extfieldsofstudy: Medicine Chemistry

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Synthesis, Properties and Biological Activity of Organotitanium Substituted Heteropolytungstates

Six new compounds [(CpTi)X, Cp=ϋ∇-c∇H∇, X=Ge, Ga, B) have been prepared and their Keggin structures determined by elementary analysis, IR, UV, ∞H NMR and ∞∀∊WNMR spectrometry. The results show that the complexes retain Keggin structure. The complexes exhibit antitumoral activity in vitro as shown by MTT experiment.

INTRODUCTION
Our recent work involves studies in both the antitumoral activity and catalytic activity of substituted heteropolyorganometallic complexes. Polyoxometalates (POMs) have attracted much attention because of their potential applications in catalysis, medicine and material science I--l. Hill has pointed out that the versatility of the POMs and grafting organic and organometallic groups on to the polyoxometalate surface can significantly increase their catalytic or medical applications. POMs containing a [CpTi]group show high anti-HIV activity I41 but, to the best of our acknowledge, there is no report on the antitumoral activity of organotitanium substituted heteropoly complexes. As part of our continuing investigat!on of POMs containing organometallic groups, we have systematically synthesized the [CpTi]"substituted heteropolytungstates and investigated their antitumoral activity in vitro.
The title complexes [(CpTi)XWO39]" (X Ge, Ga, B) are attractive targets for three reasons" first, CpzTiCI2 is a more potent antiviral agent than other organotitanium compounds, but its insolubility in water makes it less useful in chemotherapy. So modifying Cp2TiCI2 into soluble complexes is a ve,'y attractive topic. Second, POMs containing titanium exhibit high antitumoral activity I5.61, so their investigation is very useful for determining the influence of the organometallic group on antitumoral or antiviral activity.

Antitumoral activiO,
The antitumoral activity of POMs on two types of the human cancer cells was tested by the MTT experiment as described in ref. [9].

Physical measurements
H NMR spectra were recorded on a Brucker AC-80 spectrometer. S3W NMR were recorded on a Unity-Jingfu Liu et al.
Synthesis, Properties and Biological Activity of Organotitanium Substituted Heteropolytungstates 400 spectrometer. IR spectra were recorded on an Alpha Centauri FTIR spectrometer (4000-200cm range) as KBr pellets. UV spectra were recorded on a Beckman Du-8D spectrometer (solvent H20). Polarograms were obtained using a 384B polarographic analyser. W, Ti, Si, Ge were determined by a Leeman Corporation inductively coupled plasma (ICP) emission spectrograph. C, N, H were determined using a PE-2400 analyzer. Water content was determined by thermogravimetry. The IR spectra display a single, sharp absorption at 1469cm in the 1300-1500cm region. This feature is characteristic of the C-C stretch for a :rls-CH5 ligand bonded to Ti I01. At 3032, 3038cm in the 3030-3040cm region, the C-H stretches shows the existence ofrl-cyclopentadienyl ligand.

C6'mp0unds
The title anions absorb at ca. 200+2 nm and 260+2 nm. The first band at higher energy has been assigned to the Ob/-->W charge transfer band. The second band at lower energy is characteristic of 12heteropolytungstates and has been attributed to the O0->W charge-transfer band.

An'ions
Anions Table 2 The,, IR (cm") and UV(,nm) data of comp0unds, In the well-known T Keggin structure all the tungsten atoms are identical, as shown by a singlet in the 83W NMR spectra. imposed by Cs point symmetry, and the expected six lnes are obtmned m the W NMR spectra .The W NMR spectra of(CpTi)XWl confirm that the substituted [CpTi] 3+ group occupies an empty octahedral site of a XW anion and forms a polyanion with a Keggin structure.

Electrochemistry
The redox proprieties of (CpTi)XWI were investigated by polarography. The half-wave potentials in different pH values are listed in Table 4. It can be seen that: (1) The reduction process of (CpTi)XW anions involve the reduction of titanium and tungsten. The first peak is assigned to the conversion of Ti (IV) to Ti (III) t21. (2) The latter two polarographic peaks are assigned to the conversion of W (VI) to W (V), showing that the title anions undergo a two-step tungsten reduction process. They are pH-dependent reduction waves. And straight lines are obtained by logarithmic analysis of the polarographic wave. The number of electrons involve in the reduction can be calculated from the slope of the straight lines, showing that (CpTi)XWI has undergone a two-step, two-electron tungsten reduction. (3)E/2 shifts to negative potentials with increasing pH of the solution, implying that the reduction of polyanions is accompanied by protonation. From the slope of E/2 versus pH, it can be calculated that when n 2, m 2 (n and m stand for the electron and proton number, respectively) for the reduction of tungsten, two protons must be added to gain two electrons while the polyanion is reduced; this prevents charge build-up on the anion, which would otherwise be destabilized.

Antitumoral activity
The data summarized in Table 5 show that the title complexes display inhibitory action to two types of human cancer cells Hela and SSMC-7721 tumor cells.
3.6 The stability of the POMs at the physiological condition In order to assess the stability of the complexes, they were treated in the same MTT experimental conditions but without adding cancer cells. After evaporation to dryness, IR and H NMR spectra showed that the complexes retained the Keggin structure and the [CpTi] 3+ group was not eliminated from the XW moiety demonstrating that they are stable under the evaluation conditions.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

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Oxidative Modification of Tryptophan 43 in the Heme Vicinity of the F43W/H64L Myoglobin Mutant*

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catal-ysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m -chloroper-benzoic acid ( m CPBA). To identify the exact structure of the modified tryptophan in this study, the m CPBA-treated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the 1 H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan

Myoglobin (Mb), 1 a carrier of molecular oxygen, can perform oxidation reactions in the presence of hydrogen peroxide (H 2 O 2 ), although the activity is not as great as that of peroxidase (1)(2)(3). The accumulated biochemical and biophysical data allow us to utilize Mb as a heme enzyme model system, and various myoglobin mutants have been constructed to elucidate structure-function relationship on the activation of peroxides (3)(4)(5). For example, F43H/H64L Mb, one of the distal histidine relocation mutants, exhibits the enhanced reactivity with H 2 O 2 and the longer lifetime of an active intermediate, a ferryl porphyrin radical cation (OϭFe IV porphyrin ⅐ ϩ ) (6). Therefore as the results, the F43H/H64L mutant is able to catalyze the sulfoxidation and epoxidation reaction at the rate comparable with the values of peroxidases.
On the other hand, cytochromes P-450 (P-450) catalyze the hydroxylation of a wide variety of substrates, including hydrocarbons and polycyclic aromatic molecules (7,8). The variance in reactivity of Mb and P-450 could arise from differences in the active site structure and the arrangement of functional amino acid residues. The crystal structure of P-450cam with d-camphor reveals that the substrate is tightly bound in the hydrophobic heme pocket through hydrogen bonding interaction with the hydroxyl group of Tyr-96 and the carbonyl oxygen of dcamphor (Fig. 1A) (9). The distance between the heme iron and C5 of d-camphor, the hydroxylation site, is 4.2 Å. On the contrary, the active site of myoglobin is exposed to the exterior and does not provide any specific interactions for accommodating a foreign substrate with high affinity (10). Therefore, it will be difficult for a ferryl porphyrin radical cation of Mb to hydroxylate a substrate molecule, which is not bound in an appropriate position nearby the heme. We hypothesize that a ferryl oxygen atom transfer to aliphatic or aromatic molecules by Mb mutants might be possible even without a proximal thiolate ligand if the substrates were fixed nearby the heme iron.
The F43W/H64L Mb mutant, which was previously constructed to investigate effects of an electron-rich tryptophan residue on the peroxygenase activity, appears to be a good model for examining the hypothesis, because aromatic carbon atoms of tryptophan are fixed in the heme vicinity ( Fig. 1B) (12). Although the crystal structure of the F43W/H64L Mb is not available at the moment, the calculated model structure suggests that the distances of Fe-C7 and Fe-C6 are 4.9 and 5.5 Å, respectively. (A calculated structure of F43W/H64L Mb was obtained by using the Insight II molecular modeling program (Biosym MSI, San Diego, CA). The Trp-43 in the mutant is generated by replacing His-43 of the F43H/H64L Mb (6) with a tryptophan residue and minimizing the energy of the heme pocket.) The predicted values are similar to the distance between C5 of d-camphor and iron in P-450cam. Our earlier studies provided preliminary evidence that Trp-43 in F43W/ H64L Mb was oxidatively modified in the reaction with mchloroperbenzoic acid (mCPBA); however, the exact structure of the modified product(s) remains to be elucidated (12). The subject of this study is to identify the oxidized tryptophan residue to determine whether or not the F43W/H64L Mb mutant is capable of performing the oxidation of aromatic molecules.

EXPERIMENTAL PROCEDURES
The Oxidation of Trp-43 and the Isolation of Peptide Fragments Bearing Trp-43-F43W/H64L Mb (0.2 mM) in 50 mM potassium phosphate buffer at pH 7.4 was mixed with 4 equivalents of mCPBA at 4°C. The modified protein (500 mg) was digested with Lys-C (1/100 w/w) in 100 mM Tris-HCl buffer at pH 9.0 containing 2 M urea, and the mixture was incubated at 25°C for 24 h. The digestion was stopped by the addition of trifluoroacetic acid (final concentration 1%). The products were analyzed on an Ä kta FPLC system (Amersham Pharmacia Biotech) with a Vydac C-18 reverse phase column eluted typically at a flow of 1.5 ml/min with a gradient of solvent A (0.1% trifluoroacetic acid in water) into solvent B (20% acetonitrile and 0.1% trifluoroacetic acid in water) over 200 min. The eluent was monitored either at 280 nm for aromatic residues or 215 nm for amide bonds in peptide fragments. The isolated fragments A and B, which have been identified as modified Trp-43 linked with unmodified Asp-44-Arg-45-Phe-46-Lys-47 (W*DRFK in which the modified tryptophan is designated as W*) (12), were further treated with trypsin (ϳ1/100 w/w) in 100 mM Tris-HCl buffer (pH 9.0) at 30°C for 12 h and analyzed by the Ä kta FPLC system. Although A was not cleaved by trypsin, B was digested to afford a peptide fragment B (W*DR). In a control experiment, the intact F43W/ H64L mutant (250 mg) was treated with Lys-C and trypsin, and the peptide fragment bearing unmodified Trp-43 was isolated by preparative FPLC.
MS/MS Analysis of the Peptide Fragments-The fragments A and B isolated after Lys-C digestion were directly analyzed on a Voyager DESTR (PerSeptive Biosystems) for MALDI-TOF-MS. The spectrometer was calibrated with an angiotensin II (molecular weight ϭ 1046.2). 1 l of the digested samples containing ϳ10 pmol/l were mixed with 1 l of saturated ␣-cyano-4-hydroxycinnamic acid in water/acetonitrile (1:1) and applied to the sample plate by the dried-droplet method.

RESULTS AND DISCUSSION
The oxidative modification of Trp-43 in F43W/H64L Mb has been performed by adding mCPBA in 50 mM potassium phosphate buffer at pH 7.4. The transition from 5-to 6-coordinated ferric high spin state is a good indication of the protein modification (Scheme 1) (17)(18)(19)(20)(21). The mCPBA-treated F43W/H64L mutant is digested with Lys-C achromobacter, and two peptide fragments A and B, which consist of Trp-43(modified)-Asp-44-Arg-45-Phe-46-Lys-47 (W*DRFK), are purified by FPLC. Fig. 2 shows the comparison of the MS/MS spectra of unmodified WDRFK fragment and the modified fragment A. The difference in the spectra provides the direct evidence for the modified site to be Trp-43, and the increased mass number of 30 Da could correspond to the addition of two oxygen atoms and loss of two protons. The MS/MS spectrum of the fragment B is identical to that of the fragment A. Prior to the NMR analysis, A and B have been further treated with trypsin to simplify the NMR spectra. While the fragment B affords a shorter peptide defined as B (W*DR), trypsin does not cleave the carboxyl side of arginine in the fragment A. Therefore, we have performed NMR analysis of the intact WDR fragment and modified fragments A (W*DRFK) and B (W*DR).
Although the 1 H NMR spectrum of the intact WDR fragment exhibits signals derived from five tryptophan protons, only three proton signals of the tryptophan appear in the aromatic region for the modified product A (W*DRFK) (Fig. 3, Table I). The close examination of the 1 H NMR spectrum of A reveals that one proton signal at 6.60 ppm couples with two different proton signals with coupling constants of J ϭ 8.2, 2.3 Hz. Two other proton signals appear at 6.58 and 7.19 ppm with coupling constants of J ϭ 2.3 and 8.2 Hz, respectively. The only structure that could afford such a coupling pattern is a 2,6-disubstituted indole substructure as shown in Fig. 4; i.e. the resonance at 6.58 ppm, 6.60 ppm, and 7.19 ppm are unambiguously assigned to the protons at C7, C5, and C4, respectively.
The coupling between proton signals at C4 and C5 observed in the COSY spectrum also support the assignment. Furthermore, the correlation of the C4 -H with C ␣ -H (4.14 ppm) and C ␤ -H (2.65 ppm) is observed by the ROESY experiments. The combination of the COSY, ROESY, and TOCSY methods allows us the complete assignment for the fragment A as summarized in Table II. Essentially the same assignment can be applied to the interpretation for the NMR spectra of B. Two doublet signals at 6.36 ppm (J ϭ 2.5 Hz) and 6.91 ppm (J ϭ 8.5 Hz) are assigned to the C7 and C4 protons of the modified Trp-43 in B, respectively, and the resonance at 6.26 ppm (J ϭ 8.5, 2.5 Hz) is derived from C5-H (Table I). Therefore, the tryptophan residue in the fragment B (W*DR) also must have the 2,6-disubstituted indole substructure (Fig. 4). The coupling pattern in the 1 H NMR spectrum of the fragment B is essentially the same as those observed in A and B spectra.
Amino acid sequence and composition analyses provided us with a clue to identify the structural differences in the modified tryptophan of the fragment A and B. First of all, A does not react with phenylisothiocyanate, and the Edman degradation does not give us any sequence information, while the sequence analysis for B shows Trp*-43(not detected)-Asp-44-Arg-45 sequence. Second, amino acid composition analysis of A reveals that the fragment consists of Asp, Arg, Phe, and Lys; however, the tryptophan residue is not identified due to its modification. The results imply that the amino terminus of the fragment A is protected, but the residues except for Trp-43 are intact. To clarify the amino-terminal structure, we have measured 13 C NMR of A. The spectrum exhibits a unique signal at 71.1 ppm, which could be assigned as a signal from the hydroxylated carbon atom. Since the fragment A does not contain a serine nor a threonine residue, the comparison of chemical shift for the ␣ carbon in tryptophan (56.1 ppm), indole-3-lactic acid (70.6 ppm), and A (71.1 ppm) suggest that the terminal amino group in A is replaced with the hydroxyl moiety. Thus, we conclude at the moment that the indole substructure in the modified tryptophan is 2,6-dihydro-2-imino-6-oxoindole in A (XϭO, YϭNH in Fig. 4) and 2,6-dihydro-2,6-dioxoindole in B terminal (XϭYϭO). The proposed structures are consistent with the increase in the 30-Da mass unit with respect to the intact tryptophan. Although further studies are required to clarify the mechanism, a cyclic intermediate generated through the reaction of the terminal amine with the carbonyl carbon atom at the C2 position followed by hydrolysis might be involved to afford 2,6-dihydro-2-imino-6-oxoindole substructure without changing the total mass of the peptide (Scheme 2).
It should be noticed that trypsin somehow recognizes the structural difference in the amino-terminal of pentapeptide A (W*DRFK) and prevents hydrolysis of the carboxyl side of arginine to yield A (W*DR). More interestingly, the fragment A is found to be produced from B during the incubation with trypsin or Lys-C, while B is stable in aqueous solution in the absence of peptidases.  Since the mixing of mCPBA and tryptophan does not produce the modified tryptophan, a ferryl porphyrin radical cation generated in the reaction of F43W/H64L Mb with mCPBA is presumably a catalytic species to form 6-hydroxytryptophan either directly or via the epoxidation followed by hydrolysis (Scheme 3). Previous studies using Fremy's salt as the oxidant indicate that 6-hydroxy-3-methylindole is readily oxidized to yield 3-methylindole 2,6-and 6,7-dione, and 6,7-dione is reported to be a minor product due to its instability (yield of 3-methylindole 2,6-dione ϭ 63%, 6,7-dione ϭ 7%) (22). We expect that 6-hydroxytryptophan could further be oxidized with mCPBA (Scheme 3A) or compound I (Scheme 3B) by overall four electron oxidation to produce the 2,6-dihydro-2,6-dioxoindole substructure.
In summary, the indole substructure of Trp-43 in F43W/H64L Mb is oxidatively transformed into 2,6-dihydro-2,6-dioxoindole in the presence of mCPBA. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system as far as we know. The results implicate that a ferryl oxygen atom transfer to aromatic molecules would be possible by a heme enzyme with a non-thiolate ligand if the substrates were fixed nearby the heme iron. Our results would coincide with recent genetic analysis, suggesting that a hemoenzyme similar to cytochrome c peroxidase with an imidazole as the proximal ligand is involved in biosynthesis of tryptophan tryptophylquinone, a novel cofactor bearing indole 6,7-dione moiety, in methylamine dehydrogenase (11,(23)(24). In addition, we have reported herein that a unique amino-oxo exchange reaction of the amino-termnal 2,6-dihydro-2,6-dioxoindole was performed by Lys-C and trypsin.  (␦ in ppm) for the residues in the intact WDR fragment, product A (WءDRFK), and product B (WءDR) The NMR spectra were obtained in 20 mM potassium phosphate buffer (pD 7.0) in D 2 O at 25°C on a Unity Inova 600 MHz NMR and a Unity plus 600 MHz spectrometer (Varian). The chemical shifts are referred to TSP-d 4 .


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Microwave Assisted Synthesis of Six Member Ring Azaheterocycles and Their Antimycobacterial and Anticancer Evaluation

We report herein a comparative study concerning the influence of microwave (MW) irradiation comparative with conventional thermal heating (TH) in the reaction of six member ring azaheterocycles (derived from phenanthroline, quinoline, isoquinoline and phthalazine) with ethyl cyanoformate, and their anticancer and antimycobacterial activity. Under MW irradiation the reactions have some undeniable advantages: the reaction times decrease dramatically (from hours to minutes), the yields are higher, the reactions became selective in some cases (4,7-phenanthrolinium and phthalazinium ylides). The decreasing of reaction time+ from hours to minutes corroborated with the fact that in majority of cases the yield are higher and the selectivity is better, allow us to say that these reactions could be considered as eco-friendly. As far for biological activity, three of the compounds exhibited a significant antitumor activity against some cancer cell lines including prostate cancer, leukemia, renal cancer, lung cancer and breast cancer. The best potency in terms of growth inhibition of tumor cells (~45%) was shown by compound 4a against renal cancer A498 and ovarian cancer SK-OV-3 cells. The tested compounds have not shown any antimycobacterial activity against Mycobacterium tuberculosis.

Recently, our group reported the synthesis of several classes of six member ring azaheterocycles derived from phenanthroline, quinoline, isoquinoline and phthalazine, using conventional thermal heating conditions [26]. Being compounds with completely new cores, with a certain biological potential, and in continuation of our concern in the field of MW assisted reaction in medicinal and organic chemistry [5][6][7][8][9][10][11][12][13][27][28][29][30][31], we decided to develop an efficient and general method for preparation of our azaheterocycles derivatives using MW irradiation and, to evaluate their anticancer and antimycobacterial activity.

General procedure
All the reagents and solvents were purchased from commercial sources (Sigma Aldrich and Merck) and used without further purification. Melting points were recorded on an Electrothermal MEL-TEMP (Barnstead International, Dubuque, Iowa, USA) apparatus in open capillary tubes and are uncorrected. Analytical thin-layer chromatography was performed with commercial silica gel plates 60 F254 (Merck Darmstadt, Germany) and visualized with UV light. The NMR spectra were recorded on a (Bruker Vienna, Austria) Avance III 500 MHz spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C. Infrared (IR) data were recorded as films on potassium bromide (KBr) pellets on a FT-IR (Shimadzu Kyoto, Japan) Prestige 8400s spectrophotometer. For the microwave irradiation we used a monomode reactor Monowave 300 (Anton Paar Graz, Austria). This reactor can reach up to 300°C and controls the temperature via a built-in IR sensor. Some additional specifications of Monowave 300: microwave power 850 W, operation limits at 300°C and 30 bars, reaction vial Borosilicat, operation volume between 0.5-20 mL, pressure control by hydraulic system, agitation with an integrated magnetic stirrer (0 to 1200 rpm) and cooling with compressed air.

General Procedure for Synthesis of Azaheterocycles 2, 4, 6, 7, 9, 14, 18 and 19 under Conventional TH Conditions and MW Irradiation
The MW irradiation setup procedure is as follow: the mixture of reagents (the corresponding cycloimmonium salt (1 mmol, 1 equiv.) and ethyl cyanoformate (1.1 mmol, 1.1 equiv.) in 10 mL chloroform)) was placed into the reaction vessel and exposed to irradiation for 10 min. Using MW irradiation, the best results were obtained using a "temperature control" method. The "temperature control" method ensures a constant temperature (in this case 130°C) and varying the magnetron power. This method takes place in three stages. In the first step the temperature is raised as quickly as possible (within less than 1 min) by applying the maximum power. In the second step the reaction mixture is kept at a constant temperature with the control of the magnetron power. In the last step the reaction tube is cooled to 55°C by stopping the irradiation and blowing the reaction vial with compressed air. Once the heating cycle is completed, the reaction vial is removed from the reactor, and processed as indicated for TH.
The conventional TH setup procedures and characterization of all compounds were previously described in [26].

3.Results and discussions
In a previously research work, we presented a thoroughly study concerning the reactions of phenanthrolinium ylides, quinolinium ylides, isoquinolinium ylides and phthalazinium ylides (generated in situ in the presence of triehtylamine (TEA) from the corresponding salts) with ethyl cyanoformate, under conventional TH conditions [26]. Normally, the reactions of these ylides with ethyl cyanoformate occur as a Huisgen 3+2 cycloaddition reaction [5,10]. Interestingly, in the case of phenanthrolinium ylides (generated from the corresponding salts 1, 3, 5) no cycloaddition reaction took place, only nucleophilic substitution (attack of the cyanide ion in the γ-position, leading to products 2, 4, 6) and an α-oxidation take place simultaneously (Scheme 1) and, in the case of 4,7phenanthroline derivative 5b, a supplementary product 7b (γ-cyan substituted) is obtained. https://doi.org/10.37358/Rev. Chim.1949

Scheme 1. Reactions of phenanthrolinium ylides with ethyl cyanoformate
In the case of quinolinium ylides, isoquinolinium ylides and phthalazinium ylides (generated from the corresponding salts 8, 13, 17), the reactions occur differently (Scheme 2). Thus, in the case of quinolinium derivatives, a nucleophilic substitution (at the γ-position) and an α-oxidation took place simultaneously on the azine ring (leading to products 9), together with some complex chain transformation (leading to the byproducts 10, 11, 12).

Scheme 2. Reactions of quinolinium ylides, isoquinolinium ylides and phthalazinium ylides with ethyl cyanoformate
In the case of isoquinoline derivatives, the desired 3+2 cycloaddition reaction took place (leading to the fused imidazo-derivatives 14), together with some complex chain transformation (leading to small amounts of products 15 and 16). In the case of phthalazinium derivatives, again the 3+2 cycloaddition reaction took place (leading to the fused imidazo-derivatives 18), together with another concurrent 3+3 cycloaddition (leading to the dimeric structures 19).
From the data presented above, we may noticed that under conventional TH these reactions have some major disadvantages: moderate to low selectivity, side reactions, long reaction time (24 hours), moderate to low yields (around 50%), etc.
As an alternative route of this synthetic approach, we decide to study the reaction of these six member ring azaheterocycles with ethyl cyanoformate under MW irradiation, using a monomode reactor Monowave 300 (Anton Paar, Graz, Austria). This reactor is equipped with a stirring system (0 https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (3) to 1200 rpm) and can reach up to 300°C, with temperature control. The reactions take place in a closed vessel, at 30 bars maximum pressure. The optimal reaction conditions were found to be at 130°C, 5.5-6.0 bars, (Table 1). Table 1 lists the optimized reaction conditions we employed under MW irradiation and conventional TH. The results listed in table 1 show that MW irradiation has some undeniable advantages comparative with TH in the reaction of our azaheterocycles with ethyl cyanoformate, in terms of reaction time, selectivity of reaction and yields. The main advantage is the decrease of reaction times from 24 hours to 10 minutes. In the case of quinolinium ylides and isoquinolinium ylides under MW irradiation, the reactions became selective, only one product being obtained (9,14) and, the yields are higher. In the case of phthalazinium ylides the yields for imidazo-derivatives 18 are considerably increased (with 15-20%), while the yields for the dimeric structures 19 slightly decreased (with 5%). For phenanthrolinium ylides, the reactions under MW irradiation occurred almost similarly with the ones under conventional TH (only a slightly increasing of yields being observed), excepting the reaction of salt 5b that lead in this case to the formation of only compound 6b. That proves the better selectivity of this reaction under MW. The decreasing of reaction times from hours to minutes corroborated with the fact that in majority of cases the yields are higher and the selectivity is better, allow us to say that these reactions could be considered as eco-friendly.
In the final step, we investigated the biological activity, anticancer and antimycobacterial, of some of the new obtained compounds. In order to test the anticancer activity of compounds, we submitted all the synthesized compounds on the platform of National Cancer Institute, US (NCI) for the preliminary selection in order to test them on their 60 human tumor cell line panel. Seven compounds (2b, 4a, 6b,  14a, 18a, 18b, 18c) have been selected and tested for the anticancer activity by NCI, under the Developmental Therapeutics Program (DTP), at a single high dose (10 -5 M) cell assay. This assay was performed in a 60 human tumor cell line panel, representing leukemia, melanoma and cancers of lung, colon, brain, breast, ovary, kidney and prostate, in accordance with the protocol of the NCI [32][33][34][35]. The results are expressed as percentage of growth inhibition (PGI) values, and represent growth relative to the no-drug control and relative to the time zero number of cells. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). Table 2 lists a selection of the obtained results.  2b, 4a, 6b, 14a, 18a, 18b, 18c and phenstatin against NCI 60 human tumor cell lines (selection) As table 2 shows, derivatives 4a, 14a and 18b exhibit a significant antitumor growth inhibitory activity against several cell lines. Thus, compound 4a showed the best growth inhibition properties in the series, with the best values of PGI (around 45%) against renal cancer A498 and ovarian cancer SK-OV-3 cells. Also, moderate activity (35-40%) was observed against leukemia RPMI-8226, lung cancer NCI-H226, renal cancer UO-31, and prostate cancer PC-3 cells. Compound 18b exhibited a good activity against several tumor cells with the best values of PGI (~40%) against renal cancer UO-31, breast cancer T-47D and prostate cancer PC-3 cells, while compound 14a revealed moderate inhibitory activity (31-35%) against lung cancer NCI-H522, renal cancer A498 and prostate cancer PC-3 cells. Compounds 4a and 14a showed better growth percentage values against non-Small Cell Lung Cancer NCI-H226 line than Phenstatin used as positive control drug. Compound 4e showed also a better growth inhibition values against non-Small Cell Lung Cancer NCI-H226 line while compound 18b showed comparative results with Phenstatin.
Compounds 2b, 6b, 18a and 18c exhibited only weak insignificant inhibitory properties on some other tumor cells. From the obtained results, we can deduce that the presence of p-chlorobenzoyl moieties has a beneficial contribution to the anticancer activity. This finding is in agreement to some previous results obtained in our group that show a good inhibitory activity of compounds containing this structural feature [36][37][38].

4.Conclusions
In conclusion, we report herein a comparative study concerning the influence of MW irradiation comparative with conventional TH in the reaction of six member ring azaheterocycles (derived from phenanthroline, quinoline, isoquinoline and phthalazine) with ethyl cyanoformate, and their anticancer and antimycobacterial activity. Under MW irradiation the reactions have some significant advantages. As a general rule, the reaction times under MW decreased dramatically (from hours to minutes) and the yields of the main products are higher. In the case of 4,7-phenanthrolinium ylides and phthalazinium ylides under MW irradiation, the reactions became more selective. Thus, only one product has been obtained in case of 4,7-phenanthrolinium salt 5b, while in case of phthalazinium salts 17a-c, the yields for the dimeric structures 19 decreased. The decreasing of reaction times from hours to minutes corroborated with the fact that in majority of cases the yield are higher and the selectivity is better, allow us to say that these reactions could be considered as eco-friendly. Seven compounds were evaluated by NCI for the anticancer properties, three of them possessing promising activity against several cancer cell lines including leukemia, non-small cell lung cancer, ovarian cancer, renal cancer and breast cancer. The best activity was those one of compound 4a which possess a p-chlorobenzoyl moiety, with indicate a clearly beneficial contribution to the anticancer activity. Other two compounds, 18a and 18b, showing also good inhibitory properties, have as common structural feature the presence of a fused substituted imidazole, which indicate also a positive contribution of this moiety to the enhancing of anticancer activity. Seven compounds were also tested for antimycobacterial activity against Mycobacterium tuberculosis but none of them showed measurable activity.


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Crystal structures of the transposon Tn5-carried bleomycin resistance determinant uncomplexed and complexed with bleomycin.

The transposon Tn5 carries a gene designated ble that confers resistance to bleomycin (Bm). In this study, we determined the x-ray crystal structures of the ble gene product, designated BLMT, uncomplexed and complexed with Bm at 1.7 and 2.5 A resolution, respectively. The structure of BLMT is a dimer with two Bm-binding pockets composed of two large concavities and two long grooves. This crystal structure of BLMT complexed with Bm gives a precise mode for binding of the antibiotic to BLMT. The conformational change of BLMT generated by binding to Bm occurs at a beta-turn composed of the residues from Gln(97) to Thr(102). Crystallographic analysis of Bm bound to BLMT shows that two thiazolium rings of the bithiazole moiety are in the trans conformation. The axial ligand, which binds a metal ion, seems to be the primary amine in the beta-aminoalanine moiety. This report, which is the first with regard to the x-ray crystal structure of Bm, shows that the bithiazole moiety of Bm is far from the metal-binding domain. That is, Bm complexed with BLMT takes a more extended form than the drug complexed with DNA.

Bleomycin (Bm), 1 an antibiotic produced by Streptomyces verticillus, is widely employed in the treatment of several neoplastic diseases including non-Hodgkin's lymphoma, squamous cell carcinomas, and testicular tumors (1,2). The Bm⅐Fe(II) complex, in conjunction with a reducing agent and oxygen, causes nucleotide sequence-specific DNA cleavage (3). It has been suggested that "activated Bm," generated by the reductive activation of oxygen by a Bm⅐Fe(II) complex, cleaves DNA (3,4). Recent studies have provided unequivocal evidence for Bm-mediated degradation of certain RNA substrates, notably transfer RNAs and tRNA precursor transcripts (5)(6)(7).
Although Bm has not been used as an antibacterial agent, almost all strains of methicillin-resistant Staphylococcus aureus, isolated in Hiroshima University Hospital, were resistant to the drug. A Bm resistance gene, designated blmS, has been cloned from chromosomal DNA isolated from methicillin-resistant S. aureus and sequenced (11). The gene product, designated BLMS, is also a Bm-binding protein (9).
The transposon Tn5, expressed in Escherichia coli, carries Bm resistance together with kanamycin and streptomycin resistances (12)(13)(14). The nucleotide sequence analysis of the Bm resistance gene, designated ble, has suggested that it encodes a protein consisting of 126 amino acids with a molecular mass of 14,058 daltons (14). In addition to its role as the Bm resistance determinant, a ble product confers survival advantage to E. coli (15,16). The amino acid sequence of the ble gene product, designated by us as BLMT (17), shares sequence homology with the BLMA protein (21%) and the Shble protein (25%). BLMT has been determined to be a Bm-binding protein (17,18).
We have determined the crystal structure of BLMA at a high resolution of 1.5 Å by the single isomorphous replacement method including the anomalous scattering effect (19). Another group has determined the crystallographic analysis of the Shble protein at a 2.3 Å resolution (20). Both groups have independently provided a model that suggests that dimeric formation of the protein generates two pockets for binding to Bm. However, because the crystallization of BLMA and the Shble protein, which are complexed with Bm, has been unsuccessful until now, the precise binding mode between the protein and Bm has not been determined.
In this study, we successfully crystallized BLMT uncomplexed and complexed with Bm; the former and latter structures were determined at 1.7 and 2.5 Å resolution, respectively. We describe the conformational differences of BLMT in the Bm-free and Bm-bound form. Although a structural model of Bm bound to an oligonucleotide, determined by two-dimensional NMR analysis, has been proposed (21,22), we report a structural model based on x-ray crystallography.

EXPERIMENTAL PROCEDURES
Crystal Preparation-BLMT, overproduced using an E. coli host vector system, was purified according to the methods described previously (17,23). For crystallization, BLMT was adjusted to a final concentration of 21 mg/ml. Crystals were obtained by vapor diffusion at 25°C using the hanging-drop method (24) with the mother liquor of 25% PEG 6000 (polyethylene glycol), 0.1 M calcium acetate, and 0.1 M sodium cacodylate at pH 7.0. These crystals belong to the orthorhombic space group C222 1 with unit cell dimensions a ϭ 81.3 Å, b ϭ 85.0 Å, and c ϭ 78.8 Å. The crystal volume per unit of mass, V M (25), is 2.42 Å 3 /Da with 2 BLMT monomers in the asymmetric unit, which corresponds to a solvent content of 49%.
For crystallization of BLMT complexed with Bm, the protein was incubated at a final concentration of 14 mg/ml for 1 h at room temperature with a 3-fold molar excess of Bm A 2 sulfate (Fig. 1). The molar ratio of Bm A 2 was suitable for complete binding to BLMT. The crystals of the BLMT⅐Bm complex were obtained by vapor diffusion at 25°C using the hanging-drop method with the mother liquor of 15% PEG 6000, 0.1 M ammonium sulfate, 0.02 M magnesium chloride, and 0.05 M MES-NaOH at pH 5.0. These crystals belong to the orthorhombic space group P2 1 2 1 2 1 with unit cell dimensions a ϭ 115.3 Å, b ϭ 117.0 Å, and c ϭ 79.9 Å. The V M is 2.14 Å 3 /Da with four 2:2 complexes (2:2 ϭ two monomeric BLMT molecules complexed with two Bm molecules) in an asymmetric unit. The value corresponds to a solvent content of 42.5%.
Data Collection and Processing-Diffraction data for the Bm-free BLMT crystals were collected with synchrotron radiation (1.0 Å wavelength) at beam line 18B of the Photon Factory, the National Laboratory for High Energy Physics, Tsukuba, Japan. The data collection was done with three crystals. Three sets of high resolution data up to 1.7 Å resolution were recorded on Fuji imaging plates (400 ϫ 800 mm) using a Sakabe's Weissenberg camera (26). The diffraction images were processed to intensity data with the program WEIS (27). The intensity data from each imaging plate were scaled to each other by the Fox and Holmes method (28) and were then merged.
The diffraction data for a crystal of BLMT complexed with Bm was collected on a Rigaku R-AXIS IIC imaging plate detector system using the mirror monochromated CuK ␣ radiation produced by a Rigaku RU-300 rotating anode generator operated at 40 kV and 100 mA. The crystal-to-detector distance was set to 95 mm. Each frame of the 1.5°c rystal oscillation was collected for 10 min. The diffraction spots in a rotation range of 90°were recorded on a total of 60 frames. The data processing was accomplished at 2.5 Å resolution with the R-AXIS IIC data processing software package. The number of reflections having adequate intensities was small; this may be because of the noncrystallographic translational symmetry causing the systematic weakness of the intensities. Details of the data collection are summarized in Table I.
Structure Determination and Refinement of BLMT Uncomplexed with Bm-The crystal structure of BLMT was solved by the molecular replacement method using the programs in X-PLOR (29). The start model was a dimeric BLMA structure previously determined at 1.5 Å resolution (19). However, the N-terminal Met 1 and Val 2 and the Cterminal Gly 121 and Glu 122 residues in BLMA were omitted from the model, together with four turn regions (Val 20 -Trp 23 , Arg 52 -Ile 57 , Trp 78 -Ala 81 , and Glu 99 -Gly 103 ).
Atomic coordinates, obtained by the molecular replacement method, were refined against the data from 10 to 4.0 Å for 20 cycles with the entire monomer as a rigid body, resulting in a crystallographic R-factor of 56.4%. Atomic refinement of the model was performed with the combination of simulated annealing (30) and conventional restrained refinement methods (31) using the program X-PLOR. In lower resolution refinement, strict noncrystallographic symmetry restraint was imposed. Noncrystallographic symmetry restraint, gradually weakened according to the progress of refinement, was finally ignored. A subset of 10% of the reflections was used to monitor the free R-factor (R free ) (32). Individual B-factors were refined by starting with a mean B-factor of 22 Å 2 estimated from the Wilson plot (33).
The refinement was started with data from 10 to 3.5 Å resolution and finished at 2.0 Å resolution. Positional parameters and refinement for individual isotropic B-factors were included in each refinement cycle. The program Xfit in the XtalView suite (34) was used for the visualization and building of the model. In the final stage of the X-PLOR refinement, R free and the R-factor were improved to 23.8 and 20%, respectively, against the 18,250 reflections with F Ͼ 2 from 10 to 2.0 Å resolution. Further refinement was performed with the data set from 5.0 to 1.7 Å resolution using the program SHELXL-97 (35). The final R-factor was 19.1% for 26,798 reflections between 5.0 and 1.7 Å resolution.
The current model contains two protein monomers in the asymmetric unit, together with 111 water molecules, two tetraethylene glycol moieties, and one calcium ion. Because Met 1 and Leu 122 -Ser 126 were invisible on the electron density due to their higher flexibility, they were excluded from the current model. All side-chain atoms of the residues from Thr 2 to Leu 121 , except Glu 98 , are included in the model. The side-chain atoms of Glu 98 were completely invisible and, above the C␤, the atoms of the Glu 98 side-chain are excluded from the model. A Luzatti plot (36) revealed that the mean coordinate error was ϳ0.2 Å. No residues except Trp 99 are in the acceptable regions of the Ramachandran plot (37,38). A peptide bond between Tyr 88 and Pro 89 is in cisconformation. The r.m.s. deviations from the ideal values are 0.007 Å in bond length, 2.0°in bond angle, and 1.23°in improper angle (Table I).
The average B-factors are 26.3 Å 2 and 39.5 Å 2 for 1892 all-protein atoms and for 138 non-protein atoms, respectively.
Structure Determination and Refinement of BLMT Complexed with Bm-Pseudo-precession photographs indicate that the reflection intensities (I (hkl)) were systematically weak if k was an odd number. When calculated using the reflections from 10.0 to 4.0 Å resolution, the second highest peak, which is 6% lower than the origin peak, was present at (x, y, z) ϭ (0, 0.5, 0) on the Patterson map. This suggests that the noncrystallographic translational symmetry parallel to the b-axis is present in the crystal. To obtain the solutions of the rotation and the translation functions by decreasing the number of independent molecules per asymmetric unit, we made modifications as follows: the cell length of b-axis was halved. The axis transformation of the crystalline lattice was performed to satisfy the international rule for orthorhombic crystals (b Ͼ a Ͼ c). In other words, we assumed that this crystal belongs to the space group P2 1 2 1 2 with the unit cell dimensions a ϭ 79.9 Å, b ϭ 115.3 Å, and c ϭ 58.5 Å having two 2:2 complexes in an asymmetric unit. Simultaneously, the data set of the structure factor amplitude (F (hkl)) was transformed as follows: when k is an odd number, the reflection was ignored. Otherwise, if k was an even number, the Miller indices of F (hkl) were transformed as (hЈkЈlЈ) ϭ (l h k/2), where (hЈkЈlЈ) are the transformed Miller indices and (hkl) are the original ones. Initially, we solved the structure by the molecular replacement method and refined the atomic parameters using the transformed structure factor amplitudes and cell parameters with the programs in X-PLOR. The Bm-free form of dimeric BLMT was provided as a search model.
The atomic coordinate, obtained by the molecular replacement method, was refined against the data from 10.0 to 4.0 Å with F Ͼ 2 for 20 cycles with the entire monomer as a rigid body, resulting in a crystallographic R-factor of 32.8%. The atomic refinement of the model was performed in the combination of the simulated annealing and the conventional restrained refinement methods, imposing strict noncrystallographic symmetry restraint. The refinement was started with data having 10.0 -3.5 Å resolution, and the upper limit was raised to 2.5 Å resolution. The noncrystallographic symmetry restraint was weakened using the R free value as an indicator. The program Xfit was used for visualizing and rebuilding the model. In the course of the refinement, because the F o Ϫ F c map showed that Bm was present in the crystal, we constructed the model of Bm into the difference electron density and included it in the subsequent refinement cycles. Several cycles of X-PLOR refinement resulted in decreasing both the R-factor and R free to 18.6 and 24.5%, respectively, for the reflections from 10.0 to 2.5 Å resolution with F Ͼ 2 .
For refinement using the original cell parameters, the refined atomic coordinate of two 2:2 complexes was transformed as (xЈ, yЈ, zЈ) ϭ (y, z, x ϩ1/4), where (x, y, z) is the atomic coordinate in the halved crystalline lattice as (xЈ, yЈ, zЈ) is in the original one. The translational term (1/4) is necessary to correct the positional shift of the crystal origin. The atomic coordinates of the adjacent molecules, which are related by the crystallographic translational symmetry parallel to c-axis in the halved crystalline lattice, were transformed according to the above formula. The atomic coordinates of four 2:2 complexes were refined against the data between 10.0 and 3.5 Å with F Ͼ 2 for 20 cycles considering the entire 2:2 complex as a rigid body, resulting in a crystallographic R-factor of 21.7%. Because the number of reflections with adequate intensities was smaller when compared with the number of refined parameters, we paid attention to the following points: the noncrystallographic symmetry restraint imposing on the proteins and Bm was strongly set and decreased according to the R free value. After the bulk solvent correction (39) was done, reflections at low resolution together with weak reflections with F Ͻ 2 were used for the refinement. Further cycles of atomic refinement yielded the current model. The R-factor and the R free were improved to 22.1 and 30.2%, respectively, against all reflections from 30.0 to 2.5 Å resolution. When limited to the reflections with F Ͼ 2 , the final R-factor and the R free were 19.0 and 26.4%, respectively.
In the present study, we prepared the original topology and parameter files of the Bm molecule. The metal-binding domain in the Bm A 2 molecule is composed of the ␤-aminoalanine, pyrimidinyl propionamide, and ␤-hydroxyhistidine moieties. The binding domain for DNA is composed of the ␥-aminopropyl dimethylsulphonium and bithiazole moieties (Fig. 1). The parameters used for construction of a three-dimensional model of the former and latter moieties were obtained from the x-ray crystal structures of the P-3A⅐Cu(II) complex (40) and 3-(2Ј-phenyl-2,4Ј-bithiazole-4-carboxamide)propyl dimethylsulfonium iodide (41), respectively. The parameters for threonine and methyl valerate in the linker moiety and for gulose and mannose in the sugar moiety of the Bm molecule were prepared using the energy minimization structure, which is calculated by the program CAChe.
The current model contains 8 monomeric BLMT molecules, 8 Bm molecules, and 48 water molecules in the asymmetric unit. The monomeric BLMT molecule in the current model consists of the Thr 2 -Glu 120 residues, because both terminal residues, Met 1 and Glu 121 -Ser 126 , were invisible in the electron density. All of the side-chain atoms of the Thr 2 -Glu 120 residues are included in the model. In the electron density, the ␥-aminopropyl dimethylsulphonium and the ␤-aminoalanine moieties of Bm are almost invisible and poorly defined, respectively. The mean coordinate error was estimated from a Luzzati plot to be 0.32 Å. The Ramachandran plot indicates that the backbone torsion angles for 67.9% of the nonglycine residues are in the favor region, whereas those for 95.9% are in the acceptable region. Most of the remaining residues, which fall outside of the acceptable region, have poorly defined electron densities and may have large errors in the parameters. The r.m.s. deviations from the ideal values are 0.007 Å in bond length, 1.2°in bond angle, and 0.57°in improper angle (Table I). The average B-factors are 28.1 Å 2 for 7544 all-protein atoms and 44.2 Å 2 for the 768 atoms of the Bm molecule.
Measurement of Dissociation Constant of BLMT for Bm-Bm A 2 sulfate was added at a range of 0.05 to 0.5 M to 2 ml of 1 mM Tris-HCl (pH 7.5) containing 0.4 M BLMT. Experiments were done at 25°C using a spectrofluorophotometer (model RF-5000, Simadz, Japan) under the condition of excitation ϭ 280 nm, emission ϭ 350 nm, slit width at excitation ϭ 10 nm, and slit width at emission ϭ 10 nm. To estimate the concentration of protein contained in solution, the molar absorptivity (⑀ ϭ 19,800), calculated from the number of Trp and Tyr residues in the BLMT molecule, was employed. The dissociation constant of BLMT for Bm was calculated as described previously (19).
Two monomeric BLMT molecules are related by a noncrys-tallographic 2-fold axis (Fig. 3A). The ␤1-strand interacts with the partner's ␤6-strand, suggesting that the former strand plays a key role for the dimeric formation. The topology of BLMT is almost the same as BLMA (19) and the Shble protein (20). The dimer formation of BLMT, generated by the alternate arm exchange of two monomeric BLMT molecules, results in two large concavities and two long grooves. Although a noncrystallographic symmetry restraint is not imposed in the final refinement, one monomeric BLMT molecule is almost the same as the partner's monomer. The superposition of main-chain atoms on each BLMT monomer is shown in Fig. 3B. The r.m.s. positional differences between two monomeric BLMT molecules are 0.41 Å for main-chain atoms and 0.79 Å for all-protein atoms. Structural deviations occur at two turn regions, Ala 49 -Ser 58 and Leu 96 -Thr 102 , and at a long loop composed of the Gln 82 -Gly 87 residues. The conformational deviations may be generated because of the flexibility of these regions.
The overall structure of BLMT complexed with Bm is almost the same as that of the Bm-free BLMT (Fig. 4). The current model shows with accuracy that two Bm molecules bind to two Bm-binding pockets formed by the alternate arm exchange of two monomeric BLMT molecules.

Binding Mode between Bleomycin and BLMT
The bithiazole moiety of Bm is inserted into the long groove running along the dimer interface (Fig. 5A). The first thiazolium ring, which is interacted by the hydrophobic effect with the Phe 30B benzene, is also interacted by the polar effect with the Arg 65A guanidino group (Fig. 5B). The second thiazolium ring is tightly stacked with two indole rings from Trp 35B and Trp 99A (Fig. 5B). This stacking effect contributes to stabilization of the Bm molecule.
The Trp 35B indole ring is interacted by a hydrogen bond with the Glu 46B carboxylate. The Glu 46B carboxylate, interacted by two salt bridges with the Arg 65A guanidino group, is also stabilized by a hydrogen bond with the Thr 6B hydroxyl oxygen (Fig. 5B). That is, a hydrogen bonding network is extended from Trp 35B to Arg 65A . In addition, the Arg 65A and Trp 35B are interacted with the first and second thiazolium rings, respectively.
The large concavity is formed mainly by the residues from one monomer. One side of the concavity has the hydrophobic residues. The linker, metal-binding, and sugar domains of Bm are buried in the large concavity (Fig. 5A) and stabilized by a large number of hydrogen bonds to the protein atoms.
In the metal-binding domain of Bm, an amino group attached  to the pyrimidine ring is interacted by a hydrogen bond with two backbone carbonyl oxygens of Phe 60A and Gly 111A (Fig. 6). The carbonyl oxygen of the pyrimidinyl propionamide moiety of Bm forms a hydrogen bond with the Arg 90A guanidino group (Fig. 6). The terminal amide group of the pyrimidinyl propionamide moiety forms three hydrogen bonds: its oxygen atom with the Arg 115A guanidino group, its nitrogen atom with the Ser 61A hydroxyl oxygen, and the Trp 59A carbonyl oxygen (Fig.  6). However, ␤-aminoalanine and ␤-hydroxyhistidine for the metal binding make no polar interactions with the protein atoms. Because the ␤-aminoalanine moiety is poorly defined in the electron density, the conformation may be unstable. However, its amide group orients parallel to the Trp 59A indole ring and seems to be stacked by the apolar interaction. In fact, the Trp 59A side-chain is flexible in the Bm-free form but rigid in the complexed form because the side-chain is stacked with the imidazole rings of His 50B and the amide group of the ␤-aminoalanine moiety of Bm. Only mannose, but not gulose, in the sugar moiety of Bm is involved in the binding to the protein. The carbamoyl group, attached to the 3Ј hydroxyl oxygen of mannose, makes two hydrogen bonds: its nitrogen atom with the carbonyl oxygens of Pro 55A and Ser 58A (Fig. 6). The 2Ј and 4Ј hydroxyl groups form a hydrogen bond with the Leu 56A carbonyl oxygen and the Arg 90B guanidino group, respectively. The 6Ј hydroxyl group interacts by the hydrogen bond with the Arg 90B guanidino, Ser 85B carbonyl, and Gly 87B amide groups (Fig. 6).
The interaction between the protein and the linker domain of Bm is weak. Only one hydrogen bond is formed between the Arg 115A guanidino group and the carbonyl oxygen of the threonine moiety of Bm (Fig. 6). This occurrence is likely to be related with the flexibility of the domain.
The terminal ␥-aminopropyl dimethylsulphonium moiety of Bm A 2 , having the positive charge, is not well defined in the electron density. The end of the long groove, which is the expected binding site for the ␥-aminopropyl dimethylsulphonium moiety, has negatively charged residues Asp 3 , Asp 42 , Asp 67 , and Glu 120 . These residues are located around the positive charge of the ␥-aminopropyl dimethylsulphonium moiety. However, no electrostatic interactions are observed between this moiety and the negatively charged residues, suggesting that the negatively charged residues are necessary for the recognition of the ligand rather than for stabilization. Fig. 7A shows the superposition of the BLMT⅐Bm structure on the Bm-free structure. The positional r.m.s. differences between two structures are 0.53 and 0.77 Å for the main-chain atoms and all-protein atoms, respectively. The marked positional deviation occurs at the residues located on the loop and turn regions, such as His 50 -Ala 57 , Glu 83 -Gly 87 , and Gln 97 -Thr 102 . In particular, the Gln 97 -Thr 102 residues, composed of a ␤-turn between the ␤7and ␤8-strands, move about 2 Å toward the partner's monomer. The conformational change enables the Trp 99 indole ring of one monomer to approach the Trp 35 indole ring of the partner's monomer. The conformational approach results in the formation of stable structure of the long groove and strong intercalating interaction with the bithiazole moiety of Bm. However, the corresponding region of the Bm-free BLMT appears to be highly flexible with large B-factors (Fig. 7B).

Comparison with Bm-binding Proteins from Other Microbial Sources
Overall Structure-The amino acid sequence homology between BLMA and the Shble protein is ϳ60%, whereas that between BLMT and BLMA, or between BLMT and the Shble protein is only 21-25%. Nevertheless, the superposition of the main-chain atoms of BLMT on that of BLMA or the Shble protein shows that the overall structure of BLMT is almost the same as those of BLMA and the Shble protein (Fig. 8A). The r.m.s. positional differences for 114 residues of the core region composed of ␤-sheets are 1.25 Å between BLMT and BLMA and 1.35 Å between BLMT and the Shble protein. The marked structural deviation found among three Bm-binding proteins occurs away from the gravity center of dimeric molecules.
The conformation of a long loop connecting ␣2-helix and the ␤7-strand in BLMT is more stable than that in BLMA or the Shble protein. The stability is likely to be generated by the deletion of one amino acid in this region (Fig. 8B). Otherwise, the turn region between the ␤7and ␤8-strands of BLMT is highly flexible. The flexibility may be generated by the insertion of an amino acid into the long loop (Fig. 8B).
Substrate Specificity- Table II lists the dissociation constants (K d ) to Bm and phleomycin (Phm) for BLMT and other Bm-binding proteins. Phm is an analog of Bm and has a thiazolinylthiazole instead of a bithiazole moiety in Bm; that is, the second thiazolium ring in Phm is reduced and nonplanar. We have observed that the affinity between BLMT and Phm is stronger than between BLMA and Phm. Although the K d value to Phm for the Shble protein has not been determined yet, there is a report that the minimum inhibitory concentration of Phm to E. coli expressing the Shble protein is much lower than that of Bm (20). This suggests that the binding affinity of the Shble protein to Phm is lower than to Bm. The difference of the dissociation constant between Bm and Phm can be explained as follows. The turn region of BLMT composed of the Gln 97 -Thr 102 residues, which is the Bm-binding domain, is more flexible than those of BLMA and the Shble protein. The flexibility of the binding domain in BLMT is generated by the insertion of an

Structure of Tn5 Bleomycin-binding Protein⅐Bleomycin Complex
additional amino acid into the turn region (Fig. 8B). The flexibility might decrease the substrate specificity for binding.
Bm-binding Sites-Aromatic residues corresponding to Phe 30 and Trp 35 of BLMT, which interact with the bithiazole moiety of Bm, are present in BLMA, BLMS, and the Shble protein (Fig. 8B), suggesting that the aromatic rings are interacted by the hydrophobic effect with the bithiazole moiety. An amino acid corresponding to Trp 99 of BLMT, which interacts with the bithiazole moiety of Bm, is also present in BLMS and the Shble protein. The Trp residue of these proteins is likely to interact with the bithiazole moiety. BLMA is replaced by Ala instead of Trp 99 . A crystallographic analysis of BLMA complexed with Bm showed that Pro 101 interacts with the bithiazole moiety. 2 The negatively charged residues of the BLMT⅐Bm complex are located around the positive charge of the ␥-aminopropyl dimethylsulphonium moiety of Bm A 2 , but no electrostatic interactions are found between the protein and Bm. The distribution of the negatively charged residues in BLMT (Fig. 8B) resembles those in other Bm-binding proteins (19,20). These negatively charged residues might be helpful in the recognition of the ligand.

Structure of Bleomycin
Despite the powerful investigation of the x-ray crystal structure of Bm for the last two decades, the structure has not yet been determined. Therefore, the controversial and elusive points on the structure of the Bm molecule must be resolved; for example, it is not yet clear whether the two thiazolium rings in the bithiazole moiety of the Bm molecule are in cis-or trans-conformation. The crystallographic study of bithiazole derivatives has suggested that the two thiazolium rings may be in trans-conformation (41,42). Some groups have determined that the solution structure of bithiazole moiety bound to DNA and suggested that the cis-conformation is favored to account for the upfield shift of the chemical shift of the bithiazolium ring protons (43,44). In the present study, we tried to model both cis-and trans-type bithiazole moiety. From the electron density map obtained in this study, we conclude that the two thiazolium rings are in trans-conformation.
The ligands of the Bm molecule for the metal ion and its chirality also remain ambiguous. Most investigators agree on the fact that the equatorial ligands are the secondary amine of the ␤-aminoalanine moiety, the amide nitrogen of the ␤-hydroxyhistidine moiety, and the nitrogens from the pyrimidine and the imidazole rings. However, three possibilities are emphasized for the axial ligand: (i) the primary amine of the ␤-aminoalanine moiety (45)(46)(47); (ii) the carbamoyl nitrogen of the mannose moiety; and (iii) the primary amine of the ␤-aminoalanine moiety and the carbamoyl nitrogen of the mannose moiety (48 -50). Although our model of BLMT complexed with Bm does not contain the metal ion, we suggest that the primary amine of the ␤-aminoalanine moiety is suitable as an axial ligand for the metal ion. In the Bm molecule, while the car-

Structure of Tn5 Bleomycin-binding Protein⅐Bleomycin Complex
bamoyl group of the mannose moiety takes a stable conformation by forming the hydrogen bonds to the protein atoms, the conformation of ␤-aminoalanine moiety is unstable because of the lack of polar interactions with the protein atoms. Furthermore, the primary amine of the ␤-aminoalanine is just positioned over the putative equatorial plane for the metal ion with the same chirality as the propositions (45)(46)(47). Judging from these observations, the primary amine of the ␤-aminoalanine moiety may be an axial ligand.
The most interesting aspect in the research of the structure of Bm is the interpretation of the DNA-cleavage activity of Bm. Until now, models of Co(III)⅐Bm A 2 complexed with DNA have been built by NMR analysis (21,22). In the model, the bithiazole moiety is inserted into the space between two base pairs and stacked with the pyridine and pyrimidine rings. The positively charged terminal amine of Bm is buried in the major groove of DNA and is interacted with the negatively charged backbone phosphate. The linker and metal-binding domains of Bm, located in the minor groove of DNA, are stabilized by several hydrogen bonds with DNA including base pair-specific ones.
The binding mode of Bm complexed with BLMT may be similar to that with DNA for the following reasons. First, the bithiazole moiety is intercalated with the aromatic rings in both cases of DNA and BLMT. Second, the linker and metalbinding domains are buried in the minor groove of DNA or the large concavity of BLMT and stabilized by a number of hydrogen bonds. Finally, the positive charge of the terminal amine of Bm may be necessary for the electrostatic interaction with DNA or BLMT.
A model of the DNA⅐Bm complex shows that Bm exhibits a more compact form, that is, the bithiazole moiety folds back toward the metal-binding domain (21,22). However, x-ray analysis of Bm complexed with BLMT shows that the Bm molecule has a more extended form, that is, the bithiazole moiety is far from the metal-binding domain.


Synthesis of Substituted 2-Benzoylaminothiobenzamides and Their Ring Closure to Substituted 2-Phenylquinazoline-4-thiones

Acylation of 2-aminothiobenzamide or 2-methylaminothiobenzamide with substituted benzoyl chlorides has been used to synthesise the corresponding 2-benzoyl-aminothiobenzamides whose subsequent sodium methoxide-catalysed ring closure gives the corresponding quinazoline-4-thiones. These compounds were characterised by means of their 1H- and 13C-NMR spectra. The preferred tautomeric form of selected compounds has been discussed on the basis of their 13C-NMR, IR and Raman spectra. It has been found that in the given medium 1-methyl-quinazoline-4-thiones undergo a replacement of the sulphur substituent by oxygen giving 1-methyl-quinazoline-4-ones. In strong acid media, 2-benzoylaminothiobenzamide is cyclised through its sulphur atom to give 2-phenylbenzo[d-1,3]thiazin-4-one.

Introduction
Quinazoline-4-ones and their derivatives are well known for their pharmacological activity [1]. However, their synthesis mostly starts from anthranilic acid derivatives, in particular their N-acylamides [2,3]. The aim of the present work is to synthesise the sulphur analogues of Nbenzoylaminoanthranilamides and to carry out their ring closure to the corresponding 2-phenylquinazoline-4-thiones, which could show biological activity too.

Results and Discussion
Quinazoline-4-thiones can be synthesised by several ways: e.g. it is possible to transform the corresponding oxygen derivative into its sulphur analogue by treating it with phosphorus pentasulphide [4] or Lawesson's reagent [5]. It is also possible to make use of the reactivity of suitably substituted isothiocyanates [6]. In our work we have chosen the way of constructing the heterocyclic skeleton which consists in the acylation of 2-aminothiobenzamide or 2-methylaminothiobenzamide with substituted benzoyl chlorides and subsequent ring closure of the thus obtained 2benzoylaminothiobenzamides (1a-m) in basic medium to give the quinazoline-4-thiones (2a-m) (Scheme 1).

Scheme 1 -Preparation of 2-benzoylaminothiobenzamides and 2-phenylquinazoline-4-thiones
The starting 2-aminothiobenzamide and 2-methylaminothiobenzamide were prepared by two-step syntheses from 2-aminobenzamide and 2-methylaminobenzamide, respectively. In the first step we prepared the corresponding pyridinium salt by treatment with phosphorus pentasulphide [7], and in the second step the pyridinium salt was transformed into the respective thioamide by our original hydrolysis method in a toluene-water system. The benzoylation of the thioamides thus obtained was carried out by treatment with commercial benzoyl chlorides in acetone solvent. The reaction gives 2benzoylaminothiobenzamides 1a-m, which are very sensitive to both basic and acidic media as well as to air oxygen and heating (the melting temperature intervals are relatively broad, which is a consequence of the decomposition during m. p. determination). Due to its low stability it was impossible to prepare compound 1e (R 2 = 4-NO 2 ) with sufficient purity for characterisation. In the syntheses any contact with metals must be avoided, otherwise the 1 H-NMR spectra show broadened signals due to the presence of paramagnetic substances -most probably metal complexes of the thioamides. The ring closure of 2-benzoylaminothiobenzamides 1a-m to the corresponding 2-(subst. phenyl)quinazoline-4-thiones 2a-m is easily achieved in solutions of sodium methoxide even at room temperature (Scheme 1).
The newly synthesised 2-(subst.phenyl)quinazoline-4-thiones 2a-f can exist in three tautomeric forms (Scheme 2) similar to those of the parent compound, the unsubstituted quinazoline-4-thione itself, whose structure was discussed previously, but only on the basis of its UV [8] and IR spectra [9]. Therefore, we have focused our attention on interpretation of 13 C-NMR, IR, Raman and UV spectra. The IR spectra of the substances mentioned exhibit a broad band in the interval of 3150 -3300 cm -1 , which indicates the presence of NH group (valence vibrations). This finding together with the fact that the Raman spectrum does not show any band in the region of 2500 -2600 cm -1 (the valence vibration of SH group) exclude structure III. Also the chemical shift of C4 in the region of 187.9 -188.2 ppm is much higher than that of 2-aryl-4-allylthioquinazolines (157.8 -158.6 ppm) [10]. The differentiation between structures I and II is possible on the basis of comparison of the UV spectra, or better still, that of the 13 C-NMR shifts of C=S group. The absorption maximum of derivatives 2a-f is at 360 nm, while that of the derivatives 2g-m (whose methyl group prefers a tautomeric arrangement close to structure II) is at 388 nm. A similar conclusion follows from the 13 C-NMR shifts of C=S group: those of derivatives 2a-f lie in the region of 187.9 -188.2 ppm, that of 2phenyl-3-methylquinazoline-4-thione is at 189.2 ppm, and those of derivatives 2g-m lie in the region of 198.7 -199.0 ppm. The shifts mentioned indicate that the preferred tautomeric form is I as it is the case with the analogous 2-phenylquinazolin-4-ones too. When synthesising the N-methyl derivatives 2g-m we observed that the cyclisation is followed by desulphuration giving the corresponding 2-phenylquinazolin-4-one derivatives. This finding was verified on preparative scale: we refluxed 1-methyl-2-(4-chlorophenyl)quinazoline-4-thione (2j) in sodium methoxide and obtained 1-methyl-2-(4-chlorophenyl)quinazolin-4-one in 67% yield (Scheme 3). Similar desulphurations of quinazoline-4-thiones to quinazolin-4-ones were described earlier but the reaction conditions were substantially different: either a preliminary S-alkylation [11][12][13] or treatment with hydrogen peroxide [13]. Moreover, we have found that in strong acid medium 2-benzoylaminothiobenzamide (1a) undergoes ring closure through its sulphur atom, and the same reaction can also be presumed with the other derivatives (1b-f). A similar reaction was described earlier with thioureide esters [14] and selenoureide esters [15]. This finding can be interpreted by the SH group being a stronger nucleophile in acid medium than the protonated nitrogen of thioamide group, the opposite being true in basic media. When compound 1a was stirred in concentrated sulphuric acid at room temperature for three days, the products isolated from the resulting reaction mixture involved (besides the unreacted starting substance 1a) two cyclisation products: 2-phenyl-4-iminobenzo[d-1,3]thiazine and 2phenylbenzo[d-1,3]thiazin-4-one, the latter being formed by subsequent hydrolysis during isolation (Scheme 4).

Scheme 4 -Cyclisation of 2-benzoylaminothiobenzamide (1a) in acid medium
After prolonging the reaction time (to 7 days) and increasing of reaction temperature (60 °C) the reaction products again included the starting material 1a, and a new product also appeared: bis(2phenylquinazolin-4-yl)sulphide formed by a parallel reaction. In order to suppress the oxidation, the ring closure was carried out in anhydrous phosphoric acid at room temperature (14 days), which was followed by hydrolysis in 50% aqueous methanol (30 min reflux). The HPLC-MS analysis of the product showed that it contained 15% starting thioamide 1a and 85% 2-phenylbenzo[d-1,3]thiazin-4-one; the latter compound was then obtained in pure state by recrystallisation from aqueous methanol (yield 49%).

Conclusions
The 2-benzoylaminothiobenzamides and 1-methyl-2-phenyl-1H-quinazolin-4-thiones prepared by new methods have not been described yet. The previously known 2-phenyl-3H-quinazolin-4-thiones were prepared by a new method using other reagents, and the yields were comparable with those given in literature. Our synthetic approach has the advantage in that it offers the possibility of controlling the course of the cyclisation reaction that can involve the attack of carbonyl group either by sulphur atom or by nitrogen atom depending on the medium used. The reaction conditions were optimised so as to prevent the subsequent desulphuration reaction. 1-Methyl-2-phenylquinazolin-4thiones exclusively occur in their 1H thioketone form whereas the analogous 2-phenylquinazoline-4thiones are predominantly present in their 3H thioketone form, the tautomeric form being independent of the medium (solid phase, solution). The antituberculotic, antialgal and antimycotic activities of the 2-benzoylaminothiobenzamides and 2-phenylquinazoline-4-thiones have been tested, and will be reported in a specialised journal.
The 1 H-and 13 C-NMR spectra were measured at 360.14 and 90.57 MHz, respectively, at 25 °C, using a Bruker AMX spectrometer. Compounds 1a-m and 2a-m were measured as saturated solutions in hexadeuteriodimethyl sulphoxide (DMSO-d 6 ), and the chemical shifts are referenced to tetramethylsilane (δ( 1 H) = 0) and the solvent signal (δ( 13 C) = 39.6). The CH, CH 3 , and C quart groups in 13 C-NMR spectrum were differentiated by means of the APT method.
The IR spectra were measured in Nujol using a Perkin-Elmer 684 apparatus and a KBr cell. The Raman spectra were measured in dimethyl sulphoxide using a Bruker IFS 55 apparatus with an FRA 106 extension. For the source we used the Nd:YAG laser with the excitation frequency of 9394 cm -1 .
The mass spectra were measured with a VG Platform II mass spectrometer (Micromass, Manchester, UK) using chemical ionisation at atmospheric pressure (APCI) and a quadrupole analyser (0-3000 Da). Before the mass spectrometer there was inserted a separation apparatus consisting of a Waters 616 high-pressure pump, Waters 717 autosampler and a Waters 966 UV detector (all from Waters, Milford, USA), and a Separon SGX C18 octadecyl silica glass cartridge column (150 × 3 mm i.d., 7 µm particle size; Tessek Ltd., Prague, Czech Republic). A mixture of 70% acetonitrile and 30% redistilled water was used as the mobile phase. The effluent from the liquid chromatography was directly introduced into a quadrupole mass spectrometer equipped with APCI probe operated in positive ion mode. The data was acquired in the m/z range 15-600 at 1.9 s per scan. In the APCI mode, the temperature of the ion source and of the probe were 100 and 500°C, respectively. The cone voltage was set at 10 V for positive-ion APCl.

2-Benzoylaminothiobenzamides 1a-m
A 100-mL three-necked flask equipped with a magnetic stirrer, dropping funnel, and argon inlet was charged with the appropriate thioamide (10 mmol), dry acetone (40 mL), and triethylamine (10 mmol). The solution formed was treated with the corresponding benzoyl chloride (10 mmol) dissolved in acetone (10 mL), added within ca. 5 min. The reaction mixture was stirred under an argon atmosphere at room temperature 0.5-1 h. The separated triethylamine hydrochloride was collected by filtration on a pressure filter (sintered glass), and the filtrate was evaporated in vacuum at room temperature. The evaporation residue was mixed with ice-cold methanol (10 mL). The separated crystals were collected under argon and carefully washed with ice-cold methanol (3×10 mL). If the product thus obtained did not possess the required quality, it was recrystallised from methanol at room temperature (max. 25 °C). The yields, melting points, and 1 H-and 13 C-NMR spectra are summarised in Tables 1 -5.

2-Phenylquinazoline-4-thiones 2a-m
A 100-mL flask was charged with the appropriate 2-benzoylaminothiobenzamide 1a-m (2 mmol) and ethanol (50 mL). The solution formed was treated with 1M sodium methoxide (1 mL), and the reaction mixture was refluxed 1 h, whereupon it was neutralised with acetic acid to pH ~ 7 and concentrated to crystallisation. The crystalline product obtained on cooling was recrystallised from toluene, methanol, or dimethylformamide. The yields, melting points, 1 H-and 13 C-NMR spectra are summarised in Tables 6 -10.

2-Benzoylamino-N-methylthiobenzamide
This substance was prepared in the same way as the 2-benzoylaminothiobenzamides 1a-m. The yield of the product thus obtained was 1.

Ring Closure of 1a in Sulphuric Acid
A 50-mL flask was charged with 2-benzoylaminothiobenzamide (1a, 0.5 g, 1.95 mmol) together with 96% sulphuric acid (5 mL) and acetic anhydride (3 mL). The mixture was stirred at room temperature 3 days, whereupon it was poured onto crushed ice (50g). The separated crystals were collected by suction, washed with water, with 10% aqueous sodium bicarbonate (30 mL), and again with water. One major and two minor components were identified by means of HPLC-MS. The minority components were identified as: (1)

Ring Closure of 1a in Anhydrous Phosphoric Acid
A 50-mL flask was charged with 2-benzoylaminothiobenzamide (1a, 0.5 g, 1.95 mmol) together with anhydrous phosphoric acid (10 mL, 85% H 3 PO 4 + 6.7 g P 2 O 5 ). The mixture was stirred at room temperature 14 days. Then it was poured onto crushed ice (50g) and the separated crystalline solid was collected by suction, washed with water, with 10% aqueous sodium bicarbonate (30 mL), and again with water. The filter cake was suspended in 50% aqueous methanol (60 mL) and refluxed 30 min. The crystalline solid separated on cooling was collected by suction and recrystallised from methanol with addition of charcoal. Yield 0.23g (49%) long yellowish needles, m.p. 113-116 °C (ref. [19]