Structure-based design of potent CDK1 inhibitors derived from olomoucine.

Cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, constitutes a possible target in the search for new antitumor agents. Starting from the purine derivative olomoucine and following a structure-based approach, potent inhibitors of this enzyme were rapidly identified. The molecular modeling aspects of this work are described.

2,6,9-trisubstituted purines: optimization towards highly potent and selective CDK1 inhibitors.

Novel 2,6,9-substituted purine derivatives represent a class of potent and selective inhibitors of CDK1/cyclinB. The synthesis, SAR and biological profile of selected compounds are described.

New aza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: candidates for clinical development.

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

In vitro effect of alpha1-acid glycoprotein on the anti-human immunodeficiency virus (HIV) activity of the protease inhibitor CGP 61755: a comparative study with other relevant HIV protease inhibitors.

Protein binding can impair the potency of human immunodeficiency virus (HIV) protease inhibitors. Therefore, the activity of a novel compound, CGP 61755, was studied in the absence or presence of alpha1-acid glycoprotein (alpha1AGP). In MT-2 cells, the activity loss was 4-fold (EC90 without alpha1AGP, 29 nM vs. 122 nM with alpha1AGP). In primary lymphocytes, the loss was 8-fold (EC90, 45 nM vs. 364 nM). In identical experiments, the activity loss in MT-2 cells and lymphocytes was 2- and 3-fold, respectively, for indinavir, 11- and 10-fold for saquinavir, and 11- and 48-fold for ritonavir. For SC-52151, a 17-fold loss was seen in MT-2 cells, whereas no EC90 with alpha1AGP was reached in lymphocytes. This study demonstrates that the impact of alpha1AGP on in vitro activity varies greatly among different HIV protease inhibitors. The magnitude of such differences is greater in human lymphocytes than in a standard cell line.

Profile of CGP 61755: a novel and potent HIV-1 protease inhibitor that shows enhanced anti-HIV activity when combined with other antiretroviral agents in vitro.

CGP 61755 is a novel hydroxyethylene derivative produced by a high yield 10 step chemical synthesis. It is highly specific for HIV-1 protease with an IC50 of 1 nM. The ED90 in MT-2, PBLs and macrophages is infected with laboratory strains of HIV-1 or clinical isolates is 30-100 nM. In chronically infected macrophages the ED90 is 1000 nM (1000 nM for saquinavir and 10 microM for indinavir). When the antiviral activity of CGP 61755 on HIV-1 infected lymphocytes was examined using serum free medium an ED99 of 60 nM was determined, while in the presence of 10% human serum the same activity was achieved with 120 nM. When examined in combination with RT inhibitors or protease inhibitors, either in a co-culture of CEM-SS and chronically infected H9IIIB cells or in a free virus lymphocyte infection, cooperativity of the antiviral activities was observed. Dog pharmacokinetic studies comparing p.o. and i.v. data indicate that CGP 61755 has a bioavailability between 50 and 80%. Following oral administration the area under the concentration curve (AUC) values increased in a dose proportional manner. The plasma levels of the drug at 6 hours after oral administration were above the ED90. Based on these properties we believe that CGP 61755 has an attractive profile that justifies further preclinical evaluation of the drug.

Aza-peptide analogs as potent human immunodeficiency virus type-1 protease inhibitors with oral bioavailability.

A series of aza-peptide analogs with a (hydroxyethyl)hydrazine isostere has been synthesized as HIV-1 protease inhibitors using a simple synthetic scheme. Structure-activity studies based on the X-ray of a previously described inhibitor-enzyme complex led to potent inhibitors with antiviral activity in the low-nanomolar range. The S-configuration of the transition-state hydroxyl group was preferred in this series. Small modifications of the P2P3 and P2'P3' substituents had little effect on enzyme inhibition but greatly influenced the pharmacokinetic profile. As a result of these studies, the symmetrically acylated compound 8a and its close analog 24a bearing a methyl carbamate in P3 and an ethyl carbamate in P3' position were identified as potent inhibitors with plasma concentrations exceeding antiviral ED50 values 150-fold following oral application in mice.

Synthesis of potent and orally active HIV-protease inhibitors.

A series of potent HIV-protease inhibitors has been prepared. Several of the newly synthesized compounds showed high plasma even after oral administration to animals. Based on the overall biological profile, CGP 61755 was chosen for further preclinical evaluation. For this compound, a 10 step synthesis potentially suitable for large scale production was developed.

Pharmacokinetics and body distribution of liposomal zinc phthalocyanine in tumor-bearing mice: influence of aggregation state, particle size, and composition.

The pharmacokinetics and body distribution of zinc phthalocyanine (ZnPc) intravenously administered in liposomes composed of ZnPc, 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and 1,2-dioleoylphosphatidylserine (OOPS) (1:90:10 or 1:70:30 w/w) to tumor (Meth A sarcoma) bearing mice were studied. It was found that aggregation of ZnPc in the liposomes (i) increases the clearance rate of the dye from plasma, (ii) lowers the maximal dye concentration in tumor tissue, and (iii) increases the maximal dye concentration in the liver. In addition, aggregated dye is hardly eliminated from the liver and monomeric dye is eventually completely eliminated from this organ. Liposomes in the size range of 48-123 nm, containing the dye with the same aggregation state, showed the same pharmacokinetics and body distribution of the dye. The PS-content of the ZnPc liposomes (POPC alone versus POPC/OOPS 7:3) did not influence tumor, liver, and plasma pharmacokinetics during the studied time intervals. Free flow electrophoretic analysis showed in lyophilisates of ZnPc liposomes containing aggregated ZnPc the presence of two distinct populations differing in size, aggregation state of the dye, and PC/PS and ZnPc/phospholipid ratio. The liposomal formulation with monomeric ZnPc has a compositional homogeneity and demonstrated selectivity and reached high uptake in tumors, 48 h after intravenous administration and appears promising for photodynamic therapy.

Large-scale production of liposomes containing monomeric zinc phthalocyanine by controlled dilution of organic solvents.

This work describes the development of an organic solvent dilution method suitable for the large scale manufacturing of small unilamellar liposomes containing the water-insoluble photosensitizer zinc phthalocyanine in the monomeric state. N-Methyl pyrrolidone (NMP)/tert-butyl alcohol was selected as water miscible organic phase in which the phospholipids 1-palmitoyl, 2-oleoylphosphatidylcholine (POPC), and 1,2-dioeloylphosphatidylserine (OOPS) and the dye were dissolved. This organic phase was mixed with an excess of a water phase using a dynamic mixing device, yielding reproducibly unilamellar liposomes with a mean size of 50-150 nm as measured with quasielastic light scattering. After concentration, the organic solvents were efficiently removed by cross-flow filtration. The liposomes were then sterile filtered and freeze-dried. A method to measure the aggregation state of the dye in the liposomes was developed. A stable lyophilized formulation containing monomeric liposomal ZnPc could be obtained by using a solution of ZnPc in NMP (2 mg/mL) and ZnPc/phospholipid (1:100 w/w ratio) and performing concentration and dialysis at 4 degrees C and lyophilization in presence of a mixture of lactose and phospholipid (5:1 w/w ratio).

CGP 55398, a liposomal Ge(IV) phthalocyanine bearing two axially ligated cholesterol moieties: a new potential agent for photodynamic therapy of tumours.

Ge(IV) phthalocyanine (GePc) with two axially ligated cholesterol moieties was prepared by chemical synthesis and incorporated in a monomeric state into small unilamellar liposomes (CGP 55398). Upon photoexcitation with light wavelengths around its intense absorption peak at 680 nm, GePc shows an efficient photosensitising activity towards biological substrates through a mechanism which largely involves the intermediacy of singlet oxygen. GePc injected systemically into mice bearing an intramuscularly implanted MS-2 fibrosarcoma is quantitatively transferred to serum lipoproteins and localises in the tumour tissue with good efficiency: at 24 h post injection the GePc content in the tumour is 0.74 and 1.87 micrograms per g of tissue with a tumour/peritumoral ratio of 4.35 and 5.67 for injected doses of 0.76 and 1.52 mg kg-1 respectively. At this time the red-light irradiation of the GePc-loaded fibrosarcoma causes a fast and massive tumour necrosis involving both malignant cells and blood vessels.

S-adenosylmethionine decarboxylase inhibitors: new aryl and heteroaryl analogues of methylglyoxal bis(guanylhydrazone).

A series of 3-acylbenzamidine (amidino)hydrazones 7a-h, the corresponding (hetero)aromatic congeners 7i-p, and 3,3'-bis-amidino-biaryls 25a-e were synthesized. The hydrazones 7a-p were prepared by conversion of the corresponding acyl nitriles 1a,c-d,i,n-p to the imido esters 3a,c-d,i and the amidines 5a,c-d,h-i, followed by a reaction with aminoguanidine, or vice versa. Similarly, the biaryl 3,3'-dinitriles 23a-e were converted, via the imino esters 24a-c or the imino thioesters 27d-e, to the diamidines 25a-e. These new products are conformationally constrained analogues of methylglyoxal bis(guanylhydrazone) (MGBG). They are up to 100 times more potent as inhibitors of rat liver S-adenosylmethionine decarboxylase (SMDC) and generally less potent inhibitors of rat small intestine diamine oxidase (DAO) than MGBG. Some of these SAMDC inhibitors, e.g., compounds 7a, 7e, 7i, 25a, and 25d, have shown antiproliferative effects against T24 human bladder carcinoma cells. These products, whose structure-activity relationships are discussed, are of interest as potential anticancer agents and drugs for the treatment of protozoal and Pneumocystis carinii infections.

Synthesis and biological activity of 2-lactonyl penems.

A series of potent antibacterial agents have been prepared. These agents are penems carrying a lactone ring in the C-2 position. Excellent activity against Gram-positive and Gram-negative organisms--except Pseudomonas aeruginosa--was found.

Biological effects of modified colchicines. Improved preparation of 2-demethylcolchicine, 3-demethylcolchicine, and (+)-colchicine and reassignment of the position of the double bond in dehydro-7-deacetamidocolchicines.

A variety of colchicine, demecolcine, and isocolchicine derivatives were examined for their potency in the lymphocytic leukemia P388 screen in mice, for their toxicity in mice, and for their binding to microtubule protein. A qualitatively direct correlation was found between in vivo potency and toxicity; potency appeared to be less well correlated with tubulin binding. The most potent compounds were N-acylated analogues of colchicine and demecolcine. Among the monophenols, only 3-demethylcolchicine showed an appreciable effect in vitro and in vivo and was less toxic than colchicine. Improved methods were found for the preparation of 3- and 2-demethylcolchicine, which involved the use of 85% phosphoric acid and concentrated sulfuric acid, respectively. Decoupling experiments with 1H NMR proved that the double bond of dehydro-7-deacetamidocolchiceine and its derived tropolonic methyl ethers 24 and 25 was in the 5,6 position, rather than the 6,7 position formerly tentatively assigned.

Colchicine binding to antibodies.

Antibodies to colchicine have been prepared by injecting rabbits with an antigen prepared by coupling deacetylcolchicine to bovine serum albumin with a water-soluble carbodiimide. Sera of high titer were obtained with apparent dissociation constants for colchicine ranging from 5 to 17 nM. Binding was rapid and occurred at low temperatures. The chemical specificity of the binding site(s) resembled that in tubulin for the A and B rings of colchicine. Unlike tubulin, the antibody binding site tolerated numerous changes in the tropolone moiety (C ring). In contrast to tubulin, binding of colchicine to the antibody site did not promote fluorescence. The colchicine binding capacity of the antibody was high enough to prevent colchicine fluorescence in the presence of tubulin and to restore polymerization in colchicine-inhibited tubulin preparations.