It can be concluded that 1,2-mannosidase from Prozyme

It can be concluded that 1,2-mannosidase from Prozyme. Enzyme assays Assays with Class II -Mannosidases (GH family 38) The isolation and purification of recombinant Golgi (GMIIb) and lysosomal (LManII) -mannosidases was carried out as described by Nem?ovi?ov et al.[15] The -mannosidase from (Jack bean) (JBMan) was purchased from Sigma. His90, Asp92, Asp204, His471) and with an inhibitor (bidentate ligand). All potent dGMII inhibitors have to bind properly to the bottom of the active site by conversation with Zn2+ and neighboring amino acid residues normally the potency of the inhibitor is usually dropped dramatically.[9a, 9b, 9e, 9f, 9i-k, 10c, 12] The critical role of Zn2+ ion and aspartic acid residues of the catalytic subsite for the interactions with the dGMII inhibitors was recently confirmed by quantum mechanics calculations.[13] From crystal structures of available fruit travel dGMII[5b, 11a] and bovine bLMan[8a] it is evident that this active site of both enzymes are structurally and chemically almost identical in radius of 10 ? around Zn2+ ion co-factor. This is one of reasons why structurally small potent GMII inhibitors like swainsonine inhibit both enzymes effectively with no significant selectivity observed. Thus, our strategy in the design of a selective GMII inhibitor was based on a previous proposal,[5a, 12c] and Pyroxamide (NSC 696085) consists of two Rabbit Polyclonal to ZADH2 basic points: (i) design of the core unit of the inhibitor (important interactions with catalytic subsite which is usually identical in both dGMII and bLMan); and (ii) design of structural linker (specific interactions with holding or anchor subsites of dGMII which are missing in bLMan).[5a, 8a, 11a] For these purposes, polyhydroxypyrrolidines with a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt, overnight, 98% for 6, 95% for 7; c) BnNH2, 120 C, 7-8 h, 87% for 8, 97% for 9; d) 6m HCl/MeOH 1:2 (v/v), rt, overnight, 62% from 8, 68% from 9; e) 1. H2, 10% Pd-C, MeOH, rt, overnight, 2. 10% HCl, 89%. Highly efficient reductive ring opening of the lactols 2 and 3 with NaBH4 was performed in EtOH providing corresponding diols 4[20] and 5. Standard mesylation of diols 4 and 5 led efficiently to dimesylated derivatives 6[20] and 7. The cyclization of the dimesylate 6 with benzyl amine to access fully guarded 1,4-imino-l-lyxitol 8 was conducted in refluxing toluene within 24 h previously.[20] Another substrate nearly the same as 7 was cyclized to at least one 1,4-imino-l-lyxitol in nice benzyl amine under reflux for 18 h.[21] However, optimization of response conditions showed that ideal reaction period and temperature for the cyclization of dimesylates 6 and 7 in nice benzyl amine had been 7 h at 120 C. Furthermore, the work-up procedure was simplified simply by extraction of excess BnNH2 with cold 0 considerably. 5M citric acidity[22] of its tiresome removal by evaporation instead. Acid-sensitive protective organizations were stable beneath the condition utilized and the band closures had been performed on the gram size (~ 3.5 g, produce 87%). Simultaneous removal of isopropylidene and trityl/silyl protecting organizations from 9 under acidic circumstances (6m HCl/MeOH) afforded a) RNH2, 120 C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6m HCl/MeOH 1:2 (v/v), rt, over night, 52% for 15, 54% for 16, 62% for 17. Another strategy based on removing a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 C, 4h, 89% for 19, 74% for 20; c) 6m HCl/MeOH 1:2 (v/v), rt, over night, 73% for 21, 66% for 22. Biological assays Some seven polyhydroxylated pyrrolidines 10, 11, 15-17, 21 and 22 was examined towards the course II -mannosidases (GH family members 38) GMIIb, LManII and JBMan as well as the course I -mannosidases (GH family members 47) AspMan. All ideals had been exactly like IC50 ideals essentially, not differing a lot more than by 10%. A selective GMII inhibitor must show none or reduced inhibitory activity towards LMan significantly. All examined polyhydroxylated pyrrolidines had been found to become weakened LManII inhibitors with IC50 ideals in the millimolar level (IC50 in selection of 1.2 mM to >8mM). (Jack port bean) (JBMan) (EC 3.2.1.24, GH family members 38), utilized like a magic size for acidic -mannosides widely.[2b, 5f, 9e, 9q-r, 10, 12a-b] This assay provided identical outcomes when compared with LManII (Desk 1). All examined structures didn’t inhibit JBMan in the 2mM focus from the inhibitor aside from 24. These outcomes support the validity from the selectivity index from the -1 additional,2-mannosidase (AspMan) (EC 3.2.1.113, GH family members 47). All examined structures, aside from unsubstituted derivative 11 (IC50 = 1 mM), didn’t inhibit AspMan in the 1-2 mM focus from the inhibitor. These outcomes indicate how the most selective inhibitors (10.All tested constructions didn’t inhibit JBMan in the 2mM focus from the inhibitor aside from 24. neighboring amino acid residues the potency of the inhibitor can be lowered dramatically in any other case.[9a, 9b, 9e, 9f, 9i-k, 10c, 12] The critical part of Zn2+ ion and aspartic acidity residues from the catalytic subsite for the interactions using the dGMII inhibitors was recently confirmed by quantum mechanics computations.[13] From crystal structures of obtainable fruit soar dGMII[5b, 11a] and bovine bLMan[8a] it really is evident how the energetic site of both enzymes are structurally and chemically almost identical in radius of 10 ? around Zn2+ ion co-factor. That is one of explanations why structurally little powerful GMII inhibitors like swainsonine inhibit both enzymes efficiently without significant selectivity noticed. Thus, our technique in the look of the selective GMII inhibitor was predicated on a earlier proposal,[5a, 12c] and includes two basic factors: (i) style of the primary unit from the inhibitor (crucial relationships with catalytic subsite which can be similar in both dGMII and bLMan); and (ii) style of structural linker (particular interactions with keeping or anchor subsites of dGMII that are lacking in bLMan).[5a, 8a, 11a] For these reasons, polyhydroxypyrrolidines using a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt, right away, 98% for 6, 95% for 7; c) BnNH2, 120 C, 7-8 h, 87% for 8, 97% for 9; d) 6m HCl/MeOH 1:2 (v/v), rt, right away, 62% from 8, 68% from 9; e) 1. H2, 10% Pd-C, MeOH, rt, right away, 2. 10% HCl, 89%. Highly effective reductive band opening from the lactols 2 and 3 with NaBH4 was performed in EtOH offering matching diols 4[20] and 5. Regular mesylation of diols 4 and 5 led effortlessly to dimesylated derivatives 6[20] and 7. The cyclization from the dimesylate 6 with benzyl amine to gain access to fully covered 1,4-imino-l-lyxitol 8 was executed previously in refluxing toluene within 24 h.[20] Another substrate nearly the same as 7 was cyclized to at least one 1,4-imino-l-lyxitol in nice benzyl amine under reflux for 18 h.[21] However, optimization of response conditions showed that optimum reaction period and temperature for the cyclization of dimesylates 6 and 7 in nice benzyl amine had been 7 h at 120 C. Furthermore, the work-up method was considerably simplified by removal of unwanted BnNH2 with frosty 0.5M citric acidity[22] rather than its tiresome removal by evaporation. Acid-sensitive defensive groups were steady beneath the condition utilized and the band closures had been performed on the gram range (~ 3.5 g, produce 87%). Simultaneous removal of isopropylidene and trityl/silyl defensive groupings from 9 under acidic circumstances (6m HCl/MeOH) afforded a) RNH2, 120 C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6m HCl/MeOH 1:2 (v/v), rt, right away, 52% for 15, 54% for 16, 62% for 17. Another strategy based on removing a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 C, 4h, 89% for 19, 74% for 20; c) 6m HCl/MeOH 1:2 (v/v), rt, right away, 73% for 21, 66% for 22. Biological assays Some seven polyhydroxylated pyrrolidines 10, 11, 15-17, 21 and 22 was examined towards the course II -mannosidases (GH family members 38) GMIIb, LManII and JBMan as well as the course I -mannosidases (GH family members 47) AspMan. All beliefs were fundamentally the identical to IC50 values, not really differing a lot more than by 10%. A selective GMII inhibitor must exhibit non-e or significantly decreased inhibitory activity towards LMan. All examined polyhydroxylated pyrrolidines had been found to become vulnerable LManII inhibitors with IC50 beliefs on the millimolar level (IC50 in selection of 1.2 mM to >8mM). (Jack port bean) (JBMan) (EC 3.2.1.24, GH family members 38), used as a widely.Since the employed model GMIIb from fruit fly showed some distinct biochemical differences compared to mannosidase III or mannosidase IIx from other invertebrates and vertebrates,[15] additional enzymatic assays with Co2+ independent types of Golgi mannosidase II, GMII, are necessary for any future refinements of inhibitor design. Substances 1, 10, 15, 16, 21, 22 were tested because of their cytotoxic activity with an 8 cancer tumor cell lines: A549 (individual lung adenocarcinoma), CCRF-CEM (T-lymphoblastic leukemia), CEM-DNR (T-lymphoblastic leukemia, daunorubicin resistant overexpressing the LRP proteins), K562 (acute myeloid leukemia), K562-Taxes (acute myeloid leukemia, overexpressing the P-glycoprotein), HCT116 (individual colorectal cancers with wild-type p53), HCT116p53-/- (individual colorectal cancers with deleted p53), U2Operating-system (Human Bone tissue Osteosarcoma Epithelial Cells) and on both nonmalignant cell lines BJ (individual fibroblast) and MRC-5 (individual lung fibroblasts). neighboring amino acidity residues usually the strength of the inhibitor is normally dropped significantly.[9a, 9b, 9e, 9f, 9i-k, 10c, 12] The critical function of Zn2+ ion and aspartic acidity residues from the catalytic subsite for the interactions using the dGMII inhibitors was recently confirmed by quantum mechanics computations.[13] From crystal structures of obtainable fruit take a flight dGMII[5b, 11a] and bovine bLMan[8a] it really is evident which the energetic site of both enzymes are structurally and chemically almost identical in radius of 10 ? around Zn2+ ion co-factor. That is one of explanations why structurally little powerful GMII inhibitors like swainsonine inhibit both enzymes successfully without significant selectivity noticed. Thus, our technique in the look of the selective GMII inhibitor was predicated on a prior proposal,[5a, 12c] and includes two basic factors: (i) style of the primary unit from the inhibitor (essential connections with catalytic subsite which is normally similar in both dGMII and bLMan); and (ii) style of structural linker (particular interactions with keeping or anchor subsites of dGMII that are lacking in bLMan).[5a, 8a, 11a] For these reasons, polyhydroxypyrrolidines using a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt, right away, 98% for 6, 95% for 7; c) BnNH2, 120 C, 7-8 h, 87% for 8, 97% for 9; d) 6m HCl/MeOH 1:2 (v/v), rt, right away, 62% from 8, 68% from 9; e) 1. H2, 10% Pd-C, MeOH, rt, right away, 2. 10% HCl, 89%. Highly effective reductive band opening from the lactols 2 and 3 with NaBH4 was performed in EtOH offering matching diols 4[20] and 5. Regular mesylation of diols 4 and 5 led effortlessly to dimesylated derivatives 6[20] and 7. The cyclization from the dimesylate 6 with benzyl amine to gain access to fully covered 1,4-imino-l-lyxitol 8 was executed previously in refluxing toluene within 24 h.[20] Another substrate nearly the same as 7 was cyclized to at least one 1,4-imino-l-lyxitol in nice benzyl amine under reflux for 18 h.[21] However, optimization of response conditions showed that optimum reaction period and temperature for the cyclization of dimesylates 6 and 7 in nice benzyl amine had been 7 h at 120 C. Furthermore, the work-up method was considerably simplified by Pyroxamide (NSC 696085) removal of unwanted BnNH2 with frosty 0.5M citric acidity[22] rather than its tiresome removal by evaporation. Acid-sensitive defensive groups were steady beneath the condition utilized and the band closures had been performed on the gram range (~ 3.5 g, produce 87%). Simultaneous removal of isopropylidene and trityl/silyl defensive groupings from 9 under acidic circumstances (6m HCl/MeOH) afforded a) RNH2, 120 C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6m HCl/MeOH 1:2 (v/v), rt, right away, 52% for 15, 54% for 16, 62% for 17. Another strategy based on removing a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 C, 4h, 89% for 19, 74% for 20; c) 6m HCl/MeOH 1:2 (v/v), rt, right away, 73% for 21, 66% for 22. Biological assays Some seven polyhydroxylated pyrrolidines 10, 11, 15-17, 21 and 22 was examined towards the course II -mannosidases (GH family members 38) GMIIb, LManII and JBMan as well as the course I -mannosidases (GH family members 47) AspMan. All beliefs were fundamentally the identical to IC50 values, not really differing a lot more than by 10%. A selective GMII inhibitor must exhibit non-e or significantly decreased inhibitory activity towards LMan. All examined polyhydroxylated pyrrolidines had been found to become vulnerable LManII inhibitors with IC50 beliefs on the millimolar level (IC50 in selection of 1.2 mM to >8mM). (Jack port bean) (JBMan) (EC 3.2.1.24, GH family members 38), trusted being a model for acidic -mannosides.[2b, 5f, 9e, 9q-r, 10, 12a-b] This assay provided equivalent outcomes when compared with LManII (Desk 1). All examined structures didn’t inhibit JBMan on the 2mM focus from the inhibitor aside from 24. These outcomes additional support the validity from the selectivity index from the -1,2-mannosidase (AspMan) (EC 3.2.1.113, GH family members 47). All examined structures, aside from unsubstituted derivative 11 (IC50 = 1 mM), didn’t inhibit AspMan on the 1-2 mM focus from the inhibitor. These outcomes indicate the fact that most selective inhibitors (10 and 21) may maintain their specificity towards Golgi -mannosidases without inhibitory results towards acidic lysosomal and endoplasmic reticulum -mannosidases. Because the utilized model GMIIb from fruits fly demonstrated some distinctive biochemical differences compared to mannosidase III or mannosidase IIx from various other invertebrates and vertebrates,[15] extra enzymatic assays with Co2+ indie types of Golgi mannosidase II, GMII, are needed.Cytotoxic activities are presented in Desk 2. Table 2 Cytotoxic activity of the preferred pyrrolidines 10, 15, 16, 21, 22 and swainsonine 1 against 8 tumor and two nonmalignant cell lines (IC50 in M). 110 C 140 M) indicating that the pyrrolidine set ups and the typical 1 may possess similar pharmacokinetics properties. Molecular modeling The primary goals of molecular modeling were to create of the selective GMII inhibitor, also to explain observed selective binding to GMII from the synthesized polyhydroxypyrrolidines. 11a] and bovine bLMan[8a] it really is evident the fact that energetic site of both enzymes are structurally and chemically nearly similar in radius of 10 ? around Zn2+ ion co-factor. That is one of explanations why structurally little powerful GMII inhibitors like swainsonine inhibit both enzymes successfully without significant selectivity noticed. Thus, our technique in the look of the selective GMII inhibitor was predicated on a prior proposal,[5a, 12c] and includes two basic factors: (i) style of the primary unit from the inhibitor (essential connections with catalytic subsite which is certainly similar in both dGMII and bLMan); and (ii) style of structural linker (particular interactions with keeping or anchor subsites of dGMII that are lacking in bLMan).[5a, 8a, 11a] For these reasons, polyhydroxypyrrolidines using a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt, right away, 98% for 6, 95% for 7; c) BnNH2, 120 C, 7-8 h, 87% for 8, 97% for 9; d) 6m HCl/MeOH 1:2 (v/v), rt, right away, 62% from 8, 68% from 9; e) 1. H2, 10% Pd-C, MeOH, rt, right away, 2. 10% HCl, 89%. Highly effective reductive band opening from the lactols 2 and 3 with NaBH4 was performed in EtOH offering matching diols 4[20] and 5. Regular mesylation of diols 4 and 5 led effortlessly to dimesylated derivatives 6[20] and 7. The cyclization from the dimesylate 6 with benzyl amine to gain Pyroxamide (NSC 696085) access to fully secured 1,4-imino-l-lyxitol 8 was executed previously in refluxing toluene within 24 h.[20] Another substrate nearly the same as 7 was cyclized to at least one 1,4-imino-l-lyxitol in nice benzyl amine under reflux for 18 h.[21] However, optimization of response conditions showed that optimum reaction period and temperature for the cyclization of dimesylates 6 and 7 in nice benzyl amine had been 7 h at 120 C. Furthermore, the work-up method was considerably simplified by removal of unwanted BnNH2 with frosty 0.5M citric acidity[22] rather than its tiresome removal by evaporation. Acid-sensitive defensive groups were steady beneath the condition utilized and the band closures had been performed on the gram range (~ 3.5 g, produce 87%). Simultaneous removal of isopropylidene and trityl/silyl defensive groupings from 9 under acidic circumstances (6m HCl/MeOH) afforded a) RNH2, 120 C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6m HCl/MeOH 1:2 (v/v), rt, right away, 52% for 15, 54% for 16, 62% for 17. Another approach based on the removal of a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 C, 4h, 89% for 19, 74% for 20; c) 6m HCl/MeOH 1:2 (v/v), rt, overnight, 73% for 21, 66% for 22. Biological assays A series of seven polyhydroxylated pyrrolidines 10, 11, 15-17, 21 and 22 was evaluated towards the class II -mannosidases (GH family 38) GMIIb, LManII and JBMan and the class I -mannosidases (GH family 47) AspMan. All values were essentially the same as IC50 values, not differing more than by 10%. A selective GMII inhibitor is required to exhibit none or significantly reduced inhibitory activity towards LMan. All tested polyhydroxylated pyrrolidines were found to be weak LManII inhibitors with IC50 values at the millimolar level (IC50 in range of 1.2 mM to >8mM). (Jack bean) (JBMan) (EC 3.2.1.24, GH family 38), widely used as a model for acidic -mannosides.[2b, 5f, 9e, 9q-r, 10, 12a-b] This assay provided comparable results as compared to LManII (Table 1). All tested structures did not inhibit JBMan at the 2mM concentration of the inhibitor except for 24. These results further support the validity of the selectivity index of the.It should be noted that these calculations at the current state of implementation are not corrected for basis set superposition effect (BSSE).[40] Some authors argue this not being a problem at close-to-equilibrium separations of the interacting moieties.[41] General remarks around the FMO accuracy may be found elsewhere.[38b, 42] FMO-PIEDA analysis was carried out in the GAMESS package [43] using the Def2-SVP basis sets.[44] For each residue (amino acid), its own fragment was assigned and all were in the same layer (i.e. active site by conversation with Zn2+ and neighboring amino acid residues otherwise the potency of the inhibitor is usually dropped dramatically.[9a, 9b, 9e, 9f, 9i-k, 10c, 12] The critical role of Zn2+ ion and aspartic acid residues of the catalytic subsite for the interactions with the dGMII inhibitors was recently confirmed by quantum mechanics calculations.[13] From crystal structures of available fruit travel dGMII[5b, 11a] and bovine bLMan[8a] it is evident that this active site of both enzymes are structurally and chemically almost identical in radius of 10 ? around Zn2+ ion co-factor. This is one of reasons why structurally small potent GMII inhibitors like swainsonine inhibit both enzymes effectively with no significant selectivity observed. Thus, our strategy in the design of a selective GMII inhibitor was based on a previous proposal,[5a, 12c] and consists of two basic points: (i) design of the core unit of the inhibitor (key interactions with catalytic subsite which is usually identical in both dGMII and bLMan); and (ii) design of structural linker (specific interactions with holding or anchor subsites of dGMII which are missing in bLMan).[5a, 8a, 11a] For these purposes, polyhydroxypyrrolidines with a) NaBH4, EtOH, rt, 2 h, 90% for 4, 98% for 5; b) MsCl, Et3N, CH2Cl2, rt, overnight, 98% for 6, 95% for 7; c) BnNH2, 120 C, 7-8 h, 87% for 8, 97% for 9; d) 6m HCl/MeOH 1:2 (v/v), rt, overnight, 62% from 8, 68% from 9; e) 1. H2, 10% Pd-C, MeOH, rt, overnight, 2. 10% HCl, 89%. Highly efficient reductive ring opening of the lactols 2 and 3 with NaBH4 was performed in EtOH providing related diols 4[20] and 5. Regular mesylation of diols 4 and 5 led easily to dimesylated derivatives 6[20] and 7. The cyclization from the dimesylate 6 with benzyl amine to gain access to fully shielded 1,4-imino-l-lyxitol 8 was carried out previously in refluxing toluene within 24 h.[20] Another substrate nearly the same as 7 was cyclized to at least one 1,4-imino-l-lyxitol in nice benzyl amine under reflux for 18 h.[21] However, optimization of response conditions showed that ideal reaction period and temperature for the cyclization of dimesylates 6 and 7 in nice benzyl amine had been 7 h at 120 C. Furthermore, the work-up treatment was considerably simplified by removal of excessive BnNH2 with cool 0.5M citric acidity[22] rather than its tiresome removal by evaporation. Acid-sensitive protecting groups were steady beneath the condition utilized and the band closures had been performed on the gram size (~ 3.5 g, produce 87%). Simultaneous removal of isopropylidene and trityl/silyl protecting organizations from 9 under acidic circumstances (6m HCl/MeOH) afforded a) RNH2, 120 C, 7-8 h, 87% for 12, 78% for 13, 77% for 14; b) 6m HCl/MeOH 1:2 (v/v), rt, over night, 52% for 15, 54% for 16, 62% for 17. Another strategy based on removing a) H2, 10% Pd-C, MeOH, rt, 4 h, 80%; b) RBr, K2CO3, DMF, 40 C, 4h, 89% for 19, 74% for 20; c) 6m HCl/MeOH 1:2 (v/v), rt, over night, 73% for 21, 66% for 22. Biological assays Some seven polyhydroxylated pyrrolidines 10, 11, 15-17, 21 and 22 was examined towards the course II -mannosidases (GH family members 38) GMIIb, LManII and JBMan as well as the course I -mannosidases (GH family members 47) AspMan. All ideals were basically the identical to IC50 values, not really differing a lot more than by 10%. A selective GMII inhibitor must exhibit non-e or significantly decreased inhibitory activity towards LMan. All examined polyhydroxylated pyrrolidines had been found to become fragile LManII inhibitors with IC50 ideals in the millimolar level (IC50 in selection of 1.2 mM to >8mM). (Jack port bean) (JBMan) (EC 3.2.1.24, GH family members 38), trusted like a model for acidic -mannosides.[2b, 5f, 9e, 9q-r, 10, 12a-b] This assay provided identical outcomes when compared with LManII (Desk 1). All examined structures didn’t inhibit JBMan in the 2mM focus from the inhibitor aside from 24. These outcomes additional support the validity from the selectivity index from the -1,2-mannosidase (AspMan) (EC 3.2.1.113, GH family members 47). All examined structures, aside from unsubstituted derivative 11 (IC50 = 1 mM), didn’t inhibit AspMan in the 1-2 mM focus from the inhibitor. These outcomes indicate how the most selective inhibitors (10 and 21) may maintain their specificity towards Golgi -mannosidases without inhibitory results towards acidic lysosomal and endoplasmic reticulum -mannosidases. Because the used model GMIIb from fruits fly demonstrated some specific biochemical differences compared to mannosidase III or mannosidase IIx from additional invertebrates and vertebrates,[15] extra enzymatic assays with Co2+ 3rd party types of Golgi mannosidase II, GMII, are necessary for any potential refinements of inhibitor style. Substances 1, 10, 15, 16, 21, 22 had been tested for his or her cytotoxic.