Identification of human-selective analogues of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA).

BACKGROUND: Species selectivity of DMXAA (5,6-dimethylxanthenone-4-acetic acid, Vadimezan) for murine cells over human cells could explain in part the recent disappointing phase III trials clinical results when preclinical studies were so promising. To identify analogues with greater human clinical potential, we compared the activity of xanthenone-4-acetic acid (XAA) analogues in murine or human cellular models. METHODS: Analogues with a methyl group systematically substituted at different positions of the XAA backbone were evaluated for cytokine induction in cultured murine or human leukocytes; and for anti-vascular effects on endothelial cells on matrigel. In vivo antitumour activity and cytokine production by stromal or cancer cells was measured in human A375 and HCT116 xenografts. RESULTS: Mono-methyl XAA analogues with substitutions at the seventh and eighth positions were the most active in stimulating human leukocytes to produce IL-6 and IL-8; and for inhibition of tube formation by ECV304 human endothelial-like cells, while 5- and 6-substituted analogues were the most active in murine cell systems. CONCLUSION: Xanthenone-4-acetic acid analogues exhibit extreme species selectivity. Analogues that are the most active in human systems are inactive in murine models, highlighting the need for the use of appropriate in vivo animal models in selecting clinical candidates for this class of compounds.

Comparative bioactivation of the novel anti-tuberculosis agent PA-824 in Mycobacteria and a subcellular fraction of human liver.

BACKGROUND AND PURPOSE: PA-824 is a 2-nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F(420) )-dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des-nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti-mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA-824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis. EXPERIMENTAL APPROACH: PA-824 (250 µM) was incubated with the 9000 × g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis. KEY RESULTS: PA-824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des-nitro product. Six of these products, but not the des-nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn-catalyzed des-nitrification and radiolytic reduction of PA-824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem-catalyzed product of imidazole ring hydration (M2). CONCLUSIONS AND IMPLICATIONS: PA-824 was metabolized by des-nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA-824 was selectively bioactivated in M. tuberculosis and there was no evidence for 'cross-activation' by human enzymes.

Pharmacokinetics and metabolism of the nitrogen mustard bioreductive drug 5.

PURPOSE: To characterise the pharmacokinetics and metabolism in mice of 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide (SN 23862), the lead compound of a new class of bioreductive drugs in which a nitrogen mustard is activated by nitroreduction. Comparison is made with the corresponding aziridine derivative CB 1954. METHODS: Male C3H/HeN mice, bearing s.c. KHT tumours, received 3H-labelled SN 23862 or CB 1954 i.v. at 200 micromol/kg. Plasma, urine and tumour samples were assayed for total radioactivity, and for parent compounds by HPLC. Metabolites were identified by 1H-NMR and mass spectrometry. Cytotoxicity of compounds against Chinese hamster AA8 cells was determined by growth inhibition assay. RESULTS: The plasma pharmacokinetics of SN 23862 and CB 1954 were similar, with half-lives of 1.1 and 1.2 h, respectively. SN 23862 provided tumour/plasma ratios and absolute tumour AUC values almost two times higher than CB 1954. Despite this, SN 23862 was more extensively metabolised than CB 1954, the major route being sequential oxidative dechloroethylation of the nitrogen mustard moiety to the relatively non-toxic half mustard and 5-amine. The inferred chloroacetaldehyde co-product was 260 times more potent than SN 23862. A tetrahydroquinoxaline metabolite resulting from reduction of the 4-nitro group followed by intramolecular alkylation was weakly cytotoxic, while the more cytotoxic 2-amino derivative of SN 23862 was detected in trace amounts. CB 1954 was metabolised by analogous pathways, but the 4- and 2-amine nitroreduction products were the major metabolites while oxidative dealkylation was minor. CONCLUSION: The lesser propensity for SN 23862 to undergo nitroreduction in the host, relative to CB 1954, argues that dinitrobenzamide mustards may be preferable to the corresponding aziridines as bioreductive prodrugs for cancer treatment. However, the toxicological significance of oxidative metabolism of the bis(2-chloroethyl)amine moiety needs to be addressed.

Modulation of the pharmacokinetics of the antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice by thalidomide.

BACKGROUND: 5,6-Dimethylxanthenone-4-acetic acid (DMXAA), an investigative drug currently in clinical trial, acts on tumour vasculature through the induction of cytokines. Coadministration of thalidomide, a modulator of cytokine production, potentiates the antitumour activity of DMXAA against the murine Colon 38 carcinoma in mice. We wished to determine whether alteration of the pharmacokinetics of DMXAA by thalidomide could provide an explanation for this potentiation. RESULTS: Coadministration of thalidomide to Colon 38 tumour-bearing mice significantly (P < 0.05) increased the elimination half-life (t1/2) of DMXAA in plasma (413 micromol/l), liver (132 micromol/l), and spleen (77 micromol/l), and significantly (P < 0.05) increased DMXAA concentrations in Colon 38 tumour tissue (0.25-4.5 h). L-Thalidomide had a greater effect on DMXAA elimination (P < 0.01) than did D-thalidomide or the racemate. Coadministration of thalidomide increased the area under the concentration-time curve (AUC) of DMXAA by 1.8-fold in plasma, liver and spleen, and by 3.0-fold in tumour. Bile from mice given thalidomide and DMXAA contained substantially lower amounts of the glucuronide metabolite of DMXAA (DMXAA-G) than did bile from mice given DMXAA alone. CONCLUSION: Glucuronidation is a major excretory pathway for DMXAA in the mouse. Thalidomide, probably as the L-form, decreases the rate of elimination of DMXAA from plasma, spleen, liver and tumour by altering the rate of glucuronidation. The reduction in the elimination of DMXAA by thalidomide may lead to a selective increase in exposure of tumour tissue to drug, providing a basis for its potentiation of antitumour activity.

Relationships between hydrophobicity, reactivity, accumulation and peripheral nerve toxicity of a series of platinum drugs.

Previous work has shown platinum drugs to differ in their effects on the peripheral nervous system. To test whether their differential toxicity was due to differences in their partitioning into the peripheral nervous system, we correlated the hydrophobicity, reactivity, tissue accumulation and neurotoxicity of a series of eight platinum analogues. Neurotoxicity was detected by measuring sensory nerve conduction velocity (SNCV) in Wistar rats treated twice per week at the maximum tolerated dose. Tissue platinum concentrations were measured by inductively coupled plasma mass spectrometry. Hydrophobicity (log P) was measured using an octanol-aqueous shake-flask method. The half-life of platinum drug binding to plasma proteins in vitro was determined. The cumulative dose causing altered SNCV ranged from 15 to > 2050 micromol kg(-1). Ranking of the compounds by their neurotoxic potency in rats (oxaliplatin > R,R-(DACH)PtC4 > ormaplatin > S,S-(DACH)PtCl4 > S,S-(DACH)Pt oxalato > cisplatin > carboplatin > JM216) correlated with the frequency of neurotoxicity in patients (r> 0.99; P < 0.05). Ranking the compounds by their peripheral nerve accumulation was cisplatin > carboplatin > oxaliplatin > R,R-(DACH)PtCl4 = S,S-(DACH)PtCl4 and did not correlate with neurotoxicity. Log P ranged from - 2.53 to -0.16 but did not correlate with neurotoxicity. Log P correlated inversely with platinum accumulation in dorsal root ganglia (r2 = 0.99; P = 0.04), sural nerve (r2 = 0.85; P = 0.025), sciatic nerve (r2 = 0.98; P= 0.0012), spinal cord (r2 = 0.97, P= 0.018) and brain (r2 = 0.98, P= 0.001). Reactivity correlated with neurotoxicity potency in rats (r2 = 0.89, P = 0.0005) and with the frequency of neurotoxicity in patients (r2 = 0.99, P = 0.0002). The hydrophilicity of platinum drugs correlates with platinum sequestration in the peripheral nervous system but not with neurotoxicity. Differences in the reactivity of platinum complexes accounts for some of the variation in their neurotoxicity.

Structure-activity relationships for 5-substituted 1-phenylbenzimidazoles as selective inhibitors of the platelet-derived growth factor receptor.

Following an earlier discovery of 1-phenylbenzimidazoles as ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR), further structure-activity relationships for analogues (particularly 5-substituted derivatives) are reported. The data are consistent with a binding model (constructed from the homology-modeled structure of the catalytic subunit of the PDGFR using protein kinase A as the template) in which the ligand binds in the relatively narrow ATP site, with the phenyl ring pointing toward the interior of the pocket and the 5-position of the benzimidazole ring toward the mouth of the pocket. The narrow binding pocket allows a maximum torsion angle between the phenyl and benzimidazole rings of about 40 degrees, consistent with that calculated (43.6 degrees) for the minimum-energy conformation of the unsubstituted free ligand. The inactivity of 7- or 2'-substituted analogues is consistent with the greater torsion angle (and thus larger ligand cross-section) of such substituted analogues. There is substantial bulk tolerance for 5-substituents, which protrude out of the mouth of the hydrophobic pocket, with the most effective analogues being those bearing weak bases. On the basis of this model, 5-OR derivatives bearing cationic side chains were prepared as soluble analogues, and these showed sub-micromolar potencies against the isolated PDGFR enzyme. They were also moderately effective inhibitors of autophosphorylation of PDGFR in rat aortic vascular smooth muscle cells, with IC50s in the range 0.1-1 microM.

Thalidomide increases both intra-tumoural tumour necrosis factor-alpha production and anti-tumour activity in response to 5,6-dimethylxanthenone-4-acetic acid.

5,6-Dimethylxanthenone-4-acetic acid (DMXAA), synthesized in this laboratory and currently in phase I clinical trial, is a low molecular weight inducer of tumour necrosis factor-alpha (TNF-alpha). Administration of DMXAA to mice with established transplantable tumours elicits rapid vascular collapse selectively in the tumour, followed by extensive haemorrhagic necrosis mediated primarily through the production of TNF-alpha. In this report we have investigated the synthesis of TNF-alpha mRNA in hepatic, splenic and tumour tissue. Co-administration of thalidomide with DMXAA increased anti-tumour activity and increased intra-tumoural TNF-alpha production approximately tenfold over that obtained with DMXAA alone. Thalidomide increased splenic TNF-alpha production slightly but significantly decreased serum and hepatic levels of TNF-alpha induced with DMXAA. Lipopolysaccharide (LPS) induced 300-fold higher serum TNF-alpha than did DMXAA at the maximum tolerated dose, but induced similar amounts of TNF-alpha in spleen, liver and tumour. Splenic TNF-alpha activity induced with LPS was slightly increased with thalidomide, but serum and liver TNF-alpha levels were suppressed. Thalidomide did not increase intra-tumoural TNF-alpha production induced with LPS, in sharp contrast to that obtained with DMXAA. While thalidomide improved the anti-tumour response to DMXAA, it had no effect on the anti-tumour action of LPS that did not induce a significant growth delay or cures against the Colon 38 tumour. The increase in the anti-tumour action by thalidomide in combination with DMXAA corresponded to an increase in intra-tumoural TNF-alpha production. Co-administration of thalidomide may represent a novel approach to improving selective intra-tumoural TNF-alpha production and anti-tumour efficacy of DMXAA.

Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Interaction of thalidomide, phthalimide analogues of thalidomide and pentoxifylline with the anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid: concomitant reduction of serum tumour necrosis factor-alpha and enhancement of anti-tumour activity.

DMXAA (5,6-dimethylxanthenone-4-acetic acid), a novel anti-tumour agent currently undergoing clinical evaluation, appears to mediate its anti-tumour effects through immune modulation and the production of the cytokine tumour necrosis factor-alpha (TNF). Our previous studies have shown that thalidomide, a potent inhibitor of TNF biosynthesis that has numerous biological effects, including inhibition of tumour angiogenesis, unexpectedly augments the anti-tumour response in mice to DMXAA. We show here that thalidomide (100 mg kg(-1)) has no effect when administered with inactive doses of DMXAA, and that it must be given simultaneously with an active dose of DMXAA to have its maximum potentiating effect on the growth of the murine Colon 38 adenocarcinoma. To address the issue of whether inhibition of serum TNF production is important for potentiation of anti-tumour activity, we have tested three potent analogues of thalidomide. All three analogues, when co-administered with DMXAA to mice at doses lower than those used with thalidomide, inhibited TNF production and were effective in potentiating the anti-tumour activity of DMXAA against transplanted Colon 38 tumours. One of the analogues, N-phenethyltetrafluorophthalimide, was 1000-fold more potent than thalidomide and at a dose of 0.1 mg kg(-1) in combination with DMXAA (30 mg kg(-1)) cured 100% of mice, compared with 67% for the group treated with DMXAA alone. We also tested pentoxifylline and found it to suppress TNF production in response to DMXAA and to potentiate the anti-tumour effect of DMXAA. The results are compatible with the hypothesis that pharmacological reduction of serum TNF levels might benefit the anti-tumour effects of DMXAA and suggest new strategies for therapy using this agent.

Synthesis and hypoxia-selective cytotoxicity of a 2-nitroimidazole mustard.

A four-step synthesis of 5-[N,N-bis(2-chloroethyl)amino]-1-methyl-2-nitroimidazole from 1-methyl-2-nitroimidazole is described. This compound showed similar hypoxia-selective cytotoxicity to the dinitrobenzamide mustard SN 23,862 in UV4 cells (ca. 40-fold), and superior selectivity (> 7-fold) in repair-competent AA8 cells.

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor.

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.

The reproductive cycle of the female American alligator (Alligator mississippiensis).

We examined ovarian and oviducal gross morphology and collected blood samples from wild female alligators in central Florida during most of the year. Females with vitellogenic follicles were observed throughout the year, although ovaries containing follicles larger than 15 mm were seen only during the spring and early summer (March-June). We detected a poor relationship between female body size and the number of vitellogenic follicles whereas body size was significantly correlated with clutch size. Plasma samples were analyzed for (1) estradiol-17beta (E2), testosterone (T), and corticosterone by specific radioimmunoassays; (2) vitellogenin by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and (3) total protein, phosphorus, and calcium by spectrophotometric assays. Reproductively active females showed elevated plasma concentrations of E2 during the fall (September-November) and spring (March-May) whereas non-reproductively active females exhibited basal levels with no apparent peaks. Vitellogenin was detected in the plasma during the same months that plasma E2 concentrations were elevated. Elevated plasma vitellogenin and E2 were not correlated with plasma total protein but were correlated with plasma calcium concentration. During late vitellogenesis, plasma T concentrations were elevated in reproductively active females coincident with a period of intense courtship and mating. Corticosterone plasma concentrations exhibit no significant monthly variation, nor apparent changes during various stages of reproductive activity although plasma concentrations were elevated during late gravidity. Our data suggest that female reproductive activity begins in the fall with an increase in plasma E2 concentration in September or October and vitellogenesis in October. Ovarian activity slows during winter and reactivates with the onset of spring.

Tyrosine kinase inhibitors. 11. Soluble analogues of pyrrolo- and pyrazoloquinazolines as epidermal growth factor receptor inhibitors: synthesis, biological evaluation, and modeling of the mode of binding.

A new route to N-1-substituted pyrazolo- and pyrroloquinazolines has been developed from the known quinazolones 19 and 23, via conversion to the corresponding thiones, S-methylation to the thioethers, N-1-alkylation, and coupling with 3-bromoaniline. C-3-Substituted pyrroloquinazolines were prepared by Mannich base chemistry. A series of compounds bearing solubilizing side chains at these positions has been prepared and evaluated for inhibition of the tyrosine kinase activity of the isolated epidermal growth factor receptor (EGFR) and of its autophosphorylation in EGF-stimulated A431 cells. Several analogues, particularly C-3-substituted pyrroloquinazolines, retained high potency in both assays. A model for the binding of the general class of 4-anilinoquinazolines to the EGFR was constructed from structural information (particularly for the catalytic subunit of the cAMP-dependent protein kinase) and structure-activity relationships (SAR) in the series. In this model, the pyrrole ring in pyrroloquinazolines (and the 6- and 7-positions of quinazoline and related pyridopyrimidine inhibitors) occupies the entrance of the ATP binding pocket of the enzyme, with the pyrrole nitrogen located at the bottom of the cleft and the pyrrole C-3 position pointing toward a pocket corresponding to the ribose binding site of ATP. This allows considerable bulk tolerance for C-3 substituents and lesser but still significant bulk tolerance for N-1 substituents. The observed high selectivity of these compounds for binding to EGFR over other similar tyrosine kinases is attributed to the 4-anilino ring binding in an adjacent hydrophobic pocket which has an amino acid composition unique to the EGFR. The SAR seen for inhibition of the isolated enzyme by the pyrazolo- and pyrroloquinazolines discussed here is fully consistent with this binding model. For the N-1-substituted compounds, inhibition of autophosphorylation in A431 cells correlates well with inhibition of the isolated enzyme, as seen previously for related pyridopyrimidines. However, the C-3-substituted pyrroloquinazolines show unexpectedly high potencies in the autophosphorylation assay, making them of particular interest.

Mustard prodrugs for activation by Escherichia coli nitroreductase in gene-directed enzyme prodrug therapy.

Twenty nitrogen mustard analogues derived from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954, 1) were evaluated as candidate prodrugs for gene-directed enzyme prodrug therapy (GDEPT) in Chinese hamster V79 cell lines engineered to express Escherichia coli nitroreductase (NR). Structural variations within the series included the use of N-dihydroxypropyl and (N-dimethylamino)ethyl carboxamide side chains, the use of chloro, bromo, mesyl, and iodo leaving groups on the mustards, and regioisomeric changes. The compounds were assayed for cytotoxicity (IC50) with the NR-expressing and controls of non-NR-expressing cell lines. The proportion of NR-expressing cells required in a mixture for nonexpressing cells to experience 50% of their cytotoxicity (termed the TE50) was used to assess the compounds' ability to induce a bystander effect. This study suggests that 5-[N,N-bis(2-bromoethyl)amino]-2,4-dinitrobenzamide (8), 5-[N,N-bis(2-iodoethyl)amino]-2,4-dinitrobenzamide (9), 2-[N,N-bis(2-bromoethyl)-amino]-3,5-dinitrobenzamide (13), and 2-[N,N-bis(2-iodoethyl)amino]-3,5-dinitrobenzamide (14) showed considerable improvements over 1, exhibiting greater potency, higher IC50 ratios, and lower TE50s, and are thus superior prodrugs to 1 for GDEPT.

Synthesis and evaluation of 4-substituted analogues of 5-[N,N-bis (2-chloroethyl)amino]-2-nitrobenzamide as bioreductively activated prodrugs using an Escherichia coli nitroreductase.

2,4-Dinitrobenzamide mustards, exemplified by the parent compound SN 23862 (2) are activated under aerobic conditions by an Escherichia coli nitroreductase enzyme (NR2) via selective reduction of the 2-nitro group, and are thus of interest as prodrugs for antibody-directed enzyme-prodrug therapy (ADEPT). A series of related compounds 12a-12d, where the 4-nitro group of 2 was replaced by other substituents of varying electronic properties, were prepared and evaluated as potential ADEPT prodrugs. One-electron reduction potentials of the compounds correlated well with the substituent sigma m values, with the exception of the unsubstituted (4-H) analogue 13, which had a much lower value than expected on electronic grounds, due to a coplanar conformation of the mustard. The cytotoxicities of the compounds towards aerobic UV4 cells correlated positively with the electron-donating ability of the 4-substituent (measured by sigma p values), indicating that the cytotoxicities of the compounds in the absence of the NR2 enzyme are due substantially to the parent (unreduced) compounds. A positive, although less strong, correlation was seen between the electronic properties of the 4-substituent and their cytotoxicities in the presence of the NR2 enzyme, suggesting that, in this closely related series, the degree of activation by the enzyme is significantly dependent on the reduction potential of the 2-nitro group. While the 4-SO2Me derivative 12d was the next most preferred substrate after the parent 2, it was considerably less so (degree of activation as measured by IC50 ratio of 26 compared with 145), despite the similar electronic properties of the two 4-substituents.

Hypoxia-selective antitumor agents. 14. Synthesis and hypoxic cell cytotoxicity of regioisomers of the hypoxia-selective cytotoxin 5-[N,N-bis(2-chloroethyl)amino]-2,4-dinitrobenzamide.

A series of regioisomers of the novel hypoxia-selective cytotoxin (HSC) 5-[N,N-bis(2-chloroethyl)-amino]-2,4-dinitrobenzamide (2a) have been prepared by displacement of the chloro group from methyl chlorodinitrobenzoates or the corresponding carboxamides with diethanolamine, followed by dimesylation and mesylate displacement with LiCl. The compounds fall into two classes, where the two nitro groups have either a meta or an ortho (or para) disposition to each other. The four meta derivatives had one-electron reduction potentials in the range -340 to -375 mV, similar to that of the known isomer 2a, while the other isomers had much higher values (-262 to -285 mV). The meta derivatives were much less cytotoxic to AA8 cells under aerobic conditions (IC50s from 75 to 470 microM) than were the other compounds (IC50s from 1.6 to 20 microM). However, the ratios of IC50s of the compounds in repair-proficient (AA8) and repair-deficient (UV4) cell lines varied, indicating differing contributions of DNA alkylation to aerobic toxicity between the isomers, with no clear relationship between this and nitro group disposition. The hypoxic selectivities of the (dimethylamino)ethylcarboxamide analogues for each isomer were determined by clonogenic assay against both AA8 and UV4 cells. With one exception, the meta derivatives showed excellent hypoxic selectivities (ca. 45-115-fold) against UV4 cells, while the ortho or para isomers had little selectivity (ca. 2-7-fold). A possible reason may be that the latter compounds, with higher reduction potentials, undergo rapid bioreduction even under aerobic conditions. None showed hypoxic selectivities greater than 2-3-fold against AA8 cells. The 3-[N,N-bis(2-chloroethyl)amino]-2,6-dinitrobenzamide isomer (5b), which showed the highest hypoxic selectivity for UV4 cells in this series, was active against both hypoxic and aerobic cells in KHT tumors in mice at well-tolerated doses, and showed superior in vivo activity to the previously studied 2,4-dinitro isomer 2b.

Effect of substituents on the metabolism of nitracrine in rat.

1. Male Wistar rats were treated with either the antitumour agent nitracrine (1-nitro-9-(3'-dimethylamino-N-propylamino)-acridine; NC), 4-methoxy-NC, NC-aliphatic-N-oxide, 4-methoxy-NC-aliphatic-N-oxide, or NC-aromatic-N-oxide (30 mumol/kg, via the femoral vein) and the major biliary and urinary metabolites analysed by hplc. 2. No NC or 4-methoxy-NC were detected in bile or urine of rat treated with NC or 4-methoxy-NC respectively, whereas the aliphatic N-oxides of NC and 4-methoxy-NC were recovered largely unchanged in both bile and urine. 3. NC-aromatic-N-oxide was rapidly and extensively converted to a major polar biliary product. This product was also synthesised enzymatically from NC-aromatic-N-oxide using rat liver cytosol and has been identified by mass and 1H-nmr spectrometry as 1-(S-glutathionyl)-9-(3'-dimethylamino-N-propylamino)-acridine-N(10)-oxi de. 4. The equivalent 1-(S-glutathionyl) conjugate appears to be formed from NC, and excreted in bile as a minor product, but not from 4-methoxy-NC. Further experiments with cytosol indicate that direct displacement of the nitro group by GSH is mediated by GSH transferase. 5. Finally, the major biliary metabolite of NC has been provisionally identified as a glucuronide of 1-nitro-2-hydroxy-NC. 6. It is concluded that, for at least a significant fraction of NC, nitroreduction does not occur. Further, N-oxidation of the aliphatic (but not the aromatic ring) nitrogen, plus 4-methoxy substitution, decreases the overall metabolism of NC in the rat.

Tyrosine kinase inhibitors. 10. Isomeric 4-[(3-bromophenyl)amino]pyrido[d]-pyrimidines are potent ATP binding site inhibitors of the tyrosine kinase function of the epidermal growth factor receptor.

Following the discovery of the very high inhibitory ability of the 4-[(3-bromophenyl)amino]-quinazolines against the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (e.g., 3, IC50 0.029 nM), four series of related pyrido[d]pyrimidines bearing electron-donating groups at the 6- or 7-positions have been synthesized and evaluated. The compounds were prepared by nucleophilic substitution of the corresponding 6- and 7-fluoro analogues. While members of all series showed potent inhibitory activity against isolated EGFR, there were important differences between the different isomeric pyrido[d]pyrimidines and the parent quinazolines. Overall, the [3,4-d] and [4,3-d] series were the most potent, followed by the [3,2-d] compounds, with the [2,3-d] analogues being least active. Whereas in the parent quinazoline series the addition of steric bulk to a 6- or 7-NH2 substituent (i.e., NHMe and NMe2 groups) dramatically decreased potency, no such trend was discernable in the [3,2-d] series. Furthermore, in the 7-substituted pyrido[4,3-d]- and 6-substituted pyrido[3,4-d]pyrimidine series, and to a limited extent in the 7-substituted pyrido[2,3-d] series, such substitution increased potency dramatically, to the extent that the 7-(methylamino)pyrido[4,3-d]pyrimidine (5f) (IC50 0.13 nM) and 6-(methylamino)pyrido[3,4-d]pyrimidine (7f) (IC50 0.008 nM) constitute important new leads. Selected compounds were evaluated for their ability to inhibit EGFR autophosphorylation in A431 cells, and a positive quantitative correlation was found between this activity and inhibitory activity against the isolated enzyme.

Tyrosine kinase inhibitors. 9. Synthesis and evaluation of fused tricyclic quinazoline analogues as ATP site inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor.

Following the discovery of 4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (4; PD 153035) as an extremely potent (IC(50) 0.025 nM) inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), several fused tricyclic quinazoline analogues have been prepared and evaluated for their ability to inhibit the enzyme. The most potent compound was the linear imidazo[4,5-g]quinazoline (8), which exhibited an IC(50) of 0.008 nM for inhibition of phosphorylation of a fragment of phospholipase C-gamma-1 as substrate. While N-methyl analogues of 8 showed similar potency, analogous N-[2-(dimethylamino)ethyl] derivatives were less effective. The next most potent compounds were the linear pyrazoloquinazolines (19 and 20) (IC(50)s 0.34 and 0.44 nM) and pyrroloquinazoline (21) (IC(50) 0.44nM), while several other linear tricyclic ring systems of similar geometry to 8 (triazolo-, thiazolo-, and pyrazinoquinazolines) were less effective. In the imidazo[4,5-g]quinazoline and pyrroloquinazoline series, the corresponding angular isomers were also much less effective than the linear ones. These results are consistent with structure-activity relationship studies previously developed for the 4-[(3-bromophenyl)amino] quinazolines, which suggested that small electron-donating substituents at the 6- and 7-positions were desirable for high potency. Cellular studies of the linear imidazoloquinazoline 8 show that it can enter cells and rapidly and very selectively shut down EGF-stimulated signal transmission by binding competitively at the ATP site of the EGFR.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase.

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.