WO1998026660A1 - Cd4-mimetic ligands - Google Patents

Abstract

This invention relates to pharmaceutically active compounds which bind to gp120. Such compounds are useful for inhibiting the infection of HIV-1 and HIV-2, the human immunodeficiency viruses that induce acquired immunodeficiency syndrome (AIDS).

Description

CD4-Mimetic Ligands

FIELD OF THE INVENTION

This invention relates to pharmaceutically active compounds which bind to gpl20. Such compounds are useful for inhibiting the infection of HIV-l and HIV-2, the human immunodeficiency viruses that induce acquired immunodeficiency syndrome (AIDS).

BACKGROUND

The human immunodeficiency viruses, HIV-l and HIV-2, induce acquired immunodeficiency syndrome (AIDS) in humans [Barre-Sinoussi et al. Science 220, 868-871 (1983); Gallo et al. Science 224, 500-503 (1984); Desrosiers Ann. Rev. Immunol. 8, 557-578 (1990)]. Like that of other retroviruses, the entry of the human immunodeficiency viruses into target cells is mediated by the viral envelope glycoproteins. These glycoproteins, gpl20 and gp41, are derived from a gplθO precursor [Allan et al. Science 228, 1091-1093 (1985); Robey et al. Science 228:593- 595 (1985)]. Oligomers of gplόO form in the endoplasmic reticulum [Earl et al. Proc NatlAcad Sci USA £7:648-652 (1990); Pinter et al. J. Virol. 63, 2674-2679 (1995); Lu et al. Nature Structural Biol. 2, 1075-1082 (1995)]. Following oligomerization, the gpl60 glycoprotein is transported to the Golgi apparatus, where cleavage by a cellular protease generates the gpl20 and gp41 glycoproteins, which remain associated through noncovalent interactions [Kowalski et al. Science 237, 1351-1355 (1987); Earl et al. J. Virol. 63, 2674-2679 (1991)]. In mammalian host cells, but not in insect cells, addition of complex sugars to selected, probably surface-exposed, carbohydrate side chains of the envelope glycoproteins occurs in the Golgi apparatus [Leonard et al. J. Biol. Chem. 265, 10373-10382 (1990)].

The mature envelop glycoprotein complex is incorporated into virions, where it mediates virus entry into the host cell. The gpl20 exterior envelope glycoprotein binds the CD4 glycoprotein, which serves as a receptor for all of the primate immunodeficiency viruses [Klatzmann et al. Nature London 312, 767-768 (1984); Dalgleish et al. Nature 312, 763-767 (1984)]. The association of gpl20 with CD4 is believed to trigger a conformational reorganization of the envelope glycoprotein complex, leading ultimately to gp41 -mediated fusion of the viral and target cell membranes [Thali et al. J. Virol. 67, 3978-3988 (1993); Sattentau et al. J. Virol. 67, 7383-7393 (1993)]. Recent studies with soluble ectodomains suggest that gpl20-CD4 binding proceeds with complex binding kinetics, resulting in an almost irreversible interaction [Wu et al. Proc. Natl. Acad. Sci. USA, in press (1997)]. Both CD4 binding and membrane fusion have been shown to be essential for successful entry of the viral core and genetic material into the host cell [Kowalski et al. Science 237, 1351-1355 (1987); Helseth et al. J. Virol. 64, 2416-2420 (1990)]. Recent in vivo data have revealed that HIV infection involves a dynamic struggle between rapid virus production and extensive CD4-positive T cell renewal in response to massive destruction of infected host cells [Wei et al. Nature 373, 117-122 (1995); Ho et al. Nature 373, 123-129 (1995)]. The essential role of the interaction of envelop glycoprotein gpl20 and human cell surface receptor CD4 in HIV infection and AIDS makes this interaction an attractive target for therapeutic intervention.

CD4 is composed of four immunoglobulin-like domains, a transmembrane region and an intracytoplasmic tail. The amino terminal two domains of CD4 have been shown to be sufficient for HIV-l entry, when expressed on the cell surface as chimeric molecules with CD8 [Poulin et al. J. Virol. 65, 4893-4901 (1991)]. The interaction of soluble molecules containing only the amino-terminal two domains of CD4 (D1D2 sCD4) with the monomeric HIV-l gρl20 glycoprotein is similar to that of the four-domain soluble CD4 (sCD4) molecule, as judged by affinity, mutational effects, and the effect of this binding on antibody recognition of the gpl20 glycoprotein [Arthos et al. Cell 57, 469 (1989); Moore & Sodroski . J. Virol. 70, 1863-1872 (1996)].

The critical binding residues for gpl20 interaction appear to be located within the D1D2 region of CD4 molecule. Mutational analyses in the literature provide a somewhat complex picture of the critical residues within D1D2, but the consensus appears to include Lys29, located in a β-strand, Lys35, located at the end of a loop but adjacent to Phe43 and Lys46 located in another loop and Arg59 located in a large proximal loop. [Peterson & Seed Cell 54, 65-72 (1988); Arthos et al. Cell 57, 469 (1989); Brodsky et al. J. Immunol. 144, 3078-3086 (1990); Moebius et al. J. Exp. Med. 176, 507-517 (1992); and S.E. Ryu, et al. Nature (London) 348, 419-425 (1990), Structure, 2, 59-74 (1994)]. The mutational data converge on Phe43 and Arg59 as the key determinants. Substitution of Phe43 by Val, for example, leads to a large reduction of affinity for gpl20 [Arthos et al. Cell 57, 469 (1989)]. The stretch of sequence from Lys35 to Pro48 may be viewed as a long loop, as depicted in Figure 1 (note in Figure 1 that Phe43 is not at the apex of the loop but one residue in at the i+3 position of the β-turn). This topography is important since this long loop sits at the top of the Dl segment of the CD4 finger, like a fingernail. It would seem that retention of these residues could be important for gpl20 recognition. Mutational studies in this region have included Lys35, Gln40 Phe43 and Lys46, but not Thr45. Of those residues that have been evaluated, Gln40 does not appear to be critical. The limited set of mutants attempted at Ser42, suggest that it may be more important for maintaining conformation than for gpl20 interaction. Thus Gly41 and Ser42 do not appear to part of the pharmacophoric presentation to gpl20. Arg59 which appears on an adjacent loop but is located proximally to Phe43, cannot be replaced with Ala, Met, Thr or Asp, but can be replaced with Lys to give a mutant with three times the inhibitory activity of sCD4.

In Chen et al., Proc. Natl. Acad. Sci. USA 89, 5872-5876 (1992), the focus was on the Gln40-Phe43 loop and a β-turn mimetic of Compound 1 (R = H; R' = Leu-

ThrNHBn; X, X₂ = CH₂OH; X₃ = Bn) was designed. The IC₅₀ for inhibition of gpl20 binding to CD4+ cells was 6.1-30.6 uM for Compound 1, compared to 22 nM for gpl20. In a recent published patent application (WO 94/03494, published February 17, 1994) the IC₅₀ was reported to be 0.8 uM. Testing of Compound 1 in an ELISA assay it was minimally active (IC50 >100 uM). [Jarvest et al. Biorg. Med. Chem. Lett., 3, 2851-2856 (1993)] Despite this weak activity the mimetic was reported to show 50% inhibition of syncytia formation at 250 ug/mL. By contrast the linear CD4(40-45) hexapeptide was inactive at the same concentration. [Chen et al. Proc. Natl. Acad. Sci. USA 1992, 89, 5872-5876 (1992)] In a recent review, the Kahn group claims that an N-acyl derivative of this analog of Compound 1 displayed 43% oral bioavailability with a Tl/2 of 6 hr. [Qabar et al. // Farmaco, 51, 87-96 (1996)]

Finberg, et al. Science, 1990, 247, 287 (1990) described the development of a dipeptide DD-CPF, Compound 2, that the authors related to Phe-43.

Compound 2 was reported to be an irreversible inhibitor of gpl20. Testing of Compound 2 in an ELISA assay resulted in an IC50 of 300 uM. [Jarvest et al. Biorg. Med. Chem. Lett., 3, 2851-2856 (1993)] A cyclic peptide CDR2.AME(39-44), S,S-cyclo-H-Tyr-Cys-Asn-Gln-Gly-Ser-

Phe-Leu-Cys-Tyr-OH, derived from the same region of CDR2 was shown recently to be inactive at inhibition of T cell activation (LL-2 production), whereas a CDR3 peptide CDR3.AME(82-89), S,S-cyclo-H-Phe-Cys-Tyr-Ile-Cys(SH)-Glu-Val-Glu-Asp-Gln- Cys-Tyr-OH, caused 80% inhibition at 10 uM and even the neighboring peptide CDR2.AME(45-50) caused 52% inhibition at 10 uM.[Zhang, et al. , Nature

Biotechnology, 14, 472-475 (1996)] These studies suggest that CDR2(40-45) is not part of the binding surface on CD4 that is involved with T cell activation and CD4- MHC II interaction. CDR2(40-45) is, therefore, a novel motif that is recognized by

A curious off-shoot of CDR3 peptide studies began with the observation that benzylated side products from the synthesis of CDR3(81-92) inhibited syncytia formation, the most active being a tribenzylated peptide Thr(Bn)-Tyr-Ile-Cys(Bn)- Glu(Bn)-Val-Glu-Asp-Gln-Lys(Bn)-Glu-Glu-OH.[Nara et al. Proc. Natl. Acad. Sci. USA, 86, 7139-7143 (1989)]. A recent study by Prieto et al. AIDS Research and Human Retroviruses, 1996, 12, 1023-1030 (1996) of phenylalanine analogs of

CDR3(81-92) found that FYICFVED and FY1CFVEDE were the most active analogs (IC50 1-2. 1-6 uM, respectively). The authors appear to have dismissed the possibility that these peptides might no longer be mimetics of the CDR3(81-92) region of CD4, but might now be mimetics of the CDR2(40-45) region of CD4, largely on the inability of others to prepare active peptide derivatives of this region. Nonetheless, they have also shown that the single most important substitution of a phenylalanine in CDR3 81- 92 was E85F:

CDR2(40-45) -Gln-Gly-Ser-Phe-Leu-Thr CDR3(81-92) Thr-Tyr-Ile-Cys-Glu-Val-Glu-Asp...

E85F-CDR3(81-92) Thr-Tyr-Ile-Cys-Phe-Val-Glu-Asp...

As seen in these sequence alignments, they have created a sequence that, in the 81-86 region, is now similar to CDR2(40-45). While Ser substitution for Cys might be viewed as a conservative modification, this modification in CDR3(81-92) gave an inactive compound. [Lifson et al. Science, 241, 712-716 (1988)]. The opposite substitution in CDR2(40-45), i.e. S43C, has not been reported, but the E85F-CDR3(81- 82) study suggests that the E85F modification could considerably enhance the activity of CDR2(40-45). Based upon these findings it appears that the region of CD4 that these Phe-modified analogs of CDR3(81-92) mimic has not been clarified.

As noted above, the mimetic analog Compound 1 (R=H; R'=Leu-ThrNHBn, X, X2 = CH2 OH; X3 = Bn) displayed weak but significant inhibitory activity in a gpl20 binding assay and inhibition of syncytia formation. Compound 3, below, was disclosed as incorporating binding elements into Compound 1 that would mimic Arg59, with the modifications R'= homoArgOMe, X2 = CH(CH2)3- [Ramurthy et al. Biorg. Med. Chem., 2, 1007-1013 (1994)]

3, R'=homoArgOMe, X₂ = CH(CH₂)3

However, although Compound 3 inhibits viral infectivity in cell culture with an IC₅₀ of 100 uM, it was inactive in a gpl20/sCD4 binding assay. These data would suggest, that Compound 3 does not inhibit gpl20/CD4 but does something else in the viral infectivity assay. Therefore, there is a need to provide compounds which both inhibit gpl20/CD4 binding and are active in the viral infectivity assay.

SUMMARY OF THE INVENTION

This invention is directed to compounds described by formula (I) and formula (II) which have pharmaceutical activity for inhibiting the binding of gpl20 to CD4.

This invention is also directed to a pharmaceutical composition comprising compounds according to formula (I) and formula (II) and a pharmaceutical carrier.

This invention is also directed to a method of treating diseases in which the pathology may be modified by binding to a membrane receptor related to or identical to gpl20 found on the surface of HIV-l and HIV-2 and inhibiting interaction with CD4 found on human T cells. In a particular aspect, the compounds of this invention are useful for treating AIDS, and AIDS related diseases, including, but not limited to Karposi sarcoma.

DETAILED DESCRIPTION

Figure 1 depicts the CD4 loop that are mimicked by the compounds of this invention.

This invention comprises compounds of formula (I) or formula (II):

Formula (I)

Formula (II) wherein:

X is a group that is designed to mimic Arg-59 in CD4; Y is a group that is designed to mimic Phe-43 in CD4;

; or -NHCH(COQ)(CH₂)_nNH-; where Q is -OH, -OR, -NR1R2 or -NH-Lys-OR; and

W is either or W is NRχR₂;

W is NR; m = 0-14, n = 0-6; and R, Rj, and R₂, are the same or different and are hydrogen, alkyl, Bn, EtPh,

(cycloalkyl)alkyl, arylalkyl, heteroarylalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, amino acid sidechain.

Preferably, X is NH₂(CH₂)n-, NH=C(NH₂)NH(CH₂)n-, Het-(CH₂)n-. Preferably, Y is Aryl(CH₂)n-; Cyclohexyl(CH₂)n-.

Unless otherwise indicated, the terms are defined as follows: The term "alkyl" is used herein at all occurrences to mean a straight or branched chain radical of 1 to 6 carbon atoms, unless the chain length is limited thereto, including, but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert- butyl, and the like.

The terms "halo" or "halogen" are used interchangeably herein at all occurrences to mean radicals derived from the elements chlorine, fluorine, iodine and bromine. The terms "aryl" or "heteroaryl" are used herein at all occurrences to mean substituted and unsubstituted aromatic ring(s) or ring systems which may include bi- or tri-cyclic systems and heteroaryl moieties, which may include, but are not limited to, heteroatoms selected from O, N, or S. Representative examples include, but are not limited to, phenyl, benzyl, naphthyl, pyridyl, quinolinyl, thiazinyl, and furanyl. The term "Bn" is used herein at all occurrences to mean benzyl. The term "Ph" is used herein at all occurrences to mean phenyl.

Specific preferred compounds of this invention are as follows.

wherein Q = OH, m = 0; Z' = Leu-ThrNHBn;

(C)

Ser-(Bn)Lys-Leu — Thr — Lys — Gly e-NH

Gly — Gin — Asn — Gly Leu — Me Lys

(D)

Ser- (Bn)Lys-Leu — Thr Lys-e-NH

Gly — Gin — Asn — Gly Leu-D-Lys— Lys

(E) Ser— (Bn)Lys-Leu D-Pro

Gly — Gin z- -Pro

(F)

where Btd is:

Compounds of formula (I) and formula (II) can be made according to the following examples. It will be recognized by the skilled artisan that reagents and starting materials are commercially available or can be made by standard methods of peptide synthesis. It will also be recognized that when synthesizing the compounds of this invention, suitably protected amino acid intermediates which are the building blocks of compounds of formula (I) and formula (II). These amino acid intermediates are assembled to give compounds of formula (I) and formula (II) by known methods.

Synthesis of (Bn)Lys as shown in preferred compounds of formula (I) and formula (II) (B) through (F) above is described in Gander-Coquoz, M., Seebach, D., Helv. Chim. Acta, 1988, 71, 224, incorporated herein by reference.

Synthesis of Boc-NH-(Bn)Lys as shown in preferred compound of formula (I), Compound (A) above, is described in Viret, J., Gabard, J., and Collet, A., Tetrahedron, 1987, 43, 891-894, incorporated herein by reference, and is shown in the following Scheme:

a) KOCN, 60°, 4 h; KOC1, KOH; Boc₂O

When Z is the following moiety

, see, preferred compound (B) above, the synthesis is described in

Freidinger et al., J. Org. Chem., 47, pp. 104-109 (1982), incorporated herein by reference.

Synthesis of preferred Compound (A): The synthesis of preferred Compound (A) is based upon the methods of preparation of compounds described in the following, which are all incorporated herein by reference:

a. Michael Kahn, Preparation of CD4 b-Turn Mimetics WO 93/24518 Al, published December 9, 1993. b. Michael Kahn, Preparation of Conformationally Restricted Mimetics of Reverse Turns and Peptides Containing the Same, WO 94/03494 Al, published February 17, 1994. c. Ramurthy, S., Lee, M. S., Nakanishi, H., Shen, R., and Kahn, M., Peptidomimetic Antagonists Designed to Inhibit the Binding of CD4 to HIV gpl20, Biorg. Med. Chem., 1994, 2, 1007-1013. d. Chen, S., Chrusciel, R. A., Nakanishi, H., Raktabutr, A., Johnson, M. E., Sato, A., Weiner, D., Hoxie, J., Saragovi, H. U., Greene, M. I., and Kahn, M., Design and Synthesis of a CD4 β-Turn Mimetic that Inhibits Human Immunodeficiency Virus Envelope Glycoprotein gpl20 Binding and Infection of Human Lymphocytes, Proc. Natl. Acad. Sci. USA 1992, 89, 5872-5876.

In the preferred compound (F) above, moiety Btd is synthesized by the procedure described in Nagai et al., Tetrahedron, 49, pp. 3577-3592 (1993, incorporated herein by reference. The above description fully discloses the invention including preferred embodiments thereof. Modifications and improvements of the embodiments specifically disclosed herein are within the scope of the following claims. Without further elaboration it is believed that one skilled in the art can, given the preceding description, utilize the present invention to its fullest extent. Therefore any examples are to be construed as merely illustrative and not a limitation on the scope of the present invention in any way. The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows.

Claims

Claims:What is claimed is:

1. A compound of formula (I) or formula (II):

Formula (I)

Formula (II) wherein:

X is a group that is designed to mimic Arg-59 in CD4; Y is a group that is designed to mimic Phe-43 in CD4;

; or -NHCH(COQ)(CH₂)_nNH-; where Q is -OH, -OR, -NRιR₂ or -NH-Lys-OR; and

W is either ^{OΓ is NR}1^R2;

W is NR; m = 0-14, n = 0-6; and

R, R , and R2, are the same or different and are hydrogen, alkyl, Bn, EtPh,

(cycloalkyl)alkyl, arylalkyl, heteroarylalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, amino acid sidechain.