A conformational NMR analysis of methymycin aglycones: complete and unambiguous assignments of stereochemically diverse glycosylated methymycin analogs by 1D and 2D NMR techniques and molecular modeling.

The (1)H and (13)C NMR spectra of 10-deoxymethynolide (1), 8.9-dihydro-10-deoxymethynolide (2) and its glycosylated derivatives (3-9) were analyzed using gradient-selected NMR techniques, including 1D TOCSY, gCOSY, 1D NOESY (DPFGSENOE), NOESY, gHMBC, gHSQC and gHSQC-TOCSY. The NMR spectral parameters (chemical shifts and coupling constants) of 1-9 were determined by iterative analysis. For the first time, complete and unambiguous assignment of the (1)H NMR spectrum of 10-deoxymethynolide (1) has been achieved in CDCl(3), CD(3)OD and C(6)D(6) solvents. The (1)H NMR spectrum of 8,9-dihydro-10-deoxymethynolide (2) was recorded in CDCl(3), (CD(3))(2)CO and CD(3)OD solutions to determine the conformation. NMR-based conformational analysis of 1 and 2 in conjugation with molecular modeling concluded that the 12-membered ring of the macrolactones may predominantly exist in a single stable conformation in all solvents examined. In all cases, a change in solvent caused only small changes in chemical shifts and coupling constants, suggesting that all glycosylated methymycin analogs exist with similar conformations of the aglycone ring in solution.

Regioselective Synthesis of a C-4'' Carbamate,C-6'' n-Pr Substituted Cyclitol Analogue of SL0101.

An asymmetric synthesis of two analogues of SL0101 (1) has been achieved. The effort is aimed at the discovery of inhibitors of the p90 ribosomal S6 kinase (RSK) with improved bioavailability. The route relies upon the use of the Taylor catalyst to regioselectively install C-3″ acetyl or carbamate functionality. This study led to the identification of a third-generation analogue of SL0101 with a C-4″ n-Pr-carbamate and a C-3″ acetate with improved RSK inhibitory activity.

A novel descriptor of amino acids and its application in peptide QSAR.

A novel descriptor, vector of principal component scores (VSW) for weighted holistic invariant molecular index, was derived from the principal component analysis of a matrix of 99 weighted holistic invariant molecular indices of amino acids. The scale was then applied in three panels of peptide QSARs models constructed by partial least square (PLS). The correlative coefficient R(cum)(2) and the cross-validation correlative coefficient Q(LOO)(2) of three models were 0.861 and 0.835 for 58 angiotensin-converting enzyme inhibitors, 0.873 and 0.751 for 48 bitter tasting thresholds, 0.997 and 0.954 for 12 antimicrobial polypeptides, respectively. External validation was also performed to validate the predictive power of resulting models. Compared with other 2D or 3D descriptors, the VSW scales could better characterize structural features of peptides and provide more sound statistical models.

Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture. We report herein a critical redesign of the HPD chemotype featuring an additional wing at the C5 position that led to drastically improved RNase H inhibition and significant antiviral activity. Structure-activity relationship (SAR) concerning primarily the length and flexibility of the two wings revealed important structural features that dictate the potency and selectivity of RNase H inhibition as well as the observed antiviral activity. Our current medicinal chemistry data also revealed that the RNase H biochemical inhibition largely correlated the antiviral activity.

3-Hydroxypyrimidine-2,4-diones as Selective Active Site Inhibitors of HIV Reverse Transcriptase-Associated RNase H: Design, Synthesis, and Biochemical Evaluations.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains an unvalidated antiviral target. A major challenge of specifically targeting HIV RNase H arises from the general lack of selectivity over RT polymerase (pol) and integrase (IN) strand transfer (ST) inhibitions. We report herein the synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to achieve selective RNase H inhibition. Biochemical studies showed the two subtypes with an N-1 methyl group (9 and 10) inhibited RNase H in low micromolar range without significantly inhibiting RT polymerase, whereas the N-1 unsubstituted subtype 11 inhibited RNase H in submicromolar range and RT polymerase in low micromolar range. Subtype 11 also exhibited substantially reduced inhibition in the HIV-1 INST assay and no significant cytotoxicity in the cell viability assay, suggesting that it may be amenable to further structure-activity relationship (SAR) for identifying RNase H inhibitors with antiviral activity.

3-Hydroxypyrimidine-2,4-dione-5-N-benzylcarboxamides Potently Inhibit HIV-1 Integrase and RNase H.

Resistance selection by human immunodeficiency virus (HIV) toward known drug regimens necessitates the discovery of structurally novel antivirals with a distinct resistance profile. On the basis of our previously reported 3-hydroxypyrimidine-2,4-dione (HPD) core, we have designed and synthesized a new integrase strand transfer (INST) inhibitor type featuring a 5-N-benzylcarboxamide moiety. Significantly, the 6-alkylamino variant of this new chemotype consistently conferred low nanomolar inhibitory activity against HIV-1. Extended antiviral testing against a few raltegravir-resistant HIV-1 clones revealed a resistance profile similar to that of the second generation INST inhibitor (INSTI) dolutegravir. Although biochemical testing and molecular modeling also strongly corroborate the inhibition of INST as the antiviral mechanism of action, selected antiviral analogues also potently inhibited reverse transcriptase (RT) associated RNase H, implying potential dual target inhibition. In vitro ADME assays demonstrated that this novel chemotype possesses largely favorable physicochemical properties suitable for further development.

Synthesis and Structure-Activity Relationship Study of 5a-Carbasugar Analogues of SL0101.

The Ser/Thr protein kinase, RSK, is associated with oncogenesis, and therefore, there are ongoing efforts to develop RSK inhibitors that are suitable for use in vivo. SL0101 is a natural product that demonstrates selectivity for RSK inhibition. However, SL0101 has a short biological half-life in vivo. To address this issue we designed a set of eight cyclitol analogues, which should be resistant to acid catalyzed anomeric bond hydrolysis. The analogues were synthesized and evaluated for their ability to selectively inhibit RSK in vitro and in cell-based assays. All the analogues were prepared using a stereodivergent palladium-catalyzed glycosylation/cyclitolization for installing the aglycon. The l-cyclitol analogues were found to inhibit RSK2 in in vitro kinase activity with a similar efficacy to that of SL0101, however, the analogues were not specific for RSK in cell-based assays. In contrast, the d-isomers showed no RSK inhibitory activity in in vitro kinase assay.

Inhibition of histone H3K79 methylation selectively inhibits proliferation, self-renewal and metastatic potential of breast cancer.

Histone lysine methylation regulates gene expression and cancer initiation. Bioinformatics analysis suggested that DOT1L, a histone H3-lysine79 (H3K79) methyltransferase, plays a potentially important role in breast cancer. DOT1L inhibition selectively inhibited proliferation, self-renewal, metastatic potential of breast cancer cells and induced cell differentiation. In addition, inhibitors of S-adenosylhomocysteine hydrolase (SAHH), such as neplanocin and 3-deazaneplanocin, also inhibited both H3K79 methylation and proliferation of breast cancer cells in vitro and in vivo. The activity of SAHH inhibitors was previously attributed to inhibition of H3K27 methyltransferase EZH2. However, inhibition of EZH2 by a specific inhibitor did not contribute to cell death. SAHH inhibitors had only weak activity against H3K27 methylation and their activity is therefore mainly due to DOT1L/H3K79 methylation inhibition. Overall, we showed that DOT1L is a potential drug target for breast cancer.

De novo synthesis and biological evaluation of C6″-substituted C4″-amide analogues of SL0101.

In an effort to improve upon the in vivo half-life of the known ribosomal s6 kinase (RSK) inhibitor SL0101, C4″-amide/C6″-alkyl substituted analogues of SL0101 were synthesized and evaluated in cell-based assays. The analogues were prepared using a de novo asymmetric synthetic approach, which featured Pd-π-allylic catalyzed glycosylation for the introduction of a C4″-azido group. Surprisingly replacement of the C4″-acetate with a C4″-amide resulted in analogues that were no longer specific for RSK in cell-based assays.

Improving the affinity of SL0101 for RSK using structure-based design.

Enhanced activity of the Ser/Thr protein kinase, RSK, is associated with transformation and metastasis, which suggests that RSK is an attractive drug target. The natural product, SL0101 (kaempferol 3-O-(3″,4″-di-O-acetyl-α-L-rhamnopyranoside), has been shown to be a RSK selective inhibitor. However, the Ki for SL0101 is 1 µM with a half-life of less than 30 min in vivo. To identify analogues with improved efficacy we designed a set of analogues based on the crystallographic model of SL0101 in complex with the RSK2 N-terminal kinase domain. We identified an analogue with a 5″-n-propyl group on the rhamnose that has > 40-fold improved affinity for RSK relative to SL0101 in an in vitro kinase assay. This analogue preferentially inhibited the proliferation of the human breast cancer line, MCF-7, versus the normal untransformed breast line, MCF-10A, which is consistent with results using SL0101. However, the efficacy of the 5″-n-propyl analogue to inhibit MCF-7 proliferation was only two-fold better than for SL0101, which we hypothesize is due to limited membrane permeability. The improved affinity of the 5″-n-propyl analogue for RSK will aid in the design of future compounds for in vivo use.

Structure Activity Relationship Study of the Cleistriosides and Cleistetrosides for Antibacterial/Anticancer Activity.

Two known cleistriosides and six known cleistetrosides were synthesized and evaluated for anticancer and antibacterial activity. This study, for the first time, reports anticancer activity and comprehensively the antibacterial activity for these oligosaccharide natural products. In addition, two new unnatural cleistetroside analogues were synthesized and tested. Biological activities for the ten oligosaccharides against B. subtilis were found to range between 4 and >64 µM, and for NCI-H460 human lung cancer epithelial cells between 7.5 and 90.9 µM. Similar activities were found for seven of the oligosaccharides against the NCI panel of 60 cell lines. The degree of acylation and location of the specific acetate groups had significant effects on the anticancer and antibacterial activity of both the cleistriosides and cleistetrosides.

C5'-Alkyl Substitution Effects on Digitoxigenin α-l-Glycoside Cancer Cytotoxicity.

A highly regio- and stereo-selective asymmetric synthesis of various C5'-alkyl side chains of rhamnosyl- and amicetosyl-digitoxigenin analogs has been established via palladium-catalyzed glycosylation with post-glycosylated dihydroxylation or diimide reduction. The C5'-methyl group in both α-l-rhamnose and α-l-amicetose digitoxin analogs displayed a steric directed apoptosis induction and tumor growth inhibition against non-small cell human lung cancer cells (NCI-H460). The anti-tumor activity is significantly reduced when the steric hindrance is increased at C5'-stereocenter.

Synthesis of several cleistrioside and cleistetroside natural products via a divergent de novo asymmetric approach.

The de novo asymmetric syntheses of several partially acylated dodecanyl tri- and tetra-rhamnoside natural products (cleistriosides-5 and 6 and cleistetrosides-2 to 7) have been achieved (19-24 steps). The divergent route requires the use of three or less protecting groups. The asymmetry was derived via Noyori reduction of an acylfuran. The rhamno-stereochemistry was installed by a diastereoselective palladium-catalyzed glycosylation, ketone reduction and dihydroxylation.

A de novo approach to the synthesis of glycosylated methymycin analogues with structural and stereochemical diversity.

A divergent and highly stereoselective route to 11 glycosylated methymycin analogues has been developed. The key to the success of this method was the iterative use of the Pd-catalyzed glycosylation reaction and postglycosylation transformation. This unique application of Pd-catalyzed glycosylation demonstrates the breath of α/ß- and d/l-glycosylation of macrolides that can be efficiently prepared using a de novo asymmetric approach to the carbohydrate portion.