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Chemistry of the M (M=Fe, Ca, Ba)-Se-H2O Systems at 25 °C

The chemistry of the M (M=Fe, Ca, Ba)-Se-H2O systems at 25 °C is reviewed based on our previous papers. In this paper, the phase equilibria in the Fe(III)-Se(IV)-H2O, Ca-Se(IV,VI)-H2O and Ba-Se(IV,VI)-H2O systems at 25 °C are discussed. Then, the three-stage process for removal of selenium from industrial waste water [Se(IV,VI) < 1,500 mg/L] containing sulfuric acid was introduced. This seems to be a promising process for selenium removal from acidic sulfate waste water containing high concentration levels of selenium to below 0.1 mg/L.

Introduction
Selenium, atomic number 34, is a member of the Group16 elements together with oxygen, sulfur, tellurium and polonium. The most relevant oxidation states of selenium in aqueous solution are −2, 0, +4 and +6. Selenium (-II)  ) are predominant in aqueous solutions, depending on the oxidation-reduction potential and pH of the solution.
Selenium is also a significant component in ores such as chalcopyrite, galena and pyrite. In smelting of copper and lead, the majority of selenium is concentrated in the anode slime formed during electrolytic refining of copper, but significant quantities are followed in the sludge accumulating in sulfuric acid plants and in the electrostatic precipitator dust collected during the processing of ores and concentrates. These three intermediate products have been processed for recovering some valuable metals and selenium by the pyrometallurgical and/or hydrometallugical processes. The selenium-containing waste waters resulting from these processes should be treated in an environmentally acceptable manner. Five detailed reviews of the work on the removal of selenium(IV) and selenium(VI) from waste water were published by Koren et al. [1], Kapoor et al. [2], Mirza and Ramachandran [3] and Hata et al. [4] and Twidwell et al. [5], in which most works cited deal with precipitation, coprecipitation, adsorption, ion exchange, reverse osmosis elimination, chemical reduction and biological reduction of selenium entities. These processes are applicable to the removal of selenium(IV) from waste water, but not very effective to remove selenium(VI) under mild conditions. Hydrazine can reduce selenium(VI) to elemental selenium only in hot and concentrated sulfuric acid solution [6]. Although some anaerobic bacteria are capable of reducing selenium(VI) to elemental selenium [7][8][9][10][11], a weakness of this technique is that it requires days rather than minutes for the reduction.
An aqueous chemistry of selenium is closely related to the treatment of selenium-bearing waste waters and the hydrometallurgical processing of selenium-bearing intermediates. In this paper the chemistry of the M (M=Fe, Ca, Ba)-Se-H 2 O systems at 25 °C is reviewed based on our papers [12][13][14], and then a three-stage process for removal of selenium from industrial waste water [Se(IV,VI) < 1,500 mg/L) containing sulfuric acid to below 0.1 mg/L is discussed [15][16]. The BaSO 4 method used in the second stage allows removal of selenium(VI) to a level of around 2 mg/L. In order to remove selenium(VI) below 0.1 mg/L, a modification of the reduction method of selenium(VI) to elemental selenium by ferrous hydroxide in alkaline solution developed by Murphy [17] was successful, and furthermore, the use of the polyamine-type weakly basic ion exchange resin (Eporasu K-6) was attempted.

Equilibrium in the Fe(III)-Se(IV)-H 2 O System [12]
The Fe 2 O 3 -SeO 2 -H 2 O system is interesting in relation to the removal of selenium(IV) from hydrometallurgical solutions by the precipitation with certain ferric species. Selenious acid solutions suspended with the starting precipitate, i.e., amorphous ferric selenite, were mechanically shaken in an air bath kept at 25 °C for three months to allow equilibrium to be established between the solid and liquid phases. The results from the analysis of solutions, wet residues and precipitates (dried in vacuum) for 30 different runs were converted to weight percent values to enable the construction of the ternary phase diagram for the Fe 2 O 3 -SeO 2 -H 2 O system, which is shown in Figure 1. In this figure, the compositions of solution, wet residue and precipitate are given by ○, • and Δ, respectively. The graphical procedure based on the wet residue method proves the existence of two solid phases of Fe(HSeO 3 ) 3 and Fe 2 (SeO 3 ) 3 ·5H 2 O indicated by the points A and B. At the invariant point C (Fe 2 O 3 = 0.02 wt%, SeO 2 = 52.4 wt%), the two solid phases are in equilibrium. Point D (SeO 2 = 74.7 wt%) represents a saturated solution of selenious acid and is the equilibrium point between Fe(HSeO 3 ) 3 and solid selenious acid. The Fe 2 O 3 concentration of the liquid phase is very low, and it is approximately 0.02 wt%, even at the invariant point C.
The X-ray diffraction (XRD) patterns for compound A, Fe(HSeO 3 ) 3 , did not match with any compound in the International Center for Diffraction Data (ICDD) Cards, whereas the XRD patterns for compound B was similar to that in the data for the mineral mandarinoite, Fe 2 (SeO 3 ) 3 ·4H 2 O, which is quoted in the ICCD Card No. 29-718. However, the chemical analysis of the solid phase of compound B indicated that the ferric selenite had a hydration number of 5, i.e., it is a pentahydrate rather than being mandarinoite. Also, the differential thermal analysis and thermogravimetric analysis conformed that a hydration number was 5.  2 is a dual plot of Fe(III)/Se(IV) mole ratio in the solid phase and the log concentration data for the solution phase, both versus pH. These plots express the stability and solubility regions for ferric selenite as function of pH at 25 °C. It is clear from Figure 2(a) that Fe 2 (SeO 3 ) 3 .5H 2 O is stable in the pH range 0.1 to 2.5 which was considered in our work. Figure 2(b) shows that the iron concentration in solution in equilibrium with Fe 2 (SeO 3 ) 3 ·5H 2 O has a minimum value of 6.0 × 10 −5 mol/L at pH 2.2, while the selenium concentration decreases monotonously with an increase in pH. Hence, it may be concluded that selenium(IV) in solution containing ferric ion is basically removed in the form of

Equilibrium in the Fe(III)-Se(VI)-H 2 O System
The formation of ferric selenate at moderate temperature has been not reported. Only the XRD data for Fe 2 (SeO 4 ) 3 ·9H 2 O which was synthesized from an aqueous solution of Fe and selenic acid by slow evaporation over a period of several months are listed in the ICDD Card No.45 -544. [13] In the hydrometallurgical and mining waste water processes, calcium hydroxide and calcium carbonate have often been used as a neutralizing agent or pH adjustor. Sulfate ion is usually removed as CaSO 4 ·2H 2 O (gypsum) in nonferrous hydrometallurgy. Since selenium has similar properties to sulfur, it is possible for selenium to co-precipitate with gypsum. Also, selenium with sulfur may be incorporated into the sludge produced in the waste gas-desulfurization stage. The Ca-Se(IV)-H 2 O and Ca-Se(VI)-H 2 O systems are closely related to the treatment of selenium-contaminated process liquors in these hydrometallurgical applications and to the mineralogy relating to the occurrence of any calcium selenites and calcium selenates.

Equilibrium in the Ca-Se(IV)-H 2 O and Ca-Se(VI)-H 2 O Systems
Selenious acid solutions containing a suspension of CaO were held under mild shaking for one month at 25 °C. The original analytical data for the solutions, wet residues and precipitates are presented graphically in Figure 3 as the ternary phase diagram for the CaO-SeO 2 -H 2   The XRD patterns for compounds A and B formed in high and low selenious acid solutions were identical to those in ICCD Cards No. 36 -467 for Ca(HSeO 3 ) 2 ·H 2 O and No. 35 -883 for CaSeO 3 ·H 2 O, respectively. However, the XRD patterns for compound C, Ca 2 SeO 3 (OH) 2 ·2H 2 O, formed in alkaline solutions could not matched with any patterns in the ICCD Cards. Figure 4 shows the stability and solubility regions for calcium selenites as function of pH at 25 °C. It is noted from Figure 4 Figure 4(b) shows that the calcium concentration in solutions in equilibrium with the calcium selenites has a minimum value of 1.1 × 10 −3 mol/L at pH 10.6, while the selenium(IV) concentration has a minimum value of 4.4 × 10 −5 mol/L at pH 12.5 where Ca 2 SeO 3 (OH) 2 ·2H 2 O is in equilibrium with the solution of high calcium concentration. It should be noted that this lowest concentration for selenium (IV) (3.5 mg/L) is still much higher than the limit of industrial waste water regulation for selenium (0.1 mg/L) as specified by most regulatory authorities. A ternary phase diagram for the CaO-SeO 3 -H 2 O system at 25 °C is shown in Figure 5. In this equilibrium experiment, selenate acid solution (which was prepared by dissolving H 2 SeO 3 in distilled water) was used as the starting material. The XRD patterns for compound C are in fair agreement with that in ICCD Card No.40-235 for CaSeO 4 ·2H 2 O. On the other hand, the XRD data for three compounds A, B, and D, that is, CaSe 2 O 7 , CaSeO 4 and Ca 2 SeO 4 (OH) 2 are not listed in the ICCD Cards. Figure 6 shows the stability and solubility regions for calcium selenates as function of pH at 25 °C. It is noted from Figure 6 (a) that CaSeO4·2H2O having Ca/Se(VI) mole ratio of 1.0 is stable over the wide range of pH, while Ca 2 SeO 4 (OH) having Ca/Se(VI) mole ratio of 2.0 is stable in the very narrow range of pH 11.8 to 12.2. Also, this diagram shows two invariant points where CaSeO 4 ·2H 2 O and Ca 2 SeO 3 (OH) 2 coexist at pH 12.0 and Ca 2 SeO 3 (OH) 2 and Ca(OH) 2 ·H 2 O coexist at pH 12.2. As shown in Figure 6(b), the calcium concentration in solutions in equilibrium with CaSeO 4 .2H 2 O keeps constant at 0.42 mol/L in the pH range 0.6 to 12.0 and then decreases to 0.14 mol/L. The selenium(VI) concentration slightly decreases with increasing pH from 0.6 to 1.4 and then remains constant at same value of calcium concentration in the same pH range. A further increase in pH leads again to a decreased selenium(VI) concentration until it is reached to 11.1 g/L (0.14 mol/L).

Equilibrium in the Ba-Se(IV)-H 2 O and Ba-Se(VI)-H 2 O Systems [14]
A knowledge of the chemistry of the systems Ba-Se(IV)-H 2 O and Ba-Se(VI)-H 2 O is very important to the removal of selenium from wastewaters in an environmentally acceptable manner and sometimes selenium is recovered from process liquors by the precipitation of barium selenite and barium selenate.  The XRD patterns for compound A, BaSe 2 O 5 , formed in high selenious acid solutions could not be matched to any in the ICCD Cards, while the XRD patterns for compound B formed in low selenious acid solution were identical to that in ICCD Card No.8 -268 for BaSeO 3 .
The stability and solubility regions for barium selenites as a function of pH at 25 °C are shown in Figure 8. The plot in Figure 8(a) indicates that BaSe 2 O 5 having Ba/Se(IV) mole ratio = 1/2 is stable at pH below 4.6 and BaSeO 3 having Ba/Se(IV) = 1/1 in the pH range 4.6 to 13.7. Furthermore, the system Ba-Se(IV)-H 2 O has two invariant points at pH 4.6 where BaSe 2 O 5 and BaSeO 3 coexist and at pH 13.7 where BaSeO 3 and Ba(OH) 2 .H 2 O coexist. Figure 8(b) shows that the barium concentration in solutions in equilibrium with the barium selenites has a minimum value of 2.9 × 10 −4 mol/L at pH 9.8, whereas the selenium(IV) concentration has an extremely low value of 1.3 × 10 −4 mol/L in the pH range 13.5 to 13.7 where BaSeO 3 is in equilibrium with the solution of high barium concentration. It is noted that this lowest concentration for selenium (IV) (10 mg/L) is much higher than the limit of industrial wastewater regulation for selenium (0.1 mg/L).   The XRD pattern for compound A, BaSe 2 O 7 , could not be identified in the ICCD, whereas the XRD patterns for compound B were matched with that in ICCD Card No.15-374 for BaSeO 4 . Figure 10 shows the stability and solubility regions for barium selenates as function of pH at 25 °C. It is noted from Figure 10(a) that BaSeO 4 having Ba/Se(VI) mole ratio = 1/1 is stable over the wide range of pH and at the invariant point of pH 13.7, BaSeO 4 and Ba(OH) 2 ·H 2 O coexist. As shown in Figure 10

Conditional Free Energies of Formation for Calcium Selenites and Selenates, and Barium Selenites and Selenates
Thermodynamic data are very useful for the design of the treatment of waste waters and hydrometallurgical processing. From the solubility data described above, the solubility products for calcium selenites and selenates, and barium selenites and selenates were calculated, and conditional free energies of formation were estimated from the average of solubility products obtained as follows: They are not standard state values because there is no allowance for Ca-Se(IV,VI) species or Ba-Se(VI,VI) complexes in the thermodynamic calculation; however, they may be available as conditional values. Here, the solubility product constant for Fe 2 (SeO 3 ) 3 ·5H 2 O cannot be calculated, as thermodynamic data for the ferric selenite complex as FeHSeO 3 2+ proposed by Hamada [18], which is very stable in an aqueous solution containing ferric ion [15], is lacking. Also, the solubility product constant for calcium selenites and selenates, and barium selenites which exist in high acid solutions cannot be treated in the same way as the low solubility compounds.

Removal of Selenium from Industrial Waste Water
For protection of the environment from pollution, it has been of the greatest interest to engineers treating selenium-bearing materials to remove selenium from process liquors of high selenium concentration and industrial effluents of low selenium concentration to a value lower than the limit set by industrial waste water regulations (0.1 mg/L). Many published techniques are available for the removal of selenium(IV) from waste water, but they are not very effective for selenium(VI) or costly. In this paper, the removal of selenium(IV) and selenium(VI) from aqueous solution (Se < 1,500 mg/L) containing sulfuric acid by the following three-stage process has been proposed based on the experimental results.
1. Removal of selenium(IV) as ferric selenite and sulfate ion as calcium sulfate 2. Removal of selenium(VI) as barium selenate and sulfate ion as barium sulfate 3. Removal of selenium(VI) by reduction-precipitation of selenium(VI) to elemental selenium with ferrous hydroxide or by adsorption of selenium(VI) on the polyamine-type weakly basic ion exchange resin (Eporasu K-6).

Removal of Selenium with Ferric Species [15]
The residual concentration of selenium(IV) in solution is shown against the solution pH in Figure  11. In this figure, the residual concentration of selenium(VI) in solution after neutralizing the Na 2 SeO 4 -Fe 2 (SO 4 ) 3 solution is also shown. At a mole ratio of Fe(III)/Se(IV) of 0.67 corresponding to the theoretical ratio for Fe 2 (SeO 3 ) 3 ·5H 2 O, the residual concentration of selenium(IV) in the solution has a minimum value of 55 mg/L at pH 3.1. At Fe(III)/Se(IV) mole ratio in the starting solution of 10, the concentration of selenium(IV) is reduced to a level around 0.3 mg/L in the pH range 3.5 to 7.0 by the coprecipitation with ferric hydroxide. In reverse at below pH of 2.9, the residual concentration of selenium(IV) in the solution at a mole ratio of Fe(III)/Se(IV) of 10 is higher than that at a mole ratio of 0.67. This indicates that the dissolved ferric selenite complex is very stable in an aqueous solution containing a large amount of ferric ion. On the other hand, the concentration of selenium(VI) is reduced a very little at pH range 3.0 to 4.0 even at very high mole ratio of Fe(III)/Se(VI) of 10. This means that there is not ferric selenate compound in the Fe(III)-Se(VI)-H 2 O system at moderate conditions. Hence, selenium(VI) in waste water must be removed by other techniques than the precipitation with ferric hydroxide.  [15] In order to determine the limit of the removal of selenium(VI) by the BaSeO 4 method, the effect of Ba/Se(VI) mole ratio in the starting solution on the removal of selenium(VI) at pH 6 was determined, and is shown in Figure 12, in which the initial concentration of selenium(VI) was 0.01 M. An increase in Ba/Se(VI) mole ratio in the starting solution leads to a drastic decrease in concentration of selenium(VI) and a terminal concentration of selenium(VI) levels of around 2 mg/L at Ba/Se(VI) mole ratio above 2.5. It is to be noted that this concentration is still above the maximum contaminant level for selenium in waste water (0.1 mg/L) and the further removal of selenium(VI) is required.

Removal of Selenium as Barium Selenate
The process liquors resulting from the hydrometallurgical treatments of the anode slime formed during electrolytic refining of copper usually contain large amounts of selenium and sulfuric acid. In the first stage of the process under consideration, calcium oxide is added to such solutions for adjusting pH of the solution to precipitate selenium(IV) with iron(III) species and for removing sulfate ion to a level of 0.01 M as calcium sulfate. The removal of selenium(VI) from the 0.01 M Se(VI) -0.01 M H 2 SO 4 solution was determined at different concentrations of residual barium, and is shown in Figure 13. In this figure, the results for the 0.001 M and 0.01 M Se(VI) solutions are also presented. It is obvious that the concentration of selenium(VI) remaining in solution is governed by the concentration of coexisting barium ion, independently of the initial concentration of selenium(VI) and the presence of sulfate ion in the starting solution.

Removal of Selenium(VI) by Reduction-Precipitation of Selenium(vi) to Elemental Selenium with
Ferrous Hydroxide [15] In 1988 Murphy [17] developed a new method for removing selenium(VI) from waste water based on the reduction of selenium(VI) to elemental selenium by ferrous hydroxide in alkaline solution. The stoichiometric equation in the presence of excess ferrous hydroxide is given by: and magnetite is the predominant iron product. As the ratio of ferrous hydroxide to selenate decreases, maghemite is predominantly produced according to the following equation: In 1990, the use of this method was attempted to remove selenium(VI) from precious metals tailing pond water by Lien et al. [19]. It was found that an addition of a large amount of ferrous sulfate corresponding to a Fe(II)/Se(VI) mole ratio of about 1,800 is required to lower the selenium(VI) concentration from 4 mg/L to below 0.1 mg/L. This large dosage of ferrous sulfate may be the result of a significant drop in the reactivity of ferrous hydroxide with dissolved oxygen from air. In our work, the reduction of selenium(VI) with ferrous hydroxide was performed in an atmosphere of nitrogen to avoid the presence of oxygen in the reaction suspension. As described above, the minimal dosage of ferrous salts holds the key of the success of the process for reduction of selenium(VI) with ferrous hydroxide. If oxygen is dissolved in the solutions, the oxidation of iron(II) would also take place in alkaline solution. In the Fe(II)/Se(VI) mole ratio range of 10 to 50, the effect of oxygen on the removal of selenium(VI) was determined at an initial selenium(VI) concentration of 5 mg/L, pH 9.0 (where selenium(VI) is reduced most favorably), 70 °C and reaction time of 15 minutes, and is shown in Figure 14. The decrease in concentration of selenium by a normal progress of the reaction of selenium(VI) with ferrous hydroxide is observed in the oxygen-free solution degassed with nitrogen, but as predicted above, the dissolved oxygen from air leads to significant suppression of removal of selenium. Specially, this effect is most remarkable when air was bubbled into the solution. This means that in the air-bubbling solution the efficient amount of ferrous hydroxide for the reduction of selenium(VI) is diminished by a rapid progress of the oxidation of iron(II) by fully supplying oxygen into the solution and consequently the reduction reaction proceeds more slowly than in the solution exposed to air. In the removal of selenium from industrial effluents of low selenium concentration, the first and second stages in three-stage process considered in our work are not always required, if selenium(IV) contained in the effluents is effectively removed together with selenium(VI) by reduction with ferrous hydroxide. The effect of Fe(II)/Se(IV) mole ratio on the removal of selenium(IV) of an initial concentration of 50 mg/L at pH 9.0 in an atmosphere of nitrogen was determined. Here, the reaction time was 60 minutes. The reduction-precipitation of selenium(IV) to elemental selenium proceeds in the same manner at 25 °C and 70 °C, and results in a sufficiently low concentration of selenium at Fe(II)/Se(IV) mole ratio of 4. This value corresponds to the stoichiometric ratio in the reaction given by Equation (3). It should be concluded that in an atmosphere of nitrogen, selenium(IV) levels as low as 0.1 mg/L can be achieved by adding small amounts of almost stoichiometric amount of ferrous salts, independently of reduction temperature: In the same manner, the removal of selenium(VI) was determined, and is shown in Figure 15. Differently from selenium(IV) removal, a rise in temperature significantly accelerates the reduction of selenium(VI). At 25 °C even at a high mole ratio of Fe(II)/Se(VI) of 50 the selenium concentration of 0.1 mg/L is not attained, but at 70 °C selenium can be removed to a level lower than 0.1 mg/L at a ratio of 20. In our work, the similar trends were obtained for the removal of selenium(VI) at an initial concentration of 5 mg/L.

Removal of Selenium(iv,vi) from Solution Containing Sulfuric Acid by Three-Stage Process Using by Reduction-Precipitation of Selenium(vi) to Elemental Selenium with Ferrous Hydroxide in the
Third Stage [15] The operation conditions and the main reactions for each stage in the process proposed in our work were described already. The three-stages, precipitation of ferric selenite and calcium sulfate, precipitation of barium selenate and barium sulfate and reduction-precipitation of selenium(VI) to elemental selenium with ferrous hydroxide were combined for the removal of selenium(IV,VI) from solution containing sulfuric acid. This process was modeled with the feed solution of 0.01 M ( = 790 mg/L) in Se(IV), 0.01 M ( = 790 mg/L) in Se(VI) and 10 g/L in H 2 SO 4 .
At the first stage, after 5 mL of 0.5 M Fe 2 (SO 4 ) 3 solution (corresponds to a mole ratio of Fe(III)/total Se of 0.5) was added into 500 mL of the feed solution, the solution was neutralized to pH of 3.5 with calcium hydroxide and held with stirring at 25 °C for one hour. The typical analytical data for solutions of six runs are listed in Table 1, which indicates that the majority of selenium(IV) is removed as ferric selenite and a small amount of selenium(VI) is also removed. Sulfate ion may be removed to a level of 10 −1.6 mol/L (= 2.5 g/L), calculated from data for calcium concentration (0.5 g/L) and solubility product of 10 −4.4 for calcium sulfate. At the second stage, 1.0 M BaCl 2 solution of various volumes was added into the filtrates containing selenium(IV) of 10 mg/L and selenium(VI) of 740 mg/L from the first stage and the pH of solutions was adjusted to 6 and held with stirring at 25 °C for two hours. As shown in Table 2, selenium(IV) remaining in the solution from the first stage solution can be removed to a very low level as barium selenite (BaSeO 3 ) by addition of a large excess of BaCl 2 . Selenium(VI) can be removed to a level of 100 mg/L when the barium concentration in solution was kept at about 50 mg/L. Furthermore, for removing selenium(VI) to a level of 10 mg/L the residual concentration of barium is required to be about 500 mg/L. At the third stage, the two filtrates containing 4.7 mg/L and 51 mg/L selenium from the second stage were treated by dissolving ferrous sulfate of weights corresponding to mole ratios of 50 at 25 °C and 20 at 70 °C in an atmosphere of nitrogen and then by adjusting pH of solution to 9.0. Table 3 shows the analytical data for solutions after the reduction-precipitation with ferrous hydroxide for one hour. In both cases, the reduction of selenium(VI) as well as selenium(IV) with ferrous hydroxide is possible to remove selenium to below 0.1 g/L from the liquor from the second stage except the case which the filtrate containing 51 mg/L selenium(VI) was treated at Fe(II)/Se(VI) mole ratio of 50 and at 25 °C. Also, barium is precipitated with sulfate ion from ferrous sulfate added.

Adsorption of Selenium(VI) in Batch Experiments
The adsorption capacity of weakly basic ion exchange resin is generally affected by hydroxyl ion in solution. The adsorption capacity of the resin (Eporasu K-6) was determined as a function of pH for selenium(VI) by batch experiments, and is shown in Figures 16. Here, selenium(VI) concentration in the starting solution was 60 mg/L. The amount of selenium(VI) adsorbed by the resin jumps up as soon as a stable species in solution changes from HSeO 4 − to SeO 4 2− by a rise in pH and has a high value of 46 g/L-R over a wide pH range of 3 to 12. At pH 13 a decrease in adsorption is observed. This may be attributed to the depression of ionization of the functional group of the resin due to a large amount of hydroxyl ion in solution. From these results, it is noted that selenium(VI) can be effectively absorbed on the resin over a wide pH range than selenium(IV).  and Cl − on the adsorption of selenium(VI) on the resin was determined by adding various amounts of Na 2 SO 4 or NaCl to the selenium(VI) solution of 60 mg/L at pH 6.0, and is shown in Figure 17

Adsorption of Se(VI) in Column Experiments
Selenium(VI) concentrations of 10, 25 and 60 mg/L in the feed solution (pH = 6.0) were passed through the resin bed (7.6 mmφ × 110 mm) packed in the column at space velocities of 10. As shown in Figure 18, after selenium(VI) starts to be detected in the effluent, the selenium(VI) concentration in effluent leaked out curves gently up with an increase in effluent volume at selenium(VI) concentration of 10 mg /L in the feed solution, while jumps sharply up at both 25 and 60 mg/L. The amount of selenium(VI) adsorbed on the resin calculated from a permissible effluent volume for disposal is 65.1 g/L-R for selenium(VI) concentration of 10 mg /L in the feed solution, 62.5 g/L-R for 25 mg/L and 54.4 g/L-R for 60 mg/L, which indicates that it is not advisable to use the resin for removing a high level of selenium(VI).
In order to find a suitable eluent of selenium(VI) absorbed, batch experiments were carried out for hydrochloric acid and sodium hydroxide solutions of various concentrations. Here, the resin loaded by selenium(VI) at 46 g/L-R was contacted with the eluents for three hours. As shown in Figure 18, in hydrochloric acid solution the amount of selenium(IV) adsorbed by the resin decreases sharply to 6g/L-R with a slight increase in acidity and then still continues to decrease marginally as the acidity increases. In sodium hydroxide solution, the amount of selenium(IV) adsorbed decreases gradually with an increase in basicity and then stays at around 13 g/L-R over NaOH concentration range above 1.0 mol/L. These suggests that hydrochloric acid solution will acts more effectively as an eluent of selenium(VI) from the resin than sodium hydroxide solution.
The cycles of adsorption and elution of selenium(VI) were repeated three times. A decrease in loading capacity caused by recycling the resin was not observed and selenium(VI) was eluted from the resin with relatively small volumes of 1 M HCl (22 L/L-R), which indicate that the resin can be used repeatedly for the removal of selenium(VI) from waste water.

Removal of Selenium(IV,VI) from Solution Containing Sulfuric Acid by Three-Stage Process Using by Adsorption of Selenium(VI) on the Polyamine-Type Weakly Basic Ion Exchange Resin (Eporasu K-6) in the Third Stage
As noted previously, in the third stage, the remaining selenium(VI) is removed to a lower concentration than 0.1 mg/L by reduction-precipitation of selenium(VI) to elemental selenium with ferrous hydroxide at pH 9 at 70 °C in an atmosphere of nitrogen. However, this process requires very strict conditions to effectively remove selenium(VI). Replacement of the ferrous hydroxide reduction process with the adsorption process using the polyamine-type weakly basic ion exchange resin (Eporasu K-6) yields a simplified selenium removal process which is shown in Figure 19. This process seems to be promising to remove selenium from high concentration levels of selenium(IV) and selenium(VI) in acidic sulfate waste water to below 0.1 mg/L. Figure 19. Flowsheet of process for removal of selenium from industrial waste water.

Conclusions
In this paper, the chemistry of the M (M = Fe, Ca, Ba)-Se-H 2 O systems at 25 °C was described and the process for the removal of selenium from the industrial waste water was proposed. In order to develop a simpler and easier process, the compounds of lower solubility or the other reactions should be determined by further investigations.


Tris{2-methoxy-6-[(4-methylphenyl)iminiomethyl]phenolate-κ2 O,O′}tris(thiocyanato-κN)lanthanum(III)

In the title compound, [La(NCS)3(C15H15NO2)3], the metal centre is nine-coordinated by six O atoms from three zwitterionic Schiff base 2-methoxy-6-[(4-methylphenyl)iminiomethyl]phenolate (L) ligands and three terminal N atoms of the thiocyanate ions in a monocapped square-antiprismatic environment. The L ligands chelate the LaIII ion with strong La—O(deprotonated phenolic) bonds [2.435 (3)–2.464 (3) Å] and significantly longer La—O(methoxy) bonds [2.801 (3)–2.831 (3) Å]. The La—N bond lengths range from 2.541 (4) to 2.605 (4) Å.

Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: PV2228).

S1. Comment
The rare earth complexes prepared by ligands derived from o-vanillin have been reported in the past decades due to the intriguing biological activities of o-vanillin and the varied structures and behaviors of the Schiff bases (Yeap et al., 2003).
For these reasons, we have been engaged in the syntheses of new analogous Schiff bases derived from o-vanillin and their rare metal complexes. For the past few years we have synthesized and reported several Schiff complexes (Zhao et al., 2007;Liu et al., 2009). Herein, we describe the crystal structure of a new lanthanum(III) complex, (I).
The structure of the title compound is shown in Fig. 1  (phenolic) distances. The La-N bonds vary from 2.541 (4)Å to 2.605 (4) Å. These bond lengths correspond to those previously observed (Liu et al., 2009). While in contract to the isomorphous Ce III complex, the La-X (X = O/N) are slightly longer than the corresponding distances reported in the Ce III complex (Liu et al., 2009), which can be attribute to a decrease in the ionic radii from La III to Ce III due to the lanthanide contraction. Because of the geometric and chemical environment requirements of the chelating groups the coordination geometry deviates considerably from the distorted capped square antiprism geometry (Fig. 2). In the HL ligands, the protons of the phenolic groups are considered to have transferred to N-imine atoms, which are involved in intramolecular hydrogen bonds (Table 1) that probably forces them to assume nearly planar conformations.
A solid product (HL) was separated from the solution after stirring for about 10 min. and was purified by recrystallization from ethanol.
To a methanol solution of N-3-methoxysalicylidene-p-toluidine (HL) (3 mmol, 10 ml) was dropped 1 mmol La(Cl 3 ) 3 .6H 2 O (dissolved in methanol) under stirring condition and the mixture solution was still stirred at room temperature for 8 h to give a purplish red solution. The deposit was filtered out and the solution was kept for evaporating.
The red crystals of the title compound (I) were formed after several days.

S3. Refinement
The H atoms bonded to C and N atoms were positioned geometrically and refined using a riding model with C-H distances: 0.96, 0.93 and 0.86 Å for aliphatic, aromatic and imino H-atoms, respectively, and U iso (H) = 1.5U eq (C- aliphatic) or 1.2U eq (the rest of the parent atoms).

Figure 1
The molecular structure of the title complex, showing the atom-labeling scheme. Displacement ellipsoids are drawn at the 30% probability level.  The coordination environment of the lanthanum(III) atom, showing the monocapped square antiprism.

Tris{2-methoxy-6-[(4-methylphenyl)iminiomethyl]phenolate-κ 2 O,O′}tris(thiocyanato-κN)lanthanum(III)
Crystal data  Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max = 0.002 Δρ max = 0.53 e Å −3 Δρ min = −0.54 e Å −3 Special details Experimental. HL product: yield = 80%, (m.p. 373-374 K). Analysis calculated for C 15 H 15 NO 2 : C 74.66, H 6.27, N 5.81%; found: C 74.62, H 6.31, N 5.77%. Geometry. All e.s.d.s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.s are taken into account individually in the estimation of e.s.d.s in distances, angles and torsion angles; correlations between e.s.d.s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.s is used for estimating e.s.d.s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


text:
4,4′-Bis(2-methoxylstyryl)biphenyl

The title compound, C30H26O2, was prepared by the reaction of a Wittig reagent and 2-methoxybenzaldehyde. The molecule lies about an inversion centre located at the midpoint of the C—C bond between the inner benzene rings. The crystal structure is stabilized by C—H⋯π interactions.

The title compound, C 30 H 26 O 2 , was prepared by the reaction of a Wittig reagent and 2-methoxybenzaldehyde. The molecule lies about an inversion centre located at the midpoint of the C-C bond between the inner benzene rings. The crystal structure is stabilized by C-HÁ Á Á interactions.

D-HÁ
Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: AT2831). such as the optics characteristic. (Song et al., 2003). As part of our search for new ethylene biphenyl compounds we synthesized the title compound (I), and describe its structure here.
As shown in Fig. 1, the molecule has an inversion centre lied on the midpoint of the C-C bond between the inner benzene rings. The C8-C9 bond length of 1.314 (4)Å is comparable with C-C double bond [1.336 (2) Å] reported (Trueblood et al., 1982).
In the structure, there is no classcial hydrogen bonds. The crystal structure is stabilized by C-H···π interactions (Table   1).

S2. Experimental
A mixture of the Wittig-reagent (0.1 mol), and 2-methoxybenzaldehyde (0.2 mol) was stirred in refluxing N,N-dimethylformamide (20 mL) for 4 h to afford the title compound (0.084 mol, yield 84%). Single crystals suitable for X-ray measurements were obtained by recrystallization from ethanol at room temperature.

S3. Refinement
H atoms were fixed geometrically and allowed to ride on their attached atoms, with C-H = 0.93 -0.96 Å, and with U iso (H) =1.2 or 1.5U eq (C).

Special details
Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.


extfieldsofstudy: Medicine Chemistry

text:
Trichlorido[(methyl{2-[methyl(2-pyridylmethyl)amino]ethyl}amino)acetonitrile]iron(III) methanol hemisolvate

The title compound, [FeCl3(C12H18N4)]·0.5CH3OH, contains an FeIII ion in a distorted octahedral coordination environment. The neutral N,N′,N′′-tridentate ligand adopts a fac coordination mode, and chloride ligands lie trans to each of the three coordinated N atoms. In the crystal, the complexes form columns extending parallel to the approximate local threefold axes of the FeN3Cl3 octahedra, and the columns are arranged so that the uncoordinated nitrile groups align in an antiparallel manner and the pyridyl rings form offset face-to-face arrangements [interplanar separations = 2.95 (1) and 3.11 (1) Å; centroid–centroid distances = 5.31 (1) and 4.92 (1) Å]. The methanol solvent molecule is disordered about a twofold rotation axis.

The title compound, [FeCl 3 (C 12 H 18 N 4 )]Á0.5CH 3 OH, contains an Fe III ion in a distorted octahedral coordination environment. The neutral N,N 0 ,N 00 -tridentate ligand adopts a fac coordination mode, and chloride ligands lie trans to each of the three coordinated N atoms. In the crystal, the complexes form columns extending parallel to the approximate local threefold axes of the FeN 3 Cl 3 octahedra, and the columns are arranged so that the uncoordinated nitrile groups align in an antiparallel manner and the pyridyl rings form offset face-toface arrangements [interplanar separations = 2.95 (1) and 3.11 (1) Å ; centroid-centroid distances = 5.31 (1) and 4.92 (1) Å ]. The methanol solvent molecule is disordered about a twofold rotation axis.
The solvent was removed under reduced pressure, then the residue was re-dissolved in water, adjusted to pH 8 with conc.
The title compound was then prepared as follows: Anhydrous FeCl 3 (15.8 mg, 0.097 mmol) was added to the mixed ligand product from above (23.3 mg, 0.098 mmol) in methanol (1.75 ml), and a few yellow blocks of (I) were deposited overnight.  The molecular structure of (I) with displacement ellipsoids shown at 50% probability for non-H atoms. The methanol molecule (C1S-O1S) is disordered around a 2-fold rotation axis.

Figure 2
Unit-cell contents of (I) projected along the b axis, which corresponds to the stacking directions of the "columns" referred to in the Abstract.

Trichlorido[(methyl{2-[methyl(2-pyridylmethyl)amino]ethyl}amino)acetonitrile]iron(III) methanol hemisolvate
Crystal data [FeCl 3 (C 12  where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.48 e Å −3 Δρ min = −0.45 e Å −3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å 2 )
x y z U iso */U eq Occ. (


s2fieldsofstudy: Chemistry Biology

text:
Structure-activity relationship analysis of cytotoxic cyanoguanidines: selection of CHS 828 as candidate drug

Background N-(6-(4-chlorophenoxy)hexyl)-N'-cyano-N''-4-pyridyl guanidine) (CHS 828) is the first candidate drug from a novel group of anti-tumour agents – the pyridyl cyanoguanidines, shown to be potent compounds interfering with cellular metabolism (inhibition of nicotinamide phosphoribosyl transferase) and NF-κB signalling. Substituted cyanoguanidines are also found in anti-hypertensive agents such as the potassium channel opener pinacidil (N-cyano-N'-(4-pyridyl)-N''-(1,2,2-trimethylpropyl)guanidine) and histamine-II receptor antagonists (e.g. cimetidine, N-cyano-N'-methyl-N''-[2-[[(5-methylimidazol-4-yl]methyl]thio]ethyl)guanidine). In animal studies, CHS 828 has shown very promising activity, and phase I and II studies resulted in further development of a with a water soluble prodrug. Findings To study the structural requirements for cyanoguanidine cytotoxicity a set of 19 analogues were synthesized. The cytotoxic effects were then studied in ten cell lines selected for different origins and mechanisms of resistance, using the fluorometric microculture cytotoxicity assay (FMCA). The compounds showed varying cytotoxic activity even though the dose-response curves for some analogues were very shallow. Pinacidil and cimetidine were found to be non-toxic in all ten cell lines. Starting with cyanoguanidine as the crucial core it was shown that 4-pyridyl substitution was more efficient than was 3-pyridyl substitution. The 4-pyridyl cyanoguanidine moiety should be linked by an alkyl chain, optimally a hexyl, heptyl or octyl chain, to a bulky end group. The exact composition of this end group did not seem to be of crucial importance; when the end group was a mono-substituted phenyl ring it was shown that the preferred position was 4-substitution, followed by 3- and, finally, 2-substitution as the least active. Whether the substituent was a chloro, nitro or methoxy substituent seemed to be of minor importance. Finally, the activity patterns in the ten cell lines were compared. Substances with similar structures correlated well, whilst substances with large differences in molecular structure demonstrated lower correlation coefficients. Conclusion According to this structure-activity relationship (SAR) study, CHS 828 meets the requirements for optimal cytotoxic activity for this class of compounds.

Background
The anti-tumoral activity of the pyridyl cyanoguanidines was first detected in a routine in vivo screening programme in a rat model with Yoshida ascites sarcoma cell tumours. The studied compounds were synthesized as analogues to the anti-hypertensive potassium channel opener pinacidil (Fig. 1). Replacement of the side chain by longer aryl-containing side chains caused a loss of activity in this respect, and a candidate drug, N- (6-(4-chlorophenoxy)hexyl)-N'cyano-N''-4-pyridyl guanidine) (CHS 828), was selected after studies of structure-activity relationships (SARs) in vitro and preliminary evaluation in vivo [1].
As CHS 828 displays a broad spectrum of activity, low cross-resistance with standard drugs, favourable in vitro therapeutic indices [1,3,4] and IC 50 values in the nanomolar range, the drug was brought into phase I clinical trials [5,6]. A detailed description of the mechanism of action of CHS 828 was not presented until 2004 when Dr Stangelshøj Olsen and co-workers published data sup-porting inhibition of the Inhibitory κ-B kinase (IKK) complex, with an IC 50 of 8 nM [7]. The mechanism of action may, however, be dual, and may in part depend on drug concentration and exposure time as suggested by Hassan et al [8]. According to Ekelund et al [9], high concentrations (1 and 10 μM, well exceeding the IC 50 ) of CHS 828, just like meta-iodobenzyl guanidine (MIBG), inhibit mitochondrial respiration, with a subsequent increase in glycolysis to cover the energy need. Interestingly, this high concentration effect is similar in resistant U-937/CHS cells and the U-937 GTB cells [9]. In addition, it was shown that cells grown in pyruvate-supplemented, glucose-free medium are, at least partly, protected from CHS 828 toxicity, thereby indicating that the extent of glycolysis may be an important determinant of optimal CHS 828 activity [9]. The process following CHS 828 exposure is characterized by an almost normal proliferation during the first 24 hours followed by an abrupt shut-off of deoxyribonucleic acid (DNA) and protein synthesis, a modest increase in caspase-3 activity and DNA fragmentation, and the first signs of cell death [10,11]. These processes seem dependent on intact protein synthesis [10]. Addition of the adenosine diphosphate (ADP) ribosylation inhibitor 3-aminobenzamide to the medium and U-937 cells exposed to CHS 828 resulted in a 100-fold decrease in IC 50 , and also in more predominant apoptotic cell death [12]. This effect, however, appears to be unrelated to the effects on poly(ADP-ribose) polymerase (PARP), and the authors suggest that it is mediated through effects on glycolytic enzymes, such as glyceraldehyde-3-phosphate [12]. In the subsequent paper it was shown that Substituted cyanoguanidines with defined pharmacological effects  C PARP-1 inactivation in mouse fibroblasts in fact sensitizes cells to the cytotoxic action of CHS 828, and consequently, that the drug is able to activate different cellular pathways depending on PARP status [13]. During the 2007 American Association for Cancer Research/National Cancer Institute/European Organization for Research and Treatment of Cancer (AACR-NCI-EORTC) International Conference on Molecular Targets and Cancer Therapeutics, Roulston and co-workers at Gemin X Biotechnologies Inc. presented data strongly suggesting that GMX1778 (EB1627), a soluble prodrug of CHS 828, acts by inhibition of nicotinamide phosphoribosyl transferase (Nampt), an enzyme involved in nicotinamide adenine dinucleotide (oxidized) (NAD+) biosynthesis, and that nuclear factor-kappa B (NF-κB) inhibition is only a consequence of NAD+ decline [14]. This mechanism was recently confirmed in a study by Dr Høgh Olesen and coworkers, including cross-resistance to FK866, a known inhibitor of Nampt [15]. Nampt has also been presumed to be a cytokine (PBEF) or a hormone (visfatin). The crystal structure of Nampt in the presence and absence of nicotineamide mononucleotide shows that Nampt is a dimeric type II phosphoribosyltransferase and provides insights into the enzymatic mechanism [16]. Previously noted effects by CHS 828 on glycolysis and inhibition of the transcription factor NF-κB can likely be secondary to inhibition of Nampt.
The aim of this study was to establish the structural requirements for cyanoguanidine cytotoxicity. The cytotoxic effects of nineteen substituted pyridyl cyanoguanidines, with variations in pyridyl substitution, different alkyl chain linkers and end groups, were studied on a panel of ten cell lines.

Cell lines
Cytotoxicity was assayed in a panel of ten human tumour cell lines. This panel was previously established for use in the early investigation of new anti-cancer compounds, and the cell lines represent different histological origins and mechanisms of resistance [17]. The panel has successfully been used in in vitro evaluation of a wide range of different cytotoxic agents, both for clinical and for investigational purposes. A brief description of the cell lines is presented in Table 1. Cells were maintained as previously described [17].

Medium and reagents
Cell growth medium RPMI-1640 (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS) (Sigma), 2 mM glutamine, 100 μg/mL streptomycin and 100 U/mL penicillin was used for all experiments. Fluorescein diacetate (FDA) (Sigma) was dissolved in dimethylsulphoxide (DMSO) (Sigma) to a concentration of 10 mg/mL and kept frozen as a stock solution in the dark.

Test compounds
The test compounds, a set of 19 structure analogues to CHS 828 with the cyanoguanidine core preserved, was a kind gift from the Department of Chemical Research, Leo Pharma, Ballerup, Denmark. Compounds were synthesized using methods described elsewhere [1,9].

Cytotoxicity assay
The FMCA has been previously described in detail [18][19][20]. The method is based on 72-hour drug exposure in 96well microtitre plates, whereafter cells are washed and the fluorescence generated from hydrolysis of fluorescein diacetate is measured. Fluorescence is proportional to the number of living cells in the well. The method has been used extensively for experimental as well as clinical purposes, and seems valuable with most classes of chemotherapeutic drugs. For concentration-response curves, each drug was tested at five different concentrations, each concentration in triplicate. Each 96-well microtitre plate had six control (unexposed cells) and six blank (cell medium only) wells. Quality criteria for a successful assay included > 90% starting viability (judged by Trypan blue exclusion), a control signal more than ten times the blank, and, finally, a coefficient of variation in control and blank wells of < 30%.

Statistical analysis
Direct comparisons of the activity for the substances were made by using paired t-tests in GraphPad Prism, version 4 (Graphpad Software Inc., La Jolla, CA, USA).

Results
The compounds showed varying cytotoxic activity allowing the SAR analysis; however, the dose-response curves for some analogues were very shallow, in several cases ending in a plateau at a survival index of 20-80%, thereby preventing a correct determination of the IC 50 value. This characteristic dose-response profile has been previously described for CHS 828 in the fluorometric microculture cytotoxicity assay (FMCA) [3]. Prolonged assay time (violation to standard FMCA protocol) to 144 hours produced better dose-response curves and allowed determination of the IC 50 values, results were similar but slightly changed (not shown). To avoid interference the survival index at 10 μM (on the plateau) was used with FMCA assay time 72 hrs (standard protocol). In a phase I trial reported by Ravaud et al [5] CHS 828 was given orally as a singe dose every 3 weeks. Doses up to 500 mg was given, which resulted in plasma concentrations of 11 μM. The inherent order of sensitivity among the cell lines was similar to that presented previously [3], with the doxorubicin resistant small-cell lung cancer (SCLC) cell line H69AR being the most sensitive and the maternal myeloma cell line RPMI 8226S being highly sensitive, while the doxorubicinselected (p-glycoprotein (Pgp)-expressing) sub-line, 8226 Dox40, displayed a considerable degree of resistance, as did the T-cell leukaemia lines CCRF-CEM and CEM/R. This pattern of activity among the cell lines was similar for most of the compounds (see below). The reference compounds pinacidil and cimetidine ( Fig. 1) were tested in concentrations of up to 10 mM in all ten cell lines and were found to be non-toxic (not shown).
First, the pyridyl substitution pattern on the cyanoguanidine core was studied. As seen in Figure 2, 4-pyridyl substitution was significantly more efficient than was 3pyridyl substitution (p < 0.01).
Second, the linking alkyl chain length was examined. This factor was apparently of considerable importance, having optimum activity with hexyl, heptyl or octyl chains ending with a bulky end group (Fig. 3). The exact composition of this end group did not seem to be of crucial importance (Fig. 4A). When the end group was a monosubstituted phenyl ring it was shown that the preferred position was 3-substitution, followed by 2-and, finally, 4substitution, but differences were minor. The compound with an unsubstituted phenyl was as active as the 4-substituted compounds, while the trichlorinated derivative appeared less active (however, the difference was statistically not significant; p = 0.13). Whether the substituent(s) was a chloro-or methoxy-group(s) seemed to be of minor importance (Fig. 4B).
Finally, the activity patterns in the ten cell lines were compared. Substances with similar structures correlated well, whilst substances with large differences in molecular structure demonstrated lower correlation coefficients (not shown).

Discussion
The unsubstituted pyridyl moiety appears important, although it has not been studied in detail in the present study. Others have shown that a substituted pyridyl or a phe-Effects of pyridyl substitution pattern on cytotoxic activity (as survival index at 10 μM) in the ten cell lines studied Effects of linking chain length on cytotoxic activity (as survival index at 10 μM) in the cell lines studied  guanidine is a fairly potent inhibitor of the IKK activity in vitro (IC 50 22 nM v. 8.0 nM for CHS 828), which contrasts with its low anti-proliferative activity against NYH SCLC cells in vitro and in vivo [7], and consequently in this particular case the cytotoxic action and the IKK inhibitory action appear separate. Furthermore, while retaining the capacity of increasing the glycolytic activity in U-937 cells, this compound appears to be almost atoxic also in this cell line (compound 8 in Ekelund et al [9]), again leading to speculations about a dual mechanism of action. Compounds with the pyridyl ring substituted for a mono or unsubstituted phenyl ring display no IKK inhibitory or cytotoxic activity [7]. Arylcyanoguanidines with short aliphatic side chains (e.g. N-aryl-N'-cyano-N''alkyl-guanidines) are known as activators of ATP-sensitive potassium channels and inhibitors of insulin release from beta cells, but small structural changes can dramatically change the efficacy [21].
The length of the alkyl chain linking the cyanoguanidine to a bulky end group proved to be important, ideally being an hexyl to an octyl chain, in agreement with a previous report from Schou and co-workers [1]. In light of this, it is worth noting that the dodecyl derivative with a tert-butyloxycarbonylamino end group (CHS 850) nevertheless has shown high potency as an inhibitor of IKK activity, as well as proliferation of NYH SCLC cells in vitro and in vivo [7].
It has recently been shown that different Na + /H + exchange inhibitors, including the cyanoguanidine cimetidine, also inhibit chemokine production and NF-κB activation in immuno-stimulated endothelial cells [22]. For the model substance in these studies, amiloride (with no structural relationship to the cyanoguanidines), this was suggested to result from an inhibitory effect on IκB degradation [22]. Cimetidine and amiloride are both completely nontoxic in the FMCA model in the cell lines studied (Ekelund et al [23] and unpublished data), and interaction analysis between amiloride and CHS 828 in U-937 cells has shown synergistic effects [23].
In order to develop a second-generation drug candidate from CHS 828 with the aim to improve cytotoxicity and/ or selectivity, Chern and co-workers synthesized analogues with a bridge connecting the two amino nitrogens of the cyanoguanidine, thus forming a cyclic structure such as 2-cyanoimino-4-imidazodinone derivatives. Some of these drugs, especially the 5-substituted ones, displayed cytotoxic activities at sub-micromolar level. However, in contrast to the analogue series presented in this paper, the rigid analogues appeared to display a different activity spectrum compared with the model compound (namely, higher activity against colon and liver cancer cell lines, and lower activity against gastric, nasopharyngeal and breast cancer) [24]. However, it is at present unclear whether these compounds with cyclic structures displays a more favourable activity profile than the compounds described in our work.
Effects of different end groups (4A); and end group substituents (4B), on cytotoxic activity (as survival index at 10 μM) in the cell lines studied

Conclusion
The cytotoxic cyanoguanidines, with CHS 828 as the model compound, have received a fair amount of attention in the scientific literature, and early clinical trials have been performed. Further development resulted in a water soluble prodrug called EB1627 (also denoted GMX1777). Prolonged infusions of EB1627 have recently been reported to yield significant effects in myeloma, small cell lung cancer and colon cancer xenografts, which according to the authors support the design of an open-label, doseescalation trial, in which patients with refractory solid tumors and lymphomas receive 24 h infusions of EB1627 as a single agent in 3-week cycles. Furthermore, results indicate that nicotinic acid is a potent antidote to treat EB1627 overdose [25].
Despite efforts for 10 years to pinpoint details of the mechanistic events following exposure to the drug, the exact mechanism of action was not established until very recently. Malignant cells display increased demands for energy production and DNA repair, and NAD is required for both processes and is also continuously degraded by cellular enzymes. N-(6-(4-chlorophenoxy)hexyl)-N'cyano-N''-4-pyridyl guanidine most probably acts by inhibition of nicotinamide phosphoribosyltransferase (Nampt), a crucial factor in the resynthesis of NAD [14,15]. Using a functional non-clonogenic viability assay we identified structural prerequisites for cytotoxicity, and found CHS 828 to be a reasonable drug candidate.

Authors' contributions
HL and JG contributed equally to the planning, the performance of the experiments and the analysis. RB was engaged in data analysis and methodology questions, including stability issues and additional laboratory work, upon major revision of the manuscript. The manuscript was mainly written by JG and reviewed by HL and RB.

Declaration of competing interests
The authors declare that they have no competing interests.