Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

Detection of an interchain carbinolamine cross-link formed in a CpG sequence by the acrolein DNA adduct gamma-OH-1,N( 2)-propano-2'-deoxyguanosine

Spectroscopic evidence is presented for the formation of a carbinolamine interchain cross-link in 5'-CpG-3' sequences, arising from the acrolein adduct gamma-OH-PdG. This may be important in understanding biological processing of acrolein-induced DNA damage in CpG sequences.     

NMR determination of the conformation of a trimethylene interstrand cross-link in an oligodeoxynucleotide duplex containing a 5'-d(GpC) motif

Malondialdehyde interstrand cross-links in DNA show strong preference for 5'-d(CpG) sequences. The cross-links are unstable and a trimethylene cross-link has been used as a surrogate for structural studies. A previous structural study of the 5'-d(CpG) cross-link in the sequence 5'-d(AGGCGCCT), where G is the modified nucleotide, by NMR spectroscopy and molecular dynamics using a simulated annealing protocol showed the guanine residues and the tether lay approximately in a plane such that the trimethylene tether and probably the malondialdehyde tether, as well, could be accommodated without major disruptions of duplex structure [Dooley et al. J. Am Chem. Soc. 2001, 123, 1730-1739]. The trimethylene cross-link has now been studied in a GpC motif using the reverse sequence. The structure lacks the planarity seen with the 5'-d(CpG) sequence and is skewed about the trimethylene cross-link. Melting studies indicate that the trimethylene cross-link is thermodynamically less stable in the GpC motif than in the 5-d(CpG). Furthermore, lack of planarity of the GpC cross-link precludes making an isosteric replacement of the trimethylene tether by malondialdehyde. A similar argument can be used to explain the 5'-d(CpG) preference for interchain cross-linking by acrolein.     

Rearrangement of the (6S,8R,11S) and (6R,8S,11R) exocyclic 1,N2-deoxyguanosine adducts of trans-4-hydroxynonenal to N2-deoxyguanosine cyclic hemiacetal adducts when placed complementary to cytosine in duplex DNA

trans-4-Hydroxynonenal (HNE) is a peroxidation product of omega-6 polyunsaturated fatty acids. The Michael addition of deoxyguanosine to HNE yields four diastereomeric exocyclic 1,N(2)-dG adducts. The corresponding acrolein- and crotonaldehyde-derived exocyclic 1,N(2)-dG adducts undergo ring-opening to N(2)-dG aldehydes, placing the aldehyde functionalities into the minor groove of DNA. The acrolein- and the 6R-crotonaldehyde-derived exocyclic 1,N(2)-dG adducts form interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Only the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry forms interstrand N(2)-dG:N(2)-dG cross-links in the 5'-CpG-3' sequence context. Moreover, as compared to the exocyclic 1,N(2)-dG adducts of acrolein and crotonaldehyde, the cross-linking reaction is slow (Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc. 2003, 125, 5687-5700). Accordingly, the chemistry of the HNE-derived exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry has been compared with that of the (6R,8S,11R) adduct, when incorporated into 5'-d(GCTAGCXAGTCC)-3'.5'-d(GGACTCGCTAGC)-3', containing the 5'-CpG-3' sequence (X = HNE-dG). When placed complementary to dC in this duplex, both adducts open to the corresponding N(2)-dG aldehydic rearrangement products, suggesting that the formation of the interstrand cross-link by the exocyclic 1,N(2)-dG adduct of (6S,8R,11S) stereochemistry, and the lack of cross-link formation by the exocyclic 1,N(2)-dG adduct of (6R,8S,11R) stereochemistry, is not attributable to inability to undergo ring-opening to the aldehydes in duplex DNA. Instead, these aldehydic rearrangement products exist in equilibrium with stereoisomeric cyclic hemiacetals. The latter are the predominant species present at equilibrium. The trans configuration of the HNE H6 and H8 protons is preferred. The presence of these cyclic hemiacetals in duplex DNA is significant as they mask the aldehyde species necessary for interstrand cross-link formation.     

Formation of a N2-dG:N2-dG carbinolamine DNA cross-link by the trans-4-hydroxynonenal-derived (6S,8R,11S) 1,N2-dG adduct

Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.     

Retinoidal pyrimidinecarboxylic acids. Unexpected diaza-substituent effects in retinobenzoic acids

Several pyridine- and pyrimidine-carboxylic acids were synthesized as ligand candidates for retinoid nuclear receptors, retinoic acid receptors (RARs) and retinoic X receptors (RXRs). Although the pyridine derivatives, 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carbamoyl]pyri dine-3-carboxylic acid (2b) and 6-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]py ridine-3-carboxylic acid (5b) are more potent than the corresponding benzoic acid-type retinoids, Am80 (2a) and Am580 (5a), the replacement of the benzene ring of Am580 (5a), Am555 (6a), or Am55 (7a) with a pyrimidine ring caused loss of the retinoidal activity both in HL-60 cell differentiation assay and in RAR transactivation assay using COS-1 cells. On the other hand, pyrimidine analogs (PA series, 10 and 11) of potent RXR agonists (retinoid synergists) with a diphenylamine skeleton (DA series, 8 and 9) exhibited potent retinoid synergistic activity in HL-60 cell differentiation assay and activated RXRs. Among the synthesized compounds, 2-[N-n-propyl-N-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)a mino]pyrimidine-5-carboxylic acid (PA013, 10e) is most active retinoid synergist in HL-60 assay.     

Methods for the synthesis of 5,6,7,8-tetrahydro-1,8-naphthyridine fragments for alphaVbeta3 integrin antagonists

The preparation of 3-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)propan-1-amine 2a and 3-[(7R)-7-methyl-5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl]propan-1-amine 2b, key intermediates in the synthesis of alpha(V)beta(3) antagonists, is described. The syntheses rely on the efficient double Sonogashira reactions of 2,5-dibromopyridine 3 with acetylenic alcohols 4a/4b and protected propargylamines 10a-e followed by Chichibabin cyclizations of 3,3'-pyridine-2,5-diyldipropan-1-amines 9a/9b.     

Synthesis of some new pyrimidine selanyl derivatives

The targeted synthesis of 2-(methylsulfanyl)-6-(furan-2-yl)-4(3H)-selenoxo -pyrimidine-5-carbonitrile failed due to the formation 1-methyl-2-methylsulfanyl-6-oxo -4-(furan-2-yl)-1,6-dihydropyrimidine-5-carbonitrile. A new series of 5,6,7,8-tetrahydro-1-benzo thieno[2,3-d]pyrimidine-4-yl substituted selanyl derivatives were prepared by the reaction of sodium diselenide with 4-chloro-5,6,7,8-tetrahydro-1-benzothieno[2,3-d]pyrimidine followed by the reaction with chloroacetic acid derivatives such as ethyl chloroacetate, chloroacetamide or chloroacetonitrile. Hydrazinolysis of ethyl (5,6,7,8-tetrahydro-1-benzothieno[2,3-d]pyrimidine- 4-ylselanyl)acetate with hydrazine hydrate gave the corresponding hydrazino derivative. The latter reacted with ethyl acetoacetate, acetylacetone, diethyl malonate, ethoxymethylenemalononitrile or ethyl 2-cyano-3-ethoxyacetate to afford 5-methyl-2-[2-(5,6,7,8-tetrahydro-1-benzothieno [2,3-d]pyrimidine-4-ylselanyl)acetyl]-2,4-dihydropyrazol-3-one, 1-(3,5-dimethylpyrazol-1-yl)-2- (5,6,7,8-tetrahydro-1-benzothieno[2,3-d]pyrimidin-4-ylselanyl)ethanone, 1-[2-(5,6,7,8-tetrahydro -1-benzothieno[2,3-d]pyrimidine-4-ylselanyl)acetyl]-2,4-dihydropyrazolidine-3,5-dione and 5-Amino-1-[2-(5,6,7,8-tetrahydro-1-benzothieno[2,3-d]pyrimidin-4-ylselanyl)acetyl]-1H-pyrazol -4-yl substituted carbonitrile or ethyl carboxylate, respectively. The structure of the novel compounds was confirmed by spectroscopic tools (IR, ¹H NMR ¹³C NMR and mass spectra) and elemental analysis.     

Mispair-aligned N3T-alkyl-N3T interstrand cross-linked DNA: synthesis and characterization of duplexes with interstrand cross-links of variable lengths

Therapeutic bifunctional alkylating agents generate interstrand cross-links in duplex DNA. As part of our continuing studies on DNA duplexes that contain alkyl interstrand cross-links, we have synthesized a cross-link that bridges the N(3) positions of a mismatched thymidine base pair. This cross-link, which is similar to the N(3)C-alkyl-N(3)C cross-link that has been observed between mismatched cytosine base pairs, was introduced by first incorporating a cross-linked phosphoramidite unit at the 5'-end of an oligonucleotide chain. Fully cross-linked duplexes were then synthesized using an orthogonal approach to selectively remove protecting groups, thus allowing construction of the cross-linked duplex via conventional solid-phase oligonucleotide synthesis. Short DNA duplexes with alkyl cross-links of various lengths (two, four, and seven methylene units) were prepared, and their physical properties were studied via UV thermal denaturation and circular dichroism spectroscopy. These linkers were found to stabilize the duplexes by 37, 31, and 16 degrees C for the two-, four-, and seven-carbon linkers, respectively, relative to a non-cross-linked duplex. Circular dichroism spectra suggested that these lesions induce very little deviation in the global structure relative to the non-cross-linked duplex DNA control. Molecular models show that the two-carbon cross-link spans the distance between the N(3) atoms of the T-T mismatch without perturbing the helix structure, whereas the longer linkers, particularly the seven-carbon linker, tend to push the thymines apart, creating a local distortion. This perturbation may account for the lower thermal stability of the seven-carbon versus two-carbon cross-linked duplex.     

Cisplatin damage overrides the predefined rotational setting of positioned nucleosomes

Cisplatin and carboplatin are used successfully to treat various types of cancer. The drugs target the nucleosomes of cancer cells and form intrastrand DNA cross-links that are located in the major groove. We constructed two site-specifically modified nucleosomes containing defined intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links. Histones from HeLa-S3 cancer cells were transferred onto synthetic DNA duplexes having nucleosome positioning sequences. The structures of these complexes were investigated by hydroxyl radical footprinting. Employing nucleosome positioning sequences allowed us to quantify the structural deviation induced by the cisplatin adduct. Our experiments demonstrate that a platinum cross-link locally overrides the rotational setting predefined in the nucleosome positioning sequence such that the lesion faces toward the histone core. Identical results were obtained for two DNA duplexes in which the sites of platination differed by approximately half a helical turn. Additionally, we determined that cisplatin unwinds nucleosomal DNA globally by approximately 24 degree. The intrastrand cis-{Pt(NH3)2}(2+) 1,3-d(GpTpG) cross-links are located in an area of the nucleosome that contains locally overwound DNA in undamaged reference nucleosomes. Because most nucleosome positions in vivo are defined by the intrinsic DNA sequence, the ability of cisplatin to influence the structure of these positioned nucleosomes may be of physiological relevance.     

Regioselective alkylation of 2-alkyl-5,6,7,8-tetrahydro-3h-cycloheptimidazol-4-ones and 2-alkyl-3h-cycloheptimidazol-4-ones

Regioselective alkylation of 2-alkyl-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (1) and 2-alkyl-3H-cycloheptimidazol-4-one (2) was investigated. 3-[2'-(1-tert-Butyl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-2-propyl-5,6,7,8-tetrahydro-1H-cycloheptimidazol-4-one (6) was preferentially obtained under the conditions by using NaH in DMF or THF. On the other hand, 3-[2'-(1-tert-butyl-1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-2-propyl-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (5), the synthetic intermediate compound of Pratosartan, was obtained selectively in the presence of n-Bu(4)NBr in toluene by using aqueous sodium hydroxide as a base. In this reaction, it was found that the concentration of the alkaline solution influences its regioselectivity. This selectivity was observed even for aldehyde and ester derivatives.     

Interstrand and intrastrand DNA-DNA cross-linking by 1,2,3,4-diepoxybutane: role of stereochemistry

1,2,3,4-Diepoxybutane (DEB) is a bifunctional electrophile capable of forming DNA-DNA and DNA-protein cross-links. DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can then form 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) lesions. All three optical isomers of DEB are produced metabolically from 1,3-butadiene, but S,S-DEB is the most cytotoxic and genotoxic. In the present work, interstrand and intrastrand DNA-DNA cross-linking by individual DEB stereoisomers was investigated by PAGE, mass spectrometry, and stable isotope labeling. S,S-, R,R-, and meso-diepoxides were synthesized from l-dimethyl-2,3-O-isopropylidene-tartrate, d-dimethyl-2,3-O-isopropylidene-tartrate, and meso-erythritol, respectively. Total numbers of bis-N7G-BD lesions (intrastrand and interstrand) in calf thymus DNA treated separately with S,S-, R,R-, or meso-DEB (0.01-0.5 mM) were similar as determined by capillary HPLC-ESI(+)-MS/MS of DNA hydrolysates. However, denaturing PAGE has revealed that S,S-DEB produced the highest number of interchain cross-links in 5'-GGC-3'/3'-CCG-5' sequences. Intrastrand adduct formation by DEB was investigated by a novel methodology based on stable isotope labeling HPLC-ESI(+)-MS/MS. Meso DEB treatment of DNA duplexes containing 5'-[1,7, NH(2)-(15)N(3),2-(13)C-G]GC-3'/3'-CCG-5' and 5'-GGC-3'/3'-CC[(15)N(3),2-(13)C-G]-5' trinucleotides gave rise to comparable numbers of 1,2-intrastrand and 1,3-interstrand bis-N7G-BD cross-links, while S,S DEB produced few intrastrand lesions. R,R-DEB treated DNA contained mostly 1,3-interstrand bis-N7G-BD, along with smaller amounts of 1,2-interstrand and 1,2-intrastrand adducts. The effects of DEB stereochemistry on its ability to form DNA-DNA cross-links may be rationalized by the spatial relationships between the epoxy alcohol side chains in stereoisomeric N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine adducts and their DNA environment. Different cross-linking specificities of DEB stereoisomers provide a likely structural basis for their distinct biological activities.     

Competitive reactions of interstrand and intrastrand DNA-Pt adducts: A dinuclear-platinum complex preferentially forms a 1,4-interstrand cross-link rather than a 1,2 intrastrand cross-link on binding to a GG 14-mer duplex

A study of the kinetics and mechanism of the reaction between the dinuclear Pt complex [(trans-PtCl(NH(3))(2))(2)(mu-NH(2)(CH(2))(6)NH(2))](2+) (1) and the 14-mer duplex 5'-d(ATACATG(7)G(8)TACATA)-3'.5'-d(TATG(25)TACCATG(18)TAT)-3' is reported. [(1)H,(15)N]-HSQC NMR was used to follow the reaction at 298 K, pH 5.4. The product is primarily the 5'-5' 1,4-interstrand cross-link between G(8) and G(18) bases and exists in two conformational forms. No evidence for the possible 1,2-intrastrand G(7)G(8) adduct was seen, confirming the preferential formation of interstrand cross-links by these dinuclear complexes. An initial electrostatic association of (15)N-1 with the duplex is indicated by changes in its (1)H/(15)N chemical shifts, followed by aquation of 1 to form the monoaqua monochloro species 2, with a rate constant of 4.00+/-0.03x10(-5) s(-1). Monofunctional binding to the duplex occurs primarily at G(8), the 3' base of the nucleophilic GG grouping, with a rate constant of 1.5+/-0.7 M(-1) s(-1). Changes in the (1)H/(15)N shifts indicate there is an electrostatic interaction between the unbound (PtN(3)Cl) group of the monofunctional adduct and the duplex. No peaks for a transient aquated monofunctional species are seen and closure of 3 to form the 1,4-G(8)G(18) interstrand cross-link (5) was treated as direct, with a rate constant of 4.47+/-0.06x10(-5) s(-1). The G(8)G(18) cross-link was confirmed from analysis of the NOESY NMR spectrum of the final product. Structural perturbations for the 1,4-interstrand cross-link extend over approximately four base-pairs and are similar to those found for a 1,4-interstrand cross-link with a shorter 8-mer -GTAC- sequence. A major distortion was evident for the 5'T (T(17)) adjacent to the platinated G(18), consistent with the findings from the use of chemical probes to investigate the conformation of 1,4-interstrand cross-links.     

A NOVEL SYNTHESIS OF 2-SUBSTITUTED-4H-THIENO [2,3-d][1,3] OXAZIN-4-ONE AND 2,3-DISUBSTITUTED THIENO [2,3-d]PYRIMIDIN-4(3H)-ONE DERIVATIVES

Abstract

The synthesis of acetic 2-{[1-methyl-2-(4-oxo-5,6,7,8-tetrahydro-4H-benzo [4,5]-thieno [2,3-d] [1,3-oxazin-2-yl)ethylidene]amino}-4,5,6,7-tetrahydrohenzo[b]thiophene ?3-carboxylic acid anhydride 5 and 2-(oxopropyl)-5,6,7,8-tetrahydro-4H-benzo-[4,5] thieno[2,3-d][1,3]oxazin-4-one 7, has been achieved in three steps from ethyl 2-amino-4,5,6,7-tetrahydrobenzo(b]thiophene-3-carboxlate 1 via the reaction with ethyl acetoacetate followed by hydrolysis and acetic anhydride-induced cyclization. The 2-substituent in compound 5 has two functional groups i.e. active methylene and acid anhydride which are suitably located for intramolecular transformation. Thermal and/or base catalyzed intramolecular cyclization of 5 afforded 2-(4-acetoxy-(hydroxyl)-2-methyl-5,6,7,8-tetrahydrobenzo[4,5] thieno[2,3-b]pyridin-3-yl)-5,6,7,8- tetrahydro-4H-benzo[4,5]thieno[2,3-d] [1,3]oxazin-4-one 10 and 9 respectively. Treatment of 5 with hydrazine hydrate, aromatic and/or heterocyclic amines induced the same intramolecular cyclization with a concomitant oxazine-pyrimidine interconversion to give 3-amino(aryl or heteryl)-2-(4-hydroxy-5,6,7,8-tetrdhydrobenzo-[4,5]thieno[2,3-b]pyridin-3-yl)-3,4,5,6,7,8-hexahydrobenzo[4,5] thieno[2,3-d] pyrimid in-4-one 11–14 respectively.     

Synthesis and pharmacological activity of the metabolites of Pratosartan

Three hydroxylated metabolites of 2-propyl-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one (Pratosartan), which is a selective angiotensin II receptor antagonist, were synthesized in confirmation of their structures and in studies of their pharmacological properties. An MTPA ester of the human main metabolite was identified with the synthesized compound by comparing (1)H-NMR spectra, MS spectra, and HPLC retention time. The structure of the human main metabolite was confirmed to be (S)-(-)-2-(1-hydroxypropyl)-3-[2'-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-5,6,7,8-tetrahydro-3H-cycloheptimidazol-4-one ((S)-(-)-1). Also, the rat main metabolites were confirmed to be 8-hydroxylated compound (2) and 5-hydroxylated compound (3). These metabolites showed lower antagonistic activity than that of the parent compound.     

Notizen zur Synthese sulfonierter Derivate von 5,6,7,8-Tetrahydro-1-naphthylamin und 5,6,7,8-Tetrahydro-2-naphthylamin

Notes on the Synthesis of Sulfonated Derivatives of 5,6,7,8-Tetrahydro-1-naphthylamine and 5,6,7,8-Tetrahydro-2-naphthylamine Sulfonation of 5,6,7,8-tetrahydro-1-naphthylamine ( 1 ) with sulfuric acid gave a mixture of 1-amino-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 2 ), 4-amino-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 13 ) and 4-amino-5,6,7,8-tetrahydronaphthalene-1-sulfonic acid ( 3 ). The same reaction with 5,6,7,8-tetrahydro-2-naphthylamine ( 20 ) yielded 3-amino-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 21 ); formation of 2-amino-5,6,7,8-tetrahydronaphthalene-1-sulfonic acid ( 16 ) or of 3-amino-5,6,7,8-tetrahydronaphthalene-1-sulfonic acid ( 24 ) was not observed. Treatment of 4-bromo-5,6,7,8-tetrahydro-1-naphthylamine ( 4 ) or of its 4-chloro analogue 5 with amidosulfuric acid gave 1-amino-4-bromo-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 9 ) and its 4-chloro analogue 10 , respectively, which were dehalogenated to 2 . Preparations of 13 and 24 were achieved by sulfonation of 5-nitro-1,2,3,4-tetrahydronaphthalene ( 14 ) and 6-nitro-1,2,3,4-tetrahydronaphthalene ( 22 ) to 4-nitro-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 15 ) and 3-nitro-5,6,7,8-tetrahydronaphthalene-1-sulfonic acid ( 23 ), respectively, followed by Béchamp reductions. The sulfonic acid 13 was also obtained by hydrogenolysis of 4-amino-1-bromo-5,6,7,8-tetrahydronaphthalene-2-sulfonic acid ( 11 ) or of its 1-chloro analogue 12 ; compounds 11 and 12 were synthesized from N-(4-bromo-5,6,7,8-tetrahydro-1-naphthyl)acetamide ( 7 ) and from its 4-chloro analogue 8 , respectively, by sulfonation with oleum and subsequent hydrolysis. By ‘baking’ the hydrogensulfate salt of 1 or 20 compounds 3 and 21 were obtained, respectively. Synthesis of 16 was achieved by sulfur dioxide treatment of the diazonium chloride derived from 2-nitro-5,6,7,8-tetrahydro-1-naphthylamine ( 17 ) giving 2-nitro-5,6,7,8-tetrahydronaphthalene-1-sulfonyl chloride ( 18 ), followed by hydrolysis of 18 to the corresponding sulfonic acid 19 and final reduction.     

Synthesis of 5,6,7,8-tetrahydro-5-oxopyrido[2,3-d]pyrimidine-6-carbonitriles and -6-carboxylic acid esters

Dieckmann ring closure reactions of 4-[(2-cyanoethyl)substituted amino]-2-phenyl-5-pyrimidinecarboxylates (Ha-f) afforded several 5,6,7,8-tetrahydro-5-oxo-2-phenylpyrido[2,3-d]pyrimidine-6- carbonitriles (IIIa-f). The open-chain intermediates (IIa-f) were prepared by dechloroamination of 5-carbethoxy-4-chloro-2-phenylpyrimidine (1a) with several 3-substituted amino- propionitriles. Alkylation of the sodium salt of 5,6,7,8-tetrahydro-8-methyl-5-oxo-2-phenyl-pyrido[2,3-d]pyrimidine-6- carbonitrile (IIIa) with methyl iodide in DMF resulted in methylation at C-6 to afford IV. Tosylation of IIIa in pyridine gave the corresponding tosyl ester (V) of the enolic form. Oxidative dehydrogenation at the 6,7-position resulted when IIIa reacted with thionyl chloride, affording 5,8-dihydro-8-methyl-5-oxo-2-phenylpyrido[2,3-d]pyrimidine-6- carbonitrile (VII). Dechloroamination of la or 5-carbethoxy-4-chloro-2-methylthiopyrimidine (Ib) with ethyl 3-ethylaminopropionate followed by Dieckmann cyclization of the resulting open-chain intermediates gave the corresponding ethyl 5,6,7,8-tetrahydro-5-oxopyrido[2,3-d]pyrimidine-6-carboxylates IX'a and IX'b, respectively. These exist predominately in the enol form and undergo alkylation and oxidation reactions similar to IIIa.     

Synthesis of a 1,8-naphthyridin-5-one derivative via an intramolecular 1,3-dipolar cycloaddition reaction

Synthesis of a 1,8-naphthyridin-5-one derivative [(5,6,7,8-tetrahydro-(3-chloro-6-hydroxymethyl-8-methyl)-1,8-naphthyridin-5-one (9) ] is described starting from 2-chloronicotinic acid using an intramolecular 1,3-dipolar cycloaddition reaction as the key step.     

Superacidic activation of 1- and 3-isoquinolinols and their electrophilic reactions

Isomeric 1- and 3-isoquinolinols (11 and 12) when activated in CF(3)SO(3)H-SbF(5) acid system undergo selective ionic hydrogenation with cyclohexane to give 5,6,7,8-tetrahydro-1(2H)- and 5,6,7,8-tetrahydro-3(2H)-isoquinolinones (22 and 27). Under the influence of aluminum chloride similar products were also obtained along with 3,4-dihydro-1(2H)- and 1,4-dihydro-3(2H)-isoquinolinones (23 and 28), respectively. Compounds 11 and 12 also condense with benzene in the presence of aluminum halides, under mild conditions, to give 3,4-dihydro-3-phenyl-1(2H)- and 1,4-dihydro-1-phenyl-3(2H)-isoquinolinones (24 and 29), respectively. Prolonged reaction time or catalysis under strongly acidic HBr-AlBr(3) provides an alternative reaction pathway to yield 5,6-dihydro-6,8-diphenyl-1(2H)- and 5,6,7,8-tetrahydro-6,8-diphenyl-3(2H)-isoquinolinones (25 and 30), respectively. Products 24 and 29 were also found to revert back to 11 and 12 in the presence of aluminum halides in o-dichlorobenzene. The mechanism of these intriguing reactions, which involves superelectrophilic dicationic intermediates, is discussed.