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Medicine
Materials Science

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Hantzsch pre-column derivatization for simultaneous determination of alendronate sodium and its pharmacopoeial related impurity: Comparative study with synchronous fluorometry using fluorescamine

High performance liquid chromatographic (HPLC) method with a pre-column derivatization based on Hantzsch condensation reaction was applied for simultaneous determination of alendronate sodium (ALN) and its main related impurity, 4-Aminobutanoic acid (ABA) at its pharmacopeial limit. The separation of colored condensation products of ALN and ABA were achieved on Agilent Zobrax Eclipse SB-C18 analytical column (250 × 4.6 mm, 5 μm) using a mobile phase composed of acetonitrile–0.1 M acetate buffer, pH 5.0 (15:85, v/v). The flow rate was 1 mL min−1. The detection was carried out at 340 nm using photo-diode array detector. Peak areas were used for the linear regression line in the range of 10–500 and 0.2–40 μg mL−1 for ALN and ABA, respectively. Different conditions for the optimization of the derivatization reactions as well as for the HPLC measurement were studied. The proposed method was validated for linearity, precision, accuracy, specificity and robustness. This method was used to check the purity of ALN in the presence of ABA (related impurity) at the pharmacopeial limit (0.5%). For comparison purpose, another method was proposed which involves synchronous fluorescence measurement after ALN reaction with fluorescamine. In this method, the third derivative synchronous spectra were estimated as peak to peak measurement from 339 to 370 nm for ALN determination with LOD and LOQ of 24 and 73 ng mL−1, respectively, showing very high sensitivity. Both methods have been applied for determination of the alendronate sodium (ALN) in bulk and pharmaceutical preparations without interference of additives in tablets or oral solution.

Introduction
Alendronate sodium (ALN) is an amino bisphosphonate compound which is used for the treatment of a variety of bone diseases including osteoporesis, Paget's disease and hypocalcemia associated with malignancy [1].
ALN is designated chemically as (4-amino-1hydroxybutylidene) biphosphonic acid monosodium salt trihydrate [2] (Scheme 1). ALN was prepared by reacting 4aminobutanoic acid with phosphorous acid and a halophosphorous compound, in the presence of diphenyl ether to obtain alendronic acid then alendronic acid was treated with sodium hydroxide to obtain alendronate sodium [3]. So, 4aminobutanoic acid (ABA, Scheme 1) is expected to be a possible impurity in ALN raw material and pharmaceutical preparations which has a specified pharmacopeial limit.
Determination of ALN represents a real analytical challenging example because its structure lacks any chromophore, and hence it cannot be determined by direct spectrophotometric or HPLC-UV methods. ALN is official in the BP 2013 [4] and USP 36 [5]. These official methods exhibit some difficulties and need sophisticated instruments. In USP method [5], the reagent used for pre-column derivatization of ALN is very toxic and carcinogenic. Furthermore, separation from excess reagent is needed by performing an extraction step using methylene chloride. The BP method [4] depends on assay of ALN using ion exchange chromatography, which consumed time as the equilibration of the column takes a long time.
Fluorometry is considered one of the most accessible analytical techniques, because of its low cost, wide availability in most quality control laboratories and high sensitivity. Synchronous fluorescence spectroscopy has several advantages [23], such as high selectivity, simple spectra, low interference, etc. In addition, derivative synchronous fluorometry is more advantageous than differentiation of the conventional direct fluorometry in terms of sensitivity [24].
However, a comprehensive literature survey except the pharmacopeial ones revealed the lack of a selective method Scheme 1 e Schematic illustration of chemical reactions of (a) ALN and ABA with the reagents in Hantzsch condensation reaction or (b) ALN with fluorescamine. j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0 for the simultaneous determination of ALN and its related impurity, ABA. Moreover, no synchronous fluorometric method has been applied previously for the assay of ALN in pharmaceutical preparations. Furthermore, only one spectrophotometric method has been reported for the assay of ALN in oral solution [16], however, it is tedious and time consuming because of the extraction steps requirement for condensed product after derivatization reaction.
The aim of this work is to develop two validated methods for determination of ALN in bulk and pharmaceutical preparations (tablets and oral solution). The first method (method I) depends on the application of a pre-column HPLC-DAD derivatization technique based on Hantzsch condensation reaction. This proposed method allowed the simultaneous determination of ALN and its main related impurity, ABA at the pharmacopeial limit of 0.5%. The second method (method II) is achieved by applying the third derivative synchronous fluorometry following the derivatization with fluorescamine. Both methods were validated in compliance with ICH guidelines.

Apparatus
The HPLC system (Agilent, Germany) consisted of Agilent 1200 Series Quaternary pump G1311A which comprises a solvent cabinet, an Agilent 1200 Series Vacuum Degasser G1322A and a four-channel gradient pump; Agilent 1200 Series Diode Array and Multiple Wavelength detector G1315D. The LC system is equipped with Agilent 1200 Series Thermostated Column Compartment G1316A and Agilent 1200 Series Manual Injector which uses a Rheodyne 7725i7-port sample injection valve and fitted with a 20 mL sample loop. LC separations were performed on Agilent Zobrax Eclipse SB-C18 analytical column (250 Â 4.6 mm, 5 mm) column. Fluorescence spectra and measurements were carried out on a PerkineElmer LS45 Luminescence Spectrometer, equipped with 1-cm quartz cell. The instrument is interfaced to a computer loaded with FL WinLab software and the computer is connected to a HewlettePackard DeskJet 640C printer.

Preparation of standard solutions
For method I, stock solutions were prepared by dissolving ALN and ABA in distilled water to obtain stock solutions having concentrations of 2 mg mL À1 . For method II, stock solution of 1 mg mL À1 ALN was prepared in water then further dilution with water was performed to obtain 50 mg mL À1 working solution. The solutions were kept in a refrigerator at 4 C.

Preparation for reagents
2.4.1. Preparation of 0.1M acetate buffer, pH5 A weight of 13.6 g of sodium acetate trihydrate was dissolved in 800 mL distilled water. The pH was adjusted to 5 using glacial acetic acid. The volume was completed with distilled water to 1 L and the solution was mixed well.

Preparation of pre-column derivatizing reagent
The derivatizing reagent was freshly prepared by mixing 6 mL of distilled water, 1 mL of 0.1 M acetate buffer (pH 5), 1 mL of formaldehyde and 2 mL of acetyl acetone. Four drops of formaldehyde were added till the solution become clear.

Preparation of fluorescamine
Fluorescamine reagent was prepared as 0.6 mg mL -1 in acetonitrile.

Preparation of borate buffer, pH 9.8
Boric acid solution (0.1 M) was prepared by dissolving 0.6183 g in 80 mL distilled water and its pH was adjusted to 9.8 using 1M NaOH then the volume was completed to 100 mL with distilled water.

Method II (Derivative synchronous fluorometry)
Accurately measured aliquots of ALN working solution (50 mg mL À1 ) were transferred into separate 5-mL volumetric flasks to obtain a series of ALN solutions covering the working range of 0.1e1.5 mg mL À1 in the final solution. Addition of 2.5 mL borate buffer of pH 9.8 followed by 0.8 mL of fluorescamine solution (0.6 mg mL À1 ) was done to each flask. Then the flasks were completed to volume with acetonitrile. A reagent blank was prepared similarly using water instead of the ALN working stock solution.
The synchronous spectra of the ALN solutions after the derivatization reaction were obtained by scanning both monochromators at Dl ¼ 100 nm. The scan rate was kept 600 nm min À1 and the excitation and emission slits were 10 nm. Derivative spectra were generated by Excel for Microsoft Windows (Dl ¼ 20 nm).
Calibration curve was obtained by plotting the concentrations versus the third derivative of synchronous fluorometric peak amplitudes from 339 to 370 nm.

2.7.
Assay of ALN pharmaceutical preparations

Analysis of ALN in tablets
Ten Fosamax ® tablets were weighed and finely powdered. A portion of the tablet powder equivalent to 50 mg ALN was accurately weighed and transferred into a 25-mL volumetric flask using 15 mL water. The sample solution was sonicated for 20 min. Dilution was made to volume in the flask with distilled water followed by filtration through Whatman No. 1 filter paper to give solution of 2 mg mL À1 ALN. Portions of 150 mL of this solution were transferred into 10 mL screwcapped test tubes (to give final concentration of 30 mg mL À1 ALN) and the procedure was then completed as mentioned under construction of calibration curves.
For method II, further dilution was done using distilled water to prepare 50 mg mL À1 working solution from which portions of 0.1 mL were transferred into 5-mL volumetric flasks (to give final concentration of 1 mg mL À1 ) and the procedure was then completed as mentioned under construction of calibration curves.

Analysis of ALN in oral solution
For method I, an accurate volume of Borgalendro ® Oral solution equivalent to 0.3 mg was directly transferred into 10 mL screw-capped test tubes and volumes were completed to 3 mL using distilled water. Then, 1 mL of pre-column derivatizing reagent was added to each test tube. The test tube was heated in a water bath (55 C) for 15 min. The yellow coloured solutions produced were cooled, transferred quantitatively into 10 mL volumetric flasks, the volumes were completed with the 0.1 M acetate buffer to obtain 30 mg mL À1 solution of ALN. Portions of 20 mL of this solution was chromatographed using the chromatographic conditions mentioned above.
For method II, an accurate volume of Borgalendro ® Oral solution equivalent to 5 mg ALN was transferred into a 100-mL volumetric flask using 60 mL water, sonicated for 5 min (for mixing), completed to mark with distilled water giving final concentration of 50 mg mL À1 . Portions of 0.1 mL of this solution were transferred into 5-mL volumetric flasks (to give final concentration of 1 mg mL À1 ) and the procedure was then completed as mentioned under construction of calibration curves.

3.
Results and discussion

Method I (Pre-column derivatization)
Pre-column derivatization method based on Hantzsch condensation reaction was applied in which the 1 ry aliphatic amine group in both ALN and its related impurity, ABA, react with acetyl acetone and formaldehyde via Hantzsch condensation reaction to produce yellow coloured products (Scheme 1). However, both drugs exhibit maximum absorbance at 340 nm. Therefore, the application of separation technique like HPLC following the pre-column derivatization reaction was adopted to permit the separation of condensation products of ALN and ABA and their simultaneous determination using diode array detector at 340 nm.

Optimization of the pre-column derivatization reaction
The influence of different reaction conditions, such as reaction temperature, buffer pH, buffer type, reaction time and reagent volume, on the derivatization reaction was studied to achieve maximum sensitivity and selectivity of the proposed method. This was done chromatographically, by checking the effect of parameters on area and sharpness of ALN condensation product peak and retention times for peaks of condensation product of ALN and blank.
3.1.1.1. Effect of reaction temperature. Different temperatures were studied (25, 40, 55, 80 and 95 C) while keeping other parameters constant. Results indicate that at low temperatures, no reaction products were produced. While as the temperature increased, the peak area of ALN condensation product increased, and thus increasing sensitivity, however the blank shows broader peak. The optimum reaction temperature was found to be 55 C as sharp chromatographic peaks with suitable peak area that were well separated from the blank peak was obtained.
3.1.1.2. Effect of buffer pH. Various pH values of acetate buffer were tried. The optimum pH value of 5 was chosen which gave high peak area of ALN condensation product (Fig. 1a).
3.1.1.3. Effect of buffer type. Different buffer types (as acetate, phosphate and citrate) adjusted at pH 5 were tried. Both acetate and citrate buffer gave higher peak area than phosphate but acetate buffer gave the sharpest chromatographic peak.
3.1.1.4. Effect of the reaction time. Different reaction times (5, 10, 20, 30, 40 and 60 min) were tried. It was found that the best reaction time that gave maximum sensitivity and sharp peaks was 15 min. But, changing reaction time has no effect on the retention time of ALN peak (Fig. 1b).
3.1.1.5. Effect of reagent volume. Different volumes of the reagent were studied. The effects of reagent volumes on chromatographic peak were shown in (Fig. 1c). It was found that the reagent volume has great effect on peak area of ALN condensation product. On the other hand, the same retention times of both peaks were obtained for each volume used. It was found that using 1 mL of reagent gave highest chromatographic peak of ALN condensation product (Fig. 1c).

Optimization of HPLC conditions
The main target of the proposed HPLC method was to get maximum separation of closely eluting peak of ALN condensation product from ABA condensation product peak. During method development, various experimental conditions were studied and optimized such as, using different mobile phase compositions and different flow rates as followed: 3.1.2.1. Effect of mobile phase components ratio. Different ratios of acetonitrile were tried in the mobile phase consisting of acetonitrile and acetate buffer pH 5. Fig. 1d shows the retention times of the blank and condensation products of ALN and ABA peaks as a function of acetonitrile %. It was found that, at lower concentrations of acetonitrile (<15%) separation occurred but with increased retention times for all peaks. At 20% of acetonitrile, the ALN condensation product peak is well separated from blank peak but partially overlapped with ABA condensation product peak. Increasing acetonitrile concentration (>20%) led to loss of resolution and overlapped peaks. It was found that, the optimum resolution within reasonable total running time was achieved using 15% acetonitrile.
3.1.2.2. Effect of flow rate. Different flow rate values of 0.5, 1, 1.5 and 2 mL min À1 were tried. At flow rate of 0.5 mL min À1 , very broad peaks were obtained. At flow rate of 1.5 mL min À1 , ALN condensation product peak is separated from blank peak but overlapped with ABA condensation product peak. At flow rate of 2 mL min À1 , all peaks are overlapped. So, the flow rate of 1 mL min À1 was chosen for optimum chromatographic measurement. Fig. 2 shows the HPLC chromatogram of the standard mixture of 20 mg mL À1 ALN and 10 mg mL À1 ABA after pre-column derivatization using Hantzsch condensation reaction. After precolumn derivatization using the optimized parameters mentioned above, the condensation products of ALN and ABA were separated with sharp well-resolved peaks at 10.9 and 13 min, respectively that are well separated from the blank peak. 3.1.3. System suitability. Using the optimized chromatographic system, different system suitability parameters have been tested since they are important for the validation of the analytical method and they confirm the suitability and efficiency of the operating system [25]. System suitability parameters of the proposed method were as followed: retention time (R t ) (for ALN, 10.93; for ABA, 13.08), retention factor (k') (for ALN, 3.37; for ABA, 4.23). While, asymmetry factor (A f ) which used to evaluate the degree of peak asymmetry were 0.82 and 0.91 for ALN and ABA, respectively. A f for both drugs did not exceed 1.2, indicating acceptable degree of peak asymmetry. In addition, the large number of theoretical plates which are 9592 and 21428 for ALN and ABA, respectively indicating high column efficiency. The obtained system suitability parameters indicated that the proposed HPLCeDAD method permitted adequate resolution of the ALN from ABA [good resolution (R s ) (3.29) and selectivity values (a) (1.26)] within reasonable runtime and high column efficiency.

Excitation and emission spectra
Because of the absence of native fluorescence of ALN, a derivatization step using fluorogenic reagent was requisite for j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0 its fluorometric analysis. Fluorescamine was chosen as a derivatizing reagent because it forms highly fluorescent derivatives with primary amines under relatively mild reaction conditions. It was found that ALN reacts with fluorescamine to give a fluorescent derivative with maximum fluorescence intensity (l em ) at 489 nm after excitation at wavelength (l ex ) of 386 nm. Obviously, the emission spectra of ALN and the reagent blank are strongly overlapped with high blank emission reading. Therefore, ALN can be determined directly by normal fluorometry but with low sensitivity because of interference of the reagent blank fluorescence.

Synchronous fluorescence spectra and third derivative spectra
The synchronous fluorescence spectral peaks of ALN and the reagent blank are at 280 and 240 nm, respectively (Fig. 3a) Although a synchronous fluorescence technique was used, the synchronous fluorescence spectra of ALN and the reagent blank cannot be separated entirely. To resolve the problem of spectral overlap, derivative technique was used. The synchronous fluorescence spectrum of the mixture solution (ALN and the reagent blank) was derived. Obviously, peaks of ALN and the reagent blank were separated on the third derivative spectrum. ALN can be more conveniently and accurately determined by measuring the third derivative of synchronous spectrum from peak-to-peak at (339e370 nm) which correspond to the absence of any interference from reagent blank (Fig. 3b). The ratios of third derivative synchronous spectral peak amplitudes at these wavelengths were calculated for different concentrations of ALN standard solutions and pharmaceutical formulations. These ratios were used for the detection of the presence of any interferences [26].
In addition, results revealed that the synchronous fluorescence spectral peaks of ALN and the reagent blank were not well separated during the Dl below or above 20 nm. Therefore, Dl of 20 nm was selected for recording the synchronous spectrum of the mixture solution (ALN and the reagent blank).

Optimization of the reaction conditions 3.2.3.1. Effect of fluorescamine concentration. Different vol-
umes of 0.6 mg mL À1 fluorescamine reagent from 0.2 to 1.2 mL were studied using the procedure mentioned above. Optimum values of the relative fluorescence intensity (RFI) were obtained with 0.8 mL of fluorescamine reagent (Fig. 4a).

Effect of pH.
The pH effect on the reaction was studied by carrying out the reaction in borate buffer solution in the pH range of 4.0e10.5, in addition to using 0.1M NaOH with pH 13. The results shown in Fig. 4b indicated that the RFI increased initially as the pH increased and maximum readings were attained at pH 9.8 ± 0.3. At higher pH values, sharp decrease in the readings occurred. This was probably attributed to the hydrolysis of the reaction product between ALN and fluorescamine in alkaline medium. Concerning the effect of buffer volume, the highest fluorescence reading was obtained upon adding 2.5 mL buffer pH 9.8 (Fig. 4c).

Effect of time.
To study the optimum time needed for reaction completion, the derivatization reaction was performed at room temperature (25 ± 2 C) and the RFI was recorded instantly after the addition of fluorescamine and observed for 30 min. The results indicated that the reaction was very fast as the fluorophore is formed immediately. RFI was constant by time and remained stable for a minimum of 2 h at room temperature.

Effect of diluting solvent. Various solvents were tried
to choose the most appropriate diluting solvent which allows both formation and stability of ALN reaction product. These solvents were: water, DMF, acetone, methanol, ethanol, and acetonitrile. The highest fluorescence intensities were obtained with acetonitrile as a diluting solvent (Fig. 4d).

Validation
The ICH guidelines [27] for method validation were followed for the developed pre-column derivatization HPLC-DAD and

Linearity
The linearity of the proposed methods was evaluated by analyzing series of different concentrations of each of ALN alone or with ABA. According to ICH, a minimum of five points must be involved. Under the experimental conditions described, the graphs obtained by plotting peak area (for method I), third derivative synchronous values (for method II) versus concentrations (in the ranges stated in Table 1) show linear relationships. All linearity parameters obtained by the linear least squares regression treatment of the results are also given. Regression lines with high F-values (low significance F) are considered better compared to those with lower ones. Good regression lines show high values for both (r) and (F) values [28].

Limit of detection and limit of quantitation
For method I, limit of detection (LOD) is considered as the concentration which has a signal-to-noise ratio of 3:1. For limit of quantitation (LOQ), the ratio considered was 10:1 with RSD% value less than 10% [27]. For method II, LOD and LOQ were calculated using the formulae given by Miller [29] where the limit of detection, LOD ¼ 3.3 S/b and the limit of quantitation, LOQ ¼ 10 S/b, where S is the standard deviation of replicate blank responses (under the same conditions as for sample analysis) and b is the sensitivity, namely the slope of the calibration graph. Using the proposed methods, LOD and LOQ for each compound were calculated and are presented in Table 1.

Accuracy and precision
For method I, the accuracy and precision were evaluated by analyzing three laboratory-prepared mixtures of ALN and ABA at various concentration ratios within the working range of each compound (Table 2). On the other hand, for method II, solutions containing three different concentrations of ALN were prepared and analyzed in five replicates to evaluate the accuracy and precision. Good results were obtained upon checking method repeatability (intra-day precision) using the RSD% values obtained from repeating the assay five times on the same day (Table 2). Intermediate precision was evaluated by the assay of the sample sets on three different days (interday precision). Satisfactory recoveries, small percentage   j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0 relative errors (Er%) and small relative standard deviations (RSD%) were obtained, which indicated the high accuracy and precision of the proposed methods.

Robustness
Robustness of the proposed methods was evaluated by analyzing ALN alone or with ABA at the same concentration levels used for accuracy and precision studies. For method I, the parameters studied were acetonitrile % in mobile phase, pH of the acetate buffer, wavelength of detection and flow rate. However, for method II, the parameters included: volume of fluorescamine reagent, pH value of borate buffer, volume of buffer used and different lots of acetonitrile. It was found that variation in the above-mentioned parameters had no significant influence on the determination of the drug alone or with ABA using the proposed methods. The low values of RSD% of peak areas along with nearly unchanged k' values (for method I) and the low RSD% of third derivative synchronous peak values (for method II) obtained after introducing small deliberate changes in the method parameters indicated the robustness of the developed methods.

Selectivity and specificity
For method I, the selectivity was checked by analyzing synthetic mixtures containing different ratios of both drugs, where good percentage recoveries were obtained indicating that they did not interfere with each other ( Table 2). The peak purity of ALN and ABA condensation products was checked by using a G1315D photo diode array detector where Fig. 5 shows the superimposed UV spectra for the peaks of ALN and ABA condensation products for standards and samples' solutions obtained at different times through their elution indicating purity of the obtained peaks. In addition, the obtained purity angle was not exceeding the threshold limit which indicates that no additional peaks were co-eluting with each of ALN and ABA providing evidence of the ability of the method to assess the analyte of interest in the presence of potential interferences. The selectivity of method II was checked by measuring the ratio of the third derivative values of synchronous spectra for different concentrations of ALN standard solutions and pharmaceutical formulations. The value of D 3 370 nm/D 3 339 nm for ALN bulk powder was found to be 3.01 with a relative standard deviation (RSD) of 1.84% (n ¼ 5), for ALN tablet solution, this ratio was 3.00 with RSD of 2.06% and in ALN oral solution this ratio was 3.02 with RSD of 2%. This ratio value is concentration independent and of reasonable reproducibility. Furthermore, it is specific for the drug and can be used for its identification as well as for testing its purity.

Analysis of pharmaceutical formulations
The proposed methods were successfully used to determine ALN in tablets and oral solution. HPLC chromatograms of Fosamax ® tablet and Borgalendro ® oral solution are shown in Fig. 2. Five replicate determinations were performed. Satisfactory results were obtained for ALN, and they were in a good agreement with label claims (Table 3). The results obtained from the two methods were compared statistically by the use of Student's t-test (for accuracy) and the variance ratio F-test (for precision). The results in Table 3 show that the calculated t and F values were smaller than the critical values, indicating there were no significant differences between the two proposed methods.  j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0 other derivatization methods reported in literature with several advantages, such as being simple and rapid methods with no need for tedious and elaborate steps (heating, centrifugation or waiting time). On the other hand, the proposed HPLC method (method I) is the first reported method for the separation of ALN and its related impurity ABA at the pharmacopeial limit. Compared to the pharmacopeial method [4], the proposed method I is simpler where separation between ALN and ABA was done at room temperature (RT) after isocratic elution within a shorter run time and using greener reagents and solvents without the need of tedious procedure. In addition, compared to the reported HPLC-FD method [8] for  j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0  [7], RT (60 min) [8], 50 C (10 min) [9], RT [11], 100 C (60 min) [12], RT [13], 90 C (20 min) or RT (60 min) [14], 100 C (15 min) [16] RT [20,21] RT (60 min) RT RT (30 min)
-No tedious or elaborate steps.
-Rapid with no waiting time.
-Time consuming procedures -Use of specific HPLC column which adds to the cost of the procedure.
-Use of toxic mercury electrode, not ecofriendly.
-Tedious procedure for preparation of reagent.
j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 7 ( 2 0 1 9 ) 2 0 8 e2 2 0 the determination of ALN alone in tablets, the proposed method I showed wider linearity range, shorter derivatization reaction time without the need of specific column (rigid spherical styrene-divinylbenzene copolymer) or detector (fluorescence detector) which decreases the cost of analysis. Also, the use of water (method I) and acetonitrile (method II) as the diluting solvents promotes these methods to be greener alternatives to other reported methods [8,19,21]. Another advantage is the use of simple instrumentation that are readily available in most pharmaceutical laboratories.

Conclusion
This work reports the development of two methods for the analysis of ALN in raw material as well as in pharmaceutical preparations. The first method describes a fast, simple, selective and eco-friendly pre-column derivatization HPLC-DAD method which allows the sensitive determination of ALN in presence of its main related impurity, ABA, at its pharmacopeial limit (0.5%). It is worth nothing that this is the first HPLC report for this purpose. On the other hand, the second proposed method describes a simple, highly sensitive and selective third derivative synchronous fluorometric method that allows the sensitive analysis of ALN in different pharmaceutical dosage forms with no interference from various excipients. Comparing our proposed methods with other published methods reveals that the proposed methods are of comparable or better sensitivity to other derivatization methods with several advantages such as being fast ecofriendly sensitive and are independent on expensive reagents or instrumentation which makes our proposed methods to be superior to other reported methods and more suitable for routine analysis of ALN in quality control laboratories.


Exchange interactions and magnetic properties of hexagonal rare-earth-cobalt compounds

The magnetic properties of some GdxY1-xCo4A compounds with A = Co, Si or B are analysed including the pressure effects. Isomorphous structure transitions, parallelly with changes of cobalt moments from high spin states to low spin states, were shown as pressure increases. The magnetic data, obtained from band structures, were compared with those predicted by the mean field model.

Introduction
The magnetic properties of RCo5-based compounds, where R is a rare earth or yttrium, were intensively investigated both from scientific reasons as well as in connection with their technical uses, as permanent magnets [1,2]. These compounds crystallize in a CaCu5-type structure, space group P6/mmm. In this lattice, the R atoms are located in 1a site and cobalt at 2c and 3g positions [1]. The RCo4B compounds have a hexagonal structure, derived from CaCu5 one, by replacing every two layers Co2c atoms by boron. There are two sites for R(1a, 1b) and Co(2c,6i) atoms and one for boron (2d) [3]. When R is a magnetic heavy rare-earth the above compounds are ferrimagnetically ordered, while for light rare earths, a parallel alignment of R and Co moments is shown. The easy axis of magnetization, for RCo5 compounds with R = Y or Gd, is the c-axis, as imposed by the local uniaxial anisotropy of the 2c sites, higher than the planar one favoured by 3g atoms [4], YCo4B shows also uniaxial anisotropy, at ambient temperature, which changes to planar one, at TsR = 159 K [5].
The exchange interactions between magnetic rare-earths and cobalt are mediated by R5d band polarization, as suggested by the 4f-5d-3d model [6,7]. The above polarization is the result of both local 4f-5d exchange as well as due to R5d-Co3d short range interactions, hybridization effects, respectively. The exchange interactions between cobalt atoms are of short range as well as those between R5d-R5d orbitals, in addition to those by means of conduction electrons (RKKY-type) [8].
In this paper, we analyse the exchange interactions in GdxY1-xCo4A series with A = Co, Si, B in the framework of mean field model [9], as well as based on the data obtained from their band structures. The pressure dependences of the cobalt moments, for end series compounds, are theoretically investigated. A linear dependence of cobalt moments on the exchange splitting energy of their 3d bands was shown.

Experimental and computing method
The samples were prepared from high purity elements, in an induction furnace or by arc melting. An excess of rare earth (yttrium) of (2-4)% was added in order to compensate their losses during the melting. The samples were annealed at temperatures 900 o C ≤ T ≤ 1000 o C, in vacuum for one week. The X-rays analysis, at ambient temperature, evidenced the presence of only one phase. Some samples were already prepared [10,11]. Magnetic measurements were performed in the temperature range 4.2 -1000 K, and fields up to 70 kOe. The ground state electronic structures and magnetic properties have been determined in the scalarrelativistic limit, i.e. without spin-orbit coupling, by using the tight-binding linear muffin-tin orbital (TB-LMTO) method within the atomic sphere approximation (ASA) [12,13]. The local density approximation (LSDA) has been employed for the exchange and correlation potential of the electron gas, assuming Von Barth and Hedin parameterization [14]. The valence basis consists of s-, p-, and delectrons. All k-space integrations were carried out with a 16×16×16×16 k-point mesh with an accuracy of 10 -2 meV. The silicon was located in 3g sites, as previously reported [15].

Results
The partial density of states (DOS) of Co2c and Co6i atoms in YCo4B, at ambient pressure, v/v0 = 1, as well as at v/v0 = 0.88 are plotted in Fig.1. The Fermi level, EF, when v/v0=1 is situated in a peak, or nearly on a peak in the density of states in spin-up cobalt sub bands at 2c and 6i sites, respectively. In the spin down sub-bands, the EF is located in a deep in DOS. As effect of pressure, relative volumes reduction, respectively, there is a shift of the spin-up sub-bands to lower energy and of the spin down ones, at higher energy. As a result, there is a decrease of DOS at EF in spin up sub band and an increase in spin down one. When cobalt in nonmagnetic, the densities of states for spin up and spin down sub bands are near identical, in a mirror-plane. The evolution with pressure of the centres of gravity, on the energy scale, for cobalt sub-bands were determined, the exchange splitting energy of Co3d bands, ΔEs, respectively. A linear dependence of cobalt moments, at both sites, on the exchange splitting energy was shown, with a rate ΔEs/MCo=0.94 eV/B, close to the previous reported values [10,16,17]  volumes 0.965 v/v0 1.0, there is a nearly linear decrease of cobalt moments, the corresponding rates ΔM/M being higher, as the initial cobalt moments are smaller. This behaviour can be correlated with their itinerancy degree - Fig.3. At a characteristic pressure, relative volume, respectively, a collapse of the cobalt moments from high spin state to low spin one is evidenced. When the initial cobalt moments are small, as example at 6i site in YCo4B, a direct transition to nonmagnetic state is evidenced. The magnetic anomaly develops parallelly with a sudden decrease of a c/a ratio, an isomorphous phase transition, respectively. Similar behaviour has been already evidenced in YCo5 compound and attributed [18] to a first order Lifshitz transition [19] or to a spin reorientation transition [20]. The transition develops, parallelly with the change of the Fermi surface topology. A change of anisotropy from uniaxial to planar, in YCo5, has been previously suggested in connection with the diminution of cobalt moments [21][22][23][24][25]. The anisotropy of YCo4B, at ambient pressure, changes from uniaxial to planar one, at TsR = 159 K [5]. As effect of pressure, the TsR temperature decreases with a rate of 22 K/GPa [26] and thus, at pressures estimated for the isomorphous structure transition (p > 7 GPa), the uniaxial anisotropy is still present, and consequently, a possible spin reorientation to planar anisotropy.  atoms situated in the first coordination shell to an R site, zi and their magnetic moments, Mi. The ratio |M5d(d)/M4d|/∑ z i M i = 1.25×10 -2 . The same value has been already evidenced in RM5 (M = Co, Ni), compounds [7,10], being a characteristic feature of all hexagonal RCo5-based compounds. The exchange interactions, at the level of unit cell, are mediated by R5d/Y4d band polarizations. The changes of magnetic behaviour, of R and Co sublattices, as well as the implied exchange interactions, are mirrored by the R5d/Y4d band polarizations. This is also confirmed by linear dependence of the Curie temperatures on R5d/Y4d band polarizations of RCo5-based samples with different substitutions both at R or Co sites - Fig.4. The magnetic properties of ferrimagnetic GdxY1-xCo4A with A = Si or B are analysed also by using a two sublattices mean field model and the average cobalt moments compared with those obtained from band structures. The magnetizations of gadolinium and cobalt sublattices, at 4 K, compensate at x = 0.57 for A=Si and x = 0.43 when A = B - Fig.5. Assuming that gadolinium moment is 7 B, the mean cobalt moments were determined as well the parameters characterizing the interactions inside and between magnetic sublattices.  A linear dependence of mean cobalt moments on the exchange field, Hex is evidenced - Fig. 6 -with a rate ΔMCo/ΔHexch(3·10 6 ) -1 B/Oe, similar as that reported in RCo2 based compounds [27]. These data indicate that the exchange splitting of Co3d bands is nearly proportional to the internal field intensities.
As effect of pressure, or through the variations of the internal fields, the exchange splitting energies of Co3d bands and the related cobalt moments are modified. The mean field model, gives only the general trend on the evolution of cobalt moments, the band structure calculations allowing a more accurate description of magnetic moments and exchange interactions in connection with the considered cobalt sites.
The exchange interactions, in RCo5 based compounds, at the level of the unit cell, are mediated by R5d band polarizations. The Y4d band polarizations mirror all changes in the cobalt sublattice magnetizations. Thus, the M5d and M4d values are key points in describing the exchange interactions in a considered system, as also confirmed by their linear trend when plotting with respect to Curie temperatures.

Conclusions
The pressure dependences of the cobalt moments in hexagonal compounds, evidence the presence of a first order isomorphous structure transitions, parallelly with the cobalt moment collapse from high spin state to low spin one. The Fermi surface topology, at the transition, is also changed. The R5d and Y4d band polarizations mirror the changes in the magnetic properties as effect of substitutions both at R and Co sites, mediating also the exchange interactions. The mean field model can describe only the general features of the magnetic properties of the hexagonal compounds, but not at the atomic level particularly when non-equivalent sites are present in the considered structure.


extfieldsofstudy: Medicine Chemistry

text:
Dehydrogenation Properties and Catalytic Mechanism of the K2NiF6-Doped NaAlH4 System

The K2NiF6 catalytic effect on the NaAlH4 dehydrogenation properties was studied in this work. The desorption temperature was studied using temperature-programmed desorption and exhibited a lower onset hydrogen release after doped with different wt % of K2NiF6 (5, 10, 15 and 20 wt %). It was found that the NaAlH4 doped with 5 wt % K2NiF6 showed the optimal value that can reduce the onset desorption temperature of about 160 °C compared to 190 °C for the milled NaAlH4. The NaAlH4 + 5 wt % K2NiF6 sample showed faster desorption kinetics where 1.5 wt % of hydrogen was released in 30 min at 150 °C. In contrast, the milled NaAlH4 only released about 0.2 wt % within the same time and temperature. From the Kissinger analysis, the apparent activation energy was 114.7 kJ/mol for the milled NaAlH4 and 89.9 kJ/mol for the NaAlH4-doped 5 wt % K2NiF6, indicating that the addition of K2NiF6 reduced the activation energy for hydrogen desorption of NaAlH4. It is deduced that the new phases of AlNi, NaF, and KH that were formed in situ during the dehydrogenation process are the key factors for the improvement of dehydrogenation properties of NaAlH4.

■ INTRODUCTION
Hydrogen has been considered to be the most promising candidate as a suitable energy carrier as it produces only water as a byproduct of the energy generation. As an energy carrier, hydrogen holds the potential to fundamentally secure our future energy supply and draw it more environmentally favorable. However, both hydrogen production and storage are the most important issues to realize for the development of hydrogen economy, especially for the transportation application. 1−3 According to the latest US Department of Energy (DOE) targets for 2020, the fuel cell demands hydrogen storage materials with more than 4.5 wt % of hydrogen capacity and faster sorption kinetics. 4 Storing hydrogen in the solid state form benefits the onboard applications in the aspects of safety, economy, and efficiency as compared to the gas or cryogenic liquid form. 5,6 Complex metal hydride, which is NaAlH 4 has become a promising candidate material for the solid-state hydrogen storage because of the high gravimetric and volumetric hydrogen densities. It is well-known that the dehydrogenation of NaAlH 4 consists of three steps based on the reactions as follows Na AlH 2Al 3H 4 3 6 2 (1) → + + Na AlH 3NaH Al 1.5H 3 6 2 (2) The hydrogen content for NaAlH 4 is 7.4 wt % of which 5.5 wt % can be released under moderate temperature for two dehydrogenation steps. 7 However, the operation temperature is still high and sluggish de/rehydrogenation kinetics limit NaAlH 4 for practical applications. 8 A lot of methods have been used to improve the performance of NaAlH 4 such as by ball milling, 9 adding the catalyst, 10−13 and destabilizing with other hydrides. 14−16 Among them, the hydrogen storage properties of NaAlH 4 greatly enhanced by the addition of catalyst. Different types of catalysts such as metal, 10,17 metal oxide, 11,18 and metal halide 19,20 have been doped into NaAlH 4 . Recently, Khan and Jain 21 reported that TiO 2 -doped NaAlH 4 showed faster desorption kinetics with improved hydrogen capacity (3.6−5.1 wt %) at 250°C, while the study on the effect of Nb 2 O 5 on the dehydrogenation kinetics of NaAlH 4 revealed that the amount of desorbed hydrogen varied between 4.8 and 5.0 wt % at 250°C following the catalyst content. 22 In the other study on the effect of metal chlorides (TiCl 3 , PdCl 2 , and CeCl 3 ) on the hydrogen desorption of NaAlH 4 , Khan and Jain 23 found that the amount of hydrogen desorbed at a temperature below 150°C was less than 1 wt %. The amount of hydrogen desorbed increased as the dehydrogenation temperature rose and 2 wt % CeCl 3 gave the higher amount of hydrogen desorbed which was 5.4 wt % at 250°C. However, a total comprehension of the effects of catalysts on the improvement of the NaAlH 4 dehydrogenation properties is insufficient and there is room for improvements. In addition, different complex metal hydride requires a different catalyst because it will give different roles and effects. Thus, it is essential to find another type of catalyst that has a potential to enhance the NaAlH 4 dehydrogenation properties without sacrificing its hydrogen storage capacity, especially at a lower operating temperature.
To date, no research has been found that surveyed the effect of the K 2 NiF 6 additive on the hydrogen storage properties of NaAlH 4 . So far, only Sulaiman et al. 24,25 reported the applications of K 2 NiF 6 as the additive for the MgH 2 system. They claimed that the formed species, KF, KH, and Mg 2 Ni were acting synergistically and were responsible for the enhancement of the MgH 2 sorption properties. Moreover, the combination of three elements of K, Ti, and F on NaAlH 4 has been reported by Liu et al. 26 They found that the NaAlH 4 −0.025 K 2 TiF 6 sample can release about 4.4. wt % of hydrogen within 40 min at 140°C. Therefore, it can be speculated that the K 2 NiF 6 may also give a similar effect on NaAlH 4 . In addition, Ni is one of the good catalysts for MgH 2 . 27−31 The doping effect of the Ni element is more notable than Ti because of the hardness of Ni that can help to reduce the particle size of MgH 2 , thus reduced the initial dehydrogenation temperature as studied by Zhang et al. 32 Meanwhile, Yahya and Ismail 33 in their current study reported the effect of Ni on the hydrogen storage properties of the MgH 2 −SrTiO 3 composite. Their result showed that the decomposition temperature was reduced to 260°C for the MgH 2 −10 wt % SrTiO 3 −5 wt % Ni composite with a total of 6 wt % hydrogen released. Thus, it was hypothesized in this present study that K 2 NiF 6 may play a vital part as a dopant precursor which can give a synergetic catalytic impact on the hydrogen storage properties of NaAlH 4 . Therefore, the aim of this work is to investigate the dehydrogenation properties of NaAlH 4 with the addition of the K 2 NiF 6 catalyst and the possible catalytic mechanism will also be discussed.

■ RESULTS AND DISCUSSIONS
The curves of the temperature-programmed desorption (TPD) of the undoped NaAlH 4 and NaAlH 4 doped with various amounts of K 2 NiF 6 (5, 10, 15 and 20 wt %) is shown in Figure  1. Pure NaAlH 4 starts to release hydrogen at around 190°C. The dehydrogenation reaction for the first two steps has completed at 300°C with a total hydrogen release of about 5.7 wt %. This result is very close to the theoretical value and in good agreement with a previous study. 34 On the other hand, the milled NaAlH 4 has a close onset desorption temperature as the pure NaAlH 4 . This outcome shows that the 1 h ball milling process has a minimal impact on the dehydrogenation properties of NaAlH 4 . However, the dehydrogenation process completed at a lower temperature than the pure NaAlH 4 for two-step decomposition (280°C) with a total hydrogen release of 5.5 wt %. Compared to the undoped sample, the onset desorption temperature for different wt % of K 2 NiF 6doped NaAlH 4 compounds show a lower dehydrogenation temperature. The NaAlH 4 + 5 wt % K 2 NiF 6 sample starts to release hydrogen at 160°C and completed at around 260°C for the two-step dehydrogenation process. For the 10 wt % doped sample, the dehydrogenation temperature starts to decompose at about 150°C and completes at 250°C. The addition of 5 and 10 wt % K 2 NiF 6 has reduced the onset desorption temperature of NaAlH 4 by 30 and 40°C, respectively, as compared with the milled NaAlH 4 . However, the total value of hydrogen released is slightly reduced to 5.1 wt %. Meanwhile, the desorption temperature has reduced to 145°C after increasing the doping value of K 2 NiF 6 to 15 and 20 wt %. The dehydrogenation process for 15 and 20 wt % doped samples was completed at 245 and 237°C with a total hydrogen capacity at about 4.9 and 4.5 wt %, respectively. This result indicates that a higher amount of doping further reduces the onset desorption temperature and decreases the hydrogen capacity. This phenomenon is believed because of the high level of the added amount of K 2 NiF 6 that led to the excessive catalytic effect and it is almost similar to our previous work. 24,35 Figure 2 shows the hydrogen desorption curves of the milled NaAlH 4 and NaAlH 4 doped with 5, 10, 15, and 20 wt % of K 2 NiF 6 at 150°C. The hydrogen released of the milled NaAlH 4 is only at about 0.5 wt % after 120 min, demonstrating poor dehydrogenation kinetics of the undoped sample. The hydrogen has released about 1.5 wt % at 30 min after being doped with 5 wt % K 2 NiF 6 . Further increasing the doping amount to 10, 15, and 20 wt %, the result shows faster  desorption rate in which the hydrogen released about 2.1, 2.5, and 2.2 wt %, respectively within 30 min dehydrogenation. The total hydrogen capacity for the all doped NaAlH 4 is at about 3.0 wt % after 120 min dehydrogenation. Therefore, the remarkable improvement in the dehydrogenation kinetics of NaAlH 4 can be achieved by the addition of the K 2 NiF 6 additive. These results indicated that the addition of minimum amount of K 2 NiF 6 (5 wt %) can reduce the onset decomposition temperature and improve the dehydrogenation kinetics of NaAlH 4 . Thus, the NaAlH 4 + 5 wt % K 2 NiF 6 sample was selected for further analysis.
To compare the thermal properties of the doped and undoped composite, Figure 3 shows the differential scanning calorimetry (DSC) curves of the milled NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 samples. The samples were measured at 100− 350°C with a heating rate of 25°C. Three endothermic peaks are shown by the DSC curves of the undoped sample curve. The decomposition of NaAlH 4 is signified by the first strong endothermic peak at approximately 195°C. Meanwhile, the phase transition of α-Na 3 AlH 6 to β-Na 3 AlH 6 is signified by the weaker peak at 278°C. The third endothermic peak at approximately 310°C is ascribed to the decomposition of Na 3 AlH 6 into NaH and Al. The pattern of this result is almost the same as reported in the previous study by Mao et al. 20,34 In contrast, the K 2 NiF 6 -doped sample has shifted to lower temperature with only two endothermic peaks appeared. These two endothermic peaks decomposed at approximately 179 and 277°C correspond to the decomposition of NaAlH 4 and Na 3 AlH 6 , respectively. The reduction in the peak temperature of the DSC results revealed that the dehydrogenation properties of NaAlH 4 have improved with the addition of K 2 NiF 6 .
The activation energy for the dehydrogenation of NaAlH 4 + 5 wt % K 2 NiF 6 was calculated to investigate the impact of the introduction of K 2 NiF 6 on the kinetic property of NaAlH 4 . In this context, the Kissinger plot was prepared based on the Kissinger equation 36 as follows where β is the heating rate, T p is the peak temperature obtained from the DSC curve, R is the gas constant, and A is the linear constant. Thus, the activation energy can be achieved from the slope in a plot of ln[β/T p 2 ] versus 1000/T p . Figures 4 and 5 show the DSC traces at various heating rates and Kissinger plot for the first-step and second-step dehydrogenation of doped and undoped NaAlH 4 , respectively. From the Kissinger plot of the DSC data for the first-step dehydrogenation as shown in Figure 4c, the apparent activation energy of the milled NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 was found to be 114.7 and 89.9 kJ/mol. Meanwhile, Figure 5c shows the second step dehydrogenation apparent activation energy of the doped and undoped NaAlH 4 . The NaAlH 4 + 5 wt % K 2 NiF 6 sample gives an  activation energy value of about 99.6 kJ/mol, while the undoped sample has an activation energy of 125.2 kJ/mol. The activation energies for the NaAlH 4 + 5 wt % K 2 NiF 6 are reduced by 24.8 and 25.6 kJ/mol for the first and second step dehydrogenation, respectively, which are significantly lower compared to those of undoped NaAlH 4 . This result indicated that the dehydrogenation behavior of NaAlH 4 has remarkably improved by the addition of K 2 NiF 6 with the reduction of the E a value. Table 1 shows the comparison of activation energy of the undoped and different catalysts doped to NaAlH 4 . Referring to this table, all the catalysts have effectively reduced the apparent activation energy of the NaAlH 4 for the first and second step dehydrogenation. As can be seen from the table, K 2 NiF 6 -doped NaAlH 4 does not have the best reduction in the E a value as compared to the other catalysts. The NaAlH 4 -doped with NiFe 2 O 4 shows the lowest E a value which is 54.3 and 73.1 kJ/ mol, for the first and second step dehydrogenation specifically. However, the addition of K 2 NiF 6 still gives the positive effect on the reduction of the apparent activation energy of NaAlH 4 . Figure 6 shows the morphologies of the pure NaAlH 4 , pure K 2 NiF 6 , milled NaAlH 4 , and NaAlH 4 + 5 wt % K 2 NiF 6 . The scanning electron microscopy (SEM) image shows an irregular shape with the average particle size was in the range of 100 μm for the pure NaAlH 4 (Figure 6a). The pure K 2 NiF 6 without further treatment had a smaller particle size which is smaller than 100 μm (Figure 6b). In addition, after the milling process, Figure 5. DSC traces for the second-step dehydrogenation of (a) NaAlH 4 , (b) NaAlH 4 + 5 wt % K 2 NiF 6 , and (c) Kissinger plot for the second-step dehydrogenation of NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 .  Figure 6. SEM images of (a) pure NaAlH 4 , (b) pure K 2 NiF 6 , (c) milled NaAlH 4 , and (d) NaAlH 4 + 5 wt % K 2 NiF 6 .
the particles size of the doped and undoped sample reduced drastically. However, the particle size of the milled NaAlH 4 (Figure 6c) was inhomogenous and agglomerated. This may be the reason for the slightly reduced onset temperature in the TPD results for milled NaAlH 4 as shown in Figure 1. It can be observed that the particles of the NaAlH 4 -doped with 5 wt % K 2 NiF 6 ( Figure 6d) were dispersed more homogeneously and less agglomeration than those of the undoped NaAlH 4 . The particle size appeared to have a finer surface and reduced drastically as compared with the undoped sample. The particle sizes of the pure NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 are determined by Image J software and the particle size distributions are plotted in histograms as shown in Figure  7. On the basis of the histograms, the estimated average of particle sizes are 21.86 and 0.14 μm for the pure NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 , respectively. This indicates that the milling process and the addition of catalyst remarkably reduced the particle size of the compound. The particle size reduction can increase the specific surface area and reduce the diffusion length of hydrogen within the particles which can lead to the increment of the kinetic rates as reported by previous studies. 42,43 Figure 8 presents the X-ray diffraction (XRD) patterns of the pure NaAlH 4 and the pure K 2 NiF 6 . The purity of the NaAlH 4 and K 2 NiF 6 compounds are corroborated by the XRD patterns of the as-received NaAlH 4 (JCPDS card no. , as well as the as-received K 2 NiF 6 (JCPDS card no. . This result is correlated with data previously reported. 39,44 The XRD patterns as in Figure 9 exhibit the milled NaAlH 4 and the milled K 2 NiF 6 -doped with 5 and 20 wt % samples. The NaAlH 4 does not decompose during the ball milling process and it is confirmed by the XRD pattern of the milled NaAlH 4 . In that XRD pattern, only the NaAlH 4 phase has appeared. Meanwhile, the XRD pattern for NaAlH 4 -doped with 5 and 20 wt % K 2 NiF 6 after ball milling shows that only NaAlH 4 and K 2 NiF 6 peaks appeared. This demonstrates that the K 2 NiF 6 additive does not react with NaAlH 4 and remains stable throughout the whole process of milling. The present finding seems to be consistent with the result reported by Huang et al., 11 which found that no reaction had occurred between NaAlH 4 and NiFe 2 O 4 throughout the ball milling process in their study. In order to confirm whether there is a reaction occuring or not after the ball milling process, Fourier transform infrared spectra (FTIR) measurement was performed for milled NaAlH 4 and NaAlH 4 -doped with 5 and 20 wt % K 2 NiF 6 as shown in Figure 10. For milled NaAlH 4 , two intense bands appeared at 1654 and 900 cm −1 which represent the characteristic stretching mode and the bending mode of the Al−H vibration in the AlH 4 group, respectively. The pattern is almost closed with the previous studies. 12 Interestingly, after being doped with 5 and 20 wt % of K 2 NiF 6 similar stretching and bending mode are observed but slightly reduced in Figure 7. Particle size distribution histograms of pure NaAlH 4 and NaAlH 4 + 5 wt % K 2 NiF 6 .  . XRD patterns of the (a) milled NaAlH 4 , (b) NaAlH 4 + 5 wt % K 2 NiF 6 , and (c) NaAlH 4 + 20 wt % K 2 NiF 6 after 1 h ball milling. Figure 10. FTIR spectra of the milled NaAlH 4 , NaAlH 4 + 5 wt % K 2 NiF 6 , and NaAlH 4 + 20 wt % K 2 NiF 6 after 1 h ball milling.
intensity. This indicated that increasing the wt % of the additive weakening the Al−H bonds of NaAlH 4 . All the bands are still of NaAlH 4 and no Na 3 AlH 6 band is observed. Thus, it can be deduced that there is no reaction occurring between the K 2 NiF 6 and the host material of NaAlH 4 throughout the ball milling process.
The XRD measurement was carried out on the K 2 NiF 6doped NaAlH 4 sample in order to verify the phase structure after the dehydrogenation process. Figure 11 presents the XRD pattern of the NaAlH 4 -doped with 5 and 20 wt % K 2 NiF 6 after dehydrogenation at 300°C under 1 atm hydrogen pressure. The XRD analysis for the sample of 5 wt % K 2 NiF 6 ( Figure  11a) demonstrated that only NaH and Al phases appeared with which no other compound was observed. This indicated that NaAlH 4 has completed the first and second dehydrogenation step as shown in the reaction of eqs 1 and 2 in the introduction part. As an addition the sample with 20 wt % K 2 NiF 6 was analyzed (Figure 11b) in order to fully understand the phase composition of the sample as it is not sufficient only with 5 wt % K 2 NiF 6 because of the low amount of the catalyst. It can be seen that new peaks of NaF, AlNi, and KH can be observed by increasing the wt % of K 2 NiF 6 to 20 wt %. This suggested that the reaction of NaAlH 4 with K 2 NiF 6 may have occurred. The possible reaction between K 2 NiF 6 and NaAlH 4 during the dehydrogenation process is presented as follows Meanwhile, the peak corresponding to NaH and Al remains unchanged with the peak intensity gradually enhanced. This indicates that the second step of the NaAlH 4 has been accomplished.
The above results suggested that the in situ formation of AlNi, KH, and NaF phases during the dehydrogenation process is important which can play a vital role in the improvement of NaAlH 4 dehydrogenation properties. This result corroborates the studies by Li et al. 45 which found the appearance of Al−Ni peaks after dehydrogenation and demonstrated that Ni−B doped with NaAlH 4 is a promising catalyst for enhancing the dehydrogenation properties of light metal complex hydride. Meanwhile, introducing KH into NaAlH 4 pronouncedly enhances the dehydrogenation performances as well, especially for the decomposition of Na 3 AlH 6 as reported by Wang et al. 46 Additionally, the in situ formation of NaF may act as a grain refiner for NaH. It can help to promote the nucleation as well as the growth of NaH during dehydrogenation which significantly accelerates the disintegration of NaAlH 4 as suggested by previous studies. 20,47 It is likewise recommended that the in situ formed catalyst indicates higher catalytic activity and superior stability than those of the externally added catalyst in view of the better homogeneity and finer particle sizes. 48 Hence, it is evidence from the SEM result that the NaAlH 4 -doped K 2 NiF 6 compound has smaller and finer particle sizes. These smaller particle sizes give larger surface areas which can reduce the diffusion length and improved the dehydrogenation kinetics as well as lowering the onset decomposition temperature as reported in the literature. 49,50 Furthermore, it is believed that, the establishment of the new active species may facilitate the dehydrogenation process by functioning as the active sites for nucleation and growth of the dehydrogenated product associated with the shortening of the diffusion paths among the reaction ions and thus decreasing the kinetic barriers and improving the dehydrogenation kinetics. 51 Therefore, it is reasonable to believe that the in situ formed active species; AlNi, KH, and NaF will work simultaneously and work synergistically for a significant enhancement on the hydrogen storage properties of NaAlH 4 .

■ CONCLUSIONS
As a conclusion, the addition of K 2 NiF 6 has successfully lowered the decomposition temperature and boosts the kinetic performance of NaAlH 4 . The 5 wt % K 2 NiF 6 -doped compound gave the optimal value for the improvement of the dehydrogenation properties of NaAlH 4 . The sample doped with 5 wt % K 2 NiF 6 started to release hydrogen at 160°C, which is 30°C lower as compared to the milled NaAlH 4 . The dehydrogenation kinetic revealed that the doped sample showed faster hydrogen released, with 5 wt % K 2 NiF 6 can release about 1.5 wt % of hydrogen in 30 min and at 150°C. While the milled NaAlH 4 only released about 0.2 wt % within the same time and temperature. The apparent activation energy calculated from Kissinger plots were reduced from 114.7 and 125.2 kJ/mol for undoped NaAlH 4 to 89.9 and 99.6 kJ/mol after being doped with K 2 NiF 6 for the first and secondstep of dehydrogenation, respectively. In addition, the particles size also reduced and less agglomerated with the addition of K 2 NiF 6 . This can help to improve the hydrogen sorption of NaAlH 4 . These result demonstrated that the active species that formed in situ, the AlNi, NaF, and KH are responsible for the enhancement of the dehydrogenation properties of NaAlH 4 .

■ EXPERIMENTAL DETAILS
The commercial starting materials, NaAlH 4 (purity ≈ 98%) and K 2 NiF 6 (purity ≈ 99%) were obtained from Sigma-Aldrich and were utilized without further treatment. All handling of the samples were operated in a glovebox (MBraun Unilab) with an argon atmosphere to avoid from humidity and oxygen. NaAlH 4 -doped with different wt % of K 2 NiF 6 (5, 10, 15 and 20 wt %) were prepared via the planetary ball mill (NQM-0.4). This process was conducted for 1 h with a rotation speed of 400 rpm for three cycles in a different direction. Each of the sample was sealed in stainless steel jars together with stainless steel balls. Similarly, milled NaAlH 4 was also prepared in the same manner as a reference.
The decomposition temperature and dehydrogenation kinetics of the samples were investigated using a Sievert-type pressure-composition-temperature equipment from Advanced Figure 11. XRD patterns of the (a) NaAlH 4 + 5 wt % K 2 NiF 6 and (b) NaAlH 4 + 20 wt % K 2 NiF 6 after dehydrogenation at 300°C.
Material Corporation. For the TPD experiment, all the samples were heated to 300°C at a heating rate of 5°C/min. The isothermal dehydrogenation kinetics was conducted at 150°C under 30 atm hydrogen pressure. The DSC measurement was conducted on Mettler Toledo TGA/DSC 1. The samples were operated at four different heating rates starting from room temperature to the desired temperature with the influence of 50 mL/min of an argon flow.
Characterizations of the samples were carried out using a SEM (JEOL JSM-6360LA). The phase compositions were characterized using an XRD method with Cu Kα radiation (Rigaku MiniFlex X-ray diffractometer). The diffraction angle of the samples were scanned between 20 and 80°at a scan speed of 2.00°/min. The FTIR measurement of the samples after ball milled was recorded on the IRTracer-100 spectrophotometer using attenuated total reflection in the transmission mode. The spectral resolution was 4 cm −1 and the scans were taken from 750 to 4000 cm −1 .


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Bioactive Bromotyrosine-Derived Alkaloids from the Polynesian Sponge Suberea ianthelliformis

Herein, we describe the isolation and spectroscopic identification of eight new tetrabrominated tyrosine alkaloids 2–9 from the Polynesian sponge Suberea ianthelliformis, along with known major compound psammaplysene D (1), N,N-dimethyldibromotyramine, 5-hydroxy xanthenuric acid, and xanthenuric acid. Cytotoxicity and acetylcholinesterase inhibition activities were evaluated for some of the isolated metabolites. They exhibited moderate antiproliferative activity against KB cancer cell lines, but psammaplysene D (1) displayed substantial cytotoxicity as well as acetylcholinesterase inhibition with IC50 values of 0.7 μM and 1.3 μM, respectively.

Introduction
Sponges of the marine genus Suberea (order: Verongiida) are known to produce a large array of structurally diverse brominated tyrosine alkaloids, which are considered as chemotaxonomical markers [1][2][3]. Nevertheless, bromotyrosine-derived metabolites have been isolated from sponges belonging to distinct orders such as Agelasida [4] and others. In addition, an interesting recent study showed that culture of the marine sponge-derived bacterium Pseudovibrio denitrificans Ab134 isolated from the Arenosclera brasiliensis sponge (order Haplosclerida) can produce bromotyrosine-derived alkaloids as well [5]. Although misidentifications and contaminations due to misplaced specimens are possible, these reports compromise this family of compounds as a Verongiida marker and suggest staying cautious about secondary metabolites markers for sponge phylogeny [6]. The bromotyrosine derivatives' architectures vary from the simple monomeric molecules such as subereaphenol K [7], or the dimeric suberedamine A [8,9], to more complex derivatives like the well-known fistularin-3 [10].
wide scope of bioactivities including cytotoxicity against murine leukemia L1201 cells and KB cells [8,9], DNA demethylating [11], antiproliferative [12], antimicrobial [13][14][15][16], and antiplasmodial activities [17]. In continuation of our search for bioactive marine natural products from South Pacific sponges [18], in both health and chemical ecology fields, we report here the isolation, structural determination, and cytotoxicity evaluation of eight new tetrabromotyrosine alkaloids (Figure 1), along with four known compounds from the sponge Suberea ianthelliformis. The known major compound psammaplysene D (1) was also evaluated for its acetylcholinesterase inhibition activity. Although acetylcholinesterase inhibition has been mostly explored as treatment for Alzheimer's pathology, our attention has been brought to the chemical ecology importance of such enzyme inhibitors in their environment or against predation [19]. Only one publication relates fish toxicity on Gambusia affinis (western mosquitofish) and butylcholinesterase from a Latrunculia magnifica toxin [20]. We describe our observations of the inhibitory effect of psammaplysene D on acetylcholinesterase, using balneation experiments on two fish species, Poecilia reticulata (guppy) and Acanthurus triostegus (reef fish).
reported in the literature. It is worth to point out that psammaplysene D (1) was isolated by normal phase MPLC as the major compound (6.7 g) representing over 50% of the entire n-BuOH extract.
The molecular formula C23H27 79 Br2 81 Br2N2O3 of compound 2 was determined by HRESIMS (m/z 698.8675 [M + H] + ) indicating ten degrees of unsaturation. The isotopic pattern of the protonated molecule confirmed the presence of four bromines. Analysis of the NMR data recorded for 2 revealed striking similarities with the NMR data recorded for the co-isolated psammaplysene D (1), except for the absence of the signal corresponding to the propylamine group, as confirmed by the HRESIMS spectrum. In general, the 1 H NMR spectra of these families of compounds were complicated by the multiplicity of the signals due to the two rotational isomers trans/cis (6:4 ratio) along the amide bond [21]. The same behavior was already reported for tetrabromotyrosine derivatives psammaplysene A-D, isolated from the sponges Psammaplysilla and Psammoclemma [21][22][23][24][25]. The 1D and 2D NMR analysis revealed the existence of four aromatic protons comprising two singlets at δH 7.78 and 7.70, assigned to H-3/5, and another broad singlet at δH 7.57 integrating for 2 protons, attributed to H-15/17. An A-B system signal, corresponding to two olefinic protons at δH 7.39-7.36/7.38-7.35 (J = 15.5 Hz) and δH 7.15-7.11/7.02-6.99 (J = 15.5 Hz), were assigned to the E olefin -CH-7=CH-8-, satisfying the tenth degree of unsaturation. The HMBC data analysis showed correlations between H-7 and the two sp 2 aromatic carbons C-3/5 (δC 133.09/133.01) and the sp 2 non-protonated carbon C-4 (δC 130.98) confirming the connection of the trans-cinnamoyl motif to the 2,6-dibrominated phenolic ring (deduced with the chemical shifts of C-2/6 at δC 112.80 and C-1 at δC 154.10). The second olefinic proton H-8 exhibited an HMBC correlation with the non-protonated carbon C-9 at (δC 168.96/168.80) linking this end to an amidic carbonyl function. HSQC and COSY spectra analysis allowed the assignment of the signals at δH 4.11 and 4.05 (2H, 2t, J = 6.4), 3.89 and 3.74 (2H, 2t, J = 7.2 Hz), 2.22 and 2.16 (2H, 2m) to the three propylic carbon sequence N-CH2-10-CH2-11-CH2-12-O, while those at δH 3.30 (2H, m) and 3.00 (2H, m) were attributed to the ethylic chain CH2-19-CH2-20. The protons H-20 were correlated to N-Me22 and N-Me23 by HMBC analysis (Figure 2). The methylene group CH2-10 was connected to the amidic N-Me21 based on its chemical shift (δC 47.14/48.2) and the HMBC correlation to C-9 (δC 168.96/168.80) and C-21 ( (Table 1) showed similarity between compounds 1 and 2, which was easily observed by superimposition of their spectra. The A-B system of two downfield doublets of doublets at δH 6.49-6.52/6.56-6.58 (J = 12.5 Hz, cis) and δH 6.13-6.16/6.09-6.12 (J = 12.5 Hz) were assigned to -CH-7=CH-8 of the cinnamoyl moiety with a Z configuration. Furthermore, the COSY, HSQC, and HMBC analysis data ( Figure 2) showed the same correlations as previously found for 2. Thus, the tetrabrominated tyrosine 3 was assigned to psammaplysene G.   Compounds 4 and 5 were obtained in a minute quantity (0.7 mg) as a 1:1 mixture that could not be separated. Due to the minute quantity of the mixture, we decided to determine the structures of the latter two metabolites from the mixture. HRESIMS analysis revealed the molecular formulas C 27  (atomic mass unit) less than psammaplysene D (1), respectively. Direct comparison of the 1 H NMR spectra of this mixture (Table 2) and the co-isolated major compound psammaplysene D (1) showed good superimposition, including the rotameric forms. The main differences concerned the signals of  2) along with the chemical shift of C-3 , showed the presence of two methyl groups on the nitrogen N-C-3 . The chemical shift of C-20 and C-22 were the same for compound 5, but the chemical shift of C-2 (δ C 27.9), C-3 (δ C 48.7), and CH 3 -4 (δ C 34.0), as well as the HMBC correlations between C-3 and CH 3 -4 , suggested the presence of a NH-Me connected to the carbon C-3 . Therefore, our mixture was composed of two psammaplysene D derivatives lacking one and two N-methyls and named psammaplysene H (4) and psammaplysene I (5), respectively.   respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines. These NMR and mass data showed that compound 7 has one methyl group less than compound 6. Examination of the 1 H NMR and HSQC spectra (Table 3) displayed signals with similar chemical shifts to those reported for the tetrabromotyrosine compounds anomoians A and B, previously isolated from Anomoianthella popeae [26] (order: Verongiida) and from an unknown species of the genus Hexadella (order: Verongiida) collected in Indonesia [27]. 2D NMR analysis including COSY, HSQC, and HMBC (Table 3, Figure 4), allowed identification of the structures of 6 and 7 as the O-demethyl anomoian B and O-demethyl anomoian A, respectively. Thus, the new compounds were named anomoian C (6) and anomoian D (7). The 1 H NMR signals of the aromatic protons CH-3/5 and CH-15/17 of these new compounds indicated a 6:4 rotameric proportionality.  respectively, indicating nine degrees of unsaturation with an isotopic pattern of four bromines. These NMR and mass data showed that compound 7 has one methyl group less than compound 6. Examination of the 1 H NMR and HSQC spectra (Table 3) displayed signals with similar chemical shifts to those reported for the tetrabromotyrosine compounds anomoians A and B, previously isolated from Anomoianthella popeae [26] (order: Verongiida) and from an unknown species of the genus Hexadella (order: Verongiida) collected in Indonesia [27]. 2D NMR analysis including COSY, HSQC, and HMBC (Table 3, Figure 4), allowed identification of the structures of 6 and 7 as the O-demethyl anomoian B and O-demethyl anomoian A, respectively. Thus, the new compounds were named anomoian C (6) and anomoian D (7). The 1 H NMR signals of the aromatic protons CH-3/5 and CH-15/17 of these new compounds indicated a 6:4 rotameric proportionality.    (Table  4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7). Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY, HSQC, and HMBC, enabling us to set the correlations shown in Figure 5. The new compound 8 was named anomoian E.
The HRESIMS data of 9 revealed the molecular formula C30H45 79 Br2 81 Br2N4O3 (m/z 829.0259 [M + H] + ). Its 1 H NMR spectrum was very similar to anomoian E (8) spectrum, except for the two additional methyl groups at δH 2.51/2.54 integrating for six protons for 9. HSQC, COSY, and HMBC spectra analysis led to the same correlations as in compound 8 and enabled the assignment of the N-8-methyl groups through an additional correlation from the N-CH3-24/25 (δH 2.51/2.54, 6H) to the methine group CH-8 (δH 3.97/3.89, m; δC 66.79/66.58). Therefore, structure 9 was determined as anomoian F, existing as 6:4 rotamers (Table 4, Figure 5).   (Table 4) showed signals with identical chemical shifts to those reported for anomoian C (6) and D (7). Furthermore, it revealed the presence of an additional dimethylaminopropylic chain, as observed for psammaplysene D (1). This was confirmed by the 2D NMR spectra analysis, including COSY, HSQC, and HMBC, enabling us to set the correlations shown in Figure 5.  Figure 5).

Biological Activities
The isolated compounds 1-3 and 6-11 were tested for their cytotoxic activity against human epidermoid carcinoma cells (KB) cancer cell lines (Table 5) and acetylcholinesterase. The results showed of the compounds exhibited moderate cytotoxicity. The major compound psammaplysene D (1) showed the most interesting cytotoxicity with an IC50 = 0.7 μM. Anomoian E (8) and F (9), containing the N,N-dimethylaminopropyl side as psammaplysene D, but lacking the double bond -CH-7=CH-8, displayed a lower activity. Moreover, psammaplysene F (2), which bear a

Biological Activities
The isolated compounds 1-3 and 6-11 were tested for their cytotoxic activity against human epidermoid carcinoma cells (KB) cancer cell lines (Table 5) and acetylcholinesterase. The results showed of the compounds exhibited moderate cytotoxicity. The major compound psammaplysene D (1) showed the most interesting cytotoxicity with an IC 50 = 0.7 µM. Anomoian E (8) and F (9), containing the N,N-dimethylaminopropyl side as psammaplysene D, but lacking the double bond -CH-7=CH-8, displayed a lower activity. Moreover, psammaplysene F (2), which bear a trans-cinnamoyl part, was found to exhibit a lower activity than psammaplysene D (1). Therefore, from a structural  The screening of our French Polynesian sponges extract library was performed on acetylcholinesterase inhibitor (AChE) in the aim to investigate a possible chemical defense of sessile organisms on the reef against fishes, their main predators. The chemical study of the bioactive extract of Suberea ianthelliformis using AChE led to the identification of psammaplysene D (1) as its unique in vitro AChE inhibitor with an IC 50 = 1.3 µM. None of the other closely related compounds displayed any activity on this target enhancing here again the importance of the phenoxy-1-N,N-dimethyl propane chain and the trans-cinnamoyl motifs (Figure 1). Further kinetics investigation of the mode of AChE inhibition of psammaplysene D (convergence of the lines on the Lineweaver Burk plot, decreasing V max values (from 0.0036 to 0.0007 µmol·min −1 ) and increasing K M values (from 0.14 to 0.23 µM)) suggested a mixed competitive/non-competitive inhibition suggesting that psammaplysene D (1) not only binds to the free enzyme, but also to the enzyme-substrate complex. In order to try to evaluate the effect of psammaplysene D in the environment, it was further tested on fresh water fish (guppy, Poecilia reticulata) and reef fish (Acanthurus triostegus) feeding experiment using bathing with or without the bioactive compound. Guppies are fed with commercial flakes, but the wild reef fish feed on natural coral heads, and their appetite evaluated by counting the bites on the corals. Two experiments were run on guppies using Suberea ianthelliformis crude extract and purified psammaplysene D (1): an acute toxicity bioassay (100 µg/mL of crude extract or purified psammaplysene D (1), 1 h) and a chronic toxicity (5 µg/mL of crude extract or 1 µg/mL of purified psammalysene D (1), 72 h).
During the acute exposure, fish were totally confused and had uncontrolled mobility, displaying sudden jumps and balance control loss, as soon as 20 min after the addition of psammaplysene D in the tank. This result can be compared to the observation reported by Nèeman et al. with Latrunculia species toxins [20]. Exposure of guppies during 24 h of at a sub-lethal dose (5 µg/mL of crude extract or 1 µg/mL of purified psammalysene D (1) of the compound caused passivity of the fish towards the food flakes offered daily, whereas those of the controls ate normally. These observations indicate a clear link between the loss of appetite and psammaplysene D (1) toxicity. Similar activity was observed on reef fish recruits (fish larvae undergoing metamorphosis) of Acanthurus triostegus: the presence psammaplysene D (1) (1 µg/mL) in tanks significantly and readily decreased the number of bites of recruits, suggesting a rapid saturation of the environment perception sensors of the fish larvae during the 72 h of experiment ( Figure 6). Psammaplysene D (1) displayed clearly behavioral effects on both guppies and reef fish larvae: they showed a loss of appetite from the first day of experiment. Reef fish juveniles' behavior did not seem to be impacted by psammaplysene D. It is interesting to note that only A. triostegus larvae were impacted by the pure psammaplysene D, suggesting a change of sensibility towards the compound during the metamorphosis. The toxicity test at a high dose (100 μg/mL of crude extract or purified psammaplysene D) on the guppies led to their death after one hour bathing with either the crude extract or the purified psammaplysene D fraction (acute toxicity experiment). Brains and gills of these guppies were extracted and the AChE activity measured on the extracts (Figure 7a,b) [29]. The results were clearer with the purified psammaplysene D, showing a mild but significant decrease of the activity of the enzyme in the gills, suggesting an action on the olfactory organs (peripheral nervous system) that could not be further investigated there. A great number of AChE inhibitors targeting Alzheimer's disease have been isolated from natural sources [30]. Among them, 6 compounds with activities ranging from IC50 1 to 100 μM were isolated from Verongiida sponges ( Table 6). It would be interesting also to further investigate those compounds as chemical anti-predatory defenses through fish antifeedant activity, since the fish model is easier to set up than related bioassays involving sponge predators, such as nudibranchs or crustaceans. Psammaplysene D (1) displayed clearly behavioral effects on both guppies and reef fish larvae: they showed a loss of appetite from the first day of experiment. Reef fish juveniles' behavior did not seem to be impacted by psammaplysene D. It is interesting to note that only A. triostegus larvae were impacted by the pure psammaplysene D, suggesting a change of sensibility towards the compound during the metamorphosis. The toxicity test at a high dose (100 µg/mL of crude extract or purified psammaplysene D) on the guppies led to their death after one hour bathing with either the crude extract or the purified psammaplysene D fraction (acute toxicity experiment). Brains and gills of these guppies were extracted and the AChE activity measured on the extracts (Figure 7a,b) [29]. The results were clearer with the purified psammaplysene D, showing a mild but significant decrease of the activity of the enzyme in the gills, suggesting an action on the olfactory organs (peripheral nervous system) that could not be further investigated there. A great number of AChE inhibitors targeting Alzheimer's disease have been isolated from natural sources [30]. Among them, 6 compounds with activities ranging from IC 50 1 to 100 µM were isolated from Verongiida sponges ( Table 6). It would be interesting also to further investigate those compounds as chemical anti-predatory defenses through fish antifeedant activity, since the fish model is easier to set up than related bioassays involving sponge predators, such as nudibranchs or crustaceans. Psammaplysene D (1) displayed clearly behavioral effects on both guppies and reef fish larvae: they showed a loss of appetite from the first day of experiment. Reef fish juveniles' behavior did not seem to be impacted by psammaplysene D. It is interesting to note that only A. triostegus larvae were impacted by the pure psammaplysene D, suggesting a change of sensibility towards the compound during the metamorphosis. The toxicity test at a high dose (100 μg/mL of crude extract or purified psammaplysene D) on the guppies led to their death after one hour bathing with either the crude extract or the purified psammaplysene D fraction (acute toxicity experiment). Brains and gills of these guppies were extracted and the AChE activity measured on the extracts (Figure 7a,b) [29]. The results were clearer with the purified psammaplysene D, showing a mild but significant decrease of the activity of the enzyme in the gills, suggesting an action on the olfactory organs (peripheral nervous system) that could not be further investigated there. A great number of AChE inhibitors targeting Alzheimer's disease have been isolated from natural sources [30]. Among them, 6 compounds with activities ranging from IC50 1 to 100 μM were isolated from Verongiida sponges ( Table 6). It would be interesting also to further investigate those compounds as chemical anti-predatory defenses through fish antifeedant activity, since the fish model is easier to set up than related bioassays involving sponge predators, such as nudibranchs or crustaceans.   [33] a NCI: non competitive inhibition; b CI: competitive inhibition; c ND: non described.
A common side effect of AChE inhibition treatments is anorexia [34,35], which strengthens the importance of natural AChE inhibitors as potential deterrents of predators such as fishes. Furthermore, the olfactory dysfunction in Alzheimer's disease has also been demonstrated [36]. Psammaplysene D is the major compound of the sponge (0.66% w/w). It has been shown that tyrosine derived compounds are located in sphaerus cells of the sponge, allowing their release in the aquifer channels and further in the close environment of the sponge [37]. It would be thus interesting to track and quantify the concentration of this compound in the close reef environmental water of the sponges, using for example, solid phase adsorption toxin tracking (SPATT) [38], to allow the on field deterrent or reef protective activity of this compound acting softly on olfactory sense of predators in the marine environment. The importance of AChE inhibitors in the marine environment can be included in neuroecology of chemical defenses, a merging field of chemical ecology, which displays the importance of the links between ecology and evolution, secondary metabolites, behavior, and neurosciences [39].

General Procedures
Optical rotations were measured using MCP-300 polarimeter (Anton Paar, Les Ulis, France). IR spectra were recorded on a BX FT-IR spectrometer (Perkin Elmer, Courtaboeuf, France). 1D and 2D NMR spectra were recorded on a Avance 500 and 600 MHz (CNRS-ICSN, Bruker, Wissembourg, France). The chemical shifts are relative to the residual signal solvent (CD3OD: δH 3.31; δC 49.20; DMF-d7: δH 8.03, 2.92, 2.75; δC 163.2, 34.9, 29.8). High-resolution mass spectra were obtained on a LCT Premier XE spectrometer (Waters, Guyancourt, France) in electrospray ionization mode by direct  A common side effect of AChE inhibition treatments is anorexia [34,35], which strengthens the importance of natural AChE inhibitors as potential deterrents of predators such as fishes. Furthermore, the olfactory dysfunction in Alzheimer's disease has also been demonstrated [36]. Psammaplysene D is the major compound of the sponge (0.66% w/w). It has been shown that tyrosine derived compounds are located in sphaerus cells of the sponge, allowing their release in the aquifer channels and further in the close environment of the sponge [37]. It would be thus interesting to track and quantify the concentration of this compound in the close reef environmental water of the sponges, using for example, solid phase adsorption toxin tracking (SPATT) [38], to allow the on field deterrent or reef protective activity of this compound acting softly on olfactory sense of predators in the marine environment. The importance of AChE inhibitors in the marine environment can be included in neuroecology of chemical defenses, a merging field of chemical ecology, which displays the importance of the links between ecology and evolution, secondary metabolites, behavior, and neurosciences [39].

Animal Material
The sponge was collected off the coast of Nuku Hiva (8 • 55 977 S-140 • 01 17 W) between 5 and 42 m deep using SCUBA on the 28 August 2009 [40]. It was identified as Suberea ianthelliformis and a reference specimen is deposited at the Queensland Museum (Brisbane, Australia) under the accessing number G331075.

Isolation of Bioactive COMPOUNDS
The freeze-dried sponge sample (960 g) of Suberea ianthelliformis was extracted three times at room temperature with a 1:1 mixture of CH 2 Cl 2 /MeOH (1.5 L). The extracts were combined and dried under reduced pressure to afford a brown residue (50 g). Forty-five grams of this residue were partitioned between CH 2 Cl 2 and H 2 O. The resulting organic layer was collected and dried under reduced pressure to afford a brown material (10.5 g). The aqueous layer was further extracted with n-BuOH to afford the butanolic extract (13 g). The remaining aqueous fraction was dried to afford 21.2 g material. The CH 2 Cl 2 extract was then subjected to normal phase silica-gel MPLC. A gradient composed of CH 2 Cl 2 /MeOH (1:0 to 0:1), followed by a mixture of EtOAc/Acetone/H 2 O/Formic Acid (5/3/0.5/0.5) were used to afford 8 fractions F1 to F8. The n-BuOH extract was also subjected to normal phase silica gel MPLC, using the same gradient as before to afford 6 fractions F1 to F6. Promising fractions were selected based on the LC-MS analytical profile, and then each fraction was submitted for purification by preparative reversed phase HPLC (column: Waters Sunfire C 18 19 mm × 150 mm, 5 µ, H 2 O + 0.1% formic acid/CH 3 CN + 0.1% formic acid). Some sub-fractions were further purified using semi-preparative reversed phase column (Waters Sunfire C 18 , 10 mm × 150 mm, 5 µ, H 2 O + 0.1% formic acid/CH 3 CN + 0.1% formic acid).

Cell Culture and Cell Proliferation Assay
The human cell line KB originated from the National Cancer Institute (NCI) and was grown in Dulbecco's Modified Eagle Medium (D-MEM) medium supplemented with 10% foetal calf serum, in the presence of penicillin, streptomycin, and fungizone in a 75 mL flask under 5% CO 2 . A total of 600 cells were plated in 96-well tissue culture plates in 200 L of medium and treated 24 h later with 2 µL of stock solution of compounds dissolved in DMSO using a Biomek 3000 (Beckman-Coulter, Brea, CA, USA). Controls received the same volume of DMSO (1% final volume). After 72 h exposure, cell titer 96 Aqueous One solution (Promega, Madison, WI, USA) was added and incubated for 3 h at 37 • C: the absorbance was monitored at 490 nm, and results were expressed as the inhibition of cell proliferation calculated as the ratio [(1 − (OD490 treated/OD490 control)) × 100] in triplicate experiments.

Acetylcholinesterase Inhibition Assay
The crude extract of the sponge and the purified psammaplysene D (1) were dissolved in DMSO at 10 mg/mL, stored at −20 • C and diluted subsequently 100 times in H 2 O prior testing. Determination of AChE-inhibitory activity was measured according the Ellman's colorimetric method [41], in 96-well tissue culture plates. All reagents, buffers and salts were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Galantamine was used as a positive control for each analysis. Solvent controls received the same volume of water. After a 30 min incubation at 25 • C in the dark, absorbance was monitored at λ = 405 nm. Results were expressed as the percentage of enzyme inhibition calculated as the ratio: 100 × (OD control − OD treated )/(OD control − OD blank ), where the blanks are the wells without AChE. Serial dilutions of the compound or extract were prepared (100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 µg/mL) and tested according to the same procedure using 10 µL of each solution. IC 50 values were graphically determined by plotting the percentage of inhibition against the compound concentrations. Each assay was performed in triplicate and results are expressed as mean values ± S.D.

Kinetic Study
The inhibition mode of psammaplysene D on AChE was determined while measuring the enzyme activity in presence of an increasing concentration of ATCI (0.024, 0.049, 0.098, 0.19, 0.39, 0.78, 1.57, and 3.15 µM), in absence or presence of 2 µg/mL final concentration of purified psammaplysene D.
The inhibition mode, V max , and K M values were determined by double-reciprocal Lineweaver and Burk plot analysis (1934) of the data obtained.
3.5.2. Guppy Bioassay 5 to 8 cm long guppies (Poecilia reticulata) were collected at night in a private fresh water pool at Papara (Tahiti, French Polynesia) and immediately acclimated at the laboratory temperature (25 • C). After acclimatization, fishes were parted in two 2 L tanks: one for the control group treated with solvent (N = 5). Another experiment was performed using a 1 h treatment with 100 µg/mL of crude extract of the sponge or purified psammaplysene D (1) to check their acute toxicity.

Reef Fish Feeding Behavior Experiment
Manini (Acanthurus triostegus) were caught during new moon nights on the foreshore puddles at Tema'e public beach [42]. Recruits (fish larvae undergoing metamorphosis) and juveniles (larvae after the recruits stage) were collected and immediately used for the experiments. Natural lagoon sea-water was used and UV-sterilized prior use. Three conditions were tested on three groups: control, solvent, 1 µg/mL sponge extract treatment, with 4 or 5 fishes in each 3 L tank. The fishes were exposed under semi-static system for 72 h, the experimental solutions were renewed every 24 h to maintain clean experimental conditions. The experiments were performed with fish recruits and juveniles. Rubbles were disposed in each aquarium 1 h/day during 3 days. Feeding behavior experiment consists in counting the overall number of bites on those rubbles in each aquarium. Six videos sequences (N = 6) of 5 or 10 min per aquarium and per day were analyzed. Results are expressed in number of bites per fish and per hour.

Statistical Analysis
All the values of AChE activity are represented in a boxplot distribution, because of non-normality of variable distribution (N = 4 or 5). The significant differences between controls and treatments (p-value p < 0.05) were analyzed with non-parametric test of Wilcoxon Mann Whitney. In feeding deterrent assay, pairs significant differences between different conditions (p < 0.05) were tested with non-parametric Kruskal Wallis test. All statistical assays were executed using R software (R-3.2.0, The R Foundation for Statistical Computing, Vienna, Austria).

Conclusions
This paper describes the isolation of eight new bromotyrosine secondary metabolites from the Polynesian Sponge Suberea ianthelliformis. Their structures are close to those of psammaplysenes and anomoians but all displaying methylated amidic nitrogen and rotameric mixtures. Since flexible syntheses were developed for psammaplysene A [43], and iso-anomoian A [28] and other analogues, our molecules present new derivatives for further optimization of biologically interesting molecules. They all exhibited moderate antiproliferative activity against KB cancer cell lines, but psammaplysene D (1) displayed substantial cytotoxicity as well as acetylcholinesterase inhibition with IC 50 values of 0.7 µM and 1.3 µM, respectively. It would be interesting to further investigate the ecological role of the major psammaplysene D as chemical anti-predatory defenses through fish antifeedant activity. The available synthetic strategies would allow the preparation of our metabolites and a rage of their analogues for the screening of the inhibition of various cancer cell lines and SAR studies.