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Structure−activity relationships for inhibition of DNA-dependent protein kinase (DNA-PK) have been defined for substituted chromen-4-ones. For the 2-amino-substituted benzo[h]chromen-4-ones, a morpholine substituent at this position was essential for activity. Small libraries of 6- and 7-alkoxy-substituted chromen-4-ones showed that a number of 7-alkoxy-substituted chromenones displayed improved activity. Focused libraries incorporating 6-, 7-, and 8-aryl and heteroaryl substituents were prepared. In these cases, 6- and 7-substitution was disfavored, whereas 8-substitution was largely tolerated. Surprisingly, two compounds, 2-N-morpholino-8-dibenzofuranyl-chromen-4-one (NU7427, 32{38}) and the 2-N-morpholino-8-dibenzothiophenyl-chromen-4-one (NU7441, 32{26}) were excellent inhibitors (IC50 vs DNA-PK = 40 and 13 nM, respectively). The ring-saturated analogue 2-N-morpholino-8-(6‘,7‘,8‘,9‘-tetrahydrodibenzothiophene)chromen-4-one, 36, retained potent activity (IC50 vs DNA-PK = 23 nM). The dibenzothiophene 32{38} sensitized HeLa cells to ionizing radiation in vitro, with dose modification factors of 2.5 at 10% survival being observed at 0.5 μM. The cytotoxicity of the topoisomerase II inhibitor etoposide was also potentiated.

A diverse range of chromen-2-one, chromen-4-one and pyrimidoisoquinolin-4-one derivatives was synthesized and evaluated for inhibitory activity against the DNA repair enzyme DNA-dependent protein kinase (DNA-PK), with a view to elucidating structure−activity relationships for potency and kinase selectivity. DNA-PK inhibitory activity varied widely over the series of compounds evaluated (IC50 values ranged from 0.19 to >10 μM), with excellent activity being observed for the 7,8-benzochromen-4-one and pyrimido[2,1-a]isoquinolin-4-one templates. By contrast, inhibitors based on the benzochromen-2-one (coumarin) or 2-aryl-7,8-benzochromen-4-one (flavone) scaffolds were less potent. Crucially, these studies revealed a very constrained structure−activity relationship at the 2-position of the benzopyranone and pyrimido[2,1-a]isoquinolin-4-one pharmacophore, with only a 2-morpholino or 2-(2‘-methylmorpholino) group being tolerated at this position. More detailed biological studies conducted with the most potent inhibitor NU7163 (48; IC50 = 0.19 μM) demonstrated ATP-competitive DNA-PK inhibition, with a Ki value of 24 nM, and 48 exhibited selectivity for DNA-PK compared with the related enzymes ATM, ATR, mTOR, and PI 3-K (p110alpha). Compound 48 sensitized the HeLa human tumor cell line to the cytotoxic effects of ionizing radiation in vitro, a dose modification factor of 2.3 at 10% survival being observed with an inhibitor concentration of 5 μM. This study identified these structural classes as novel DNA-PK inhibitors and delineated initial structure−activity relationships against DNA-PK.

The adenosine 5‘-triphosphate (ATP) competitive cyclin-dependent kinase inhibitor O⁶-cyclohexylmethylguanine (NU2058, 1) has been employed as the lead in a structure-based drug discovery program resulting in the discovery of the potent CDK1 and -2 inhibitor NU6102 (3, IC50 = 9.5 nM and 5.4 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). The SAR for this series have been explored further by the synthesis and evaluation of 45 N²-substituted analogues of NU2058. These studies have confirmed the requirement for the hydrogen bonding N²-NH group and the requirement for an aromatic N²-substituent to confer potency in the series. Additional potency is conferred by the presence of a group capable of donating a hydrogen bond at the 4‘-position, for example, the 4‘-hydroxy derivative (25, IC50 = 94 nM and 69 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), 4‘-monomethylsulfonamide derivative (28, IC50 = 9 nM and 7.0 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively), and 4‘-carboxamide derivative (34, IC50 = 67 nM and 64 nM vs CDK1/cyclinB and CDK2/cyclinA3, respectively). X-ray crystal structures have been obtained for key compounds and have been used to explain the observed trends in activity.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumors in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and in rat hepatocytes in culture. These arise primarily from its metabolite α-hydroxytamoxifen, and may also arise, in part, from another metabolite, 4-hydroxytamoxifen. We have prepared two model compounds for the potential reactive metabolite formed from 4-hydroxytamoxifen in rat liver. One of these was its α-acetoxy ester. This was much more reactive than that from tamoxifen, and could not be isolated in pure form. It reacted with DNA in the same way that α-acetoxytamoxifen did, to give adducts which were isolated by hydrolysis and chromatography, and identified as alkyldeoxyguanosines. The second derivative was α,β-dehydro-4-hydroxytamoxifen. This also reacts with DNA in vitro, to give the same products as those from α-acetoxy-4-hydroxytamoxifen. Reaction probably proceeds through the same resonance-stabilized carbocation in either case. However, when primary cultures of rat hepatocytes were treated with either 4-hydroxytamoxifen, 4,α-dihydroxytamoxifen, or α,β-dehydro-4-hydroxytamoxifen at a concentration of 10 μM, no adducts could be detected in their DNA by the ³²P-postlabeling technique. Similarly, no adducts could be found in the liver DNA of female Fischer F344 rats treated orally (at 0.12 mmol kg^-1) with the same substances. If 4-hydroxytamoxifen is metabolized to 4,α-dihydroxytamoxifen in rat liver, then either this substance is not converted to reactive esters or they are rapidly detoxified.

A series of homologs of idoxifene [1a, (E)-1-[4-(N-pyrrolidinoethoxy)phenyl]-1-(4-iodophenyl)-2-phenyl-1-butene] and selected homologs of 4-iodotamoxifen [2a, (E)-1-[4-[(N-dimethylamino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phenyl-1-butene] with the side chain (CH2)n varying in length from n = 3 (1b, 2b) to n = 10 (1i, 2i) have been synthesized and tested for antagonism of the calmodulin-dependent activity of cAMP phosphodiesterase and for binding affinity to rat uterine estrogen receptor. Compared with 1a (IC50 = 1.5 μM), the homologs showed a progressive increase in calmodulin antagonism with a maximum inhibition at n = 7−9 (1f−h) (IC50 = 0.2 μM), declining at n = 10 (1i) to IC50 = 1.6 μM. In the pyrrolidino series, estrogen receptor binding affinity peaked at n = 3 (1b, RBA = 23; estradiol = 100), declining by n = 10 (1i) to RBA = 0.4, but the homolog n = 8 (1g, RBA = 3.5) was still comparable to tamoxifen (RBA = 3.9). A similar pattern of activity was seen for the dimethylamino counterparts. These compounds represent a new class of antiestrogens with potent calmodulin antagonism.

The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the α-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-b ut-1-ene (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 μM idoxifene, after analysis by the ³²P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3 -buten-2-ol (α-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the α-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than α-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N²-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug.

Tamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1,2-diphenylbut-1-ene], a nonsteroidal antiestrogen, induces liver tumors in rats by a genotoxic mechanism. The mechanism of DNA adduct formation is believed to proceed via the formation of a reactive carbocation at the α-position from the α-hydroxylated metabolite. Molecular mechanics calculations [Kuramochi, H. (1996) J. Med. Chem. 39, 2877−2886] have predicted that 4-substitution will affect the stability of the carbocation and thus will alter its reactivity toward DNA. We have synthesized the putative α-hydroxylated metabolites of 4-hydroxytamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-3-hydroxy-2-phenylbut-1-ene] and idoxifene [(Z)-1-(4-iodophenyl)-3-hydroxy-2-phenyl-1-(4-(2-(N-pyrrolidino)ethoxy)phenyl)but-1-ene] and compared their reactivities with DNA with that of α-hydroxytamoxifen [(E)-1-(4-(2-(N,N-dimethylamino)ethoxy)phenyl)-3-hydroxy-1,2-diphenylbut-1-ene]. As predicted, the bis-hydroxylated compound reacted with DNA in aqueous solution at pH 5 to give 12-fold greater levels of adducts than α-hydroxytamoxifen, whereas α-hydroxyidoxifene gave one-half the number of adducts. The results demonstrate that idoxifene presents a significantly lower genotoxic hazard than tamoxifen for the treatment and prophylaxis of breast cancer.

The metabolic activation of tamoxifen and its metabolite alpha-hydroxytamoxifen in primary cultures of rat, mouse and human hepatocytes has been compared. The extent of formation of DNA adducts in these cells was measured by 32P-postlabelling, using either nuclease P1 digestion or sorbent extraction of DNA digests to enhance the sensitivity of the assay. DNA adducts were readily detected in rat hepatocytes treated with 1 or 10 microM tamoxifen (mean levels 18.2 and 89.8 adducts/10(8) nucleotides respectively). Similar levels of adducts were formed by mouse hepatocytes (15.0 +/- 1.8 adducts/10(8) nucleotides, 10 microM tamoxifen). However DNA adducts were not detected in tamoxifen-treated human hepatocytes with a detection limit for the assay of 4 adducts/10(10) nucleotides. Treatment of rat cells with alpha-hydroxytamoxifen resulted in 15- to 63-fold higher levels of adducts than with comparable concentrations of tamoxifen. A similar level of adducts was also seen in mouse hepatocytes treated with alpha-hydroxytamoxifen at the 1 microM concentration (173.9 +/- 4.1 adducts/10(8) nucleotides). Treatment of human cells with alpha-hydroxytamoxifen resulted in DNA adduct formation at levels (1.94 +/- 0.89 and 18.9 +/- 17.9 adducts/10(8) nucleotides at 1 and 10 microM respectively) approximately 300-fold lower than those in rat hepatocytes. The presence of alpha-hydroxytamoxifen in the culture medium from experiments where cells were incubated with tamoxifen was monitored by mass spectrometry. Concentrations were found to be approximately 50-fold lower in the medium from human hepatocytes than from rat and mouse hepatocytes.

Experimental results on the performance comparison of two dispersion-managed fibre types in a 500 4;km (6×84.5 4;km) 16-channel 40 4;Gbit/s system are reported. The PDF(+D)-NDF(−D)-PDF(+D) configuration showed superior Q performance and nonlinearity tolerance over a broad range of signal power and Raman gain, compared to the PDF(+D)-NDF(−D) arrangement.