Background of the invention

This invention relates to an improvement in enantiomers separation and detection by means of a chiral reagent and high performance liquid chromatography (HPLC). More specifically it relates to an optically active fluorenyl chloroformate which forms stable diastereomeric carbamates with primary and secondary amines in a quantitative way. This product can be detected by fluorimetry or by absorptiometry after separation on HPLC column .

The separation of optically active amino-containing substances is of great importance in biological research and pharmaceutical chemistry. These substances are common in pharmaceuticals and many of them are enantiomers ocurring as racemic mixtures. In many cases the biological activity of these enantiomers is attributed to the conformation of one particular optically active antipode. Therefore it is very important to distinguish or resolve the enantiomers.

For quantitive purpose chromatographic methods are to be used since they offer many advantages such as small sample size, simple pretreatment and high sensitivity of detection.

Since the first chromatographic separation of optically active isomers in 1951 with paper chromatography technique a great number of such methods have been introduced and they have recently been reviewed in books (Souter, R.W. "Chromatographic Separation of Stereoisomers" ; CRC Press, Boca Raton, 1985 and Jacques, J. ; Collet, A. ; ilen, S.H. "Enantiomers, Racemates and Resolutions"; John Wiley & Sons New York, 1981).

The resolution of enantiomers requires the intervention of a chiral agent. Chromatographic resolving methods have been developed in two major directions:

1. Direct resolution of enantiomers in columns with either a chiral stationary phase (CSP) or a chiral component in the mobile phase.

2. Indirect resolution of formed diastereomers after derivatization with a chiral reagent.

The liquid chromatographic direct separation of enantiomers on chiral stationary phases has received a great deal of attention and nowadays there are some commercially available chiral columns. The efforts have been focused on to make a chiral stationary phase feasable to separate as many different types of chiral substances as possible. Still there are very few a ine and amino acids that have been directly resolved on chiral columns of any type, without previous derivatization.

Tamegai et al, J. Liq. Chromatography, 1979, 2, 1229 have reviewed methods based on indiredt resolution of enantiomers by derivatization with a chiral reagent to form diastereomers which are resolved on conventional columns. Furukawa et al, Chem. Pharm. Bull. 1975, 23, 1623 were the first to separate enantiomeric amino acids using readily available (+)-10- camphor sulphonyl chloride as a chiral reagent. The carbonyl residue was then transformed in a second step into the p-nitrobenzylester which resulted in derivatives sensitive to UV-detection .

The most common chiral reagents for selective amine function derivatization are those based on isothiocyanate which give UV sensitive thiourea derivatives with primary and secondary amines. The reaction is effected under weakly basic conditions for about 20 minutes at room temperature without formation of by-products .

The most successful reagent of this kind is 2,3,4,6 -tetra-o-acetyl-p-d-gluco-pyranosyl isothiocyante (GITC) used for both amino acids and amines. The resulting thiourea derivatives of most protein amino acids were resolved on a

conventional reversed phase column within two hours. The resolution obtained is preferably due to the lipophilic nature of the sugar residue combined with conformational rigidity.

Fluorescence chiral derivatization reagents for amino containing compounds are rare and only reagents based on o-phthalaldehyde combined with chiral thiols (OPA) are reported, JP 60 38,652 (85 38,652). The different chiral thiol compounds which have been used are N-acetyl-L-cysteine , BOC-L-cysteine all these yield diastereomeric isoindols with primary amino groups. The reaction is selective and completed within a minute at room temperature direct in a buffered water mixture without racemization . The formed diastereomers are however not stable.

These are some essential and desirable conditions such a reagent should have.

* It has to be optically pure

* It has to contain a chromophore or a fluorophor

* The reaction conditions should be mild, otherwise the risk of potential racemization will increase.

* The reagent should react with primary and secondary amino groups .

* The resulting diastereomers should be stable.

* The resulting diastereomers should separate on conventional HPLC columns .

* The method should be applicable on a preparative scale.

* The method should be feasable to automatize.

Nowhere, there has however been disclosed or suggested a chiral reagent combined with a method which effectively derivatize, detect and separate racemic mixtures of primary and secondary amino-containing compounds.

Summary of the invention

The object of the present invention is to provide a chiral reagent and a method for indirect resolution of primary and

secondary amino-containing compounds on conventional reversed phase HPLC columns. The reagent should give fluorescent derivatives which are stable, and detectable by fluorometry or absorptiometry after separation by liquid chromatography. A stable derivative facilitates quantitation and automation. Furthermore the reaction should occur rapidly under mild conditions in aqueous phase and without racemization.

This has been achieved by derivatizing the amino function of said compounds by adding an optically active regent of the formula

wherein X is a halogen, an azide group or a succinimidyl group and wherein R is an alkyl group or a tri luormethyl group, to form diastereomeric carbamates which are determined by per se known methods, e.g. fluorimetry or absorptiometry, after separation by liquid chromatography.

The reagent performs:

1. The determination of optical purity in resolved amines and amino acids .

2. The determination of different amounts of enantiomeric amino-containing compounds in biological systems.

3. Preparative resolution of amino-containing racemic mixtures .

Description of the drawings

Fig.1 shows a reagent and reaction scheme.

Fig.2 is a graph showing reaction rates and yields to form

fluorescent diastereomeric carbarmates of different amino acids .

Fig.3 is a graph showing reaction yields vs. pH for asparagine acid and glutamine acid.

Fig. is a chromatogram showing the resolution of 17 amino acids as fluoresecent labeled diastereomeric carbamates performed with reversed phase liquid chromatography.

Fig.5 is a chromatogram showing the resolution of racemic metoprolol .

Fig.6 is a chromatogram showing derivatization and resolution of commercially available "optically pure" L-glutamine.

Description of the invention

The invention is based on the rapid reaction of chloroformates with different amines to form stable carbamates during Schotten-Baumann conditions. The reaction is performed in one step within seconds at room temperature. In a certain pH range the reaction is selective to amino functions and the only formed by-product is the corresponding alcohol. The reagent excess is easy to handle thereby the interference in the following separation step is omitted.

The 9-fluorenylmethylchloroformate reagent (FMOC) is fluorescent with excitation emission wavelengths 270/315 mm.

Thus the formed products are selectively detected at this wavelengths combination. This property together with the selective reaction lead to that very complex mixtures e.g. body fluids can be handled without any clean-up or pretreatment procedure.

The invention is to synthezise an optically active reagent which can be used in the same way as the achiral FMOC-reagent with its many advantages so to form diastereomers with racemic amino-containing compounds. The diastereomers should be

feasable to resolve on a conventional separation system typically reversed phase HPLC, the most common and effective LC separation system for trace analysis and preparative separation. Making the reagent optically active is certainly not a guarantee for obtaining resolvable diastereomers. The introduction of an asymmetric carbon atom must be carried out at a position so that the reagent properties are maintained.

The invention was to create chirality of the FMOC reagent by introducing an asymmetric carbon atom at the 9-fluorenyl-1 position. This type of compound has not been described before in the literature. This position is strategic while it is close to the formed amid function as well as to the rigid cyclic arrangement representing the fluorene moiety. It should be pointed out that these are still no general rules to design chiral reagents amenable for succesful resolution of diastereomers. Therefore it is not possible to make predictions. Thus optical resolution is still based on trial and error experiments. However, surprisingly the 9-fluorenyl-1 -ethylchlorofornate reagent (FLEC) yields diastereomers with most amines resolvable on reversed phase LC colums. Furthermore the (+)-1 -( 9-fluorenyl) ethylchloroformate gave diastereomers of the rare D-amino acids which were separated in prior of the main L-form ideally is rare determinations. The properties of the new reagent were maintained compared to achiral FMOC reagent. As a conclusion a new reagent is described which will be very practical and useful for the determination of optically active amino containing compounds in complex mixtures and for the preparative resolution of amines.

The reaction of FLEC with amino acids proceeds as shown in Fig. 1. The reagent also reacts with water to yield the corresponding alcohol as a hydrolysis product.

Synthesis of (+)- 1 -( 9-fluorenyl )ethyl chloroformate (FLEC)

Synthesis of 1 -( 9-fluorenyl)ethanol: To a solution of 8.3 g fluorene in 100 ml of dry ether 31 ml BuLi (1.6 M in hexane)

was added. The mixture was refluxed for 30 min. and then cooled in an ice bath. To the resulting mixture a solution of 2.8 ml acetaldehyde in 40 ml dry ether was added during 15 - min. and then refluxed for 1 h. Water (100 ml) was added and the ether layer collected in a separatory funnel, dried (MgSO ) and evaporated. The product was further purified by flash-chromatography and recrystallized from ligroin (b.p.

0 0

80-110 C) to give white needles, m.p. 101-103 C, mass spectrum

M*m/e 210.15, Η NMR(CDC1 ₃ ) , 0.87(d,3H) , 1.70 (S, 1H) , 4.09

(d, 1H) , 4.35-4.65 (m, 1H) , 7.15-7.85 (m, 8H).

Optical resolution of 1 -(9-fluorenyl)ethanol: To a solution of

4.0 g 1 -( 9-fluorenyl) ethanol in 25 ml anhydrous pyridine, an equimolar amount of ( - ) -camphanic acid chloride (4.12 g) was added, and the mixture was stirred at room temperature for 3 h. The solution was poured into ice-water and extracted with

CH Cl . The CH Cl -layer was washed with dilute hydrochloric 2 2 2 2 acid, dried (MgSO ) and evaporated to dryness. The crude ester was dissolved in 200 ml MeOH and cooled to -15 C. Deposited crystals (fraction A) were crystallized twice from the same solvent giving 1.0 g of the less soluble diastereomeric ester in pure form, (m.p. 159-160 C) , (racematemp. 152 C) . The optical purity of the diastereomeric ester was checked on a chiral stationary phase ( ( - ) -dinitrobenzoylphenylglycine coupled to aminopropylsilica , Pirkle's phase) and it was 99"/.

Hydrolysis of the ester: To a solution of the optically pure diastereomeric ester (1.0 g) in 50 ml dry ether, 0.8 g LiAlH was added. The mixture was stirred at room temperature for 1 h giving the alcohol after work up, m.p. 91-93 C (decomp. ).

( + )-1- (9-fluorenvHethvl chloroformate: To a solution of phosgene (0.8, 8.1 mol) in 15 ml of dry toluene cooled to 0 C, a solution of optically pure alcohol (0.44 g, 2.1 mmol) and triethylamine (0.30 ml, 2.1 mmol) in 20 ml dry toluene, was added dropwise. After addition was completed, stirring was continued for 2 h at 0 C. the triethylaminehydrochloride was then removed by filtration and the filtrate was concentrated at reduced pressure giving an oil.

[α] ²⁵ =+67.9° (CH ₂ C1 ₂ . C=1)

Cα] ²⁵ =+70.5° (CH Cl , C=1) 578

¹ H NMR (CDC1 ₃ ): 0.76 (d, 3H) , 4.30 (d, 1H) , 5.47-5.75 (m. 1H) , 7.18-7.75 (m, 8H)

Anal, calcd for C H 0 Cl: C, 70.46; H, 4.80; Cl , 13.00

16 13 2

Found: C, 70.64; H, 4.80; Cl, 13.13

Experimental conditions

Solvents and reagents: Acetonitrile , tetrahydrofurane and acetone were purchased from Rathburn (Walkerburn, U.K. ) . The amino acid standards and the FM0C-C1 reagent were obatained from Sigma (St. Louis, MO, U.S.A. ). The elution buffer was made of acetic acid in double distilled water (3 mL/L) , titrated to the appropriate pH with sodium hydroxide. The FLEC-Cl reagent was dissolved in acetonitrile: acetone (1:3) and had a concentration of 15 mmol/L. The reaction buffer was made of boric acid (1M) and the pH was adjusted with sodium hydroxide .

Derivatization: Sample (0.4 mL ) and buffer (0.1 mL , pH ) are mixed in a 3 ml reaction vial. The reagent (0.5 mL ) is added and allowed to react. After 1 minute the vial is almost filled with pentane and the reaction mixture is extracted to remove excess reagent. The extraction is repeated twice, and then the aqueous phase is ready for injection.

Reaction rate: The rate of the reaction of the amino acids with the FLEC-Cl reagent is shown in Fig. 2 and was determined by dervatization of the amino acids (one at a time) in the usual manner with the reagent at pH 8.03 (sample plus buffer). After a certain time interval the reaction was stopped by addition of acetic acid, and the excess of the FLEC-Cl reagent was removed by pentane extractions. The amount that remained unreacted was determined by precolumn dervatization with o-phtalaldehyde/mercaptoethanol, followed by liquid

chromatography. The results were compared with a solution treated in in the same way, but without the FLEC-Cl reagent (original amount of amino acid) , from which the conversion to a FLEC-derivative could be calculated. Each point is a mean of two measurements.

The effect of pH on the reaction rates of aspartic acid and glutamic acid is shown in Fig. 3 and was carried out in the same way as described above with 1 minute reaction time. Measurements were were made at pH 8.01 , 8.46, 9.01 , 9.47 and 10.23. The pH was measured in the reaction solution after the addition of the buffer.

The reactive reaction rates of the FLEC-Cl and the FM0C-C1 reagents with valine, glutamic acid, proline and lysine were determined by comparison of the yield obtained when a standard amino acid solution was dervatized with each of the reagents, with the results when a mixed FLEC/FMOC reagent was employed. The derivatives were separated with chromatography and the peak areas compared.

In fig. 4 is shown the resolution of 17 amino acids as fluorescent labelled diastereomeric carbamates performed with reversed phase liquid chromatography.

Experimental conditions were:

The FLEC-Cl reagent is dissloved in acetonitrile/acetone

(1/4) , 15mM. The reaction buffer is a borate buffer, 1M, pH

6.5.

0.4 ml sample and 0.1 ml borate buffer are mixed. To this solution is added 0.5 ml of the reagent. After 1 min. reaction time the mixture is extracted 3 times with pentane. The pentane extracts are discarded and the aqueous phase is ready for injection. When derivatizing relatively hydrophobic amines not having an acid group, the extraction step is omitted and the excess reagent is removed by reaction with a hydrophilic

amine, typically hydroxyproline or hydrazine.

Table I

Gradient elution: Time (min) ZAcN 7.THF '/.Buffer

0 8 17 75

8 8 17 75

22 0 30 70

70 0 50 50

Separation conditions (amino acids) :

Column: 150 x 4.6 mm packed with Spherisorb octyl material, d=3μm. Elution buffer: Acetic acid 3 promille, pH 4.35. Flow rate : 0.3 ml/min .

In table II below are stated the k'-values and resolution factors, α-values for some amino-containing pharmaceuticals.

Table II

Amine k ' 1 k " 2 α

Metoprolol 6.8 7.5 1.1 (1 )

Tokainid 9.4 10 1.06 (2)

Norephedrine 5.03 5.28 1.05 (3)

Column: 250 x 4.6 mm, packed with Spherisorb® octyl material, d=5μm. Flow rate: 1.2 ml/min.

Elution conditions: (1 ) 607. Acetonitrile, 402 water

(2) 507 Acetonitrile, 507 water

(3) 557 THF ( tetrahydrofurane) . 45'/ water

In Fig. 5 is shown the resolution of a racemic solution of metoprolol, a cardioselective β-adrenoceptor antagonist (β-blocker), dervatized with a racemic FLEC-Cl reagent. Column: 4.6 x 250 mm. packed with 5μm octyl material (Spherisorb): Eluent: 607. acetonitrile, 407 water.

In Fig. 6 is shown the derivatization and resolution of commercially available "optically pure" L-glutamine to determine the combined effect of optical purity of the amino acid standard, the optical purity of the reagent and the racemization of the derivatization. L-glutamine (1mmol/l) dervatized with FLEC-Cl. The D-glutamine peak amounts to 0.6 promille of the L-glutamine, determined by standard addition of a small amount of D-glutamine (2μmol/l).

In Table III below are stated the k' and resolution factors, α-values, for the common peptide amino acids separated on reversed phase column.

Table III

Amino acid ^' ι α ' ₂

Aspargine 1.34 1.46 1 .09 I

Glutamine 1.29 1.46 1 .13

Serine 2.01 2.09 1 .04

Aspartic acid 2.37 2.54 1 .07

Threonine 2.94 3.31 1 .13

Glutamic acid 3.26 3.69 1 .13

Arginine 3.54 4.03 1 .14

Alanine 5.63 6.20 1 .10 Proline 5.51 5.51 1 . nU nU

Tyrosine 3.29 3.91 1 .19 II

Methionine 3.57 4.29 1 .20

Phenylalanine 4.83 6.09 1 .26

Valine 5.00 5.91 1 .18

Isoleucine 8.17 9.69 1 .19 Leucine 8.46 10.20 1 . __.1

Histidine 3.37 4.77 1 .42 III

Ornithine 5.37 6.69 1 .25 Lysine 7.23 8.37 1 .1 b

Eluent composition:

I 357 THF, 657 Acetic acid buffer (37. pH 4.35)

II 407 THF, 607 - " - III 457 THF, 557 - " -

Column: 4.6 x 250 mm, packed with 5 μm octyl material (Spherisorb)

As indicated above reagent excess can be removed by different methods. One method, prefarably used when separating amines, is to react reagent excess with an amine yielding a product which does not interfere during the separation with the compounds to be determined. The amine as well as the derivative should be water-soluble. Examples of such amines are hydroxyproline and hydrazine.

Another method, preferably used when separating amino acids or amino sugars forming hydrophilic amino derivatives, is to extract reagent excess with a solvent of low polarity, e.g pentane or hexane.

The reagent according to the present invention is also applicable for preparative resolution of amino-containing racemic mixtures. After separation of the derivatives by HPLC the amino-containing compunds are regained by conventional methods, such as acid or basic hydrolysis or by hydrogenolysis.

In table IV below a comparison of current precolumn derivatizing agants and FLEC-Cl is made which illustrates the superiority of the FLEC-reagent .

Table IV

PITC Dansyl

FLEC GITC OPA Dabsyl

Primary and secondary Yes Yes No Yes amino compounds

Automated Yes No Yes No

Stable derivatives Yes No No No

MDQ 100-200 1-10 100-200 100-200 femtomoles picomoles femtomoles femtomoles

Major interferences No No No

Although 1 -( 9-fluorenyl) ethyl chloroformate is the only reagent described by way of examples there are a number of structural analogues that may be used as well. Methyl as R in the general formula in claim 1 could be substituted by trifluormethyl or a higher alkyl group. Instead of chlorine X could be bromine, an azide group or a succinimidyl group, which have been tested for the FMOC-reagent and found to be good leaving groups.