CN106349343A - Norvancomycin derivatives and preparation and purification method thereof - Google Patents

Abstract

The invention relates to a preparation and purification 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 by virtue of the method. The invention further relates to pharmaceutical application of the norvancomycin derivatives (compounds 1-3).

Description

Norvancomycin derivant and its prepare purification process

Technical field

The invention belongs to medicinal chemistry art, specifically, the present invention relates to norvancomycin derivant and its preparation Purification process.

Background technology

Norvancomycin is class glycopeptide antibioticss, in China nearly 40 years of commercial applications, clinical main It is applied to treat the serious infections such as endocarditiss, osteomyelitis¹.Norvancomycin mainly has to gram positive bacteria Effect, particularly methicillin-resistant staphylococcus aureus, Methicillin-resistant Staphylococcus epidermidis and penicillin-fast enterobacteria, lung Scorching streptococcus and clostridium.The producing strains of norvancomycin are nineteen fifty-nine Lie group etc.²Separate from Soils of Guizhou The actinomycetes going out ten thousand-No. 23, were named as Amycolatopsis orientalis (amycolatopsis orientalis) later.Nor- through the ages Mycin has the chemical constitution substantially similar with another glycopeptide compound vancomycin (vcm) and biological activity³, they are equal The heptapeptide parent being become by three ring groups and a disaccharidase substituent group being made up of glucose-amino sugar, difference is The n- end of first vancomycin lacks a n- methyl (Fig. 1) compared with vancomycin.In document form heptapeptide aminoacid respectively with Aa-1 to aa-7 represents, five aromatic rings are from a-e number consecutively, in addition, the numbering of three big rings numbers composition a- with aromatic rings B, c-o-d and d-o-e, compound structure parsing for convenience, the application will continue to use the method for numbering serial of document.

In early days, the initial operational phase of vancomycin, finds during Clinical practice that it causes multiple negative responses, such as nephrotoxicity With ototoxicity etc., it is that its purity is low the reason possible, the reason impurity content is high.Subsequently, people use high performance liquid chromatography (hplc)^4-7, capillary electrophoresis (ce)⁸And liquid chromatography mass spectrometric-mass spectrum technology used in conjunction (lc-ms/ms)^4,9The relevant thing to vancomycin Matter carried out system in-depth study, was therefrom found that eight impurity are dechlorination vancomycin a and b respectively¹⁰, go double chlorine mould through the ages Element¹⁰, vancomycin crystallization process catabolite cdp-i_mAnd cdp-i_m ¹¹, remove disaccharidase vancomycin¹², the sugar that deaminizes is mould through the ages Element¹²And norvancomycin.Further pharmacologically active experimental studies have found that, containing cdp-i_mAnd cdp-i_mVancomycin medicine Clinical treatment failure can be caused¹³, and the antibacterial activity that three dechlorination vancomycins are similar to thing reduces 2 to 10 than vancomycin Times³, the antibacterial activity in vivo of vancomycin of the sugar that deaminizes is greatly reduced, and mic is about five times of vancomycin.Therefore, " Europe Continent pharmacopeia "¹⁴" American Pharmacopeia "¹⁵To deaminize-, go disaccharidase base-, and demethyl vancomycin is defined as vancomycin raw material Three major impurities, and specify its single miscellaneous must not exceed 4%, always miscellaneous must not exceed 7%.

Comparatively speaking, norvancomycin due to its impurity component indefinite, " Chinese Pharmacopoeia "¹⁶Regulation is with integrating peak areas Calculate, always miscellaneous must not exceed 12%, single miscellaneous is less than 4%.Accordingly, it would be desirable to more efficiently purification and identification norvancomycin Method, for material composition structure relevant in clear and definite norvancomycin crude drug, also needs to norvancomycin raw material meanwhile Medicine carries out the chemical constitution study of more system, excavates the analog of its new construction.

List of references:

1.wu,x.j.,et al.establishment of norvancomycin fluorescence polarization immunoassay for therapeutic drug monitoring.j antibiot(tokyo) .65,35-39(2012).

2.li q.,s.a.l.,liu j.r.,&wang x.y.actinomycetes van 23-vancomycin producing strain (shanghai scientific&technical publishers,shanghai,china, 1962).

3.nagarajan,r.structure-activity relationships of vancomycin-type glycopeptide antibiotics.j antibiot(tokyo).46,1181-1195(1993).

4.diana,j.,visky,d.,hoogmartens,j.,van schepdael,a.&adams, e.investigation of vancomycin and related substances by liquid chromatography/ion trap mass spectrometry.rapid commun mass spectrom.20,9 (2006).

5.diana,j.,visky,d.,roets,e.&hoogmartens,j.development and validation of an improved method for the analysis of vancomycin by liquid chromatography selectivity of reversed-phase columns towards vancomycin components.j chromatogr a.996,115-131(2003).

6.inman,e.l.determination of vancomycin related substances by gradient high-performance liquid chromatography.j chromatogr.410,363-372 (1987).

7.jesus valle,m.j.,lopez,f.g.&navarro,a.s.development and validation of an hplc method for vancomycin and its application to a pharmacokinetic study.j pharm biomed anal.48,835-839(2008).

8.kang,j.w.,van schepdael,a.,roets,e.&hoogmartens,j.analysis of vancomycin and related impurities by micellar electrokinetic capillary chromatography.method development and validation.electrophoresis.22,4(2001).

9.hadwiger,m.e.,sommers,c.d.,mans,d.j.,patel,v.&boyne,m.t.2nd.quality assessment of u.s.marketplace vancomycin for injection products using high- resolution liquid chromatography-mass spectrometry and potency assays.antimicrob agents chemother.56,7(2012).

10.harris,c.m.,kannan,r.,kopecka,h.&harris,t.m.the role of the chlorine substituents in the antibiotic vancomycin:preparation and characterization of mono-and didechlorovancomycin.journal of the american chemical society.107,6652-6658(1985).

11.harris,c.m.,kopecka,h.&harris,t.m.vancomycin:structure and transformation to cdp-i.journal of the american chemical society.105,6915- 6922(1983).

12.nagarajan,r.&schabel,a.a.selective cleavage of vancosamine, glucose,and n-methyl-leucine from vancomycin and related antibiotics.journal of the chemical society,chemical communications.1306-1307(1988).

13.somerville,a.l.,wright,d.h.&rotschafer,j.c.implications of vancomycin degradation products on therapeutic drug monitoring in patients with end-stage renal disease.pharmacotherapy.19,702-707(1999).

14.the european pharmacopoeia commission.the european pharmacopoeia 8th edn.(council of europe:strasbourg,2008).

15.the united states pharmacopeia commission(the united states pharmacopeia convention,inc,rockville,md,usa,2012).

16.chinese pharmacopoeia commission,pharmacopoeia of the people's republic of china(in chinese)2015edn(china medical science and technology press,beijing,china,2015).

Content of the invention

The present invention separates from norvancomycin crude drug and obtains three glycopeptide compounds, by means of 1d, 2d nmr Determine three compound structures with methods such as hresims, be norvancomycin analog, including a d-o-e ring expansion and Two are removed glycosyl derivatives.

Present invention firstly relates to a kind of hplc preparative separation method of norvancomycin and its derivant, methods described For:

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

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

(3) norvancomycin raw material aqueous solution is crossed chromatographic column, flow velocity is 1.0～5.0ml/min, Detection wavelength 280nm, described norvancomycin crude drug is preferably norvancomycin hydrochloride Starting material medicine,

(4) collect main peak product and obtain final product norvancomycin sterling, collection secondary peak product obtains final product norvancomycin and derives Thing.

In methods described, chromatographic column is preferably c18 chromatographic column or phenyl post.

The invention still further relates to the norvancomycin derivant obtaining, described norvancomycin are separated by methods described Derivant is: compound 1 (structure is as shown in following formula 1), compound 2 or 3 (structure is as shown in following formula 2,3),

The invention still further relates to application in preparing medicine for the described compound 1-3, described medicine is resisting gram-positive bacteria Medicine, described gram positive bacteria is preferably: methicillin-resistant staphylococcus aureus, methicillin-resistant epidermis Fructus Vitis viniferae ball Bacterium, penicillin-fast enterobacteria, streptococcus pneumoniae and clostridium.

Brief description

Fig. 1 compound 1-3, the structure of norvancomycin and vancomycin, the curved arrow of the related dotted line of hmbc of compound 1 Head represents.

The hplc chromatography figure of Fig. 2 norvancomycin crude drug.

2a, uses the hplc collection of illustrative plates of " Chinese Pharmacopoeia " method；

The uv-spectrogram stacking chart of 2b, a in figure a, b, c and norvancomycin；

2c, hplc collection of illustrative plates after method optimization；

2d, c in figure impurity 1,2,3,4 and the uv-spectrogram stacking chart of norvancomycin.

(parent ion is [m+h] to Fig. 3 norvancomycin⁺M/z 1434, Fig. 3 a) and compound 1 (parent ion is [m+2h]²⁺ M/z 724.5, Fig. 3 c) esims/ms figure and both lytic pathway (3b be norvancomycin, 3d be compound 1).

Specific embodiment

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

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

Chromatographic grade acetonitrile and methanol are purchased from adamas company (Chinese Shanghai)；

dmso-d₆(cambridge, ma, usa.) is produced by cambridge isotope company；

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

The instrument using, including p-2000 polariscope (jasco, tokyo, japan), jascop-650 uv-spectrophotometric Meter (jasco), sys 600mhz nuclear magnetic resonance spectrometer (palo alto, ca, usa), finnigan ltq xt ion trap mass spectrometer (finnigan, san jose, ca), finnigan ltq orbit ion trap spectrometer (finnigan), Shimadzu lc-20 liquid chromatograph Instrument (shimadzu, japan).

Nuclear-magnetism assay method:

All samples¹H and¹³C nmr measures all on the sys 600mhz nuclear magnetic resonance spectrometer with cryogenic probe, and solvent is deuterated dmso.¹H and¹³C nmr chemical shift is all using tms (δ=0.00ppm) and dmso-d₆(δ=39.5ppm) solvent does internal standard.

The preparation of embodiment 1 compound 1～3 with isolate and purify

First, we used in " Chinese Pharmacopoeia " norvancomycin crude drug hplc method to analyze sample (see figure 2a and 2b), find in retention time a main peak for 21.44min, integral area accounts for the 95.5% of all peak area summations, Peak position is gone out for 18～22min thus it is speculated that this material is exactly norvancomycin according to norvancomycin in pharmacopeia, in addition, such as Shown in Fig. 2 a, also there are the less impurity peaks of at least three integral areas in retention time before and after this main peak, and retention time is divided Not Wei 10.31 (a), 12.30 (b), 14.73 (c) min, their online uv absorption peak type is also similar with norvancomycin Seemingly, as shown in Figure 2 b.However, employing phosphate due in the method, and because in sample after preparation process or preparation Phosphatic residual meeting severe jamming Mass Spectrometer Method, therefore, this method is not suitable for carrying out liquid chromatograph mass spectrography to sample (lc-ms).

We, in order to tentatively obtain the corresponding informations such as these derivant impurity molecule amounts by means of lc-ms, establish a set of Mobile phase salt-free hplc method.When we carry out hplc analysis using methanol -0.1% aqueous formic acid, find phase The main peak answered and impurity peaks can obtain good peak type, and between impurity and impurity, also have relatively between impurity and main peak Good separating degree, as shown in Figure 2 c, derivant impurity 1,2,3,4 peak areas account for the 3.4% of the gross area respectively, and 1.91%, 0.96%, and 2.02%, main peak is 87.97%.

(1) the hplc analysis method optimizing is as follows:

agilent c₁₈Chromatographic column (150 × 4.6mm i.d., 5 μm),

Mobile phase: methanol: 0.1% aqueous formic acid,

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

1.0ml min^-1Flow velocity, 280nm wavelength detecting, column temperature and detection cell temperature are room temperature (25 DEG C),

3.0mg ml^-1Norvancomycin raw material aqueous solution, sample size is 20 μ l.

(2) the hplc preparation method optimizing is as follows:

Ymc-pack phenyl post (150 × 20mm i.d., 20 μm, ymc co., ltd, kyoto, japan),

Flowing phase configuration is consistent with analysis experiment with gradient elution method.

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

100mg ml^-1Norvancomycin raw material aqueous solution, single sample size is 300 μ l.

Using the salt-free hplc method optimizing, each impurity is carried out with system and separated preparation, through vacuum distillation recovered solvent Afterwards, separate from 5.0g norvancomycin crude drug and obtained compound 1 (5.0mg), compound 2 (5.0mg), compound 3 (1.5mg) and 4.5g norvancomycin sterling (purity be more than 95%).

Embodiment 2 compound 1～3 structure elucidation

Compound 1,2,3 uv absorption is similar to norvancomycin (see Fig. 2 d), points out them may be nor- ten thousand Ancient mycin analog.

Compound 1, colorless oil, [α]²⁰ _d- 44.5 (c 0.22, meoh), high-resolution electrospray ionization mass spectrometry (hresims) provide its quasi-molecular ion peak [m+2h]²⁺M/z 724.7505, molecular weight is 1447da, in conjunction with nmr data-speculative Its molecular formula is c₆₆h₇₅o₂₄n₉cl₂, degree of unsaturation is 33, points out to contain multiple aromatic rings and other unsaturation structure in its molecule. By the molecular formula of compound 1 and norvancomycin (c₆₅h₇₃o₂₄n₉cl₂) compare, find only have methylene segment ch₂ Difference, thus it is speculated that compound 1 be probably norvancomycin methylate.By compound 1 electron spray second order mses (esi-ms/ms) collection of illustrative plates collection of illustrative plates corresponding to norvancomycin is compared (Fig. 3), finds its fragment ion peak such as m/z1305, 1143 also have identical difference with the fragment ion peak (m/z 1291,1129) of norvancomycin.Further two are cracked Approach is analyzed (Fig. 3), finds that compound 1 and norvancomycin have identical disaccharidase structure fragment, therefore, methylates Site is only possible to occur on the precursor skeleton of heptapeptide.

Using nuclear-magnetism heteronuclear list quantum correlation (hsqc) collection of illustrative plates by compound 1¹H nmr and¹³The corresponding signal one of c nmr One is corresponding, and according to two dimension with nuclear proton Correlated Spectroscopy (hhcosy), the data such as the long-range Correlated Spectroscopy of nuclear-magnetism heteronuclear Multiple-quantum (hmbc) Parsing, by the corresponding amino acid assignments forming heptapeptide in compound 1 structure out, including three phenylglycines, a bright ammonia Acid, two dibasic Phenylalanine of 3,4- and an agedoite.Because compound 1 is probably the methyl of norvancomycin Change product, therefore by compound 1 and norvancomycin (dmso-d in same solvent₆) nmr data be compared (table 1), find that having the maximum position of chemical shift change concentrates on the agedoite and Phenylalanine fragment of d-o-e ring.Such as In compound 1, h- (aa-3) -2 and h- (aa-3) -3a and norvancomycin relevant position Chemical shift comparison are respectively to High-Field Displacement δ δ_h- 0.37 and -0.12ppm, and h- (aa-3) -3a to low field displacement δ δ_h+0.4ppm.These concentrate on Radix Asparagi The change of the nuclear magnetic data on histidine residue (includes cdp-i with cdp-i_mAnd cdp-i_m, c-cl key only on e ring for the difference takes To)¹¹It is consistent with vancomycin difference.Cdp-i is the product of vancomycin d-o-e expansion, so speculating compound 1 It is also likely to be the analog of d-o-e ring expansion.

Especially in 2d-noesy collection of illustrative plates, h₂- (aa-3) -3 and h- (aa-2) -5, h- (aa-3) -2, h- (aa-2) -6 phase Close, and h- (aa-2) -5 is related to h- (aa-4) -2 shows that these protons are in plane of a loop side；H- (aa-2) -2 and h- (aa- 2) -7, h- (aa-2) -8, h- (aa-4) -6 is related, and h- (aa-4) -6 is related to h- (aa-4) -7, shows that these protons are in flat The opposite side in face, and the orientation of c-cl key on e ring and norvancomycin, vancomycin and cdp-i_mIdentical.And its Remaining part divides nmr data with norvancomycin appropriate section difference less, shows that compound 1 has identical with norvancomycin A-b, c-o-d ring ingredient.

In compound¹H and¹³It is in δ in c nmr_h3.17 (3h, s) and δ_cIn the signal instruction compound of 48.5ppm Containing a n- methyl structural fragment.By analysis of compounds 1 and water under norvancomycin and vancomycin the same terms The esi-ms data validation of solution product, this n- methyl is located at the n- end of leucine fragment in structure.In esi-ms, chemical combination Thing 1 has and vancomycin hydrolyzate identical n- methylleucine quasi-molecular ions (m/z 146 [m+h]⁺).Therefore, compound 1 Structure be confirmed as the n- methyl norvancomycin of d-o-e ring expansion.

Compound 1 and norvancomycin¹H and¹³C nmr data see table 1.

Table 1

Note: nh- (aa-1) -1, nh- (aa-2) -9, nh- (aa-4) -8, nh- (aa-5) -8 and nh- (aa- in compound 1 6) -9 chemical shift is respectively δ_h8.59,8.43,8.88,8.50 and 8.50ppm；And n-ch₃Nmr numerical value be respectively δ_h 3.17ppm, δ_c48.5ppm.

The spectroscopic data of compound 2 shows that it is also one analog of norvancomycin, by the nmr data of compound 2 (table 2) is compared with norvancomycin, and the disaccharide moiety finding norvancomycin is by a glucosyl group of compound 2 Replace, and other positions data does not have big change.Particularly¹Vancosaminyl in norvancomycin is belonged in h nmr 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, Compound 2 disappears.And¹³The chemical shift more nor- ten thousand of the c-2 position of glucosyl group of c nmr data display compound 2 Ancient mycin relevant position is to High-Field displacement (δ δ_c4.4ppm), these data are relatively gone with reference to compound in hresims 2 molecular weight First vancomycin is low by 143, illustrates that compound 2 is exactly the sugar compounds that deaminize of norvancomycin.Therefore, compound 2 is determined For the sugared norvancomycin that deaminizes.

The ultraviolet of compound 3 and nuclear magnetic data show the analog that it is compound 2, carefully compare both nmr data, special It is not¹H nmr data, display compound 3 be exactly 2 remove glucosyl group analog, in addition, the hresims spectrogram of compound 3 is given Go out one [m+h]⁺Quasi-molecular ion peak in m/z 1129.2780, calculate its molecular formula be c₅₂h₅₀o₁₇n₈cl₂, and And this molecular weight fewer than compound 2 162, demonstrate further compound 3 be exactly compound 2 remove glucosyl group compound, So compound 3 is confirmed as disaccharidase norvancomycin.

Compound 2¹H and¹³C nmr data and compound 3¹H nmr data see table 2.

Table 2

Embodiment 3 compound 1～3 determination of activity

The minimum inhibitory concentration (mic) of compound is using the plating dilutions being provided by U.S. clinical and laboratory standards institute Method, is used vancomycin as positive control, and all of test bacterium is atcc reference culture or Clinical isolation. Muller-hinton (m-h) steamed beef soup is used as examining and determine culture medium, and inoculum concentration is 10,000cfu/spot, each sample Concentration range be 0.5 to 128 μ g ml^-1, cultivate 18 hours under 35 DEG C of condition of culture, with the invisible thalli growth of naked eyes be Minimum inhibitory concentration mic.

Compound 1～3 and vancomycin (vcm), norvancomycin (nvcm) crude drug and sterling (purity > 95%) Antibacterial activity relatively see table 3.

Table 3

^a: purity > 95% norvancomycin sample.Atcc, American Type Culture Collecti；Mrsa, methicillin-resistant gold Staphylococcus aureus；Mssa, MSSA；Mrse, Methicillin-resistant Staphylococcus epidermidis； Msse, methicillin-sensitivity staphylococcus epidermidiss；Visa, vancomycin intermediary staphylococcus aureuses；Vre, vancomycin resistance Enterococcus；Vse, vancomycin sensitive enterococcus.

Result shows, the antibacterial activity of norvancomycin sterling and norvancomycin crude drug and vancomycin phase When obtained noval chemical compound is all active to gram positive bacteria especially fastbacteria mrsa and mrse.The antibacterial activity of compound 1 Substantially weaken compared with norvancomycin；Compound 2 shows the anti-mrsa and mrse energy suitable or lower slightly with norvancomycin Power；And compound 3 shows the antibacterial ability suitable with norvancomycin, part bacterial strain is slightly better than nor- mould through the ages Element.Additionally, the leather such as all test compounds equal nonreactive escherichia coli (escherichia coli) including positive control Lan Shi negative bacterium and the ability of vancomycin-resistant enterococcus.

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

Claims (7)

1. a kind of hplc preparative separation method of norvancomycin derivant, methods described is:

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

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

(3) norvancomycin raw material aqueous solution is crossed chromatographic column, flow velocity is 1.0～5.0ml/min, Detection wavelength 280nm, Described norvancomycin crude drug is preferably norvancomycin hydrochloride Starting material medicine,

(4) the secondary peak product component eluent beyond collection main peak, obtains final product norvancomycin derivant pure after vacuum distillation Product.

2. method according to claim 1 it is characterised in that used in methods described chromatographic column be c18 chromatographic column or Phenyl post.

3. separate, by claim 1 or 2 methods describeds, the norvancomycin derivant obtaining.

4. norvancomycin derivant according to claim 3 is it is characterised in that described norvancomycin derives Thing is: compound 1 (structure is as shown in following formula 1), compound 2 or 3 (structure is as shown in following formula 2,3).

Formula 1

R=β-d-glucosyl, formula 2；

R=h, formula 3.

5. application in preparing medicine for the norvancomycin derivant described in claim 3 or 4.

6. application according to claim 5 is it is characterised in that described medicine is the medicine of resisting gram-positive bacteria, described Gram positive bacteria be preferably: methicillin-resistant staphylococcus aureus, Methicillin-resistant Staphylococcus epidermidis, penicillin resistant Enterobacteria, streptococcus pneumoniae and clostridium.

7. the hplc purification process of a kind of norvancomycin, methods described is:

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

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

(3) norvancomycin raw material aqueous solution is crossed chromatographic column, flow velocity is 1.0～5.0ml/min, Detection wavelength 280nm, Described norvancomycin crude drug is preferably norvancomycin hydrochloride Starting material medicine,

(4) collect main peak product component eluent, obtain final product norvancomycin sterling after vacuum distillation.