Apoptosis,also called programmed cell death,is a biological process to develop and maintain the homeostasis between cell growth and death by programmedly clearing individual old or damaged eukaryotic cells. The balance of Bcl-2 family proteins performs a crucial role in the regulation of apoptosis of cells ^{[1, 2]}. Excessive apoptosis causes hypotrophy,such as in ischemic damage,whereas an insufficient apoptosis results in uncontrolled cell proliferation,such as HIV progression and cancer development ^[3]. Reactivation of the function of pro-apoptotic Bcl-2 members by disruption of the pro-apoptotic protein vs antiapoptotic protein interaction using a non-peptide small molecule inhibitor is now recognized as a novel and promising strategy for the design of anticancer drugs. A range of small-molecular inhibitors of Bcl-2 family of proteins has been reported in recent years,such as ABT-263 ^[4] and gossypol ^[5] which have already entered clinical trials ^[6].

Recently,a variety of indolin-2-one-derived compounds, known for the pharmacophore of indolin-2-one,had been documented as having a broad spectrum of biological activities ^{[4, 5, 6, 7]} with an especial potential for use in targeted cancer therapy, and eliciting their effects through inhibition of the over-expression of the Bcl-2 family of proteins ^{[8, 9]}.

We used substructure search techniques to identify small organic molecules that contain an indolin-2-one ring. Among the small organic molecules we have identified,there are a number of small organic molecules,such as SOID8 ^[10] and Z24 ^[11] which contain an indolin-2-one core structure,and have been reported as inhibitors for anti-apoptotic Bcl-2 proteins. Therefore,the indolin-2-one core structure may be used as the starting point for the design of a new class of anti-apoptotic Bcl-2 proteins inhibitors. Moreover,1,3,4-thiadiazole derivatives ^[12],having the 1,3,4-thiadiazole moiety,also achieved good effects in promoting apoptosis and inhibiting expression in the blood vessels.

Different anticancer biotargets and mechanisms of indolin-2-one or 1,3,4-thiadiazole derivatives encourage us to design hybrids containing these two moieties within their structures.

To our knowledge,there have been reported few related compounds which have the structural combination of indolin-2-one and 1,3,4-thiadiazole moieties at the 5-position of the 1,3,4-thiadiazole ring. Thus,a series of 5'-phenyl-3'H-spiro[indoline-3,2'-^{[1, 3, 4]}oxadiazol]-2-one analogs (1,Fig. 1),all of which have both indolin-2-one and 1,3,4-thiadiazole motif,were designed,synthesized and screened for their Bcl-2 protein inhibitory activities.

Fig. 1

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Fig. 1. Structures of gossypol,ABT-263,SOID8,Z24,1,3,4 thiadiazole derivatives and target compounds.

Melting points were determined on a RDCSY-I capillary apparatus and were uncorrected. The compounds synthesized were purified by column chromatography using silica gel (200-300 mesh) except for recrystallization and thin-layer chromatography (TLC) using silica gel 60 F254 plates (250μm; Qingdao Ocean Chemical Company,China). The 1H NMR spectra were recorded on a 500 MHz Bruker AC-300 spetrometer. Chemical shifts are reported in parts per million (δ ) using internal TMS standard. The mass spectrum was obtained on Hewlett-Packard 1100 LC/ MSD spectrometer.

The synthesis of the target compounds has been carried out as depicted in Scheme 1 and the details of the key intermediates 6a-p and target compounds 1a-p are showed in Table 1. Benzothiohydrazide (4) was obtained from 2-(phenylcarbonothioylthio) acetic acid which was prepared by the Grignard reaction of bromobenzene and CS₂,and then prepared via nucleophilic substitution reaction with H₂NNH₂ at room temperature for 24 h ^{[13, 14]} in good yield. Isatin or isatin derivatives (6a-i) were synthesized from aniline or substituted aniline with chloral hydrate,anhydrous sodium sulfate and hydroxylamine hydrochloride ^[15]. Subsequently,an N-substitution reaction of 6a-i with alkylating agent in the presence of K2CO3gave 6j-p ^[16]. Starting from intermediates 6a-p,various types of derivatives of 1a-p could be prepared ^[17].

Table 1

Table 1 Details of the key intermediates 6a-p and target compounds 1a-p

Table 1 Details of the key intermediates 6a-p and target compounds 1a-p

The spectral data of all the target compounds are in full agreement with the proposed structures as shown in Ref. ^[18].

Scheme 1

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Scheme 1. (a) (i) Mg,reflux,12 h; (ii) CS2,chloroacetic acid,r.t.,12 h; (b) 80% hydrazine hydrate,2-4℃; (c) chloral hydrate,Na2SO4,H2NOH-HCl,90℃,2-3 min; (d) BnBr or CH3I,K2CO3,tetrabutylammonium bromide,r.t.,12 h; and (e) EtOH, 40℃,20 h.

Benzothiohydrazide(4):Bromobenzene (2,62.8 g,0.40 mol) and a grain of iodine were added to 9.75 g (0.40 mol) of magnesium turnings covered with 250 mL of anhydrous tetrahydrofuran. The bromobenzene was added at a sufficient rate to maintain a gentle reflux and upon completion of addition was refluxed for 2 h until the magnesium turnings disappeared. After being cooled to 0℃, CS₂(34.2 g,0.45 mol) was added slowly,the solution was stirred at room temperature for 12 h after the addition. Then the mixture was poured into 1000 g of ice water and chloroacetic acid (37.7 g, 0.40 mol),anhydrous sodium carbonate (21.0 g,0.20 mol) was added respectively and stirred for 24 h at room temperature to obtain a solution of sodium salt of 3. The liquid was adjusted to pH 2 with concentrated hydrochloric acid and a red solid (3) was gotten by filtration.

The red crystals (2.0 g) obtained from toluene after recrystallization was dissolved in 10 mL NaOH (1 mol/L) and 10 mL water and cooled to 2-4℃. Then 2 times the amount of hydrazine hydrate was added,kept at the temperature and stirred for another 2 h. After being adjusted to pH 2 with concentrated hydrochloric acid,the mixture was subjected to stirring for 1 h. Compound 4 was obtained after filtration and recrystallization from petroleum ether: ethyl acetate (3:1,v/v) with a yield of 24.0%.

Isatin or isatin derivatives (6a-i): To a solution of 120 mL water, chloral hydrate (8.93 g,0.054 mol),anhydrous sodium sulfate (130 g,0.40 mol),aniline or substituted aniline 5 (0.05 mol), hydroxylammonium chloride (10.98 g,0.158 mol) and concentrated hydrochloric acid (43 mL) were added respectively. Subsequently,the resulting suspension was heated to 90℃ for 5 min and cooled to room temperature. After filtration and washing with water (2×20 mL),the crude material was obtained. The crude was added in batches to a 250 mL,three mouth flask filled with concentrated sulfuric acid (32.6 mL) at the temperature of 65℃ and then heated up to 80℃ for 20 min. The reaction solution was cooled to room temperature and poured onto 500 g of ice water and stirred vigorously for 4 h. Intermediates 6a-i were isolated by filtration,washing with water,and recrystallization from ethanol with yields from 25.7% to 73.2%.

N-substitution isatin derivatives 6j-p: In the presence of K₂CO₃ (1 mol) and tetrabutylammonium bromide (1 mol),isatin or substituted isatin 6a-i(0.5 mol) was reacted with BnBr or CH3I (1 mol) in DMF (6 L) and the reaction mixture was stirred 12 h at room temperature. Intermediates 6j-p were obtained by filtration and purified by recrystallization from acetic acid with high yield (72.7%-85.1%).

5'-Phenyl-3'H-spiro[indoline-3,2'-^{[1, 3, 4]}thiadiazol]-2-one  analogs (1a-p): To a solution of benzothiohydrazide 4(0.1 mol) in 50 mL absolute ethanol,the isatin derivatives (6a-p) (0.1 mol) were added in absolute ethanol (150 mL). Then the mixture was heated to 40℃ for 12 h. Target compounds 1a-p were obtained by evaporation and recrystallized from a solution of dichloromethane: ethanol (2:3,v/v) with yields from 33.6% to 62.3%.

The Bcl-2 protein inhibitory activities of compounds 1a-p were evaluated with Bcl-2,Bcl-xL and Mcl-1 proteins by the standard fluorescence-polarization-based (FP-based) assay,in vitro,with ABT-263 and gossypol,as the positive control. The target proteins and small organic subject molecules waiting determination were added to phosphate buffered saline (PBS-1X) buffer and after mixing well,incubated for 30 min in the dark at room temperature. Then Bid-BH₃ polypeptide labeled by 5-carboxyfluorescein (5-FAM) was added to make the total volume of each solution to 200μL and incubated for 20 min. The resulting solution gotten above and the calibration solution were transferred to black 384 orifice plate (three parallel samples) and measured immediately using the enzyme mark instrument with the excitation wavelength of 485 nm,emission wavelength of 460 nm,and the polarization value of the calibration solution was stetted at 20 mP. Primary screening of all the compounds was determined at three typical concentrations 1μmol/L,10μmol/L,50μmol/L,with each compound determined by 3 parallel wells on the same plate and the polarization value obtained by calculating the average value. The inhibition rate was obtained from the polarization values of the negative,positive control,and subject compounds. Concentration of target proteins in the measurement commonly used is 300~500 nmol/L and the polypeptide labeled was usually 5-FAM-Bid-BH₃ polypeptide and the positive control,gossypol or ABT-263. If the result of the test indicates the inhibition rate of the compound is more than 50% at the concentration of 50mmol/L, meanwhile,its inhibition rate shows a dose-dependent manner at three different concentrations,the compound was thought to have specific binding affinities to target proteins and further determination of a more accurate IC₅₀ value is needed (Table 2). The compounds which obviously showed binding activity to target proteins were determined at seven different concentrations 1 nmol/L,10 nmol/L,100 nmol/L,1μmol/L,10μmol/L,50μmol/L and 100μmol/L. Each compound is determined by three parallel wells on the same plate and the polarization value obtained by calculating the average value. The IC50 value was obtained by data processing and graph drawing with the help of GraphPad Prism software. Competitive inhibition constants Ki were calculated from the total concentration of protein,the total concentration of polypeptide labeled,dissociation constant of protein-polypeptide complex,and IC50 of compounds tested following the method described in the reference ^[19]. The biological results expressed as IC50 and Ki were summarized in Table 3.

The synthesis route of the target compounds 1a-p has been illustrated in Scheme 1. The indolin-2-ones 6a-p were synthesized from corresponding anilines according to the reported procedures ^{[15, 16]}. Benzothiohydrazide 4 was synthesized via reaction of bromobenzene,carbon disulfide and chloroacetic acid under basic conditions followed by nucleophilic substitution reaction with 80% hydrazine hydrate. Various types of derivatives of 1a-p were prepared from intermediates 6a-p. All the target compounds and intermediates were confirmed by their spectral data shown in Ref. ^[18].

Using FP-based binding assays for Bcl-2 and Bcl-xL proteins,the inhibitory activities of Bcl-2 protein by selection of new compounds were evaluated by their binding affinities to the anti-apoptotic Bcl-2 protein. For comparison purposes,the inhibitory activities of ABT-263 and gossypol were evaluated under the same conditions.

According to the result shown in Table 2 and Table 3,a number of the new compounds exhibited potential anti-apoptotic Bcl-2 protein inhibitory activities. Compound 1k  showed good binding affinities to Bcl-xL and Mcl-1,with inhibition constants of 8.9μmol/L and 3.4μmol/L,respectively. While compound 1c exhibited the most anti-apoptotic Bcl-2 protein inhibitory activity with the binding affinities to Bcl-xL (Ki=0.16μmol/L) and the inhibit rate of approximately up to 100% at a concentration of 100μmol/L,which is comparable to the positive contrasted compounds ABT-263 or gossypol at a concentration of 10μmol/L.

Table 2

Table 2 Inhibition rate of the designed compounds to Bcl-xL,Bcl-2,and Mcl-1 proteins.

Table 2 Inhibition rate of the designed compounds to Bcl-xL,Bcl-2,and Mcl-1 proteins.

Table 3

Table 3 Binding affinities of the designed and positive compounds to Bcl-xL,Bcl-2,and Mcl-1 proteins in fluorescence polarization assays.

Table 3 Binding affinities of the designed and positive compounds to Bcl-xL,Bcl-2,and Mcl-1 proteins in fluorescence polarization assays.

Introduction of electron-withdrawing halogens of the indolin-2-one ring was more favorable for increasing inhibitory activity against anti-apoptotic Bcl-2 proteins (1c,1g). In contrast, introduction of a methyl or methoxy group (electron-donating group) reduced their inhibitory activity (1i,1h). However, compound 1c exhibited better activity against Bcl-xL proteins than compounds 1b and 1e. These results suggested that a change in the electron-withdrawing chloro-substitution at the 5-position of indolin-2-one ring to other positions of the indolin-2-one ring led to a clear loss of activity against Bcl-xL proteins. Replacement of the electron-withdrawing group resulted in compound 1a possessing less inhibitory activity against anti-apoptotic Bcl-2 proteins. Interestingly,compound 1k exhibited the most potent inhibitory activities against all the three anti-apoptotic Bcl-2 proteins. It suggested that substitution at the 1-position of indolin-2-one ring increased inhibitory activity remarkably. Therefore,we tentatively concluded that the electronic influences of the substituent at the 5-position of the indolin-2-one ring appear to play an important role in activity and that this behavior depends on the targeted protein.

In conclusion,a series of 5'-phenyl-3'H-spiro[indoline-3,2'-[1,3, 4]thiadiazol]-2-one analogs have been synthesized and tested against three Bcl-2 family of proteins in anin vitro binding assay.

In view of the result,1c achieved tight binding to Bcl-xL (Ki= 0.16mmol/L). Subject compound 1k also showed good binding affinities to Bcl-xL and Mcl-1,with inhibition constants of 8.9 and 3.4μmol/L,respectively. They were the most potent compounds studied and could serve as new lead compound for further development of antitumor agents. A practical advantage of these compounds is that their synthesis is relatively simple,and substituent groups can be conveniently introduced onto different sites. Definitive structure-activity relationships could not be confirmed by our work and further studies on biological evaluation,structure activity relationships and mechanisms of these new classes of compounds are currently in progress and the results will be reported in due course.