Prazomicin impurity H and preparation method thereof

Abstract

The invention provides a prazomicin impurity H. The prazomicin impurity H is characterized in that the structure of the prazomicin impurity H is as follows: # imgabs0 #. The invention also provides a preparation method of the prazomicin impurity. A reference substance of the impurity has very important significance on quality research and quality control of prazomimicin.

Description

Technical Field

[0001] The invention relates to the field of drug synthesis, and in particular to a prazomicin process impurity and a preparation method thereof. Background Art

[0002] Plazomicin (Zemdri), chemical name is (2R,3R,4R,5R)-2-[(1′S,2′S,3′R,4′S,6′R)-4′-amino-6′-[(2″′S)-4″′-amino-2″′-hydroxybutyramido]-3′-[(2″S,3″R)-3″-amino-6″-[(2-hydroxyethylamino)methyl]-3″,4″-dihydro-2H-pyran-2″-oxy]-2′-hydroxy-cyclohexyloxy]-5-methyl-4-(methylamino)tetrahydro-2H-pyran-3,5-diol.

[0003]

[0004] This drug is a new type of aminoglycoside antibiotic jointly developed by Ibis Therapeutics and Achaogen. It was granted Fast Track qualification and Qualified Infectious Disease Product (QIDP) designation for complicated urinary tract infection and bloodstream infection by the U.S. FDA in 2012 and 2014 respectively. On June 25, 2018, this drug was officially approved by the U.S. FDA for the treatment of complicated urinary tract infection (cUTI), pyelonephritis, etc.

[0005] Plazomicin is derived from the chemical structure modification of sisomicin. Its preparation process mainly involves introducing a hydroxy-aminobutyric acid substituent at the 6′ amino group of sisomicin and a hydroxyethyl substituent at the 7′ amino group, thereby obtaining a new type of aminoglycoside antibiotic. Sisomicin after structural modification can avoid being destroyed by aminoglycoside antibiotic inactivating enzymes (AMEs), thereby maintaining antibacterial activity. At present, some adverse reactions commonly found in aminoglycoside drugs such as nephrotoxicity and ototoxicity have not been found, so it has very good market application prospects.

[0006] As the active ingredient of a new aminoglycoside antibacterial drug, the chemical synthesis method of prazomicin has also received extensive attention and reports, mainly focusing on process development and optimization in the selection of protective group types and reaction sequence during the synthesis process. The existing reported routes are difficult to avoid the shortcomings of long synthesis routes, many side reactions, complicated post-treatment, and many types of impurities, which poses a great challenge to the quality research and control of prazomicin.

[0007] Therefore, it is very important to identify new impurities of prazolamide and obtain high-purity impurity control samples. Summary of the invention

[0008] The present invention aims to overcome the above-mentioned defects, provides a new impurity H, and obtains a high-purity impurity H sample by synthetic means.

[0009] The present invention provides a prazomicin impurity H, characterized in that:

[0010] The structure of the above-mentioned plazomicin impurity H is shown below:

[0011]

[0012] The present invention also provides a method for preparing the impurity H of prazomicin, characterized in that:

[0013] Using sisomicin as a raw material, compound A is formed by aminoazidation reaction;

[0014] The above compound A is degraded to form compound B;

[0015] The compound B is subjected to a reduction reaction to reduce the azide group thereon to an amino group to form a compound C; the compound C is subjected to a selective protection and condensation reaction to form a compound D;

[0016] The compound D is selectively deprotected from the amino group protected by PNZ to form a compound E; the compound E is subjected to a substitution reaction to form a compound F;

[0017] The compound F is subjected to amino deprotection reaction to form the target product;

[0018] The specific synthesis route is as follows:

[0019]

[0020] It is worth noting that any of the above compounds AF can be used as a raw material for synthesizing impurity H.

[0021] In addition, the process of preparing compound D from compound C includes a selective amino protection reaction of p-nitrobenzyl carbonate, a selective amino protection reaction of Boc, and a condensation reaction, which are specifically as follows:

[0022] The intermediate raw material undergoes a selective amino protection reaction of the nitrobenzyl carbonate group to form the transition product 1;

[0023] The transition product 1 is subjected to a selective amino protection reaction of Boc to form a transition product 2;

[0024] The transition product 2 undergoes a condensation reaction to form a compound D;

[0025] Among them, the structure of the intermediate raw material is as follows:

[0026]

[0027] The structure of transition product 1 is shown below;

[0028]

[0029] The structure of transition product 2 is shown below;

[0030]

[0031] In addition, the present invention also provides the reaction conditions of the above reaction, which are as follows:

[0032] The reaction conditions for forming compound A via aminoazidation reaction are:

[0033] Under alkaline conditions, sisomicin and an azidation reagent are reacted at a temperature of 0°C to 10°C for 4-8 hours; and / or

[0034] The reaction conditions for compound A to form compound B through degradation reaction are:

[0035] Compound A is reacted under the action of a strong oxidant at a temperature of 25°C to 80°C for 40-60 hours;

[0036] and / or

[0037] The reaction conditions for compound B to form compound C through reduction reaction are:

[0038] Compound B reacts in the presence of a reducing agent for 4-8 hours;

[0039] and / or

[0040] The reaction conditions for selectively removing the protecting group of the amino group protected by PNZ from compound D to form compound E are:

[0041] Under alkaline conditions, compound D is reacted at a temperature of 0°C to 10°C for 1-12 hours;

[0042] and / or

[0043] The reaction conditions for the formation of compound F by substitution reaction of compound E are:

[0044] Under alkaline conditions, compound E reacts with a halogenating agent at a temperature of 10° C. to 50° C. for 25-50 hours; and / or

[0045] The reaction conditions for compound F to form the target through amino deprotection reaction are:

[0046] Under acidic conditions, compound F is reacted at a temperature of 0°C to 30°C for 2-6 hours. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples, but the present invention is not limited to the above-mentioned embodiments. The present invention is further described in detail below in conjunction with the examples. These specific embodiments only show the implementation technical solutions selected to achieve the purpose of the present application, and the improvements to the technical solutions of the present application in combination with the prior art all belong to the scope of protection of the present application.

[0048] The implementation conditions used in the following examples can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments. Among them, the chemical reagents used in the examples are all commercially available chemical reagents.

[0049] This embodiment provides a plazomicin process impurity H:

[0050]

[0051] The impurity H is formed by the specific impurity of the raw material sisomicin free base, that is, compound C, which participates in the reaction derivatization in the synthesis route of prazomicin and can be detected by HPLC and LCMS methods.

[0052] This embodiment also provides a method for preparing the impurity H. When compound F is used as a raw material, the method comprises the following steps: in a solvent, in the presence of an acid, compound F is subjected to a deprotection reaction as shown below to obtain a compound as shown in formula H;

[0053]

[0054] The above-mentioned deprotection reaction conditions and steps can be conventional conditions and steps in the art, and the following conditions are particularly preferred:

[0055] In the above-mentioned deprotection reaction, the above-mentioned solvent can be a conventional solvent in the art. Preferably, the above-mentioned solvent is one or more of an ether solvent, a nitrile solvent and a ketone solvent; more preferably, the above-mentioned solvent is one or more of tetrahydrofuran, acetonitrile, 1,4-dioxane and acetone.

[0056] In the above deprotection reaction, the acid of the above deprotection reaction can be an acid conventionally used in this field for such deprotection reactions. Preferably, the above acid is one or more of hydrochloric acid, sulfuric acid, phosphoric acid and trifluoroacetic acid.

[0057] In the above deprotection reaction, the reaction temperature may be a conventional temperature for such deprotection reactions in the art. Preferably, the reaction temperature is 0°C to 30°C; more preferably, the reaction temperature is 20°C to 25°C.

[0058] In the above deprotection reaction, the disappearance of the above compound F is regarded as the reaction endpoint. Preferably, the reaction time is 2 hours to 6 hours, and more preferably, the reaction time is 4 hours.

[0059] The above compound F can be obtained by subjecting compound E and a halide to a substitution reaction as shown below in a solvent in the presence of a base:

[0060]

[0061] The conditions and steps of the above-mentioned substitution reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0062] In the above-mentioned substitution reaction, the above-mentioned solvent can be a conventional solvent for such substitution reaction in the art, preferably an anhydrous solvent, preferably, the above-mentioned solvent is one or more of an ether solvent, a nitrile solvent and a ketone solvent; more preferably, the above-mentioned solvent is one or more of tetrahydrofuran, acetonitrile, 1,4-dioxane and acetone.

[0063] In the above-mentioned substitution reaction, the above-mentioned halogenated substance may be a conventional halogenating agent for such substitution reaction in the art. Preferably, the above-mentioned halogenating agent is one or more of iodoethanol, bromoethanol and chloroethanol. More preferably, the above-mentioned halogenating agent is iodoethanol.

[0064] In the above substitution reaction, the above base can be a base conventionally used in this type of substitution reaction in the art. Preferably, the above base is sodium bicarbonate.

[0065] In the above substitution reaction, the molar ratio of the above compound E to the above halogenating agent can be a conventional ratio for such substitution reactions in the art, preferably 1:05 to 1:1.5, more preferably 1:1.1.

[0066] In the above substitution reaction, the reaction temperature may be a conventional reaction temperature for such substitution reactions in the art. Preferably, the above reaction temperature is 10°C to 50°C, and more preferably, the above reaction temperature is 30°C to 40°C.

[0067] In the above substitution reaction, the above catalyst may be a base conventionally used in this type of substitution reaction in the art. Preferably, the above base is triethylenediamine.

[0068] In the above substitution reaction, the disappearance of the above compound E is regarded as the reaction endpoint. Preferably, the reaction time is 25 hours to 50 hours, and more preferably, the reaction time is 32 hours.

[0069] The compound E can be subjected to the PNZ protecting group removal reaction as shown below with the compound D in a solvent in the presence of a base;

[0070]

[0071] The conditions and steps for the above-mentioned PNZ protecting group removal reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0072] In the above-mentioned PNZ protecting group removal reaction, the above-mentioned solvent can be a conventional solvent for such deprotection reactions in the art. Preferably, the above-mentioned solvent is an alcohol solvent, and more preferably, the above-mentioned solvent is methanol.

[0073] In the above-mentioned PNZ protecting group removal reaction, the above-mentioned base can be a base conventionally used in this type of deprotection reaction in the art. Preferably, the above-mentioned base is sodium hydroxide.

[0074] In the above-mentioned PNZ protecting group removal reaction, the above-mentioned reducing agent may be a reducing agent conventionally used in this type of deprotection reaction in the art. Preferably, the above-mentioned reducing agent is sodium dithionate.

[0075] In the above-mentioned PNZ protecting group removal reaction, the reaction temperature can be a conventional temperature for such deprotection reactions in the art. Preferably, the above-mentioned reaction temperature is 0 to 10°C, and most preferably, the above-mentioned reaction temperature is 0 to 5°C.

[0076] In the above deprotection reaction, the disappearance of the above compound D is regarded as the reaction endpoint. Preferably, the reaction time is 1 hour to 12 hours.

[0077] The preparation method of the compound shown in Formula H may further comprise the following three steps (step 1, step 2, step 3):

[0078]

[0079] Step 1:

[0080] In a solvent, in the presence of a p-nitrobenzyl carbonate protecting agent, compound C is subjected to a p-nitrobenzyl carbonate protecting group reaction as shown below to obtain compound C-1;

[0081]

[0082] The conditions and steps for the p-nitrobenzyl carbonate protecting group reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0083] In the above-mentioned p-nitrobenzyl carbonate protecting group reaction, the above-mentioned solvent can be a solvent conventionally used in this type of protecting group reaction in the art. Preferably, the above-mentioned solvent is one or more of a halogenated alkane solvent, a nitrile solvent, an alcohol solvent and an ether solvent. More preferably, the above-mentioned solvent is one or more of dichloromethane, acetonitrile, methanol and tetrahydrofuran. Most preferably, the above-mentioned solvent is dichloromethane and methanol.

[0084] In the above-mentioned upper p-nitrobenzyl carbonate protecting group reaction, the above-mentioned upper protecting group reagent can be a reagent conventionally used in this type of upper protecting group reaction in the art. Preferably, the above-mentioned upper p-nitrobenzyl carbonate protecting reagent is norbornene-2,3-dicarboxyimino p-nitrobenzyl carbonate.

[0085] In the above-mentioned p-nitrobenzyl carbonate protecting group reaction, the molar ratio of the above-mentioned compound C to the above-mentioned protecting group reagent can be a conventional ratio for such protecting group reactions in the art, preferably 1:0.95 to 1:2.

[0086] In the above-mentioned p-nitrobenzyl carbonate protecting group reaction, the reaction temperature can be the conventional reaction temperature of this type of protecting group reaction in the art. Preferably, the above-mentioned reaction temperature is 0°C to 10°C, and more preferably, the above-mentioned reaction temperature is 0°C to 5°C.

[0087] In the above-mentioned p-nitrobenzyl carbonate protecting group reaction, the above-mentioned protecting group reaction takes the above-mentioned compound C as the reaction end point. Preferably, the reaction time is 1 hour to 3 hours.

[0088] Step 2:

[0089] In a solvent, in the presence of di-tert-butyl dicarbonate reagent, compound C-1 is subjected to a Boc protecting group reaction as shown below to obtain compound C-2;

[0090]

[0091] The conditions and steps for the above-mentioned Boc protecting group reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0092] In the above-mentioned Boc protecting group reaction, the above-mentioned solvent can be a solvent conventionally used in this type of protecting group reaction in the art. Preferably, the above-mentioned solvent is one or more of a nitrile solvent, an alcohol solvent and an ether solvent. More preferably, the above-mentioned solvent is one or more of acetonitrile, methanol and tetrahydrofuran. Most preferably, the above-mentioned solvent is methanol.

[0093] In the above-mentioned Boc protecting group reaction, the above-mentioned Lewis acid reagent can be Zn(OAc) which is a reagent conventionally used in this type of protecting group reaction in the art. 2 、ZnCl 2 or Zn(OPiv) 2 Preferably, the Lewis acid reagent is Zn(OAc) 2 .

[0094] In the above-mentioned Boc protecting group reaction, the above-mentioned alkaline reagent can be triethylamine or N,N-diisopropylethylamine, which are reagents conventionally used in this type of protecting group reaction in the art. Preferably, the above-mentioned Lewis acid reagent is triethylamine.

[0095] In the above-mentioned Boc protecting group reaction, the above-mentioned protecting group reagent can be the reagent Boc used conventionally in this type of protecting group reaction in the art. 2 O or Boc-ONb, preferably, the Boc group protecting agent is Boc 2 O.

[0096] In the above-mentioned Boc protecting group-adding reaction, the molar ratio of the above-mentioned compound C to the above-mentioned protecting group-adding reagent can be a conventional ratio for such protecting group-adding reactions in the art, preferably 1:3 to 1:5.

[0097] In the above-mentioned Boc protecting group reaction, the reaction temperature can be the conventional reaction temperature of this type of protecting group reaction in the art. Preferably, the above-mentioned reaction temperature is 30°C to 50°C, and more preferably, the above-mentioned reaction temperature is 40°C.

[0098] In the above-mentioned Boc protecting group reaction, the above-mentioned protecting group reaction takes the above-mentioned compound C as the reaction end point, and preferably, the reaction time is 1 hour to 5 hours.

[0099] Step 3:

[0100] In a solvent, in the presence of a condensation reagent, compound C-2 undergoes a condensation reaction as shown below to obtain compound D;

[0101]

[0102] The conditions and steps of the above-mentioned condensation reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0103] In the above condensation reaction, the molar ratio of the above compound C-2 to (2S)-4-tert-butyloxycarbonylamino-2-hydroxybutyric acid (Boc-(S)-HABA) can be 1:2 to 1:5.

[0104] In the above-mentioned condensation reaction, the above-mentioned solvent can be a solvent conventionally used in this type of protecting group reaction in the art. Preferably, the above-mentioned solvent is one or more of a nitrile solvent, a halogenated alkane solvent and an ether solvent. More preferably, the above-mentioned solvent is one or more of acetonitrile, dichloromethane and tetrahydrofuran. Most preferably, the above-mentioned solvent is dichloromethane.

[0105] In the above condensation reaction, the above condensation reagent may be HOBt / EDCI or HOAt / EDCI, which are reagents commonly used in this type of condensation reaction in the art.

[0106] In the above condensation reaction, the molar ratio of the above compound C to the above HOBt / EDCI can be a conventional ratio in this type of protecting group reaction in the art, preferably 1:1.1:1.1 to 1:1.3:1.3.

[0107] In the above condensation reaction, the reaction temperature can be the conventional reaction temperature of this type of condensation reaction in the art. Preferably, the above reaction temperature is 20°C to 40°C, and more preferably, the above reaction temperature is 30°C.

[0108] In the above condensation reaction, the above condensation reaction is carried out for the above reaction time, preferably, the reaction time is 12 hours to 24 hours.

[0109] The preparation method of the compound represented by the formula H may further comprise the following steps: in a solvent, in the presence of a reducing agent, compound B undergoes a reduction reaction as shown below to obtain the compound C;

[0110]

[0111] The conditions and steps of the above reduction reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0112] In the above-mentioned reduction reaction, the above-mentioned solvent can be a solvent conventionally used for such reduction reactions in the art. Preferably, the above-mentioned solvent is one or more of a nitrile solvent, an alcohol solvent and an ether solvent. More preferably, the above-mentioned solvent is one or more of acetonitrile, methanol and tetrahydrofuran. Most preferably, the above-mentioned solvent is tetrahydrofuran.

[0113] In the above reduction reaction, the above reducing agent may be a tetrahydrofuran solution of trimethylphosphine which is a reagent commonly used in this type of reduction reaction in the art.

[0114] In the above reduction reaction, the above reduction reaction is carried out with the above compound B as the reaction time, preferably, the reaction time is 4 hours to 8 hours.

[0115] The preparation method of the compound represented by the formula H may further comprise the following steps: in a solvent, in the presence of a strong oxidant, compound A undergoes a degradation reaction as shown below to obtain the compound B;

[0116]

[0117] The conditions of the above-mentioned degradation reaction can refer to the conditions described in the document Chem. Commun., 2010, 46, 2013-2015. The present invention particularly prefers the following conditions:

[0118] In the above-mentioned degradation reaction, the above-mentioned solvent can be a solvent conventionally used for such degradation reactions in the art. Preferably, the above-mentioned solvent is one or more of nitrile solvents and alcohol solvents. More preferably, the above-mentioned solvent is one or more of acetonitrile and methanol. Most preferably, the above-mentioned solvent is methanol.

[0119] In the above-mentioned degradation reaction in the literature, the oxidation system used in the above-mentioned degradation reaction can be a commonly used reagent system of sodium periodate, 1,1-dimethylhydrazine hydrochloride and acetic acid.

[0120] In the above-mentioned degradation reaction, the above-mentioned degradation reaction is carried out for the above-mentioned reaction time, preferably, the reaction time is 40 hours to 60 hours.

[0121] In the above-mentioned degradation reaction, the above-mentioned degradation reaction is carried out at the above-mentioned reaction temperature, preferably, the reaction temperature is 25°C to 80°C.

[0122] The preparation method of the compound represented by the above formula H may further comprise the following steps: in a solvent, in the presence of an azidating agent, sisomicin is subjected to an azidation reaction as shown below to obtain the above compound A;

[0123]

[0124] The conditions and steps of the above-mentioned azidation reaction can be conventional conditions and steps in the art, and the present invention particularly prefers the following conditions:

[0125] In the above-mentioned azidation reaction, the above-mentioned azidation reagent can be sodium azide or 1H-imidazole-1-sulfonyl azide hydrochloride, which are reagents conventionally used in this type of azidation reaction in the art. Preferably, 1H-imidazole-1-sulfonyl azide hydrochloride is selected.

[0126] In the above-mentioned azidation reaction, the above-mentioned catalyst reagent can be the reagent Cu(OAc) conventionally used in this type of azidation reaction in the art. 2 , CuCl 2 CuSO 4 Preferably, the catalyst reagent is CuSO4 .

[0127] In the above-mentioned azidation reaction, the above-mentioned alkaline reagent can be sodium bicarbonate, sodium carbonate, potassium bicarbonate or potassium carbonate, which are reagents conventionally used in this type of azidation reaction in the art. Preferably, the above-mentioned catalyst reagent is sodium bicarbonate.

[0128] In the above-mentioned azidation reaction, the reaction temperature may be a conventional temperature for such azidation reactions in the art. Preferably, the above-mentioned reaction temperature is 0°C to 10°C; more preferably, the above-mentioned reaction temperature is 0°C to 5°C.

[0129] In the above-mentioned azidation reaction, the disappearance of the above-mentioned sisomicin is regarded as the reaction endpoint. Preferably, the reaction time is 4 to 8 hours, and most preferably, it is 6 hours.

[0130] The most preferred embodiment uses the following equations and conditions:

[0131]

[0132] Example 1 Preparation of Compound A

[0133]

[0134] At room temperature, sisomicin sulfate (24.0 g, 43.9 mmol), sodium bicarbonate (21.0 g, 249.9 mmol), copper sulfate (2.32 g, 9.3 mmol) and water (250 mL) were added to the reaction bottle in sequence. The mixture was cooled to 0°C, 1H-imidazole-1-sulfonyl azide hydrochloride (16.0 g, 76.2 mmol) was added in batches, and the mixture was naturally heated to room temperature and stirred for 5 h. The mixture was extracted with dichloromethane (150 mL*3), washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and subjected to silica gel column chromatography to obtain 25.0 g of intermediate compound A (2R, 3R, 4R, 5R)-2-(((1S, 2S, 3R, 4S, 6R)-4, 6-diaza-3-(((2S, 3R)-3-azido-6-(azidomethyl)-3, 4-dihydro-2H-pyran-2-yl)oxy)-2-hydroxycyclohexyl)oxy)-5-methyl-4-(methylamino)tetrahydro-2H-pyran-3, 5-diol (S-1). The purity was 93% as determined by LCMS, and the yield was 84.5%.

[0135] 1H NMR (400MHz, DMSO) δ5.79(d,J=2.3Hz,1H),5.12(d,J=3.5Hz,1H),5.00(dd,J=4.6,2.8Hz,1H),3.9 7~3.88(m,2H),3.73(d,J=4.4Hz,1H),3.70(s,1H),3.67(dd,J=11.0,7.7Hz,2H),3.61(dd,J=7.9, 4.4Hz,3H),3.56(dd,J=9.4,4.2Hz,2H),3.42~3.23(m,4H),3.16(d,J=11.9Hz,1H),2.87(s,1H),2 .32(ddd,J=17.7,11.8,7.4Hz,3H),2.24~2.15(m,2H),1.37(dd,J=24.6,12.3Hz,1H),1.10(s,3H).

[0136] 13C NMR (101MHz, DMSO) δ145.79,98.63,97.88,96.82,79.98,79.50,74.05,71.07, 68.61,67.91,64.03,60.15,59.75,54.49,51.98,37.62,32.89,23.70,21.50. MS-ESI(m / z): 552.2[M+H]+.

[0137] Example 2 Preparation of Compound B

[0138]

[0139] At room temperature, compound A (20.0 g, 36.3 mmol), NaIO4 (25.0 g, 116.8 mmol) and methanol (250 mL) were added to the reaction bottle in sequence, and the mixture was reacted at room temperature for 16 h. Ethanol (50 mL) was added and stirred for 0.5 h. The mixture was filtered and the filtrate was concentrated. Ethanol (200 mL) was added and stirred to dissolve. The mixture was filtered again. 1,1-dimethylhydrazine hydrochloride (37.5 g, 388.4 mmol) was added to the filtrate. The pH was adjusted to about 6 with aqueous NaOH solution. Acetic acid (36 mL) was added and the mixture was heated to reflux for 24 h. The mixture was concentrated and extracted with ethyl acetate (500 mL). The mixture was washed with 2N ethyl acetate. The reaction mixture was added with HCl solution and washed with saturated NaHCO3 solution. The organic phase was dried over anhydrous sodium sulfate, concentrated, and chromatographed on a silica gel column to obtain 10.0 g of a yellow solid intermediate compound B (1S, 2R, 3R, 4S, 6R)-4,6-diazide-3-(((2S, 3R)-3-azido-6-(azidomethyl)-3,4-dihydro-2H-pyran-2-yl)oxy)cyclohexane-1,2-diol (S-2). The purity was 91% as determined by LCMS and the yield was 78%.

[0140] 1H NMR (400MHz, DMSO) δ5.81(d,J=2.4Hz,1H),5.61(d,J=5.8Hz,1H),5.51(d,J= 5.5Hz,1H),4.98(t,J=3.8Hz,1H),3.86(d,J=13.9Hz,1H),3.70(d,J=13.9Hz ,1H),3.62~3.49(m,3H),3.49~3.36(m,2H),3.18(td,J=9.4,5.8Hz,1H),2.3 8~2.21(m,2H), 2.02(dt,J=12.3,4.2Hz,1H), 1.19(dd,J=24.4,12.3Hz,1H).

[0141] 13C NMR (101MHz, DMSO) δ145.77,98.68,96.80,80.04,76.54,76.42,60.60,60.21,54.46,52.04,32.67,21.54. MS-ESI(m / z): 393.2[M+H]+.

[0142] Comparative Example 2 Preparation of Compound C

[0143]

[0144] At room temperature, compound B (10 g, 25.25 mmol), THF (100 mL), piperidine (125 g, 145.66 mmol), trimethylphosphine (126 mL) and aqueous ammonia (15 mL) were added to the reaction flask in sequence, and the reaction was continued for 4-5 h. The reaction was stopped, ethyl acetate was added for extraction, and the mixture was washed with 5% NaCl aqueous solution. The organic phase was dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography to obtain 5.0 g of off-white solid compound C (1S, 2R, 3R, 4S, 6R)-4,6-diamino-3-((2S, 3R)-3-amino-6-(aminomethyl)-3,4-dihydro-2H-pyran-2-yl)oxy)cyclohexane-1,2-diol (S-3). The purity was 94.30% by HPLC, and the yield was 68%.

[0145] 1HNMR(400MHz,D2O)δ5.29(d,J=2.3Hz,1H),4.85(dd,J=4.9,2.4Hz,1H),3.45~3.35(m,2H),3.15(s,2H),3.13~2.99(m,2H),2.80~2 .70(m,1H),2.65(ddd,J=12.0,9.7,4.1Hz,1H),2.16(tt,J=14.4,7.2Hz,1H),1.94(ddd,J=12.9,8.7,3.0Hz,2H),1.23~1.09(m,1H).

[0146] 13C NMR (101MHz, D2O) δ148.51,99.94,96.70,84.27,77.77,76.06,50.34,49.36,46.49,42.29,35.42,24.59.

[0147] MS-ESI (m / z): 289.2 [M+H]+

[0148] Example 3 Preparation of Compound D

[0149]

[0150] At room temperature, weigh the intermediate compound C (4.8 g, 16.65 mmol) and dissolve it in a mixed solvent of DCM (100 mL) and methanol (100 mL), and cool it to 0-5°C. Add PNZ-ONB (4.72 g, 15.8 mmol), react at 0-5°C for 1 h, evaporate dichloromethane under reduced pressure to obtain a crude compound C-1, add 100 mL of methanol, add triethylamine (5.1 g, 50.40 mmol) and zinc acetate (9.15 g, 49.8 mmol), react at 40°C for 1 h, and dropwise add Boc 2O (10.89 g, 49.89 mmol), keep warm at 40 ° C for 3 h, evaporate methanol under reduced pressure, add 25% ammonia water (30 mL) and DCM (100 mL) for separation, extract the aqueous phase with DCM (200 mL) twice, combine the organic phases, and concentrate to dryness to obtain crude compound C-2, add DCM (100 mL), add Boc-(S)-HABA (4.6 g, 20.8 mmol), HOBt (3.38 g, 3.33 mmol), EDCI hydrochloride (4.8 g, 25.0 mmol) and react at room temperature for 16 h. After the reaction, add 100 mL of water, separate the liquids, and extract twice with DCM (40 mL). Combine the organic phases, concentrate and pass through silica gel column chromatography to obtain the product white intermediate compound D 4-Nitrobenzyl (((2S,3R)-3-((tert-butoxycarbonyl)amino)-2-((1R,2R,3S,4R,6S)-6-((tert-butoxycarbonyl)amino)-4-((S)-4-((tert-butoxycarbonyl)amino)-2-hydroxybutyramido)-2,3-dihydroxycyclohexyl)oxy)-3,4-dihydro-2H-pyran-6-yl)methyl)carbamate (PL-1) 2.93 g, purity 70% by LCMS, yield 20%. MS-ESI (m / z): 869.2 [M+H] + .

[0151] Example 4 Preparation of Compound E

[0152]

[0153] At room temperature, weigh the intermediate compound D (2.6 g, 3.01 mmol) and dissolve it in methanol (100 mL) for later use. Prepare NaOH (1.2 g, 30.1 mmol) in another reaction bottle, add 100 mL of water to dissolve, cool to 0-5 °C, and add sodium hydroxide. 2 S 2 O 4(3.65 mg, 21.06 mmol), after stirring and dissolving, the temperature was lowered to 0 ° C, the methanol solution of compound 2 was added dropwise to the reaction solution, the temperature was controlled at 0-5 ° C for 1 h, the temperature was naturally raised to room temperature for 12 h, EtOAc (100 mL) was added and extracted three times, the organic phases were combined, concentrated and subjected to silica gel column chromatography to obtain the product white solid intermediate compound E tert-butyl ((1S, 2R, 3R, 4S, 5R)-2-(((2S, 3R)-6-(aminomethyl)-3-((tert-butoxycarbonyl)amino)-3,4-dihydro-2H-pyran-2-yl)oxy)-5-((S)-4-((tert-butoxycarbonyl)amino)-2-hydroxybutyramido)-3,4-dihydroxycyclohexyl)carbamate (PL-2) 1.75 g, LCMS detection purity 65%, yield 87.5%. MS-ESI (m / z): 690.4 [M+H]+.

[0154] Example 5 Preparation and Synthesis of Compound F

[0155]

[0156] At room temperature, weigh the intermediate compound E (1.40 g, 2.03 mmol) and dissolve it in acetone (50 mL). Add NaHCO 3 (0.487 g, 5.8 mmol) and 2-iodoethanol (438.8 mg, 2.552 mmol) were stirred at 35°C for 16 h, and triethylenediamine (0.715 g, 6.375 mmol) was added and reacted for 16 h. Ethyl acetate (50 mL) and water (10 mL) were added to the reaction solution, stirred and separated, and the aqueous phase was extracted twice with ethyl acetate (90 mL). The organic phases were combined, washed once with saturated brine, concentrated and subjected to silica gel column chromatography to obtain 1.2 g of the product white solid intermediate compound F tert-butyl ((1S, 2R, 3R, 4S, 5R)-5-((S)-4-((tert-butoxycarbonyl)amino)-2-hydroxybutyramide)-2-(((2S, 3R)-3-((tert-butoxycarbonyl)amino)-6-(((2-hydroxyethyl)amino)methyl)-3,4-dihydro-2H-pyran-2-yl)oxy)-3,4-dihydroxycyclohexyl)carbamate (PL-3). LCMS detected a purity of 80% and a yield of 80%. MS-ESI (m / z): 734.2 [M+H]+.

[0157] Example 6 Preparation and Synthesis of Impurity H (i.e., Compound of Formula H)

[0158]

[0159] Under nitrogen protection and stirring conditions, intermediate compound F (0.9 g, 1.224 mmol) and dichloromethane (50 mL) were added, cooled to below 10 ° C in an ice-water bath, trifluoroacetic acid (10 mL) was added and stirred, and the temperature was slowly raised to room temperature for 1 h. LCMS showed that the reaction was complete and the solvent was evaporated under reduced pressure. The product was separated and purified by preparative HPLC, and the product was collected and freeze-dried to obtain 213 mg of white solid impurity H(S)-4-amino-N-((1R,2S,3R,4R,5S)-5-amino-4-(((2S,3R)-3-amino-6-(((2-hydroxyethyl)amino)methyl)-3,4-dihydro-2H-pyran-2-yl)oxy)-2,3-dihydroxycyclohexyl)-2-hydroxybutyramide (PL-4), with a purity of 94.23% and a yield of 40% by HPLC.

[0160] 1H NMR (400MHz, D 2 O) δ5.32(dt,J=28.1,12.3Hz,1H),4.92(t,J=10.5Hz,1H),4.21(ddd,J=33.6,8.0,4.0Hz,1H),3.92~3.59(m,3H),3.57~3.35(m,3H),3. 31~3.07(m,3H),3.06~2.90(m,2H),2.75(ddt,J=18.3,12.7,7.5Hz,3H),2.30~2.14(m,1H),2.11~1.66(m,4H),1.37(q,J=12.3Hz,1H).

[0161] 13C NMR (101MHz, D 2 O) δ175.68,145.79,99.72,99.03,83.53,76.17,74.42,69.70,59.74,49.71,49.52,49.18,49.01,46.40,36.61,33.63,32.22,24.61. MS-ESI(m / z): 434.3[M+H]+.

Claims

1. A prazomicin impurity H, characterized in that: The structure of the plazomicin impurity H is shown below:

2. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: Using sisomicin as a raw material, compound A is formed by aminoazidation reaction; The compound A forms compound B through degradation reaction; The compound B is subjected to a reduction reaction to reduce the azide group thereon to an amino group to form a compound C; The compound C is subjected to selective protection and condensation reaction to form compound D; The compound D selectively removes the protecting group of the amino group protected by PNZ to form a compound E; the compound E undergoes a substitution reaction to form a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of sisomicin is shown below: The structure of the compound A is shown below: The structure of the compound B is shown below: The structure of the compound C is shown below: The structure of the compound D is shown below: The structure of the compound E is shown below: The structure of the compound F is shown below:

3. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: Using azido-sisomicin as a raw material, compound B is formed through degradation reaction; The compound B is subjected to a reduction reaction to reduce the azide group thereon to an amino group to form a compound C; The compound C is subjected to selective protection and condensation reaction to form compound D; The compound D selectively removes the protecting group of the amino group protected by PNZ to form a compound E; the compound E undergoes a substitution reaction to form a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of the azidated sisomicin is shown below: The structure of the compound B is shown below: The structure of the compound C is shown below: The structure of the compound D is shown below: The structure of the compound E is shown below: The structure of the compound F is shown below:

4. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: The compound shown in the following structure is used as a raw material, and the azide group thereon is reduced to an amino group through a reduction reaction to form a compound C; the compound C is subjected to selective protection and condensation reaction to form a compound D; The compound D selectively removes the protecting group of the amino group protected by PNZ to form a compound E; The compound E, through a substitution reaction, forms a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of the raw material is as follows: The structure of the compound C is shown below: The structure of the compound D is shown below: The structure of the compound E is shown below: The structure of the compound F is shown below:

5. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: The compound shown in the following structure is used as a raw material, and a compound D is formed through selective protection and condensation reaction; the compound D is selectively deprotected from the amino group protected by PNZ to form a compound E; the compound E is subjected to a substitution reaction to form a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of the raw material is as follows: The structure of the compound D is shown below: The structure of the compound E is shown below: The structure of the compound F is shown below:

6. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: The compound shown in the following structure is used as a raw material, and the protecting group of the amino group protected by PNZ is selectively removed to form a compound E; the compound E is subjected to a substitution reaction to form a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of the raw material is as follows: The structure of the compound E is shown below: The structure of the compound F is shown below:

7. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: The compound shown in the following structure is used as a raw material, and a substitution reaction is performed to form a compound F; The compound F is subjected to an amino deprotection reaction to form a target product; The structure of the raw material is as follows: The structure of the compound F is shown below:

8. The method for preparing a prazomicin impurity H according to claim 1, characterized in that: The compound shown in the following structure is used as a raw material, and the target product is formed by amino deprotection reaction; The structure of the raw material is as follows:

9. A method for preparing a prazomicin impurity H as claimed in any one of claims 2 to 5, characterized in that: The selective protection and condensation reaction comprises: a selective amino protection reaction of p-nitrobenzyl carbonate group, a selective amino protection reaction of Boc, and a condensation reaction, which are specifically as follows: The intermediate raw material undergoes a selective amino protection reaction of the nitrobenzyl carbonate group to form the transition product 1; The transition product 1 is subjected to a selective amino protection reaction of Boc to form a transition product 2; The transition product 2 undergoes a condensation reaction to form a compound D; Among them, the structure of the intermediate raw material is as follows: The structure of transition product 1 is shown below; The structure of transition product 2 is shown below; 10. The method for preparing a prazomicin impurity H according to claim 2, characterized in that: The reaction conditions for forming compound A via aminoazidation reaction are: Under alkaline conditions, sisomicin and an azidation reagent react at a temperature of 0°C to 10°C for 4-8 hours; and / or The reaction conditions for compound A to form compound B through degradation reaction are: Compound A is reacted under the action of a strong oxidant at a temperature of 25°C to 80°C for 40-60 hours; and / or The reaction conditions for compound B to form compound C through reduction reaction are: Compound B is reacted at room temperature for 4-8 hours in the presence of a reducing agent; and / or The reaction conditions for selectively removing the protecting group of the amino group protected by PNZ from compound D to form compound E are as follows: under alkaline conditions, compound D is reacted at a temperature of 0°C to 10°C for 1-12 hours; and / or The reaction conditions for the formation of compound F by substitution reaction of compound E are: Under alkaline conditions, compound E reacts with a halogenating agent at a temperature of 10° C. to 50° C. for 25 to 50 hours; and / or The reaction conditions for compound F to form the target through amino deprotection reaction are: Under acidic conditions, compound F is reacted at a temperature of 0°C to 30°C for 2-6 hours.

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