Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: design, synthesis, biological evaluation, and docking studies.

We report the design and synthesis of a series of dipeptide-type inhibitors with novel P3 scaffolds that display potent inhibitory activity against SARS-CoV 3CLpro. A docking study involving binding between the dipeptidic lead compound 4 and 3CLpro suggested the modification of a structurally flexible P3 N-(3-methoxyphenyl)glycine with various rigid P3 moieties in 4. The modifications led to the identification of several potent derivatives, including 5c-k and 5n with the inhibitory activities (Ki or IC50) in the submicromolar to nanomolar range. Compound 5h, in particular, displayed the most potent inhibitory activity, with a Ki value of 0.006 µM. This potency was 65-fold higher than the potency of the lead compound 4 (Ki=0.39 µM). In addition, the Ki value of 5h was in very good agreement with the binding affinity (16 nM) observed in isothermal titration calorimetry (ITC). A SAR study around the P3 group in the lead 4 led to the identification of a rigid indole-2-carbonyl unit as one of the best P3 moieties (5c). Further optimization showed that a methoxy substitution at the 4-position on the indole unit was highly favorable for enhancing the inhibitory potency.

Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study.

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 µM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.

Preformulation and formulation of newly synthesized QNT3-18 for development of a skin whitening agent.

New molecules having the structure of (E)-2-(4-tert-butylbenzylidene) hydrazinecarbothioamide (QNT3-18) or 4-tert-butylphenylthiourea (QNT3-20) was synthesized and presupposed to inhibit melanogenesis through the inhibition of tyrosinase, which is involved in melanin formation. Therefore, we seek to develop these new molecules as skin whitening agents in topical formulations based on preformulation studies. QNT3-18 or QNT3-20 showed a strong single endothermic peak at 159.34°C with 10.79 µm-sized or at 150.69°C with 9.0 µm-sized aggregated particles, respectively. Both QNT3-18 and QNT3-20 did not show cytotoxicity at effective concentration range (0.4 µM) against keratinocyte cells and QNT3-18 was more retained than QNT3-20 in the skin instead of permeating through the skin. QNT3-18 or QNT3-20 was practically insoluble in water; the aqueous solubility was 3.8 ± 0.37 or 130.6 ± 2.52 µg/mL, respectively. Also, the partition coefficient value (log P) corresponding to the quotient between aqueous and octanol concentration of the molecule was 3.9 or 2.6, respectively. The skin retention amount of QNT3-18 was 1.7-fold higher than that of QNT3-20. When the optimal SLN cream (J3 formulation) containing 4 µM QNT3-18 was applied on the backs of hairless rats for 4 days after UV irradiation for 7 days and the skin color was checked by reflectance spectrophotometer, the rat skin treated with SLN cream with QNT3-18 quickly recovered to normal compared to skin treated with SLN cream without QNT3-18. Taken together, this study suggests that topical formulations such as creams including SLNs with QNT3-18 might be appropriate carriers for skin whitening agents.

Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety.

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL(pro). A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL(pro) motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1' site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC(50) or K(i) values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K(i) values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL(pro) may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1'-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.

Structure-activity relationship of naphthaldehydethiosemicarbazones in melanogenesis inhibition.

2-(Naphthalen-1-ylmethylene)hydrazinecarbothioamide (14, IC(50)=1.1µM) was discovered as a highly potent inhibitor of melanogenesis. To define the role of hydrogens (at N1 and N3) and sulfur in 14, a series of analogs 15a-p were synthesized and evaluated for anti-melanogenic activity using melanoma B16 cells under the stimulus of α-MSH. It was observed that replacement of either of these hydrogens at N1 or N3 by substituents increases the activity significantly. Conversely, concomitant substitutions decrease the inhibitory potency. In addition, the presence of sulfur in thiosemicarbazone is essential for the activity.

Structural requirement of phenylthiourea analogs for their inhibitory activity of melanogenesis and tyrosinase.

Effect of a series of 1-phenylthioureas 1a-k and 1,3-disubstituted thioureas 2a-k were evaluated against melanin formation in melanoma B16 cell line and mushroom tyrosinase. Inhibitory activity of tyrosinase of 1-phenylthioureas 1a-k is parallel to their melanogenic inhibition. Thus, the melanogenic inhibition in melanoma B16 cells of 1-phenylthioureas could be the result of inhibition of tyrosinase. However, 1,3-diaryl or 1-phenyl-3-alkylthioureas, 2a-k, appears as melanogenic inhibitor without inhibition of tyrosinase. The molecular docking study of 1e and 2b to binding pocket of tyrosinase provided convincing explanation regarding the necessity of direct connection of planar phenyl to thiourea unit without N'-substitution of phenylthioureas 1 as tyrosinase inhibitor and 2 as non-tyrosinase inhibitor.

Identification of indoline-2-thione analogs as novel potent inhibitors of α-melanocyte stimulating hormone induced melanogenesis.

Based on the hits, 3,4-dihydroquinazoline-2-thione (1) and benzimidazole-2-thione (2), a series of indole-2-thione (3) and indole-2-thiol inhibitors (4) of melanogenesis were designed, synthesized and evaluated in melanoma B16 cells under the stimulant of α-melanocyte stimulating hormone (α-MSH). The indole-2-thione compounds (3a-g) exhibited an effective inhibitory activity on melanin synthesis. The Structure-Activity Relationship (SAR) studies of 2 have revealed that in potent inhibitor 3a (>100% inhibition at 30 µM, IC50=1.40 µM) the role of nitrogen (3-N) at 3-position is insignificance. In addition, the hydrophobic substituents of 3 were better than the hydrophilic one. However, conversion of thione (-C=S, 3) to thiol (-C-SH, 4) led to decrease in the potency.

Ketonethiosemicarbazones: structure-activity relationships for their melanogenesis inhibition.

A series of 2-(1-phenylalkylidene)hydrazinecarbothioamides 2, 2-(1-phenylalkyl)hydrazinecarbothioamides 3, 2-(3,4-dihydronaphthalen-1(2H)-ylidene)hydrazinecarbothioamide (4), and 2-(1-(thiophen-2-yl)ethylidene)hydrazinecarbothioamide (5) were synthesized for their melanogenesis inhibition in melanoma B16 cells. The SAR of these ketonethiosemicarbazones revealed that the benzylidene hydrogen in aldehydethiosemicarbazones 1 can be replaced by hydrophobic moiety and substitutions with alkyl group for the terminal amino hydrogen of ketonethiosemicarbazones improved the activity appreciably. In addition, the double bond in thiosemicarbazones is an important factor for the increment of hydrophobicity. Thus hydrophobic ketonethiosemicarbazones are excellent inhibitors of melanogenesis like aldehydethiosemicarbazones.

Structure--activity relationship studies of novel arylsulfonylimidazolidinones for their anticancer activity.

To define the SAR, a series of novel N-arylsulfonylimidazolidinone derivatives were evaluated for their in vitro anticancer activity against five human tumor cell lines, including A549, COLO205, KATO III, K562, SK-OV-3 and murine leukemia (P288D1) cell line. Among them, N-(2-chloroacetyl)-6-(2-oxo-4-phenylimidazolidin-1-ylsulfonyl)-3,4-dihydroquinoline-1(2H)-carboxamide (4m) and N-cyclohexyl-6-(2-oxo-4-phenylimidazolidin-1-ylsulfonyl)-3,4-dihydroquinoline-1(2H)-carboxamide (4n) exhibited comparable in vitro anticancer activity to doxorubicin against A549, KATO III and K562 cell lines and gave superior xenographic results against NCI-H23 and SW620 cancer cell lines. Regarding the structure-activity relationship, two critical points were discovered; the steric congestion at 4-position of N-arylsulfonylimidazolidinone scaffold abolishes the activity and the bulkiness or hydrophobicity of acyl groups at 3,4-dihydroquinoline of 4, especially with carbamoyl moiety, enormously enhances the activity.

Structural requirements of (E)-6-benzylidene-4a-methyl-4,4a,5,6,7,8-hexahydronaphthalen-2(3H)-one derivatives as novel melanogenesis inhibitors.

Chalcone type compound 1a ((E)-6'-benzylidene-4a'-methyl-4',4a',7',8'-tetrahydro-3'H-spiro[[1,3]dithiolane-2,2'-naphthalen]-5'(6'H)-one) was discovered as an potent inhibitor in melanogenesis. To define its structure-activity relationship, a series of analogs 1b-n, dithiolane truncated 2a-b and ring A removed 3a-e were prepared and evaluated. The electron donating substitution on the phenyl ring (ring C) rather than an electron withdrawing group and dithiolane motif of 1 are needed for the activity enhancement. The scaffold containing both rings A and B associated with α,ß-unsaturated system connected to phenyl of 1 was essential for antimelanogenesis.

Structural characteristics of thiosemicarbazones as inhibitors of melanogenesis.

A series of thiosemicarbazones 2(e-s) have been synthesized and studied their structure-activity relationship as melanogenesis inhibitor. Among them, (Z)-2-(naphthalen-1-ylmethylene)hydrazinecarbothioamide (2q, >100% inhibition at 10 µM, IC(50)=1.1 µM, ClogP=3.039) showed the strongest inhibitory activity. Regarding their structure-activity relationship, the hydrophobic substituents regardless the position on phenyl ring of benzaldehyde thiosemicarbazones enhance the inhibitory activity. Furthermore, the aromatic group of benzaldehydethiosemicarbazones can be replaced with sterically bulky cyclohexyl. Thus, hydrophobicity of the aryl or alkyl group on hydrazine of thiosemicarbazones is determinant factor for their inhibitory activity in melanogenesis rather than planarity.

Novel benzo[d]imidazole-2(3H)-thiones as potent inhibitors of the alpha-melanocyte stimulating hormone induced melanogenesis in melanoma B16 cells.

In order to determine the optimum size of heterocycle of lead compound 1 (6-methyl-3-phenethyl-3,4-dihydro-1H-quinoline-2-thione; IC(50)=0.8 microM) for inhibition of melanogenesis, we have synthesized and evaluated some benzimdazole-2(3H)-thiones 5a-e. The preliminary bioassay has shown that the benzimdazole-2(3H)-thione motif of 5 is essential structural unit for their inhibitory activity. Among all thiones 5a-e, the compound 5d strongly inhibited the formation of melanin with IC(50) value of 1.3 microM.

Refinement of the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones for their anti-melanogenesis activity.

In order to define the structural requirements of quinazoline-2(1H)-thiones 1 for their inhibitory activity on melanogenesis, a novel series of 3,4-dihydroquinazoline-2(1H)-thiones (3a-h) were prepared and screened for their melanogenesis inhibition on melanoma B16 cell line under the stimulant of alpha-MSH. The anti-melanogenesis activity of 3 is mainly mediated by the hydrogen bonding ability of thioamide unit in addition to complexation ability of thione and the hydrophobic binding power of side chain substitutions at 3-position. Thus, the pharmacophore of 3,4-dihydroquinazoline-2(1H)-thiones for their anti-melanogenesis activity could be refined as 3-hydrophobic substituted quinazolinethione.

Synthesis and evaluation of novel chromone analogs for their inhibitory activity against interleukin-5.

A novel series of chromone analogs were synthesized and evaluated for their inhibitory activity against interleukin-5. Among them compounds 5-Cyclohexylmethoxy-3-(4-hydroxybenzyl)-4H-chromen-4-one (6a, 98% inhibition at 30 microM, IC50<3.0 microM) and 5-Cyclohyxylmethoxy-3-(hydroxymethyl)-4H-chromen-4-one (8a, 84% inhibition at 30 microM, IC50=7.6 microM) showed most potent activity. The structural requirement of chromone analogs possessing the inhibitory activity against IL-5 could be summarized as: (i) importance of hydrophobic group such as cyclohexylmethoxy at 5th position of ring A, (ii) requirement of ring B with small size of hydrogen bonding group with electron donating property such as phenolic hydroxyl group at 4th position and (iii) planarity of the chromen-4-one ring.