European Journal of Pharmaceutical Sciences 14 (2001) 209–216www.elsevier.nl / locate /ejps

Synthesis, in vitro and in vivo cytotoxicity, and prediction of the intestinalabsorption of substituted 2-ethoxycarbonyl-imidazo[2,1-b]benzothiazoles

*Giuseppe Trapani , Massimo Franco, Andrea Latrofa, Antonia Reho, Gaetano Liso` `Dipartimento Farmaco-Chimico, Facolta di Farmacia, Universita degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy

Received 13 February 2001; received in revised form 15 June 2001; accepted 20 June 2001

Abstract

The imidazobenzothiazole compounds 3–17 together with the imidazobenzoxazole 18, and the imidazobenzoimidazole 19 wereprepared and their cytotoxic activity evaluated at the National Cancer Institute (NCI) for testing against a panel of approximately 60tumor cell lines. Compounds 5, 7, 8, and 16 exhibited interesting in vitro cytotoxic activity. The most active imidazobenzothiazolederivative 8 was further evaluated as a cytotoxic agent in the hollow fiber assay and showed a score greater than the minimum values forxenograft testing together with a net cell kill. Comparison with the results displayed in the in vivo assay by standard antitumor drugs inclinical use revealed a significant in vivo activity of the benzothiazole compound. COMPARE analyses for compounds 4–19 against theNCI’s standard agent database show poor or no correlation, and it might suggest for these compounds a mechanism of action unrelated tothat of any known drug. Furthermore, the benzothiazole 8 did not show significant antitumor activity in a panel of two xenotransplantedtumors (i.e. colon and non-small cell lung tumors). By computing the polar surface area of compounds 3–19 with the MAREA computerprogram it was established that the most active compounds 5, 7, 8, and 16 should experience good intestinal permeability. 2001Elsevier Science B.V. All rights reserved.

Keywords: Imidazobenzoimidazoles; Cytotoxic activity; Hollow fiber assay; Xenograft test; COMPARE analysis; Intestinal permeability

1. Introduction develop useful new lead compounds of simple structure,exhibiting optimal in vivo antitumor potency and new

Cancer is an important public health concern and in mechanisms of action. In this regard, it should be empha-developed countries it represents the second leading cause sized that the National Cancer Institute (NCI) in vitroof death, after cardiovascular diseases (Unger, 1997). primary anticancer drug screen represents a valuableAlthough considerable advances have been achieved over research tool to facilitate the discovery of new structural /recent decades in the research and development of various mechanistic types of antitumor agents (Boyd and Paull,cancerostatic drugs, current antitumor chemotherapy still 1995).suffers from two major limitations. The first is the lack of Among the antitumor agents discovered in recent years,selectivity of conventional chemotherapeutic agents for the identification of various 2-(4-aminophenyl)benzo-cancer tissues, bringing about unwanted side effects. The thiazoles (Fig. 1) as potent and selective antitumor drugssecond is the acquisition by cancer cells of multiple-drug against breast, ovarian, colon and renal cell lines hasresistance. Unwanted side effects of antitumor drugs could stimulated remarkable interest (Shi et al., 1996; Chua etbe overcome with agents capable of discriminating tumor al., 1999; Kashiyama et al., 1999; Hutchinson et al., 2001).cells from normal proliferative cells. The resistance to Moreover, there is experimental evidence suggesting achemotherapeutic antitumor agents by cancer cells could novel mechanism of action for these benzothiazole com-be minimized using a combination of drugs with different pounds (Shi et al., 1996). The interesting antitumorand complementary mechanisms of action (Menta and properties of these compounds as well as those of otherPalumbo, 1997). Therefore, there is a need to discover and molecules containing the benzothiazole nucleus (El-Sher-

beny, 2000; Ryu et al., 2000; Beneteau et al., 1999)prompted us to investigate in some detail the potential*Corresponding author. Tel.: 139-080-544-2764; fax: 139-080-544-cytotoxic activity of previously prepared compounds pos-2724.

E-mail address: trapani@farmchim.uniba.it (G. Trapani). sessing benzothiazole moieties and already evaluated as

0928-0987/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0928-0987( 01 )00173-7

210 G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216

resolution spectrometer. All compounds showed appro-1priate IR, H NMR and mass spectra. Elemental analyses

were carried out with a Carlo Erba model 1106 analyzerand results were within 60.40% of the theoretical values.Silica gel 60 (Merck 70–230 mesh) was used for columnchromatography. All the following reactions were per-formed under a nitrogen atmosphere. The starting 2-amino-benzothiazole compounds are commercially available ex-cept for 5,6-dimethoxy-2-aminobenzothiazole and 5,6,7-trimethoxy-2-aminobenzothiazole prepared as below de-scribed. Compounds 3–9, 12–14, 16 were prepared fol-lowing literature procedures (Clements-Jewery et al., 1988;Trapani et al., 1996; Grandolini et al., 1993).

2.1.1. General procedure for preparation of 2-aminobenzothiazoles

Potassium thiocyanate (0.055 mol) was added to asolution of the appropriate aniline (0.027 mol) in glacialacetic acid (40 ml). To this mixture a solution of bromine(0.055 mol) in acetic acid (8 ml) was added dropwise andthe temperature was maintained below 358C with a waterbath. After the addition was complete, the reaction mixturewas stirred at room temperature for 16 h. Then, thereaction mixture was poured into water, neutralized withNaHCO , and extracted with CHCl (3330 ml). Evapora-3 3

tion of the solvent gave a residue, which was purified bycolumn chromatography on silica gel [light petroleumether /ethyl acetate 8 /2 (v /v) as eluent] to give therequired 2-aminobenzothiazoles.

5,6-Dimethoxy-2-aminobenzothiazole: Yield 66%; m.p.225–2278C (from CHCl / light petroleum ether); IR (KBr)3

21 13360, 3300, 1640 cm ; H NMR (CDCl ) d 3.90 (s, 3H,3

OCH ), 3.86 (s, 3H, OCH ), 5.32 (s, 2H, NH ), 7.10 (s,Fig. 1. Chemical structures of antitumor 2-(4-aminophenyl) ben- 3 3 21zothiazoles. 1H, Ar), 7.16 (s, 1H, Ar); MS m /z 210 (M , base).

Analysis, found: C, 51.09; H, 5.16, N, 13.38%;benzodiazepine receptor ligands (Trapani et al., 1996). In C H N O S requires: C, 51.42; H, 4.80; N, 13.33%.9 10 2 2

this paper we describe the cytotoxicities of some im- 5,6,7-Trimethoxy-2-aminobenzothiazole: Yield 21%;21 1idazobenzothiazole compounds 3–17 (Fig. 1) and our m.p. 198–1998C; IR (KBr) 3400, 3280, 1630 cm ; H

efforts to explore the structure–activity relationships NMR (acetone d ) d 3.80 (s, 3H, OCH ), 3.86 (s, 3H,6 3

(SAR) in such new series of potentially antitumor agents. OCH ), 4.00(s, 3H, OCH ), 6.68 (s, 2H, NH ), 6.86 (s,3 3 21Furthermore, the effects of the most active compound 8 in 1H, Ar); MS m /z 240 (M , 74), 225 (base). Analysis,

an in vivo animal model are also presented. found: C, 50.37; H, 5.36; N, 11.36%; C H N O S10 12 2 3

requires: C, 50.00; H, 5.04; N, 11.66%.

2. Materials and methods 2.1.2. General procedure for preparation of ethylimidazo[2,1-b]benzothiazole-2-carboxylates 15 and 17

2.1. Chemistry Ethyl bromopyruvate (20 mmol) was added dropwise toa stirred solution of the appropriate 2-aminobenzothiazole

Melting points were determined in open capillary tubes (15 mmol) in DMF (20 ml) and the mixture was stirred for¨with a Buchi apparatus and are uncorrected. IR spectra 24 h. EtOH (10 ml) was added and the solution was heated

were obtained on a Perkin–Elmer 283 spectrophotometer at reflux for 2 h. Solvent was evaporated under reduced1(KBr pellets for solid or nujol for liquid). H NMR spectra pressure, and the residue was dissolved in CHCl (30 ml)3

were determined on a Varian 390 or Bruker 300 MHz and washed with 5% NaHCO . Evaporation of the solvent3

instrument. Chemical shifts are given in d values down- gave a residue, which was purified by column chromatog-field from Me Si as internal standard. Mass spectra were raphy on silica gel [light petroleum ether /ethyl acetate 8 /24

recorded on a Hewlett–Packard GC–MS 6890-5973 low (v/v) as eluent] to give the imidazobenzothiazole 15 or 17.

G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216 211

Ethyl 6,7-dimethoxy-imidazo[2,1-b]benzothiazole-2-car- cytotoxicity of test agents in their panel of 60 cell linesboxylate (15): m.p. 193–1968C (from EtOAc); IR (KBr) (Boyd and Paull, 1995). Briefly, cell lines were inoculated

21 1 into a series of 96-well microtiter plates, with varied1700 cm ; H NMR (CDCl ) d 1.40 (t, J57 Hz, 3H,3

seeding densities depending on the growth characteristicsCH ), 3.92 (s, 3H, OCH ), 3.96 (s, 3H, OCH ), 4.39 (q,3 3 3

of particular cell lines. Following a 24-h drug-free incuba-J57 Hz, 2H, OCH ), 7.12 (s, 2H, Ar), 8.25 (s, 1H, Imid.);21 tion, test agents were added routinely at five 10-foldMS m /z 306 (M , 72), 234(base). Analysis, found: C,

24dilutions with a maximum concentration of 10 M. After55.06; H, 4.89; N, 9.04%; C H N O S requires: C,14 14 2 4

48 h of drug exposure, the change in protein stain optical54.90; H, 4.61; N, 9.15%.density allowed the inhibition of cell growth to be ana-Ethyl 6,7,8-trimethoxy-imidazo[2,1-b]benzothiazole-2-lyzed. The cell lines used are derived largely from solidcarboxylate (17): m.p. 128–1298C (from CHCl /petro-3

21 1 tumors, including non-small cell lung, central nervousleum ether); IR (KBr) 1700 cm ; H NMR (CDCl ) d3

system, renal, ovarian, prostate, and breast cancer, along1.40 (t, J57 Hz, 3H, CH ), 3.90 (s, 3H, OCH ), 3.96 (s,3 3

with some leukemia cell lines. From this screen emerges3H, OCH ), 4.12 (s, 3H, OCH ), 4.46 (q, J57 Hz, 2H,3 3

the profile of cellular response for each tested agent, whoseOCH ), 7.00 (s, 1H, Ar), 8.30 (s, 1H, Imid.); MS m /z 33621 antitumor activity is presented for each cell line by three(M , base). Analysis, found: C, 53.20; H, 5.00; N, 8.36%;

parameters, namely logGI value (GI 5molar concen-C H N O S requires: C, 53.57; H, 4.80; N, 8.33%. 50 5015 16 2 5

tration of the compound that inhibits 50% net cell growth),log TGI value (TGI5molar concentration of the com-2.1.3. Preparation of ethyl 7-amino-imidazo[2,1-b]-pound leading to total inhibition of net cell growth), andbenzothiazole-2-carboxylate (10)log LC value (LC 5molar concentration of the com-50 50A solution of 6 (2 g, 6.9 mmol) in EtOH (50 ml) andpound leading to 50% net cell death). Furthermore, a meancatalytic amount of Pd/C was hydrogenated at 1 atm ofgraph midpoint (MG MID) is calculated for each of thehydrogen for 60 h. The reaction mixture was filtered andparameters mentioned, giving an averaged activity parame-the catalyst was washed with ethanol. The combinedter over all cell lines. Selectivity of a compound withfiltrates were evaporated under reduced pressure to give arespect to one or more cell lines of the screen is character-residue which was purified by silica gel column chroma-ized by a high deviation (D) of the particular cell linetography [light petroleum ether /ethyl acetate 8 /2 (v /v) asparameter compared to the MG MID value. D is consid-eluent] to give 0.5 g (16%yield) of 10: m.p. 188–1918C;

21 1 ered low if ,1, moderate if .1 and ,3, high if .3.IR (KBr) 3100, 3030, 1700 cm ; H NMR (CDCl ) d3

1.41 (t, J57 Hz, 3H, CH ), 3.86 (br s, 2H, NH ), 4.40 (q,3 2 2.2.2. Hollow fiber assay for preliminary in vivo testingJ57 Hz, 2H, OCH ), 6.74 (dd, J53 and 8 Hz, 1H, Ar),2For this assay, human tumor cells were cultivated in6.94 (d, J53 Hz, 1H, Ar), 7.40 (d, J58 Hz, 1H, Ar), 8.22

1 polyvinylidene fluoride hollow fibers, and a sample of each(s, 1H, Imid.); MS m /z 261 (M , 60), 189 (base).cell line was implanted intraperitoneally (IP) and subcuta-Analysis, found: C, 55.49; H, 4.13; N, 15.87%;neously (SC) in mice. Each test mouse received a total ofC H N O S requires: C, 55.17; H, 4.24; N, 16.09%.12 11 3 2six fibers (three IP and three SC) representing threedistinct cancer cell lines. Three mice were treated with the

2.1.4. Preparation of ethyl 7-acetamido-imidazo[2,1- tested compound at two dosage levels by the intraperi-b]benzothiazole-2-carboxylate (11) toneal route using a qd34 treatment schedule. Vehicle

A solution of 10 (0.2 g, 0.8 mmol) in acetic anhydride controls consisted of six mice receiving the compound(30 ml) was stirred at reflux for 3 h. Then, evaporation of diluent only. The fiber cultures were collected on the daythe solvent under reduced pressure gave a yellow solid following the last day of treatment. To assess anticancer(0.06 g, 27% yield) identified as compound 11: m.p. effects, viable cell mass was determined for each of the21 1278–2808C; IR (KBr) 3320, 3110, 1706, 1680 cm ; H cell lines using a formazan dye (MTT) conversion assay.NMR (CDCl 1 CD CN) d 1.29 (t, J57 Hz, 3H, CH ),3 3 3 Furthermore, it was noted whether a net cell kill of one or1.85 (s, 3H, CH ), 4.27 (q, J57 Hz, 2H, OCH ), 7.39 (dd,3 2 more cell lines occurs. To simplify evaluation, a score of 2J52 and 7 Hz, 1H, Ar), 7.51 (d, J57 Hz, 1H, Ar), 7.40 was assigned for each compound dose that resulted in a(d, J58 Hz, 1H, Ar), 8.09 (s, 1H, Imid.), 8.10 (s, 1H, Ar); 50% or greater reduction in viable cell mass. In the NCI1MS m /z 303 (M , 68), 231 (72), 189 (base). Analysis, criteria, compounds with a combined IP1SC score .20, afound: C, 55.35; H, 4.67; N, 13.53%; C H N O S14 13 3 3 SC .8 or a net cell kill of one or more cell lines arerequires: C, 55.44; H, 4.32; N, 13.86%. referred for xenograft testing.

2.2. Biology 2.2.3. In vivo antitumor evaluation of compound 8Studies were conducted according to standard protocols

2.2.1. In vitro cytotoxicity at NCI. Tumors were transplanted subcutaneously (SC) inCompounds 4–19 were submitted to the NCI for testing. nude athymic female mice. Compound 8 was suspended in

The NCI uses the sulforhodamine B assay for assessing the saline containing Tween 80 (0.05%). Suspensions were

212 G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216

prepared freshly for each drug administration and injected Compounds 18 and 19 (Fig. 1) were prepared as previouslyintraperitoneally (IP) in a volume of 0.1 ml /10 g body reported (Trapani et al., 1996).weight. Drug efficacy is expressed as T /C, where T and Care the values of median tumor weight of treated and 3.1.2. In vitro studiescontrol mice. To gain information on the SAR in the imidazoben-

zothiazole series, the C-7 substituted congeners (i.e., 4–11)were examined first. While compounds 4, 6, 9–11, as theparent compound 3, were essentially found devoid of

3. Results and discussion activity in in vitro antitumor screenings, compounds 5, 7and 8 exhibited an interesting activity, since they were

3.1. Chemistry characterized by log MG MID GI values of 24.74,50

24.80, and 24.87, respectively. Selected results of the inThe general synthetic procedure employed to prepare the vitro cytotoxic activity of compounds 5, 7 and 8 are

imidazo[2,1-b]benzothiazoles 3–9, 12–17 is shown in summarized in Table 1. As can be seen, the screenScheme 1. It involves the reaction of the appropriate revealed that leukemia, colon, and breast cancer cell lines2-aminobenzothiazole 2 with ethyl bromopyruvate in are, to some extent, sensitive to compounds 5, 7 and 8.refluxing DMF affording an intermediate hydrobromide Examination of the data obtained for C-7 substitutedwhich was subsequently cyclized to the desired compounds congeners (i.e., 4–11) indicates that the introduction of theby prolonging the reflux or in boiling EtOH. No commer- hydrophilic and strongly electron-withdrawing NO group2

cially available 2-aminobenzothiazoles (i.e., 5,6-dimethoxy- (6) or the hydrophilic and strongly electron-donating NH2

2-aminobenzothiazole and 5,6,7-trimethoxy-2-aminobenzo- group (10) led to the disappearance of any detectablethiazole) were prepared following the general method for cytotoxic activity, just as found for the parent compoundtheir preparation involving the reaction of the appropriately (3). On the other hand, introduction of a lipophilic andsubstituted aniline with thiocyanogen (SCN) . This reagent strongly electron-withdrawing substituent such as the2

was normally generated in situ by the action of bromine on trifluoromethyl group (9) resulted in a little increase ofKSCN in CH COOH as solvent (Kaufmann, 1928; cytotoxic activity. The best results were obtained for3

Clements-Jewery et al., 1988; Trapani et al., 1996; Gran- electron-donating substituents such as OCH and CH3 3

dolini et al., 1993). Whereas the thiocyanation reaction of groups (i.e., 8 and 7, respectively), and for the lipophilic3,4-dichloro-aniline allowed the preparation of both re- electron-withdrawing substituent Cl (i.e., 5).gioisomers (i.e., 5,6- and 6,7-dichloro-2-aminobenzo- The effect of the introduction of a further Cl, methyl andthiazole, respectively) (Alaimo, 1971), it should be methoxy group into the structure of 5, 7 and 8, respective-noted that starting from 3,4-dimethoxyaniline, only the ly, was then evaluated. Thus, further substitution with Cl5,6-dimethoxy-2-aminobenzothiazole was isolated. The atom at the 5-position of the monochloro derivative 5 gaveamine 10 was obtained by catalytic hydrogenation of the 13, which showed a marked decrease in activity whilenitrobenzothiazole 6 using 10% Pd/C in ethanol. Succes- introduction of the Cl atom at the 8-position affordingsive treatment of 10 with acetic anhydride gave the amide compound 16 caused a slight enhancement of activity, with11. Structural assignments for new compounds 10, 11, 15 16 resulting nearly as active as compound 8 (log MG MID

1and 17 are based on IR, H NMR and mass spectral data. GI 524.89). Di- and tri-methoxy derivatives, 15 and 17,50

Scheme 1.

G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216 213

Table 1aSelected results of the in vitro antitumor screening

bCell line log GI (M)50

5 7 8 9 14 15 16 17

LeukemiaCCRF-CEM 25.26 25.11 n.d. .24.00 24.62 24.12 25.30 24.20K-562 25.54 25.60 25.43 24.72 24.84 .24.00 25.40 24.30MOLT-4 25.53 25.46 25.34 24.49 24.54 24.36 25.06 24.40RPMI-8226 25.07 25.45 25.39 24.57 24.59 24.27 25.30 24.57SR 25.81 n.d. n.d. 25.16 25.17 24.64 25.59 24.55

Non-small cell lung cancerNCI-H522 24.51 25.13 24.74 24.56 24.51 26.59 25.39 24.63

Colon cancerCOLO 205 25.26 25.34 25.47 24.48 24.58 .24.00 25.21 24.20HCT-116 25.47 25.39 25.16 24.58 24.75 24.08 25.25 24.31SW-620 25.32 25.33 25.51 24.44 24.70 .24.00 25.29 24.17

CNS cancerSF-539 25.24 25.70 25.53 24.54 24.84 .24.00 25.17 24.33

MelanomaM14 24.74 .24.00 25.28 24.40 24.58 .24.00 25.02 24.24SK-MEL-5 25.11 25.33 25.56 24.47 24.78 24.01 25.17 24.58

Ovarian cancerOVCAR-3 24.85 25.22 25.42 24.64 24.76 .24.00 25.00

Renal cancer786-0 25.18 n.d. 25.20 24.34 24.72 .24.00 25.12 24.10SN12C 24.94 24.33 25.13 24.06 24.72 24.19 25.46 24.29

Prostate cancerPC-3 24.51 24.43 24.82 24.19 24.67 .24.00 24.74 .24.00DU-145 24.97 25.26 25.39 24.44 24.67 .24.00 24.75 .24.00

Breast cancerMDA-MB-435 25.25 25.59 25.93 24.82 24.69 .24.00 25.48 24.70MDA-N 25.57 25.57 25.58 24.49 25.22 .24.00 25.81 24.53T-47D 24.24 24.12 n.d. 24.27 24.59 .24.00 6.04 24.22MG MID 24.74 24.80 24.87 24.38 24.65 24.14 24.89 24.26

a Data obtained from the NCI’s in vitro human tumor cells screen.b Log of molar concentration that inhibits 50% net cell growth.

respectively, showed reduced cytotoxic activity compared activity was observed in the case of the correspondingto the parent mono-methoxy compound 8. However, the oxazole and imidazole compound (e.g., 18 and 19). Itstrong cytotoxicity of 15, at submicromolar level, against should be noted, however, that 18 is strongly cytotoxic atNCI-H5, a non-small cell lung cancer cell line is notewor- submicromolar level (logGI value526.25 and D52.21)50

thy. The cytotoxicity of the 6,7-dimethyl derivative 14 is against the leukemia cell line K-562.slightly lower than that of the parent compound 7, their log A further result of this study is a poor correlationMG MID GI values being 24.65 and 24.80, respective- between the calculated lipophilicity (Clog P) and cytotox-50

ly. icity expressed by log MG MID GI . This may be an50

As far as the selectivity of the most active compounds 5, indication that other factors should be considered to7, 8 and 16 is concerned, results are quite low, with D account for the cytotoxicity of imidazobenzothiazole com-values being ,1 for compounds 7 and 16, 1 and 1.07 for 8 pounds.and 5, respectively. The SAR observed for antitumor 2-(4-aminophenyl)ben-

Besides studying the influence of the aromatic substitu- zothiazoles and our imidazobenzothiazole compounds ledtion, we also explored the effect of the change of the us to suggest that the cytotoxic activity of these com-heteroatom at position 9 of the imidazobenzothiazole ring pounds should be ascribed to the benzothiazole nucleus. Asystem. In comparison to 3, no improvement in anticancer similar conclusion has been recently drawn for the 2-(4-

214 G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216

Table 2 demonstrated that 8 possesses good activity in the NCI’s inaAnticancer activity of compound 8 in the hollow fiber assay vivo hollow fiber assay. In particular, it should be noted

Compound IP SC Total Cell that, in this test, compound 8 shows a score comparablebscore score score kill with that of cis-platinum or L-PAM, and higher than that

8 12 10 22 Y of Chlorambucil, Cyclophosphamide, and BleomycinChlorambucil 10 10 20 Y (Hollingshead, personal communication). It should beCyclophosphamide 0 18 18 N remembered, however, that the hollow fiber assay is notMitomycin C 26 14 40 Y

equivalent to drug assays against tumor implants, whereVinblastine sulfate 18 10 28 Yangiogenesis of metastases can occur. Examination ofCisplatin 14 8 22 Y

Bleomycin 10 0 10 Y hundreds of new synthetic and natural products in theTaxol 24 8 32 Y hollow fiber screen revealed that the highest score is 64 forL-PAM 16 6 22 Y a natural product. Many of these compounds with a highBCNU 8 16 24 Y

score may not perform well in human xenograft assays anda Polyvinylidene fluoride hollow fibers containing various cancer cell often are very toxic. On the other hand all compounds

lines were implanted intraperitoneally (IP) or subcutaneously (SC) in displaying high activity against tumor implants are activemice. Data for reference compounds were obtained from DTP, NCI.

b in the hollow fiber assay (Hollingshead et al., 1999).A net cell kill at one or more of the implant sites is indicated with aThe in vivo activity of the benzothiazole compound 8 inY.

a panel of two xenotransplanted tumors (i.e., colon andnon-small cell lung tumors) is recorded in Table 3. In both

aminophenyl)benzothiazoles by Hutchinson et al. (2001) cases, however, 8 did not show significant antitumorwho also stated that the 4-aminophenyl moieties increase activity. Pharmacokinetic reasons may be invoked tothe potency and the spectrum of activity. explain this outcome. For instance, because of its high

lipophilicity this compound could be predominantly bound3.1.2. In vivo studies to plasma proteins or possess a large volume of dis-

The most active imidazobenzothiazole derivative 8 was tribution reducing so its in vivo potency.evaluated by the Developmental Therapeutics Program(DTP, DCTD, NCI) as an anticancer agent in a preliminary 3.3. COMPARE correlationsin vivo test, in which polyvinylidene fluoride hollow fiberscontaining various cancer cell cultures were implanted Although rigorous evidence of the mechanism of actionintraperitoneally (IP) and subcutaneously (SC) into mice of the imidazobenzothiazoles is still lacking, an explorat-(Hollingshead et al., 1995). Compound 8 was administered ory effort in this direction was made. Our imidazoben-by the IP route and its effects on reduction in viable cancer zothiazole compounds represent a novel class of potentialcell mass were determined and compared to those of cytotoxic agents and differences between other clinicallycontrols. The results obtained are listed in Table 2. used drugs and these compounds were apparent fromCompound 8 showed both a combined IP1SC score COMPARE correlations (Boyd and Paull, 1995; Paull etgreater than 20 (i.e., 22) and a SC score greater than 8 al., 1989). COMPARE was developed at the NCI to(i.e., 10), which are considered, in the DTP criteria, the compare the patterns of cytotoxicity in 60-cell line cancerminimum values for xenograft testing. Moreover, com- screens. The COMPARE computer program allows thepound 8 showed a net cell kill at one or more of the user to calculate Pearson correlation coefficients betweenimplant sites, which is an additional favorable factor, in the data for seed compounds and those for standard agents inDTP evaluations, for xenograft testing. Comparison with the database. Antitumor agents with identical mechanismsthe results displayed in the hollow fiber assay by standard of action possess identical or nearly identical cytotoxicityantitumor drugs in clinical use (Table 2) revealed a patterns (correlation coefficient .0.8). COMPARE analy-significant in vivo activity of compound 8. These results ses for compounds 4–19 against NCI’s standard agent

Table 3In vivo evaluation of compound 8 (IP administered) against colon (HCT-116) and non-small cell lung (NCI-H23) tumor xenografts

bCompound (dose) Tumor Schedule T /C % (day) Net log cell killa8 (200 mg/kg) NCI-H23 q4dx3,day 15 71 (41) 20.70b8 (134 mg/kg) NCI-H23 q4dx3,day 15 68 (41) 20.50

b8 (90 mg/kg) NCI-H23 q4dx3,day 15 43 (34) 20.30b8 (120 mg/kg) HCT-116 qdx5,day 8 73 (33) 20.50

b8 (80 mg/kg) HCT-116 qdx5,day 8 96 (33) 20.60b8 (54 mg/kg) HCT-116 qdx5,day 8 98 (33) 20.70

a Five mice examined.b Six mice examined.

G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216 215

database show poor or no correlation, and it might suggest absorbed whereas a good permeability is expected forfor these compounds a different mechanism of action drugs characterized by PSA values in the range 62 –100

2˚unrelated to that of any known drug. In particular, the A .highest correlation to compound 8 was found in DUP785 In conclusion, some imidazobenzothiazole compounds(Brequinar) and methotrexate (Pearson correlation coeffi- display promising in vitro cytotoxicity when tested againstcient 0.705 and 0.636, respectively). a panel of approximately 60 tumor cell lines. Compound 8

showed significant antitumor activity in the hollow fibertest. Although this interesting activity was not confirmed

3.4. Computational approach to predict the intestinal against NCI-H23 and HCT-116 tumor xenografts, 8 can beabsorption of compounds 3 –19 considered as a new and promising lead compound worthy

of further development, especially considering that itIt is well known that many lead compounds fail to should be characterized by a good intestinal permeability.

progress into the clinic because they are lacking appro-priate pharmaceutical properties, such as oral bioavail-ability. Hence, there is a need to estimate at the preclinical Acknowledgementsstage molecular transport properties and particularly in-testinal absorption (Blake, 2000). To test the possibility of We thank members of the Screening and Pharmacologyan oral administration of compounds 3–19, among which Group of the European Organisation for Research andthere are possible leads, we used computational methods Treatment of Cancer (EORTC) for many useful discus-based on the determination of polar surface area (PSA) for sions. We are grateful to Dr R.J. Schultz, Dr S. Radtke, Drprediction of their intestinal permeability. In this regard, it V. Steele and their colleagues at NCI for the productiveis known (Palm et al., 1996; Clark, 1999; Ertl et al., 2000) collaboration to this project. We acknowledge the De-2˚that (i) compounds which possess PSA values $140 A velopmental Therapeutics Program’s contribution (particu-should be poorly absorbed, (ii) consideration of only a larly that of Dr M. Hollingshead) to these studies. Wesingle conformer when computing PSA gives an excellent thank the Division of Pharmaceutics, Department of Phar-correlation with intestinal absorption data. Calculation of macy, Uppsala University, Sweden, for providing us thepolar surface properties of compounds 3–19 was per- computer program MAREA v. 1.4. Thanks are also due toformed using the algorithm implemented in the MAREA Peakdale Fine Chemical, Derbyshire (UK) for providing uscomputer program (Palm et al., 1997; Stenberg et al., the NCI’s data on compound 3. We thank Antonio1999). The resulting PSA values are shown in Table 4 Palermo, Nicola Di Masi and Dr Antonio Carrieri (Di-together with the calculated octanol–water partition co- `partimento Farmaco-Chimico, Universita di Bari) for theirefficients (Clog P). From the reported results it can be help in recording NMR spectra and in performing PSAdeduced that the most active compounds 5, 7, 8, and 16 calculations, respectively.should experience a good intestinal permeability since it is

2˚known that drugs having a PSA561 A are completely

ReferencesTable 4Calculated polar surface area and lipophilicity of compounds 3–19

Alaimo, R.J., 1971. The preparation and characterization of 2-amino-5,6-2 a˚Compound PSA (A ) Clog P dichloro and 2-amino-6,7-dichlorobenzothiazole. J. Heterocyclic

Chem. 8, 309–310.3 78.23 4.25

Beneteau, V., Besson, T., Guillard, J., Leonce, S., Pfeiffer, B., 1999.4 78.23 4.39

Synthesis and in vitro antitumor evaluation of benzothiazole-2-car-5 78.23 4.96

bonitrile derivatives. Eur. J. Med. Chem. 34, 1053–1060.6 121.41 4.02

Blake, J.F., 2000. Chemoinformatics. Predicting the physicochemical7 78.23 4.75

properties of ‘drug-like’ molecules. Curr. Opin. Biotechnol. 11, 104–8 98.20 4.29

107.9 78.23 5.13

Boyd, M.R., Paull, K.D., 1995. Some practical considerations and10 110.91 3.21

applications of the national cancer institute in vitro anticancer drug11 112.25 3.44

discovery screen. Drug Dev. Res. 34, 91–109.12 76.99 4.75

Chua, M.-S., Shi, D.-F., Wrigley, S., Bradshaw, T.D., Hutchinson, I.,13 76.22 5.68

Shaw, P.N., Barrett, D.A., Stanley, L.A., Stevens, M.F.G., 1999.14 78.23 5.20

Antitumor benzothiazoles.7. Synthesis of 2-(4-aminophenyl)ben-15 113.37 3.73

zothiazoles and investigations into the role of acetylation in the16 78.23 5.56

antitumor activities of the parent amines. J. Med. Chem. 42, 381–392.17 123.31 3.12

Clark, D.E., 1999. Rapid calculation of polar molecular surface area and18 104.69 3.76

its application to the prediction of transport phenomena. 1. Prediction19 107.79 3.49

of intestinal absorption. J. Pharm. Sci. 88, 807–814.a Calculated using CLog P 3.54 software, Pomona College Medicinal Clements-Jewery, S., Danswan, G., Gardner, C.R., Matharu, S.S., Mur-

Chemistry Project, Claremont, CA, USA. doch, R., Tully, W.R., Westwood, R., 1988. (Imidazo[1,2-a]pyrimidin-

216 G. Trapani et al. / European Journal of Pharmaceutical Sciences 14 (2001) 209 –216

2-yl)phenylmethanones and related compounds as potential nonseda- Palm, K., Luthman, K., Ungell, A.-L., Strandlund, G., Artusson, P., 1996.tive anxiolytics. J. Med. Chem. 31, 1220–1226. Correlations of drug absorption with molecular surface properties. J.

El-Sherbeny, M.A., 2000. Synthesis of certain pyrimido[2,1-b]ben- Pharm. Sci. 85, 32–39.zothiazole and benzothiazolo[2,3-b]quinazoline derivatives for in vitro Palm, K., Stenberg, P., Luthman, K., Artursson, P., 1997. Polar molecularantitumor and antiviral activities. Arzneim.- Forsch. 50, 848–853. surface properties predict the intestinal absorption of drugs in humans.

Ertl, P., Rohde, B., Selzer, P., 2000. Fast calculation of molecular polar Pharm. Res. 14, 568–571.surface area as a sum of fragment-based contributions and its Paull, D.K., Shoemaker, R.H., Hodes, L., Monks, A., Scudiero, D.A.,application to the prediction of drug transport properties. J. Med. Rubinstein, L., Plowman, J., Boyd, M.R., 1989. Display and analysisChem. 43, 3714–3717. of differential activity of drugs against human tumor cell lines:

Grandolini, G., Ambrogi, V., Perioli, L., De Caprariis, P., Palagiano, F., development of mean graph and COMPARE algorithm. J. Natl. CancerRimoli, M.G., Lampa, E., D’Amico, M., 1993. Synthesis and pharma- Inst. 81, 1088–1092.cological activity of imidazo[2,1-b]benzothiazole acids. Il Farmaco 48, Ryu, C.-K., Kang, H.-Y., Lee, S.K., Nam, K.A., Hong, C.Y., Ko, W.-G.,31–43. Lee, B.-H., 2000. 5-arylamino-2-methyl-4,7-dioxobenzothiazoles as

Hollingshead, M.G., Alley, M.C., Camalier, R.F., Abbott, B.J., Mayo, inhibitors of cyclin-dependent kinase 4 and cytotoxic agents. Bioorg.J.G., Malspeis, L., Grever, M.R., 1995. In vivo cultivation of tumor Med. Chem. Lett. 10, 461–464.cells in hollow fibers. Life Sci. 57, 131–141. Shi, D.-F., Bradshaw, T.D., Wrigley, S., McCall, C.J., Lelieveld, P.,

Hollingshead, M., Plowman, J., Alley, M., Mayo, J., Sausville, E., 1999. Fichtner, I., Stevens, M.F.G., 1996. Antitumor benzothiazoles. 3.Relevance of Tumors Models in Anticancer Drug Development. Synthesis of 2-(4-aminophenyl)benzothiazoles and evaluation of theirContribution To Oncology. Karger Verlag, Germany. activities against breast cancer cell lines in vitro and in vivo. J. Med.

Hutchinson, I., Chua, M.-S., Browne, H.L., Trapani, V., Bradshaw, T.D., Chem. 39, 3375–3384.Westwell, A.D., Stevens, M.F.G., 2001. Antitumor benzothiazoles. 14. Stenberg, P., Luthman, K., Ellens, H., Lee, C.P., Smith, P.L., Lago, A.,Synthesis and in vitro biological properties of fluorinated 2-(4-amino- Elliott, J.D., Artusson, P., 1999. Prediction of the intestinal absorptionphenyl)benzothiazoles. J. Med. Chem. 44, 1446–1455. of endothelin receptor antagonists using three theoretical methods of

Kashiyama, E., Hutchinson, I., Chua, M.-S., Stinson, S.F., Phillips, L.R., increasing complexity. Pharm. Res. 16, 1520–1526.Kaur, G., Sausville, E.A., Bradshaw, T.D., Westwell, A.D., Stevens, Trapani, G., Franco, M., Latrofa, A., Carotti, A., Genchi, G., Serra, M.,M.F.G., 1999. Antitumor benzothiazoles. 8. Synthesis, metabolic Biggio, G., Liso, G., 1996. Synthesis and benzodiazepine receptorformation, and biological properties of the C- and N-oxidation binding of imidazo- and pyrimido[2,1-b]benzothiazoles. Eur. J. Med.products of antitumor 2-(4-aminophenyl)benzothiazoles. J. Med. Chem. 31, 575–587.Chem. 42, 4172–4184. Unger, C., 1997. New therapeutic approaches in cancer treatment. Drug

Kaufmann, H.P., 1928. Die bildung von thiazol-derivaten bei der Future 22, 1337–1345.rhodanierung von aminen. Arch. Pharm. 266, 197–218.

Menta, E., Palumbo, M., 1997. Novel antineoplastic Agents. Exp. Opin.Ther. Patents 7, 1401–1426.