Highly Enantioselective Semisynthesis of (+)/(-)-Gossypol Schiff Base Derivatives from Ground Plant Material.

We have recently developed a one-pot process for simultaneous extraction and chemical modification (SECheM) on Cienfuegosia digitata, a Mauritanian Malvaceae called locally "Izide". On the basis of this innovative methodology that consisted of using ground plant roots as starting material in gossypol Schiff base semisynthesis, we now report how this concept can be used to access enantiomerically pure Schiff base atropisomer derivatives of gossypol in only two steps. This study has been envisioned since enantiomerically pure Schiff base atropisomer derivatives of gossypol are generally more potent biologically when compared to racemic gossypol Schiff bases.

Tautomerism and stereodynamics in Schiff bases from gossypol and hemigossypol with N-aminoheterocycles.

Tautomerism plays a pivotal role in structural stabilization and reactivity. Herein we investigate in detail, aided by DFT simulations, the case of gossypol, a naturally occurring atropisomeric dialdehyde showing promising properties as a male contraceptive and an antineoplasic agent. Its toxicity linked to reactive aldehydo groups can be reduced through amino conjugation. The occurrence of either imino or enamino structures is puzzling indeed and a clear-cut rationale is missing yet. N-enamine-N-enamine structures are prevalent or exclusive tautomers for Schiff bases from gossypol, while their corresponding hydrazones only possess N-imine-N-imine structures both in solution and the solid state. The modification of interactions between the lone pairs on the nitrogen atoms by altering the steric hindrance of the non-iminic nitrogen can favor enamine tautomers. This assumption has now been confirmed and, in the solid state, hydrazones from N-aminopiperidine and their cis-2,6-dimethylderivative present bis-imine and bis-enamine structures, respectively. In solution, these compounds exist in equilibrium between both structures. The tautomerization mechanism, analysis of axial chirality and aromaticity in such H-bonded pseudorings are discussed as well.

Synthesis, structure and antimicrobial evaluation of a new gossypol triazole conjugates functionalized with aliphatic chains and benzyloxy groups.

Synthetic limitations in the copper-catalyzed azide alkyne cycloaddition (CuAAC) on gossypol's skeleton functionalized with alkyne (2) or azide (3) groups have been indicated. Modified approach to the synthesis of new gossypol-triazole conjugates yielded new compounds (24-31) being potential fungicides. Spectroscopic studies of triazole conjugates 24-31 have revealed their structures in solution, i.e., the presence of enamine-enamine tautomeric forms and π-π stacking intramolecular interactions between triazole arms. Biological evaluation of the new gossypol-triazole conjugates revealed the potency of 30 and 31 derivatives, having triazole-benzyloxy moieties, comparable with that of miconazole against Fusarium oxysporum. The results of HPLC evaluation of ergosterol content in different fungi strains upon treatment of gossypol and its derivatives enabled to propose a mechanism of antifungal activity of these compounds.

Synthesis and antiviral, insecticidal, and fungicidal activities of gossypol derivatives containing alkylimine, oxime or hydrazine moiety.

Gossypol is a part of the cotton plant's defense system against pathogens and herbivorous insects. To discover gossypol analogs with broad spectrum and high activity, a series of gossypol alkylamine Schiff base, oxime and hydrazone derivatives were synthesised and bioassayed. The biological results indicated that most of these derivatives exhibited higher anti-TMV activity than gossypol. Interestingly, the activities of compounds 10, 15, 18, 20, 23 and 26 were much higher than that of ribavirin. Furthermore, compound 26, which was low toxicity to rat, showed better activity than control plant virus inhibitors in the field. Additionally, allyl amine Schiff base (9) displayed remarkable insecticidal activities against Mythimna separata, Helicoverpa armigera and Ostrinia nubilalis, whereas (pyridin-3-yl)methanamine Schiff base (13) showed excellent activity against Culex pipiens pallens. The fungicidal results revealed that all of compounds exhibited good activity against Physalospora piricola.

Design, synthesis, and biological activities of aromatic gossypol Schiff base derivatives.

A series of aromatic gossypol Schiff bases have been successfully synthesized via a feasible chemical modification. The antiviral activity against tobacco mosaic virus (TMV) of these gossypol Schiff bases has been tested for the first time. The bioassay studies indicated most of these derivatives exhibited excellent anti-TMV activity, in which o-trifluoromethylaniline Schiff base (19) displayed the best antiviral activities. Furthermore, compound 19 exhibited an eminent anti-TMV effect in the field and low toxicity to mice. These results suggest it is a promising candidate for the inhibitor of plant virus.

Synthesis and anti-H5N1 activity of chiral gossypol derivatives and its analogs implicated by a viral entry blocking mechanism.

A series of chiral gossypol derivatives and its analogs were synthesized and tested in vitro for their anti-H5N1 activity. Interestingly, (+)-gossypol derivatives and its analogs were more active against H5N1 than the corresponding (-)-gossypol derivatives and its analogs. Through a simple chemical modification with amino acids, less active chiral gossypol could be converted into more active derivatives, and most of chiral gossypol derivatives were more potent against H5N1 than 1-adamantylamine. With regard to the mechanism of action, chiral gossypol derivatives and its analogs might impair the virus entry step of cell infection, likely targeting to HA2 protein.

Synthesis and antiviral activities of novel gossypol derivatives.

In this study, a series of novel gossypol derivatives were synthesized and screened in vitro for their anti-HIV-1 and anti-H(5)N(1) activities, respectively. Replacing the aldehyde groups of gossypol with some amino acids not only reduced the cytotoxicity but also enhanced the activities against HIV-1 and H(5)N(1). Compounds 13-17 showed more potent activities against HIV-1 and H(5)N(1) than the other gossypol derivatives. Meanwhile, these compounds also exhibited more potent activities against H(5)N(1) than 1-adamantylamine. The absence of the COONa group in gossypol derivatives resulted in a loss of anti-HIV-1 activity, suggesting that this group might play an important role in mediating the antiviral activity. Time-of-addition assays indicated that compounds 13-17 had the similar mechanism of anti-HIV-1 action with T20. Molecular modeling analysis demonstrated that compounds 13-17 could fit inside the gp41 hydrophobic pocket through hydrogen bonding network, hydrophobic contacts and strong electrostatic interactions.

Novel O-glycosidic gossypol isomers and their bioactivities.

Novel glycosidic gossypol analogs, 7,7'-gossypol diglucoside tetraacetate GS1, 6,7'-gossypol diglucoside tetraacetate GS2, 7,7'-gossypol diglycoside GS1', 6,7'-gossypol diglycoside GS2' were obtained by the ultrasound-assisted reaction of the potassium salt of gossypol with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide under PTC conditions and were fully characterized by 1D NMR ((1)H NMR, (13)C NMR, DEPT, 1D NOE), 2D NMR (HMBC, HMQC), FTIR, HRMS and HPLC. The anticancer activities, cytotoxic effects as well as anti-trypanosomal activities of these novel glycosidic gossypols were explored and suggest that gossypol glycosides could be used to develop new candidates for anticancer drugs and anti-trypanosomal agents.

Synthesis and biological evaluation of Apogossypolone derivatives as pan-active inhibitors of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins.

Overexpression of antiapoptotic Bcl-2 family proteins is commonly related with tumor maintenance, progression, and chemoresistance. Inhibition of these antiapoptotic proteins is an attractive approach for cancer therapy. Guided by nuclear magnetic resonance (NMR) binding assays, a series of 5,5' substituted compound 6a (Apogossypolone) derivatives was synthesized and identified pan-active antagonists of antiapoptotic Bcl-2 family proteins, with binding potency in the low micromolar to nanomolar range. Compound 6f inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, and Mcl-1 with IC(50) values of 3.10, 3.12, and 2.05 µM, respectively. In a cellular assay, 6f potently inhibits cell growth in several human cancer cell lines in a dose-dependent manner. Compound 6f further displays in vivo efficacy in transgenic mice and demonstrated superior single-agent antitumor efficacy in a PPC-1 mouse xenograft model. Together with its negligible toxicity, compound 6f represents a promising drug lead for the development of novel apoptosis-based therapies for cancer.

A novel water-soluble gossypol derivative increases chemotherapeutic sensitivity and promotes growth inhibition in colon cancer.

Compound 1 ((-)-gossypol) has been long known as a chemical anticancer agent. With its low water solubility and toxicity, it is not widely used as a commercial drug. To overcome these disadvantages, several novel derivatives of gossypol were designed, synthesized, and analyzed. One of the derivatives, compound 7 (6-aminopenicillanic acid sodium-gossypolone), was identified with great water solubility and anticancer property, suggested by inducing a dramatically decrease in Bcl-2 and Bcl-xL protein expression level found in vitro and growth inhibition of murine colon tumor in vivo. Furthermore, it was also recognized with less toxicity than compound 1 in vivo and significantly increased chemotherapeutic sensitivity against colon cancer in combination with traditional chemotherapeutic agent 5-fluorouracil. Therefore, it is concluded that compound 7 is superior to parent compound 1, and further preclinical studies of compound 7 is necessary for colon cancer therapy.

[Synthesis, structures, and acute toxicity of gossypol nonsymmetrical aldehyde derivatives].

Nonsymmetrical aldehyde derivatives of gossypol, a yellow polyphenolic pigment of cottonseed, were synthesized by reactions with ammonia, aniline, 4-aminoantipyrine, and barbituric acid. Their structures were determined by UV spectrophotometry and IR and (1)H NMR spectroscopy methods. Their acute toxicities in white mice were compared with those of gossypol and the corresponding symmetrical analogues. It was demonstrated that in general, the fewer free aldehyde groups that contained the gossypol derivative, the lower its acute toxicity. Only in the case of a nonsymmetrical gossypol derivative bearing a 4-aminoantipyrine residue did we observe a deviation from the above correlation: its symmetrical counterpart was even more toxic, but still less toxic than gossypol.

BI-97C1, an optically pure Apogossypol derivative as pan-active inhibitor of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2) family proteins.

In our continued attempts to identify novel and effective pan-Bcl-2 antagonists, we have recently reported a series of compound 2 (Apogossypol) derivatives, resulting in the chiral compound 4 (8r). We report here the synthesis and evaluation on its optically pure individual isomers. Compound 11 (BI-97C1), the most potent diastereoisomer of compound 4, inhibits the binding of BH3 peptides to Bcl-X(L), Bcl-2, Mcl-1, and Bfl-1 with IC(50) values of 0.31, 0.32, 0.20, and 0.62 microM, respectively. The compound also potently inhibits cell growth of human prostate cancer, lung cancer, and lymphoma cell lines with EC(50) values of 0.13, 0.56, and 0.049 microM, respectively, and shows little cytotoxicity against bax(-/-)bak(-/-) cells. Compound 11 displays in vivo efficacy in transgenic mice models and also demonstrated superior single-agent antitumor efficacy in a prostate cancer mouse xenograft model. Therefore, compound 11 represents a potential drug lead for the development of novel apoptosis-based therapies against cancer.

Synthesis, crystal structures and antibacterial activity studies of aza-derivatives of phytoalexin from cotton plant--gossypol.

Using Gossypol (GOS) extracted from cotton seeds, a series of its Schiff bases (1-18) and hydrazones (19-27) have been synthesized and evaluated for their antimicrobial activities against Gram-positive bacteria, Gram-negative bacteria and yeast-like organisms. Of the 27 aza-derivatives of gossypol, 11 have been found active against the microorganisms tested, similar to gossypol. Crystal structures of the new Schiff base (compound 7) and hydrazone (compound 25) of gossypol reveal the presence of two different tautomers within two types of the aza-derivatives studied. The newly synthesized aza-derivatives of gossypol are characterized by the FT-IR, NMR and MS methods.

Apogossypol derivatives as antagonists of antiapoptotic Bcl-2 family proteins.

Guided by a combination of nuclear magnetic resonance binding assays and computational docking studies, we synthesized a library of 5,5' substituted Apogossypol derivatives as potent Bcl-XL antagonists. Each compound was subsequently tested for its ability to inhibit Bcl-XL in an in vitro fluorescence polarization competition assay and exert single-agent proapoptotic activity in human cancer cell lines. The most potent compound BI79D10 binds to Bcl-XL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively, and potently inhibits cell growth in the H460 human lung cancer cell line with an EC50 value of 680 nmol/L, expressing high levels of Bcl-2. BI79D10 also effectively induces apoptosis of the RS11846 human lymphoma cell line in a dose-dependent manner and shows little cytotoxicity against bax-/-bak-/- mouse embryonic fibroblast cells, in which antiapoptotic Bcl-2 family proteins lack a cytoprotective phenotype, implying that BI79D10 has little off-target effects. BI79D10 displays in vivo efficacy in transgenic mice, in which Bcl-2 is overexpressed in splenic B cells. Together with its improved plasma and microsomal stability relative to Apogossypol, BI79D10 represents a lead compound for the development of novel apoptosis-based therapies for cancer.

Design and synthesis of a gossypol derivative with improved antitumor activities.

A novel chemical process has been devised for the synthesis of a new derivative of gossypol, 6,7,6',7'-tetrahydroxy-5,5'-diisopropyl-3,3'-dimethyl-[2,2']binaphthalenyl-1,4,1',4'-tetraone (Apogossypolone). This new process has only four steps, with a shorter synthesis span, a simple purification process, and improved yield and quality. The structure of apogossypolone was characterized by( 1)H-nuclear magnetic resonance, (13)C-nuclear magnetic resonance, mass spectroscopy, infrared spectroscopy, and elemental analysis. Cell-cytotoxicity assay demonstrates that apogossypolone is three- to six-fold more potent than the parent compound, (-)-gossypol, in inhibiting the human prostate tumor cell lines PC-3 and DU-145 as well as the human breast cancer cell line MDA-MB-231. The colony-formation assay with DU-145 cells showed that apogossypolone inhibited more than 70% of colony formation at 1 muM, whereas (-)-gossypol at 10 muM only inhibited less than 50% of colony formation. The results indicate that apogossypolone exerts strong antitumor activities in human prostate and breast cancer cells, and thus represents a promising cancer therapeutic.

Antifungal activity of alkyl and heterocyclic aza-derivatives of gossypol as well as their complexes with NaClO4 against Fusarium oxysporum f. sp. lupini.

Eight alkyl and six heterocyclic aza-derivatives of gossypol (2-15) have been synthesized using gossypol (1) extracted from Gossypium Herbaceum cottonseeds. The ability of gossypol aza-derivatives to form complexes with NaClO(4) has been investigated by electrospray ionisation (ESI) mass spectra recorded in the positive and negative ion detection modes. The gossypol aza-derivatives have been characterized by FT-IR, (1)H and (13)C NMR spectroscopic methods and subsequently tested for their antifungal properties against Fusarium oxysporum. Four alkyl aza-derivatives (2-5), present in the enamine-enamine tautomeric form, have shown activity comparable or higher than that of gossypol against this fungus. To improve the antifungal activity the complexes of the most active compounds 2-5 with NaClO(4) were prepared. Complexes of 2 and 5 with NaClO(4) have shown antifungal activity higher than that of the uncomplexed compounds.

Synthesis and evaluation of Apogossypol atropisomers as potential Bcl-xL antagonists.

Anti-apoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-X(L) have been recently validated as targets for the discovery of novel anti-cancer agents. We previously reported that racemic (+/-) Apogossypol, a semi-synthetic compound derived from the natural product Gossypol, binds and inhibits Bcl-2 and Bcl-X(L)in vitro and in cell. Given that (+) and (-) Gossypol display different proapoptotic activities, here we report on the synthesis of (+) and (-) Apogossypol and the evaluation of their in vitro and cellular activity.

Structure-based design of potent small-molecule inhibitors of anti-apoptotic Bcl-2 proteins.

A structure-based approach was employed to design a new class of small-molecule inhibitors of Bcl-2. The most potent compound 5 (TW-37) binds to Bcl-2 with a K(i) value of 290 nM and also to Bcl-xL and Mcl-1 with high affinities. Compound 5 potently inhibits cell growth in PC-3 prostate cancer cells with an IC(50) value of 200 nM and effectively induces apoptosis in a dose-dependent manner.

Synthesis of gossypol atropisomers and derivatives and evaluation of their anti-proliferative and anti-oxidant activity.

Gossypol 1, gossypolone 2, and a series of bis 3 and half Schiff's bases 4 of gossypol were synthesised and tested for anti-proliferative and anti-oxidant activity. (-)-Gossypol (-)-1 was the most potent inhibitor of the proliferation of the HPV-16 keratinocyte cell line (using an MTT viability assay) with a GI50 of 4.8 microM. The bis Schiff's base of (-)-gossypol with L-tyrosine ethyl ester (-)-3b was the most potent inhibitor of iron/ascorbate dependent lipid peroxidation (using the thiobarbituric acid test), with an IC50 of 11.7 microM, with (-)-gossypol being the next most potent of the series, with an IC50 of 13.1 microM. The results from these initial assays suggest that gossypol, as either a racemic mixture rac-1, or the individual atropisomers (-)-1 or (+)-1, has potential for the treatment of psoriasis.

Rational design and real time, in-cell detection of the proapoptotic activity of a novel compound targeting Bcl-X(L).

Antiapoptotic Bcl-2-family proteins Bcl-2 and Bcl-X(L) have been recently validated as drug discovery targets for cancer. Here, by using a combination of molecular modeling, NMR-based structural analysis, fluorescence polarization assays, and cell-based assays, we have designed and characterized a novel proapoptotic compound targeting these proteins. Our compound, Apogossypol, is capable of binding and inhibiting Bcl-2 and Bcl-X(L) with high affinity and induces apoptosis of tumor cell lines. Mechanistic studies on the action of our compound were also performed via confocal microscopy that provided real-time detection of the interaction with Bcl-X(L) in intact cells. Finally, preliminary data on cells freshly isolated from patients affected by chronic lymphocytic leukemia strongly suggest potential applications of Bcl-2 antagonists as chemosensitizers in cancer therapy.