Jasim Al-Rawi

Synthesis, DNA-PK inhibition, anti-platelet activity studies of 2-(N-substituted-3-aminopyridine)-substituted-1,3-benzoxazines and DNA-PK and PI3K inhibition, homology modelling studies of 2-morpholino-(7,8-di and 8-substituted)-1,3-benzoxazines

A number of new 2-(pyridin-3-ylamino)-4H-(substituted) benz[e]-1,3-oxazin-4-ones were synthesized 10a-g. These were then reacted with the hydro-halogen salt of 2, 3 and 4-(halo-methyl) pyridine in the presence of Cs(2)CO(3) to give eighteen new 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11a-i, 13a-c, and 15a-f). X-ray crystallography was used to confirm that the 2-N-substituted structures 11 and 13 were formed rather than the 3-N-substitution analogues 12 and 14. Eleven of the new compounds were tested for their effect on collagen induced platelet aggregation and it was found that the most active inhibitory compound was 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC(50) of 10 ± 2 μM. DNA-dependent protein kinase (DNA-PK) inhibition data for 12 previously prepared 2-morpholino substituted-1,3-benzoxazines (compounds 19-31) were measured and showed high to moderate activity where the most active compound was compound 27 with an IC(50) of 0.28 μM. Furthermore DNA-PK inhibition data for six newly prepared 2-(N-substituted (pyridin-3-ylmethyl) amino)-substituted-1,3-benzoxazines (compounds 11b, 13a-b, 15a-b and 15e) and 8-methyl-7-(pyridin-3-ylmethoxy)-3-(pyridin-3-ylmethyl)-2H-benz[e]-1,3-oxazin-2,4(3H)-dione 17d were measured and moderate to low inhibitory activity was observed, with the most active of the compounds in this series being 8-methyl-2-(pyridin-3-yl(pyridin-3-ylmethyl)amino)-7-(pyridin-3-ylmethoxy)-4H-benz[e]-1,3-oxazin-4-one 15e with an IC(50) of 2.5 μM. PI3K inhibition studies revealed that compound 27 is highly potent (IC(50) for PI3Kα = 0.13 μM, PI3Kβ = 0.14 μM, PI3Kγ = 0.72 μM, PI3Kδ = 2.02 μM). Compound 22 with 7-[2-(4-methylpiperazin-1-yl)ethoxy] group shows greater inhibition of DNA-PK over PI3K. Docking of some 2-morpholino-substituted-1,3-benzoxazine compounds 19-31 within the binding pocket and structure-activity relationships (SAR) analyses were performed with results agreeing well with observed activities.

Synthesis, structural elucidation and DNA-dependant protein kinase and antiplatelet studies of 2-amino-[5, 6, 7, 8-mono and 7, 8-di-substituted]-1, 3-benzoxazines

A number of new 2-amino-[5, 6, 7 and 8]-O-substituted 1,3-benzoxazines, and 2-amino 8-methyl-7-O-substituted-1,3-benzoxazines were synthesized. Thirty one new compounds were tested for their effect on collagen induced platelet aggregation and it was found that the most active compounds were 8-methyl-2-morpholin-4-yl-7-(pyridin-3-ylmethoxy)-4H-1,3-benzoxazin-4-one 9f and 8-methyl-2-morpholin-4-yl-7-(pyridin-4-ylmethoxy)-4H-1,3-benzoxazin-4-one 9j with IC(50) = 2 ± 1.5 and 4 ± 2 μM respectively. Inhibition of DNA-PK activity at concentrations of 1.6-4 μM were tested for 9 products 5i, 7a-e and 9b, 9f and 9j.

Generalized Method for the Production of 1,3‐Benzoxazine, 1,3‐Benzothiazine, and Quinazoline Derivatives from 2‐(Hydroxy, Thio, or Amino) Aromatic Acids Using Triphenylphosphine Thiocyanogen

Abstract A modified one‐pot method was developed for the synthesis of 1,3‐benzoxazines, in which the preparation of unstable thiocyanogen was omitted. The method was found to be general for substituted (methyl, methoxy, halo, and hydroxy) 2‐hydroxy benzoic acids and 2‐hydroxy naphthoic acids. The method was extended to 2‐thio, 2‐amino, and N‐methyl aminobenzoic acid with which the synthesis of 1,3‐benzothiazine and quinazoline derivatives has been achieved, respectively. It was also found that 3‐hydroxypyridine‐2‐carboxylic acid and 2‐hydroxynicotinic acid using a modified method gave 2‐thioxo‐2,3‐dihydro‐4H‐pyrido[2,3‐e][1,3]oxazin‐4‐one and 2‐thioxo‐2,3‐dihydro‐4H‐pyrido[3,2‐e][1,3]oxazin‐4‐one, respectively. The structures of the new compounds were confirmed by the analysis of their IR, 1H, and 13C NMR spectra.

Use of Diphenyliodonium Bromide in the Synthesis of Some N-Phenyl α-Amino Acids

The N-phenyl methyl esters 4 of glycine, alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, serine, threonine, tyrosine, aspartic acid, and glutamic acid have been synthesized in good to excellent yields using diphenyliodonium bromide, AgNO3, and a catalytic amount of CuBr starting from the relevant amino acid ester. The chiral integrity of the amino acids 5 was maintained during these reactions, which were confirmed by the synthesis of dipeptide for each N-phenyl amino acid. The structures of the new compounds were confirmed by the analysis of their IR, 1H, and 13C NMR spectra in addition to CHN microanalysis or high-resolution mass spectrometry for the new N-phenyl amino acids 5 and the esters 4.

Synthesis, structural elucidation, DNA-PK inhibition, homology modelling and anti-platelet activity of morpholino-substituted-1,3-naphth-oxazines

A number of new angular 2-morpholino-(substituted)-naphth-1,3-oxazines (compound 10b), linear 2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 13b-c), linear 6, 7 and 9-O-substituted-2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 17-22, 24, and 25) and angular compounds 14-16 and 23 were synthesised. The O-substituent was pyridin-2yl-methyl (15, 18, and 21) pyridin-3yl-methyl (16, 19, and 22) and 4-methylpipreazin-1-yl-ethoxy (23-25). Twelve compounds were tested for their inhibitory effect on collagen induced platelet aggregation and it was found that the most active compounds were compounds 19 and 22 with IC(50)=55±4 and 85±4 μM, respectively. Furthermore, the compounds were also assayed for their ability to inhibit DNA-dependent protein kinase (DNA-PK) activity. The most active compounds were 18 IC(50)=0.091 μM, 24 IC(50)=0.191 μM, and 22 IC(50)=0.331 μM. Homology modelling was used to build a 3D model of DNA-PK based on the X-ray structure of phosphatidylinositol 3-kinases (PI3Ks). Docking of synthesised compounds within the binding pocket and structure-activity relationships (SAR) analyses of the poses were performed and results agreed well with observed activity.

Chemo-sensitisation of HeLa cells to Etoposide by a Benzoxazine in the absence of DNA-PK inhibition

SummaryThe benzoaxines have been developed from structurally similar chromones as specific inhibitors of the PI3K family to sensitize cancer cells to the effects of chemotherapeutic agents; most have been shown to do this through specific inhibition of DNA-PK and DNA repair mechanisms. In this study we examined the benzoxazine, 2-((3-methoxybut-3en-2-yl)amino)-8methyl-4H-benzo[1,3]oxazin-4one (LTUSI54). This compound had no DNA-PK or PI3K inhibitory activity but still sensitized HeLa cells to the effects of Etoposide. LTUSI54 works synergistically with Etoposide to inhibit growth of HeLa cells and sub G1 analysis indicates that this is not due to an increase in apoptosis. LTUSI54 neither enhances DSB formation due to Etoposide nor does it delay the repair of such damage. Cell cycle analysis shows a clear G2 block with Etoposide alone while, in combination with LTUSI54 there is an additional S phase arrest. Phospho-kinase analysis indicated that LTUSI54 engages key regulators of cell cycle progression, specifically p38α, p53 and ERK 1/2. From our results we hypothesize that LTUSI54 is promoting the cell cycle arrest through activation of p38α pathways, independent of p53 mechanisms. This results in a decrease in p53 phosphorylation and hence, restricted apoptosis. Changes in cell number appear to be the result of p38α pathways disrupting cell cycle progression, at the S and G2 checkpoints. Further investigation into the finer mechanisms by which LTUSI54 effects cell cycle progression would be of great interest in assessing this compound as a chemosensitising agent.

Radiosensitizing activity of a novel Benzoxazine through the promotion of apoptosis and inhibition of DNA repair

SummaryThe DNA dependant protein kinase (DNA-PK) enzyme plays a major part in the repair of double stranded breaks induced by radiation and hence in the radio-resistance of tumour cells. Inhibitors of DNA-PK have been tested successfully in the past for their ability to sensitize cancer cells to the effects of radiation. Here we present a novel benzoxazine, 8-methyl-2-(morpholine-4yl)-7-(pyridine-3-methoxy)-4H-1,3-benzoxacine-4-one (LTU27) and analyse its ability to cause sensitization of lung cancer and colon cancer cells to radiation. There was a significant reduction in survival rate, increase in apoptosis and inhibition in autophosphorylation of DNA-PK and AKT1 after treating them concomitantly with both radiation and LTU27. The mechanism of action appears to be through inhibition of DNA-PK leading to delayed DNA repair and promotion of apoptosis.

Synthesis of linear and angular aryl-morpholino-naphth-oxazines, their DNA-PK, PI3K, PDE3A and antiplatelet activity

To continue our study of 2-morpholino-benzoxazine based compounds, which show useful activity against PI3K family enzymes or antiplatelet activity, we designed and synthesized a series of linear 6.7-fused, 5,6-angular fused and 7,8-angular fused-aryl-morpholino-naphth-oxazines. The compounds were prepared from substituted 2-hydroxynaphthoic acid to give the corresponding thioxo analogues 8, 9, 15 and 19. The thioxo products were then converted to the morpholino substituted analogue. The aryl group was introduced by Suzuki coupling of bromo precursors. The products were evaluated for activity at PI3K family enzymes and as platelet aggregation inhibitors and compared to reported unsubstituted analogues. The linear 6.7-fused product 13a and 13b were moderated potent but selective PI3Kδ isoform inhibitors (IC50=7.7 and 5.61μM). Good antiplatelet activity was noticed for the angular 7,8-fused compounds 22a, b, k and l with IC50=3.0,14.0, 2.0 and 5.0μM respectively. The antiplatelet activity is independent of PDE3.

Reaction of Ph3P(SCN)2 with Further Orthohydroxy Carboxylic Acid Systems, Including Substituted β-Keto Acids: Synthesis of Novel 2-Thio-1,3-oxazines and Their Subsequent Transformation with Amines

Abstract We now report the first reaction of Ph3P(SCN)2 with 4,6-dihydroxy-5-methylisophthalic acid to give 10-methyl-2,8-dithio-1,3-oxazino-1,3-benzoxazine-4,6-dione. Also, the enol tautomer has been utilized in the reaction of β-keto acids with Ph3P(SCN)2 to give novel 2-thio-1,3-oxazines. Subsequent reaction of the 2-thio-1,3-oxazines with benzylamine resulted in opening of the oxazine ring and gave novel dibenzylamino-enamides, which could be cyclized to thiouracils. The reaction of 2-thio-1,3-oxazines with morpholine at low temperature led to the production of unstable 2-Mercapto-2-morpholino-1,3-oxazines. 2-Mercapto-2-morpholin-4-yl-2,3,5,6,7,8-hexahydro-4H-1,3-benzoxazin-4-one was observed to lose H2S at room temperature to give 2-morpholin-4-yl-5,6,7,8-tetrahydro-4H-1,3-benzoxazin-4-one, which was subsequently tested and found to exhibit some antiplatelet activity.

Radiosensitizing activity of novel small molecule BRCA1 and DNA-PK inhibitors in lung and colon carcinoma

Abstract The DNA dependant protein kinase (DNA-PK) enzyme plays a major part in the repair of double stranded breaks induced by radiation and hence in the radio-resistance of tumour cells. Inhibitors of DNA-PK have been tested successfully in the past for their ability to sensitize cancer cells to the effects of radiation. Here we present two novel benzoxazines (LTU28 and LTU31) and analyse their ability to cause sensitization of cancer cells to radiation. There was a significant reduction in survival rate, increase in apoptosis and inhibition in autophosphorylation of DNA-PK and AKT1 after treating them with LTU28 concomitantly with radiation. The mechanism of action by LTU28 appears to be through inhibition of DNA-PK leading to delayed DNA repair and promotion of apoptosis. LTU31 showed an inhibition in the phosphorylation of BRCA1, thereby blocking DNA repair by homologous recombination.