Ritterostatin GN 1N , a Cephalostatin-Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78.

Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine-cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine-cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin GN 1N , a cephalostatin-ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.

Quantitative analysis of F-actin redistribution in astrocytoma cells treated with candidate pharmaceuticals.

Actin fibers (F-actin) control the shape and internal organization of cells, and generate force. It has been long appreciated that these functions are tightly coupled, and in some cases drive cell behavior and cell fate. The distribution and dynamics of F-actin is different in cancer versus normal cells and in response to small molecules, including actin-targeting natural products and anticancer drugs. Therefore, quantifying actin structural changes from high resolution fluorescence micrographs is necessary for further understanding actin cytoskeleton dynamics and phenotypic consequences of drug interactions on cells. We applied an artificial neural network algorithm, which used image intensity and anisotropy measurements, to quantitatively classify F-actin subcellular features into actin along the edges of cells, actin at the protrusions of cells, internal fibers and punctate signals. The algorithm measured significant increase in F-actin at cell edges with concomitant decrease in internal punctate actin in astrocytoma cells lacking functional neurofibromin and p53 when treated with three structurally-distinct anticancer small molecules: OSW1, Schweinfurthin A (SA) and a synthetic marine compound 23'-dehydroxycephalostatin 1. Distinctly different changes were measured in cells treated with the actin inhibitor cytochalasin B. These measurements support published reports that SA acts on F-actin in NF1(-/-) neurofibromin deficient cancer cells through changes in Rho signaling. Quantitative pattern analysis of cells has wide applications for understanding mechanisms of small molecules, because many anti-cancer drugs directly or indirectly target cytoskeletal proteins. Furthermore, quantitative information about the actin cytoskeleton may make it possible to further understand cell fate decisions using mathematically testable models.

A convergent total synthesis of the potent cephalostatin/ritterazine hybrid -25-epi ritterostatin GN1N.

The convergent synthesis of 25-epi ritterostatin GN1N is described for the first time, starting from hecogenin acetate (HA). Stereoselective dihydroxylation employing the chiral ligand (DHQ)2PHAL was used as the key step to introduce the C25 epi-stereocenter on the north 1 segment. The title compound was obtained through a coupling reaction between the C3-keto-azide (cstat North 1) and North G.

Fluoride-mediated elimination of allyl sulfones: application to the synthesis of a 2,4-dimethyl-A-ring vitamin D3 analogue.

A coupling strategy for the synthesis of 2,4-dimethyl-1α,25(OH)(2)D(3) is achieved which involves methylation of a pro-A ring vinyl sulfone and in situ traping of the allyl sulfonyl anion with a CD ring allyl chloride. TBAF-promoted 1,2-eliminative desulfonylation and concomitant silyl ether deprotection gives the vitamin D(3) analogue.

Studies on the synthesis of apoptolidin: synthesis of a C1-C27 fragment of apoptolidin D.

Synthesis of a C(1)-C(27) fragment, a key intermediate in the synthesis of apoptolidin D, is reported. The synthesis involves a combination of Heck coupling and Horner-Wadsworth-Emmons reaction for the C(1)-C(7) trienoate portion and an efficient Suzuki cross-coupling protocol for the C(10)-C(13) diene portion.

Reagent-directed allylic quadraselection. Chemoselective anti- and syn-Lawton S(N)2' methylation of seven-membered epoxyvinylsulfones.

Methods have been developed for regio- and stereoselective 1,4-syn or 1,4-anti methylation of seven-membered epoxyvinylsulfones. 1,4-Syn addition is achieved via the combination of Me(2)Zn and catalytic Li(2)CuCl(4), a hitherto unexplored reagent combination. The complementary 1,4-anti addition relies on Cu(I) catalyzed methyl Grignard addition or (CH(3))(3)Al assisted CH(3)Cu addition. The methods described were assayed on four diastereomeric stereodiads and on their parent epoxide.

Lactol-directed osmylation. Stereodivergent synthesis of four C-19,20 apoptolidin diols from a single allylic hemiacetal.

A synthetic approach to prepare four Apoptolidin C-19,20 diastereomeric diol derivatives was developed. Two diastereomers were obtained from the (Z)-form, which is converted to the (E)-form, followed by dihydroxylation to deliver two more diastereomers. The (E)-allylic hemiacetal and methoxyacetal showed opposite diastereoselectivity.

Antitumor agent 25-epi Ritterostatin GN1N induces endoplasmic reticulum stress and autophagy mediated cell death in melanoma cells.

Metastatic melanoma is the most aggressive of all skin cancers and is associated with poor prognosis owing to lack of effective treatments. 25-epi Ritterostatin GN1N is a novel antitumor agent with yet undefined mechanisms of action. We sought to delineate the antitumor mechanisms of 25-epi Ritterostatin GN1N in melanoma cells to determine the potential of this compound as a treatment for melanoma. Activation of the endoplasmic reticulum (ER) stress protein glucose-regulated protein 78 (GRP78) has been associated with increased melanoma progression, oncogenic signaling, drug resistance, and suppression of cell death. We found that 25-epi Ritterostatin GN1N induced cell death in melanoma cells at nanomolar concentrations, and this cell death was characterized by inhibition of GRP78 expression, increased expression of the ER stress marker CHOP, loss of mitochondrial membrane potential, and lipidation of the autophagy marker protein LC3B. Importantly, normal melanocytes exhibited limited sensitivity to 25-epi Ritterostatin GN1N. Subsequent in vivo results demonstrated that 25-epi Ritterostatin GN1N reduced melanoma growth in mouse tumor xenografts and did not affect body weight, suggesting minimal toxicity. In summary, our findings indicate that 25-epi Ritterostatin GN1N causes ER stress and massive autophagy, leading to collapse of mitochondrial membrane potential and cell death in melanoma cells, with minimal effects in normal melanocytes. Thus, 25-epi Ritterostatin GN1N is a promising anticancer agent that warrants further investigation.

An efficient C-H oxidation protocol for alpha-hydroxylation of cyclic steroidal ethers.

[reaction: see text] Various C-16 hydroxy steroids have been prepared with the aid of CrO(3)/Bu(4)NIO(4). Out of the two possible reaction courses, transition state B is favored because of less steric interference between substrate and CrO(4). Thus, C-H bonds at C-16 are oxidized selectively.

Efficient protocol for ring opening of spiroketals using trifluoroacetyl trifluoromethanesulfonate (TFAT).

[reaction: see text] Ring opening of steroidal spiroketals under exceptionally mild conditions is smoothly achieved via reaction with trifluoroacetyl trifluoromethanesulfonate (TFAT). The new spiroketal ring-opening protocol provides omega-trifluoroacetyl vinyl ethers in good yield and avoids difficulties that attended previously employed vigorous reaction conditions.

Polyphosphoric acid trimethylsilyl ester promoted intramolecular acylation of an olefin by a carboxylic acid: convenient construction of C-18-functionalized delta14-hecogenin acetate.

[reaction: see text]. Polyphosphoric acid trimethylsilyl ester (PPSE)-promoted intramolecular Friedel-Crafts reactions on a nonaromatic carboxylic acid system have been investigated. Studies led to the synthesis of C-18 functionalized steroidal compounds 5 and 9a-d with strict retention of the spiroketals. Isomerization of spiroketal 9e was studied.

New oxidative tools for the functionalization of the cephalostatin north 1 hemisphere.

Dimethyldioxirane (DMDO) C-H oxidation of ketone 17 to hemiketal 18 (82%), bis-dehydration to vinyl ether 21 (77%), and DMDO again provides C-23 axial alcohol 23 (99%). Routine processing, including a double-stereoselective Sharpless AD reaction (de >98%), gives alcohols 7 and 32. C-23 deoxy substrate 7 undergoes Suarez hypoiodite oxidative cyclization to (natural) beta spiroketal 34, but compound 32, bearing a C-23 silyl ether, generates unnatural spiroketal 33. [reaction: see text]

An efficient synthesis of the C-23 deoxy, 17 alpha-hydroxy South 1 hemisphere and its cephalostatin 1 analog.

[reaction: see text] Methods for functionalization of the D ring of diene 1 were investigated. This study led to efficient syntheses of 3 and the subsequent 23'-deoxy,17'alpha-hydroxy cephalostatin 1 analog (4). The bioactivity data of 2 and 4 are in the low nanomolar range for a 10-cell-line minipanel.

A new protocol for in situ dioxirane reactions: stoichiometric in oxone and catalytic in fluorinated acetophenones.

[reaction: see text] Dioxiranes made in situ from the commercially available tetrafluoroacetophenones (7, 8) and pentafluoroacetophenone (9) are reported for highly efficient epoxidation of olefins for the first time. Studies showed that ketone 7, 8, or 9 can be used in catalytic amount (0.2 equiv) with only 0.6 equiv of Oxone (equal to 1.2 equiv of peroxymonosulfate) to selectively oxidize diene 1 to epoxide 2. The epoxidation reactions of dioxiranes of fluoroacetophenones are compared with the recently described complementary aliphatic acyclic fluorinated ketones.

The first total synthesis of (corrected) ritterazine M.

Hecogenin acetate was converted to ritterazine M in 16 operations with an average yield per opearation of 87%. The overall linear yield was 12%. This confirmed 1 as the corrected structure for ritterazine M by total synthesis.

Synthesis of termini-differentiated 6-carbon stereotetrads: an alkylative oxidation strategy for preparation of the c21-c26 segment of apoptolidin(1).

[reaction: see text] Two methods have been developed for the synthesis of epoxide 36. The first uses (+)-pulegone 25 as an enantiopure starting material and introduces the requisite intricacy of target 22 in 12 operations. The second method employs an enantiospecific catalytic Jacobsen epoxidation of 1a and is five operations shorter. The second sequence features an oxygen-directed alkylative oxidation reaction that re-establishes the dienyl sulfone functionality with concomitant 1,3-transposition of the sulfone moiety.

Redox refunctionalization of steroid spiroketals. Structure correction of ritterazine M.

The structure of the North spiroketal moiety of ritterazine M has been corrected from 1a to 1b. This was accomplished by comparison of published spectra of the natural product with five synthetic spiroketal-alcohols. Synthesis of these models was efficiently accomplished by reductive cleavage of the spiroketal and Sharpless asymmetric dihydroxylation of an isopentyl, methyl 1,1-disubstituted olefin, followed by Suarez iodine[III] oxidative spirocyclization of monoprotected 1 degree,3 degree 1,2 diols.

Intramolecular methylation of an allyl sulfone via lithium alkoxyaluminate; application to the enantioselective synthesis of the CD ring of vitamin D3.

Alcohol-directed intramolecular methylation of an enantiopure allyl sulfone using AlMe(3) provides a trans-hydrindane CD ring alcohol. The substrate cis-CD ring allyl sulfone alcohol is prepared via intramolecular allyl sulfonyl anion addition to aldehyde using Ba(OH)(2).