CN1865251A

CN1865251A - Leucogen diastereoisomeride structural confirmation and determination method

Info

Publication number: CN1865251A
Application number: CN 200510022543
Authority: CN
Inventors: 张宏业
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-12-23
Filing date: 2005-12-23
Publication date: 2006-11-22

Abstract

This invention discloses a structure identification and determination method of lekejun diastereoisomer, comprising: likejun(alias:leucogen) has for diastereoisomers which keep transforming in the solution until balancing, so they can be determined quantitatively by the sum of the four peak areas. The chromatography condition and system adapted test of the likejun determination are: octadecyl silicane gel as bulking agent, different proportions of water-acetonitrile-glacial acetic acid or water-acetonitrile as mobile phase, column temperature 30Deg C, detection wavelength 210nm. This invention is characterized of overcoming the content determination disadvantage in the current likejun agent quality testing method with simple operation, higher accuracy, sensitivity and reliability than the original method.

Description

The structural confirmation of leucogen diastereomer and measuring method

Invention field;

The present invention relates to field of pharmaceutical preparations, disclose the structural confirmation and the measuring method of leucogen diastereomer.

Background technology:

(former name: the leucogen) chemistry is by name: 2-(α-phenyl-α-ethoxycarbonyl-methyl) thiazolidine-4-carboxylic acid is drug for increasing white cells to leucogen.Structural formula is seen Fig. 1, and Fig. 1 is the leucogen structure.

Molecular formula: C ₁₄H ₁₇O ₄NS

Molecular weight: 295.36

Leucogen is used to white corpuscle, the thrombocytopenia of preventing and treating a variety of causes to cause, clinically is mainly used in the white corpuscle that tumor chemoradiotherapy, endocrine disturbance medicine etc. cause, the treatment of thrombocytopenia.Because of leucogen has three chiral carbon, so a plurality of four diastereomers are arranged.And national drug standards analytical procedure is a nonaqueous titrations at present, also there is research to use directly and the report of Indirect UV spectrophotometry, first derivative spectrophotometry, indirect atomic absorption method, single-sweep oscillographic polarography, portable injection chemiluminescence method, flow injection spectrophotometric etc. in addition, but these methods all lack specificity, can not reflect the medicine inner quality really, might influence the result of treatment of medicine, the popularization of clinical use is restricted.

The invention provides a kind of existing leucogen textural defect that overcome, the leucogen diastereomer has been carried out structural confirmation, and measuring method is provided.

Goal of the invention

Purpose of the present invention at first is that the leucogen diastereomer has been carried out structural confirmation, and secondly, another object of the present invention provides measuring method.

Summary of the invention

The present invention finds that in research process national drug standards analytical procedure mensuration content is the leucogen sample more than 99%, measure with the HPLC method immediately after being mixed with solution, a plurality of peaks are arranged in the color atlas, after solution is placed, each chromatographic peak area can change, and (see Fig. 2, Fig. 2 is a leucogen reference substance color atlas; See Fig. 3, LC-MS color atlas when Fig. 3 reaches balance for the placement of leucogen solution).For this reason, at first carried out the structural research of leucogen under the solution state and related substance thereof; In order to study the reason that above-mentioned a plurality of peaks produce under solution state, in this research integrated application HPLC-MS/MS, HPLC-DAD, HPLC/ ¹Research methods such as H-NMR, the phenomenon that the conclusive evidence leucogen exists diastereomer to transform mutually in solution, and set forth the theoretical foundation of its transformation with quantum chemistry calculation result.For the HPLC method of setting up leucogen and related substance thereof is measured provider's science of law foundation.

1. instrument and reagent

Day island proper Tianjin LC-10A VP of company type highly effective liquid phase chromatographic system, SPD-10A VP type UV-detector; The intelligent information Graduate School of Engineering N2000 of Zhejiang University chromatographic working station; The U.S. Finnigan company's T SQ type Quantum Ultra AM type LC-MS/MS combined instrument, electric spray ion source (ESI) and Xcalibur 2.1 data systems.Agilent 1100 Series high performance liquid chromatographs, Agilent 1100 Series diode matrix detectors, the Inova-500LC-NMR combined instrument of Chemstations.Varian company, wherein high performance liquid chromatograph is Prostar-230.Gaussian03 software is used for quantum chemistry calculation.

2.HPLC-MS/MS

Study with HPLC-MS/MS: the mass scanning scope: m/z 100-300, no matter be just to have prepared or placed to reach equilibrated solution, the mass spectral m/z of one-level at four peaks, back (LC-MS color atlas when Fig. 3 reaches balance for the placement of leucogen solution) is 295.76, all with leucogen [M+H] ⁺Corresponding [see Fig. 4, Fig. 4 is leucogen (back four a peaks) one-level mass spectrum]; Their second order ms fragment peak is also identical [sees Fig. 5, Fig. 5 is leucogen (back four peaks) second order ms figure], the m/z of fragmention abundance maximum is 131.80, detect gained color atlas and detect the chromatographic peak shape of gained and ratio all identical [see Fig. 6, Fig. 6 places for leucogen solution and selects fragmention (m/z131.80) to scan color atlas after reaching balance] with this fragmention with parent ion peak.

3.HPLC-DAD

Use HPLC-DAD, detect under the wavelength, the ultraviolet spectrogram unanimity (see Fig. 7, Fig. 7 is leucogen color atlas and spectrogram) at chromatographic peak point place at 210nm.Peak upward slope, summit and 3 different sites of peak descending that write down each component compare in that the spectrum of 200-400nm wave band is superimposed, the spectrogram at four peaks is identical, detects through DAD, and the three-dimensional collection of illustrative plates at each peak is similar (sees Fig. 8, Fig. 8 is the three dimensional chromatogram of leucogen), the purity at peak is all very high.

4.HPLC- ¹H-NMR

Use HPLC- ¹H-NMR studies because solvent peak suppresses not exclusively, the embedding of water peak the hydrogen on No. 2 carbon.1% propionitrile is arranged in the acetonitrile, influential to the hydrogen on No. 6 carbon.Fig. 9 is the reference substance leucogen ¹The H-NMR spectrum is isolated number and the chemical shift comparison that four peak collection of illustrative plates carry out hydrogen with it and HPLC-NMR, finds the collection of illustrative plates basically identical.Difference is that the chemical shift of the hydrogen on isomer 3 and No. 4 chiral carbon changes, and can confirm that four components have identical structure, and just sterie configuration is different, and Figure 10 be that four component peaks of leucogen (1-4) hydrogen is composed (HPLC- ¹H-NMR) chiral carbon links to each other, and (3-H, 4-H) spectrum is amplified collection of illustrative plates to hydrogen.

5. diastereomer

Above-mentioned three kinds of coupling techniques can confirm that these four peaks produce for its diastereomer.Three chiral carbon are arranged in the leucogen molecule, and wherein No. 5 chiral carbon are defined as the R configuration in synthetic, also have 3,4 to be two chiral carbon, and common property is given birth to four diastereomers (3S, 4R, 5R; 3R, 4R, 5R; 3R, 4S, 5R; 3S, 4S, 5R sees Figure 11, Figure 11 is four diastereomers of leucogen).

6. quantum chemistry calculation (Quantum Calculation)

For four diastereomers studying leucogen transform reason, we calculate respectively in gas phase and acetonitrile solvent four kinds of configurations with Gaussian03 software.Method of calculation: at first use hf/6-31g (d) (hf method, use 6-31g (d) base group) carries out geometric configuration optimization, optimize on the good structure at hf/6-31g (d) then and carry out mp2/6-31g (d) (mp2 method, use 6-31g (d) base group) unit calculating, obtain the energy following (seeing Table 1) of configuration separately:

Four diastereomer ground state energies of table 1 leucogen and relative energy (is unit with a.u.)

	3R4R5R	3S4R5R	3R4S5R	3S4S5R
	3R4R5R	3S4R5R	3R4S5R	3S4S5R	In gas phase (in the gas phase)
E △E △E(kcal/mole)	-1291.49870 0.00231 1.45	-1291.49546 0.00555 3.48	-1291.50101 0 0	-1291.49880 0.0022l 1.39	In gas phase (in the gas phase)
E △E △E(kcal/mole)	-1291.49870 0.00231 1.45	-1291.49546 0.00555 3.48	-1291.50101 0 0	-1291.49880 0.0022l 1.39	In acetonitrile solvent (ε in the acetonitrile solution=36.64)
E △E △E(kcal/mole)	-1291.53012 0.00196 1.23	-1291.52625 0.00583 3.66	-1291.53208 0 0	-1291.53014 0.00194 1.22

Show by above-mentioned quantum chemistry calculation: the stability order of leucogen diastereomer is: 3R4S5R＞3S4S5R＞3R4R5R＞3S4R5R.In gas phase, the 3R4S5R energy is minimum, its stable best in isomer.Under solid state, leucogen should mainly exist with the 3R4S5R form, and this has just explained that leucogen why is a reason based on a kind of isomer at the dissolving initial stage.When leucogen is dissolved in the acetonitrile solution, the energy difference of four diastereomers obviously diminishes, so it is easy to transform in solution.Experiment shows: dissolve leucogen with acetonitrile, treat that diastereomer transforms balance after, use N ₂Solution is dried up, get solid sample and dissolve back injection liquid chromatography, find color atlas and dissolve preliminary phase seemingly, this has just proved the existence that transforms in the equilibrium process, and experimental result conforms to quantum chemistry calculation.According to concerning between diastereomer energy height and the outflow chromatographic peak area size, can determine that four chromatographic peaks are followed successively by 3S4S5R, 3R4R5R, 3R4S5R, 3S4R5R, Theoretical Calculation is confirmed and transforms for four diastereomers theoretical foundation is provided.

Diastereomer is running balance in solution, HPLC-NMR studies show that: separate the individual isomer that obtains through liquid chromatography, rest in the trace probe, for the nuclear magnetic resonance measuring sampling, after adding up 12 hours, discovery is the peak value of mixture in collection of illustrative plates, illustrates that isomer ceaselessly transforms in solution, up to balance.

Have only five peaks on the solution initial stage color atlas, peak 1 is an impurity, through the area normalization fractional analysis: impurity peaks 1 accounts for 3.4%, four peaks, back are not isomorphism type of four kinds of leucogen molecules, 2,3,4,5 account for 3.0%, 1.0%, 92.0%, 0.6% respectively, can transform mutually in solution, available four peak area sums are come quantitatively.

7. measuring method

Strong to close silica gel be weighting agent to the test of chromatographic condition and system suitability with octadecylsilane; Water-acetonitrile in varing proportions-Glacial acetic acid or water-acetonitrile are moving phase; Column temperature is 30 ℃; The detection wavelength is 210nm.

Assay method is got the about 10mg of leucogen, and accurate the title decides.Put in the 100ml measuring bottle, add that acetonitrile or phosphate buffered saline buffer (pH6.8)-acetonitrile is an amount of, supersound process makes the leucogen dissolving, is diluted to scale with moving phase, shakes up, and filters, and precision is measured subsequent filtrate 20 μ l and injected liquid chromatograph, the record color atlas; It is an amount of that other gets the leucogen reference substance, accurate claims surely, adds that acetonitrile or phosphate buffered saline buffer (pH6.8)-acetonitrile is an amount of, and supersound process makes the leucogen dissolving, makes the solution that contains 100 μ g among every 1ml with the moving phase dilution, measures with method.Calculate with peak area (four diastereomer peak area sums) by external standard method, promptly.

Claims

1, it is characterized in that leucogen (former name: the leucogen) form by four diastereomers.

2, four diastereomers of the leucogen of claim 1 ceaselessly transform in solution, to reaching running balance.

3, claim 1 and four diastereomers of leucogen of 2 ceaselessly transform in solution, reach running balance, and the area sum at its liquid chromatography peak is constant, so available four peak area sums are carried out assay.

4, the chromatographic condition measured of claim 1 and 2,3 leucogen and system suitability test are to be weighting agent with octadecylsilane chemically bonded silica; Water-acetonitrile in varing proportions-Glacial acetic acid or water-acetonitrile are moving phase; The detection wavelength is 210nm.