WO2008023273A2

WO2008023273A2 - Method for improving pharmacokinetics

Info

Publication number: WO2008023273A2
Application number: PCT/IB2007/003593
Authority: WO
Inventors: Jinzi J. Wu; Michael Ge; Chandra J. Panchal
Original assignee: Ambrilia Biopharma Inc.
Priority date: 2006-07-17
Filing date: 2007-07-16
Publication date: 2008-02-28
Also published as: WO2008023273A3; AU2007287353A1; WO2008023273A8; EP2109461A2; JP2010502569A; CA2657316A1; AU2007287353A8

Abstract

The present invention provides pharmaceutical compositions and methods for improving the pharmacokinetics of drugs which may be metabolized by cytochrome P450 monooxigenase, the method may comprise co-administering a compound of formula I, with the drug; when the compound of formula I comprises an amino group, pharmaceutically acceptable ammonium salts thereof, wherein R1 may be, for example, H1 (HO)2P(O)-, (NaO)2P(O)-, alkyl-CO- or cycloalkyl-CO-, wherein X may be, for example, F, Cl, and Br, and wherein R2 and R3 are as defined herein.

Description

METHOD FOR IMPROVING PHARMACOKINETICS

TECHNICAL FIELD OF THE INVENTION

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. In particular, the present invention relates to methods for improving the pharmacokinetics of HIV protease inhibitors by co-administering PPL-100 with such protease inhibitors.

BACKGROUND OF THE INVENTION

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.

Examples of improved compounds have been developed in the form of prodrugs of aspartyl protease inhibitors such as described, for example, in U.S. patent no. 6,436,989 to Hale et al, the entire content of which is incorporated herein by reference. This patent shows a novel class of molecules characterized by favourable aqueous solubility, high oral bioavailability and facile in vivo generation of the active ingredient. However, it is well known that HIV has the ability to develop resistance to the currently available drugs. Thus, there is a need for alternative HIV protease inhibitors active towards wild-type and resistant viral strains. Thus, molecules derived from current HIV protease inhibitors showing enhanced solubility and bioavailability is desirable to fight resistant viral strains.

A unique class of aromatic derivatives which are inhibitors of aspartyl proteases is described in U.S. patent no. 6,632,816 to Stranix et al, the entire content of which is incorporated herein by reference. This patent includes, more particularly, /V-synthetic amino acid substituted L-lysine derivatives possessing potent aspartyl protease inhibitory properties. However, it would be advantageous to improve these derivatives by enhancing aqueous solubility and bioavailability in order to reduce the pill burden and to favour patient's compliance. Since it is challenging to generate active protease inhibitors, specifically toward wild-type and resistant strains, the formation of derivatives of original HIV protease inhibitors such as inhibitors described in U.S. patent no. 6,632,816 to Stranix βt al, known to be active toward resistant strains represents a viable route with considerable advantages. More particularly, generation of compounds and formulations with enhanced aqueous solubility, oral bioavailability, time of duration and formulation properties along with other advantages is desirable in the development of an effective drug. Protease inhibitors with improved pharmacokinetics are therefore desirable.

SUMMARY OF THE INVENTION

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.

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). 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. 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.

Some of the 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. VX-478, AG1343, DMP-323, XM-450, BILA2011 BS, BILA 1096 BS, BILA 2185 BS₁ BMS 186,318, LB71262, SC-52151, SC-629 (N,N-dimethylglycyl-N-(2-hydroxy-3-(((4-methoxyphenyl)sulfonyl)(2-methylpropyl) amino)- 1-(phenylmethyl)propyl)-3-methyl-L-valinamide), KNI-272, CGP 53437, CGP 57813 and U-103017).

Exemplary embodiments of compounds encompassed by the present invention and which may be used for inhibiting CYP450 (e.g., CYP3A4) 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),

wherein n may be, for example, 3 or 4,

wherein X and Y, the same or different, may be selected, for example, from the group consisting of H₁ 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, -CF₃, -OCF₃, - CN, -NO₂, -NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄, and -CH₂OH or X and Y together define an alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH₂O- and an ethylenedioxy group of formula - OCH₂CH₂O-,

wherein R₆ 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,

wherein R₃ 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. methyl, ethyl-, propyl, /so-propyl, butyl, /so-butyl, te/t-butyl, tert- butyl-CH₂-, 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-CH₂-, cyclohexyl-CH₂-, etc.), an alkyloxy group of 1 to 6 carbon atoms (e.g. CH₃O-, CH₃CH₂O-, /so-butylO-, terf-butylO- (Boc), etc.), tetrahydro-3-furanyloxy, -CH₂OH, -CF₃, -CH₂CF₃, - CH₂CH₂CF₃, pyrrolidinyl, piperidinyl, 4-morpholinyl, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl- OCH₂CH₂-, HO₂CCH₂-, 3-hydroxyphenyl, 4-hydroxyphenyl, 4-CH₃OC₆H₄CH₂-, CH₃NH-, (CH₃)₂N-, (CH₃CH₂)₂N-, (CH₃CH₂CHz)₂N-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, C₆H₅CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl-, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

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

and a group of formula,

wherein X' and Y', the same or different, may be selected, for example, from the group consisting of H₁ 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, -CF₃, -NO₂, - NR₄R₅, -NHCOR₄. -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH,

wherein R₄ and R₅, the same or different, 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 R₂ may be selected, for example, from the group consisting of a diphenylmethyl group of formula IV

a naphthyl-1 -CH₂- group of formula V

a naphthyl-2-CH₂- group of formula Vl

VI a biphenylmethyl group of formula VII

and an anthryl-9-CH₂- group of formula VIII

VIII

wherein R₁ 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.

In accordance with the present invention, Ri may be selected, for example, from the group consisting of (HO)₂P(O) and (MO)₂P(O), wherein M is an alkali metal (e.g. Na, K, Cs₁ etc) or alkaline earth metal (Ca, Mg, etc.).

Further in accordance with the present invention, R₁ 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-CH₂-, 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-CH₂-, cyclohexyl-CH₂-, etc.), an alkyloxy group of 1 to 6 carbon atoms (e.g. CH₃O-, CH₃CH₂O-, /so-butylO-, tert-butylO- (Boc), etc.), -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, (CHa)₂NCH₂-, (CHa)₂CHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, 2- pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 1-methyl-1 ,4-dihydro-3-pyridyl, 2-pyrazinyl, 2- quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyI, a phenyl group of formula

HI a picolyl group selected from the group consisting of

(4-picolyl)

a picolyloxy group selected from the group consisting of

(2-picolyloxy) (3-picolyloxy) (4-picolyloxy)

a substituted pyridyl group selected from the group consisting of

(substituted 2-pyridyl) (substituted 3-pyridyl) (substituted 4-pyridyl)

and a group of formula,

wherein X¹, Y¹, R₄ and R₅ are as defined herein.

The compounds mentioned herein may thus be used for improving the pharmacokinetics of drugs in the methods, pharmaceutical compositions, kits and uses described herein, More particularly, the present invention relates to 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;

wherein n may be 3 or 4,

wherein X and Y, the same or different, may be selected, for example, from the group consisting of H₁ 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, -CF₃, -OCF₃, - CN, -NO₂, -NR₄R₅. -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄, and -CH₂OH 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 -OCH₂O- and an ethylenedioxy group of formula -OCH₂CH₂O-,

wherein R₈ 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,

wherein R₃ 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, -CH₂OH₁ -CF₃, -CH₂CF₃, -CH₂CH₂CF₃. pyrrolidinyl, piperidinyl, 4-morpholinyl, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 3-hydroxyphenyl, 4-hydroxyphenyl, 4-CH₃OC₆H₄CH₂-, CH₃NH-, (CHa)₂N-, (CH₃CH₂)₂N-, (CH₃CH₂CH₂)₂N-. HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, C₆H₅CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl-, 2-pyrazinyl, 2- quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

10 a picolyl group which may be selected from the group consisting of

i s 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

20

and

a group of formula

wherein X¹ and Y", the same or different, 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, -CF₃, -NO₂, - NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH₁

wherein R₄ and R₅, the same or different, 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,

wherein R₂ may be selected from the group consisting of a diphenylmethyl group of formula IV

a naphthyl-1 -CH₂- group of formula V

a naphthyl-2-CH₂- group of formula Vl

a biphenylmethyl group of formula VII

and an anthryl-9-CH₂- group of formula VIII

VIII wherein R₁ 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.

In accordance with the present invention, Ri may be selected, for example, from the group consisting of H₁ (HO)₂P(O) and (MO)₂P(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, -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 2-hydroxyphenyl, 3-hydroxyphenyl, 4- hydroxyphenyl, (CHa)₂NCH₂-, (CH₃)₂CHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 1-methyl-1 ,4-dihydro-3-pyridyl, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

III

a picolyl group selected from the group consisting of

(2-picolyl) (3-picoIyl) (4-picolyl)

a picolyloxy group selected from the group consisting of

(2-picolyloxy) (3-picolyloxy) (4-picolyloxy)

a substituted pyridyl group selected from the group consisting of

(substituted 2-pyiϊdyl) (substituted 3-pyridyl) (substituted 4-pyridyl)

and a group of formula,

wherein X", Y¹, R₄ and R₅ are as defined herein.

More particularly, the present invention provides in one aspect thereof, 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;

wherein n may be 3 or 4,

wherein X and Y, the same or different, 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, F₁ Cl, Br, I, -CF₃, -OCF₃, -CN, -NO₂, -NR₄Rs, - NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄, and -CH₂OH 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 -OCH₂O- and an ethylenedioxy group of formula - OCH₂CH₂O-,

wherein R₃ 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 R₃A-CO-, where R₃A 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, -

CH₂OH, -CF₃, -CH₂CF₃, -CH₂CH₂CF₃, pyrrolidinyl, piperidinyl, 4-morpholinyl, CH₃O₂C-,

CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 3-hydroxyphenyl, 4-hydroxyphenyl, 4-

CH₃OCeH₄CH₂-, CH₃NH-, (CH₃)₂N-, (CH₃CH₂)₂N-, (CH₃CH₂CH₂)₂N-, HOCH₂CH₂NH-,

CH₃OCH₂O-, CH₃OCH₂CH₂O-, C₆H₅CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl-, 2- pyrazinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

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

a group of formula

wherein X' and Y', the same or different, 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, F₁ Cl, Br, I, -CF₃, -NO₂, -NR₄Rs, - NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH,

a naphthyl-1-CH₂- group of formula V

a naphthyl-2-CH₂- group of formula Vl

VI a biphenylmethyl group of formula VII

and an anthryl-9-CH₂- group of formula VIII

VIII

wherein R₁ may be H or a physiologically cleavable unit, whereby upon physiological conditions (in vivo) the compound may be converted into an active protease inhibitor.

In accordance with the present invention, R₁ may be selected, for example, from the group consisting of H, (HO)₂P(O) and (MO)₂P(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, -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl- OCH₂CH₂-, HO₂CCH₂-, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxy phenyl, (CHs)₂NCH₂- , (CH₃)_ZCHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-. CH₃OCH₂CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 1-methyl-1 ,4-dihydro-3-pyridyl, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

III

a picolyl group which may be selected from the group consisting of

(2-picolyl) (3-picolyl)

a picolyloxy group which may be selected from the group consisting of

(2-picolyloxy) (3-picolyl oxy) (4-picolyloxy)

a substituted pyridyl group which may be selected from the group consisting of

(substituted 4-pyridyl)

and a group of formula,

wherein X', Y¹, R₄ and R₅ are as defined herein.

In accordance with an embodiment of the present invention, pharmaceutical compositions which comprise compounds of formula Il wherein R₆ may be, for example, /so-butyl and n may be 3 are encompassed herewith. In accordance with an additional embodiment of the present invention, pharmaceutical compositions which comprise compounds of formula Il wherein R₈ may be, for example, /so-butyl and n may be 4 are also encompassed herewith.

In a particular embodiment of the present invention, Ri may be selected, for example, from the group consisting of H, (HO)₂P(O) and (NaO)₂P(O).

In another particular embodiment of the present invention, Ri may be selected, for example, from the group consisting of CH₃CO, 3-pyridyl-CO, (CH₃)₂NCH₂CO and

In accordance with another particular embodiment of the present invention, pharmaceutical compositions which comprise compounds of formula Il wherein R₃ may be selected, for example, from the group consisting of CH₃CO, CH₃O-CO, (CH₃)₂N-CO, 3- pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO are encompassed by the present invention.

In accordance with an embodiment of the present invention, X may be 4-NH₂ and Y may be H or F.

In accordance with an embodiment of the present invention, X' and Y' may both be H.

In accordance with a particular embodiment of the present invention, pharmaceutical compositions which comprise compounds of formula Il wherein R₂ may be selected, for example, from the group consisting of a diphenylmethyl group of formula IV₁ a naphthyl-1- CH₂- group of formula V, a naphthyl-2-CH₂- group of formula Vl, a biphenylmethyl group of formula VII and an anthryl-9-CH₂- group of formula VIII are encompassed herewith.

For example, R₂ may, more particularly, be selected from the group consisting of a diphenylmethyl group of formula IV, a naphthyl-1-CH₂- group of formula V, and a naphthyl- 2-CH₂- group of formula Vl.

In a further aspect, the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R₆ is /so-butyl, n is 4 and R₂ is a diphenylmethyl group of formula IV. In accordance with an embodiment of the present invention, R₁ may be selected from the group consisting of H, (HO)₂P(O) and (NaO)₂P(O).

In accordance with a further embodiment of the present invention, R₁ may be selected from the group consisting of CH₃CO, 3-pyridyl-CO, (CH₃J₂NCH₂CO and (CH₃)₂CHCH(NH₂)CO.

In accordance with an additional embodiment of the present invention, R₃ may be selected, for example, from the group consisting of CH₃CO, CH₃O-CO₁ (CH₃)₂N-CO, 3- pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO.

More particularly, in accordance with an embodiment thereof, the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X may be, for example, 4-NH₂, Y may be H, X' may be H, Y' may be H and R₃ may be CH₃O-CO.

In accordance with a particular embodiment of the present invention, Ri may be (HO)₂P(O).

In accordance with another particular embodiment of the present invention, R₁ may be (NaO)₂P(O).

In accordance with a further particular embodiment of the present invention, R₁ may be H.

More particularly, in accordance with a further embodiment thereof, the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X may be 4-NH₂, Y may be 3-F, X' may be H, Y' may be H and R₃ may be CH₃O- CO.

In accordance with a particular embodiment of the present invention, R₁ may be (HO)₂P(O). In accordance with another particular embodiment of the present invention, R₁ may be (NaO)₂P(O).

In accordance with a further particular embodiment of the present invention, Ri may be H.

More particularly, in accordance with an additional embodiment thereof, the present invention provides a pharmaceutical composition which may comprise a compound of formula II, wherein X is 4-NH₂, Y is H or 3-F, X¹ is H, Y' is H and R₃ is CH₃CO.

In accordance with another particular embodiment of the present invention, Ri may be (NaO)₂P(O).

More particularly, in accordance with an embodiment thereof, the present invention further provides a pharmaceutical composition which may comprise a compound of formula II, X is 4-NH₂, Y is H or 3-F, X^* is H₁ Y' is H and R₃ is 4-morphoHne-CO.

In accordance with an embodiment of the present invention X may be 4-NH₂, Y may be H, X¹ may be H, Y^* may be H and R₃ may be CH₃O-CO.

In accordance with a particular embodiment of the present invention R₁ may be 3-pyridyl- CO.

In accordance with another particular embodiment of the present invention R₁ may be (CH₃)₂NCH₂CO.

In accordance with yet another particular embodiment of the present invention R₁ may be (CHa)₂CHCH(NH₂)CO.

In accordance with an additional particular embodiment of the present invention R₁ may be CH₃CO. In an additional embodiment the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R₈ is /sobutyl, n is 4, X' and Y" are both H, R₂ is Naphtyl-1-CH₂-, X is 4-NH₂, Y is H, R₃ is 4-morpholine-CO and R₁ may be selected, for example, from the group consisting of H, (HO)₂P(O) and (NaO)₂P(O).

In a further embodiment, the present invention provides pharmaceutical compositions comprising a compound of formula II, wherein R₆ is /so-butyl, n is 4, X¹ and Y' are both H, R₂ is Naphtyl-2-CH₂-, X is 4-NH₂, Y is H₁ R₃ is CH₃O-CO and Ri may be selected, for example, from the group consisting of H, (HO)₂P(O) and (NaO)₂P(O).

More particularly, the present invention provides a pharmaceutical composition which may comprise; a) a compound of formula Ha

and pharmaceutically acceptable salts thereof,

wherein X and Y, the same or different, 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, -CF₃, -OCF₃, - CN, -NO₂, -NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄. and -CH₂OH or X and Y together define an alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH₂O- and an ethylenedioxy group of formula - OCH₂CH₂O-,

wherein X' and Y', the same or different, 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, -CF₃, -NO₂, - NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH₁ wherein n, R₁, R₃, R₄, R₅ and R_β are as defined herein;

b) a drug which may be metabolized by a cytochrome P450 monooxigenase, and;

c) a pharmaceutically acceptable carrier.

In accordance with an embodiment of the present invention, R₁ may be selected from the group consisting of H, (HO)₂P(O) and (MO)₂P(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, -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, ACeIyI-OCH₂CH₂-, HO₂CCH₂-, 2-hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, (CH₃)₂NCH₂-, (CHa)₂CHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, 2-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl. 1 -methyl- 1 ,4-dihydro-3-pyridyl, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

a picolyl group selected from the group consisting of

(2-picolyl) (3-picolyl) (4-picoIyl)

a picolyloxy group selected from the group consisting of

(2-picolyloxy) (3-picolyloxy) (4-picolyloxy)

a substituted pyτidyl group selected from the group consisting of

(substituted 2-pyridyl) (substituted 3-pyridyl) (substituted 4-pyridyl)

and a group of formula,

wherein X', Y', R₄ and R₅ are as defined herein.

In accordance with another embodiment of the present invention, R₆ may be feo-bυtyl.

In accordance with yet another embodiment of the present invention, n may be 4.

In accordance with a further embodiment of the present invention, R₁ may be selected from the group consisting of H, (HO)₂P(O) and (NaO)₂P(O).

Further in accordance with an embodiment of the present invention, Ri may be selected, for example, from the group consisting of CH₃CO, 3-pyridyl-CO, (CH₃J₂NCH₂CO and (CH₃)₂CHCH(NH₂)CO.

Also in accordance with an embodiment of the present invention, R₃ may be selected from the group consisting of CH₃CO, CH₃O-CO₁ (CH₃)₂N-CO, 3-pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO. In accordance with another embodiment of the present invention, R₃ may be selected from the group consisting of CH₃CO, CH₃O-CO, (CH₃)₂N-CO, 3-pyridyl-CO, 4-pyridyl-CO and 4-morpholine-CO.

In accordance with another embodiment of the present invention, X may be 4-NH₂ and Y may be H or F.

Also in accordance with an embodiment of the present invention, X may be 4-NH₂, Y may be H or 3-F, X¹ may be H, Y' may be H and R₃ may be CH₃CO.

In accordance with another embodiment of the present invention, X may be 4-NH₂, Y may be H or 3-F, X' may be H, Y' may be H and R₃ may be 4-morpholine-CO.

In accordance with a particular embodiment of the present invention, X may be 4-NH₂, Y may be H₁ X' may be H, Y" may be H, R₃ may be CH₃O-CO and R₁ may be (HO)₂P(O), (NaO)₂P(O) or H.

In accordance with another particular embodiment of the present invention, X may be 4- NH₂, Y may be 3-F, X' may be H, Y' may be H₁ R₃ may be CH₃O-CO and Ri may be (HO)₂P(O). (NaO)₂P(O) or H.

In accordance with a further particular embodiment of the present invention, X may be 4- NH₂, Y may be H or 3-F, X^* may be H, Y' may be H₁ R₃ may be CH₃CO and Ri may be (HO)₂P(O)₁ (NaO)₂P(O) or H.

In accordance with another particular embodiment of the present invention, X may be 4- NH₂, Y may be H or 3-F, X¹ may be H, Y' may be H and R₃ may be 4-morpholine-CO.

In accordance with an additional embodiment of the present invention, X may be 4-NH₂, Y may be H₁ X" may be H, Y' may be H₁ R₃ may be CH₃O-CO and R₁ may be 3-pyridyl-CO, (CH₃)₂NCH₂CO, (CH₃)₂CHCH(NH₂)CO or CH₃CO. In accordance with another embodiment of the present invention, X may be 4-NH₂, Y may be 3-F, X' may be H, Y' may be H, R₃ may be CH₃O-CO and Ri may be 3-pyridyl-CO, (CHg)₂NCH₂CO or (CH₃)₂CHCH(NH₂)CO.

Other compounds which may be used to carry out the present invention may include, for example, a compound of formula MA;

IIA

wherein Y, π, R₁, R₂, R₃, X" and Y' are as defined herein.

In accordance with the present invention, R₁ may be, for example, H₁ (HO)₂P(O) or (NaO)₂P(O). Further in accordance with the present invention, n may be 4. Y may be, for example, H. R₃ may be, for example. CH₃O-CO. R₂ may be, for example, a diphenylmethyl group of formula IV, where X' and Y¹ may be, for example H₁

Compounds of formula HA' as well as pharmaceutically acceptable salts and derivatives thereof may be used to carry out the present invention,

ILV

such as, for example, compound of formula HA' wherein R₁ is H, or (HO)₂P(O) or, 5 compound of formula MA' wherein Ri is (NaO)₂P(O).

For example, pharmaceutical compositions, methods, uses and kits encompassed by the present invention may comprise one or more of the following compounds and combination thereof; I O

-a compound of formula Ha wherein n is 4, R₁ is (HO)₂P(O), X is 4-NH₂, Y is H, X' is H, Y¹ is H, R₆ is /so-butyl and R₃ is CH₃O-CO,

- a compound of formula Ha wherein n is 4, R₁ is (NaO)₂P(O), X is 4-NH₂, Y is H, X' is H, 15 Y" is H, R_β is /so-butyl and R₃ is CH₃O-CO,

- a compound of formula Ua wherein n is 4, R₁ is (HO)₂P(O), X is 4-NH₂, Y is H, X' is H, Y' is H, R₆ is /so-butyl and R₃ is CH₃CO, 0 - a compound of formula Ha wherein n is 4, R₁ is (HO)₂P(O), X is 4-NH₂, Y is 3-F, X' is H, Y¹ is H, R₆ is /so-butyl and R₃ is CH₃O-CO,

- a compound of formula Ha wherein n is 4, Ri is CH₃CO₁ X is 4-NH₂, Y is H, X' is H, Y' is H, R₆ is /so-butyl and R₃ is CH₃O-CO,

25

- a compound of formula Ha wherein n is 4, R₁ is 3-pyridyl-CO, X is 4-NH₂, Y is H, X' is H, Y' is H, R₆ is /so-butyl and R₃ is CH₃O-CO₁

- a compound of formula Ha wherein n is 4, R₁ is (CHa)₂NCH₂CO₁ X is 4-NH₂, Y is H, X' is 30 H₁ Y^* is H₁ R₆ is /so-butyl and R₃ is CH₃O-CO₁ - a compound of formula Ha wherein n is 4, R₁ is (CHa)₂CHCH(NH₂)CO, X is 4-NH₂, Y is H, X' is H, Y" is H, R₆ is /so-butyl and R₃ is CH₃O-CO,

- a compound of formula lib wherein n is 4, R₁ is (HO)₂P(O), X is 4-NH₂, Y is H, X" is H, Y' is H, R₈ is /so-butyl and R₃ is CH₃O-CO and wherein the naphthyl group is a naphthyl-2- CH₂ group,

- a compound of formula Hb wherein n is 4, R₁ is (HO)₂P(O), X is 4-NH₂, Y is H₁ X" is H, Y' is H, R₆ is /so-butyl and R₃ is 4-morpholine-CO and wherein the naphthyl group is a naphthyl-1-CH₂ group, or

- a combination of any of the above mentioned compounds.

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. For example, compounds being able to release or generate (either in vivo or in vitro) an active ingredient of the following formula;

are 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.

In addition, compounds which able to release (either in vivo or in vitro) an active ingredient of the following formula;

may be used to carry out the present invention and are therefore encompassed by the present invention.

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.).

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.).

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.

The term "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. The term "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. It is also to be understood herein that 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. In the case of the present invention, 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.

The terms "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.

The terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable adjuvant" and "physiologically acceptable vehicle" refer 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. For example, 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).

The term "derivative" refers to a compound which has been chemically synthesized from an original compound. For example, when considering the chemical synthesis of PL-461 , PL-461 is a derivative of PL-100.

The term "consisting essentially of means that the pharmaceutical composition includes the specified materials and may include other material that does not materially affect the basic characteristics of the pharmaceutical composition.

Pharmaceutically acceptable derivatives of the compounds of formula I (such as compounds of formulae I, II, Ha, lib, Uc, MA and HA¹) and where applicable 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

(directly or indirectly) a an active compound or an antivirally active metabolite or residue thereof.

It is to be understood herein that a "straight alkyl group of 1 to 6 carbon atoms" includes for example, methyl, ethyl, propyl, butyl, pentyl, hexyl.

It is to be understood herein that a "branched alkyl group of 3 to 6 carbon atoms" includes for example, without limitation, /so-butyl, tert-butyl, 2-pentyl, 3-pentyl, etc.

It is to be understood herein, that a "cycloalkyl group having 3 to 6 carbon" includes for example, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclocyclohexyl (i.e., C₆H₁₁).

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. Examples of such 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, nitrate, oxalate, pamoate, pectinate, perchlorate. persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate.

Compounds which are encompassed by the present invention also envisions the quaternization of any basic nitrogen containing groups of the compounds disclosed herein. The basic nitrogen 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.

It is to be understood herein, that if a "range" or "group of substances" is mentioned with respect to a particular characteristic (e.g., temperature, concentration, time and the like) of the present invention, the present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges or sub-groups therein whatsoever. Thus, 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. Thus, for example, - with respect to the number of carbon atoms, 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.;

- and similarly with respect to other parameters such as concentrations, elements, etc... It is in particular to be understood herein that 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".

It is also to be understood herein that "g" or "gm" is a reference to the gram weight unit and "C", or " ⁰C " is a reference to the Celsius temperature unit.

The compounds described herein may easily be prepared using conventional techniques from readily available starting materials. The detailed descriptions of these approaches are presented, for example, in schemes 1 to 5 discussed below.

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).

Note: a) R₂ and R₃ are as defined herein.

The synthesis of 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.

III

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.

Note: a) n, X, Y₁ R₂, R₃ and Re are as defined herein.

Sc

UA

IA

1) TMS-Br DCM

2) H₂O

IUA

The synthesis of phosphate monoester IHA is performed as described for the preparation of /// (scheme 1).

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).

Note: a) R₂ and R₃ are as defined herein.

As shown in scheme 2, the phosphate monoester derivative /// was obtained from (3S)-3- isobutylamino-azepan-2-one (/V) in a seven-step reaction sequence. Initially, (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). Then, coupling of the free amino group present on intermediate IX with a variety of synthetic amino acid in the presence of 1-hydroxybenzotriazole (HOBt) and 1-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride (EDAC) led to derivative // in good to excellent yields. Finally, addition of trimethysilyl bromide in dichloromethane (DCM) gave compound /// in good to excellent yields.

Scheme 2

BocHN

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 moπoester 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.

Thus, the treatment of PL-100 with Selectfluor™ in acetonitrile gave derivative X in 38% yield. The introduction of the phosphate monoester group was performed as described previously in scheme 1 and 2. First, the diethyl phosphotriester intermediate was obtained in good yield upon treatment with diethyl chlorophosphate and sodium hydride in a mixture of tetrahydrofuran and triethylphosphate. Secondly, addition of trimethysilyl bromide in dichloromethane (DCM) gave the phosphate monoester compound in good to excellent yields. The final product X/ was easily obtained upon treatment of the phosphate monoester with a solution of sodium hydroxide with good yields.

Scheme 3

PL-100 (example 1 , step F)

1) Diethylchlorophospphnaatiee,,

Xl

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. In this case, 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.

Note: a) R₂ and R₃ are as defined herein.

Briefly, treatment of derivative // with Selectfluor™ in acetonitrile gave derivative XII in good yields. Then, addition of trimethysilyl bromide in dichloromethane (DCM) gave the phosphate monoester compound XIII in good to excellent yields. If desired, the final product XIII may be easily transformed into the phosphate monoester sodium salt analog as described before in scheme 3.

Sc

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. In this scheme R₂ is set as a diphenylmethyl group. However, 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.

Note: a) 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. Esterification of (1S)-{4-[(5-tert-butoxycarbonylamino-1-hydroxymethyl-pentyl)- isobutyl-sulfamoyl]-phenyl}-carbamic acid tert-butyl ester (VII) with a variety of acid in the presence of 1-hydroxybenzotriazole (HOBt) and 1-[3-(dimethylamino)propyl]-3- ethylcarbodiimide hydrochloride (EDAC) led to the desired esters XIV in excellent yields. The acetyl ester was obtained quantitatively using acetic anhydride in the presence of Λ/,Λ/-dimethylaminopyridine (DMAP) in dichloromethane (DCM). Cleavage of the Boc protective group was achieved quantitatively upon treatment with trifluoroacetic acid (TFA) in DCM. 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. If necessary, catalytic hydrogenation of a benzyloxycarbonyl group is performed using 10% palladium on carbon to give the final compound XVII.

Scheme 5

VII (R_1A = CH₃) XIV

TFA, DCM, 99%

XVII

As it may be appreciated by the person skilled in the art, the above synthetic schemes are not intended to be a comprehensive list of all means by which the compound described in this application may be synthesized but only represent exemplification of synthesis methods among others. Further methods will be evident to those of ordinary skill in the art.

The compounds described herein may be modified by appending appropriate functionalities to enhance selective biological properties. Such 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.

Pharmaceutical 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.

It is therefore understood herein that oral administration or administration by injection are encompassed by the present invention. For example, compounds of the present invention, may, for example, be orally administered in an aqueous solution. The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

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.

In the description herein, the following abbreviations are used:

Abbreviation Meaning

Ac Acetyl

AcOH Acetic acid APCI Atmospheric pressure chemical ionization

AIDS Acquired Immunodeficiency Syndrome APV Amprenavir

ATV Atazanavir

AZT 3-Azido-3-deoxythymine (Zidovudine)

Boc Benzyloxycarbonyl t-Butyl tert-Butyl

CAM Cerium ammonium molybdate

DCM Dichloromethane

DMAP Λ/,Λ/-dimethylaminopyridine

DMSO Dimethylsulfoxide

DMF Dimethylformamide

DNA Deoxyribonucleic acid

EDAC 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride

EtOAc Ethyl acetate

EtOH Ethyl alcohol g Gram h hour

HIV-1 , -2 Human immunodeficiency virus type 1 , type 2

HOBt 1 -Hydroxybenzotriazole

HPLC High performance liquid chromatography

HTLV-I, -Il Human T-cell lymphotropic virus type I, type Il

IL-2 lnterleukin-2

IDV Indinavir

Kg Kilogram

L Liter

LC-MS Liquid chromatography-mass spectrometry

LPV Lopinavir

M Molar

MeOH Methyl alcohol mg Milligram mp Melting point min Minute

Moc Methoxycarbonyl mol Mole mL Milliliter mmol Millimole NFV Nelfinavir nm Nanometer nM Nanomolar po Orally rEPO Recombinant erythropoietin

RTV Ritonavir

SQV Saquinavir

TLC Thin layer chromatography

3TC 2\3'-Dideoxy-3-thiacytidine

TFA Trifluoroacetic acid

THF Tetrahydrofuran

EXAMPLES This section describes the synthesis of lysine based compounds able to release an HIV aspartyl protease inhibitors as described herein. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way. This section presents the detailed synthesis of compounds no. 1 to 10 of this invention.

Exemplary synthesis schemes of the active ingredients have been disclosed in U.S. patent no. 6,632,816 to Stranix et a/,.

Materials and Methods-preparation of compounds

Analytical thin layer chromatography (TLC) was carried out with 0.25 mm silica gel E. Merck 60 F₂₅₄ plates and eluted with the indicated solvent systems. Preparative chromatography was performed by flash chromatography, using silica gel 60 (EM Science) with the indicated solvent systems and positive air pressure to allow proper rate of elution. Detection of the compounds was carried out by exposing eluted plates (analytical or preparative) to iodine, UV light and/or treating analytical plates with a 2% solution of p-anisaldehyde in ethaπol containing 3% sulfuric acid and 1% acetic acid followed by heating. Alternatively, 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^₆Mo₇O₂₄ and 8.3 g Ce(SO₄J₂ 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.

Semi-preparative HPLC conditions for purification of test compounds: 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 Column: Alltech (#96053) Hyperprep PEP, C-18, 100 Aa, 8 μm, 22 x 250 mm Flow: 15 mL/min Solvents: A: H₂O; B: CH₃CN Gradient: 25% to 80% of B over 40 min Detector: absorbance; λ: 210 & 265 nm

The crude material dissolved in acetonitrile to a concentration of around 50 to 80 mg / 2 mL were injected in each run. Fractions were collected in amounts of 9 mL pertaining absorbance was detected at the UV detector.

Unless otherwise indicated, all starting materials were purchased from a commercial source such as Aldrich Co. or Sigma Co.

Melting points (mp) were determined on a Bϋchi 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.

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AMX-ll-500 equipped with a reversed or QNP probe. Samples were dissolved in deuterochloroform

(CDCI₃), deuteroacetone (acetone-d_β), deuteromethanol (CD₃OD) or deuterodimethylsulfoxide (DMSO-d₈) for data acquisition using tetramethylsilane as internal standard. Chemical shifts () are expressed in parts per million (ppm), the coupling constants (J) are expressed in hertz (Hz) whereas multiplicities are denoted as s for singlet, d for doublet, 2d for two doublets, dd for doublet of doublets, t for triplet, q for quartet, quint, for quintet, m for multiplet, and br s for broad singlet. DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES:

Specific examples for the preparation of derivatives of general formula I

The preparation of PL-100, PL-337 and other compounds of this class is presented in U.S. patent no. 6,632,816 to Stranix et al.

The following compounds were prepared from L-lysine derivatives using the procedures summarized in schemes 1 , 1A, 2, 3, 4 and 5 of this invention.

Example 1. Preparation of (1S,5S)-<1-{5-[(4-amino-benzenesulfonyl)-isobutyl- amino]-6-phosphonooxy-hexylcarbamoyl}-2,2-diphenyl-ethyl)-carbamic acid methyl ester (PL-461)

The preparation of the title compound is based on schemes 1 and 2 of this invention.

Step A. Preparation of (3S)-3-isobutylamino-azepan-2-one (IV)

L-α-amino_τ-caprolactam (22.0 g) was dissolved in cold dichloroethane (DCM₁ 200 ml_). isobutyraldehyde (12.6 g) was added slowly and stirred until the heat evolved was dissipated (water forms at the surface). The cold solution was added to 46.5 g of powdered NaBH(OAc)₃ in DCM (0.5 L). AcOH (70 mL) was added to the solution. The slightly turbid mixture was stirred at 20 ⁰C for 4 h. A 500 mL solution of 2M NaOH was added slowly to the turbid mixture and the pH adjust to 11 using a concentrated NaOH solution, and then the mixture stirred for a further 20 min. After extraction, the DCM layer was dried with MgSO₄, filtered and evaporated. The oil thus obtained crystallizes slowly on standing (27.8 g, 85%) and was used without further purification in the next step.

¹H NMR (CDCI₃): δ 0.93 (d, J = 6.5, 3H), 0.97 (d, J = 6.5. 3H), 1.39 (t, J = 9.8, 1H), 1.47 (m, 1H), 1.78-1.65 (m, 2H), 2.00-1.93 (m, 2H), 2.32-2.2 (m, 2H), 2.38 (t, J = 9.7, 1H), 3.16 (m, 3H), 6.62 (s, 1H (NH)). mp 52-54 ⁰C (hexanes).

A small sample was converted to the S-methyl benzyl urea by adding the solid to a solution of S-methyl benzyl isocyanate in MeCN. NMR gives 98% ee Step B. Preparation of Mx-isobutyl-/Vα-(4-acetamidobenzenesulfonyl)-L-α- amino— caprolactam (V)

Λ/o-isobutyl-L-α-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.

¹H NMR (DMSO-d_β): . 0.93 (d, J = 6.0, 3H), 0.96 (d, J = 6.0, 3H), 1.39 (t, J = 12.0, 1 H), 1.85-1.65 (m, 3H), 2.08-2.18 (m and s, 6H), 2.90-2.97 (m, 1H), 3.00-3.06 (m, 2H), 3.35 (dd, J = 14.2, 8.5. 1H), 4.65 (d, J = 8.7, 1H)₁ 6.3 (s, 1H), 7.42 (d, J = 8.8, 2H), 7.6 (d, J = 8.8, 2H). mp 230-233 ⁰C (EtOH).

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/)

4.2 g of Λ/α-isobutyl-Λ/α-(4-acetamidobenzenesulfonyl)-L-α-aminor-caprolactam (V) was suspended in 30 mL MeCN and briefly sonicated to break up any large chunks. To this white suspension was added 6.7 g (3 eq.) of di-tert-butyl pyrocarbonate in 10 mL MeCN. The suspension was stirred with a magnetic bar and a 120 mg portion of DMAP was added. The solution becomes a clear light yellow after a few minutes. TLC (EtOAc) reveals 1 product Rf 0.9 (starting material Rf at 0.4). The solution is poured in distilled water 20 mL and extracted with ether, dried with Na₂SO₄ and evaporated yielding 6.90 g. A sample was recrystallized from hexanes.

¹H NMR (DMSO-Cf₆): . 0.68 (d, J = 6.0, 3H), 0.85 (d, J = 6.0, 3H), 1.39 (s, 10H), 1.47 (s, 9H)₁ 1.85-1.65 (m. 3H). 2.15 (S₁ 3H)₁ 2.80 (q. J = 4, 1H)₁ 3.10-3.36 (m, 2H)₁ 4.01 (d, J = 8.0, 1 H), 4.85 (d, J = 8.7, 1 H)₁ 7.32 (d, J = 8.8, 2H)₁ 7.87 (d, J = 8.8, 2H). mp 123-124 ⁰C

Step D. Preparation of (1S)-4-amino-/V-(5-amino-1-hydroxymethyl-pentyl)-Λ/- isobutyl-benzenesulfonamide (V7/-de protected) (reductive ring opening and deprotection) A 3.0 g portion of (SSJ-S-IH-tøcetyl-tert-butoxycarbonyl-aminoJ-benzenesulfonyll-isobutyl- amino}-2-oxo-azepane-1-carboxylic acid tert-butyl ester (W₁ step C) is dissolved in 40 mL EtOH followed by 750 mg NaBH₄. Brief heating with a heat gun gives a clear solution. TLC reveals one streaky spot after 20 min (EtOAc). The solution is concentrated to a paste, poured in 40 mL 1 N NaOH and extracted with ethyl acetate, the organic phase dried with NaSO₄ and evaporated to give 2.8 g of product intermediate (W/); (1S)-{4-[(5-tert- butoxycarbonylamino-i-hydroxymethylφentyO-isobutyl-sulfamoylJ-phenyty-carbamic acid tert-butyl ester (W/).

The above product intermediate is dissolved in 5 mL EtOH and 5 mL 12 N HCI is added. Vigorous gas evolution is observed for a few minutes. After 2 h the solution is evaporated and rendered basic with concentrated KOH and extracted with EtOAc yielding 1.75 g of a white powder.

¹H NMR (DMSO-cfe): . 0.82 (m, 6H), 0.97-1.12 (m, 2H)₁ 1.15-1.30 (m, 3H), 1.57 (m, 1H)₁ 1.84 (m, 1H)₁ 2.40 (t, J = 7.8, 2H)₁ 2.75 (m, 1H), 2.85 (m, 1H)₁ 3.21 (m, 1H)₁ 3.44 (d, J = 6.4, 2H), 5.92 (br s. 2H), 6.59 (d. J = 8.0, 2H), 7.39 (d, J = 8.0, 2H).

Step E. Preparation (2S)-2-methoxycarbonylamino-3,3-diphenyl-propionic acid

To a solution of L-diphenylalanine (241 mg, 1.0 mmol) (Peptech Corp.) in 5 mL 1N NaOH and 0.5 mL saturated Na₂CO₃ (resulting solution at pH 10) was added methoxycarbonyloxysuccinimide (carbonic acid 2,5-dioxo-pyrrolidin-i-yl ester methyl ester) (180 mg, 1.1 mmol) dissolved in 5 mL. Afterwards, the reaction mixture was stirred at room temperature for 2 h. The alkaline solution was extracted once with ether (10 mL) and the aqueous phase was acidified with 1N HCI. This was extracted twice with 20 mL EtOAc₁ and the combined organic phases were washed with 50 mL 1 N HCI. The organic phase was dried over Na₂SO₄. filtered and evaporated to an oil, which solidifies to yields 250 mg (83%) of the desired material. This derivative was used as such in the next step.

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)

The title compound was prepared from (1 S)-4-amino-Λ/-(5-amino-1-hydroxymethyl-pentyl)- Λ/-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).

LC-MS : 625.3 (M+H)^*, 95% pure

¹H NMR (CD₃OD): δ 0.71-0.85 (m, 2H), 0.88 (d, J = 6.3, 5H), 0.91-0.96 (m, 2H), 1.29- 1.34 (m, 1H), 1.41-1.52 (m, 1H) 1.82-1.92 (m, 1H)₁ 2.61-2.68 (m, 1H), 2.81-2.85 (m, 2H)₁ 2.94-3.05 (m, 2H), 3.38-3.40 (t, J = 5.0, 1H)₁ 3.50-3.51 (m, 1H), 3.52 (s, 3H), 4.28 (d, J = 11.0 1H), 4.87 (d, J = 11.0, 1H), 6.69 (d, J = 8.0, 2H), 7.15-718 (m, 2H), 7.20-7.31 (m, 6H)₁ 7.33 (d, J = 7.9, 2H), 7.47 (d, J = 7.5, 1H).

¹³C NMR (CD₃OD): δ 20.0, 20.1, 23.3, 25.4, 28.1 , 28.5, 28.9, 38.1 , 40.0, 51.2, 51.6, 53.1 , 57.2. 57.4, 59.5, 61.9, 62.4, 112.6, 125.7, 126.2, 126.3, 127.9, 128.1 ,128.15, 128.2, 128.4, 128.7, 141.3, 141.9, 152.4, 155.9, 169.9, 172.5.

Step G. Preparation of (1S,5S)-{1-[5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6-

(diethoxy-phosphoryloxy)-hexylcarbamoyl]-2,2-diphenyl-ethyl}-carbamic acid methyl ester

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 N₂ 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,

0.423 mmol) was added at 0 ⁰C. The solution was stirred for 1 h at 0 ⁰C and 12 h at room temperature. 20 mL of Amberlite XAD-2 was added to the solution and the beads were thoroughly mixed with the solvent. To the mixture was added ice water 2 mL, and the THF evaporated off. The beads were then washed with distilled water 6 times 100 mL then extracted three times with ethyl acetate (30 mL). The combined phase was evaporated and the residue was dried under high vacuum. The crude product was purified by flash chromatography using ethyl acetate/hexane (8/2), then EtOAc 100% as eluent. The yield of this reaction is 152 mg 61%.

LC-MS: 761.2 (M+H)⁺, 90% pure

¹H NMR (CD₃OD): <5 0.68-0.75 (m, 1H), 0.75-0.84 (m, 1H), 0.84-1.10 (m, 9H), 1.21-1.50 (m, 8H), 1.88 (m. 1H), 2.58-2.71 (m, 1 H)₁ 2.80-2.89 (m, 1H)₁ 2.89-3.08 (m, 2H), 3.49-3.60

(S₁ 3H)₁ 3.65-3.74 (m, 1H)₁ 3.85-3.95 (m, 1H), 3.97-4.02 (m. 1H), 4.07-4.21 (m, 4H), 4.29 (d, J = 10.8, 1H), 6.71 (d, J = 8.0, 2H). 7.10-7.20 (m, 2H)₁ 7.20-7.32 (m, 5H), 7.32-7.45 (m, 3H), 7.50 (d, J = 7.5. 2H), 7.86 (br s, 1 H).

³¹P NMR (CD₃OD): δ 1.62

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)

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 ⁰C. 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.

LC-MS: 705.2 (M+H)⁺, 95% pure

¹H NMR (CD₃OD): δ 0.65-0.73 (m, 1H), 0.75-0.83 (m, 1H), 0.89 (d, J = 5.6, 8H), 1.27- 1.38, (m, 1H). 1.42-4.55 (m, 1H), 1.82-1.94 (m, 1H), 2.57-2.68 (m, 1H), 2.78-2.90 (m, 1H), 2.91-3.09 (m, 2H), 3.54 (s, 3H), 3.60-3.72 (m, 1 H), 3.87-4.05 (m, 1H), 4.00 (m, 1H)₁ 4.29 (d, J = 11.3. 1H), 4.90 (d, J = 11.4, 1H)₁ 6.73 (d, J = 8.0, 2H), 7.13-7.22 (m, 2H), 7.22-7.33 (m, 6H), 7.33-7.45 (m, 2H), 7.51 (d, J = 7.5, 2H).

³¹P NMR (CD₃OD): δ 2.80

Example 2. Preparation of (1 S,5S)-(1-{5-[(4-amino-benzenesulfonyl)-isobutyl- amino]-6-phosphonooxy-hexylcarbamoyJ}-2,2-diphenyl-ethyl)- carbamic acid methyl ester sodium salt (PL-462)

70.7 mg of the final product of example 1 is added to 1 ml_ 0.1 N NaOH and diluted with 1 mL of distilled water. The Solution is then frozen and lyophilized. Yields 67.2 mg (92%) of the desired material with 95% purity.

¹H NMR (CD₃OD): δ 0.72-0.83 (m, 1H), 0.90 (d, J = 5.8, 9H), 1.26-1.38 (m, 1H), 1.53- 1.65 (m, 1H), 1.88-2.00 (m, 1 H), 2.60-2.70 (m, 1H), 2.79-2.89 (m, 1 H), 2.98-3.00 (m, 1H),

3.00-3.08 (m, 1H), 3.54 (s. 3H), 3.58-3.71 (m, 1H)₁ 3.72-3.83 (m, 1 H)₁ 3.84-3.95 (m. 1H). 4.28 (d, J = 11.1, 1H), 4.91 (d, J = 11.0, 1H)₁ 6.70 (d, J = 7.6, 2H), 7.12-7.22 (m, 2H), 7.22-7.32 (m, 6H), 7.33-7.40 (m, 2H), 7.50 (d, J = 7.7. 2H).

³¹P NMR (CD₃OD): δ 3.13

Example 3. Preparation of (1S,5S)-(1-{5-[(4-amino-benzenesulfonyl)-isobutyl- amino]-6-phosphonooxy-hexylcarbamoyl}-2-naphthalen-2-yl-ethyl)- carbamic acid methyl ester (PL-507)

The preparation of the title compound is based on scheme 2 of this invention.

Step A. Preparation of (1S)-(4-{[5-tert-butoxycarbonylamino-1-

(diethoxyphosphoryloxymethyl)-pentyl]-isobutyl-sulfamoyl}-phenyl)-carbamic acid tert- butyl ester (VIII)

2.00 g (3.7 mmol) (1 S)-{4-[(5-tert-butoxycarbonylamino-1-hydroxymethyl-pentyl)-isobutyl- sulfamoyl]-phenyl}-carbamic acid tert-butyl ester (VII) (example 1, step D) is dissolved in 0.63 ml. triethylphosphate and 10 mL THF at 0 ⁰C under inert argon atmosphere. 0.63 mL (4.44 mmol) diethylchlorophosphate is added and then 0.25 g (6.2 mmol), NaH 60% in oil is added in port ion wise. The mixture is allowed to warm to room temperature and left to stir for 2 h (LC-MS showed completion after 1 h). To the solution is added 20 mL of Amberlite XAD-2 resin and the slurry thoroughly mixed and added to 200 mL ice water. After stirring for 15 min. the resin suspension is filtered and the resin washed several times with distilled water (500 mL). The desired product is desorbed from the resin with acetone (5 X 50 mL), EtOAc (5 X 50 mL), the organic phase is then dried over Na₂SO₄. After evaporation of the solvent 2.66 g (89%) of clear oil is obtained. The crude product contains a fraction with two diethyl phosphates and is used as is in the next step.

¹H NMR (CD₃OD): δ 0.91 (d, J = 6.3, 6H), 1.11-1.21 (m, 2H), 1.33 (t, J = 6.9, 10H), 1.43 (s, 9H), 1.53 (S, 10H), 1.90-1.97 (m, 1H), 2.88-2.96 (m, 3H), 2.96-3.04 (m, 1H), 3.81-3.90 (m, 1H), 3.91-3.99 (m, 1H), 4.01-4.14 (m, 4H), 7.61 (d, J = 8.3, 2H), 7.72 (d, J = 8.4, 2H).

³¹P NMR (CD₃OD): δ 1.59

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 (WW₁ 2.66 g) is dissolved in 12 ml_ EtOH. 4 mL of HCI co_ne- is added and heated briefly to 70 ⁰C 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. The thick slurry obtained is extracted with EtOAc (3 X 50 mL) and the organic phase dried over Na₂SO₄. After filtration of the drying agent the organic phase is evacuated to yield 1.84 g (98%) of the desired product (/X). 0 LC-MS: 480.2 (M+H)^*, 95% pure.

¹H NMR (CD₃OD): δ 0.91 (s, 6H), 1.11-1.26 (m, 3H), 1.28-1.43 (m, 8H), 1.45-1.51 (m, 1H), 1.52-1.61 (m, 1H), 1.89-1.96 (m, 1H), 2.56 (t, J = 6.7, 2H), 2.85-2.91 (m, 1H), 2.98- 3.11 (m. 1H), 3.79-3.99 (m, 1H), 3.94 (d, J = 5.3, 1H)₁ 4.09-4.11 (m, 4H), 6.69 (d, J = 7.9,5 2H). 7.50 (d, J = 7.9, 2H).

31 P NMR (CD₃OD): 5 1.61

Step C. Preparation of (2S)-2-methoxycarbonylamino-3-naphthalen-2-yl-0 propionic acid (or L-Moc-2-naphthylalanine)

To a solution of L-2-naphthylalanine (215 mg, 1 mmol) (Peptech Corp.) in 5 mL 1 N NaOH and 0.5 mL saturated Na₂CO₃ (resulting solution at pH 10) was added methoxycarbonyloxysuccinimide (187 mg, 1.1 mmol) dissolved in 5 mL. Afterwards, the5 reaction mixture was stirred at room temperature for 2 h. The alkaline solution was extracted once with ether (10 mL) and the aqueous phase was acidified with 1N HCI. This was extracted twice with 20 mL EtOAc₁ and the combined organic phases were washed with 50 mL 1N HCI. The organic phase was dried over Na₂SO₄. filtered and evaporated to an oil, which solidifies to yields 200 mg (73%) of the desired material. This intermediate0 (referred as the Λ/-substituted amino acid) was used without further purification in the next step.

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. Then, 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 K₂CO₃ 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. The residue was taken up in 50 mL DCM and re- evaporated. The residue was again taken up in 50 mL DCM and 0.5 mL of TMSBr was added. The solution was left overnight (16 h). The DCM was evacuated and a solution of ice cold MeOH: Water 1:1 was added, stirred briefly and evacuated. The residue was triturated with ether then dissolved in 1N NaOH. The clear solution was extracted with ether and the aqueous phase acidified with 6N HCI. The white precipitated was then collected by filtration and dried in vacuo overnight. Yields 88 mg of the title compound.

LC-MS: 679.8 (M+H)\ 95% pure.

¹H NMR (CD₃OD): δ 0.89-0.98 (m, 8H), 1.15 (m, 2H). 1.35 (m, 1H), 1.45 (m, 1H)₁ 1.88 (m, 1H), 2.84 (m, 2H), 2.98 (m, 1H), 3.01 (m, 2H), 3.24 (m, 1H), 3.56. (s, 3H), 3.60 (m, 1 H), 3.81 (m, 1H), 3.99 (m, 1 H), 4.39 (t, J = 6.8, 1H), 6.91 (d, J = 8.0, 2H), 7.34 (d, J= 8.0, 1H), 7.45 (m. 2H). 7.58 (d. J = 8.0, 2H). 7.66 (s, 1H)₁ 7.70-7.82 (m, 3H).

³¹P NMR (CD₃OD): δ 2.56

Example 4. 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)

Step A. Preparation of (2S)-2-[(morpholine-4-carbonyl)-amino]-3-naphthalen-1-yl- propionic acid

To a solution of L-1-naphthylalanine (215 mg, 1 mmol) (Peptech Corp.) in 5 mL 1N NaOH and 0.5 mL saturated Na₂CO₃ (resulting solution at pH 10) was added morpholine-4- carbonyl chloride (150 mg, 1.0 mmol) dissolved in 5 mL. Afterwards, the reaction mixture was stirred at room temperature for 2 h. The alkaline solution was extracted once with ether (10 mL) and the aqueous phase was acidified with 1N HCI. This was extracted twice with 20 mL EtOAc, and the combined organic phases were washed with 50 mL 1N HCI. The organic phase was dried over Na₂SO₄, filtered and evaporated to an oil, which solidifies to yields 125 mg (38%) of the desired material. This compound was used as S such in the next step.

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

This compound was made as for the preparation of the product of example 3 (step D) with 100 mg of (2S)-2-[(moφholine-4-carbonyl)-amino]-3-naphthalen-1-yl-propionic acid (step A of this example). The resulting precipitated residue was further purified by reverse phase preparative HPLC. Yields 41 mg of the final compound. 5

LC-MS: 734.8 (M+H)⁺, 95% pure.

¹H NMR (CD₃OD): δ 0.83-0.98 (m, 8H), 1.00-1.25 (m, 4H), 1.45-1.52 (m, 1H), 1.52-1.66 (m, 1H), 1.88-1.99 (m, 1H), 2.77-2.92 (m, 2H), 2.98-3.16 (m, 3H), 3.40-3.49 (m, 1H), 3.50-0 3.56 (m, 6H), 3.67-3.69 (m, 1H), 3.81-3.89 (m, 1H), 3.99-4.05 (m, 1H), 4.59 (t, J = 6.0, 1H)₁ 6.75 (d, J = 8.0, 2H), 7.30-7.60 (m, 7H), 7.75 (d, J = 8.0, 1H), 7.90 (d, J= 7.8. I H), 8.23 (d. J=7.8 2H).

³¹P NMR (CD₃OD): δ 2.1 Λ 5

Example 5. Preparation of (2S,2S)-phosphoric acid mono-{6-(2-acetylamino-3,3- diphenyl-propionylamino)-2-[(4-amino-benzenesulfonyl)-isobutyl- amino]-hexyl> ester (PL-504) 0

Step A. Preparation (2S)-2-acetylamino-3,3-diphenyl-propionic acid

To a solution of L-diphenylalanine (100 mg, 0.4 mmol) (Peptech Corp.) in 5 mL 1 N NaOH and 0.5 mL saturated Na₂CO₃ (resulting solution at pH 10) was added acetyl chloride (0.55 mmol) dissolved in 5 mL. Afterwards, the reaction mixture was stirred at room temperature for 2 h. The alkaline solution was extracted once with ether (10 mL) and the aqueous phase was acidified with 1N HCI. This was extracted twice with 20 mL EtOAc, and the combined organic phases were washed with 50 mL 1N HCI. The organic phase was dried over Na₂SO₄. filtered and evaporated to an oil, which solidifies to yields 70 mg (60%) of the desired material. This crude intermediate was used as such in the next step.

Step B. Preparation of (2S,2S)-phosphoric acid mono-{6-(2-acetylamino-

3,3-diphenyl-propionylamino)-2-[(4-amino-benzenesulfonyl)-isobutyl-amino]-hexyl} ester (PL-504)

This compound was made as for the preparation of the product of example 3 (step D) with 100 mg of (2S)-2-acetylaminc~3,3-diphenyl-propionic acid (this example step A). The final product was obtained in 30% yield (30 mg).

LC-MS: 689.3 (M+H)⁺, 95% pure.

¹H NMR (CD₃OD): δ 0.77-1.04 (m, 9H), 1.10-1.17 (m, 1H), 1.23-1.49 (m, 1H), 1.46-1.57 (m, 1H), 1.78 (S, 3H)₁ 1.88-1.99 (m, 1H), 2.80-2.92 (m, 2H), 2.92-3.08 (m, 2H), 3.63-3.75 (m, 1H), 3.79-3.95 (m, 1H), 4.00 (m, 1H)₁ 4.34 (d. J = 11.3, 1H). 5.19-5.28 (m, 1H), 6.77- 6.85 (m, 2H), 7.10-7.20 (m, 2H), 7.27-7.33 (m, 6H), 7.32-7.41 (m, 2H)₁ 7.49-7.62 (m, 2H).

³¹P NMR (CD₃OD): δ 2.70

Example 6. Preparation of (1 S,5S)-(1 -{5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-amino]-6-phosphonooxy-hexylcarbamoyl}-2,2-diphenyl- ethyl)-carbamic acid methyl ester (PL-515)

First methodology: The preparation of the title compound is based on scheme 3 of this invention.

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)

The product of example 1, step F (0.624 g, 1 mmol) is dissolved in 5 mL MeCN at 24 ⁰C. 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. LC-MS: 643.3 (M+H)\ 99% pure.

¹H NMR (MeOD): δ^" 0.71-0.85 (m 2H), 0.88 (d, J = 6.3, 6H)₁ 0.91-0.96 (m, 2H), 1.21-1.29 (m, 1H), 1.41-1.52 (m, 1H) 1.82-1.92 (m, 1H), 2.61-2.68 (m, 1H), 2.81-2.85 (m, 2H), 2.94- 3.05 (m, 2H), 3.38-3.40 (t, J = 5. 1H), 3.49-3.52 (m, 5H), 4.28 (d, J = 10, 1H), 4.87 (d, J = 10, 1H) 6.90 (t, J = 8.3. 1H), 7.20 (m, 2H), 7.28 (m, 3H), 7.33 (m, 3H), 7.39 (m_t 4H).

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

The product of step A was phosphorylated with chlorodiethylphosphate following the procedure described in example 1, step G. Yields 157 mg, 68%.

LC-MS: 779.3 (M+H)\ 95% pure.

¹H NMR (CD₃OD): δ 0.82 (m, 1H)₁ 0.92 (d, J = 6.2, 8H), 0.96 (m, 3H). 1.36 (d, J = 3.7, 6H), 1.90 (m, 1H). 2.69 (m, 1H), 2.89 (iti, 1H), 2.98 (m, 2H), 3.56 (s, 3H), 3.74 (m, 1H)₁ 3.93 (m, 1H)₁ 4.03 (m, 1H)₁ 4.12 (q, J = 7.5 and 14.8. 4H). 4.32 (d. J = 11.4, 1H). 4.92 (d, J = 11.4, 1H), 6.90 (t, J = 8.3. 1H). 7.20 (m. 2H), 7.28 (m, 3H). 7.33 (m, 3H), 7.39 (m. 4H).

³¹P NMR (CD₃OD): δ 1.65

Step C. Preparation of (1S,5S)-(1-{5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol-β-phosphonooxy-hexylcarbamoyl^.∑-diphenyl-ethylj-carbamic acid methyl ester (XQ (PL-515)

Deprotection was effected using the procedure described in example 1 , step G. Yields 101 mg.

LC-MS: 723.2 (M+H)\ 95% pure.

¹H NMR (CD₃OD): δ 0.65-0.77 (m. 1H). 0.77-0.85 (m, 1H), 0.85-1.05 (m, 9H), 1.25-1.39 (m, 1 H), 1.40-1.52 (m, 1H), 1.82-1.98 (m, 1H), 2.58-2.72 (m, 1 H)₁ 2.82-2.92 (m, 1H). 2.92-

3.05 (m, 2H)₁ 3.54 (s, 3H)₁ 3.64-3.75 (m, 1H)₁ 3.80-3.92 (m, 1H), 3.91-4.04 (m, 1H), 4.29 (d, J = 11.4, 1 H)₁ 7.19 (t, J = 6.6, 1H), 7.13-7.21 (m, 2H), 7.22-7.33 (m, 6H), 7.34-7.38 (m, 2H), 7.39-7.48 (m, 2H).

³¹P NMR (CD₃OD): δ 2.74

Second methodology: The preparation of the title compound is based on scheme 4 of this invention.

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). To the solution was added 1.17 g of (2S)-phosphoric acid 6-amino-2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-hexyl ester diethyl ester (OQ (example 3, step B) and the mixture stirred for 3 h. 20 g of Amberlite XAD-2 resin was then added and the beads were left to soak for 10 min. The resin was transferred into a glass filter and washed thoroughly with distilled water (400 ml_) and 200 mL of 1M NaHCO₃. The beads were then washed with 4 X 50 ml portions of MeOH then EtOAc 200 ml_. The organic phase was evaporated. The residue was adsorbed onto silica gel and passed through a short silica gel column (EtOAc) to yield 2.4 g (83%) of white solid after evaporation.

NMR identical as in example 1 , step H.

Step B. Preparation (1 S,5S)-{1-[5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol-β-tdiethoxy-phosphoryloxyj-hexylcarbamoyll^^-diphenyl-ethyl^carbamic acid methyl ester (XII)

The product of step A above, (1 S,5S)-(1-{5-[(4-amino-benzenesulfonyl)-isobutyl-amino]-6- phosphonooxy hexylcarbamoyl}-2,2-diphenyl-ethyl)-carbamic acid methyl ester (0.555 g, 0.73 mmol) was dissolved in 5 ml_ MeCN. Selectfluor (0.26 g, 0.7 mmol) was added and the mixture stirred for 30 min. The mixture was purified by reverse phase preparative HPLC and lyophilized to yield 278 mg (48% yield) white solid.

¹H NMR identical as previous entry, see first methodology above. Step C. Preparation (1 S,5S)-(1-{5-[(4-amino-3-fluoro-benzenesulfonyl)- isobutyl-aminol-β-phosphonooxy-hexylcarbamoyl^^-diphenyl-ethyO-carbamic acid methyl ester (XIlI, in this specific case is compound Xl) (PL-515)

The procedure make this derivative was as described in the deprotection step for the methodology above. Yields 139 mg 70% after reverse phase HPLC.

¹H NMR identical as previous entry, see first methodology above.

Example 7. Preparation of (2S,2S)-acetic acid 2-[(4-amino-benzenesulfonyl)- isobutyl-amino]-6-{2-methoxycarbonylamino-3,3-diphenyl- propionylamino)-hexyl ester (PL-S21)

The preparation of the title derivative is based on scheme 5 of this invention.

Step A. Preparation of (2S)-acetic acid 6-tert-butoxycarbonylamino-2-[(4-tert- butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]-hexyl ester {XIV, R_1A = CH₃)

To a stirred solution of (1 S)-{4-[(5-tert-butoxycarbonylamino-1-hydroxymethyl-pentyl)- isobutyl-sulfamoyl]-phenyl}-carbamic acid tert-butyl ester (intermediate product (VII) of example 1 , step D, 97 mg, 0.18 mmol) in anhydrous CH₂CI₂ (3 ml_) was added N₁N- dimethylaminopyridine (22 mg, 0.18 mmol) and acetic anhydride (0.014 ml_, 0.18 mmol).

The mixture was stirred at room temperature for 1 hour. The solvent was evaporated.

Ethyl acetate (50 ml_) was added and the organic layer was washed with water (30 mL), then dried with Na₂SO₄ and concentrated. The residue was purified by flash chromatography eluting with ethyl acetate. The yield obtained was quantitative (100 mg).

LC-MS: 586.2 (M+H)⁺, 95% pure

Step B. Preparation of (2S)-acetic acid 6-amino-2-[(4-amino- benzenesulfoπyl)-isobutyl-amino]-hexyl ester (XV, Ri_A = CH₃)

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.

LC-MS: 386.2 (M+H)*, 95% pure

Step C. Preparation of (2S,2S)-acetic acid 2-[(4-amino-benzenesulfonyl-

)isobutyl-amino]-6-(2-methoxycarbonylamino-3,3-diphenyl-propionylamino)-hexyl ester (XW. R_1A = CH₃) (PL-521)

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).

LC-MS: 667.3 (M+H)\ 95% pure

¹H NMR (acetone-dβ): δ 0.85-0.97 (m, 12H), 1.21-1.41 (m, 2H), 1.88-2.00 (s, 3H), 2.59- 2.69 (m, 1 H), 2.83-2.90 (m, 1H), 2.90-3.01 (m, 1 H), 3.01-3.10 (br s. 1 H), 3.45-3.60 (s, 3H), 3.70-3.80 (m. 1 H), 3.93-4.00 (m, 1 H), 4.00-4.11 (m, 1 H), 4.38-4.45 (d, J = 11.0, 1H), 4.89- 4.98 (t, J = 10.0, 1H), 5.43-5.58 (br s, 1 H), 6.28-6.48 (d, J = 8.9, 1H), 6.72-6.83 (d, J = 8.0, 2H), 6.85-6.93 (br s, 1 H), 7.12-7.22 (t, J = 7.4, 1 H), 7.21-7.31 (d, J = 7.0, 4H), 7.31-7.45 (m, 5H), 7.48-7.57 (d, J = 8.0. 2H).

Example 8. Preparation of (2S,2S)-nicotinic acid 2-[(4-amino-benzenesulfonyl)- isobutyl-amino]-6-(2-rnethoxycarbonylamino-3,3-diphenyl- propionylamino)-hexyl ester (PL-520)

Step A. Preparation of (2S)-nicotinic acid 6-tert-butoxycarbonylamino-2-[(4-tert- butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]-hexyl ester (XIV, Ri_A = 3-pyridyl)

(1 S)-{4-[(5-tert-butoxycarbonylamino-1-hydroxymethyl-pentyl)-isobutyl-sulfamoyl]-phenyl}- carbamic acid tert-butyl ester (intermediate product (VII) of example 1 , step D, 130 mg, 0.24 mmol) was dissolved in anhydrous DMF (1 mL) and treated with 0.066 mL (0.48 mmol) of triethylamine followed by EDC (120 mg, 0.65 mmol), HOBt (88 mg, 0.65 mmol) and nicotinic acid (27 mg, 0.22 mmol). The mixture was stirred overnight at room temperature. The product was extracted with ethyl acetate (40 mL) and water (40 ml_). The organic phase was separated and dried with Na₂SO₄, then evaporated to give 200 mg of crude product. This compound was purified by flash chromatography with ethyl acetate as the eluent. A clear oil was obtained in 100% yield (150 mg).

LC-MS: 649.3 (M+H)\ 95% pure

¹H NMR (acetone-dβ): δ 0.90-1.14 (d, J = 5.9, 6H), 1.31-1.42 (m, 2H), 1.48 (s, 9H), 1.51- 1.55 (m, 2H), 1.59 (s, 9H), 1.62-1.69 (m, 1H), 1.72-1.83 (m, 1 H), 3.00-3.11 (m, 2H). 3.11- 3.17 (m, 1H), 3.19-3.27 (m, 1H), 4.15-4.24 (m, 1H), 4.35-4.44 (t, J = 9.1, 1H), 4.50-4.58 (dd, J = 4.4 and 11.5, 1H), 5.89-5.99 (br s, 1H), 7.53-7.60 (m, 1H), 7.70-7.77 (d, J = 8.2, 2H), 7.80-7.87 (d, J = 8.2, 2H)₁ 8.24-8.31 (d, J = 7.3, 1H), 8.75-8.82 (m, 1H), 8.82-8.88 (m, 1 H), 9.12-9.18 (br s, 1H).

Step B. Preparation of (2S)-nicotinic acid 6-amino-2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-hexyl ester (XV, Ri_A = 3-pyridyl)

The product of step A, (2S)-nicotinic acid 6-tert-butoxycarbonylamino-2-[(4-tert- butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]-hexyl ester (150 mg, 0.23 mmol), was dissolved in CH₂CI₂ (5 mL) and trifluoroacetic acid (1 mL) was added. The mixture was stirred during 2 hours at room temperature. The solvent was evaporated and the residue was extracted with ethyl acetate (40 mL) and NaOH 1M (40 mL) (pH = 10). The organic portion was separated, dried with Na₂SO₄ and evaporated. The residue (100 mg) was used for the next reaction without further purification. The yield was quantitative.

LC-MS: 449.2 (M+H)^*. 95% pure

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)

The product of 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. The product was extracted with ethyl acetate (40 mL) and water (40 mL). The organic layer was separated and dried with Na₂SO₄, then evaporated to give 160 mg of crude oil. The residue was purified by flash chromatography with a mixture of eluents hexane/ethyl S acetate (2/8). The title compound was obtained as a clear oil in 20% yield (25 mg).

LC-MS: 730.2 (M+H)\ 95% pure

¹H NMR (acetone-dβ): δ 0.80-0.97 (m, 9H), 0.97-1.13 (m, 2H), 1.26-1.40 (m, 1H), 1.40-0 1.57 (m, 1H), 2.61-2.73 (m, 1H), 2.86-2.98 (m, 2H), 3.00-3.17 (m, 2H). 3.45-3.59 (s, 3H). 3.91-4.00 (m, 1H), 4.24-4.34 (m. 1H), 4.34-4.47 (m. 2H). 4.90-4.99 (t, J = 9.7, 1 H), 6.35- 6.44 (m, 1H), 6.68-6.79 (d, J = 7.9, 1H)₁ 6.91-7.00 (br s, 1H), 7.13-7.22 (m, 2H), 7.22-7.31 (m. 3H). 7.35-7.48 (m. 4H), 7.49-7.64 (m, 2H). 7.75-7.84 (m, 1H), 8.25-8.36 (m, 1H), 8.76- 8.88 (br s, 1 H), 9.12-9.26 (br s, 1 H). 5

Example 9. Preparation of (2S,2S)-dimethylamino-acetic acid 2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3,3- diphenyl-propionylamino)-hexyl ester (PL-534) 0 Step A. Preparation of (2S)-dimethylamino-acetic acid 6-tert-butoxycarbonylamino-

2-[(4-tert-butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]-hexyl ester (XIV, R_1A = (CH₃J₂NCH₂-)

This title compound was obtained from (1 S)-{4-[(5-tert-butoxycarbonylamino-1-5 hydroxymethyl-pentylj-isobutyl-sulfamoylj-phenylj-carbamic acid tert-butyl ester (intermediate product (VW) of example 1, step D) as described example 15, step A using Λ/,Λ/-dimethylglycine. The clear oil was obtained in 100% yield (150 mg).

LC-MS: 629.3 (M+H)\ 95% pure 0

¹H NMR (acetone-dβ): δ 0.81-0.95 (d, J = 6.1, 6H)₁ 1.18-1.30 (m, 2H), 1.32-1.43 (s, 9H), 1.43-1.52 (s, 8H), 1.52-1.62 (m, 1H)₁ 1.93-2.00 (m, 1H), 2.19-2.29 (s, 4H), 2.69-2.80 (m, 4H)₁ 2.90-3.05 (m, 6H)₁ 3.60-3.65 (m. 1H), 3.85-3.97 (m, 1H), 3.98-4.08 (m, 1H), 4.08- 4.14 (m, 1H)₁ 5.78-5.88 (m. 1 H)₁ 7.68-7.80 (m, 3H), 8.80-8.88 (br s, 1H). 5 Step B. Preparation of (2S)-dimethylamino-acetic acid 6-amino-2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-hexyl ester (XV₁ R_1A = (CH₃^NCH₂-)

The title derivative was prepared from (2S)-dimethylamino-acetic acid 6-tert- butoxycarbonylamino^-^-tert-butoxycarbonylamino-benzenesulfonyO-isobutyl-amino]- hexyl ester as described in example 15, step B. The final product (100 mg) was used as such in the next step.

LC-MS: 429.3 (M+H)^*, 90% pure

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 prepared from (2S)-dimethylamino-acetic acid 6-amino-2-l(4- amino-benzeπesulfonyl)-isobutyl-amino]-hexyl ester as described in example 15, step C. The crude product was purified by LC-preparative. The final compound was obtained in 10% yield (10 mg).

LC-MS: 710.3 (M+H)⁺, 92% pure

¹H NMR (acetone-d_β): δ 0.81-0.98 (m, 12H), 1.14-1.30 (m, 2H), 1.31-1.45 (m, 1H), 2.58- 2.77 (m, 2H), 2.79-2.90 (m, 2H)₁ 3.42-3.56 (s, 3H)₁ 3.75-3.85 (m, 1H), 3.99-4.17 (m, 3H)₁ 4.23-4.35 (m, 1H). 4.36-4.45 (m, 1H), 4.86-4.96 (m, 1H). 6.33-6.42 (m, 1H), 6.74-6.83 (m. 1H)₁ 6.85-6.90 (m, 1 H)₁ 7.12-7.22 (m, 3H), 7.23-7.31 (m, 4H), 7.31-7.44 (m, 5H)₁ 7.47- 7.55 (m, 1H), 7.73-7.80 (m, 1H).

Example 10. Preparation of (2S,2S)-2-amino-3-methyl-butyric acid 2-[(4-amiπo- benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3,3- diphenyl-propionylamino)-hexyl ester (PL-530)

Step A. Preparation of (2S)-2-benzyloxycarbonylamino-3-methyl-butyric acid 6-tert- butoxycarbonylamino-2-[(4-tert-butoxycarbonylamino-benzenesulfonyl)-isobutyl-amino]- hexyl ester (XIV, R_1A = (CH₃)₂CHCH(NH₂)-) 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 hexane/ethyi acetate (1/1). The yield obtained was 100% (150 mg).

LC-MS: 777.3 (M+H)\ 95% pure 0 ¹H NMR (acetone-dβ): δ 0.80-1.00 (m, 14), 1.13-1.28 (s, 2H), 1.30-1.44 (s, 11H), 1.45- 1.56 (S₁ 10), 1.58-1.67 (m, 1H), 2.87-3.04 (m, 4H), 3.84-3.97 (m, 1H), 3.97-4.12 (m, 2H), 4.12-4.21 (m, 1H), 4.99-5.14 (m, 2H), 5.78-5.89 (m, 1 H), 6.38-6.52 (m, 1H), 7.24-7.34 (m, 1H), 7.34-7.41 (m, 2H), 7.65-7.83 (m, 4H), 8.77-8.86 (m, 1H). 5 Step B. Preparation of (2S)-benzyloxycarbonylamino-3-methyl-butyric acid

6-amino-2-[(4-amino-benzenesulfonyl)-isobutyl-amino]-hexyl ester (XV, R_1A = (CH₃)₂CHCH(NH₂)-)

This derivative was prepared from (2S)-2-benzyloxycarbonylarnino-3-methyl-butyric acid0 6-tert-butoxycarbonylamino-2-[(4-tert-butoxycarbonylamino-benzenesulfonyl)-isobutyl- amino]-hexyl ester (product of step A) as described in example 15, step B. The final compound was obtained in quantitative yield (110 mg) and used for the next step without purification. 5 LC-MS: 577.3 (M+H)^*, 90% pure

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

The title compound was obtained from (2S)-benzyloxycarbonylamino-3-methyl-butyric acid 6-amino-2-[(4-amino-benzenesulfonyl)-isobutyl-amino]-hexyl ester (product of step B) as described in example 15, step C. The clear oil was obtained in 86% yield (120 mg). 5 LC-MS: 858.3 (M+H)^*, 95% pure Step D. Preparation of (2S,2S)-2-amino-3-methyl-butyric acid 2-[(4-amino- benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3.3-diphenyl- propionylamino)-hexyl ester (PL-530)

To a stirred solution of (2S,2S)-2-benzyloxycarbonylamino-3-methyl-butyric acid 2-[(4- amino-benzenesulfonyl)-isobutyl-amino]-6-(2-methoxycarbonylamino-3,3-diphenyl- propionylamino)-hexyl ester (step C. 120 mg, 0.14 mmol) in anhydrous THF (8 mL), under nitrogen atmosphere, was added palladium 10% wt. on activated carbon (160 mg). The mixture was reacted under hydrogen atmosphere overnight, at room temperature. The solution was filtered and the palladium on carbon was washed with THF (50 mL). The solvent was evaporated and the residue (110 mg) was purified by flash chromatography using ethyl acetate as the eluent. The clear oil was obtained in 47% yield (47 mg).

LC-MS: 796.4 (IvHH)⁺, 95% pure

¹H NMR (acetone-d_β): δ 0.84-0.97 (m, 12H), 0.97-1.08 (m, 2H)₁ 1.27-1.43 (m, 3H), 1.49- 1.62 (m, 4H), 1.80-1.93 (m, 1H), 1.94-2.00 (m, 1H), 2.36-2.46 (m, 1H), 2.58-2.74 (m, 2H), 2.86-2.96 (m, 3H), 2.99-3.10 (m, 2H), 3.46-3.52 (s, 3H). 3.52-3.60 (m, 2H), 3.75-3.87 (m, 2H)₁ 3.95-4.04 (m, 1H), 4.10-4.18 (m, 1H), 4.37-4.44 (m, 1H), 4.89-4.97 (m, 1H), 5.40- 5.48 (m, 1H)₁ 6.30-6.40 (m, 1H), 6.76-6.83 (d, J = 8.2, 1 H), 6.87-7.03 (m, 2H)₁ 7.14-7.22 (m, 1H), 7.23-7.34 (m, 3H)₁ 7.35-7.45 (m, 4H)₁ 7.50-7.56 (m. 1H), 7.57-7.65 (m, 1H).

Example 11. In vitro inhibition of CYP450

Human Liver Microsomes

Pooled human liver microsomes (from 15 donors) were purchased from In vitro Technologies, Inc., Baltimore, Maryland, USA.

PL-100 and Ritonavir solutions preparation

Stock solution of PL-100 was prepared in methanol at 25 mM. For the CYP3A4 assay, 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 25μM and diluted in methanol to 2.5 and 0.25μM and further diluted 250 fold for microsomal incubation giving final concentrations of 100, 10 and 1nM.

Stock solution of Ritonavir was prepared in methanol at 25 mM. A sub-stock solution was prepared in methanol at 25 μM. This sub-stock solution was diluted in methanol to 2.5 and 0.25 μM 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.

Testosterone 6β-Hydrolase (TESH) Activity

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 μL ice-cold acetonitrile and the 6β -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 6β- hydroxytestosterone per mg of protein per min. Data were captured with Millenium chromatographic data management and storage system (version 4.0).

Michaelis-Menten Kinetics

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

To determine the mode of inhibition, a transformation of fitted 1/V values were employed to generate a linear regression of 1/V vs. 1/[S] using EnzFitter software. The nature of the curves was utilized as an indication of the type of inhibition. 5

Ki was calculated using the equation corresponding to the type of inhibition:

No inhibitor: V= Vmax*[S]/ (Km +[S])

l o Competitive inhibition: V= Vmax*[S] / (Km (1 +[I] / Ki) +[S])

Km'= Km* (1+[I] / Ki )

Non-competitive inhibition: V= Vmax*[S] / (Km (1 +[I] / Ki ) +[S] (1 +[I] / Ki )) = 15

= (Vmax / (1+[I] / Ki )* [S]) / (Km+[S])

Vmax'= Vmax / (1+[l] / Ki)

20 Uncompetitive inhibition: V= Vmax*[S] / (Km+[S](1 +[I] / Ki)) =

= (Vmax / (1+ [I] / Ki) *[S]) / (Km/(1+[l] / Ki) +[S])

Km' = Km / (1+[I] / Ki) 25

Vmax' = Vmax / (1+[I] / Ki)

V : Velocity of the enzyme reaction in the presence of inhibitor

Vmax : Maximal velocity

30 Vmax' : Maximal velocity in the presence of inhibitor

[S] : Substrate concentration

[I] : Inhibitor concentration

Km : dissociation constant of the enzyme-substrate complex

Km' : dissociaton constant of the enzyme-substrate complex in the presence of

35 inhibitor Ki : dissociation constant of the enzyme-inhibitor complex

HPLC methods 6β- hydroxytestosterone

Column : C18, 4μm, 3.9 x 150 mm

Detection: UV@ 254 nm

Mobile phase (gradient) : A: Methanol, Water, Acetonitrile (39:60:1)

B: Methanol, Water, Acetonitrile (80:18:2) Flow rate: 1m Um in.

Data calculation and Statistical analysis

Numerical data obtained during the study were subjected to calculation group mean values, standard deviations, where appropriate, using Excel software (Microsoft). Enzyme kinetic parameters (Km and Vm ax) and the 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 6β-hydrolase activity, a selective marker for CYP3A4/5, was assessed in the presence of PL-100. With a Ki of 0.445 ± 0.050 μM (R² = 0.98: Figs. 1 to 4), PL-100 could be considered as a strong competitive inhibitor of human CYP3A4 although this compound was approximately 13 fold less potent than Ritonavir.

In agreement with litterature data, Ritonavir showed a strong competitive inhibition of CYP3A4 activity with a Ki of 0.034 ± 0.005μM (R² = 0.98: Figs. 1 and 2).

A Ki of 0.052 ± 0.012 μM was calculated for ketoconazole, a strong competitive inhibitor of CYP3A4 activity (R² = 0.99: Figs. 1 to 3).

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. Example 12. In vivo inhibition of CYP450

Preparation of PPL-100 mixture (40 mg/mL)

An appropriable amount of test article, PPL-100, 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 40 mg/mL. Purity of the test article was taken into account for calculation of the concentration.

Preparation of 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.

Preparation of Dosing Solutions

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).

Animals

Sprague Dawley rats (Rattus πorvβgicus) 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.

Animals were dosed with a mixture containing Atazanavir (5 mg/mL) and PPL-100 (20 mg/mL) at a dose volume 5 mL per kg body weight. A summary of treatment is illustrated in Table A below.

Table A: Treatment Groups

Blood samples were collected on 6 occasions. Rats were bled as per outline in Table B. For the purpose of collection of the samples indicated above, each rat were bled from the orbital sinus. Each blood sample (approximately 0.4 ml_) was collected into a tube containing the anticoagulant K₂ EDTA. The time (actual time, in conjunction with the day and time of dosing) was recorded for each sample. Following collection, the samples was inverted for approximately 10 minutes and then, the samples were centrifuged under refrigeration (2 - 8^βC) for 20 minutes at 2000 rpm. The plasma obtained from each sample was recovered and stored frozen (at approximately -80⁰C) pending analysis.

A the time of analysis, 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).

Table B.

Results of this analysis presented in Fig. 5 and Fig. 6 indicate that PL-100 (PPL-100) decreases the metabolism of Atazanavir in vivo. PL-100 (PPL-100) succesfully increases the plasma concentration of Atazanavir at 24 hours.

Example 13. Microsomal Stability

In vitro studies were conducted to demonstrate that PL-100 enhances protease inhibitors' stability in microsomes.

The protease inhibitors which were tested were: Atazanavir (ATV), Lopinavir (LPV), Saquinavir (SQV), Amprenavir (APV), Nelfinavir (NFV) and Indinavir (IDV).

The concentration of the protease inhibitors and PL-100 was 10 μM, except for the experiment involving Atazanavir, in which Atazanavir was at concentration of 1.0 μM and PL-100 was at a concentration of 2.0 μM. After each protease inhibitor was incubated with microsomes, 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.

The results of the experiment (Figure 7) demonstrate that PL-100 can enhance the stability of all tested protease inhibitors in microsomes. Thus, PPL- 100 is a potential booster of these protease inhibitors in vivo.

Claims

We claim:

1. A pharmaceutical composition comprising:

a) a lysine-based compound of formula I

or a pharmaceutically acceptable salt thereof, wherein n is 3 or 4, wherein X and Y, the same or different, are 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, F₁ Cl₁ Br, I, -CF₃, -OCF₃, - CN. -NO₂, -NR₄R₅, -NHCOR₄. -OR₄. -SR₄, -COOR₄, -COR₄. and -CH₂OH or X and Y together define an alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH₂O- and an ethylenedioxy group of formula -OCH₂CH₂O-, wherein R₆ is 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, wherein R₃ is 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, and a group of formula R_3A-CO-, R₃A being 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, - CH₂OH, -CF₃, -CH₂CF₃, -CH₂CH₂CF₃, pyrrolidinyl, piperidinyl, 4-morpholinyl, CH₃O₂C-, CH₃O₂CCH₂-, ACeIyI-OCH₂CH₂-, HO₂CCH₂-, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-CH₃OC₆H₄CH₂-, CH₃NH-, (CH₃)₂N-, (CH₃CH₂)₂N-, (CH₃CH₂CHz)₂N-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, C_βH_sCH₂O-, 2- pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl-, 2-pyraziπyl, 2-quinolyl, 3-quinolyl, A- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

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 group of formula

wherein X¹ and Y', the same or different, are selected from the group consisting of H₁ 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, I₁ -CF₃, -NO₂, - NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH.

wherein R₄ and R₅, the same or different, are 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,

wherein R₂ is selected from the group consisting of a diphenylmethyl group of formula IV

a naphthyl-1-CH₂- group of formula V

V a naphthyl-2-CH₂- group of formula Vl

VI

a biphenylmethyl group of formula VII

and an anthryl-9-CH₂- group of formula VIII

VIII

wherein R₁ is H or a physiologically cleavable unit;

b) a drug which is affected by cytochrome P450 monooxigenase; and

c) a pharmaceutically acceptable carrier;

wherein the ratio (w/w) of the lysine-based compound, or pharmaceutically acceptable salt thereof, to said drug which is affected by cytochrome P450 monooxigenase is between about 4:1 and about 1:1.

2. A pharmaceutical composition according to claim 1 wherein the lysiπe-based compound is a compound of formula II,

or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition of claim 1 or claim 2, wherein R₁ is selected from the group consisting of H, (HO)₂P(O) and (MO)₂P(O), and a group of formula R_1A- CO-, R_1A being 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, -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 2- hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, (CH₃)₂NCH₂-, (CHs)₂CHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, 2-pyrrolyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, 1-methyl-1 ,4-dihydro-3-pyridyl, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

a picolyl group selected from the group consisting of

(2-picolyl) (3-picolyl) (4-picolyl)

a picolyloxy group selected from the group consisting of

(2-picolyloxy) (3-picolyloxy) (4-picolyloxy)

a substituted pyridyl group selected from the group consisting of

(substituted 4-pyridyl)

and a group of formula,

wherein M is an alkali metal or alkaline earth metal and wherein X', Y', R₄ and Rs are as defined in claim 1.

4. The pharmaceutical composition of claim 1 , wherein X, Y, n, R1 , R2, R3, R6, X' and Y' are set forth for each of the lysine-based compounds of formula I below:

5. The pharmaceutical composition of any one of claims 1 to 4, wherein said drug which is affected by cytochrome P450 monoxigenase is selected from the group consisting of Atazanavir (ATV), Lopinavir (LPV)₁ Saquinavir (SQV), Amprenavir (APV)₁ Nelfinavir (NFV) and Indinavir (IDV).

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the ratio (w/w) of said lysine-based compound and said drug which is affected by cytochrome P450 monooxigenase is about 4:1.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein said cytochrome P450 monoxignease is CYP3A4.

8. A pharmaceutical combination comprising:

a) a lysine-based compound of formula I

or a pharmaceutically acceptable salt thereof,

wherein n is 3 or 4,

wherein X and Y, the same or different, are 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, F₁ Cl₁ Br₁ 1₁ -CF₃, -OCF₃, - CN, -NO₂, -NR₄R₅, -NHCOR₄, -OR₄, -SR₄, -COOR₄. -COR₄, and -CH₂OH or X and Y together define an alkylenedioxy group selected from the group consisting of a methylenedioxy group of formula -OCH₂O- and an ethylenedioxy group of formula

-OCH₂CH₂O-.

wherein R₆ is 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,

wherein R₃ is 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, and a group of formula R_3A-CO-, R_3A being 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, - CH₂OH, -CF₃, -CH₂CF₃, -CH₂CH₂CF₃, pyrrolidinyl, piperidinyl. 4-morpholinyl,

CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCH₂CH₂-, HO₂CCH₂-, 3-hydroxyphenyl, 4- hydroxyphenyl, 4-CH₃OC₆H₄CH₂-, CH₃NH-, (CH₃)₂N-, (CH₃CH₂)₂N-, (CH₃CH₂CH₂)₂N-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, C₆H₅CH₂O-, 2- pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl-, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, A- quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

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 group of formula

wherein X' and V₁ the same or different, are 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, Cl, Br, I, -CF₃, -NO₂, - NR₄R₅, -NHCOR₄. -OR₄, -SR₄, -COOR₄, -COR₄ and -CH₂OH,

a naphthyl-1-CH₂- group of formula V

a naphthyl-2-CH₂- group of formula Vl

VI

a biphenylmethyl group of formula VII

and an anthryl-9-CH₂- group of formula VIII

VIII

wherein Ri is H or a physiologically cleavable unit; and

b) a drug which is affected by cytochrome P450 monooxigenase;

wherein the ratio (w/w) of said lysine-based compound, or pharmaceutically acceptable salt thereof, to said drug which is affected by cytochrome P450 monooxigenase is between about 4:1 and about 1 :1.

9. A pharmaceutical combination of claim 8 wherein the lysine-based compound is a compound of formula II,

or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical combination of claim 8 or claim 9, wherein R₁ is selected from the group consisting of H₁ (HO)₂P(O) and (MO)₂P(O), and a group of formula R_1A- CO-, R_1A being 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, -CH₂OH, CH₃O₂C-, CH₃O₂CCH₂-, Acetyl-OCHzCHz-, HO₂CCH₂-. 2- hydroxyphenyl, 3-hydroxyphenyl, 4-hydroxyphenyl, (CH₃)₂NCH₂-, (CH₃)₂CHCH(NH₂)-, HOCH₂CH₂NH-, CH₃OCH₂O-, CH₃OCH₂CH₂O-, 2-pyrrolyl, 2- pyridyl, 3-pyridyl, 4-pyridyl, 1-methyl-1,4-dihydro-3-pyridy1, 2-pyrazinyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 1-isoquinolyl, 3-isoquinolyl, 2-quinoxalinyl, a phenyl group of formula

III

a picolyl group selected from the group consisting of

(2-picolyl) (3-picolyl)

a picolyloxy group selected from the group consisting of

(2-picolyloxy) (3-picolyloxy) (4-picolyloxy)

a substituted pyridyl group selected from the group consisting of

(substituted 2-pyridyl) (substituted 3-pyridyl) (substituted 4-pyridyl)

and a group of formula,

wherein M is an alkali metal or alkaline earth metal and wherein X', Y', R₄ and Rs are as defined in claim 8.

11. The pharmaceutical combination of claim 8, wherein X, Y₁ n, R1 , R2, R3, R6, X' and Y' are set forth for each of the lysine-based compounds of formula I below:

12. The pharmaceutical combination of any one of claims 8 to 11 , wherein said drug which is affected by cytochrome P450 monoxigenase is selected from the group consisting of Atazanavir (ATV). Lopinavir (LPV)₁ Saquinavir (SQV), Amprenavir

(APV), Nelfinavir (NFV) and Indinavir (IDV).

13. The pharmaceutical combination of any one of claims 8 to 12, wherein the ratio (w/w) of said lysine-based compound and said drug which is affected by cytochrome P450 monooxigenase is about 4: 1.

14. The pharmaceutical combination of any one of claims 8 to 13, wherein said cytochrome P450 monoxignease is CYP3A4.

15. The use of at least one lysine-based compound of formula I

or a pharmaceutically acceptable salt thereof and a drug which is affected by cytochrome P450 monooxigenase in the manufacture of a drug for the treatment or prevention of an HIV infection or for treatment or prevention of AIDS, wherein n, X, Y, X', Y', R₁, R₂, R₃, R₄, R₅ and R₆ are as defined in claim 1;

and wherein the ratio (w/w) of the lysine-based compound and the drug which is affected by cytochrome P450 monooxigenase is between about 4:1 and about 1:1.

16. A method of treating or preventing an HIV infection or of treating or preventing

AIDS, the method comprising administering either the pharmaceutical composition as defined in claim 1, or the pharmaceutical combination as defined in claim 8, to a mammal in need thereof.

17. A method of treating or preventing an HIV infection or of treating or preventing AIDS, which comprises administering

a) a lysine-based compound of formula I

wherein n, X, Y, X', Y", R₁, R₂, R₃, R₄, Rs and R₈ are as defined in claim 1 or a pharmaceutically acceptable salt thereof, and

b) one or more drugs which are affected by cytochrome P450 monoxigenase,

wherein said lysine-based compound is in an amount which is sufficient to reduce the metabolism of said one or more drugs which are affected by cytochrome P450 monoxigenase, and wherein the ratio (w/w) of said lysine based compound to said one or more drugs is between about 4:1 and about 1 :1.

18. A method for improving the pharmacokinetics of a drug which is affected by cytochrome P450 monoxigenase, the method comprising administering to a human in need thereof an amount of the lysine-based compound of formula I of claim 1 effective to inhibit cytochrome P450 monoxigenase and the drug which is affected by cytochrome P450 monoxigenase, wherein the ratio (w/w) of the lysine- based compound to said drug is between about 4:1 and about 1:1.