Design, synthesis, and biological evaluation of N-arylpiperazine derivatives as interferon inducers.

Type I Interferon (IFN) signaling plays an important role in the immune defense system against virus infection and in the innate immune response, thus IFNs are widely used as anti-viral agents and treatment for immune disorder or cancer. However, there is a growing demand for novel small-molecule IFN inducer due to tolerance, toxicity, or short duration of action following direct administration of IFNs. In this study, we assessed arylpiperazine (ARP) as a new core skeleton of IFN inducer. To investigate structure-activity relationship, we designed and synthesized a series of ARP analogues and evaluated the ability to stimulate IFN response in THP-1 human monocyte cells. Compound 5i was identified as a potent type I IFN inducer as it significantly increased cytokine secretion and increased expression of various IFN-stimulating genes which are representative biomarkers of type I IFN pathway. Our results suggested a beneficial therapeutic potential of 5i as an anti-viral agent.

An Effective Synthesis Method for Tilorone Dihydrochloride with Obvious IFN-α Inducing Activity.

Tilorone dihydrochloride (1) has great potential for inducing interferon against pathogenic infection. In this paper, we describe a convenient preparation method for 2,7-dihydroxyfluoren-9-one (2), which is a usual pharmaceutical intermediate for preparing tilorone dihydrochloride (1). In the novel method, methyl esterification of 4,4'-dihydroxy-[1,1'-biphenyl]-2-carboxylic acid (4) was carried out under milder conditions with higher yield and played an important role in the preparation of compound 2. The structures of the relative intermediates and target compound were characterized by melting point, IR, MS, and ¹H-NMR. Furthermore, the synthesized tilorone dihydrochloride exhibited an obvious effect on induction of interferon-α (IFN-α) in mice within 12 h, and the peak level was observed until 24 h. This fruitful work has resulted in tilorone dihydrochloride becoming available in large-scale and wide application in clinics, which has a good pharmaceutical development prospects.

Dose-dependent IFN-stimulating and immunomodulating properties of 6H-indolo[2,3-B] quinoxaline derivatives.

Two 6H-indoloquinoxaline derivatives were studied in different doses and schemes of application for their INFgamma-inducing potential and ability to effect functional activity of phagocytic cells. Tested compounds were shown to possess comparable or higher activity than reference drug Amixin in analogous doses. One indoloquinoxaline significantly elevated metabolic activity of macrophages and increased their potential for phagocytosis. Application of multiple treatments and higher doses allowed us to reveal differences between studied derivatives that were not obvious in previous in vivo experiment. Capacity of 6H-indoloquinoxalines to induce vast IFN amounts on in vivo level was demonstrated for the first time.

Discovery of a highly potent series of TLR7 agonists.

The discovery of a series of highly potent and novel TLR7 agonist interferon inducers is described. Structure-activity relationships are presented, along with pharmacokinetic studies of a lead molecule from this series of N9-pyridylmethyl-8-oxo-3-deazapurine analogues. A rationale for the very high potency observed is offered. An investigation of the clearance mechanism of this class of compounds in rat was carried out, resulting in aldehyde oxidase mediated oxidation being identified as a key component of the high clearance observed. A possible solution to this problem is discussed.

[The interferon-induced and antiviral activities of dipeptides].

Five phosphodipeptides were synthesized; two of them (H-Lys-Ala(P) and H-Pro-Ala(P) had interferon-induced activity. These dipeptides at millimolar concentrations (10(-4)) and 10(-5) M) induced the synthesis of late (40-hour) interferon in human peripheral blood lymphocytes. The dipeptides H-Lys-Ala(P) and H-Pro-Ala(P) showed a protective antiviral activity in in vivo studies when singly intraperitoneally administered to mice 2 hours before inoculation with murine encephalomyocarditis virus.

[Interferonogenic activity of amixin analogs and diphenyl derivatives].

Properties of interferon productivity of several inducers of interferon, analogs of amixin, have been studied in vivo. It has been shown, that under the influence of injected agents the content of endogenic interferon in the blood serum of laboratory animals increased and their non-fractionated cells of the peripheral blood (splenocites and macrophages) synthesized alpha- and gamma-interferon.

Synthesis and biological evaluation of novel 9-substituted-8-hydroxyadenine derivatives as potent interferon inducers.

Recently we reported the adenine derivatives 3a-d as novel interferon (IFN) inducers. In the present study, we conducted a detailed structure-activity relationship study of analogues of 3a-d with respect to their IFN-inducing activity, mainly focusing on the N(9)-position of the adenine. From this study, we found that introduction of the 3-pyridylmethyl moiety was effective to increase in vitro activity, and compound 9ae was identified as being the most potent IFN inducer. This compound gave a minimum effective concentration (MEC) of 3 nM, which is comparable with that of R-848, a second generation IFN inducer. Compound 9ae also demonstrated potent IFN-inducing activity at a dose of 0.1 mg/kg by oral administration in mice. Furthermore, compound 9ae induced IFN in monkeys in a dose dependent manner, with a potency superior to that of R-848. In addition, 9ae did not cause emesis in ferrets even at a dose of 30 mg/kg. In this study the maximum plasma concentration of 9ae was 1019 ng/mL (ca. 3.1 microM), which was approximately 1000-fold higher than the MEC value. Therefore, with respect to both the efficacy and the safety margin, compound 9ae (SM-276001) is considered to be a promising compound as an orally active IFN inducer.

Synthesis and structure-activity-relationships of 1H-imidazo[4,5-c]quinolines that induce interferon production.

1H-Imidazo-[4,5-c]quinolines were prepared while investigating novel nucleoside analogues as potential antiviral agents. While these compounds showed no direct antiviral activity when tested in a number of cell culture systems, some demonstrated potent inhibition of virus lesion development in an intravaginal guinea pig herpes simplex virus-2 assay. We have determined that the in vivo antiviral activity can be attributed to the ability of these molecules to induce the production of cytokines, especially interferon (IFN), in this model. Subsequently, we found that the compounds also induce in vitro production of IFN in human peripheral blood mononuclear cells (hPBMCs). The in vitro results reported herein and the in vivo results reported previously led to the discovery of imiquimod, 26, which was developed as a topical agent and has been approved for the treatment of genital warts, actinic keratosis, and superficial basal cell carcinoma.

Prodrugs of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine: potent interferon inducing agents in monkeys.

In order to improve the oral bioavailability of 9-benzyl-8-hydroxy-2-(2-hydroxyethylthio)adenine (SM-295072), a potent interferon (IFN) inducing agent, we synthesized prodrugs of it by utilizing the hydroxy groups at the C(2)-side chain and/or the C(8)-position. The carbonate prodrug at the C(8)-position was more effective than that at the C(2)-side chain for oral absorption in rats. Among the compounds prepared, compound 6 demonstrated the most preferable prodrug properties, and the maximum plasma concentration of 6 was approximately 4-fold higher than that of SM-295072. Furthermore, compound 6 was dose-dependently absorbed in monkeys by oral administration, and exhibited a potent IFN-inducting activity that correlated well with its plasma drug concentration.

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents.

Recently, we have reported the 8-hydroxyadenine derivatives (2-4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).

Synthesis and structure-activity relationships of 2-substituted-8-hydroxyadenine derivatives as orally available interferon inducers without emetic side effects.

Recently we reported the adenine derivatives (2-4) as new interferon (IFN) inducers. In the present study, we conducted a detailed structure and activity relationship study of 4 and its related derivatives on IFN inducing activity. From this study, we found that compound 4 exhibited the most potent IFN inducing activity in vitro with a minimum effective concentration of 0.01 microM, and 4 also showed strong IFN-inducing activity at doses of more than 0.3mg/kg by oral administration in mice. This potency was 10-fold stronger than that of Imiquimod. Moreover, 4 did not cause emesis in ferrets even at doses as high as 10mg/kg, whereas, 80% of animals were emetic when orally administered with the same dose of Imiquimod. These results indicate that compound 4 is superior to Imiquimod with respect to efficacy and safety.

Synthesis of 6-substituted 9-benzyl-8-hydroxypurines with potential interferon-inducing activity.

Various 6-substituted 9-benzyl-8-hydroxypurines were synthesized in order to investigate the structure-activity relationship at the 6-position of 9-benzyl-8-hydroxyadenine (1), which is a lead compound for the screening of interferon (IFN)-inducing activity. 6-Unsubstituted, mercapto-, methylthio- and hydroxy-9-benzyl-8-hydroxypurines (2-5) were prepared from 5-amino-1-benzyl-4-cyano-2-hydroxyimidazole (9). Synthesis of a 6-methoxy analog (6) was conducted from 5-amino-4-benzylamino-6-chloropyrimidine (13). 6-Alkylamino and acylaminopurines (7 and 8) were also prepared by alkylation and acylation of 1, respectively. Since these compounds (2-8) indicated no activity, it was found that a free amino group of 1 is required for the expression of IFN-inducing activity.

Discovery of 8-hydroxyadenines as a novel type of interferon inducer.

9-Benzyl-8-hydroxyadenine (6) was found to possess interferon-inducing activity in vitro as a lead compound. Although replacement of the 9-benzyl group of 6 did not improve the activity, the introduction of a substituent such as alkyl, alkylthio, alkylamino, and alkoxy groups into the 2-position of the adenine ring resulted in a remarkable increase in the activity. The 2-alkylthio (30-32), 2-butylamino (41), and 2-butoxy (47) analogues indicated the highest activities by oral administration to mice.

[Toxicological characterization of a novel inductor of interferon, PK-43, synthesized on cotton cellulose]. / Toksikologicheskaia kharakteristika novogo induktora interferona, sintezirovannogo na osnove khlopkovoi tselliulozy PK-43.

Acute and subacute toxicity of a novel inductor of interferon [symbol: see text]-43 synthesized on the base of the cotton cellulose has been studied in white mice and rats. It has been established that a single administration of [symbol: see text]-43 even in a dose of 3000 mg/kg does not result in a death of mice. No apparent deviations were noted in the systemic condition and behavior of rats, in parameters for the peripheral blood and activity of transaminases with daily administration of the preparation over 30 days at doses 50,100 and 200 mg/kg. In addition, the drug does not have hepatotoxic and nephrotoxic effects. The derivatives of the cotton cellulose [symbol: see text]-43 are considered to be low toxic compounds.

[Synthesis and interferon-inducing activity studies on the antiviral compounds of 2,5,6-trisubstituted-4(3H) pyrimidinone derivatives].

In order to search for more ideal antiviral drugs, 21 substituted pyrimidinone derivatives were designed and synthesized, among which 11 were not reported before. The chemical structures were characterized by elemental and spectral analysis. The serum interferon-inducing activity was tested in mice. All 2-amino-5-bromo-6-substituted-4-(3H)pyrimidinone compounds were shown to have interferon inducing activity. The corresponding substituted pyrimidine thiones were less active. The new compounds of 6-sulfophenyl derivatives are soluble in water, but the interferon-inducing activity are not higher than the original compound of ABPP.

Alkylpurines as immunopotentiating agents. Synthesis and antiviral activity of certain alkylguanines.

Several simple 8-substituted 9-alkyl- and 7,8-disubstituted 9-alkylguanine derivatives were synthesized as potential antiviral agents. These were tested for antiviral protection against a lethal Semliki Forest virus (SFV) infection in mice, and their antiviral properties were evaluated from a structure-activity standpoint. In this model system, 9-alkylguanines with the alkyl chain consisting of four to six carbons were found to be the most active. Substitution of the 8-position of the purine ring did not enhance activity, with the exception of the 7-alkyl-8-oxo substituent. These data were found to support the hypothesis that guanines need not contain an intact carbohydrate moiety in order to exhibit antiviral activity by virtue of immune potentiation. Hence, phosphorylation of guanosine analogs that exhibit antiviral activity by a similar mechanism does not play a significant role.

[Antiviral agents. 28. Aliphatic substituted chlordihexylamino-1,3,5-triazine]. / Antivirale Wirkstoffe. 28. Mitteilung: Aliphatisch substituierte Chlor-dihexylamino-1,3,5-triazine.

The nucleophilic substitution of one chlorine atom in 2,4-dichloro-6-(dihexylamino)-1,3,5-triazine (1) by primary (2a-b) or secondary (2c) amines leads to the aliphatically substituted chloro-dihexylamino-1,3,5-triazines (3a-c) Structures of type 3 may be characterized by the spectroscopic data. In the mass spectra, the complete degradation of the alkyl groupings is striking and manifested in a series of consecutive peaks with differences of 14 between each other. Antiviral activity is exhibited by 3a through an inhibitory effect towards vaccinia virus and by 3b as interferon inductor towards encephalomyocarditis virus. Moreover, 3a and 3b exhibit a strong activity towards Trichomonas vaginalis. Additionally, 3a is capable of exerting temperature lowering and anthelminthic effects.

Induction of interferon in mice by sodium salt of 9-oxo-10-acridineacetic acid: specific enhancement by analogs.

9-oxo-10-acridineacetic acid bearing the common name of 10-carboxymethyl-9-acridanone or CMA (6) was found to be a very potent interferon (IFN) inducer in adult Balb/c mice. Seven structural analogs of CMA were synthetized and assayed for the interferon inducing ability. Three of the compounds had new chemical structures. The analogs were shown to be either weak or inactive interferon inducers. However, some of the analogs administered intraperitoneally (i.p.) or orally (p.o.) either 2 h before CMA or together with the active inducer enhanced by 10 to 60-fold the serum interferon response. We suggest that CMA induces interferon indirectly via a specific protein receptor. The specific enhancement of the serum interferon response to CMA by its inactive analogs may be explained in terms of the competition of the compounds for binding sites at the acceptor or transporting protein molecules. In the presence of an analog of CMA greater amount of free CMA may be available for the receptors in the target cells than when CMA acts alone. Only CMA bound to the receptor would be biologically active whereas the complexes of the compounds with the acceptor are biologically inert.

Preparation and biological properties of a highly active poly(G) X poly(C) inducer of interferon.

Experimental conditions necessary for the full expression of interferon-inducing activity by a complex of polyguanylic acid and polycytidylic acid included: (i) a sufficiently high molecular size of each homopolymer; (ii) annealing conditions which insured complete denaturation of polyguanylic acid self-structure; and (iii) the specific biological assay employed to assay interferon-inducing potency. The complex of polyguanylic acid and polycytidylic acid possessed several properties that suggested it may be an atypical polynucleotide interferon inducer. For instance, it was inactive in primary rabbit kidney cell cultures, usually exquisitely sensitive to polynucleotide interferon inducers, unless it was incubated on the cell cultures for prolonged times or in the presence of DEAE-dextran. Polyguanylic acid X polycytidylic acid could induce interferon in rabbits and mice but gave a more protracted response than did poly(I) X poly(C). Finally, poly(G) X poly(C) was, without any modification, resistant to degradation by serum nucleases.

Biological activities of analogues of lipid A based chemically on the revised structural model. Comparison of mediator-inducing, immunomodulating and endotoxic activities.

Lipid A analogues were chemically synthesized based on the model structure recently revised, and biological activities of the analogues were tested. The analogue, (beta-1,6)-linked glucosamine disaccharide carrying ester-bound 3-hydroxytetradecanoic acids at 3 and 3' position of reducing and nonreducing glucosamine in addition to amide-bound 3-hydroxytetradecanoic acids and glycosidic-linked and ester-linked phosphate groups, showed much stronger activities for mediator inducing and immunomodulating as well as endotoxic activities than those exhibited by the previously synthesized analogues based on the old model. Among the activities tested, induction of interferon and tumor necrosis factor as well as mitogenicity, adjuvanticity and pyrogenicity were, however, not expressed so strongly as natural lipid A used as controls. In contrast, the analogue exhibited comparable activities to those of control lipid A in the test of lethal toxicity to mice and gelating activity of Limulus amebocyte lysate. Other synthetic analogues carrying a phosphate group showed comparable, slightly stronger or weaker activities depending on the test, but nonphosphorylated analogue exhibited no apparent or only very weak activities.