stirred 5 h. The resulting thick slurry was poured into 500 mL 0.5 M HCI and shaken vigorously. The solid in the biphasic solution was filtered out and washed with cold acetone to give 7.3 g (87%) of clean product.
• Step C Preparation of (3S)-3- ⁇ [4-(acetyl-tert-butoxycarbonyl-amino)- benzenesulfonyl]-isobutyl-amino ⁇ -2-oxo-azepane-1-carboxylic acid tert-butyl ester (Boc activation) (V/)
• Step D Preparation of (1S)-4-amino-/V-(5-amino-1-hydroxymethyl-pentyl)- ⁇ /- isobutyl-benzenesulfonamide (V7/-de protected) (reductive ring opening and deprotection)
• V7/-de protected reductive ring opening and deprotection
• a 3.0 g portion of (SSJ-S-IH-t ⁇ cetyl-tert-butoxycarbonyl-aminoJ-benzenesulfonyll-isobutyl- amino ⁇ -2-oxo-azepane-1-carboxylic acid tert-butyl ester (W 1 step C) is dissolved in 40 mL EtOH followed by 750 mg NaBH 4 . Brief heating with a heat gun gives a clear solution.
• Step F Preparation of (1S,5S)-(1- ⁇ 5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6- hydroxy-hexylcarbamoyl ⁇ -2,2-diphenyl-ethyl)-carbamic acid methyl ester (PL-100)
• step D The title compound was prepared from (1 S)-4-amino- ⁇ /-(5-amino-1-hydroxymethyl-pentyl)- ⁇ /-isobutyl-benzenesulfonamide (W/-deprotected) (step D) and (2S)-2- methoxycarbonylamino-S ⁇ -diphenyl-propionic acid (step E) using the coupling procedure with HOBt and EDAC described in example 3 (step D). The final product was obtained in 67% yield (121 mg).
• Step G Preparation of (1S,5S)- ⁇ 1-[5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6-
• the PL-100 compound (product of step F, 203 mg, 0.325 mmol) was dissolved in dry tetrahydrofuran (3 ml.) and 0.2 mL triethylphosphate under N 2 atmosphere. The mixture was stirred at this temperature for 15 min, followed by the addition of diethyl chlorophosphate (0.061 mL, 0.423 mmol). Sodium hydride (60% in mineral oil) (17 mg,
• Step H Preparation of (1 S,5S)-(1- ⁇ 5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6- phosphonooxy-hexylcarbamoyl ⁇ -2,2-diphenyl-ethyl)-carbamic acid methyl ester (PL-461)
• step G The product of step G prepared above (152 mg) was dissolved in anhydrous dichloromethane (3.0 ml_). Trimethylsilyl bromide (0.5 ml_) was added at 0 0 C. The mixture was stirred during 1h at this temperature and overnight at room temperature. The solvent was evaporated and 0.2 ml_ water was added to the residue. 3 ml_ EtOH was added mixed and evaporated. This step was repeated three times and the residue dried in vacuo. Yields 98 mg 70% of the title derivatives of this first example.
• Step B Preparation of (2S)-phosphoric acid 6-amino-2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-hexyl ester diethyl ester (IX)
• the crude product obtained in the previous step (WW 1 2.66 g) is dissolved in 12 ml_ EtOH. 4 mL of HCI co n e- is added and heated briefly to 70 0 C then left at room temperature for 3h. The solvent is evacuated and the residue triturated with 50 m L ether. The thick residue is S then dissolved in 3 mL ice water and the pH adjusted to 12 with 50% NaOH.
• Step C Preparation of (2S)-2-methoxycarbonylamino-3-naphthalen-2-yl-0 propionic acid (or L-Moc-2-naphthylalanine)
• Step D Preparation of (1 S,5S)-(1 - ⁇ 5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6- phosphonooxy-hexylcarbamoyl ⁇ -naphthalen ⁇ -yl-ethyO-carbamic acid methyl ester (PL-5 507) 100 mg L-Moc-2-naphthylalanine (step C) was activated with 100 mg EDAC and 57 mg HOBt in 1.5 mL DMF for 30 minutes.
• step B 100 mg of phosphoric acid 6-amino-2-[(4- amino-benzenesulfonyl)-isobutyl-amino]-hexyl ester diethyl ester (step B) was added and left to stir at room temperature for 1 h. 40 mL of 1M K 2 CO 3 was added to the DMF solution and left for 10 min. 50 mL of EtOAc was then added and the mixture was then agitated vigorously. Separation of the EtOAc phase was effected, followed by extraction with 5% citric acid (50 mL) once, then water (50 mL) 3 times and finally brine. The organic phase was the separated and evaporated.
• Step B Preparation of (2S.2S) Phosphoric acid mono-(2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-6- ⁇ 2-[(morpholine-4-carbonyl)-amino]-3-naphthalen-1-yl- propionylamino ⁇ -hexyl) ester (PL-498) 0
• Step A Preparation of (1- ⁇ 5-[(4-amino-3-fluoro-benzenesulfonyl)-isobutyl-amino]-6- hydroxy-hexylcarbamoyl ⁇ -2,2-diphenyl-ethyl)-carbamic acid methyl ester (X) (PL-337)
• step F (0.624 g, 1 mmol) is dissolved in 5 mL MeCN at 24 0 C.
• SelectFluor 0.35 g (1 mmol) is added in one portion and stirred for 1 h.
• 1 mL of water is added and the solution was injected directly into a preparative reverse-phase HPLC.
• the product was collected and lyophilized to give 250 mg (38%) yield of a white solid.
• Step B Preparation of (1S,5S)- ⁇ 1-[5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-amino]-6-(diethoxy-phosphoryloxy)-hexylcarbamoyl]-2,2-diphenyl-ethyl ⁇ -carbamic acid methyl ester
• step A The product of step A was phosphorylated with chlorodiethylphosphate following the procedure described in example 1, step G. Yields 157 mg, 68%.
• Step C Preparation of (1S,5S)-(1- ⁇ 5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol- ⁇ -phosphonooxy-hexylcarbamoyl ⁇ . ⁇ -diphenyl-ethylj-carbamic acid methyl ester (XQ (PL-515)
• Step A Preparation (1S,5S)-(1- ⁇ 5-[(4-amino-benzenesulfonyI)-isobutyl-amino]-6- phosphonooxy-hexylwrbamoyl ⁇ -2,2-diphenyl-ethyl)-carbamic acid methyl ester (PL-461)
• (2S)-2-methoxycarbonylamino-3,3-diphenyl-propionic acid ((example 1 , step E) 0.9 g, 3 mmol) was activated in DMF (5 ml_) with EDAC (1.7 g, 9 mmol) and HOBt (1.2 g, 9 mmol).
• EDAC 1.7 g, 9 mmol
• HOBt 1.2 g, 9 mmol
• Step B Preparation (1 S,5S)- ⁇ 1-[5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol- ⁇ -tdiethoxy-phosphoryloxyj-hexylcarbamoyll ⁇ -diphenyl-ethyl ⁇ carbamic acid methyl ester (XII)
• Step C Preparation (1 S,5S)-(1- ⁇ 5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol- ⁇ -phosphonooxy-hexylcarbamoyl ⁇ -diphenyl-ethyO-carbamic acid methyl ester (XIlI, in this specific case is compound Xl) (PL-515)
• This derivative was prepared from (2S)-acetic acid 6-tert-butoxycarbonylamino-2-[(4-tert- butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]-hexyl ester as described in example 15, step B.
• the yellow solid (66 mg) was used for the next reaction without purification.
• This derivative was prepared from (2S)-acetic acid 6-amino-2-[(4-amino-benzenesulfonyl)- isobutyl-amino]-hexyl ester (product of step B) as described in example 15, step B.
• the final product was purified by flash chromatography with a mixture of eluents hexane/ethyl acetate (2/8). A yellow solid was obtained in 70% yield (70 mg).
• Step C Preparation of (2S,2S)-nicotinic acid 2-[(4-amino-benzenesulfonyl)- isobutyl-amino]-6-(2-methoxycarbonylamino-3,3-diphenyl-propionylamino)-hexyl ester (PL-520)
• step B (2S)-nicotinic acid 6-amino-2-[(4-amino-benzenesulfonyl)-isobutyl- amino]-hexyl ester (100 mg, 0.22 mmol) was dissolved in anhydrous DMF (2 mL) and treated with 0.062 mL (0.45 mmol) of triethylamine followed by EDC (100 mg, 0.56 mmol), HOBt (75 mg, 0.56 mmol) and (2S)-2-methoxycarbonylamino-3,3-diphenyl-propionic acid (56 mg, 0.19 mmol). The mixture was stirred overnight at room temperature.
• Step C Preparation of (2S,2S)-dimethylamino-acetic acid 2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3,3-diphenyl- propionylamino)-hexyl ester (PL-534)
• This title compound was obtained from (1S)- ⁇ 4-[(5-tert-butoxycarbonylamino-1- hydroxymethyl-pentyl)-isobutyl-sulfamoyl)-phenyl ⁇ -carbamic acid tert-butyl ester (intermediate product (VW) of example 1 , step D) as described in example 15, step A using (2S)-2-benzyloxycarbonylamino-3-methyl-butyric acid.
• the crude product was purified by S flash chromatography eluting with a mixture of
• Step C Preparation of (2S,2S)-2-benzyloxycarbonylamino-3-methyl-butyric acid 2-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3,3- diphenyl-propionylamino)-hexyl ester 0
• Stock solution of PL-100 was prepared in methanol at 25 mM.
• the 25 mM stock solution was diluted with methanol to 0.25 and 0.125 mM.
• the 25 mM stock solution and the 0.25 and 0.125 mM solutions were diluted 250 fold for microsomal incubation to a final concentration of 200, 500 and 1000 nM.
• a sub-stock solution of PL- 100 was prepared in methanol at 25 ⁇ M and diluted in methanol to 2.5 and 0.25 ⁇ M and further diluted 250 fold for microsomal incubation giving final concentrations of 100, 10 and 1nM.
• Control incubations were performed in the presence of an equal volume of methanol. The final methanol concentration in incubation mixtures was ⁇ 1%. Selective inhibitors of CYP450 were tested in parallel as a positive control.
• the TESH activity was determined by HPLC-UV analysis following incubation of microsomes with testosterone as. a substrate.
• Liver microsomes (at a final concentration of 0.30 mg/ml protein) were incubated for 10 minutes at 37°C with various concentrations of testosterone in 0.5 ml reaction mixture, containing 0.1 M phosphate buffer (pH 7.4), 1 mM EDTA, and 3mM magnesium chloride with a NADPH-generating system, PL-100, Ritonavir or Ketoconazole, at appropriate concentrations, were present in the incubation medium with the probe substrate before the reaction was started by addition of NADPH- generating system.
• the reaction was terminated with 500 ⁇ L ice-cold acetonitrile and the 6 ⁇ -hydroxytestosterone formation was quantified using 6 point standard curve (range 2 to 20 nmol/mL).
• the analysis of 3 quality controls (LQC, MQC and HQC) was also performed.
• the associated enzyme activity was expressed as nmol of 6 ⁇ - hydroxytestosterone per mg of protein per min. Data were captured with Millenium chromatographic data management and storage system (version 4.0).
• EnzFitter software (Biosoft) was used for the calculation of Km, Vmax of probe substrates and apparent Km', Vmax' in the presence of various concentration of PL-100, Ritonavir or selective inhibitors and Ki of PL-100, Ritonavir or selective inhibitors.
• the cytochrome P450 activities were plotted against substrate concentrations.
• a nonlinear regression analysis was generated for Michaelis-Menten models in the presence and absence of PL- 100, Ritonavir or selective inhibitors. Lineweaver-Burk Plots
• Ki was calculated using the equation corresponding to the type of inhibition:
• V Vmax*[S] / (Km (1 +[I] / Ki) +[S])
• Km' Km* (1+[I] / Ki )
• Vmax' Vmax / (1+[l] / Ki)
• Km' Km / (1+[I] / Ki) 25
• Vmax' Vmax / (1+[I] / Ki)
• V Velocity of the enzyme reaction in the presence of inhibitor
• inhibitor Ki dissociation constant of the enzyme-inhibitor complex
• Enzyme kinetic parameters Km and Vm ax
• Ki values of test articles and selective inhibitors were calculated by nonlinear regression analysis using EnzFitter software.
• EnzFitter software was utilized also for the fitting of the Michaelis-Menten curves and for the generation of the Lineweaver-Burk plots, from the fitted values of Michaelis-Menten curves, allowing the identification of the type of inhibition were appropriate.
• the testosterone 6 ⁇ -hydrolase activity a selective marker for CYP3A4/5, was assessed in the presence of PL-100.
• PL-100 could be considered as a strong competitive inhibitor of human CYP3A4 although this compound was approximately 13 fold less potent than Ritonavir.
• PL-100 is thus considered a strong inhibitor of CYP3A4/5, but with a Ki value 13 fold higher than Ritonavir and 8.5 fold higher than ketoconazole.
• Ki value 13 fold higher than Ritonavir and 8.5 fold higher than ketoconazole.
• Example 12 In vivo inhibition of CYP450
• test article PPL-100
• concentration 40 mg/mL.
• Purity of the test article was taken into account for calculation of the concentration.
• Atazanavir mixture (10 mg/mL) Atazanavir was dissolved in a mixture of 20% ethanol, 50% propylene glycol, 0.1% Tween and 30% water (v/v/v/v), to obtain a concentration of 10 mg/mL.
• Dosing solutions were prepared shortly prior to dosing as follows: Briefly 4 mL of PPL- 100 mixture (40 mg/mL) was mixed with 4 mL of Atazanavir mixture (10 mg/mL).
• Sprague Dawley rats (Rattus ⁇ orv ⁇ gicus) aged 7 — 8 weeks at start of dosing were used for the study (Charles River Canada Inc., Montreal, PQ). Animals received Atazanavir (25 mg/kg) and PPL-100 (100 mg/kg). Six female rats were used on each time point.
• protein was extracted from the plasma samples and PL-100 (the active ingredient released rom PPL-100 under physiological conditions) or atazanavir were detected by HPLC-UV-MSD (1100 series, Hewlett Packard).
• the concentration of the protease inhibitors and PL-100 was 10 ⁇ M, except for the experiment involving Atazanavir, in which Atazanavir was at concentration of 1.0 ⁇ M and PL-100 was at a concentration of 2.0 ⁇ M.
• the percent of parent (original protease inhibitor) remaining was measured. The measurements were conducted at time point 60 minutes. The percent of parent remaining quantifies the extent of stability of protease inhibitors, as protease inhibitors are susceptible to being metabolized into daughter molecules in microsomes, decreasing the amount of the original protease inhibitor. The greater the percent of parent remaining, the greater the stability of the protease inhibitor in microsomes.
• the percent of parent remaining was measured for each protease inhibitor in the presence or absence of PL-100. As a control, the percent of protease inhibitor remaining was also measured in the presence of Ritonavir (RTV) to compare the boosting effect between PL-100 and RTV.
• RTV Ritonavir

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