Anal. Chem. 1994,66,4283-4287
Determination of the Active Hydrogen Content in Pyridine-Borane Complex by Schiff Base Reduction and HighmPerformance Liquid Chromatography James A. Morley,* Lee Elrod, Jr., and John F. Bauer PPD Physical Analytical Chemistry Department, Abbott Laboratories, 1401 Sheridan Road, North Chicago, Illinois 60064
Borohydrides and borane complexes have been widely used as selective reducing agents in many chemical applications. In particular, the pyridine-borane complex (PBC, C5Hfl-BH3) is very stable to hydrolysis and is an attractive reagent for large-scalereductions that are common in the pharmaceutical industry. This report describes a simple, rapid, and reproducible method for quantitating the active hydrogen content in PBC. The method exploits the ease with which Schiff bases are reduced by amine-boranes and uses high-performance liquid chromatography to quantitate the reduction product. The method is not affected by small amounts of extraneous moisture and can be d e d out in common glassware. The potential application of the analytical procedure to other amine-borane complexes is also discussed. The leukotrienes have been associated with the mediation of allergic and inflammatory responses in humans.' The 5lipoxygenase enzyme is a crucial component in the biosynthesis of leukotrienes via the metabolism of arachidonic acid.2 Therefore, the inhibition of Slipoxygenase is considered a potential approach for the treatment of inflammation and allergy. Zileuton (N-(1benzo [b]thien-2-ylethyl)-N-hydroxyurea or ABT-077) is a potent inhibitor of 51ipoxygenase3and is currently in clinical trials for treatment of allergic asthma and inflammation. An intermediate step in the current synthesis of deuton employs the pyridineborane complex (PBC) on a production scale to reduce an oxime to a hydroxylamine. This necessitates the storage of PBC in large quantities for extended periods. The reduction is carried out with an excess of the complex and results in good yields. Our interests were twofold: to develop a rapid and reliable method for the measurement of active hydrogen to support the synthesis and to develop a stability indicating procedure to assure the quality of warehoused PBC. We report here a simple, rapid, and reproducible method for the quantitation of active hydrogen in PBC via the reduction of N-benzylideneaniline and high-performanceliquid chromatographic quantitation (HPLC) of the product, N-phenylbenzylamine. The procedure does not require specialized equip ment. (1) Samuelson, S. Science 1983, 220, 565. (2) Borgeat, P. J.Med. Chem. 1 9 8 1 , 2 4 , 1 2 1 . Cashman, J. R; Oliva, S. L. Drugs Today 1988,24, 723. (3) Bell, R L.; Young, P. R; Albert, D.; Lanni, C.; Summers, J. B.; Brooks, D. W.; Rubin, P.; Carter. G. W. Znt. J. Immunophannacol. 1992, 14(3), 505. 0003-2700/94/0366-4283$04.50/0 0 1994 American Chemical Society
Diborane and borane complexes have been used extensively as selective reducing agents in organic ~ynthesis.~ The amineborane complexes have a broad range of physical properties, solubilities, and reactivities that depend on the nature of the amine.5*6For example, the N-arylamine-borane complexes are hydrolyzed by water and alcohols, whereas the complexes of several N-alkylamines are water soluble and exhibit moderate to remarkable stabilities to acid hydr~lysis.~Weakly basic or hindered amines give complexes wherein boron is more electrophilic, whereas stronger bases form stronger complexes that are more stable to hydrolytic conditions and behave more like borohydrides6 For example, at 25 "C, the morpholine-borane complex is slowly hydrolyzed (120 min) in 50% 1 N HC1/50% ethylene glycol, while the N-phenylmorpholine- borane complex rapidly hydrolyzes (2 min) .5 Many amine-borane complexes including PBC are commercially available and, unlike diborane or tetrahydrofuran-borane complex, can be easily handled in the laboratory. Alternately, PBC can be prepared in high yield by the treatment of pyridine hydrochloride with sodium b~rohydride.~ The active hydrogen, reducing power, boron content, and pyridine content of material prepared in this manner is consistent with the proposed complex. Typical reductions with boranes use an excess of reagent and generally do not require a precise knowledge of the active hydrogen content. The vendors' label claims are sufficient in these instances. Nonetheless, there are several accounts in the literature of methods to determine the analytical concentration of hydride in borohydride and amine-borane compounds. The classical method is the hydrogen evolution or gasometric measurement which quantitates the active hydrogen as evolved hydrogen after hydrolysis of the boron compound? This approach has also been used to quantitate amines by complexation with excess diborane and subsequent determination of the remaining d i b ~ r a n e .Other ~ methods include volumetric determination of active hydrogen by the oxidation of the borohydride or amineborane complex with iodine,1° hypochlorite,ll silver nitrate,12and (4) Brown, H.C. Boranes in Organic Chemistty; Comell University Press: Ithaca,
NY, 1972. (5) Lane, C. F. Aldrichim. Acta 1973, 6(3), 51. (6) Pelter, A; Rosser, R M . j . Chem. Soc., Perkin Trans. 1 1984, 717. (7) Taylor, M. D.; Grant, L. R; Sands, C. A J. Am. Chem. Soc. 1955, 77,1506. (8) Chaikin, S. W.; Brown, W. G.J. Am. Chem. SOC.1949, 71,122. Schlesinger,
H. I. Bid. 1953, 75, 215. (9) Lyle, R. E.;Southwick, E.W. Anal. Chem. 1968, 40, 2201. (10) Mathews, M. B. J. Biol. Chem. 1948, 176, 229. Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4283
iodate.13 A voltammetric procedure has also been recently described for sodium b0r0hydride.l~ The hydrogen evolution method, though accurate, is not a practical procedure for the routine determination of the active hydrogen content in PBC in a quality control environment. This procedure is time consuming and requires specialized equipment. Of the remaining methods, the oxidation with iodate (iodometric titration) has generally been considered the most accurate alternative to the gasometric determination. However, this titration cannot be readily automated; the samples must be titrated immediately, or low results are obtained due to the atmospheric oxidation of iodide to iodine. The iodometric titration also cannot distinguishbetween hydride and other species capable of iodate reduction. Therefore,titrating a sample of PBC does not indicate directly the reagent's potency for carrying out a synthetic organic reduction. In a laboratoryscale synthesis, this may not be a critical parameter, but in those cases where strict stoichiometric addition of active hydrogen is mandatory, the potency of the reagent must be known with accuracy. This is espically critical when many resources are expended to produce a compound in large batches for pharmaceutical or industrial use. Preliminary efforts aimed at directly chromatographing PBC either destroyed the phase (gas chromarography on a poly(ethy1ene glycol) phase) or gave results significantly lower than iodometric titration values (HPLC on a polystyrene resin). Both approaches are severely limited by the lack of a readily available reference standard and the potentially high reactivity of the compound. EXPERIMENTAL SECTION
Chromatographic Conditions and Instrumentation. The HPLC system used in this work consisted of a ternary pump, Model SP-8800 m e r m o Separation Products, Santa Clara, CA); an autosampler, Model SIL6B (Shimadzu Corp., Kyoto, Japan); and a UV detector, Spectra Physics Model SP-100 (Therm0 Separation Products). Chromatograms were processed using a Model C-R4A Chromatopac (Shimadm Corp.). A Nucleosil CISchromatographiccolumn (5 pm, 4 mm x 12.5 cm, lOC-A pore size; Machery-Nagel, Duren, Germany) was used in this method. The HPLC buffer consisted of a 0.05 M phosphate buffer (13.6 g of monobasic potassium phosphate in 2 L of distilled water) adjusted to pH 2.7 (fO.l) with phosphoric acid. Isocratic elution was carried out with a mobile phase of 60%buffer/40% acetonitrile. The flow rate was 1.0 mL/min with UV detection at 254 nm (0.1 AUFS). All chromatography was performed at ambient temperature. Materials. Caution! Although PBC is normally a very stable compound, care should be taken in storing or handling any hydride- or hydrogen-producing reagent. PBC is a liquid and can easily be transferred by syringe or glass pipet. All solvents used in this study were reagent grade or HPLC grade (EM Science, Gibbstown, NJ). Commercial PBC was used in all experiments. The N-benzylideneaniline, N-phenylbenzylamine (free base), acetic anhydride, and other amine-borane complexes were used as received (Aldrich Chemical Co., Milwaukee, Wl). (11) Chaikin, S. W. Anal. Chem. 1953,25,831.Pecsok, R L.J. Am. Chem. SOC. 1953,75,2862. (12) Brown, H. C.; Boyd, A C., Jr. Anal. Chem. 1955,27, 150. (13) Lyttle, D. A; Jensen, E. H.; Struck, W. A Anal. Chem. 1952,24,1843. (14) Mirkin, M. V.; Bard, A J. Anal. Chem. 1991,63, 532.
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The N-phenylbenzylamine hydrochloride used as a reference standard was prepared by bubbling gaseous hydrochloric acid through a hexane/THF (92/8) solution of the free base. The resulting off-white solid was collected by vacuum filtration and washed with hexane. The hydrochloride salt gave satisfactory 'H NMR, MS, and IR spectra. The punty (199.5%) of the salt was determined by titration (AgN03 and HClOd), HPLC, and Karl Fischer moisture. The following data were also obtained: mp (uncorrected) = 203-205 "C; molar absorptivity (246 nm) = 12 174 M-' cm-' (c = 0.01, CH3CN/HzO(l:l)). Elemental analysis (theory): C, 71.06 (71.07); H, 6.14 (6.42); N, 6.36 (6.38); C1, 16.0 (16.1). The hydrochloride salt turns slightly green on exposure to light and air. This does not significantly affect the potency of the material. Procedure for the Determination of Active Hydrogen Content. Stock solutions of PBC and N-benzylideneaniline in tetrahydrofuranwere prepared by dissolving 250 mg of PBC and 2 g of N-benzylideneaniline in separate 50-mL volumetric flasks. Five milliliter aliquots of each stock solution were combined in a 1WmL volumetric flask, and 10 mL of glacial acetic acid was added. The resulting solution was mechanically shaken for 30 min followed by dilution to volume with diluent (HPLC buffer/ acetonitrile, 1:l). After being further diluted 4.0 mL to 100.0 mL with diluent, the sample preparation was assayed versus a standard preparation of approximately 60 pg/mL N-phenylbenzylamine (free base concentration). The identity of the product of Nbenzylideneaniline reduction was confirmed by isolating the material from a sample preparation run at twice the above scale. The crude N-phenylbenzylamine was converted to the hydrochlcride salt with HCl gas and recrystallized from ethanol/hexane to give material with spectral properties ('H NMR, IR) consistent with N-phenylbenzylamine hydrochloride. The isolated material coeluted with the standard preparation when spiked into the assay standard,further confirmiig the identity of the reduction product. The iodometric titrations used for comparison were carried out according to an established procedure'3 with the slight modification that PBC was dissolved in methanol prior to the determination. RESULTS AND DISCUSSION
Active hydrogen in PBC is determined using the following reaction:
l/3 Py-BH3 (PW
+
pJCHII
rt, 30 min
u
NH I
THFklOAc
(N-Bcnzylideneamline)
(N-Phenylknzylamne)
This reaction is a moditication of a literature procedure where the reduction of Schiff bases to secondary amines was observed with the dimethylamine-borane complex.15 The room temperature reduction of the Schiff base N-benzylideneanilineby PBC in a mixed solvent of tetrahydrofuran (THF)/glacial acetic acid (1: 1)proceeds smoothly to give N-phenylbenzylaminein quantitative yields. The imine is used in a large excess to insure the complete (15) Billman, J. H.; McDowell. J. W. J. 0%.Chem. 1961,26,1437.
l.OEt6
, .
/'T
7.5Et5
0
%
\
33 Mol % PBC
e
0.025 aufs
P
s
4
!
1
Y
m
n 1
0
10
'
1
1
20
30
'
1
40
'
50
mol % PBC Figure 1. Peak area response of N-phenylbenzylamineversus mol
Yo of added PBC.
consumption of PBC. The reduction does not occur in the absence of PBC, and no significant hydrolysis of the imine to benzaldehyde was observed (no benzyl alcohol produced). The presence of insoluble boric acid, a PBC hydrolysis product, did not significantly affect the assay. It was not necessary to heat the reaction, as was previously noted in the reduction of several Schiff bases by dimethyl- and trimethylamine-borane complexes.15JGThe lower temperature used here also avoided any formation of side products such as acylated material that was observed in the reductions with trimethylamine-borane complex.lG Furthermore, stressing the reaction mixture by heating on a steam bath gave only a 6% lower yield of N-phenylbenzylamine with no significant amount of acylated material present.17 The stability of PBC to hydrolysis in acetic acid has previously been investigated.'j These investigators found that the complex does not appreciably decompose after 1h in acetic acid. The THF in the solvent mixture was originally present to insure complete solubility of the reactants. However, the role of this solvent may be much more than solubility related. The partial conversion of the PBC to a more reactive THF-borane moiety may be possible in an acidic media. The stoichiometry of the reaction was examined by carrying out the reduction at varying mole ratios (PBC/imine) and plotting the resulting N-phenylbenzylaminepeak area versus the mole ratio of PBC. As shown in Figure 1, the peak area for the product amine plateaus after the addition of 33 mol % of PBC. This finding is consistent with the 1:3 mole ratio of borane:imine found previously for Schiff base reduction by dimethylamine-borane complex15 ((acyloxy)borohydride species have been implicated as likely intermediates in the reduction of imines, iminium salts, and enamines with sodium borohydride or amine-borane complexes in carboxylic acid solvents18and have also been suggested in the reductive alkylations of indoles,'g quinolines,20and secondary amines21by sodium borohydride in carboxylic acid solvents). However, the data presented here suggest that no intermediate (acy1oxy)borohydride species is formed with the acetic acid prior to reduction. This would have resulted in a lower than expected (16)Billman, J. H.;McDowell, J. W. J. 0%.Chem. 1962,27,2640. (17) Crude N-acylated phenylbenzylamine was prepared according to a general procedure: Morley, J. A; Elrod, L. E., Jr. Chromatographia 1993,37(5/ 61,295. (18)Hutchins, R 0.;Su,W.-Y.; Sivakumar, R; Cistone, F.; Stercho, Y. P. J. Org. Chem. 1983,48,3412.
t-----paration
Synthetic Mixture
-w -01 N-
Blank Preparation
Figure 2. Representative chromatograms of a synthetic mixture, sample preparation, and a blank preparation (20-pL injections; the sample preparation is described in the text; blank, HPLC buffer/ acetonitrile, 1:l). Peak identities: (a) pyridine; (b) PBC; (c) N benzylideneaniline; (d) Nphenylbenzylamine.
borane:imine ratio. No further investigation into the mechanism of the reduction was done. The N-phenylbenzylamine produced by the reduction of N-benzylideneanilineis quantitated versus an authentic standard of N-phenylbenzylamine hydrochloride. Although the free base is commercially available, the hydrochloride salt of N-phenylbenzylamine provides a more stable and easily purified reference standard. The active hydrogen content (mmol of hydrogen/g of sample) in the sample is determined from the stoichiometry of the reaction (theory = 32.3 mmol of hydrogen/g of PBC). Shown in Figure 2 are representative chromatograms of a synthetic mixture, a typical assay solution, and a blank preparation. The effect of variations in the HPLC eluent on the chromatographic behavior of N-phenylbenzylamine was studied. The N-phenylbenzylamine standard preparation was chromatographed using eluents which contained 30-50% acetonitrile and using eluents containing buffers at pH 2.1-3.1. Our system suitability requirements (i.e., capacity factors of 10-20 and tailing factors 51.4) were satisfied when the pH was 22.6 and the acetonitrile concentration was 540%. The experiments indicated that small variations in the eluent composition would have negligible consequences on the chromatographic finish of the method. (19)Gribble, G. W.; Lord, P. D.; Skotnicki,J.; Dietz, S.E.; Eaton, J. T.; Johnson, J. L. J. Am. Chem. SOC.1974,96,7812. (20) Gribble, G. W.; Heald, P. W. Synthesis 1975,650. (21)Gribble, G.W.; Jasinski, J. M.; Pellicone, J. T.; Panetta, J . ASynthesis 1978, 766.
Analytical Chemistry, Vol. 66,No. 23, December 1, 1994
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Table 1. Precision Data for the Determination of Active Hydrogen in PBC
active hydrogen (mmol/g of sample) analyst 1 1 1 2 2 2
meanu SD RSD
A
B
C
30.4 30.1 30.2 30.3 30.8 31.6 30.6 (30.5) 0.56 11.8
29.7 29.9 29.7 30.2 30.2 30.2 30.0 (29.8) 0.25 10.83
19.1 19.2 18.8 19.7 19.6 19.2 19.3 (22.2) 0.33 f1.7
Table 2. Solutlon Stabillty Data (in pg/mL) for Standard and Sample Preparatlonsa
time (h) 0 9 11 13 15 17 19
mean SD RSD
standardb 60.7 61.1 60.1 62.5 62.0 61.6 61.3 0.878 f1.43
time @)I
sampleC
0 10 12 14 16 18 20
59.1 59.1 58.3 58.4 62.5 58.8 59.6 59.4 1.44 12.42
“The value in parentheses is by iodometric titration (average of two determinations by a single analyst).
Standard and sample preparations quantitated versus an initial standard preparation. 60.7 pg/mL theory. c 59.1 pg/mL theory.
The detector response for the reduction product, N-phenylbenzylamine, is linear from 30.2 to 90.5 ,ug/mL (free base concentration corresponding to 50-150% of the standard preparation). The equation of the line (four data points) was found to be y = (11 140 186)x 451 ( i ~ l l 9 1 5with ) a correlation coefficient (r) of 0.9996. Similar results were obtained for peak height response. The reduction reaction was found to be linear from 5.1 to 35.6 mg of PBC (corresponding to 20-142% of the described sample preparation). The plot of N-phenylbenzylamine versus PBC added (five data points) gave a correlation coefficient of 0.9997 with the equation y = (25 926 f 426)x 11863 (f9948). The precision of the analytical method was determined by two analysts on different instruments using three lots of PBC. The results are shown in Table 1. Lots A and B were typical incoming bulk material, whereas lot C was material that had decomposed to an orange-red, viscous liquid on storage. Also included in the table are data generated by the iodometric titrati011.l~ The data indicate that there is good agreement between the two methods for typical lots of PBC (lots A and B). These lots were clear and colorless samples that contained no precipitate indicative of hydrolysis. However, examination of an atypical lot, lot C, gave significantly higher values with the iodometric titration. The THF stock solution of lot C contained copious amounts of precipitate (presumably boric acid). We subsequently determined that boric acid did not interfere in the reduction of N-benzylideneaniline.In contrast, lot C was completely soluble under the iodometric titration conditions (essentially alkaline methanol), and boric acid did not interfere in this assay, either. The results suggest that some other reducing agent has formed on decomposition that does not contain active hydrogen but is capable of reducing iodate to iodide. This would account for higher values with the titration procedure. The source of the assay bias for lot C was not investigated any further. Several of the parameters of the reduction reaction were investigated. The effect of extraneous water present in the reaction mixture was examined by performing the reaction in the presence of small amounts of added water. Samples were prepared as described in the Experimental Section with 0, 0.05, 0.1, and 1 mL of water added to the 5-mL aliquots of Nbenzylideneaniline used for the sample reduction. M e r the usual workup, the amount of N-phenylbenzylamine was quantitated versus a standard preparation. At these addition levels, residual water had no effect on the amount of reduction product formed. The results indicate that the rigorous drying of solvent and
glassware sometimes necessary in reductive chemistry is not necessary for this method. The effect of reaction time was demonstrated by shaking the reaction mixtures up to 60 min at room temperature. Integrated peak areas of N-phenylbenzylamine did not increase after 10 min. The chromatograms also showed complete consumption of PBC at this time (10 min), indicating complete reduction. The amount of acetic acid used in the sample preparation was varied from 1 to 15 mL (10-150% of the assay levels), and the reaction mixtures were worked-up as described in the Experimental Section. Examination of the integrated peak areas of N-phenylbenzylamine revealed no dif[erence for the 5, 1@,and l5mL acetic acid reactions (corresponding to 50, 100, and 150% of the assay preparation). However, the reaction containing 1mL of acetic acid (10% assay preparation) gave a significantly lower (-30%) peak area for N-phenylbenzylamine. Since no significant byproducts or impurities were observed using the stated conditions, no further optimization of the acetic acid content was pursued. Similarly, the amount of N-benzylideneaniline was found to have essentially no effect on the amount of N-phenylbenzylamine formed when compared at 50,100, and 150%of the assay levels. Solution stability data were obtained to determine the feasibility of using an automated HPLC finish in the method. A sample preparation and a standard preparation were made using the described procedure for solutions containing 59.1 and 60.7 ,ug/ mL of N-phenylbenzylamine, respectively. Repetitive injections of these preparations demonstrated solution stability for both solutions to at least 19-20 h at room temperature. The data are summarized in Table 2 and demonstrate that automated HPLC runs are acceptable. Attempts were made to apply this methodology, along with some variations, to other amine-borane complexes. The complexes were chosen such that a wide range of hydrolytic stability was surveyed (Table 3). The experiments in Table 3 were not optimized and represent an initial trial of developing a general procedure for active hydrogen determination in amine-borane complexes. It was found that the complexes of 2,&lutidine, morpholiie, and fert-butylamine were less soluble in THF than previously reported5 and therefore gave low yields of N-phenylbenzylamine under the conditions described in the Experimental Section. Although soluble in THF, the trimethylamine complex required an extended reaction (shake) time to go to completion. This result is consistent with reports that this complex is highly
+
+
4286 Analytical Chemistry, Vol. 66,No. 23,December 1, 1994
Table 3. Determination of Active Hydrogen in Amine-Borane Complexes.
amine trimethylamine 2,Glutidine morpholiine tert-butylamine N-phenylmorpholine
theory method Ab method Be method Cd 41.1 24.8 29.7 34.5 16.9
39.3e 14.1 24.0 26.3
f
20.2 15.1 i5.9 16.4
f
37.9 15.6 27.6 27.3
f
The values are presented as mmol of active hydrogen/g of sample. The determination as described in the Experimental Section, except where noted. The amine-borane complexes were dissolved in pyridine. The remainder of the determination was as described in the Experimental Section. The amine-borane complexes were dissolved in glacial acetic acid. The determinations were performed as in the ExperimentalSection, except that 10 mL of THF/acetic acid (1:l) was used in place of 10 mL of neat acetic acid and the reaction was heated for 1 h on a steam bath. e The reaction (shake) time was extended to 3 h. f No N-phenylbenzylamine was detected.
stable to hydrolysis5and should therefore be less reactive under the described conditions. The complexes were found to be soluble in pyridine (method B, Table 3) and glacial acetic acid (method C, Table 3) at active hydrogen concentrations similar to that described for PBC in the Experimental Section. The results for the trimethylamine and morpholine complexes (method C, Table 3) are promising and suggest that a general procedure may require only the fine-tuning of the reaction conditions. The N-phenylmorpholine complex gave no indication of imine reduction and was apparently rapidly hydrolyzed. A slight bubbling indicative of hydrogen evolution was observed when this complex was added to acetic acid. This finding is consistent with the known hydrolytic instability of N-arylamine-borane complexes.
CONCLUSIONS A simple, rapid, and quantitative method for the determination of the active hydrogen content of PBC has been described. The gasometric procedure, under the proper conditions and attention by the analyst, is the most direct way of quantitating active hydrogen but is not a practical method for rapid analysis. The method described herein is also a direct means for determining the organic reducing power of PBC because the reduction of the Schiffbase, N-benzylideneaniline, will not take place in the absence of a source of active hydrogen. Volumetric methods, such as the iodometric titration, quantitate the overall reducing power of a compound and do not differentiate between reducing species.The method described here uses an automated HPLC finish which allows for a high throughput of samples with little analyst time. Neither the gasometric nor the volumetric procedure can be readily automated. Preliminary experiments for applying this procedure to other amine-borane complexes gave promising results with the trimethylamine complex. Development of a general assay for the amine-borane complexes was outside of the scope of this method; however, this work has laid the foundation on which a more universal assay may be built for determining the active hydrogen in borohydrides and borane complexes. ACKNOWLEDGMENT The authors thank Diane Horgen for her assistance in the preparation of the manuscript. Received for review May 20, 1994. Accepted August 24,
1994.a e Abstract
published in Advance ACS Abstracts, October 1, 1994.
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