WO2009020603A2

WO2009020603A2 - Upp amphiphilic alpha-helix mimetics

Info

Publication number: WO2009020603A2
Application number: PCT/US2008/009417
Authority: WO
Inventors: Shannon Biros; Julius Rebek, Jr.; Lionel Moisan
Original assignee: The Scripps Research Institute
Priority date: 2007-08-08
Filing date: 2008-08-05
Publication date: 2009-02-12
Also published as: WO2009020603A9; WO2009020603A3

Abstract

Functionalized pyridazine derivatives having a low molecular weight and pharmaceutical compositions thereof are useful as alpha-helical mimetics for efficiently disrupting protein-protein interactions such as Bak/Bcl-XL, p53/HDM2, calmodulin/smooth muscle myosin light-chain kinase, and gp41 assembly and for treating conditions and/or disorders mediated by disruption of alpha-helix-binding receptors and proteins.

Description

UPP AMPHIPHILIC α-HELIX MIMETICS

Description

Government Rights:

The invention described herein was supported in part by grant numbers GM50174 and GM27932 from the National Institutes of Health. The government has certain rights to this invention.

Field of Invention:

The present invention relates to compounds, intermediates and methods for the preparation and uses thereof, and pharmaceutical compositions comprising the compounds. The novel compounds are useful as alpha-helical mimetics for efficiently disrupting protein-protein interactions such as Bak/Bcl-X_L, p53/HDM2, calmodulin/smooth muscle myosin light-chain kinase, and gp41 assembly. Methods for treating diseases or conditions which are modulated through disruption of interactions between alpha helical proteins and their binding sites are other aspects of the invention

Background:

The alpha-helix constitutes one of the principal architectural features of peptides and proteins. It is a rod-like structure wherein the polypeptide chain coils around like a corkscrew to form the inner part of the rod and the side chains extend outward in a helical array. Approximately 3.6 amino acid residues make up a single turn of an alpha-helix; thus the side chains that are adjacent in space and make up a "side" of an alpha-helix occur every three to four residues along the linear amino acid sequence. The alpha-helix conformation is stabilized by steric interactions along the backbone as well as hydrogen bonding interactions between the backbone amide carbonyls and NH groups of each amino acid. Nearly a third of the residues in known proteins form alpha-helices and such helices are important structural elements in various biological recognition events, including ligand-receptor interactions, protein-DNA interactions, protein-RNA interactions, and protein- membrane interactions. Given the importance of alpha-helices in biological systems, it would be desirable to have available small organic molecules that act as mimics of alpha-helices. Such compounds would be useful not only as research tools, but as therapeutics to treat conditions mediated by alpha-helix binding enzymes and receptors. Yet, despite the wealth of research on other aspects of alpha-helices, relatively little research has been devoted to identifying small molecule alpha-helix mimetics and there remains a need in the art for such compounds.

The α-helix is one of the most abundant secondary protein structures and is often a key feature for protein-protein recognition. Side chains in positions /, /+3//+4, /+7, and /+11 appear on the same face of the helix are frequently crucial for the interaction (Davis, J. M.; et al. Chem. Soc. Rev. 2007, 36, 326; Fletcher, S.; Hamilton, A. D. J. R. Soc. Interface 2006, 3, 215; Yin, H.; Hamilton, A. D. Angew. Chem. Int. Ed. 2005, 44, 4130; Jain, R.; et al. MoI. Divers. 2004, 8, 89; Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 700, 2479.). Hamilton and co-workers pioneered the synthesis of non-peptidic α-helix mimetics based on terphenyl, terephthalamide, and oligopyridine scaffolds that display side chains in a manner that closely resembles those in position /^', /+4, and /+7 of an α-helix (Kutzki, O.; et al. J. Am. Chem. Soc. 2002, 124, 11838; Ernst, J. T.; et al. Angew. Chem. Int. Ed. 2003, 42, 535; Yin, H.; et al. J. Am. Chem. Soc. 2005, 127, 5463.). They were shown to efficiently disrupt protein-protein interactions such as Bak/Bcl-Xi. (Yin, H.; et al. J. Am. Chem. Soc. 2005, 727, 10191.), p53/HDM2 (Yin, H.; et al. Angew. Chem. Int. Ed. 2005, 44, 2704.), calmodulin/smooth muscle myosin light-chain kinase (Omer, B. P.; et al. J. Am. Chem. Soc. 2001 , 723, 5382.), and gp41 assembly (Ernst, J. T.; et al. Angew. Chem. Int. Ed. 2002, 41, 278.). During efforts towards the design of inhibitors of protein-protein interactions (Davis, C. N.; et al. Proc. Natl. Acad. ScL USA 2006, 703, 2953; Bartfai, T.; et al. Proc. Natl. Acad. Sci. USA 2004, 707, 10470; Bartfai, T; et al. Proc. Natl. Acad. Sci. USA 2003, 700, 7971.), methodology was developed for structurally similar molecules featuring more hydrophilic components and a facile synthetic route (Biros, S. M.; et al. Bioorg. Med. Chem. Lett. 2007, 77, 4641.). Bak and Bcl-x_L belong to the Bcl-2 family of proteins, which regulate cell death through an intricate balance of homodimer and heterodimer complexes formed within this class of proteins (M. C. Raff, Science 1994, 264, 668-669; D. T. Chao, S. J. Korsmeyer, Annu. Rev. Immunol. 1998, 16, 395-419; C. B. Thompson, Science 1995, 267, 1456-1462; L. L. Rubin, K. L. Philpott, S. F. Brooks, Curr. Biol. 1993, 3, 391-394). Overexpression of anti-apoptotic proteins such as BCI-XL and Bcl-2 prevent cells from triggering programmed death pathways and has been linked to a variety of cancers. Bcl-2 protein plays a critical role in inhibiting anticancer drug- induced apoptosis, which is mediated by a mitochondria-dependent pathway that controls the release of cytochrome c from mitochondria through anion channels. Constitutive overexpression of Bcl-2 or unchanged expression after treatment with anticancer drugs confers drug resistance not only to hematologic malignancies but also to solid tumors (R. Kim et al. Cancer 2004, 101 , 2491-2502). A current strategy for developing new anticancer agents is to identify molecules that bind to the Bak- recognition site on BCI-XL, disrupting the complexation of the two proteins and therefore antagonizing BCI-XL function ( O. Kutzki et al. J. Am. Chem. Soc. 2002, 124, 11 , 832-1 1 , 839). The structure determined by NMR spectroscopy (M. Sattler et al. Science 1997, 275, 983-986) shows the 16 residue BH3 domain peptide from Bak (aa 72 to 87, K_d~300 nM) bound in a helical conformation to a hydrophobic cleft on the surface of Bcl-x_L, formed by the BH1 , BH2, and BH3 domains of the protein. The crucial residues for binding were shown by alanine scanning to be V74, L78, 181 , and I85, which project in an /, /+4, /+7, /+1 1 arrangement from one face of the α-helix. The Bak peptide is a random coil in solution but adopts an α-helical conformation when complexed to Bcl-X|_. Studies utilizing stabilized helices of the Bak BH3 domain have shown the importance of this conformation for tight binding. (J. W. Chin, A. Schepartz, Angew. Chem. 2001 , 113, 3922-3925; Angew. Chem. Int. Ed. 2001 , 40, 3806-3809.)

Small molecule mimetics of alpha-helices are of immense pharmaceutical interest and would circumvent the problems associated with the use of peptidic agents. Accordingly, there is a need in the art for small molecule compounds that can modulate the activity of alpha-helix mediated interactions and therefore would be useful in the treatment of a variety of diseases mediated by these proteins.

Disclosed herein is a new class of low-molecular-weight α-helix mimetics featuring a pyridazine ring and hydrophobic amino-acid side chains.

Summary:

One aspect of the invention is directed to an alpha-helix i, i+3/i+4, and i+7 mimetic represented by formula (I):

Formula (I)

In Formula (I), R₁, R₂ and R₃ are radicals independently selected from the group of radicals consisting of side chains of naturally occurring amino acids and homologs thereof; and R₄ is a radical selected from the group consisting of -H and -(C₁-C₆ alkyl). Optionally, the alpha-helix i, i+3/i+4, and i+7 mimetic may include solvates and/or pharmaceutically acceptable salts therewith. As a further option, there may be a proviso that none of R₁, R₂ and R₃ is hydrogen; or alternatively that, at most, one of R₁, R₂ and R₃ is hydrogen. In a preferred embodiment, R₁, R₂, and R₃ are radicals independently selected from the group of radicals consisting of -CH₃, - CH₂CH₃,

-CH(CHa)₂, -CH₂CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH(CH₃)CH₂CH₃, -CH₂OH, -CH₂SH, -CH₂CH₂SCH₃, -CH(OH)CH₃, -CH₂Ph, -CH₂C₆H₄OH, -CH₂C₆H₂I₂OH, -CH₂(3-indole), -CH₂C(O)NH₂, -CH₂COOH, -CH₂CH₂C(O)NH₂, -CH₂CH₂COOH, - CH₂CH₂CH₂CH₂NH₂, -CH₂(4-imidazole), -CH₂CH₂CH₂NHC(NH)NH₂, and homologs thereof. Preferred embodiments of this aspect of the invention, exemplifying preferred amino acid side chains and homologs thereof for R₁, R₂ and R₃, include the following species:

Another aspect of the invention is directed to methods for synthesizing the compounds of the first aspect and intermediates thereof.

Another aspect of the invention is directed to a process for disrupting a protein- protein interaction selected from the group consisting of Bak/Bcl-Xi., p53/HDM2, calmodulin/smooth muscle myosin light-chain kinase, and gp41 assembly comprising the step of contacting a compound of claim 1 with sufficient concentration to disrupt the protein-protein interaction.

Another aspect of the invention is directed to a process for treating conditions and/or disorders mediated by the disruption of the protein-protein interaction of claim 39 comprising the step of administering a sufficient amount to a compound of claim 1 to a patient to the disruption of the protein-protein interaction.

The synthesis of the desired α-helix mimetics has been performed in few steps. While specific derivatives are prepared and disclosed here, the methodology reported is applicable for a broader, more general decoration of the scaffold to provide a diversity of compounds within the scope of the invention. A library of over thirty compounds that bear common hydrophobic amino-acid side chains was synthesized with this strategy. These compounds are disclosed to have utility, inter alia, as inhibitors of the protein-protein interactions discussed above.

Brief Description of Drawings:

Figures 1a and 1b illustrate an overlay of the target molecules with an α-helix and the general retrosynthetic approach to these molecules, respectively.

Figure 2 illustrates a scheme showing the inverse electron demand Diels-Alder reaction of dimethyl-1 ,2,4,5-tetrazine-dicarboxylate 5 with suitable dienophiles (Boger, D. L. Tetrahedron 1983, 39, 2869.), 6, 7 or 8 to give compound 2 with different R₂ groups. Tetrazine 5 was synthesized from ethyl diazoacetate following the procedure of Boger and co-workers (Boger, D. L.; et al. Org. Synth. 1992, 70, 79.).

Figure 3 illustrates the synthetic scheme for the final products 1a-d, derived from the starting compounds 2a-c.

Figure 4 illustrates the structures of 20 UPP compounds that were synthesized and exemplifies therein preferred amino acid side groups and homologs thereof.

Figure 5 illustrates the structures of 18 further UPP compounds that were synthesized and exemplifies therein preferred amino acid side groups and homologs thereof.

Detailed Description:

The target molecules are shown in Figure 1 along with an overlay with an α- helix (Figure 1a). The scaffold mimics the position /, /+4, and /+7 of the α-helix. To ensure the water solubility of the inhibitors a pyridazine ring was the basis of the scaffold. These molecules may be thought of as synthetic counterparts to amphiphilic α-helices; they are intended to present both a hydrophobic surface for recognition and a hydrophilic "wet edge" that is rich in hydrogen bond donors and acceptors.

The general retrosynthetic approach to these molecules is laid out in Figure 1 b. The major disconnections from the final urea-pyridazine-piperazine 1 (UPP) are made at the amide and urea bonds to give a pyridazine diester 2, an amine 3, and a piperazine 4. This synthesis is modular, many amines are commercially available as are a variety of Boc-protected 2-substituted piperazines presenting standard hydrophobic side chains (i.e. benzyl, isobutyl, etc.). The central pyridazine ring is readily forged from the inverse electron demand Diels-Alder reaction of dimethyl- 1 ,2,4,5-tetrazine-dicarboxylate 5 and a suitable dienophile (Boger, D. L. Tetrahedron 1983, 39, 2869.).

Tetrazine 5 was synthesized from ethyldiazoacetate following the procedure of Boger and co-workers (Boger, D. L.; et al. Org. Synth. 1992, 70, 79.) and reacted with a range of dienophiles to introduce the side chain R₂ and give the corresponding pyridazines (Figure 2). Three kinds of dienophiles were employed depending on the structure of the desired R₂ group. A few suitable alkynes such as 6 are commercially available and were used. Enamines are also known to be good dienophiles for the cycloaddition with disubstituted tetrazines (Boger, D. L. Tetrahedron 1983, 39, 2869.). Thus R₂ = /Pr was introduced via the cycloaddition of the enamine 7 obtained by the condensation of isovaleraldehyde and pyrrolidine following the Mannich procedure (Stork, G.; et al. J. Am. Chem. Soc. 1963, 85, 207.). This procedure is suitable to introduce short, low molecular weight side-chains such as /Pr for which the corresponding alkynes are often a gas and more difficult to handle. In the case where R₂ = CH₂-Boclndole, methyl enol ether 8 was used as the dienophile. The enol ether was obtained by methylenation of the corresponding methyl ester with Tebbe's reagent (Pine, S. H.; et al. J. Am. Chem. Soc. 1980, 102, 3270.) and directly subjected to reaction with tetrazine 5 without any further purification. This procedure expanded the scope of the possible side chains to the wide range of methyl esters commercially available, especially those derived from natural amino-acids.

Installation of the piperazine group was performed by using either a peptide coupling (Method A) or direct coupling of the methyl ester function (Method B) (Figure 3). A few examples were first carried out with non-substituted N-Boc protected piperazines (R₃ = H). The corresponding final compounds present only two side chains but provide clear NMR spectra for characterization. The synthesis was then applied to substituted piperazines yielding compounds presenting three side chains for which the NMR spectra are more complex due to the conformational isomers of the piperazine around the tertiary amide bond. The 6-position methyl ester of the pyridazine 2a was selectively saponified using LiOH in a THF/water mixture at 0 ⁰C. The piperazine 4a (R₃ = H) was coupled under standard peptide coupling conditions with PyBroP (Method A) in good yields to give pyridazine- piperazine 9a. In the case of pyridazines 9b,c the coupling with piperazines 4a or 4b (R₃ = /Bu) was performed directly using Weinreb amidation (Basha, A.; et al. Tetrahedron Lett. 1977, 48, 4171.) in the presence of AIMe₃ (Method B) to give only the desired regioisomer.

To install the urea function in the 3-position, the remaining methyl ester was first converted to the acyl hydrazide 10a-c under mild conditions using an excess of hydrazine in methanol. The acyl hydrazide function was then diazotized to the acyl azide 11 using the Curtius method (sodium nitrite in acidic conditions) (J. Meienhofer, The Peptides, Analysis, Synthesis, Biology', ed. by E. Gross, J. Meienhofer; Academic Press, Inc., 1979; Vol. 1 , pp. 197-228.). Acyl azides were not purified and directly transformed to the corresponding isocyanates 12 by heating at 70 ⁰C. Ureas 13a-d were obtained by trapping these isocyanates with various amines. Final removal of the N-Boc group was accomplished using TFA, to give the desired UPP 1a-d that present three side chains to mimic the /, /+4, /+7 positions of an α-helix. EXPERIMENTAL

Solvents and reagents were of reagent-grade, purchased from commercial suppliers, and used without further purification unless otherwise stated. Substituted N-Boc-protected piperazines were purchased from Anaspec. Thin-layer chromatography (TLC) was performed on Kieselgel 60 F₂₅₄ coated plates (Merck). ¹H and ¹³C NMR spectra were recorded on Bruker 250 MHz, 300 MHz, or 600 MHz spectrometers. Chemical shifts are expressed in ppm (δ), referenced to the protio impurity of the solvent as internal standard for ¹H and ¹³C nuclei. High resolution mass spectra were recorded on an Agilent ESI-TOF mass spectrometer by Scripps Center for Mass Spectrometry.

Dimethyl 4-isopropylpyridazine-3,6-dicarboxylate (2a): To a stirred solution of isovaleraldehyde (1 ml_, 9.2 mmol) and K₂CO₃ (1.5 g, 11.1 mmol) in CH₂CI₂ (50 mL) was added dropwise over 1 h pyrrolidine (780 μl_, 9.3 mmol) at 0 ⁰C. The mixture was vigorously stirred under nitrogen for 18 h. The mixture was then filtered and the solvent removed in vacuo. The crude enamine was then used directly without any further purification and added to a solution of tetrazine dimethylester 5 (1 g, 5.05 mmol) in dioxane (50 mL) at 0 °C. The mixture was stirred for 4 h at rt, heated at 90 ⁰C for 16 h, and then concentrated in vacuo. The crude residue was purified on silica (CH₂CI₂/Ac0Et 1/0 to 9/1 ) to afford compound 2a (800 rng, 3.3 mmol, 67%) as a pale yellow solid; ¹H NMR: (250 MHz, CDCI₃) δ 8.15 (s, 1 H), 4.05 (s, 3H), 4.02 (s, 3H), 3.38 (hept, J = 6.9 Hz, 1 H), 1.27 (d, J = 6.8 Hz, 6H); ¹³C NMR: (62.5 MHz, CDCI₃) δ 165.2, 164.2, 154.2, 151.8, 148.2, 125.5, 53.4, 53.2, 28.8, 22.5; HRMS: (ESI-TOF) CnH₁₄N₂O₄H⁺ expected: 239.1026. found: 239.1024.

Dimethyl 4-((1-(ferf-butoxycarbonyl)-7H-indol-3-yl)methyl)pyridazine-3,6- dicarboxylate (2b): To a solution of methyl (1-tert-butyloxycarbonyl-indol-3- yl)acetate (Davies H. M. L.; Townsend, R. J. J. Org. Chem. 2001 , 66, 6595.) (3 g, 10 mmol) in THF (30 mL) cooled at -40 ⁰C was added Tebbe's reagent (22 mL, 1.1 eq, 0.5 M in toluene) after 30 min temperature was raised to ambient over a period of 2 h. The mixture was then cooled to -10 ⁰C and the reaction was quenched by the dropwise addition of NaOH (2.4 mL, 2 M solution). Reaction mixture was then allowed to warm to rt. The dark green solution was then diluted with excess ether and filtered through a plug of Celite. Solvent was removed under reduced pressure and the crude residue was directly diluted in dioxane (40 ml_) and added to a solution of tetrazine 5 (2 g, 10 mmol) in dioxane (10 ml_). After 18 h at rt, the volatiles were removed and the crude residue was purified on silica gel (CH₂CI₂/ AcOEt 1/0 to 15/1 ) to afford pyridazine 2b (1.3 g, 3 mmol, 31%) as a solid; ¹H NMR: (600 MHz, CDCI₃) 6 8.14 (bs, 1 H), 8.02 (s, 1 H)₁ 7.46 (s, 1 H)₁ 7.35 (m, 1 H)₁ 7.30 (m. 1 H), 7.21 (m, 1H), 4.40 (s, 2H), 4.07 (s, 3H), 4.03 (s, 3H), 1.69 (s, 9H); ¹³C NMR: (150 MHz, CDCI₃) δ 165.1 , 164.0, 153.7, 151.8, 141.3, 129.3, 128.6, 124.9, 124.8, 122.8, 118.1 , 116.7, 1 15.5, 115.1 , 84.0, 53.4, 28.1 , 27.1 ; HRMS: (ESI-TOF) C₂₂H₂₃N₃O₆H⁺ expected: 426.1660. found: 426.1670.

Dimethyl 4-isobutylpyridazine-3,6-dicarboxylate (2c): To a solution of tetrazine 5 (500 mg, 2.52 mmol) in anhydrous 1 ,4-dioxane (12.5 ml.) was added 4- methylpentyne (234 mg, 2.85 mmol), the reaction vessel was then sealed and heated to 90 °C for 18 h. The volatiles were removed under reduced pressure, and the crude product was purified by silica gel chromatography (CH₂CI₂/AcOEt 95/5) to give 2c (423 mg, 67% yield) as a yellow solid. ¹H NMR: (600 MHz, CDCI₃) δ 8.07 (s, 1 H), 4.10 (s, 3H), 4.07 (s, 3H), 2.83 (d, J = 7.3 Hz, 2H), 1.97 (m, 1 H), 0.95 (d, J = 6.6 Hz, 6H); ¹³C NMR: (150 MHz, CDCI₃) δ 165.4, 164.5, 154.8, 151.6, 142.6, 129.9, 53.7, 53.5, 40.8, 29.7, 22.5; MS: (ESI-TOF) Ci₂Hi₆N₂O₄H⁺ expected: 253.1 183, found 253.1187.

Procedures for the coupling of pyridazine with piperazines:

Method A:

Methyl 6-(4-(ferf-butoxycarbonyl)piperazine-1-carbonyl)-4-isopropylpyridazine-

3-carboxylate (9a):

To a stirring solution of pyridazine 2a (719 mg, 3.02 mmol) in THF (10 mL) at 0 ⁰C was added dropwise a cold solution of LiOH (monohydrate, 139 mg, 3.3 mmol) in water (5 mL). The reaction was stirred at 0 ⁰C until disappearance of the starting material on TLC. The pH was then made acidic (pH 1-2) with careful addition of a 3% HCI solution. The solution was extracted with AcOEt (3x30 mL), the fractions were combined, dried with Na₂SO₄, filtered and the solvent was removed in vacuo. The desired monosaponified pyridazine was obtained as a pale yellow solid. The carboxylic acid was then directly involved in the next step without any further purification. To a solution of the acid (340 mg, 1.5 mmol) in CH₂CI₂ (15 mL) was added NEt₃ (422 μl_, 3 mmol), N-Boc-piperazine (350 mg, 1.5 mmol) and PyBroP (707 mg, 1.5 mmol). After 18 h under nitrogen, the reaction mixture was diluted with CH₂CI₂ (50 mL) and washed with a solution of HCI (0.1 M, 10 mL) and then a saturated aqueous solution of NaHCO₃ (10 mL). The organic layer was then dried with Na₂SO₄, filtered and concentrated under reduced pressure. The crude residue was purified by column chromatography (Hexane/AcOEt 1/0 to 6/4) and the desired piperazine adduct 9a (452 mg, 69%) was obtained as a pale yellow foam; ¹H NMR: (250 MHz, CDCI₃) Mixture of conformers δ 7.88 (s, 1 H), 4.03 (s, 3H), 3.79 (m, 2H)₁ 3.70 (m, 2H), 3.59-3.40 (m, 5H), 1.44 (s, 9H), 1.28 (d, J = 6.5 Hz, 6H); ¹³C NMR: (62.5 MHz, CDCI₃) δ 165.1 , 164.5, 156.8, 154.4, 152.8, 149.0, 125.8, 80.3, 53.2, 47.3, 43.6, 43.3 (broad), 42.7, 28.8, 28.2, 22.5; HRMS: (ESI-TOF) C₁₉H₂₈N₄O₅H⁺ expected: 393.2132. found: 393.2126.

Method B:

To a solution of piperazine (1 equivalent) in anhydrous CH₂CI₂ (5 mL) was added slowly AIMe₃ (1 equivalent, 2.0 M solution in hexanes). The mixture was allowed to stir at room temperature for approximately 10 min. To the aluminum- amide solution was added slowly a solution of pyridazine (1.05 equivalent) in anhydrous CH₂CI₂ (2.5 mL). The schlenk flask was sealed, and the yellow solution heated to 41 ⁰C. After 24 h, the now orange solution was cooled to room temperature, and quenched with slow addition of water (3 mL) with vigorous stirring. The aqueous layer was extracted with CH₂CI₂ (3x5 mL). The organic fractions were collected, dried over MgSO₄, and evaporated to dryness under reduced pressure. The crude residue was purified by flash chromatography on silica gel.

Tert-Butyl 3-((6-(4-(fert-butoxycarbonyl)piperazine-1-carbonyl)-3- (methoxycarbonyl)pyridazin-4-yl)methyl)-7/7-indole-1-carboxylate (9b); Starting from 2b (398 mg, 0.93 mmol), column Hexane/AcOEt 7/3, white solid, yield: 331 mg, (61%);¹H-NMR (600 MHz, CDCI₃) Mixture of conformers δ 8.15 (bs, 1 H), 7.67 (bs, 1 H), 7.48 (bs, 1 H)₁ 7.33 (dd, J = 7.2, 8.4 Hz, 1 H), 7.29 (d, J = 7.8 Hz, 1 H), 7.19 (dd, J = 7.2, 8.4 Hz, 1 H), 4.41 (s, 2H), 4.08 (s, 3H), 3.74 (bs, 2H), 3.63 (bs, 2H), 3.53 (bs, 2H)₁ 3.48 (bs, 2H), 1.68 (s, 9H), 1.46 (s, 9H); ¹³C-NMR (150 MHz, CDCI₃) δ: 165.0, 164.3, 156.9, 154.5, 152.2, 149.5, 142.2, 129.4, 128.8, 125.1 , 124.9, 122.8, 118.6, 115.6, 1 15.0, 84.1 , 80.4, 53.4, 47.4, 42.7, 28.4, 28.2, 27.2; HRMS: (ESI-TOF) C₃₀H₃₇N₅O₇H⁺ expected: 580.2766, found: 580.2759.

(S)-Methyl 6-(4-(feft-butoxycarbonyl)-3-isobutylpiperazine-1-carbonyl)-4- isobutylpyridazine-3-carboxylate (9c); Starting from 2c (160 mg, 0.63 mmol), column Hexane/AcOEt 1/1 , white foam, yield: 200 mg, (68%); ¹H NMR: (600 MHz, CDCI₃) Mixture of conformers δ 7.76 (s, 1 H), 4.64 (m, 0.4H), 4.58 (m, 0.6H), 4.36 (m, 0.4H), 4.22 (m, 0.5H), 4.15 (m, 0.5H), 4.08 (m, 0.6H), 4.05 (s, 1.4H), 4.04 (s, 1.6H),

3.95 (m, 0.4H), 3.45 (m, 0.4H), 3.24 (m, 0.4H), 3.20-3.07 (m, 1.8H), 3.04 (m, 0.6H),

2.96 (m, 0.4H), 2.88-2.74 (m, 2H), 1.94 (m, 1 H), 1.62-1.52 (m, 1 H), 1.47 (s, 4.4H), 1.46 (s, 4.6H), 1.44 (m, 1 H)₁ 1.23 (m, 1 H), 0.96-0.91 (m, 9.5H), 0.82 (m, 2.5H); ¹³C NMR: (150 MHz, CDCI₃) 5 165.0, 164.9, 156.5, 154.4, 153.1 , 152.9, 143.1 , 142.8, 129.9, 129.8, 80.2, 53.2, 50.2, 47.6, 45.8, 42.7, 40.6, 37.9, 29.3, 28.3, 24.6, 24.4, 22.8, 22.6, 22.4, 22.3, 22.2; HRMS: (ESI-TOF) C₂₄H₃₈N₄O₅H⁺ expected: 463.2915. found: 463.2915.

General procedure for the hydrazinolysis of the methyl ester pyridazines:

To a solution of pyridazine methyl ester (1 equivalent) in MeOH (30 ml_) was added hydrazine monohydrate (10 equivalents). The reaction was stirred for 20 h at rt under an atmosphere of nitrogen. The volatiles were evaporated under reduced pressure, and the product was purified on silica gel.

Tert-Butyl 4-(6-(hydrazinecarbonyl)-5-isopropylpyridazine-3- carbonyl)piperazine-1-carboxylate (10a) : Starting from 9a (148 mg, 0.37 mmol), column CH₂CI₂/Me0H 97/3, white foam, yield : 130 mg, (88%); ¹H NMR: (600 MHz, CDCI₃) Mixture of conformers δ 8.95 (m, 1 H), 7.94 (s, 1 H), 4.23 (hept, J = 6.8 Hz, 1 H), 4.16 (m, 2H), 3.85 (m, 2H), 3.70 (m, 2H), 3.61 (s, 2H), 3.56 (m, 2H), 1.49 (s, 9H), 1.33 (d, J = 6.8 Hz, 6H); ¹³C NMR: (150 MHz, CDCI₃) δ 164.7, 164.4, 157.2,

154.4, 151.1 , 151.0, 126.5, 80.4, 47.3, 43.6 (broad), 42.6, 28.3, 27.9, 22.6; HRMS: (ESI-TOF) Ci₈H₂₈N₆O₄H⁺ expected: 393.2245. found: 393.2239.

Terf-Butyl 3-((6-(4-(ferf-butoxycarbonyl)piperazine-1-carbonyl)-3- (hydrazinecarbonyl)pyridazin-4-yl)methyl)-7W-indole-1-carboxylate (10b);

Starting from 9b (164 mg, 0.28 mmol), column CH₂CI₂/Me0H 97/3, pale yellow foam, yield: 130 mg, (79%); ¹H NMR (250 MHz, CDCI₃) Mixture of conformers δ: 9.13 (bs, 1H), 8.15 (bs, 1 H), 7.61 (s, 1 H)₁ 7.50 (s, 1H), 7.33 (m, 2H), 7.18 (dd, J = 7.0, 7.8 Hz, 1 H), 4.69 (s, 2H), 3.72 (m, 2H), 3.52 (m, 4H), 3.44 (m, 2H), 1.67 (s, 9H), 1.45 (s, 9H); ¹³C NMR (150 MHz, CDCI₃) δ: 164.4, 164.2, 157.2, 154.4, 150.7, 143.2, 129.7,

129.5, 125.1 , 124.9, 124.8, 122.7, 118.8, 115.6, 1 15.5, 84.0, 80.4, 47.2, 42.5, 28.3, 28.2, 27.0; HRMS: (ESI-TOF) C₂₉H₃₇N₇O₆H⁺ expected: 580.2878, found: 580.2871.

(S)-Terf-Butyl 4-(6-(hydrazinecarbonyl)-5-isobutylpyridazine-3-carbonyl)-2- isobutylpiperazine-1-carboxylate (10c); Starting from 9c (219 mg, 0.47 mmol), column CH₂CI₂/Ac0Et 1/1 , white foam, yield: 180 mg, (82%); ¹H NMR (600 MHz, CDCI₃) Mixture of conformers δ 9.03 (d, J = 9.6 Hz, 1 H), 7.73 (s, 0.6H), 7.71 (s, 0.4H), 4.65 (d, J = 12.5 Hz, 0.4H), 4.59 (d, J = 13.0 Hz, 0.6H), 4.16-4.07 (m, 3H), 3.94 (d, J = 13.2 Hz, 0.6H), 3.46 (dd, J = 13.6, 3.7 Hz, 0.4H), 3.20-2.94 (m, 4H), 2.00 (m, 1 H), 1.60 (m, 1 H), 1.53 (m, 1 H), 1.47 (s, 5H), 1.46 (s, 4H), 1.20 (m, 1 H), 0.96- 0.92 (m, 9H), 0.83 (t, J = 6.8 Hz, 3H); ¹³C NMR (150.9 MHz, CDCI₃) δ 165.3, 165.0, 156.8, 156.7, 151.5, 144.4, 144.3, 131.0, 130.7, 80.4, 53.4, 50.1 , 47.5, 45.7, 42.6, 40.8, 38.0, 29.5, 28.3, 24.7, 24.6, 22.8, 22.7, 22.6, 22.3; HRMS: (ESI-TOF) C₂₃H₃₈N₆O₄H⁺ expected: 463.3027, found 463.3026.

General procedure for the synthesis of the ureas :

To a solution of sodium nitrite (2 equivalents) in H₂O (0.7 ml_) was added at 0 °C a solution of HCI (1 M, 4 equivalents) and a solution of acetic acid (3 equivalents). After 5 min under vigorous stirring, a solution of acyl hydrazide (1 equivalent) in THF (6 ml.) was added dropwise. The mixture was stirred for another 20 min at 0 ⁰C. Saturated aqueous Na₂CO₃ solution was then added until pH is basic, and the organic phase is extracted with Et₂O (3x10 mL). The organic fractions were collected, dried over Na₂SO₄ and concentrated under reduced pressure to give the desired acyl azide as yellow oil. The azide was then dissolved in anhydrous toluene (1 mL) and stirred for 1 h at 70 °C to give the corresponding isocyanate. The reaction mixture was cooled to rt and an amine (1.1 equivalent) was added. The resulting mixture was stirred over night at rt, concentrated in vacuo and the residue purified by preparative TLC.

Terf-Butyl 4-(6-(3-isopentylureido)-5-isopropylpyridazine-3- carbonyl)piperazine-1-carboxylate (13a); Starting from 10a (55 mg, 0.14 mmol), Prep. TLC CH₂CI₂/Me0H 97/3, white solid, yield: 34 mg, (52%); ¹H NMR: (250 MHz, CDCI₃) Mixture of conformers δ 9.69 (m, 1 H), 8.04 (s, 1 H), 7.66 (s, 1 H), 3.76 (m, 4H), 3.52 (m, 4H), 3.39 (m, 2H), 3.14 (hept, J = 6.7 Hz₁ 1 H), 1.67 (m, 1 H), 1.53 (m, 2H), 1.46 (m, 9H), 1.29 (d, J = 6.7 Hz, 6H), 0.93 (d, J = 6.5 Hz, 6H); ¹³C NMR: (62.5 MHz, CDCI₃) δ 165.2,

154.6, 154.5, 154.2, 151.0, 136.8, 125.6, 80.3, 47.3, 42.6, 38.6, 28.3, 26.6, 25.9, 22.4, 21.2; HRMS: (ESI-TOF) C₂₃H₃₈N₆O₄H⁺ expected: 463.3027. found: 463.3035.

Terf-Butyl 4-(5-isopropyl-6-(3-(3-(methylthio)propyl)ureido)pyridazine-3- carbonyl)piperazine-1-carboxylate (13b); Starting from 10a (55 mg, 0.14 mmol), Prep. TLC CH₂CI₂/MeOH 97/3, white solid, yield: 35 mg, (52%); ¹H NMR: (250 MHz, CDCI₃) Mixture of conformers δ 9.80 (m, 1 H), 7.90 (s, 1 H), 7.68 (s, 1 H), 3.78 (m, 4H), 3.62-3.42 (m, 6H), 3.09 (hept, J = 6.7 Hz, 1 H), 2.57 (m, 2H), 2.10 (s, 3H), 1.93 (m, 2H), 1.46 (s, 9H), 1.30 (d, J = 6.7 Hz, 6H); ¹³C NMR: (62.5 MHz, CDCI₃) δ 165.1 ,

154.7, 154.5, 154.1 , 151.2, 136.7, 125.8, 80.3, 47.3, 43.4 (broad), 42.6, 39.1 , 31.5, 29.1 , 28.3, 26.6, 21.2, 15.5; HRMS: (ESI-TOF) C₂₂H₃₆N₆O₄SH⁺ expected: 481.2591. found: 481.2590.

Tert-Butyl 3-((6-(4-(fert-butoxycarbonyl)piperazine-1-carbonyl)-3-(3- isopentylureido)pyridazin-4-yl)methyl)-7H-indole-1-carboxylate (13c); Starting from 10b (47 mg, 0.08 mmol), Prep. TLC CH₂CI₂/Me0H 97/3, white solid, yield: 16 mg, (31%); ¹H NMR: (250 MHz, CDCI₃) Mixture of conformers δ 9.63 (m, 1 H), 8.15 (m, 1 H), 8.04 (s, 1 H), 7.49 (s, 1 H), 7.42 (s, 1 H), 7.39-7.15 (m, 3H), 4.06 (s, 2H)₁ 3.70 (m, 4H), 3.47 (m, 4H), 3.37 (m, 2H), 1.68 (s, 9H), 1.61 (m, 1 H), 1.51 (m, 2H), 1.46 (s, 9H)₁ 0.87 (d, J = 6.4 Hz₁ 6H); ¹³C NMR: (62.5 MHz₁ CDCI₃) δ 164.9, 154.8, 154.4, 151.0, 149.3. 135.6, 129.2, 128.9, 125.0, 122.8, 118.5, 115.6, 113.1 , 84.2, 80.3, 47.3, 43.5 (broad), 42.5, 38.6, 28.3, 28.1 , 25.9, 25.6, 22.4; HRMS: (ESI-TOF) C₃₄H₄₇N₇O₆H⁺ expected: 650.3660. found: 650.3654.

(S)-Tert-Butyl 2-isobutyl-4-(5-isobutyl-6-(3-isopentylureido)pyridazine-3- carbonyl)piperazine-1-carboxylate (13d); Starting from 10c (86 mg, 0.18 mmol), Prep. TLC Hexane/AcOEt 1/2, white solid, yield: 25 mg, (25%); ¹H NMR: (600 MHz, CDCI₃) Mixture of conformers δ 9.58 (m, 1 H), 7.59 (s, 1 H)₁ 7.17 (d, J = 19.6 Hz₁ 1 H), 4.62 (d, J = 12.5 Hz, 0.5H), 4.57 (d, J = 13.2 Hz, 0.5H)₁ 3.98-4.35 (m, 3H), 2.91-3.48 (m, 5H)₁ 2.48 (m, 2H), 2.03 (m, 1 H), 1.69 (m, 1 H)₁ 1.61 (m, 0.6H)₁ 1.54 (m, 2.4H), 1.48 (s, 4.5H), 1.47 (s, 4.5H), 1.30-1.42 (m, 2H), 0.93-1.00 (m, 15H)₁ 0.87 (t, J = 5.8 Hz, 3H); ¹³C NMR: (150 MHz₁ CDCI₃) δ 165.4, 154.9, 154.4, 154.2, 150.9, 150.8, 138.3, 129.8, 129.6, 80.3, 50.1 , 47.5, 45.6, 42.8, 38.8, 38.7, 38.1 , 28.4, 26.8, 26.0, 25.9, 24.8, 24.7, 22.9, 22.8, 22.6, 22.5, 22.4; HRMS: (ESI-TOF) C₂₈H₄₈N₆O₄H⁺ expected: 533.3810. found: 533.3814.

General procedure for the Boc deprotection:

To a solution of the urea in CH₂CI₂ (0.75 ml_) at 0 ⁰C was added TFA (0.25 ml_). The mixture was stirred for 3 h at rt and then concentrated in vacuo. The crude residue was purified on a short plug of silica if necessary to afford the desired amines as trifluoroacetic acid salts. In the case of compound 1c /Pr₃SiH was added for the deprotection of the indole.

1-lsopentyl-3-(4-isopropyl-6-(piperazine-1-carbonyl)pyridazin-3-yl)urea 2,2,2- trifluoroacetate (1a): Starting from 13a (34 mg, 0.07 mmol), Column CH₂CI₂/Me0H 9/1 , white solid, yield : 34 mg, (97%); ¹H NMR: (600 MHz, CDCI₃ZCD₃OD 95/5) Mixture of conformers δ 7.69 (s, 1 H), 4.06 (m, 4H), 3.75 (m, 4H), 3.35 (m, 2H), 3.04 (hept, J = 6.7 Hz, 1 H), 1.63 (hept, J = 6.7 Hz, 1 H), 1.48 (m, 2H), 1.25 (d, J = 6.7 Hz, 6H), 0.89 (d, J = 6.6 Hz₁ 6H); ¹³C NMR: (150 MHz, CDCI₃ZCD₃OD 95/5) δ 164.9, 154.6, 154.1 , 150.1 , 137.6, 125.9, 44.2, 43.5, 42.9, 39.4, 38.4, 38.3, 26.5, 25.7, 22.1 , 20.8; HRMS: (ESI-TOF) Ci₈H₃₀N₆O₂H⁺ expected: 363.2503. found: 363.2448.

1-(4-lsopropyl-6-(piperazine-1-carbonyl)pyridazin-3-yl)-3-(3- (methylthio)propyl)urea 2,2,2-trifluoroacetate (1b); Starting from 13b (35 mg, 0.07 mmol), Column CH₂CVMeOH 9/1 , white solid, yield : 34 mg, (94%); ¹H NMR: (600 MHz, CDCI₃/CD₃OD 95/5) Mixture of conformers δ 7.71 (s, 1 H), 4.07 (m, 4H), 3.60-3.40 (m, 6H), 3.04 (hept, J = 6.7 Hz₁ 1 H)₁ 2.54 (m, 2H), 2.07 (s, 3H)₁ 1.90 (m, 2H), 1.27 (d, J = 6.7 Hz₁ 6H); ¹³C NMR: (150 MHz₁ CDCI₃/CD₃OD 95/5) δ 164.8, 154.8, 154.1 ,

150.2, 137.5, 126.0, 44.2, 43.5, 39.5, 38.9, 31.3, 28.8, 26.6, 20.8, 15.2; HRMS: (ESI- TOF) C₁₇H₂₈N₆O₂SH⁺ expected: 381.2067. found: 381.2052.

1-(4-((fH-lndol-3-yl)methyl)-6-(piperazine-1-carbonyl)pyrϊdazin-3-yl)-3- isopentylurea (1c); Starting from 13c (16 mg, 0.02 mmol), Column saturated solution of NH₃ in CH₂CI₂, white solid, yield : 8 mg, (73%); ¹H NMR: (600 MHz, CDCI₃) Mixture of conformers δ 9.53 (m, 1 H), 8.58 (s, 1 H), 7.59 (s, 1 H), 7.44 (s, 1 H), 7.41 (d, J = 8.1 Hz, 1 H)₁ 7.37 (d, J = 8.1 Hz, 1 H), 7.24 (m, 1 H), 7.13-7.09 (m, 2H), 4.09 (s, 2H), 3.79 (m, 2H), 3.72 (m, 2H), 3.40 (m, 2H), 2.99 (m, 2H), 2.91 (m, 2H)₁ 1.67 (hept, J = 6.7 Hz, 1 H), 1.53 (m, 2H)₁ 0.93 (d, J = 6.6 Hz, 6H); ¹³C NMR: (150 MHz, CDCI₃) δ 164.9, 154.7, 151.4, 136.6, 129.3, 129.0, 126.5, 123.4, 122.8, 120.0,

118.3, 1 1 1.7, 108.0, 48.7, 46.4, 45.7, 43.6, 38.7, 38.6, 26.8, 25.9, 22.4; HRMS: (ESI- TOF) C₂₄H_3IN₇O₂H⁺ expected: 450.2612. found: 450.2610.

(S)-1-(4-lsobutyl-6-(3-isobutylpiperazine-1-carbonyl)pyridazin-3-yl)-3- isopentylurea (1d); Starting from 13d (25 mg, 0.04 mmol), washed with saturated NaHCO₃ solution, white solid, yield : 18 mg, (90%); ¹H NMR (600 MHz, CDCI₃) Mixture of conformers δ 9.61 (m, 1 H), 7.56 (s, 1 H), 7.29 (d, J = 12.9 Hz₁ 1 H)₁ 4.61 (d, J = 12.3 Hz, 1 H), 4.20 (m, 1 H), 3.41 (m, 2H), 2.86-3.32 (m, 4H), 2.62 (m, 0.6H), 2.48 (d, J = 7.0 Hz, 2H)₁ 2.30 (m. 0.4H)₁ 2.04 (m, 1 H), 1.77 (m, 0.4H), 1.68 (m, 1 H), 1.62 (m, 0.6H)₁ 1.53 (m, 2H), 1.35 (m, 1 H)₁ 0.88-0.99 (m, 18H); ¹³C NMR: (75 MHz₁ CDCI₃) δ 164.8, 164.7, 154.8, 154.1 , 150.9, 136.8, 129.7, 129.6, 54.0, 53.2, 47.8, 45.0, 42.8, 38.8, 38.7, 31.9, 30.3, 29.7, 29.4, 26.7, 26.0, 25.9, 24.3, 24.2, 22.7, 22.5, 22.4; HRMS: (ESI-TOF) C₂₃H₄₀N₆O₂H⁺ expected 433.3285. found 433.3277.

Definition of Terms:

Side chains of amino acids are the groups attached to the alpha carbon of alpha-amino acids. For example the side chains of glycine, alanine, and phenylalanine are hydrogen, methyl, and benzyl, respectively. The side chains may be of any naturally occurring or synthetic alpha amino acid. Naturally occurring alpha amino acids include those found in naturally occurring peptides, proteins, hormones, neurotransmitters, and other naturally occurring molecules. Synthetic alpha amino acids include any non-naturally occurring amino acid known to those of skill in the art. Representative amino acids include, but are not limited to, glycine, alanine, serine, threonine, arginine, lysine, ornithine, aspartic acid, glutamic acid, asparagine, glutamine, phenylalanine, tyrosine, tryptophan, leucine, valine, isoleucine, cysteine, methionine, histidine, 4-trifluoromethyl-phenylalanine, 3-(2- pyridyl)-alanine, 3-(2-furyl)-alanine, 2,4-diaminobutyric acid, and the like.

Pharmaceutically acceptable salts include a salt with an inorganic base, organic base, inorganic acid, organic acid, or basic or acidic amino acid. As salts of inorganic bases, the invention includes, for example, alkali metals such as sodium or potassium, alkali earth metals such as calcium and magnesium or aluminum, and ammonia. As salts of organic bases, the invention includes, for example, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine. As salts of inorganic acids, the instant invention includes, for example, hydrochloric acid, boric acid, nitric acid, sulfuric acid, and phosphoric acid. As salts of organic acids, the instant invention includes, for example, formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid. As salts of basic amino acids, the instant invention includes, for example, arginine, lysine and ornithine. Acidic amino acids include, for example, aspartic acid and glutamic acid. Certain compounds within the scope of Formula I are derivatives referred to as prodrugs. The expression "prodrug" denotes a derivative of a known direct acting drug, e.g. esters and amides, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process; see Notari, R.E., "Theory and Practice of Prodrug Kinetics," Methods in Enzymology 112:309-323 (1985); Bodor, N., "Novel Approaches in Prodrug Design," Drugs of the Future 6:165-182 (1981); and Bundgaard, H., "Design of Prodrugs: Bioreversible-Derivatives for Various Functional Groups and Chemical Entities," in Design of Prodrugs (H. Bundgaard, ed.), Elsevier, New York (1985), Goodman and Gilmans, The Pharmacological Basis of Therapeutics, 8th ed., McGraw-Hill, Int. Ed. 1992. The preceding references are hereby incorporated by reference in their entirety.

Tautomers refers to isomeric forms of a compound that are in equilibrium with each other. The concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, ketones are typically in equilibrium with their enol forms. Thus, ketones and their enols are referred to as tautomers of each other. As readily understood by one skilled in the art, a wide variety of functional groups and other structures may exhibit tautomerism, and all tautomers of compounds having Formula I are within the scope of the present invention.

Compounds of the present invention include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention.

"Treating" within the context of the instant invention, means an alleviation, in whole or in part, of symptoms associated with a disorder or disease, or halt of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder. Similarly, as used herein, a "therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with a disorder or disease, or halts of further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disease or disorder. Treatment may also include administering the pharmaceutical Formulations of the present invention in combination with other therapies. For example, the compounds of the invention can also be administered in conjunction with other therapeutic agents against bone disease or agents used for the treatment of metabolic disorders.

Detailed Description of Figures:

Figures 1a and 1b show an overlay of the target molecules with an α-helix and the general retrosynthetic approach to these molecules, respectively. The scaffold mimics the position /, /+4, and /+7 of the α-helix. To ensure the water solubility of the inhibitors a pyridazine ring was the basis of the scaffold. These molecules may be thought of as synthetic counterparts to amphiphilic α-helices; they are intended to present both a hydrophobic surface for recognition and a hydrophilic "wet edge" that is rich in hydrogen bond donors and acceptors. The major disconnections from the final urea-pyridazine-piperazine 1 (UPP) are made at the amide and urea bonds to give a pyridazine diester 2, an amine 3, and a piperazine 4.

Figure 2 is a scheme showing the inverse electron demand Diels-Alder reaction of dimethyl-1 ,2 ,4,5-tetrazine-dicarboxylate 5 with suitable dienophiles (Boger, D. L. Tetrahedron 1983, 39, 2869.), 6, 7 or 8 to give compound 2 with different R₂ groups. Tetrazine 5 was synthesized from ethyl diazoacetate following the procedure of Boger and co-workers (Boger, D. L.; et al. Org. Synth. 1992, 70, 79.). Three kinds of dienophiles were employed depending on the structure of the desired R₂ group. A few suitable alkynes such as 6 are commercially available and were used. Enamines are also known to be good dienophiles for the cycloaddition with disubstituted tetrazines (Boger, D. L. Tetrahedron 1983, 39, 2869.). Thus R₂ = /Pr was introduced via the cycloaddition of the enamine 7 obtained by the condensation of isovaleraldehyde and pyrrolidine following the Mannich procedure (Stork, G.; et al. J. Am. Chem. Soc. 1963, 85, 207.)- This procedure is suitable to introduce short, low molecular weight side-chains such as /Pr for which the corresponding alkynes are often a gas and more difficult to handle. In the case where R₂ = Chk-Boclndole, methyl enol ether 8 was used as the dienophile. The enol ether was obtained by methylenation of the corresponding methyl ester with Tebbe's reagent (Pine, S. H.; et al. J. Am. Chem. Soc. 1980, 102, 3270.), and directly subjected to reaction with tetrazine 5 without any further purification. This procedure expanded the scope of the possible side chains to the wide range of methyl esters commercially available, especially those derived from natural amino-acids.

Figure 3 shows the synthetic scheme for the final products 1a-d, derived from the starting compounds 2a-c. Installation of the piperazine group was performed by using either a peptide coupling (Method A) or direct coupling of the methyl ester function (Method B). A few examples were first carried out with non-substituted N- Boc protected piperazines (R₃ = H). The corresponding final compounds present only two side chains but provide clear NMR spectra for characterization. The synthesis was then applied to substituted piperazines yielding compounds presenting three side chains for which the NMR spectra are more complex due to the conformational isomers of the piperazine around the tertiary amide bond. The 6- position methyl ester of the pyridazine 2a was selectively saponified using LiOH in a THF/water mixture at 0 °C. The piperazine 4a

(R₃ = H) was coupled under standard peptide coupling conditions with PyBroP (Method A) in good yields to give pyridazine-piperazine 9a. In the case of pyridazines 9b, c the coupling with piperazines 4a or 4b (R₃ = /Bu) was performed directly using Weinreb amidation (Basha, A.; et al. Tetrahedron Lett. 1977, 48, 4171.) in the presence of AIMβ₃ (Method B) to give only the desired regioisomer. To install the urea function in the 3-position, the remaining methyl ester was first converted to the acyl hydrazide 10a-c under mild conditions using an excess of hydrazine in methanol. The acyl hydrazide function was then diazotized to the acyl azide 11 using the Curtius method (sodium nitrite in acidic conditions) (J. Meienhofer, The Peptides, Analysis, Synthesis, Biology¹, ed. by E. Gross, J. Meienhofer; Academic Press, Inc., 1979; Vol. 1 , pp. 197-228.). Acyl azides were not purified and directly transformed to the corresponding isocyanates 12 by heating at 70 ⁰C. Ureas 13a-d were obtained by trapping these isocyanates with various amines. Final removal of the Λ/-Boc group was accomplished using TFA, to give the desired UPP 1a-d that present three side chains to mimic the /, /+4, /+7 positions of an α-helix.

Figure 4 shows the structures of 20 UPP compounds that were synthesized and exemplifies therein preferred amino acid side groups and homologs thereof. Numbering, quantities, molecular weights and molecular formulas are listed for each compound.

Figure 5 shows the structures of 18 UPP compounds that were synthesized and exemplifies therein preferred amino acid side groups and homologs thereof. Numbering, quantities, molecular weights and molecular formulas are listed for each compound.

Claims

What is claimed is:

1. An alpha-helix i, i+3/i+4, and i+7 mimetic represented by formula (I):

Formula (i)

wherein:

R₁, R₂ and R₃ are radicals independently selected from the group of radicals consisting of side chains of naturally occurring amino acids and homologs thereof; and

R₄ is a radical selected from the group consisting of -H and -(C₁-C₆ alkyl); and optionally including solvates and/or pharmaceutically acceptable salts therewith; with an optional proviso that none of Ri, R₂ and R₃ is hydrogen; or alternatively that, at most, one of Ri, R₂ and R₃ is hydrogen.

2. An alpha-helix i, i+3/i+4, and i+7 mimetic represented by Formula (I) according to claim 1 , wherein:

R₁, R₂, and R₃ are radicals independently selected from the group of radicals consisting of -CH₃, -CH₂CH₃, -CH(CH₃)₂, -CH₂CH(CH₃)₂, -CH₂CH₂CH₂CH₃, -CH(CH₃)CH₂CH₃, -CH₂OH, -CH₂SH, -CH₂CH₂SCH₃, -CH(OH)CH₃, -CH₂Ph, -CH₂C₆H₄OH, -CH₂C₆H₂I₂OH, -CH₂(3-indole), -CH₂C(O)NH₂, -CH₂COOH, -CH₂CH₂C(O)NH₂, -CH₂CH₂COOH, -CH₂CH₂CH₂CH₂NH₂, -CH₂(4-imidazole), -CH₂CH₂CH₂NHC(NH)NH₂, and homologs thereof.

3. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^'

4. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

5. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

6. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

7. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

8. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

9. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

10. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

1 1.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

12. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^'

13.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

14. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

15.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

16.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

17. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

18. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

19.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

20. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

21.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

22. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

23. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

24. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

25.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

26.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

27.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

28.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

29. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

30. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

31.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

32. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

33.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

34. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

35. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

36.An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

CF₃CO₂ ^"

37. An alpha-helix i, i+3/i+4, and i+7 mimetic according to claim 1 having the following structure:

38. A process for synthesizing any of the compounds of claims 1-37 and intermediates thereof.

39. A process for disrupting a protein-protein interaction selected from the group consisting of Bak/Bcl-X|_, p53/HDM2, calmodulin/smooth muscle myosin light- chain kinase, and gp41 assembly comprising the step of contacting a compound of claim 1 with sufficient concentration to disrupt the protein-protein interaction.

40. A process for treating conditions and/or disorders mediated by the disruption of the protein-protein interaction of claim 39 comprising the step of administering a sufficient amount to a compound of claim 1 to a patient to the disruption of the protein-protein interaction.