CN105585617A - Norvancomycin derivatives and preparation purifying method thereof - Google Patents

Abstract

The invention relates to a preparation purifying method of norvancomycin and norvancomycin derivatives. The method is an HPLC method in which a mobile phase does not contain salt. The invention further relates to the norvancomycin derivatives (compounds 1-3) obtained through the method. The invention further relates to pharmaceutical application of the norvancomycin derivatives (compounds 1-3).

Description

Norvancomycin derivative and prepare purification process

Technical field

The invention belongs to pharmaceutical chemistry field, particularly, the present invention relates to Norvancomycin derivative and preparation thereofPurification process.

Background technology

Norvancomycin is a class glycopeptide antibiotics, in nearly 40 years of commercial applications of China, clinical mainBe applied to the severe infections diseases such as treatment endocarditis, osteomyelitis. Norvancomycin is mainly effective to gram-positive bacteria,Particularly methicillin-resistant staphylococcus aureus, methicillin-resistant MRSE and penicillin-fast enterobacteria, pneumoniaStreptococcus and clostridium. The generation bacterium of Norvancomycin be nineteen fifty-nine Lie group etc. isolated from Soils of GuizhouActinomyces ten thousand-No. 23, afterwards called after Amycolatopsis orientalis (Amycolatopsisorientalis). NorvancomycinHave and another glycopeptide compound vancomycin (VCM) basic similarly chemical constitution and biologically active, they are by oneSeven peptide parents and the disaccharide substituting group being made up of glucose-amino sugar of individual three ring compositions, difference is first ten thousandThe N-end of ancient mycin lacks a N-methyl (Fig. 1) compared with vancomycin. In document, form the amino acid of seven peptides respectively with AA-1Represent to AA-7, five aromatic rings are from A-E number consecutively, and in addition, the numbering of three large rings is with aromatic rings numbering composition A-B, C-O-D and D-O-E, resolve for convenient purification laminate structures, and the application will continue to use the method for numbering serial of document.

In early days, the initial operational phase of vancomycin, finds in clinical use procedure that it causes multiple negative reaction, as renal toxicityWith ototoxicity etc., possible reason is that its purity is low, the reason that impurity content is high. Subsequently, people use high performance liquid chromatography(HPLC), Capillary Electrophoresis (CE) and liquid phase mass spectrum-mass spectrum technology used in conjunction (LC-MS/MS) enter vancomycin related substanceThe research that the system of going is deep, has therefrom found that eight impurity are respectively dechlorination vancomycin A and B, removes two chlorine vancomycins, ten thousandAncient mycin process catabolite CDP-I_MAnd CDP-I_m, removing disaccharide vancomycin, sugared vancomycin and go first mould through the ages deaminizesElement. Further pharmacologically active experimental studies have found that, contains CDP-I_MAnd CDP-I_mVancomycin medicine can cause clinical treatment loseLose, and the antibacterial activity of three similar things of dechlorination vancomycin has reduced by 2 to 10 times than vancomycin, the mould through the ages of sugar deaminizesThe antibacterial activity in vivo of element also reduces greatly, and MIC is about five times of vancomycin. Therefore, " European Pharmacopoeia " and " American Pharmacopeia "To deaminize-, go disaccharide base-, and demethyl vancomycin is defined as three major impurities of vancomycin raw material, and specifies itSingly must not mix and exceed 4%, always must not mix and exceed 7%.

Comparatively speaking, Norvancomycin is because its impurity component is indefinite, and therefore, Chinese pharmacopoeia regulation is long-pending with peak areaDivide and calculate, always must not mix and exceed 12%, single mixing is less than 4%. Therefore, need more efficiently purifying and qualification NorvancomycinMethod, meanwhile, for related substance constituent structure in clear and definite Norvancomycin bulk drug, can also be to NorvancomycinBulk drug carries out the more chemical constitution study of system, excavates the analogue of its new construction.

Summary of the invention

The present invention separates and obtains three glycopeptide compounds from Norvancomycin bulk drug, by means of 1D, and 2DNMRDetermine three compound structures with the method such as HRESIMS, be Norvancomycin analog, comprise a C-O-D open loop andTwo desaccharification radical derivatives.

The HPLC that first the present invention relates to a kind of Norvancomycin and derivative thereof prepares separation method, described methodFor:

(1) mobile phase: methyl alcohol: 0.05～0.2% aqueous formic acid,

(2) methyl alcohol rises to 70% gradually from 10% in gradient elution step: 30～50min,

(3) the Norvancomycin bulk drug aqueous solution is crossed to chromatographic column, flow velocity is 1.0～5.0ml/min, detects wavelength280nm, described Norvancomycin bulk drug is preferably Norvancomycin hydrochloride bulk drug,

(4) collect main peak product and obtain Norvancomycin sterling, it is derivative that collection secondary peak product obtains NorvancomycinThing.

In described method, chromatographic column is preferably C18 chromatographic column or phenyl post.

The invention still further relates to by described method and separate the Norvancomycin derivative obtaining, described NorvancomycinDerivative is: compound 1 (structure is as shown in the formula shown in 1), compound 2 or 3 (structure is as shown in the formula shown in 2,3),

R=β-D-glucosyl, formula 2;

R=H, formula 3

The invention still further relates to described compound 1-3 in the application of preparing in medicine, described medicine is resisting gram-positive bacteriaMedicine, described gram-positive bacteria is preferably: methicillin-resistant staphylococcus aureus, methicillin-resistant epidermis grape ballBacterium, penicillin-fast enterobacteria, streptococcus pneumonia and clostridium.

Brief description of the drawings

Fig. 1 compound 1-3, the structure of Norvancomycin and vancomycin, the HMBC of compound 1 is relevant with the curved arrow of dotted lineHead represents.

The HPLC chromatography figure of Fig. 2 Norvancomycin bulk drug.

2A, the HPLC collection of illustrative plates of use " Chinese pharmacopoeia " method;

2B, a in A figure, b, the uv-spectrogram stacking chart of c and Norvancomycin;

2C, HPLC collection of illustrative plates after method optimization;

2D, impurity 1,2 in C figure, 3,4 and the uv-spectrogram stacking chart of Norvancomycin.

(parent ion is [M+H] to Fig. 3 Norvancomycin⁺M/z1434, Fig. 3 A) and compound 1 (parent ion is [M+H]⁺m/Z1452, Fig. 3 C) ESIMS/MS figure and both lytic pathways (3B is Norvancomycin, and 3D is compound 1).

Detailed description of the invention

Norvancomycin hydrochloride is provided by North China pharmacy (Hebei China Shijiazhuang City);

Vancomycin standard items are purchased from National Institute for Food and Drugs Control;

Chromatographic grade acetonitrile and methyl alcohol are purchased from Adamas company (Chinese Shanghai);

DMSO-d₆Produce (Cambridge, MA, USA.) by CambridgeIsotope company;

All other reagent (formic acid, phosphoric acid, hydrochloric acid, trifluoroacetic acid, ammoniacal liquor (being 25～28% containing ammonia mass fraction),Triethylamine, oxolane) be chemical grade.

The instrument using, comprises P-2000 polarimeter (JASCO, Tokyo, Japan), JASCOP-650 uv-spectrophotometricMeter (JASCO), SYS600MHz nuclear magnetic resonance spectrometer (PaloAlto, CA, USA), FinniganLTQXT ion trap mass spectrometer(Finnigan, SanJose, CA), FinniganLTQ track ion trap spectrometer (Finnigan), Shimadzu LC-20 liquid chromatogramInstrument (Shimadzu, Japan).

Nuclear-magnetism assay method:

All samples¹H and¹³CNMR all measures on the SYS600MHz nuclear magnetic resonance spectrometer with cryogenic probe, and solvent is deuteratedDMSO。¹H and¹³CNMR chemical shift is all used TMS (δ=0.00ppm) and DMSO-d₆(δ=39.5ppm) solvent does interior mark.

Preparation and the separation and purification of embodiment 1 compound 1～3

First, we have used in " Chinese pharmacopoeia " Norvancomycin bulk drug HPLC method to carry out analytic sample (to see figure2A and 2B), find a main peak that is 21.44min in retention time, integral area accounts for 95.5% of all peak area summations,Go out peak position according to Norvancomycin in pharmacopeia and be set to 18～22min, infer that this material is exactly Norvancomycin, in addition, asShown in Fig. 2 A, also there are at least three impurity peaks that integral area is less in retention time before and after this main peak, and retention time is dividedBe not 10.31 (a), 12.30 (b), 14.73 (c) min, their online ultraviolet absorption peak type also with Norvancomycin phase classSeemingly, as shown in Figure 2 B. But, owing to having used phosphate in the method, and because in the rear sample of preparation process or preparationPhosphatic residual meeting severe jamming Mass Spectrometer Method, therefore, this method is not suitable for sample to carry out liquid chromatograph mass spectrography(LC-MS)。

We,, in order tentatively to obtain the corresponding informations such as these derivative impurity molecule amounts by means of LC-MS, have set up a set ofThe salt-free HPLC method of mobile phase. In the time that we use methyl alcohol-0.1% aqueous formic acid to carry out HPLC analysis, find phaseThe main peak of answering and impurity peaks can obtain good peak type, and between impurity and impurity, between impurity and main peak, also haveGood separating degree, as shown in Figure 2 C, derivative impurity 1,2,3,4 peak areas account for respectively 3.4%, 1.91% of the gross area,0.96%, and 2.02%, main peak is 87.97%.

(1) the HPLC analytical method of optimizing is as follows:

AgilentC₁₈Chromatographic column (150 × 4.6mmi.d., 5 μ m),

Mobile phase: methyl alcohol: 0.1% aqueous formic acid,

Gradient elution step: in (1) 40min, methyl alcohol rises to 70% gradually from 10%.

1.0mlmin^-1Flow velocity, 280nm wavelength detects, and column temperature and detection cell temperature are room temperature (25 DEG C), 3.0mgml^-1The Norvancomycin bulk drug aqueous solution, sample size is 20 μ l.

(2) the HPLC preparation method who optimizes is as follows:

YMC-Pack phenyl post (150 × 20mmi.d., 20 μ m, YMCCo., Ltd, Kyoto, Japan), flows and matchesPut consistent with analysis experiment with gradient elution method.

Flow velocity is 5.0mlmin^-1, single elution time is 120min.

100mgml^-1The Norvancomycin bulk drug aqueous solution, single sample size is 300 μ l.

Utilize the salt-free HPLC method of optimizing to carry out Standard multi-component to each impurity, through vacuum distillation recovered solventAfter, from 5.0g Norvancomycin bulk drug, separate and obtained compound 1 (5.0mg), compound 2 (5.0mg), compound 3(1.5mg) and 4.5g Norvancomycin sterling (purity is greater than 95%).

Embodiment 2 compound 1～3 structure elucidations

Compound 1,2,3 UV absorption similar to Norvancomycin (seeing Fig. 2 D), points out them may be for going first ten thousandAncient mycin analogue.

Compound 1, colorless oil, [α]²⁰ _D-44.5 (c0.22, MeOH), high-resolution electrospray ionization mass spectrometry(HRESIMS) provide its quasi-molecular ion peak [M+H]⁺M/z1452.4253 molecular weight is 1542.4253, pushes away in conjunction with NMR dataSurveying its molecular formula is C₆₅H₇₅O₂₅N₉Cl₂, degree of unsaturation is 31, points out and in its molecule, contains multiple aromatic rings and other unsaturated knotStructure. By the molecular formula of compound 1 and Norvancomycin (C₆₅H₇₃O₂₄N₉Cl₂) compare, find only to have a part H₂O's is poorDifferent, therefore, infer that compound 1 may be hydrolysis or the water addition compound product of Norvancomycin. By compound 1 electron spray secondaryMass spectrum (ESI-MS/MS) collection of illustrative plates collection of illustrative plates corresponding to Norvancomycin compares (Fig. 3), finds that its fragment ion peak is as m/Z1308,1146 also have corresponding difference with the fragment ion peak (m/z1291,1130) of Norvancomycin. Further rightTwo lytic pathways are analyzed (Fig. 3), find that hydrolysis site only may occur on the precursor skeleton of seven peptides.

Utilize relevant (HSQC) collection of illustrative plates of nuclear-magnetism heteronuclear list quantum by compound 1¹HNMR and¹³The corresponding signal one of CNMROne correspondence, and according to two dimension with nuclear proton Correlated Spectroscopy (HHCOSY), the data such as nuclear-magnetism heteronuclear Multiple-quantum distant relation spectrum (HMBC)Resolve, the corresponding amino acid that forms seven peptides in compound 1 structure is belonged to out, comprise three phenylglycines, a bright ammoniaAcid, two 3, the dibasic phenylalanine of 4-and an asparagine. Because compound 1 may be the hydrolysis of NorvancomycinProduct, therefore by compound 1 and Norvancomycin (DMSO-d in same solvent₆) NMR data compare (table 1),Discovery has chemical shift and changes maximum position and concentrate on C ring and D ring. Such as H-(AA-6)-5 in compound 1, C-(AA-4)-1 and C-(AA-4)-2 and Norvancomycin relevant position Chemical shift comparison all to high field displacement Δ δ_H-0.20 and Δδ_C– 3.0ppm, particularly C-(AA-6)-2, C-(AA-6)-5 and C-(AA-4)-3 are subject to deshielding effect to High-Field displacement respectivelyΔδ_C+ 0.3 ,+5.6 and+1.0ppm, this explanation compound 1 should be Norvancomycin C-O-D cyclizing hydrolysis open-loop products. CauseThis, compound 1 is confirmed as C-O-D open loop Norvancomycin.

Compound 1 and Norvancomycin¹H and¹³CNMR data see the following form 1.

Table 1

The spectroscopic data of compound 2 shows that it is also analog of Norvancomycin, by the NMR data of compound 2(table 2) compares with Norvancomycin, finds a glucosyl group of the combined thing 2 of disaccharide part of NorvancomycinReplace, and other position data does not have large change. Particularly¹In HNMR, belong to vancosaminyl in NorvancomycinThe methyl signals δ of fragment_H1.29 (s) and 1.06 (d, J=6.6Hz), and glycosyl end group signal δ 5.23 (m) of this fragment,In compound 2, disappear. And¹³First ten thousand is gone in the chemical shift of the C-2 position of the glucosyl group of CNMR data demonstration compound 2Ancient mycin relevant position is to high field displacement (Δ δ_C– 4.4ppm), these data are gone in conjunction with 2 molecular weight of compound in HRESIMSLow 143 atomic weight of first vancomycin, illustrate that compound 2 is exactly the sugar compounds that deaminizes of Norvancomycin. Therefore, compound2 are decided to be the sugared Norvancomycin that deaminizes.

The ultraviolet of compound 3 and nuclear magnetic data show that it is the analog of compound 2, carefully compare both NMR data, spyBe not¹HNMR data, show compound 3 be exactly 2 remove glucosyl group analog, in addition, the HRESIMS spectrogram of compound 3 is givenGo out one [M+H]⁺Quasi-molecular ion peak at m/z1129.2780, calculating its molecular formula is C₅₂H₅₀O₁₇N₈Cl₂, andAnd this molecular weight ratio compound 2 has lacked 162 atomic weight, further verify that compound 3 is exactly the glucosyl that goes of compound 2Compound, so compound 3 is confirmed as disaccharide Norvancomycin.

Compound 2¹H and¹³CNMR data and compound 3¹HNMR data see the following form 2.

Table 2

Embodiment 3 compound 1～3 determinations of activity

The minimum inhibitory concentration (MIC) of compound is used flat board clinical by the U.S. and that laboratory standards institute provides to diluteMethod, is used vancomycin as positive control, and all test bacterium are ATCC reference culture or clinical isolates strain.Muller-Hinton (M-H) steamed beef soup is used as calibrating culture medium, and inoculum concentration is 10,000cfu/spot, each sampleConcentration range be 0.5 to 128 μ gml^-1, under 35 DEG C of condition of culture, cultivate 18 hours, taking the invisible thalli growth of naked eyes asMinimum inhibitory concentration MIC.

Compound 1～3 and vancomycin (VCM), Norvancomycin (NVCM) bulk drug and sterling (purity > 95%)Antibacterial activity relatively sees the following form 3.

Table 3

^a: purity > 95% Norvancomycin sample. ATCC, American Type Culture Collecti; MRSA, methicillin-resistant goldStaphylococcus aureus; MSSA, MSSA; MRSE, methicillin-resistant MRSE;MSSE, methicillin-sensitivity MRSE; VISA, vancomycin intermediary staphylococcus aureus; VRE, vancomycin resistanceEnterococcus; VSE, the responsive enterococcus of vancomycin.

Result shows, the antibacterial activity of Norvancomycin sterling and Norvancomycin bulk drug and vancomycin phaseWhen, the noval chemical compound that obtains all has activity to gram-positive bacteria especially drug-fast bacteria MRSA and MRSE. The antibacterial activity of compound 1Obviously weaken compared with Norvancomycin; Compound 2 demonstrate with Norvancomycin quite or lower slightly anti-MRSA and MRSE energyPower; And compound 3 demonstrates the antibacterial ability suitable with Norvancomycin, on part bacterial strain, be slightly better than first mould through the agesElement. In addition the leather such as equal nonreactive Escherichia coli of all test compounds including positive control (Escherichiacoli),The ability of Lan Shi negative bacterium and vancomycin-resistant enterococcus.

Finally it should be noted that, above embodiment is only as helping skilled in the art to understand technical side of the present inventionThe essence of case, and be not used as limiting the scope of the present invention.

Claims (7)

1. the HPLC of Norvancomycin derivative prepares a separation method, and described method is:

(1) mobile phase: methyl alcohol: 0.05～0.2% aqueous formic acid,

(2) methyl alcohol rises to 70% gradually from 10% in gradient elution step: 30～50min,

(3) the Norvancomycin bulk drug aqueous solution is crossed to chromatographic column, flow velocity is 1.0～5.0ml/min, detects wavelength 280nm,Described Norvancomycin bulk drug is preferably Norvancomycin hydrochloride bulk drug,

(4) collect main peak secondary peak product component eluent in addition, after decompression distillation, obtain Norvancomycin derivative pureProduct.

2. method according to claim 1, is characterized in that, the chromatographic column using in described method for C18 chromatographic column orPhenyl post.

3. separate by method described in claim 1 or 2 the Norvancomycin derivative obtaining.

4. Norvancomycin derivative according to claim 3, is characterized in that, described Norvancomycin is derivativeThing is: compound 1 (structure is as shown in the formula shown in 1), compound 2 or 3 (structure is as shown in the formula shown in 2,3).

5. the Norvancomycin derivative described in claim 3 or 4 is in the application of preparing in medicine.

6. application according to claim 5, is characterized in that, the medicine that described medicine is resisting gram-positive bacteria, described inGram-positive bacteria be preferably: methicillin-resistant staphylococcus aureus, methicillin-resistant MRSE, penicillin resistantEnterobacteria, streptococcus pneumonia and clostridium.

7. a HPLC purification process for Norvancomycin, described method is:

(1) mobile phase: methyl alcohol: 0.05～0.2% aqueous formic acid,

(2) methyl alcohol rises to 70% gradually from 10% in gradient elution step: 30～50min,

(3) the Norvancomycin bulk drug aqueous solution is crossed to chromatographic column, flow velocity is 1.0～5.0ml/min, detects wavelength 280nm,Described Norvancomycin bulk drug is preferably Norvancomycin hydrochloride bulk drug,

(4) collect main peak product component eluent, after decompression distillation, obtain Norvancomycin sterling.