Robert Michael Williams - US grants

The objective of the proposed research is the total synthesis of the novel antibiotic bicyclomycin in optically active form and of select bicyclomycin analogs to be used in evaluating the chemical mechanism of antibacterial action of bicyclomycin. New synthetic methods for constructing the bicyclomycin ring system will be investigated. The synthetic strategem is of sufficient bravity and versatility to allow the elaboration of bicyclomycin and all synthetic analogs from a few common intermediates. By these methods, the chemical structural and dynamic properties of the bicyclomycin ring system as a "latent" Alpha,Beta-unsaturated pryuvamide (Michael acceptor) will be investigated.

Amino acids are the building blocks of proteins, and thus they, and their chemistry, occupy an important position in biochemistry. Nature is able to efficiently make amino acids in a highly specific fashion because it employs enzymes. However, many amino acids of potential use are not synthesized by nature, and so the chemist is in need of tools to make these compounds with the same specificity as found in nature. This project in the Organic and Macromolecular Chemistry Program is developing such reactions for which the chemistry community will have great use. The overall objective of the proposed research is to develop efficient and practical methodology for the asymmetric synthesis of alpha-amino acids based on carbon-carbon bond constructions of electrophilic glycine-derived templates. In addition, the free radical and electrocyclic couplings to these glycine-templates will be investigated. On the practical side, access to either D- or L- configured amino acids in high optical purity and chemical purity will be the primary utility of this research. These selective coupling reactions will be achieved by a careful study of the fundamental chemistry of hemi-amino acetals as substrates for cationic and radical reactions with a variety of organometallic reagents. A new organotin acetylide coupling reaction has been discovered and will be investigated as a means for preparing vinyl and alkynyl alpha-amino acids which have potent biological activities. Several second-generation chiral auxilliaries will be investigated to broaden the scope of the electrophilic glycine template concept. The new methodology will be demonstrated with total syntheses of several representative, complex amino acids.

The chemical synthesis, mechanistic and biological evaluation of several clinically useful and/or biomedically important natural products is proposed. A mechanism of action for the recently introduced antibiotic bicyclomycin, is proposed. New synthetic methods are being developed to prepare bicyclomycin analogs that will be used to test these hypotheses. An interface of synthetic organic chemistry, mechanistic chemistry and protein (enzyme) chemistry will be used to conduct the bicyclomycin investigation. In addition, new synthetic methods to construct the [3.1.1] bicyclic acetal ring system of TXA2 using polymer-supported organometallic reagents will be studied. The chemical properties of this ring system will be eluciated in detail. Novel syntheses of C-nucleoside antibiotics such as showdomycin will be developed. Other synthetic programs related to Beta-lactam antibiotics such as thienamycin and antifungal antibiotics represented by the novel ansa piperazinomycin are being pursued.

The objective of the proposed research is to develop a unique and mild synthetic technique to construct the labile [3.1.1.] bicyclic acetal ring system found in thromboxane A2 (TXA2). Thromboxane A2 is an unstable (t1/2 30-40 s at 37C) major metabolite of the arachadonic acid cascade in human platelets with potent thrombotic and vasoconstricting properties; the structure proposed has not been rigorously verified. This proposal outlines a mild and practical method for the overall dehydrative ring closure of 1,3-dihydroxypyronosides to the corresponding bicyclic [3.1.1.] acetals using polymer-supported reagents. In this way, the labile products can be cleanly recovered from the polymer-bound reaction by-products without a chromatographic separation. The present methodology is readily adaptable to the conversion of TXB2 (the stable hydrolysis product of TXA2) into TXA2. This shall provide a non-aqueous preparation of TXA2 for structure determination and that of simpler analogs for biological investigation.

This proposal is a request for funding to study the total synthesis of the recently discovered antitumor antibiotic quinocarcin (1,DC-52). Being the simplest member of the saframycin/naphthyridinomycin class of antibiotics, the synthetic protocol developed will focus on the asymmetric preparation of the isoquinoline nuclei and the bicyclic piperazines such that synthetic entries to naphthyridinomycin will be realized. It is the purpose of this proposal to establish the functional significance of the bicyclic piperazine moiety in imparting chemical stability manifested as biological reactivity in the DC-52 molecule. A mechanism of action is proposed and synthetic protocol will be established to probe both the covalent modification of DNA as well as the reported oxygen-dependent DNA strand scission by DC-52.

biomedical equipment purchase;

The overall objective of the proposed research is to develop efficient and practical methodology for the asymmetric synthesis of alpha-amino acids based on carbon-carbon bond constructions of electrophilic glycine-derived templates. In addition, the free radical and electrocyclic couplings to these glycine-templates will be investigated. On the practical side, access to either D- or L- configured amino acids in high optical purity and chemical purity will be the primary utility of this research. These selective coupling reactions will be achieved by a careful study of the fundamental chemistry of hemi-amino acetals as substrates for cationic and radical reactions with a variety of organometallic reagents. A new organotin acetylide coupling reaction has been discovered and will be investigated as a means for preparing vinyl and alkynyl alpha-amino acids which have potent biological activities. Several second-generation chiral auxiliaries will be investigated to broaden the scope of the electrophilic glycine template concept. The new methodology will be demonstrated with total syntheses of several representative, complex amino acids.

This proposal is a continuing request to study cyclization reactions leading to the formation of medium and large rings (8-16 members) by the generation of a new carbon-carbon bond. The palladium catalyzed coupling reaction of an organic electrophile with an organotin group is an ideal reaction for this construction since it is a fast, high yield reaction that takes place stereospecifically under mild conditions and tolerates a wide variety of functional groups. Thus a prime objective of this research is the development of the intramolecular cyclization of a molecule containing an organic electrophile and a vinyltin unit at the termini. A knowledge of the phosphine type as well as the preferred solvent is critical to designing a catalyst supported on a highly cross-linked polymer, the purpose of which is to optimize the intramolecular reaction and reduce the intermolecular oligomerization. Thus, another goal is the rational design of a polymer supported catalyst. It is also the purpose of this research to determine which electrophilic types and organotin partners will participate in the coupling to give high yields of cyclized products. Broadening the scope of the cyclization reaction, particularly to the coupling reactions of vinyl electrophiles with organostannanes, will permit the synthesis of a wider variety of products. Finally, a key objective is the utilization of these cyclization procedures in the synthesis of a variety of biologically active ring compounds. In particular the ring systems of the polyoxo-macrolides produced by streptomyces microorganisms such as methynolide (12-membered), ingramycin (14-membered), erythronolides and mycinolides, tylonolides, or carbonolides (16-membered) as well as the ring systems of the beta-resorcyclic acid macrolide group such as zearalenone (14-membered) or milbemycin beta3 are potential targets. The synthesis of an important new class of antitumor antibiotics containing the conjugated enediyne chromaphore to which neocarzinostatin, calichemicin and esperamicins belong is especially attractive, since these molecules are highly functionalized yet are unstable, particularly in the presence of nucleophiles.

This award is being made by the Chemistry Division to enable the participation of 13 younger U.S. scientists in the Bilateral NSF- SERC Joint Workshop on Asymmetric Synthesis which will be held at the Pingree Park facility of Colorado State University, Pingree Park, Colorado, July 3-8, 1990. They will participate in the Workshop with 12 young scientists from Great Britain. The purposes of the workshop are (1) to expose them to the broader community of international scholarship, and (2) to build long-term bridges for productive international researh collaboration and knowledge exchange. Asymmetric synthesis has become an area of intensive research interest and significance in chemistry. The strict demands placed on the chemical and optical purity of new marketable drugs makes the discovery and implementation of new and efficient asymmetric chemical constructions a primary concern in chemistry.

This proposal is a continuing request to study the biomechanism of the anti-tumor antibiotic Quinocarcin (DC-52). Quinocarcin has been shown to effect oxygen-dependent lesions in DNA by a novel, and as yet, unknown mechanism. The primary objective of this program is to elucidate the mechanism of this process, and develop a structural hypothesis embracing this reaction that could be extended to other systems. Related to this investigation is the objective of sorting out whether quinocarcin and related anti-tumor antibiotics have multiple modes of action such as sequence-specific binding and/or intercalation; alkylation of DNA; and oxidative scission of DNA. We propose to study and elucidate the sequence specificity (if any) of the interaction of quinocarcin with DNA and to further elucidate which type of interaction is responsible for cytotoxicity. We have rigorously established that pure quinocarcin plus oxygen alone can effect the scission of DNA that is independent of added metal ions or reducing agents; these results confirm earlier reports by Tomita, et. al. It is proposed that the labile and crucial oxazolidine moiety is the reductant capable of producing oxygen and/or hydroxyl free radicals and is also likely responsible for alkylation of DNA. Experiments will be aimed at elucidating the mechanistic details of this novel reaction and to determine the significance of this reaction to the anti-tumor properties of the drug. A second, and unrelated (to the above) portion of this proposal is a continuing request to study the total synthesis and biogenesis of the natural mycotoxins, paraherquamide and marcfortines A-C. It has recently been learned that paraherquamide displays potent anti-parasitic properties and has been the subject of intensive investigation at Merck. The purpose of this investigation is to prepare that natural products, establish their biogenetic pathways and synthesize both hypothetical shunt metabolites as well as analogs to develop a structure-activity profile of this complex class of alkaloids.

This award will support the participation of six U.S. scientists in a joint seminar on "Selectivity in Synthetic and Bio-organic Chemistry," to be held in Tokyo, Japan, June 2-7, 1991. The focus of the meetings will be on the dynamic scientific advances in the area of asymmetric synthesis, which spans the entire spectrum of organic and inorganic chemistry. A U.S.-Japan seminar on this subject in 1981 served as an opportunity for research groups in the two countries to share ideas and information. During the last decade, however, there has been a virtual explosion in the discovery of reactions that deliver levels of stereocontrol once thought to be impossible to achieve via non-enzymatic means. The impact of these new tools, coupled with the new enzymatic and microbial technologies, has dramatically redefined the way absolute stereochemical, regiochemical and functional group selectivity issues in synthesis are being addressed in both academic and industrial environments. A multitude of new chiral reagents and catalysts now exist that are capable of exerting near-perfect control over those bond constructions where new stereochemical relationships are established. The development of these new technologies are based on recent, deep understanding of fundamental issues of selectivity and molecular recognition. The enormous importance of this area is manifested in the highly international and cross-disciplinary nature of the primary contributions in the literature. The co-organizers of the seminar are Professor Robert M. Williams of Colorado State Unversity and Professor Kenji Koga of the University of Tokyo. The main topics to be discussed are new asymmetric synthetic methodologies; enzymatic, whole cell and cell-free bio-reactor technologies; total synthesis of natural products; biosynthesis; catalytic antibodies; and reaction mechanisms. In addition, a poster session will be organized for young Japanese industrial chemists.

The objective of this program is to synthesize and study the mechanism of action of the recently discovered anti-tumor antibiotic FR-900482. This substance is a natural product isolated from the fermentation extracts of Streptomyces sandaensis No. 6897 at Fujisawa Pharmaceutical Co. in Japan. FR-900482 and its derived triacetate FK973 display potent cytotoxic and anti-tumor activity against murine and human tumors in vitro and in vivo. Structurally related to the well-known mitomycin C anti-tumor antibiotic, these new compounds are exciting due to their unique, demonstrated ability to cross-link double-stranded DNA and also form DNA-protein cross-links. Unlike mitomycin C and most other antitumor drugs, FK 973 does not cause oxidative damage to DNA. FK 973, now in advanced clinical trials in Japan, is significantly less toxic than mitomycin C and is three-fold more potent. The purpose of this investigation is to synthesize FR-900482 and some simple analogs to probe the unique mechanism of DNA-DNA and DNA-protein cross-link formation by this important new drug.

Quinocarcin (1) and a newly discovered, related natural product tetrazomine (2), naphthyridinomycin (3) and the recently discovered bioxalomycins (4) are potent anti-tumor antibiotics that have demonstrated potency against a range of human tumor cell lines. Most significantly, quinocarcin has excellent in vivo activity against human mammary MX-1 carcinoma with all tumors cured at i.v. doses of 4.4 mg/kg/day and p.o. doses of 262 mg/kg/day given daily for 7 days. It is the purpose of this program to exploit the mechanistic knowledge that has been uncovered during the first grant cycle in designing and synthesizing more potent and selective anti-tumor antibiotics. The primary hypothesis to be explored concerns separately evaluating, via chemical synthesis, the efficacy of quinocarcin (1), tetrazomine (2), naphthyridinomycin (3) and bioxalomycin (4) derivatives that lack the capacity to cause non-specific oxidative damage to cellular macromolecules but retain the enhanced capacity to alkylate DNA. As with many other anti-tumor antibiotics that display multiple modes of action, quinocarcin has the demonstrated capacity to both aIkylate DNA and, through the slow generation of superoxide, mediate Fenton-type oxidative damage to DNA and other cellular components in a non-specific fashion. It is the generation of the non-specific oxidative species derived from superoxide that is likely responsible for high levels of host toxicity that have made it so difficult for many promising anti- tumor drug candidates to successfully pass phase three human clinical trials and toxicological scrutiny. This program will endeavor to chemically attenuate or obviate an entire mechanistic manifold (superoxide release) exhibited by this class of compounds while at the same time, enhancing a complementary manifold (DNA alkylation). The efficient chemical synthesis of tetrazomine, naphthyridinomycin and the bioxalomycins is proposed as well as efficient chemical syntheses of several mechanism-based analogs of these potent anti-tumor antibiotics.

The primary objective of the proposed research is to study the mechanism of action of clinically significant new anti-tumor FR900482 (1a), FK973 (1b) and FR66979 (1c) (FK973, 1b was the first derivative to go to clinical trials). FK973 has been shown to cross-link double-stranded DNA and cross-link DNA to DNA-binding proteins in L1210 cells. Efforts will be directed at elucidating in complete mechanistic detail, the precise mechanism of the in vitro reductive activation of FR900482 that results in covalent modification of DNA. In the previous funding period, we have determined the sequence specificity of DNA cross-link formation by FR900482 and the natural reduction product FR66979 and have determined that reduction of these substances leads to the production of a mitosene derivative that preferentially cross-links DNA at 5'-dC-dG3' boxes. The interaction of FK973 with DNA complexed to various DNA-binding proteins will also be examined in detail. Synthetic DNA substrates will be constructed and incubated with their respective DNA-binding proteins in the presence of FK793 (or FR900482); subsequent enzymatic digestion of the cross-linked nucleotide-drug-amino acid adduct will be examined to isolate and characterize the structure of the covalent cross-link. Synthetic methodology developed during the first funding cycle will be utilized to complete a total synthesis of FR900482 (1a) and FR66979 (2); this technology will be applied to the synthesis of an isotopically-labeled form of the drug for use in elucidating the structure of the DNA-drug- protein cross-link. A new class of "latent" triggered mitosenes will be synthesized and utilized as potential new anti-cancer drugs and probes for the macromolecular cross-links.

biosynthesis; drug design /synthesis /production; indoles; paclitaxel; alkaloids; primary aminoacid; plant physiology; cyclization; chemical addition; stereochemistry; Diels Alder reaction; tissue /cell culture; Conifera;

The focus of this research is the development of synthetic techniques for the preparation of alpha-amino acids. The main classes of methodology to be explored include: 1) allyl and crotyl silane/stannane additions to oxazinal-based oxonium ions to access hydroxymethylene peptide isostere; 2) an asymmetric (1,3) dipolar cycloaddition approach to spirotryprostatin; 3) unsymmetrically beta, beta-disubstituted alpha-amino acids via inter- and intramolecular SN2' reactions and via radical additions to E- and Z-alpha, beta-dehydro oxazinones; 4) preparation of Z-beta, gamma-unsaturated-alpha-amino acids and Z-1-aminocyclopropanecarboxylic acids. With this renewal award, the Organic and Macromolecular Chemistry Program is supporting the research of Dr. Robert M. Williams of the Department of Chemistry at Colorado State University. Professor Williams will focus his work on developing efficient, selective and practical methodology for the construction of biologically important alpha-amino acids and their derivatives in optically pure form.

The SIR provided [1_13C, 90%proline; . 15g [1_13C, 98%]tryp; .35g [U_13C, ?9]isoleucine;. 159g The paraherquamides are complex, heptacyclic, toxic mold metabolites, isolated from various Penicillium sp.The brevianamides A and B are structurally related natural products that display potent insecticidal activity. The brevianamides are also produced by Penicillium sp. molds. We are pursuing the identification of a new mechanistic class of enzymes that are involved in constructing the bicyclo [2.2.21 nucleus of the brevianamides/paraherquamides. We have obtained compelling experimental evidence that this unique ring system is very possible constructed via an enzyme-catalyzed intramolecular Diels-Alder cycloaddition reaction. Despite its widespread use in synthetic organic chemistry, the Diels-Alder cycloaddition reaction does not occur frequently in nature and there is not a single documented example of an enzyme that catalyzes this most ubiquitous synthetic ring-forming reaction. The Penicillium sp. that produces the brevianamides/paraherquamides may be a rare, but vitally important example of the existence of Diels-Alderases. One of the most significant objectives of this project is to characterize this rare catalyzed, biosynthetic construction. Using amino acids provided to us by the SIR, we have established that the 0-methyl proline ring in paraherquamide is derived from L-He and not via the SAM-methylation of L-proline. In addition , we have recently shown that L-tryptophan is the biosynthetic precursor to the dioxepin oxindole half of paraherquamide and that the N-methyl group is derived from L-Met. We have previously synthesized the tritiated piperazinedione and have demonstrated that this substance is efficiently incorporated into brevianamides A and B. Our current efforts are directed toward synthesizing the P-methyloproline derivatives containing 13C-labels for biosynthetic feeding experiments. We wish to establish the exact pathway by which the L-Ile is converted into P-methyloproline and thence, into paraherquamide A.

The goals of this program are to study the interaction of the clinically relevant antitumor antibiotics, including FR900482, FR66979 and several synthetic compounds with DNA interstrand cross-linking ability, with cellular nucleic acids and DNA-binding proteins at the molecular level. The synthesis of members of this class of antitumor drugs will continue to be developed with the objective of harnessing the synthetic methodology developed to make new, less toxic, more selective and more potent antitumor drugs. A new class of antitumor pro-drugs, that can be selectively activated by chemical or photochemical means will be synthesized and their interaction with cellular nucleic acids and DNA-binding proteins will be studied. During the coming funding cycle, we plan to address the following objectives: (1) Synthetic methodology developed during the current funding cycle will be utilized to complete an efficient asymmetric, stereocontrolled total synthesis of FR900482 (1) and FR66979 (2). (2) The synthetic methodology we have developed in the total synthesis endeavor shall be utilized to prepare mitosene progenitors based on the FR900482 structure, that can be triggered by alternative chemical and biochemical means. (3) The interaction of FK973 with DNA complexed to several DNA-binding proteins that associate with DNA in the minor groove will be examined in detail. DNA substrates will be constructed and incubated with the respective peptide binding domains (BD) of the High Mobility Group I/Y (HMG-I/Y) DNA-binding proteins in the presence of FR66979*; subsequent enzymatic digestion of the cross-linked nucleotide-drug-amino acid adduct will be examined to isolate and fully characterize the structure of the covalent cross-link. (4) DNA cross-linking studies with full-length DNA-binding proteins of the High Mobility Group I/Y (HMG-I/Y) will be conducted with several synthetic DNA substrates. (5) In collaboration with Prof. Raymond Reeves (Washington State University), we plan to examine the DNA-protein cross-linking capacity of FR66979 in vivo in neoplastically transformed cells. (6) A new class of "latent" triggerable progenitors of mitosenes, pyrrolizidine alkaloids and substances related to the anthramycins will be synthesized and utilized as potential new anti-cancer drugs and probes for the macromolecular cross-links.

DESCRIPTION: (Principal Investigator's Abstract) The specific aims of this program are to study the interaction of the antitumor antibiotics, including quinocarcin, tetrazomine, the bioxalomycins and ecteinascidin 743 with cellular nucleic acids. The DNA-alkylating capacity of these drugs compared with their ability to cause oxidative damage to nucleic acids will be explored. The synthesis of members of this class of antitumor drugs will continue to be developed with the objective of harnessing the synthetic methodology developed to make new, less toxic, more selective and more potent antitumor drugs. In addition, the tools of synthesis will be exploited to synthesize mechanistic probes for the interaction of these substances with cellular nucleic acids and proteins that bind to cellular nucleic acids. We have recently discovered that bioxalomycin alpha2 specifically cross-links duplex DNA at 5'CG3' steps. We propose to elucidate the exact molecular structure of the covalent adduct. This finding has inspired new design concepts for simpler analogs based on the quinocarcin/bioxalomycin/Et 743 core that may be capable of cross-linking DNA. Coupled to these studies, we propose to examine the cross-linking of DNA to DNA-binding proteins by bioxalomycin, Et 743 and several synthetic hybrids; the proteins of interest are the High Mobility Group (HMG) nonhistone chromosomal proteins that associate with DNA in the minor groove.

[unreadable] DESCRIPTION (provided by applicant): The primary objectives of the proposed research are to study the synthesis and mechanism of action of the clinically significant antitumor drugs FR900482, FR66979, FK973, and FK317 (FK973 was the first derivative to go to clinical trials however, the semi-synthetic derivative FK317 is currently in human clinical trials in Japan). These substances are structurally and mechanistically related to the widely used antitumor drug mitomycin C (MMC). Specific Aims for the forthcoming grant period include the following: 1. Completion of the first asymmetric total synthesis of mitomycin C, mitomycin K and mitomycin B. 2. We plan to study the biosynthesis of FR900482 and mitomycin C in collaboration with Prof. David Sherman's laboratory (University of Michigan). In particular, our laboratory will synthesize isotopically labeled putative biosynthetic intermediates on these pathways as a means for identifying the structure and mechanism of several key steps. 3. In collaboration with Prof. Raymond Reeves (Washington State University), we plan to continue our investigation of several aspects of the cell biology of these antitumor drugs on neoplastically transformed human cells. In particular, we propose to address the following questions: A. What are the relative effects of MMC, FR900482 and FK317 on IL-2 expression? B. What oncogenes and other metabolically important genes are up-regulated or down-regulated by these drugs? 4. In collaboration with Prof. Karolin Luger (Colorado State University) we plan to investigate the cross-linking of nucleosomes by FR900482 and congeners. 5. A new class of "latent" triggerable progenitors of mitosenes, pyrrolizidine alkaloids and substances related to the anthramycins will be synthesized and utilized as potential new anti-cancer drugs and probes for the macromolecular cross-links. 6. The synthetic methodology we have developed in the total synthesis endeavors shall be utilized to prepare mitosene progenitors based on the FR900482 and MMC structures that can be triggered by alternative chemical and biochemical means. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The purpose of this application is to delve into amino acid problems to which poor or less effective methodologies exist. This proposal describes plans to further develop the utility of the oxazinone-based glycine templates developed in our laboratory for the asymmetric synthesis of complex, densely functionalized amino acids and derivatives in optically pure form. The specific targets that have been selected for the upcoming grant cycle have been chosen by the following criteria: (1) the synthetic targets all contain 2-substitution (in some cases, 2,3-polysubstitution) in the amino acid side chain;(2) the synthetic targets are biomedically interesting and important;and (3) methodologies to access these types of substances are, in general, lacking in the literature. During the coming grant period, plans are described to develop new synthetic methodologies necessary to achieve the asymmetric total synthesis of the following natural products: (1) nitrone dipolar cycloaddition reactions as applied to the asymmetric total synthesis of putative metabolically activated derivatives of cylindrospermopsin;(2) development of novel intramolecular azomethine ylid dipolar cycloaddition reactions for application to the asymmetric total synthesis of palau'amine and congeners;(3) diastereoselective aldol and lactone functionalization methods for application to a short, asymmetric total synthesis of quinine featuring a facially selective, Pd-catalyzed intramolecular SN2'cyclization reaction;(4) utilization of novel diastereoselective azomethine ylide dipolar cycloaddition strategies for a concise, asymmetric total synthesis of nakadomarin A and the manzamine alkaloids;(5) manipulation of a facially selective intramolecular SN2'cyclization reactions for the asymmetric total synthesis of spiroquinazoline and alantrypinone;(6) intramolecular Pauson-Khand reactions on functionalized oxazinones to construct tuberostemoninol, a member of the stemona alkaloid family;and (7) Mannich-based approaches to the total synthesis of zetikitoxin and saxitoxin. In each area, biological studies of synthetic analogs of the biologically active natural products will be undertaken. PUBLIC HEALTH RELEVANCE The purpose of this application is to delve into amino acid problems to which poor or less effective methodologies exist. This proposal describes plans to further develop the utility of synthetic technology to construct amino acids for the asymmetric synthesis of complex, densely functionalized amino acids and derivatives in optically pure form. This technology has been utilized extensively by academic and industrial laboratories all over the world to make nitrogen-containing compounds of biomedical relevance.

DESCRIPTION (provided by applicant): The primary focus of this application is the total synthesis of complex, biomedically significant natural products constituted of tetrahydroisoquinolines. The synthetic chemistry that will be developed shall be utilized to prepare analogues of the natural substances as biological and mechanistic probes. This proposal is primarily hypothesis-driven, and extensively employs new synthetic methodologies developed in this laboratory for the construction of such agents. The specific aims of this program are to study the interaction of the natural antitumor antibiotics and mechanistically inspired synthetic analogs, including ecteinascidin 743 (Et-743), saframycin A, jorumycin, tetrazomine, lemonomycin, renieramycin H and the bioxalomycins with cellular nucleic acids. The DNA alkylating capacity of these drugs compared with their ability to cause oxidative damage to nucleic acids will be explored. The synthesis of members of this class of antitumor drugs will continue to be developed with the objective of harnessing the synthetic methodology developed to make new, less toxic, more selective and more potent antitumor drugs. In addition, the tools of synthesis will be exploited to synthesize mechanistic probes for the interaction of these substances with cellular nucleic acids and oncoproteins that bind to cellular nucleic acids.

[unreadable] DESCRIPTION (provided by applicant): The Chemistry Department at Colorado State University is requesting funds to purchase a new time-of-flight mass spectrometer (LC/MSD TOF) equipped with a high performance liquid chromatography (HPLC) system. The instrument will be managed within the Department's Central Instrumentation Facility (GIF). At the present time, we do the greatest portion of our mass spectrometry work on two heavily used instruments, one of which is well past its prime. The newer instrument (a ThermoElectron LCQ Duo interfaced to an older Hewlett-Packard 1100 HPLC) operates in our open-access laboratory and is absolutely saturated with users. The other instrument is a thirteen year old Fisons VG AutoSpec set up to perform nominal and accurate mass measurements mostly by Liquid Secondary Ion MS. The new LC/MSD TOF will address the need for more HPLC/MS capacity as well as more accurate mass measurement capacity. We anticipate that for certain routine samples our throughput will increase by three to five times, maybe even more. Most importantly, the process can be automated and set to run during periods of lower instrument demand. The new LC/MSD TOF system will also allow us to measure all ions of interest (impurities, various adducts and fragments) to the required accuracy in only one operation. Finally, many of these measurements will be made in an open access environment. [unreadable] [unreadable]

high performance liquid chromatography; mass spectrometry; university

[unreadable] DESCRIPTION (provided by applicant): The Chemistry Department at Colorado State University is requesting funds to purchase a new 400 MHz NMR spectrometer that will replace our ten year old 300 NMR. The instrument will be managed within the Department's Central Instrumentation Facility (CIF). At the present time, we do the greatest portion of our NMR work on three heavily used instruments, one of which - the subject of this proposal - is well past its prime. This instrument is used for routine NMR studies in a walk-up mode. The new, 400 NMR is needed at this time to replace the aging and technically limited 300 NMR. It offers significantly better sensitivity, resolution and performance compared with the 300 NMR that it replaces. Indeed, the NMR performance is sufficiently good that we expect to do some kinds of NMR, e.g. C and fast 2D, on this new 400 NMR rather than on the more powerful 13 instruments in the CIF as we do at present. Finally, the actively shielded magnet provides for a much smaller overall system footprint and will allow us to make better use of valuable laboratory space. The 400 MHz NMR spectrometer requested provides analytical support to NIH funded projects that discover new synthetic strategies for producing pharmacologically important drug materials efficiently and in high purity. The instrument also supports NIH funded projects that discover new natural products that may be tomorrow's wonder drugs for some of the world's worst diseases including tuberculosis and leprosy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The primary focus of this application is to study the total synthesis and biosynthesis of complex, biomedically significant natural products constituted of tetrahydroisoquinolines. The synthetic chemistry that will be developed shall be utilized to prepare analogues of the natural substances as biological, biosynthetic and mechanistic probes. This proposal is primarily hypothesis-driven, and extensively employs new synthetic methodologies developed in this laboratory for the construction of such agents. The specific aims of this program are to study the interaction of the natural antitumor antibiotics and mechanistically inspired synthetic analogs, including ecteinascidin 743 (Et-743), saframycin, jorumycin, tetrazomine, lemonomycin and the bioxalomycins with cellular nucleic acids. The DNA-alkylating capacity of these drugs compared with their ability to cause oxidative damage to nucleic acids will be explored. The synthesis of members of this class of antitumor drugs will continue to be developed with the objective of harnessing the synthetic methodology developed to make new, less toxic, more selective and more potent antitumor drugs. In addition, the tools of synthesis will be exploited to synthesize mechanistic probes for the interaction of these substances with cellular nucleic acids and proteins that bind to cellular nucleic acids. We have recently discovered that bioxalomycin 12 specifically cross-links duplex DNA at 54dCpG34 steps. We propose to elucidate the exact molecular structure of the covalent adduct. This finding has inspired new design concepts for simpler analogs based on the tetrahydroisoquinoline core that may be capable of alkylating and cross-linking DNA as well as potentially cross-linking DNA to DNA-binding proteins. The antiproliferative activity of this family of alkaloids is intimately associated with the capacity of these agents to bind to and covalently modify DNA. This is particularly evident in the case of Et-743. Plans are presented to mechanistically separate the DNA alkylation and cross-linking chemistry of these agents from their capacity to inflict oxidative damage on cells. The investigation of the biosynthesis of Et-743 with a particular focus on the late-stage assembly of the macrocyclic sulfide-bridged spiro-tetrahydroisoquinoline ring system will be pursued. We plan to apply high throughput genome sequencing methods using 454 technology to identify the Et-743 biosynthetic gene cluster from Ecteinascidia turbinata. The ultimate goals of the biosynthesis studies are to genetically engineer a fermentable microorganism to produce Et-743 for clinical use. PUBLIC HEALTH RELEVANCE: The purpose of this application is to utilize the tools of chemical synthesis to study the molecular details of how Nature biosynthesizes anti-cancer drugs. In particular, this program will deploy new technologies to produce the clinically relevant anticancer drug ecteinascidin 743 (Et-743) on industrially practical scale through genetic engineering of a microbial host. In addition, the chemical technologies being developed will be applied to the design and synthesis of new anti-cancer drugs.

DESCRIPTION (provided by applicant): The primary focus of this application is to develop more potent and less toxic, isoform-selective histone deacetylase (HDAC) inhibitors (HDACi) for use as anti-myeloma and anti-cancer drug candidates, biological tools for investigation of specific functions of individual HDACs and extension of this chemical class beyond antineoplastic indications. Specifically, we plan to optimize the structures of macrocyclic peptide analogues of largazole, and related naturally occurring HDAC inhibitors for HDAC class- selectivity by altering the amino acid sequence and zinc-binding functionality in accord with parameters derived from crystal structures of the target enzymes deploying additional computational and empiric insights. Analogues will be assayed for inhibitory activity against HDACs 1-11 in the laboratories of co- investigator Dr. James E. Bradner at the Broad Institute of Harvard-MIT and the Dana-Farber Cancer Institute and Dr. James R. Berenson at the Institute for Myeloma and Bone Cancer Research. Assessment of the safety and pharmacodynamic effect will be performed by Prof. Douglas Thamm and co- workers at the CSU College of Veterinary Medicine and Biomedical Sciences. Insight from the results of these assays will guide further alterations of the candidate structures. A computational co-investigator, Prof. Olaf Wiest, at the University of Notre Dame, will deploy homology model-based docking studies to further guide the design and optimization of new synthetic HDACi's. We have developed scaleable solution-phase syntheses of the known, highly potent natural HDACi's largazole and FK228 as well as the corresponding peptide isosteres and numerous synthetic analogs of these potent and naturally occurring HDACi's. Structural diversity within the analogs will be explored by varying three parameters: (a) amino acid sequence and constitution;(b) macrocycle size;and (c) zinc-binding arms. Particularly potent and/or isoform-selective macrocyclic peptide inhibitors that are produced by this approach will be targeted for re-synthesis as the corresponding depsipeptide congeners and profiled for potency and specificity in biochemical and cellular assays. Select isoform-specific analogs will be evaluated for in vivo tolerability and antineoplastic activity. PUBLIC HEALTH RELEVANCE: The purpose of this application is to develop new classes of drugs that target a new and exciting biochemical target recently recognized as being essential to the cancer cell cycle. The new target is a class of enzymes that exist inside all cells called histone deacetylase enzymes (HDAC's). Our multidisciplinary project brings together synthetic organic chemistry, computational modeling, chemical biology and clinical evaluation of potential inhibitors of HDAC's.

Project Summary Induction of normal, but developmentally silenced, fetal globin expression reduces anemia and ameliorates clinical severity in the beta hemoglobinopathies. HDACs 1,2, and 3, are components of the NURD repressor complex, which promotes silencing of fetal globin in adult cells. Prior generation HDAC inhibitors have increased fetal globin in patients, but had limitations for pharmaceutical application and required intermittent administration due to anti-proliferative effects. Cetya has generated a library of high potency HDAC inhibitors, which can be readily modulated in chemical structure, and has demonstrated their efficacy in inducing fetal globin induction in normal human erythroid cells, including a select few candidates which do not shown undesirable growth inhibitory effects at fetal globin inducing concentrations. The laboratory of Dr. Perrine at Boston University will evaluate these candidates in progenitor cells cultured from sickle cell patients. Dr. Perrine has extensive experience in developing treatments for sickle cell and beta thalassemia. This collaboration will explore this new high-potency generation in HDAC chemistry to advance a therapeutic class to the clinic. The studies proposed will identify an optimal HDAC inhibitor for subsequent development to an IND. Aim 1 - To evaluate HDAC inhibitors for magnitude of fetal globin induction and anti- proliferative effects in sickle cell erythroid progenitors and identify an optimal candidate for preclinical development. Aim 2 - To increase the production scale of the selected candidate to support testing in IND-required toxicology studies.  

Project Summary The ?-hemoglobinopathies are prevalent genetic blood diseases with few treatment options. It is estimate that 7% of the world's population carries an abnormal hemoglobin gene with 400,000 infants born annually with a severe life threatening hemoglobinopathy. Drug mediated induction of normal, but developmentally silenced, fetal hemoglobin (HbF) expression reduces anemia and ameliorates clinical severity in the ?- hemoglobinopathies. Histone deacetylase (HDAC) 1, 2, and 3, are components of the NURD repressor complex, which promotes silencing of the fetal ?-globin genes in adult erythroid cells. Prior generation HDAC inhibitors increase HbF in patients with ?-hemoglobinopathies, but had limitations for pharmaceutical application and/or required titration to reduce anti-proliferative effects. Cetya has generated a library of high potency HDAC inhibitors, one of which, CT-101, has demonstrated efficacy in inducing HbF expression in ?- thalassemia and sickle cell erythroid progenitors and in the ?-YAC mouse model that contains the normal human ?-globin gene locus,without significant anti-proliferative effects. We will test the central hypothesis that CT-101 has a sufficiently wide margin of activity, without significant inhibition of erythroid cell growth, to be developed as a new agent for treatment of ?-hemoglobinopathies. We propose to conduct studies to evaluate CT-101 in the ?-YAC model to optimize dose and schedule, and then confirm efficacy in the Townes sickle cell disease mouse model. Cetya will scale the CT-101 manufacturing process and produce sufficient quantities to support the murine studies and oral formulation development. The goal of this project is to develop the new high-potency HDAC inhibitor CT- 101 for an IND and clinical therapeutics. To test our hypothesis the following aims will be completed. Aim 1. Test the hypothesis that CT-101 induces HbF expression in the preclinical ?- YAC and Townes sickle cell disease mouse models through epigenetic histone modifications. Aim 2. Scale the manufacturing processes and produce sufficient quantities of CT-101 to conduct IND-enabling studies required for a Phase I clinical study. Aim 3. Develop an oral dosage formulation of CT-101 suitable for human administration. The expected outcome of this Phase 2 project is to develop CT-101 as an oral effective HbF inducer. Our experimental approach rests on the scientifically validated concept of the inhibition of sickle hemoglobin polymerization by HbF and red blood cell sickling by reversing a well-known epigenetic ?-globin gene silencing mechanism. Development of the potent HDAC inhibitor CT-101 will impact the field and addresses an unmet need for additional disease modifying therapy for ?-hemoglobinopathies.