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Keywords:

  • benzoxazolyl hydrazones;
  • benzimidazolyl hydrazones;
  • antitumor agents;
  • colon cancer.

Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Here we describe the effects of novel benzoxazol-2-yl and benzimidazol-2-yl hydrazones derived from 2-pyridinecarbaldehyde and 2-acetylpyridine. The IC50 values for inhibition of cell proliferation in KB-3-1, CCRF-CEM, Burkitt's lymphoma, HT-29, HeLa, ZR-75 and MEXF276L by most of the novel compounds are in the nanomolar range. In colony-forming assays with human tumor xenografts the compounds 2-actylpyridine benzoxazol-2-ylhydrazone (EPH52), 2-acetylpyridine benzoimidazol-2-ylhydrazone (EPH61) and 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone (EPH116) exhibited above-average inhibition of colon carcinoma (IC50 = 1.3–4.56 nM); EPH52 and EPH116 also exhibited above-average inhibition of melanoma cells. As shown with human liver microsomes, EPH116 is only moderately metabolized. The compound inhibited the growth of human colon cancer xenografts in nude mice in a dose-dependent manner. Thiosemicarbazones derived from 2-formylpyridines have been shown to be inhibitors of ribonucleotide reductase (RR). The following results show that RR is not the target of the novel compounds: cells overexpressing the M2 subunit of RR and resistant to the RR inhibitor hydroxyurea are not cross-resistant to the novel compounds; inhibition of RR occurs at 6- to 73-fold higher drug concentrations than that of inhibition of cell proliferation; the pattern of cell cycle arrest in S phase induced by the RR inhibitor hydroxyurea is not observed after treatment with the novel compounds; and a COMPARE analysis with the related compounds 2-acetylpyrazine benzothiazol-2-ylhydrazone (EPH95) and 3-acetylisoquinoline benzoxazol-2-ylhydrazone (EPH136) showed that the pattern of these compounds is not related to any of the standard antitumor drugs. Therefore, these novel compounds show inhibition of colon cancers and exhibit a novel mechanism of action. © 2001 Wiley-Liss, Inc.

Theoretically, approximately 50% of the patients diagnosed with cancer can be cured by surgery and radiation therapy since their tumors have not spread. Of the remaining 50%, about 10% are curable with systemic chemotherapy, including children with leukemia and sarcomas and adults with testicular cancer and choriocarcinoma. However, most metastatic cancers are currently not curable by chemotherapy. Half of all cancer patients fail to respond to chemotherapy or relapse from the initial response and ultimately die from their metastatic disease.1 Colorectal cancer affects about 1 person in 20 in Western populations, representing 15% of all cancers.2 Because the survival rate is low, new drugs for this type of cancer are desired.2 The hope for improvement in treatment outcome resides in continued research designed to optimize the administration of currently available agents and to discover novel therapeutic products.1 Taxanes and camptothecins, for example, are new classes of compounds showing promising results in ovarian, breast and colon cancers.1 To obtain additional compounds for cancer therapy, drugs directed to novel targets would be important.

Hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RR), is in clinical use as an anticancer agent.3, 4 Thiosemicarbazones (TSCs) derived from 2-formyl and 2-acetyl pyridine (Fig. 1, structures 1a,b) represent the most active inhibitors of RR identified to date,5, 6 but unfortunately have shown high in vivo toxicity.7, 8 It has been proposed that the in vivo toxicity of the α-(N)-heteroaromatic TSCs might be caused by the release of toxic H2S during the metabolism of such compounds.9 Using this information, we designed compounds in which the thiocarbamoyl substructure of the compounds 1a and 1b was replaced by a benzothiazole system.10 It is envisaged from these compounds (Fig. 1, structures 2a,b) that release of H2S during metabolism is unlikely to occur. In an in vitro antiproliferative assay, compounds 2a and b (Fig. 1) and congeners were found to be 80- to 100-fold more active than compounds 1a and b.10 These analogues of the compounds 2a and b (Fig. 1) were also more potent than HU and showed no cross-resistance to the HU-resistant cell line K562-DFMOr, which overexpresses the RR M2 protein subunit.10

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Figure 1. Structures of the new compounds.

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Based on the promising results obtained with compounds 2a and b (Fig. 1) and their analogues, we investigated the effects of replacement of the benzothiazole ring by the isosteric heterocycles benzoxazole and benzimidazole. Here we report on the synthesis of these compounds (Fig. 2) and their effects on tumor cells in vitro and in vivo.

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Figure 2. Synthetic pathway of the novel hydrazones.

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MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Chemicals

2-Chlorobenzoxazole [3a], 2-chloro-1(3)H-benzimidazole [3b], pyridine-2-carbaldehyde [5a] and 2-acetylpyridine [5b] were purchased from Sigma Aldrich Chemicals (Vienna, Austria) and were used without further purification. 2-Chloro-1-methyl-1H-benzimidazole [3c],11 2-hydrazinobenzoxazole [4a],12 and 2-hydrazino-1H-benzimidazole [4b]13 were synthesized according to published procedures.

2-Hydrazino-1-methylbenzimidazole [4c] was obtained through the following procedure: 2-Chloro-1-methyl-benzimidazole [3c] (10 g, 0.06 mol) and 95% hydrazine hydrate (10 equivalents) were heated at 120°C for 2 hr. After cooling to room temperature the precipitate was filtered off and washed several times with water and dried to give 7.79 g (80%) of buff white crystals. Because of the instability of 4c, the product thus obtained was used in subsequent reactions without purification. 1H-NMR [dimethyl sulfoxide (DMSO)-d6: δ = 3.47 (s, 1H, CH3), 4.27 (br s, 2H, NH2), 6.89–7.05 (m, 2H, arom. H), 7.02–7.29 (m, 2H, arom. H), 7.76 (br s, 1H, NH).

Synthesis of the novel hydrazones

A mixture of the carbonyl compound 5a or b (10 mmol) and the hydrazine 4a or b (10 mmol) in methanol (10 ml) containing 3 drops of glacial acetic acid was heated to reflux until thin layer chromatography monitoring indicated no further conversion (approximately 15 hr). The resulting hydrazones precipitated on cooling to 5°C overnight. They were filtered off and recrystallized from an appropriate solvent. A slightly modified procedure was used to prepare 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone (EPH116) and pyridine-2-carbaldehyde 1-methylbenzoimidazol-2-ylhydrazone (EPH117). The reactions were carried out at ambient temperature and took 3 days. The products that precipitated out as the reaction proceeded were collected by filtration and recrystallized from methanol/water.

(E)-Pyridine-2-carbaldehyde benzoxazol-2-ylhydrazone (EPH51; Fig. 2).

EPH51 was recrystallized from MeOH to give 2.10 g (90% yield) of white crystals; m.p. 243°–245°C; 1H-NMR (DMSO-d6): δ = 7.09–7.57 (m, 5H, arom. H), 7.83–8.04 (m, 2H, arom. H), 8.26 (s, 1H, N=CH), 8.61 (ddd, 1H, pyridine-H6, J = 4.9, 1.6, 1.0 Hz), 12.25 (br s, 1H, NH); ms: 238.1 (M+, 84%), 210.1 (36%), 160.0 (29%), 133.9 (53%), 104.9 (51%), 79.0 (100%), 51.0 (49%).

(E)-2-Acetylpyridine benzoxazol-2-ylhydrazone (EPH52; Fig. 2).

EPH52 was recrystallized from EtOH/DIPE to give 2.14 g (85% yield) of colorless needles; m.p. 192°–194°C; 1H-NMR (DMSO-d6): δ = 2.42 (s, 3H, CH3), 7.06–7.52 (m, 5H, arom. H), 7.85 (ddd, 1H, pyridine-H4, J = 8.0, 7.3, 1.8 Hz), 8.22 (br d, 1H, arom. H), 8.60 (ddd, 1H, pyridine-H6, J = 4.8, 1.8, 0.9 Hz), 11.42 (br s, 1H, NH); ms: 252.1 (M+, 80%), 174.0 (27%), 148.0 (46%), 104.9 (43%), 79.0 (100%), 78.0 (92%), 51.0 (47%).

(E)-Pyridine-2-carbaldehyde benzimidazol-2-ylhydrazone (EPH60; Fig. 2).

EPH60 was recrystallized from EtOAc to give 1.78 g (75% yield) of cream crystals; m.p. 269°–271°C; 1H-NMR (DMSO-d6): δ = 6.93–7.02 (m, 2H, arom. H), 7.21–7.35 (m, 3H, arom. H), 7.84 (ddd, 1H, pyridine-H4, J = 8.0, 7.3, 1.8 Hz), 8.05 (s, 1H, N=CH), 8.22 (br d, 1H, arom. H), 8.54 (ddd, 1H, pyridine-H6, J = 4.8, 1.6, 1.0 Hz), 11.65 (br s, 2H, NH); ms: 237.1 (M+, 68%), 208.1 (26%), 159.0 (85%), 133.0 (64%), 104.9 (100%), 78.0 (29%), 51.0 (24%).

(E)-2-Acetylpyridine benzimidazol-2-ylhydrazone (EPH61; Fig. 2).

EPH61 was recrystallized from EtOAc/DIPE to give 1.76 g (70% yield) of white crystals; m.p. 162°–165°C; 1H-NMR (DMSO-d6): δ = 2.39 (s, 3H, CH3), 6.94–7.04 (m, 2H, arom. H), 7.20–7.36 (m, 3H, arom. H), 7.82 (ddd, 1H, pyridine-H4, J = 8.1, 7.4, 1.9 Hz), 8.48 (br d, 1H, arom. H), 8.56 (ddd, 1H, pyridine-H6, J = 4.9, 1.8, 0.8 Hz), 10.85 (br s, 1H, NH), 11.51 (br s, 1H, NH); ms: 251.1 (M+, 89%), 236.1 (35%), 173.0 (100%), 147.0 (47%), 132.0 (49%), 105.0 (55%), 78.0 (54%), 51.0 (29%).

(E)-Pyridine-2-carbaldehyde 1-methylbenzimidazol-2-ylhydrazone (EPH117; Fig. 2).

EPH117 was recrystallized from DIPE to give 1.51 g (60% yield) of colorless crystals; m.p. 119°–203°C; 1H-NMR (DMSO-d6): δ = 3.46 (s, 3H, NCH3), 6.97–7.30 (m, 5H, arom. H), 7.74–7.84 (m, 1H, arom. H), 8.16 (s, 1H, N=CH), 8.23 (br d, 1H, arom. H), 8.52 (ddd, 1H, pyridine-H6, J = 4.8, 1.6, 1.0 Hz), 11.24 (br s, 1H, NH); ms: 251.1 (M+, 100%), 222.1 (20%), 173.0 (71%), 146.0 (31%), 131.0 (22%), 119.0 (23%), 92.0 (20%).

(E)-2-Acetylpyridine 1-methylbenzimidazol-2-ylhydrazone (EPH116; Fig. 2).

EPH116 was recrystallized from DIPE to give 1.46 g (55% yield) of yellow crystals; m.p. 131°–133°C; 1H-NMR (DMSO-d6): δ = 2.41 (s, 3H, CH3), 3.47 (s, 3H, NCH3), 6.95–7.30 (m, 5H, arom. H), 7.76 (ddd, 1H, pyridine-H4), 8.46 (br d, 1H, arom. H), 8.53 (ddd, 1H, pyridine-H6, J = 5.0, 1.6, 1.0 Hz), 11.03 (br s, 1H, NH); ms: 265.1 (M+, 100%), 250.1 (36%), 187.0 (89%), 161.0 (28%), 146.0 (38%), 133.0 (31%), 118.9 (31%), 104.0 (22%), 78.0 (23%).

The stereochemistry at the -C=N- of the isolated compounds was determined as described previously using 1H-NMR spectroscopy and homonuclear NOE-difference experiments.14 The most characteristic difference in the chemical shifts of the E- and Z-isomeric forms are: in the E-form δ(NH) = 9–12 ppm and in the Z-form δ(NH) = 14–15 ppm. Thus considering these findings, E configuration is assigned to compounds EPH 51, 52, 60, 61, 116 and 117 with δ(NH) ≈11.03–12.25 ppm.

Cell lines and tissue culture

KB-3-1 (human oral epidermoid carcinoma), and multidrug resistant KB-C1 cells15 were obtained from Dr. M. M. Gottesman (National Cancer Institute, Bethesda, MD). KB cells resistant to HU (KB-HU)16 were kindly donated by Dr. Y.-C. Cheng (Yale University, New Haven, CT). All KB cell lines were grown in Dulbecco's modified Eagle's medium (4.5 g glucose/L). To the stock cultures of KB-C1 cells 1μg colchicine/ml medium, and to KB-HU cells 1 mM HU was added every other week. CCRF-CEM (acute lymphoblastic leukemia, ATCC CCL 119), Burkitt's lymphoma (CA 46, ATCC CRL 1648), HeLa (epithelioid cervix carcinoma, ATCC CCL 2), ZR-75-1 (breast carcinoma, CRL 1500) and MEXF 276L (melanoma) were grown in RPMI 1640 medium. HT-29 cells (colon adenocarcinoma, ATCC HTB 38) were grown in McCoy's 5A medium. The media were supplemented with 10% fetal calf serum (except Burkitt's lymphoma with 15%), 2 mM glutamine and 50 μg gentamicin/ml.

Inhibition of cellular proliferation

The cells were seeded into 96-well plates. After an initial incubation for 4 hr, various concentrations of the compounds were added to the cells and exposed continuously at 37°C in a humidified atmosphere of 95% air and 5% CO2 for 72 hr. The compounds were dissolved in DMSO. The concentration of DMSO was 0.5% and this was not toxic. Inhibition of cell proliferation of HeLa, HT-29, ZR-75-1, KB and MEXF 276L cells was determined by the sulforhodamine B assay.17 In this assay 3,000–10,000 cells in 200 μl medium were seeded per well into 96-well plates. Dose–response curves for CCRF-CEM and Burkitt's lymphoma cells were determined with an MTT assay18 from Boehringer Mannheim (Mannheim, Germany). Approximately 10,000 cells/100 μl were seeded in 96-well plates. After an incubation period of 72 hr the absorption was detected by a microplate reader (Model 3550, Bio-Rad, Hercules, CA).

Colony-forming assay

Solid human tumor xenografts growing in nude mice were removed, mechanically disaggregated and subsequently incubated with an enzyme cocktail consisting of collagenase (1.2–1.8 U/ml), DNAse (375 U/ml) and hyaluronidase (29 U/ml) in RPMI 1640 medium at 37°C for 30 min. The cell mixture was passed through sieves of 200- and 50-μm mesh size and washed thereafter twice with phosphate-buffered saline (PBS). The percentage of viable cells was determined in a Neubauer counting chamber using Trypan blue exclusion.

The clonogenic assay was performed according to a modified 2-layer soft agar assay introduced by Hamburger and Salmon.19 The bottom layer consisted of 0.2 ml of Iscove's modified Dulbecco's medium with 20% fetal calf serum and 0.75% agar. Then 8 × 103 to 1.6 × 104 cells were added to 0.2 ml of the same culture medium containing 0.4% agar and plated in 24-multiwell dishes onto the base layer. The compounds were applied 1 day after cell seeding in 0.2 ml medium (continuous exposure). Every dish included 6 control wells containing only the vehicle and drug-treated groups in triplicate at 6 concentrations. Cultures were incubated at 37°C and 7% CO2 in a humidified atmosphere for 7–15 days depending on the doubling time of the tumor stem cells. Tumor colonies with a diameter of 50 μm were counted with an automatic image analysis system. Twenty-four hours before evaluation, vital colonies were stained with a sterile aqueous solution of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (1 mg/ml, 100 μl/well). Colony growth inhibition was expressed as treated/control values in percentage and used for the determination of IC70 values.

Metabolism studies

Pooled human liver microsomes (Gentest, Woburn, MA; 0.4 mg protein/ml), NADPH (1 mM), isocitric acid (5 mM) and isocitric dehydrogenase (Sigma, Vienna, Austria) were pre-incubated at 37°C in 0.1 M phosphate buffer pH 7.4 (final volume 250 μl). The reaction was started by the addition of EPH116 (final concentration 100 μM) in DMSO (concentration of not more than 0.75% v/v) and the metabolism was assessed from 0 to 60 min. The reaction was stopped by the addition of 500 μl methanol, samples were centrifuged (10.000g, 5 min) and 100 μl of the clear supernatant was injected onto the high performance liquid chromatography (HPLC) column. HPLC was performed using a Shimadzu liquid chromatograph consisting of a LC-6A pump, a SIL-6B autoinjector and a SPD-6AV UV-VIS detector that was set at a wavelength of 368 nm. A Nina data system (Nuclear Interface, Muenster, Germany) determined retention times and peak areas. Chromatographic separations were performed on a Hypersil BDS-C18 (5 μm, 250 × 4.6 mm; 3 mm ID, Astmoor, England) preceded by a Hypersil BDS-C18 precolumn (5 μM, 10 × 4.6 mm ID) at a flow rate of 1.0 ml/min. The mobile phase consisted of heptanesulfonic acid (5 mM) in potassium phosphate (50 mM, pH 4.0 with phosphoric acid) and of methanol (6:4, v/v).

Human tumor xenografts in nude mice

Male nude mice (6–8 weeks old) of NMRI genetic background were used. CXF 280 colon tumor cells were implanted subcutaneously in both flanks of athymic nude mice. Treatment was started as soon as the tumors reached a median diameter of 6 mm. Mice were randomly assigned to treatment and control groups (5 animals per group). About 5–7 tumors were evaluable per treated group. The compounds were dissolved in DMSO and administered intraperitoneally on days 1 and 5. Tumor size was quantified by 2-dimensional measurement with calipers at the times indicated in Figure 6. Tumor volumes were calculated according to the formula V = (a × b2)/2, where a is the larger diameter and b the smaller. Data evaluation was performed using specifically designed software by plotting relative tumor volumes against time. Toxicity was assessed by measurement of the body weight of the animals.

Incorporation of [2-14C]-cytidine into DNA

Incorporation of [2-14C]cytidine into DNA was used as an indicator for inhibition of RR. The assay was performed in intact Burkitt's lymphoma cells by a modification of the procedure described previously.20 In brief, 3 ml (1–2 × 106/ml) of exponentially growing cells were incubated (37°C) in medium with various drug concentrations of each compound. After 90 min, 0.7 μCi [2-14C]cytidine (2–10 Ci/mmol, ICN Biomedicals, Meckenheim, Germany) were added to each sample and incubated for another 60 min. Subsequently, the cells were washed twice with ice-cold PBS, resuspended in 1 ml ice-cold trichloroacetic acid and transferred to Eppendorf tubes. After an incubation period on ice for 20 min, the disrupted cells were centrifuged at 3,000g for 5 min (+4°C). The pellet was resuspended in 800 μl of 80 mM Tris-HCl (pH 8) to which 20 μl DNase-free RNase (10 mg/ml) was added. After a 2-hr incubation at 37°C, the solution was cooled on ice, and 200 μl 50% trichloroacetic acid was added. The solution was incubated at + 4°C overnight. The trichloroacetic acid-precipitable material was obtained by centrifuging at 4,000g (+4°C) for 5 min. The pellet was washed 4 times with 1 ml of ice-cold 5% trichloroacetic acid and dissolved in 0.4 ml of 1.25 M NaOH overnight and counted in 2 ml scintillation fluid (Ultima Gold, Packard, Meriden, CT) to evaluate the DNA-incorporated [2-14C]cytidine. The IC50 was determined by using untreated controls as 100%.

Cell cycle analysis and apoptosis

Cell cycle analysis was performed as described previously.21 Logarithmically growing Burkitt's lymphoma cells were treated with concentrations corresponding to the 2-fold IC50 of the compounds for 24 and 48 hr. Subsequently 250 μg/ml propidium iodide (Sigma, Vienna, Austria) dissolved in 5% Triton-100 and 10 mg/ml RNase A (Sigma) were added. After incubation at room temperature for 1 hr, the cell cycle was analyzed by a FACscan (Becton Dickinson, Mountain View, CA). In the same experiment the percentage of apoptotic cells was quantified by analysis of the hypodiploid DNA peak as described elsewhere.22

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Screening studies

Initial screening studies in tissue culture cells revealed that the new compounds exhibit antiproliferative activity at very low concentrations. The cells most sensitive to these compounds are Burkitt's lymphoma and CCRF-CEM cells (Fig. 3). For example, the IC50 values for inhibition of cell proliferation by EPH52 and EPH116 for Burkitt's lymphoma was 9 and 4.3 nM, respectively, and by EPH116 for CCRF-CEM the IC50 value was 7 nM. Interestingly, the compounds do not show any significant cross-resistance to HU-resistant KB-HU and to mdr1-expressing multidrug-resistant KB-C1 cells. This panel of cell lines was used because it reflects in a small scale the situation of clinical anticancer treatment. In general, leukemic cells (CCRF-CEM and Burkitt's lymphoma) are more sensitive to antiproliferative agents compared with colon carcinoma and melanoma cells, which are less sensitive.10, 23 However, the IC50 values of HeLa cervix carcinoma and HT-29 colon carcinoma cells to some of the new compounds are similar to that of CCRF-CEM or Burkitt's lymphoma cells. The hydrazones derived from 2-acetylpyridine (EPH52, EPH61 and EPH116) generally exhibited higher antiproliferative activity than those derived from 2-formylpyridine (EPH51, EPH60 and EPH117). For this reason these compounds were selected for further investigations.

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Figure 3. IC50 values of the novel compounds for the indicated cell lines. The IC50 values (indicated in μM) were determined as described in Material and Methods. The means of at least 3 independent experiments in which duplicate determinations were taken within each experiment (±SEM) are indicated. [KB-3-1, human oral epidermoid carcinoma; KB-HU, KB cells resistant to hydroxyurea; KB-C1, multidrug resistant cells; CCRF-CEM, acute lymphoblastic leukemia cells; Burkitt, Burkitt's lymphoma cells; HT-29, colon adenocarcinoma cells; HeLa, epithelioid cervix carcinoma cells; ZR-75-1, breast carcinoma cells and MEXF276L, melanoma cells; EPH51, (E)-pyridine-2-carbaldehyde benzoxazol-2-ylhydrazone; EPH52, 2-actylpyridine benzoxazol-2-ylhydrazone; EPH60, (E)-pyridine-2-carbaldehyde benzimidazol-2-ylhydrazone; EPH61, 2-acetylpyridine benzoimidazol-2-ylhydrazone; EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone; EPH117, pyridine-2-carbaldehyde 1-methylbenzoimidazol-2-ylhydrazone.]

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Detailed in vitro studies

To obtain more detailed information about the antiproliferative activity of the compounds they were tested in clonogenic assays using human tumor xenografts. An excellent correlation of drug response in clonogenic assays and in patients has been found.24 The antiproliferative effects of the new compounds were tested with human bladder, colorectal, non-small cell lung, small cell lung, mammary, melanoma, prostate and renal tumor cells. A comparison with HU showed that the new compounds exhibit potent antiproliferative activity (Fig. 4). This assay also showed that the compounds exhibited unusual sensitivity to each of the 3 colon carcinoma cell lines tested (Fig. 4, bars extending to the left represent sensitivity of the cell line in excess of the average sensitivity of all tested cell lines). The 3 compounds also inhibited the proliferation of the small cell lung carcinoma cells, and inhibition by EPH52 and EPH116 of both melanoma cell lines were above the average.

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Figure 4. Effects of EPH52, EPH61, EPH116 and hydroxyurea in the colony-forming assay. Variations of individual IC70 (drug concentration needed to reduce colony formation to 30% of control value) from mean value are expressed as bars in the logarithmically scaled axis. Bars to the left demonstrate IC70 lower than the mean value (cells are more sensitive than the average). Bars to the right demonstrate less sensitivity than the average of all cells. Arrows show that with the concentrations indicated the IC70 value was not achieved. The means of 2 independent experiments in which triplicate determinations were taken within each experiment are indicated. (EPH52, 2-actylpyridine benzoxazol-2-ylhydrazone; EPH61, 2-acetylpyridine benzoimidazol-2-ylhydrazone; EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone.)

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EPH116 metabolism

Biotransformation may lead to a fast inactivation of drugs and reduction of the therapeutic effects in vivo. To gain an insight into the extent of the metabolism of EPH116, a metabolism study was undertaken using human liver microsomes as an in vitro model. As shown in Figures 5a and b, EPH116 was only moderately metabolized to 2 not yet identified metabolites. Sixty minutes after the addition of EPH116 to the incubation medium, 83% of unmetabolized drug could be quantified, indicating that the compound was not degraded within a short period of time and may be useful in in vivo experiments. As metabolite formation was strongly dependent on NADPH and as metabolic activity was inhibited by CO (data not shown), cytochrome P450 isoenzyme(s) may therefore catalyze EPH116 biotransformation. Efforts are currently underway to isolate and elucidate the structures of the metabolites M1 and M2.

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Figure 5. Metabolism of EPH116. High performance liquid chromatography (ultraviolet) profiles of EPH116 and its metabolites are shown. Pooled human liver microsomes were incubated with 100 μM EPH116 at 37°C for 60 min (a) without and (b) in the presence of NADPH. Data represent the mean of 2 independent determinations. (EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone; M1, metabolite 1; M2 = metabolite 2.)

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Human colon xenografts in nude mice

To investigate whether the novel compounds exhibit antitumor activity in vivo, we treated nude mice with human CXF 280 colon tumor xenografts. As shown in Figure 6a, EPH116 inhibited the tumor growth. The dose of 20 mg EPH116/kg body weight given on days 1 and 5 after randomization led to a reduction of the tumor to 62% of the untreated control on day 35 after treatment. This concentration did not decrease the body weight of the animals (Fig. 6b), indicating that it is not toxic; 40 mg/kg led to a reduction of tumor growth to 36% (day 35) compared with the untreated controls. The mean body weight was reduced to 94% on day 28 and to 96% on day 35 compared with untreated controls (Fig. 6b). From these data, it seems that the compound is not toxic. However, 1 animal died on day 6 and 1 on day 33 after randomization, indicating that 40 mg/kg is above the maximally tolerated dose. Although the treatment was on days 1 and 5, the highest antitumor effect of the compound was observed on days 28 and 35 after randomization. Reasons for the delayed effect might be that the active agent is one of the metabolites or that the compound is bound to plasma proteins from which it is released gradually. A concentration of 80 mg/kg led to an inhibition of tumor growth to 26% (day 14) of untreated controls (data not shown). However, all the animals died by day 16. Treatment of the mice xenografted with human MAXF 401 mammary carcinoma cells with 40 mg EPH116/kg body weight did not lead to a reduction of tumor growth (data not shown). This finding is in agreement with the results obtained by clonogenic assays in which colon carcinomas were more sensitive than mammary carcinomas (Fig. 4).

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Figure 6. Tumor volume (a) and body weight (b) after treatment with EPH116. Nude mice (5 per group) were xenotransplanted with colon carcinoma cells and treated with the indicated doses of EPH116 on days 1 and 5 after randomization. The means ± SD in (a) are indicated. The means of the relative body weights are shown in (b). (EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone.)

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Incorporation of [2-14C]cytidine into DNA

To obtain information about the mechanism of action of the novel compounds, incorporation of [2-14C]cytidine into DNA was measured in intact Burkitt's lymphoma cells to provide evidence that the novel compounds are capable of inhibiting RR activity in situ. Table I shows that EPH52, EPH61 and EPH116 inhibited [2-14C]cytidine incorporation. In contrast to HU, the IC50 for cell proliferation is lower than that of [2-14C]cytidine incorporation into DNA. The ratios of IC50 [2-14C]cytidine incorporation/IC50 cell proliferation were markedly different between HU and the new compounds (Table I). This finding is an indication that RR seems not to be the primary target of the new compounds.

Table I. Comparison of Inhibition of Cell Proliferation and 14C-Cytidine Incorporation into DNA
CompoundIC50 (μM) cell proliferationIC5014C-cytidine incorporationIC5014C-cytidine incorpor./IC50 cell proliferation
  1. IC50 values for cell proliferation were taken from Figure 3 (Burkitt's lymphoma cells). IC50 for 14C-cytidine incorporation was determined as described in Material and Methods. The means of 2 independent experiments in which duplicate determinations were taken within each experiment were used for the calculations of the IC50 values. The ratio was calculated by IC50-cytidine incorporation/IC50-cell proliferation.–EPH52, 2-actylpyridine benzoxazol-2-ylhydrazone; EPH61, 2-acetylpyridine benzoimidazol-2-ylhydrazone; EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone.

Hydroxyurea14037.20.26
EPH520.0090.333.3
EPH610.0440.36.8
EPH1160.0040.375.0

Cell cycle analysis

Inhibitors of ribonucleotide reductase lead to a p53-independent arrest of cells in the S phase (DNA synthesis) of the cell cycle.25 Therefore, cell cycle analysis (arrest in S phase) can indicate whether RR is involved in antiproliferative activity. For this reason the newly synthesized compounds were tested for their ability to inhibit cell cycle progression. A concentration corresponding to a 2-fold IC50 of cell proliferation (280 μM) of the RR-inhibitor HU increased the percentage of cells in S phase in Burkitt's lymphoma cells (Fig. 7a). However, treatment of these cells with 2-fold doses of EPH52 or EPH61 did not arrest cells in S phase significantly (Fig. 7b, c). A modest increase in the percentage of S-phase cells was observed after treatment with EPH116 (Fig. 7d). The pattern observed after treatment with HU was not similar to that after treatment with the new compounds, indicating that the mechanism of action was different from that of HU.

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Figure 7. Cell cycle analysis after treatment with hydroxyurea, EPH52, EPH61 and EPH116. Burkitt's lymphoma cells were treated with concentrations corresponding to the 2-fold IC50 for 24 or 48 hr. Cell cycle analysis was performed as described in Material and Methods. The means ± SD of 2 independent experiments in which duplicate determinations were taken within each experiment are indicated. (EPH52, 2-actylpyridine benzoxazol-2-ylhydrazone; EPH61, 2-acetylpyridine benzoimidazol-2-ylhydrazone; EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone.)

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Apoptosis

It has been shown that many antitumor agents are inducers of apoptosis. We quantified apoptotic cells in Burkitt's lymphoma cells after treatment with the new compounds. To compare the effects of different compounds, 2-fold IC50 concentrations were used for induction of apoptosis. EPH52 or EPH61 induced apoptosis to a slightly lesser extent than HU and camptothecin (Fig. 8). However, EPH116 is a potent inducer of apoptosis (Fig. 8).

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Figure 8. Induction of apoptosis by the novel compounds. Burkitt's lymphoma cells were treated with concentrations corresponding to 2-fold IC50 of the drugs indicated. Apoptotic cells were determined by the use of propidium iodide.22 Data represent the means ± SD of 2 independent experiments in which 2 samples were taken within each experiment. (EPH52, 2-actylpyridine benzoxazol-2-ylhydrazone; EPH61, 2-acetylpyridine benzoimidazol-2-ylhydrazone; EPH116, 2-acetylpyridine 1-methylbenzoimidazol-2-ylhydrazone.)

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

A variety of anticancer drugs are currently in clinical use. Some of these compounds can be applied successfully for the treatment of several neoplastic diseases such as leukemias or testicular cancer. However, the effect of anticancer drugs on solid tumors has been poor. Because the response of colon cancers to available anticancer chemotherapy has been poor,2 new drugs with improved efficacy are desired. The present results show that the novel hydrazones may be good candidates. In colony-forming assays with human tumor xenografts, which exhibit an excellent correlation with the tumor response in patients,24 high antiproliferative activity in colon carcinoma, small cell lung carcinoma and melanoma cell lines was observed (Fig. 4). The compounds seem not to be transported by the mdr1-encoded P-glycoprotein, because mdr1-expressing KB-C1 cells15 are not resistant to the compounds. Cells resistant against HU because of the overexpression of the M2 subunit of RR16 also do not show cross-resistance to the new compounds (Fig. 3). Because EPH116 is only moderately metabolized to 2 yet unknown biotransformation products (Fig. 5), we performed in vivo experiments with this compound. Treatment of mice with human colon carcinoma xenografts led to a dose-dependent inhibition of tumor growth (Fig. 6a).

Additional experiments were performed to obtain information about the mechanism of action of the new compounds. It has been shown that the parental structures (Fig. 1, structures 1a,b) are potent inhibitors of RR.5, 6 However, the results presented here illustrate that the major mechanism responsible for the antiproliferative activity of the novel compounds is not inhibition of RR. This hypothesis is substantiated by the following results: (i) Cells over-expressing the M2 unit of RR, which are resistant to HU,16 but not cross-resistant to the novel compounds. (ii) Inhibition of RR, as measured by incorporation of [2-14C]cytidine into DNA, occurred at higher concentrations compared with inhibition of cell proliferation. The ratio of IC50 cytidine incorporation/IC50 cell proliferation of the novel compounds was not comparable to that of the RR inhibitor HU (Table I). (iii) Cell cycle arrest in S phase induced by inhibitors of RR such as HU25 cannot be observed after treatment with the novel compounds (Fig. 7). (iv) The COMPARE program has proved useful in finding agents with activity patterns similar to that of a “seed compound.”26 At the National Cancer Institute the compounds 1-(2-pyrazinyl)-1-ethanone 1-(1,3-benzothiazol-2-yl)hydrazone (EPH95) and 1-(3-isoquinolyl)-1-ethanone 1-(1,3-benzoxazol-2-yl)hydrazone (EPH136) from this series were screened in vitro against the panel of 60 different human cell lines. A correlation coefficient of 0.55–0.6 is considered the lowest correlation that suggests a relationship with another compound.26 Using EPH95 as seed, the highest correlation coefficient with standard agents was 0.424 with 2,6-piperazinedione (ICRF-1), and that of EPH136 was 0.437 with 6-diazo-5-oxo-L-norleucine (DON) (see the results at http://dtp.nci.nih.gov/; Search; NSC No. 693631 for EPH95, NSC No. 693638 for EPH136; Cancer Data; Run COMPARE; Database: Standard Agents). The 2 compounds show good correlation coefficients with related compounds of this class—thiosemicarbazones derived from 2-acylpyridines and acetyldiazines, which do not inhibit RR,26,26 and 2-pyridylhydrazones derived from 2-benzoylpyridines and benzoyldiazines.23 Because all the correlation coefficients with the standard agents are below 0.55, these results illustrate that the mechanism of action of the novel hydrazones is different from that of the standard antitumor drugs. Therefore, their antitumoral activity is caused by a new and unknown mechanism. The major mechanism responsible for the antiproliferative activity of the novel compounds seems also not to be cell cycle arrest (Fig. 7). As shown in Figure 8, induction of apoptosis seems to be involved in the antitumor activity. To know the target of the novel compounds would be important because it could indicate why colon carcinoma cells are so responsive to these compounds. Knowledge of the target could also be used to obtain compounds with improved activity against colon cancers because this type of tumors shows poor responsiveness to the known drugs.2 However, it is beyond the scope of this report to explain the mechanism of action of these compounds.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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