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Led by the structural information of the screening hit with mDM2 protein, a structure modification of Leu26 moiety of the novel p53–hDM2 inhibitors was conducted. A structure–activity relationship study of 4-substituted piperidines revealed compound 20t with good potencies and excellent CYP450 profiles.

We describe successful efforts to optimize the in vivo profile and address off-target liabilities of a series of BACE1 inhibitors represented by 6 that embodies the recently validated fused pyrrolidine iminopyrimidinone scaffold. Employing structure-based design, truncation of the cyanophenyl group of 6 that binds in the S3 pocket of BACE1 followed by modification of the thienyl group in S1 was pursued. Optimization of the pyrimidine substituent that binds in the S2'-S2″ pocket of BACE1 remediated time-dependent CYP3A4 inhibition of earlier analogues in this series and imparted high BACE1 affinity. These efforts resulted in the discovery of difluorophenyl analogue 9 (MBi-4), which robustly lowered CSF and cortex Aβ40 in both rats and cynomolgus monkeys following a single oral dose. Compound 9 represents a unique molecular shape among BACE inhibitors reported to potently lower central Aβ in nonrodent preclinical species.

The synthesis of a series of iminoheterocycles and their structure–activity relationships (SAR) as inhibitors of the aspartyl protease BACE1 will be detailed. An effort to access the S3 subsite directly from the S1 subsite initially yielded compounds with sub-micromolar potency. A subset of compounds from this effort unexpectedly occupied a different binding site and displayed excellent BACE1 affinities. Select compounds from this subset acutely lowered Aβ40 levels upon subcutaneous and oral administration to rats.

Tertiary hydroxyl class of C-imidazole bridgehead azaheptapyridine FPT inhibitors were prepared in an attempt to block in vivo oxidation of secondary hydroxyl series. One representative compound 5a exhibited potent enzyme (IC50 = 1.4 nM) and cellular activities (soft agar IC50 = 1.3 nM) with excellent oral pharmacokinetic profiles in rats, mice, monkeys and dogs. The in vivo study in wap-ras TG mouse models showed dose dependent tumor growth inhibition and regression.

In the present paper, design, synthesis, X-ray crystallographic analysis, and HIV-1 protease inhibitory activities of a novel class of compounds are disclosed. Compounds 28-30, 32, 35, and 40 were synthesized and found to be inhibitors of the HIV-1 protease. The crucial step in their synthesis involved an unusual endo radical cyclization process. Absolute stereochemistry of the three asymmetric centers in the above compounds have been established to be (4S,2'R,3'S) for optimal potency. X-ray crystallographic analysis has been used to determine the binding mode of the inhibitors to the HIV-1 protease.

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

Fragment-based NMR screening, X-ray crystallography, structure-based design, and focused chemical library design were used to identify novel inhibitors for BACE-1. A rapid optimization of an initial NMR hit was achieved by a combination of NMR and a functional assay, resulting in the identification of an isothiourea hit with a K(d) of 15 microM for BACE-1. NMR data and the crystal structure revealed that this hit makes H-bond interactions with the two catalytic aspartates, occupies the nonprime side region of the active site of BACE-1, and extends toward the S3 subpocket (S3sp). A focused NMR-based search for heterocyclic isothiourea isosteres resulted in several distinct classes of BACE-1 active site directed compounds with improved chemical stability and physicochemical properties. The strategy for optimization of the 2-aminopyridine lead series to potent inhibitors of BACE-1 was demonstrated. The structure-based design of a cyclic acylguanidine lead series and its optimization into nanomolar BACE-1 inhibitors are the subject of the companion paper

Guided by structure-based design, we synthesized two novel series of potent inhibitors of BACE1 and generated extensive SAR around both the prime and non-prime side binding pockets. The key feature of both series is a cyclic amine motif specifically crafted to achieve interactions with both the flap and with the S2′ pocket.

The structures of both the native holo-HCV NS3/4A protease domain and the protease domain with a serine 139 to alanine (S139A) mutation were solved to high resolution. Subsequently, structures were...

Farnesyltransferase inhibitors identified from an ECLiPS® library were optimized using solution-phase synthesis. X-ray crystallography of inhibited complexes was used to identify substructures that coordinate to the active site zinc. The X-ray structures were ultimately used to guide the design of second-generation analogs with FTase IC50s of less than 1.0 nM.

Modification of the ethano bridge of the core structure of the antitumor agent, SARASAR® (SCH66336) with concomitant introduction of a sulfonamide moiety off the distal piperidine afforded inhibitor 9-(S-), a compound with greatly improved PK profile. Other compounds with enhanced FPTase inhibitory activity were obtained as exemplified by amide 10-(S-) and urea 11-(S-): these compounds demonstrated activity in picomolar range.

Protein farnesyl transferase (PFTase) catalyzes the reaction between farnesyl diphosphate and a protein substrate to form a thioether-linked prenylated protein. The fact that many prenylated proteins are involved in signaling processes has generated considerable interest in protein prenyl transferases as possible anticancer targets. While considerable progress has been made in understanding how prenyl transferases distinguish between related target proteins, the rules for isoprenoid discrimination by these enzymes are less well understood. To clarify how PFTase discriminates between FPP and larger prenyl diphosphates, we have examined the interactions between the enzyme and several isoprenoid analogues, GGPP, and the farnesylated peptide product using a combination of biochemical and structural methods. Two photoactive isoprenoid analogues were shown to inhibit yeast PFTase with K(I) values as low as 45 nM. Crystallographic analysis of one of these analogues bound to PFTase reveals that the diphosphate moiety and the two isoprene units bind in the same positions occupied by the corresponding atoms in FPP when bound to PFTase. However, the benzophenone group protrudes into the acceptor protein binding site and prevents the binding of the second (protein) substrate. Crystallographic analysis of geranylgeranyl diphosphate bound to PFTase shows that the terminal two isoprene units and diphosphate group of the molecule map to the corresponding atoms in FPP; however, the first and second isoprene units bulge away from the acceptor protein binding site. Comparison of the GGPP binding mode with the binding of the farnesylated peptide product suggests that the bulkier isoprenoid cannot rearrange to convert to product without unfavorable steric interactions with the acceptor protein. Taken together, these data do not support the "molecular ruler hypotheses". Instead, we propose a "second site exclusion model" in which PFTase binds larger isoprenoids in a fashion that prevents the subsequent productive binding of the acceptor protein or its conversion to product.

SCH 66336 is a trihalo tricyclic compound that is currently undergoing Phase II clinical trials for the treatment of solid tumors. Modifications of SCH 66336 by incorporating such groups as amides, acids, esters, ureas and lactams off the first or the distal piperidine (from the tricycle) provided potent FPT inhibitors some of which exhibited good cellular activity. A number of these compounds incorporate properties that might improve pharmacokinetic stability of these inhibitors by virtue of their increased solubility or by their change in log P.

The 10-bromobenzocycloheptapyridyl farnesyl transferase inhibitor (FTI) Sch-66336 (1) is currently under clinical evaluation for the treatment of human cancers. During structure-activity relationship development leading to 1, 10-bromobenzocycloheptapyridyl FTIs were found to be more potent than analogous compounds lacking the 10-Br substituent. This potency enhancement was believed to be due, in part, to an increase in conformational rigidity as the 10-bromo substituent could restrict the conformation of the appended C(11) piperidyl substituent in an axial orientation. A novel and potent class of FTIs, represented by indolocycloheptapyridine Sch-207758 [(+)-10a], have been designed based on this principle. Although structural and thermodynamic results suggest that entropy plays a crucial role in the increased potency observed with (+)-10a through conformational constraints and solvation effects, the results also indicate that the indolocycloheptapyridine moiety in (+)-10a provides increased hydrophobic interactions with the protein through the addition of the indole group. This report details the X-ray structure and the thermodynamic and pharmacokinetic profiles of (+)-10a, as well as the synthesis of indolocycloheptapyridine FTIs and their potencies in biochemical and biological assays.

A series of novel N-cyanoguanidine tricyclic farnesyl protein transferase (FPT) inhibitors was prepared. Replacement of a piperidine amide-group with a N-cyanoguanidine functionality increased FPT activity. X-ray crystal structure determination of 42 complexed with FPT revealed differences in the interactions of the amide and N-cyanoguanidine groups with the protein.

Protein prenylation is a posttranslational lipid modification in which C(15) and C(20) isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.

The discovery of C-linked imidazole azaheptapyridine bridgehead FPT inhibitors is described. This novel class of compounds are sub nM FPT enzyme inhibitors with potent cellular inhibitory activities. This series also has reduced hERG activity versus previous N-linked imidazole series. X-ray of compound 10a bound to FTase revealed strong interaction between bridgehead imidazole 3N with catalytic zinc atom.

Several fragment-based methods have been applied to the discovery of new lead sources for inhibitors of BACE1, an important therapeutic target for Alzheimer's disease. Among the most common fragment hits were various amidine-containing molecules in which the amidine engaged in discrete H-bond donor-acceptor interaction with the BACE1 catalytic dyad. Structure and medicinal chemistry knowledge-based optimization with emphasis on ligand efficiency resulted in identification of a key pharmacophore comprising a non-planar cyclic amidine scaffold directly attached to a phenyl group projecting into S1. This key pharmacophore is a common feature of known clinical candidates and has dominated the recent patent literature. A structural comparison of the non-planar cyclic amidine motif with other BACE1 pharmacophores highlights its uniqueness and distinct advantages.

On the basis of our observation that the biaryl substituent of iminopyrimidinone 7 must be in a pseudoaxial conformation to occupy the contiguous S1-S3 subsites of BACE1, we have designed a novel fused bicyclic iminopyrimidinone scaffold intended to favor this bioactive conformation. Strategic incorporation of a nitrogen atom in the new constrained ring allowed us to develop SAR around the S2' binding pocket and ultimately resulted in analogues with low nanomolar potency for BACE1. In particular, optimization of the prime side substituent led to major improvements in potency by displacement of two conserved water molecules from a region near S2'. Further optimization of the pharmacokinetic properties of this fused pyrrolidine series, in conjunction with facile access to a rat pharmacodynamic model, led to identification of compound 43, which is an orally active, brain penetrant inhibitor that reduces Aβ(40) in the plasma, CSF, and cortex of rats in a dose-dependent manner.

Arginase (EC 3.5.3.1) is an aminohydrolase that acts on l-arginine to produce urea and ornithine. Two isotypes of the enzyme are found in humans. Type I is predominantly produced in the liver and is a homotrimer of 35 kDa subunits. Human arginase (hArginase) I is seen to be up-regulated in many diseases and is a potential therapeutic target for many diverse indications. Previous reports of crystallization and structure determination of hArginase have always included inhibitors of the enzyme: here, the first case of a true apo crystal form of the enzyme which is suitable for small-molecule soaking is reported. The crystals belonged to space group P212121 and have approximate unit-cell parameters a = 53, b = 67.5, c = 250 A. The crystals showed slightly anisotropic diffraction to beyond 2.0 A resolution.

Protein prenylation is a posttranslational lipid modification in which C(15) and C(20) isoprenoid units are linked to specific protein-derived cysteine residues through a thioether linkage. This process is catalyzed by a class of enzymes called prenyltransferases that are being intensively studied due to the finding that Ras protein is farnesylated coupled with the observation that mutant forms of Ras are implicated in a variety of human cancers. Inhibition of this posttranslational modification may serve as a possible cancer chemotherapy. Here, the syntheses of two new farnesyl diphosphate (FPP) analogues containing photoactive benzophenone groups are described. Each of these compounds was prepared in six steps from dimethylallyl alcohol. Substrate studies, inhibition kinetics, photoinactivation studies, and photolabeling experiments are also included; these experiments were performed with a number of protein prenyltransferases from different sources. A X-ray crystal structure of one of these analogues bound to rat farnesyltransferase illustrates that they are good substrate mimics. Of particular importance, these new analogues can be enzymatically incorporated into Ras-based peptide substrates allowing the preparation of molecules with photoactive isoprenoids that may serve as valuable probes for the study of prenylation function. Photoaffinity labeling of human protein geranylgeranyltransferase with (32)P-labeled forms of these analogues suggests that the C-10 locus of bound geranylgeranyl diphosphate (GGPP) is in close proximity to residues from the beta-subunit of this enzyme. These results clearly demonstrate the utility of these compounds as photoaffinity labeling analogues for the study of a variety of protein prenyltransferases and other enzymes that employ FPP or GGPP as their substrates.

A number of novel amidine containing heterocycles were designed to reproduce the unique interaction pattern, revealed by X-ray crystallography, between the BACE-1 catalytic diad and a weak NMR screening hit (3), with special attention paid to maintaining the appropriate basicity and limiting the number of H-bonding donors of these scaffolds. The iminohydantoin cores (10 and 23) were examined first and found to interact with the catalytic diad in one of two binding modes (A and B), each with the iminohydantoin core flipped 180 degrees in relation to the other. The amidine structural motif within each core forms a bidentate interaction with a different aspartic acid of the catalytic diad. Both modes reproduced a highly conserved interaction pattern between the inhibitors and the catalytic aspartates, as revealed by 3. Potent iminohydantoin BACE-1 inhibitors have been obtained, validating the molecular design as aspartyl protease catalytic site inhibitors. Brain penetrant small molecule BACE inhibitors with high ligand efficiencies have been discovered, enabling multiple strategies for further development of these inhibitors into highly potent, selective and in vivo efficacious BACE inhibitors.

Based on lead compound 1 identified from the patent literature, we developed novel patentable BACE-1 inhibitors by introducing a cyclic amine scaffold. Extensive SAR studies on both pyrrolidines and piperidines ultimately led to inhibitor 2f, one of the most potent inhibitors synthesized to date.

The crystallization of TNF-alpha converting enzyme (TACE) has been useful in understanding the structure-activity relationships of new chemical entities. However, the propensity of TACE to undergo autoproteolysis has made enzyme handling difficult and impeded the identification of inhibitor soakable crystal forms. The autoproteolysis of TACE was found to be specific (Y352-V353) and occurred within a flexible loop that is in close proximity to the P-side of the active site. The rate of autoproteolysis was found to be proportional to the concentration of TACE, suggesting a bimolecular reaction mechanism. A limited specificity study of the S(1)' subsite was conducted using surrogate peptides and suggested substitutions that would stabilize the proteolysis of the loop at positions Y352-V353. Two mutant proteases (V353G and V353S) were generated and proved to be highly resistant to autoproteolysis. The kinetics of the more resistant mutant (V353G) and wild-type TACE were compared and demonstrated virtually identical IC(50) values for a panel of competitive inhibitors. However, the k(cat)/K(m) of the mutant for a larger substrate (P6 - P(6)') was approximately 5-fold lower than that for the wild-type enzyme. Comparison of the complexed wild-type and mutant structures indicated a subtle shift in a peripheral P-side loop (comprising the mutation site) that may be involved in substrate binding/turnover and might explain the mild kinetic difference. The characterization of this stabilized form of TACE has yielded an enzyme with similar native kinetic properties and identified a novel crystal form that is suitable for inhibitor soaking and structure determination.

Benzocycloheptapyridine tricyclic compounds with piperazine or substituted piperidine moieties extending either from the 5- or 6-position of the tricyclic bridgehead exhibited enhanced FTase activity: this resulted from favorable binding of the ligand nitrogen with the catalytic zinc found in the FTase. A single isomer at C-11 with piperazine adduct extending from the 6-position, compound 24 , exhibited excellent FTase activity with IC 50 = 0.007 μM, soft agar IC 50 = 72 nM, and Rat AUC(PO, 10 mpk) = 4.0 μM · h. X-ray of (−)-[8-chloro-6-(1-piperazinyl)-1 H -benzo[5,6]]cyclohepta[1,2- b ]pyridine-11-yl]-1-(methylsulfonyl)piperidine 24 bound to Ftase revealed favorable interaction between piperazine nitrogen and catalytic zinc atom.

Successful efforts to make farnesyl transferase (FT) inhibitors with appropriately tethered ligands designed to interact with a catalytic zinc that exist in the enzyme have been realized. Thus, by introducing either a pyridylmethylamino or propylaminolimidazole amide moieties off the 2-position of the piperidine ring, FT inhibitors with activities in the picomolar range have been achieved as exemplified by compounds 12a and 12b. An X-ray structure of 11b bound to FT shows the enhanced activity is a result of interacting with the active-site zinc.

Crystallographic and thermodynamic studies of farnesyl protein transferase (FPT) complexed with novel tricyclic inhibitors provide insights into the observed SAR for this unique class of nonpeptidic FPT inhibitors. The crystallographic structures reveal a binding pattern conserved across the mono-, di-, and trihalogen series. In the complexes, the tricycle spans the FPT active site cavity and interacts with both protein atoms and the isoprenoid portion of bound farnesyl diphosphate. An amide carbonyl, common to the tricyclic compounds described here, participates in a water-mediated hydrogen bond to the protein backbone. Ten high-resolution crystal structures of inhibitors complexed with FPT are reported. Included are crystallographic data for FPT complexed with SCH 66336, a compound currently undergoing clinical trials as an anticancer agent (SCH 66336, 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-yl)-1-piperidinyl]-2-oxoethyl]-1-piperidinecarbo xamide ). Thermodynamic binding parameters show favorable enthalpies of complex formation and small net entropic contributions as observed for 4-[2-[4-(3,10-dibromo-8-chloro-6,11-dihydro-11H-benzo[5, 6]cyclohepta[1, 2-b]pyridin-11-ylidene)-1-piperidinyl]-2-oxoethyl]pyridine N-oxide where DeltaH degrees bind = -12.5 kcal/mol and TDeltaS degrees bind = -1.5 kcal/mol.

The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0.42 nM, crystallographic R factor to 1.8 Å resolution, 19.6%) complexed with human α-thrombin shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3′ position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 Å resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 Å resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 Å resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 Å resolution = 16.9%) HPBK analogs complexed with thrombin revealed two binding modes for the closely related com-pounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3′ substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3′ methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, thrombin and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.

A series of imidazole-containing peptidomimetic PFTase inhibitors and their co-crystal structures bound to PFTase and FPP are reported. The structures reveal that the peptidomimetics adopt a similar conformation to that of the extended CVIM tetrapeptide, with the imidazole group coordinating to the catalytic zinc ion. Both mono- and bis-imidazole-containing derivatives, 13 and 16, showed remarkably high enzyme inhibition activity against PFTase in vitro with IC50 values of 0.86 and 1.7 nM, respectively. The peptidomimetics were also highly selective for PFTase over PGGTase-I both in vitro and in intact cells. In addition, peptidomimetics 13 and 16 were found to suppress tumor growth in nude mouse xenograft models with no gross toxicity at a daily dose of 25 mg kg−1.

The enzyme trypanothione reductase (TR), together with its substrate, the glutathione-spermidine conjugate trypanothione, plays an essential role in protecting parasitic trypanosomatids against oxidative stress and is a target for drug design. Here we show that a naturally occurring spermine derivative, the antihypertensive agent kukoamine A [N1N12-bis(dihydrocaffeoyl)-spermine] inhibits TR as a mixed inhibitor (Ki = 1.8 microM, Kii = 13 microM). Kukoamine shows no significant inhibition of human glutathione reductase (Ki > 10 mM) and thus provides a novel selective drug lead. The corresponding N1N8-bis(dihydrocaffeoyl)spermidine derivative was synthesized and acted as a purely competitive inhibitor with Ki = 7.5 microM. A series of mono- and di-acylated spermines and spermidines were synthesized to gain an insight into the effect of polyamine chain length, the nature and position of the acyl substituent and the importance of conformational mobility. These compounds inhibited TR with Ki values ranging from 11 to 607 microM.