WO2005105257A1 - Phase stationnaire chromatographique pour une separation de cyclodextrine et de derives de celle-ci - Google Patents

Phase stationnaire chromatographique pour une separation de cyclodextrine et de derives de celle-ci Download PDF

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Publication number
WO2005105257A1
WO2005105257A1 PCT/HU2005/000043 HU2005000043W WO2005105257A1 WO 2005105257 A1 WO2005105257 A1 WO 2005105257A1 HU 2005000043 W HU2005000043 W HU 2005000043W WO 2005105257 A1 WO2005105257 A1 WO 2005105257A1
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stationary phase
alkyl
group
cooh
optionally substituted
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Gábor VARGA
Julianna SZEMÁN
Lajos Szente
Péterné CSABAI
Krisztina Kékesi
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Chiroquest Kiralis Technologiakat Fejleszto Kft
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Chiroquest Kiralis Technologiakat Fejleszto Kft
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • B01J20/289—Phases chemically bonded to a substrate, e.g. to silica or to polymers bonded via a spacer

Definitions

  • the present invention relates to chromatographic stationary phases for examination of cyclodextrines (CDs) and their derivatives and for preparative separation thereof. Further subjects of this invention are the use of the above stationary phases, the starting compounds applied in the preparation of the characteristic surface of the above stationary phases and the use thereof.
  • CDs are known since about a century as enzyme-modified starch-derivatives having toroidal shape and consisting of 6, 7 or 8 glucopyranose units. There are CDs with higher number of glucopyranose units (delta etc.), but they are rarely used in practice.
  • the 6- member CD is named as ⁇ -CD
  • the 7-member CD is named as ⁇ -CD
  • the 8-member which has the greatest cavity, is named as ⁇ -CD.
  • the CD molecules have an hydrophobic inner cavity due to the position of glucopyranose units, while the outer surface has hydrophilic character due to the high number of hydroxyl groups.
  • This feature makes CD molecules capable to encapsulate apolar, therefore poorly water soluble, mainly aromatic guest molecules into the apolar cavity, making possible the preparation of homogenous aqueous solutions containing the guest molecules in high concentration.
  • the solubility of the guest molecules is modified, the presence of CDs influences other physical and chemical properties of the guest molecules, which may result in that chemically stable solutions can be prepared from compounds which are, otherwise, ready for decomposition ( J. Szejtli, Cyclodextrines and Their Inclusion Complexes, Akademiai kiad ⁇ , Budapest, 1982.). This phenomenon can be exploited for example in the following fields:
  • CDs ensure the controlled release of the active agents.
  • CDs and their complexes can be prepared in pure and standard form, which can be characterized easily, this statement is not true for CD-derivatives, due to several reasons coming from the chemical structure of CDs.
  • the number of the hydroxyl groups, standing in position 2, 3 and 6 in each glucopyranose unit, is 6x3 in ⁇ -CDs, 7x3 in ⁇ -CD and 8x3 in ⁇ -CD, which are mainly randomly substituted, depending on the reaction conditions (the unsubstituted ⁇ -CDs, ⁇ -CDs and ⁇ -CDs are summarized in the name "basic-CDs" in the following parts).
  • the aggregate substitution is described by the term "degree of substitution" (DS).
  • This parameter relates to the average number of substituted hydroxyl groups per CD molecule.
  • the monosubstituted derivatives can be prepared as a relatively uniform product (but in this case the yield is low) or a uniform product can be prepared, if all the hydroxyl groups are modified. If a substitution degree is aimed to achieve being between the above mentioned two extremities, the product will be the mixture of isomers (wherein several hundred isomers may present). The determination of the ratio of the isomers has not been solved yet because there is no such high performance analytical method which ensures the separation of such a great number of materials having very similar physical-chemical properties. Further analytical problem can be the quantification of the unsubstituted basic-CDs being present in small amount in the product.
  • HPLC high pressure liquid chromatography
  • GC gas chromatography
  • CE capillary electrophoresis
  • Aminopropyl silica column is one of the most frequently applied columns for analysis of carbohydrates and CDs using acetonitrile- water eluent.
  • the authorized method No. USP26-NF21 requires this method for determination and qualification of ⁇ -CDs and for the examination of other impurities (USP26-MF21, Official Monograph, pp. 2698-2699).
  • This normal phase HPLC method is suitable for characterization of hydroxypropyl- substituted ⁇ -CD-derivatives and for the separation of ⁇ -CD contamination (G. Liu, D. M. Goodall, J.S. Loran; Chirality, 5, p. 220 , 1993; N. Rabearimonjy, S. Ounnar, M. Righezza, M.
  • Reverse phase chromatography can be applied with success for the characterization of methylated CDs where octyl (C8) or octadecyl (C18) stationary phase is applied
  • octyl (C8) or octadecyl (C18) stationary phase is applied
  • Y. Kubota T. Tanimoto, S. Horiyama, K. Koizumi: Carbohydr. Res., 192, p. 159, 1989; G. Schomburg, A. Deege, H. Hinrichs, E. H ⁇ binger: J. High Resolut. Chromatogr., 15, p. 579, 1992; I.
  • the novelty of the stationary phases according to the present invention comes from the special groups covering the surface of the carrier (support).
  • These covalently bonded groups contain a nitrogen-containing, preferably carbamide-containing spacer part (on the one hand, having a distance keeping function and, on the other hand, being responsible for the selectivity) and connected to this part a group having an aromatic part (which is responsible for the strength of the selective connection, called as selector in the following parts).
  • the interaction of these two parts improves the efficiency of the separation in an unexpected manner.
  • Silica-based adsorbents are known, too, wherein an aromatic group, for example a nitrophenyl group, is bonded to a spacer consisting of propyl group and amide group [see Si- Nitrobenzamide (R64030B) mentioned in catalogue "Synthesis 2003-04" of SiliCycle Inc., wherein a 3-(4-nitrobenzamido)propyl group is attached to the silica].
  • this adsorbent is proposed for immobilization of enzymes having essentially different structure, and not for separation.
  • an adsorbent is proposed for immobilization of cationic detergents, which contains 3-carbamidopropyl group (product No.: R670030B).
  • the separation of the components of statistically substituted CD-derivatives can be solved much better by the use of the stationary phases according to the present invention, acknowledged here the fact that the absolute separation of the isomers is theoretically impossible.
  • the fingerprint chromatograms obtained by the separation of the main homologue/isomer groups can be applied for quick and simple analysis, which can be useful for identification of batches, carrying out stability assays, optimization of technological steps etc.
  • the stationary phases according to the present invention can be applied effectively both analytical and preparative purposes.
  • the first subject of the present invention is providing a chromatographic stationary phase for the separation of cyclodextrines and derivatives thereof and their impurities, which can be characterized by the general formula M-BG-SP-SE, wherein M is a chromatographic carrier matrix; BG is a group of general formula (Z) 3 Si, wherein Z is, independently from each other in every occurrence, hydroxyl, C ⁇ - 6 alkyl, C ⁇ - 6 alkoxy group or a valence bond binding to M; SP is C ⁇ - 6 alkylene, optionally substituted with one or more substituents selected from the following group: -NH 2 , -COOH and -SO 3 H and salts thereof and - [N(R ⁇ ) 3 ] + X " , wherein Ri is, independently from each other in every occurrence, C ⁇ - 6 alkyl or hydroxy(C ⁇ -6 alkyl), X " is negatively charged counter ion; SE is -W-R 2 -A1".
  • the X " counter ion is a monovalent anion, e.g. halogenide ion.
  • the meaning of C5- 1 6 aromatic group embraces the phenyl, naphthyl, anthryl and phenanthryl, where the phenyl is preferred. Further groups belonging to this meaning are the groups derived from pyrene and fluoroanthrene. Preferred halogen atoms are the chloro and bromo atoms.
  • M is a silica (silicon dioxide) based chromatographic carrier
  • SP is propylene group
  • W is -NH-CO-N-
  • Ar is phenyl or naphthyl optionally substituted with -NO 2 or -OH
  • the preparation of stationary phases according to the present invention can be carried out by processes which are well-known from the prior art. Basically the following two methods are used in the practice for the preparation of chromatographic stationary phases. 1. Covering the surface of the carrier with an appropriate silylating agent containing a reactive group suitable for the formation of the spacer, which reactive group is able to react with an appropriate group of the selector, which latter is an aromatic ring system in this case. 2. The silylating agent used in the previous method is coupled at first with the aromatic compound and the surface of the silica is covered with this derivative. In our present work we applied the second method, because this makes possible the homogeneous formation of a chemically modified surface.
  • Method A) disclosed in example 1 can be characterized by the reaction of an aromatic amine having the appropriate substituents and basicity with a 3-isocyanatoalkyl- trialkoxysilane, preferably 3-isocyanatopropyl-triethoxysilane (see Reaction Scheme 1 with the preferred compounds). In general, this reaction is fast, quantitative and selective with respect to the amino group, even in that case, too, when the aromatic group contains hydroxyl substituent.
  • Method B) disclosed in example 2 can be applied when electron-withdrawing substituents are connected to the aromatic ring, therefore the basicity of the amino group is decreased which results in reacting with isocyanates slowly and with poor yield.
  • Silylating agents prepared by the above mentioned methods are reacted with the appropriately pretreated carrier (preferably silica) preferably in an aprotic solution (preferably in toluene), at a temperature between 50 °C and the boiling point of the solution (for instance at 110 °C), for 10 to 36 hours, preferably for apprx. 24 hours.
  • the pretreatment is carried out preferably for apprx.
  • BG'-SP-SE general formula BG'-SP-SE
  • BG' is a group of general formula (Z') 3 Si, wherein Z' is, independently from each other in every occurrence, leaving group or C ⁇ - 6 alkyl
  • SP is C ⁇ -6 alkylene, optionally substituted with one or more substituents selected from the following group: -NH 2 , -COOH and -S0 3 H and salts thereof and - [N(R ⁇ ) 3 ] + X " , wherein Ri is, independently from each other in every occurrence, C ⁇ - 6 alkyl or hydroxy(C ⁇ -6 alkyl), X " is negatively charged counter ion
  • SE is -W-R 2 -Ar, wherein W is -NH-CO-NH-, -NH-CO- or -NH-CO-O-, R 2 is C ⁇ -6 alkylene, optionally substituted with one or more substituents selected from the following group: -NH 2 , -COOH and - SO
  • the phrase "leaving group” denotes a group which split off during the reaction of the silylating agent and the carrier, thus the silylating agent is able to react with the silanol groups of the carrier.
  • Halogens and C1-6 alkoxy groups are preferred groups, chloro atom, methoxy and ethoxy groups are more preferred.
  • Preferred silylating agents are the covering groups of the above mentioned preferred stationary phases with the further note that preferred leaving group is the ethoxy group.
  • Example 1 Method A) 0.420 g (2,64 mmol) 3-hydroxy- ⁇ -naphthylamine was dissolved and stirred in 10 cm " water and peroxide free tetrahydrofurane. Thereafter 0.653 g (2.64 mmol) 3- isocyanatopropyl-triethoxysilane was slowly added to the above stirred solution at 25 °C. The reaction mixture was stirred at room temperature for further 30 minutes under inert gas atmosphere. The solution was clarified with active carbon, if necessary, and the tetrahydrofurane was removed by distillation under reduced pressure.
  • Example 2 Method B 0.442 g (2.0 mmol) 3 -Aminopropyltri ethoxy silane was dissolved in 10 cm 3 tetrahydrofurane. Thereafter 0.328 g (2.0 mmol) of 4-nitro-phenylisocyanate was dissolved in 10cm 3 tetrahydrofurane and this solution was slowly added to above amine solution. The tetrahydrofurane was water and peroxide free in both solutions. The reaction mixture was stirred at room temperature for further 30 minutes under inert gas atmosphere. The solution was clarified with active carbon, if necessary, and the tetrahydrofurane was removed by distillation under reduced pressure.
  • Example 3 Preparation of silica based stationary phase Silylating agents prepared according to Example 1 or 2 were reacted with pretreated silica in toluene at 110 °C for 24 hours (the silica was dried before for 24 hours in vacuum at 110 °C).
  • the silica based stationary phases prepared by the above method can be packed into HPLC-column after filtration, washing and sedimentation
  • the following stationary phases were prepared by this method Stationary phase 3a, where the carrier is Hypersil Silica-HS (5 ⁇ m) and the covering group derives from compound la Stationary phase 3b, where the carrier is Hypersil Silica-HS (5 ⁇ m) and the covering group derives from compound lb Stationary phase 3 c, where the carrier is Hypersil Silica-HS (5 ⁇ m) and the covering group derives from compound 2a Stationary phase 3d, where the carrier is Hypersil Silica-HS (5 ⁇ m) and the covering group derives from compound 2b
  • FIG. 1 illustrates the retention times of unsubstituted basic-CDs on different stationary phases, using methanol-water 30 70 v/v as eluent
  • RI refraction index
  • the detection was carried out with a refraction index (RI) detector and the flow rate was 1,0 cmVmin
  • One grade on the time axis represents 1 minute in all chromatograms
  • the upmost chromatogram illustrates a separation which was performed on a stationary phase having 4-nitrophenyl-amide selector which embodies the closest structure known from the prior art (The preparation of this stationary phase was carried out according to Examples 2 and 3, among the same conditions applied for the preparation of the other stationary phases In case of the other Figures this type of column was applied when a column having 4- nitrophenyl-amide selector is mentioned )
  • Chromatograms 2 to 5 illustrate separations performed on different stationary phases (containing urea group) according to this invention (the stationary phases are as follows 3d, 3a, 3
  • Figures 2a to 2c illustrate chromatograms of acetyl- ⁇ -CD performed on different columns.
  • Figure 2b Stationary phase Nucleosil C18 (Macherey-Nagel GmbH & Co.), 5 ⁇ m, 4.6x250 mm, eluent: 50 v/v% methanol, flow rate: 1.0 cmVmin, temperature: 30°C.
  • FIG. 2c Stationary phase: stationary phase 3b according to this invention containing 2- naphthyl-urea selector, 5 ⁇ m, 4x250 mm, eluent: 58 to 42 v/v% methanol-water, flow rate: 1.0 cm 3 /min, temperature: 30°C. It can be seen that the resolution is significantly better on the stationary phase according to the invention (the peaks are separated at the base-line, i.e. the overlapping is less), moreover, as an unexpected effect, the contaminant basic-CD ( ⁇ -CD) can be found at the start of the chromatogram (it elutes with small elution time).
  • ⁇ -CD contaminant basic-CD
  • Figures 4a and 4b illustrate comparative chromatograms for the determination of composition of hydroxypropyl- ⁇ -CD.
  • Figure 4a Stationary phase: Nucleosil with nitrophenylpropyl selector (Macherey-Nagel GmbH & Co.), 5 ⁇ m, 4x250mm, eluent: 35 v/v% acetonitrile, flow rate: 1.0 cn ⁇ Vmin, temperature: 30°C.
  • Figure 4b Stationary phase: stationary phase 3c according to invention having 4-nitrophenyl-urea selector, 5 ⁇ m, 4x250 mm, eluent: methanol-dioxane- water 35:5:60, flow rate: 1.0 cm 3 /min, temperature: 30°C.
  • FIG. 5a and 5b illustrate the influence of the substituents in case of two selectors according to the invention through the examination of randomly methylated ⁇ -CD.
  • FIG. 5b Stationary phase: stationary phase 3b according to invention having (2-naphthyl)urea selector, 5 ⁇ m, 4x250 mm, eluent: acetonitrile-water 26:74, flow rate: 1.0 cm 3 /min, temperature: 30°C. It can be seen that the presence of the hydroxyl substituent improved the separation.
  • Figures 6a and 6b illustrate the influence of the N-containing group bonded to the spacer in case of two selectors according to the invention through the examination of randomly methylated ⁇ -CD.
  • Figure 6 a Stationary phase: 4-nitrophenylamide containing selector, 5 ⁇ m, 4x250 mm, eluent: acetonitrile-water 26:74, flow rate: 1.0 cm 3 /min, temperature: 30°C.
  • Figure 6b Stationary phase: stationary phase 3c according to invention having 4-nitrophenyl-urea selector, 5 ⁇ m, 4x250 mm, eluent: acetonitrile-water 26:74, flow rate: 1.0 cm 3 /min, temperature: 30°C.
  • the unexpected effect observed in Figures 2a to 2c is proven by these chromatograms: a minimal change in the structure caused a substantial increase in the effectiveness of the separation (much more peaks which are much sharper).
  • Figure 7 demonstrates that the stationary phase according to the invention can be applied effectively for the examination of the degradation of randomly methylated ⁇ -CD.
  • Figure 7a chromatogram of randomly methylated ⁇ -CD containing no contamination. Eluent: acetonitrile-water 20:80, flow rate: 1.0 cirrVmin, temperature: 30°C.
  • Figure 7b chromatogram of randomly methylated ⁇ -CD artifically degraded with hydrochloric acid. Eluent: acetonitrile-water 26:74, flow rate: 1.0 cm 3 /min, temperature: 30°C.

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

L'invention concerne une phase stationnaire chromatographique caractérisée par la formule M-BG-SP-SE. Dans cette formule, M désigne une matrice de support chromatographique; BG désigne un groupe de (Z)3Si, Z désignant un groupe hydroxyle, alkyle, alcoxy ou une liaison se liant à M; SP désigne un alkylène C1-6 éventuellement substitué, SE désigne -W-R2-Ar, W désignant -NH-CO-NH-, -NH-CO- ou -NH-CO-O-, R2 désignant un alkylène C1-6 éventuellement substitué, Ar désignant un groupe aromatique éventuellement substitué C5-16 contenant des atomes O, N et S; à la condition que, si SP désigne propylène et que Ar désigne phényle éventuellement substitué par -NO2, alors W est différent de -NH-CO-. L'invention concerne également l'utilisation des phases stationnaires susmentionnées, les composés de départ appliqués dans la préparation de la surface caractéristique des phases stationnaires susmentionnées et l'utilisation de ceux-ci.
PCT/HU2005/000043 2004-04-30 2005-04-29 Phase stationnaire chromatographique pour une separation de cyclodextrine et de derives de celle-ci Ceased WO2005105257A1 (fr)

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HU0400900A HUP0400900A2 (en) 2004-04-30 2004-04-30 Chromatogrphic stationary phase for separation of cyclodextrine derivatives thereof

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4322310A (en) * 1980-06-12 1982-03-30 Uop Inc. Chiral supports for resolution of racemates
US4512898A (en) * 1982-09-30 1985-04-23 Sumitomo Chemical Company, Limited Packing materials for chromatographic use and a method for analysis of an enantiomer mixture using the same
US4781977A (en) * 1985-10-31 1988-11-01 Sanraku Incorporated Cyclodextrin adsorbing-material
US4867884A (en) * 1988-02-24 1989-09-19 The United States Of America As Represented By The Secretary Of Agriculture Separation of cyclodextrins by affinity chromatography
US4997966A (en) * 1988-12-20 1991-03-05 Ciba-Geigy Corporation Enantiomeric silanes, modified packing material, and use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4322310A (en) * 1980-06-12 1982-03-30 Uop Inc. Chiral supports for resolution of racemates
US4512898A (en) * 1982-09-30 1985-04-23 Sumitomo Chemical Company, Limited Packing materials for chromatographic use and a method for analysis of an enantiomer mixture using the same
US4781977A (en) * 1985-10-31 1988-11-01 Sanraku Incorporated Cyclodextrin adsorbing-material
US4867884A (en) * 1988-02-24 1989-09-19 The United States Of America As Represented By The Secretary Of Agriculture Separation of cyclodextrins by affinity chromatography
US4997966A (en) * 1988-12-20 1991-03-05 Ciba-Geigy Corporation Enantiomeric silanes, modified packing material, and use thereof

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HUP0400900A2 (en) 2006-09-28

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