Method for semi-solid-phase synthesis of polycyclic compound and intermediate

Abstract

The invention relates to a method for semi-solid-phase synthesis of a polycyclic compound and an intermediate, a long chain is synthesized through solid phase, then two-step ring closing is performed to form a key intermediate, and simple derivation is performed on the basis of the key intermediate to obtain a PCSK9 antagonist compound. According to the invention, starting materials for solid-phase synthesis are selected, steric hindrance is effectively avoided, and solid-phase reaction and subsequent liquid-phase correlation cyclization can be smoothly carried out, so that the yield is improved, and the cost is reduced.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis, specifically to a method for synthesizing polycyclic compounds, and more specifically to a semi-solid-phase synthesis method for compound MK-0616. Background Technology

[0002] MK-0616 is a PCSK9 antagonist compound developed by Merck & Co. for the treatment of cardiovascular diseases and conditions associated with PCSK9 activity, such as atherosclerosis, hypercholesterolemia, coronary heart disease, metabolic syndrome, acute coronary syndrome, or related cardiovascular and cardiovascular metabolic conditions. This compound was first described in WO2019246349A1 and its Chinese counterpart CN112313243A, and is currently in Phase III clinical trials, indicating significant market potential.

[0003] The molecular formula of MK-0616 is shown below, which is compound 1-1 referred to in this invention.

[0004]

[0005] There are few existing descriptions of synthetic methods for this type of compound. The synthetic route described by Merck in patent CN112313243A (priority date June 21, 2018) is shown below. As shown, the ring-closing sequence in this route is "right-middle-left". The literature does not record the yield; the inventors found that ring-closing steps 89 to 90 of the compound was difficult, resulting in a very low yield when repeating the experiment.

[0006]

[0007] Another synthetic route also originated from Merck & Co., see J. Am. Chem. Soc. 2025, 147, 11036-11048. Based on common knowledge in the field, this route was an improvement made by Merck for mass production after the compound entered the clinical stage, and its technical performance should be superior to the compound patent. As shown below, its ring-closing sequence can be described as "left-middle-right". In this method, the yield is only 40% when ring-closing is done on the right side (i.e., when compound 64 is used to prepare compound 65), and 70% when ring-closing is done in the middle (i.e., when compound 63 is used to prepare compound 64), resulting in an overall ring-closing yield of only 28%.

[0008]

[0009] These molecules possess multiple macrocycles and multiple chiral centers, making their synthesis relatively difficult. During the synthesis process, after the formation of the polycyclic structure, derivatization and modification of functional groups can be carried out according to the methods described in CN112313243A or other common knowledge in the field. The main technical challenge lies in how to form the polycyclic structure in high yield and at low cost, i.e., to prepare the intermediate compound II-e. Summary of the Invention

[0010] Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art, thereby providing a method for synthesizing a compound of formula I or a pharmaceutically acceptable salt thereof, wherein the compound of formula I has the following structure

[0011]

[0012] in:

[0013] R 1 Selected from:

[0014] (a)-H; or

[0015] (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for:

[0016] (i)-H;

[0017] (ii)–NH2;

[0018] (iii)-N + H3;

[0019] (iv)-N + (CH3)3;

[0020] (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0021] (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0022] (vii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C For -O-(CH2) 3-4 -N + (CH3)3; and

[0023] (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for:

[0024] (ai)–O-(CH2)2-N + (CH3)3;

[0025] (aii)-N + (CH3)3;

[0026] (aiii) The following part:

[0027]

[0028] R 2 Selected from:

[0029] (a)-H; and

[0030] (b)-(CH2)zR 14A Where: z is 1-6, and R 14A Selected from:

[0031] (i)-H;

[0032] (ii)–NH2;

[0033] (iii)-N + H3;

[0034] (iv)-N + (CH3)3;

[0035] (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0036] (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3;

[0037] (vii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C -O-(CH2)3-4-N + (CH3)3; and

[0038] (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for:

[0039] (ai)–O-(CH2)2-N + (CH3)3;

[0040] (aii)-N + (CH3)2R 14ca , where R 14ca For –CH3 or –(CH2) 1-4 -OCH3;

[0041] (aiii) The following part:

[0042]

[0043] (aiv) The following part:

[0044]

[0045] Where R 14Cb and R 14Cc The range is 1 to 4;

[0046] A is selected from: -CH2CH2- or -CH=CH-;

[0047] R 8 Selected from: C1-6 alkyl groups, wherein the C1-6 alkyl group can be -OR 8a Instead, the R 8a Selected from H and straight-chain, branched, or cyclic alkyl groups with no more than 6 carbon atoms;

[0048] X is selected from H, F, Cl, or Br;

[0049] The term "salt" as used herein, and its use in the phrase "pharmaceutically acceptable salt," includes any of the following: acid salts formed with inorganic and / or organic acids, base salts formed with inorganic and / or organic bases, zwitterions, and quaternary ammonium complexes. Salts of the compounds of the present invention can be formed by methods known to those skilled in the art, for example by reacting the compounds of the present invention with a certain amount (e.g., a certain equivalent) of an acid or base in a medium such as salt precipitation or an aqueous medium, followed by lyophilization.

[0050] Examples of pharmaceutically acceptable acid salts include, but are not limited to, acetates (including trifluoroacetate), adipates, alginates, ascorbic acid salts, aspartate salts, benzoates, benzenesulfonates, hydrogen sulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, diglucuronates, dodecyl sulfates, ethanesulfonates, fumarates, glucohepate, glycerophosphates, hemisulfates, heptarates, hexanoates, hydrochlorides, hydrobromide, hydroiodates, 2-hydroxyethanesulfonate, lactates, maleates, methanesulfonates, methyl sulfates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, dihydroxynaphthalate, pectates, persulfates, 3-phenylpropionate, phosphates, picrates, neopentanoates, propionates, salicylates, succinates, sulfates, sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates (also known as tosylates, undecanoates, etc.).

[0051] Examples of pharmaceutically acceptable alkali salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium, lithium, and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts, aluminum salts, and zinc salts), salts containing organic bases (e.g., organic amines) (such as benzathines, diethylamine, dicyclohexylamine, hydrabamines (formed from N,N-bis(dehydrorosinyl)ethylenediamine), N-methyl-D-glucosamine, N-methyl-D-glucosamide, tert-butylamine, piperazine, phenylcyclohexylamine, choline, and tromethamine), and salts containing amino acids (such as arginine and lysine). Basic nitrogen-containing groups can be converted into ammonium ions or quaternized with agents such as: lower alkyl halides (e.g., methyl, ethyl, propyl and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl and dipentyl sulfates), long-chain halides (e.g., decyl, dodecyl, tetradecyl and octadecyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides) and other agents.

[0052] The term "pharmaceutically acceptable anion" refers to an anion suitable for forming pharmaceutically acceptable salts.

[0053] The method comprises the following steps:

[0054] (1) Preparation of intermediate compound II-a, wherein intermediate compound II-a has the following structure:

[0055]

[0056] In formulas II-a and II-b, the sphere represents the solid phase support;

[0057] R 1a Selected from:

[0058] (a)-H; or

[0059] (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for:

[0060] (i)-H;

[0061] (ii)–NH-amino protecting group;

[0062] (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B It is a -NH-amino protecting group;

[0063] (iv)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B It is a -NH-amino protecting group;

[0064] (v)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for:

[0065] (ai) The following part:

[0066]

[0067] (2) The Fmoc protecting group was removed to obtain intermediate compound II-b, which has the following structure:

[0068]

[0069] (3) The solid support is removed to obtain intermediate compound II-c, which has the following structure:

[0070]

[0071] (4) The intermediate compound II-c undergoes a two-step cyclization process to obtain the intermediate compound II-d, which has the following structure:

[0072]

[0073] (5) Optionally, the C=C double bond formed in step (4) is reduced to obtain intermediate compound II-e, which has the following structure:

[0074]

[0075] (6) Use intermediate compounds II-d or II-e to prepare compounds of formula I.

[0076] Preferably, the R 1 Selected from:

[0077] (a)-H; or

[0078] (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for:

[0079] (i)-H;

[0080] (ii)-N + (H3C)3;

[0081] (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3;

[0082] Preferably, the R 2 Selected from:

[0083] (a)-H; and

[0084] (b)-(CH2)zR 14A Where: z is 1-6, and R 14A Selected from:

[0085] (i)-H;

[0086] (ii)–NH2;

[0087] (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: or -N+ (CH3)3;

[0088] (iv)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C For -O-(CH2) 3-4 -N + (CH3)3; and

[0089] (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for:

[0090] (ai)–O-(CH2)2-N + (CH3)3;

[0091] (aii)-N+(CH3)2R 14ca , where R 14ca For –CH3 or –(CH2) 1-4 -OCH3;

[0092] (aiii) The following part:

[0093]

[0094] Preferably, the R 8 For -CH3 or the following part:

[0095]

[0096] Where R 8a It is -H, or a straight-chain, branched, or cyclic alkyl group with up to four carbon atoms.

[0097] More preferably, the compound of formula I is compound 1-1.

[0098] In step (1), the solid support is preferably a resin, more preferably a 2-Cl-Trt resin.

[0099] Step (1) is a solid-phase synthesis, and the target product has multiple amide bonds, which can be carried out by those skilled in the art using conventional methods and reagents to catalyze the reaction. Preferably, HATU and DIEA reagents are used to generate amide bonds through a condensation reaction, and compound II-a is obtained through solid-phase synthesis.

[0100] The present invention also provides intermediate compounds II-a, II-b, II-c, II-d and II-e, wherein, preferably, compound II-a is compound II-a-1 and compound II-b is compound II-b-1.

[0101]

[0102]

[0103] The present invention also provides a method for preparing a compound of formula I or a pharmaceutically acceptable salt thereof using any one of intermediate compounds II-a, II-b, II-c, II-d, II-e, with the preferred compound of formula I being compound 1-1.

[0104] The present invention also provides a method for preparing compound II-c, namely, preparing compound II-c using the first (3) step of the aforementioned method for preparing compound I.

[0105] The present invention also provides a method for preparing compound II-e, namely, using compound II-c as a raw material and using steps (4) and (5) of the aforementioned method for preparing compound I to prepare compound II-e.

[0106] Those skilled in the art will readily understand that, after obtaining compound II-e, based on the specific structure of the desired compound of formula I, reference can be made to CN112313243A, and existing methods and reagents can be used to prepare R. 2 R 1a and R 8 The double bond at point A may be restored or not, and the methods of modification and alteration will not be detailed in this invention.

[0107] In this invention, unless otherwise specified, "fragment" refers to a part of an organic compound. For example, when referring to an amino acid fragment, it refers to the residue after removing one H of the amino group and the OH of the carboxyl group.

[0108] Those skilled in the art will readily understand that "Compound of Formula I" and "Compound I" have the same meaning, and the compound designations used in this invention are for convenience only and do not constitute a substantive limitation. The scope of protection of the claims should be determined by the structure of the compound rather than its designation.

[0109] Those skilled in the art will readily understand that, in order to avoid undesirable side reactions, R in this invention... 1a The "amino protecting group" appearing on the substituent refers to an amino protecting group other than Boc and Fmoc. Optional amino protecting groups include, but are not limited to, Trt, Alloc, Teoc, meth / ethoxycarbonyl, Pht, Tfa, Dmb, and PMB.

[0110] In this invention, only the hydrochloride salt of compound 1-1 was prepared. Those skilled in the art can add other acids as needed to obtain the desired salt.

[0111] The technical solution of this invention has the following advantages:

[0112] 1. The semi-solid phase synthesis method provided by this invention uses solid phase synthesis to obtain long-chain polypeptide structures, and performs cyclization in the liquid phase, which improves the yield, shortens the reaction time, and reduces the cost.

[0113] 2. The intermediate provided by this invention, as a key intermediate in semi-solid phase synthesis, can be obtained simply by filtration, which is convenient for purification and can effectively improve the yield and reduce the impurity content when synthesizing this type of compound.

[0114] 3. This invention selects the starting materials for solid-phase synthesis, effectively avoiding steric hindrance, so that the solid-phase reaction and subsequent liquid-related loops can proceed smoothly.

[0115] 4. In this invention, when closing the loop, the outer loop is closed first, and the middle double bond is connected last, resulting in a yield that exceeds expectations. Attached Figure Description

[0116] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some exemplary embodiments of the present invention.

[0117] Figure 1 This is a flowchart of the solid-phase synthesis in Embodiment 3 of the present invention;

[0118] Figure 2 This is the 1H NMR spectrum of compound 1-1 of the present invention;

[0119] Figure 3 This is the C10 NMR spectrum of compound 1-1 of the present invention. Detailed Implementation

[0120] Unless otherwise specified, all chemicals used in this application were purchased directly from reagent companies. Reactions without specific instructions were conducted under argon protection. Purification reagents and solvents were performed according to the Purification of Laboratory Chemicals. Reaction progress was monitored by TLC using HSGF 254 silica gel plates (0.2 mm). The silica gel plates were observed under UV light, and color development was performed in phosphomolybdic acid ethanol solution or ninhydrin solution. The silica gel used for column chromatography was Qingdao Marine Silica Gel 60 (300-400 mesh). The eluent used for column chromatography separation was generally a petroleum ether / ethyl acetate or dichloromethane / methanol system, prepared by volume ratio. 1 H NMR and 13C10 NMR was performed using a Bruker Avance 400MHz and a Bruker Avance 600MHz NMR spectrometer, with deuterated chloroform, deuterated methanol, and deuterated DMSO as solvents. High-resolution mass spectrometry was performed using a Q Exactive Focus (Thermo) under ESI conditions.

[0121] The abbreviations and sources of the reagents involved in this invention are shown in Table 1. Those whose preparation methods are not described in the specification are commercially available reagents, or are prepared from commercially available reagents through simple group protection.

[0122]

[0123] Example 1

[0124] Synthesis of compounds 1-6.

[0125]

[0126] The commercially available compound 3-10 (400 mg, 1.01 mmol, 1.0 eq.) was dissolved in DMF (10 mL, 0.1 M). The reaction flask was cooled to 0 °C in an ice bath. DIPEA (0.35 mL, 2.02 mmol, 2.0 eq.) and AllylBr (0.13 mL, 1.52 mmol, 1.5 eq.) were added. After removing the ice bath, the mixture was allowed to rise naturally to room temperature and stirred for 3 hours. The experiment was monitored by TLC column chromatography. When the starting material disappeared, water (50 mL) was added, and the mixture was extracted with ethyl acetate (2 × 50 mL). The organic phase was washed with water (3 × 50 mL) and saturated brine (50 mL), and dried over anhydrous Na₂SO₄. After filtration and concentration under reduced pressure, the crude product was obtained. After column chromatography, the purified colorless oily product (410 mg, yield 93.8%) was finally obtained. TLC: R f =0.2 (silica gel, ethyl acetate / petroleum ether = 1:10). 1H NMR(400MHz,Chloroform-d)δ7.75(d,J=7.5Hz,2H),7.63(t,J=7.8Hz,2H),7.38(t,J=7.6Hz,2H ),7.31(t,J=7.5Hz,2H),5.99–5.84(m,1H),5.64(d,J=9.7Hz,1H),5.34(d,J=17.1Hz,1H),5.25 (d,J=8.5Hz,1H),4.68(dd,J=13.4,5.6Hz,1H),4.54(dd,J=13.6,6.0Hz,1H),4.42(ddd,J=9.8, 7.2, 2.3Hz, 1H), 4.40–4.29 (m, 1H), 4.30–4.05 (m, 3H), 1.27–1.21 (m, 3H), 1.13 (d, J = 2.5Hz, 9H). 13 C NMR (101MHz, CDCl3) δ170.9,156.8,143.8,141.3,131.6,127.7,127.1,125.2,120.0 ,119.0,74.1,67.4,67.2,66.0,60.0,47.2,28.4,21.0.HRMS:(ESI,m / z);calculated for C 26 H 31 NO5Na + [M+Na] + :460.2094,found:460.2031.

[0127] The product obtained in the previous step (410 mg, 0.94 mmol, 1.0 eq.) was dissolved in MeCN (10 mL, 0.09 M), and Et₂NH (0.19 mL, 1.86 mmol, 2.0 eq.) was added. The mixture was stirred overnight at room temperature. The experiment was monitored by TLC column chromatography. When the starting material spot disappeared, the reaction solution was concentrated under reduced pressure using a vacuum water pump to obtain the crude product, which was used directly in the next step.

[0128] The product obtained in the previous step was dissolved in DMF (10 mL, 0.09 M). The reaction flask was cooled to 0 °C in an ice bath. Compounds 1-2 (611 mg, 0.75 mmol, 0.8 eq.), DIPEA (0.39 mL, 2.26 mmol, 2.4 eq.), and HATU (429 mg, 1.13 mmol, 1.2 eq.) were added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour. The experiment was monitored by TLC column chromatography. When the starting material spot disappeared, a saturated NaHCO3 aqueous solution was added to quench the reaction. The mixture was extracted with ethyl acetate (2 × 50 mL), and the organic phase was washed with water (3 × 50 mL) and saturated brine (50 mL). The mixture was dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the crude product was obtained. After column chromatography, the purified yellow amorphous product (830 mg) was obtained. TLC: R f =0.2 (silicagel, dichloromethane / methanol = 50:1).

[0129] The compound obtained in the previous step (830 g, 0.82 mmol, 1.0 eq.) was dissolved in anhydrous DCM (10 mL, 0.08 M). The reaction flask was cooled to 0 °C in an ice bath. PhSiH3 (0.50 mL, 4.10 mmol, 5.0 eq.) and Pd(PPh3)4 (95 mg, 0.08 mmol, 0.1 eq.) were added. The reaction was maintained at 0 °C and stirred for 40 minutes. The experiment was monitored by TLC thin-layer column chromatography. When the starting material spot disappeared, the reaction solution was concentrated under reduced pressure to obtain the crude product. After column chromatography separation, the purified yellow amorphous compound 1-6 (758 mg, total yield of three steps 83.4%) was obtained. TLC: R f =0.2 (silica gel, dichloromethane / methanol / formic acid = 10:1:0.01). 1H NMR(400MHz,Chloroform-d)δ7.98(d,J=22.7Hz,2H),7.77(d,J=7.6Hz,2H),7.64(d,J=5.4Hz,2H),7.39(t,J=7.6Hz,2H),7.36–7.20(m,3H),7.08(dt,J=23.4,11.6Hz,4H),6.97–6.82(m,2H),6.82–6.68(m,2H),5.84(ddt,J=16.8,11.0,5.4Hz,1H),5.62(d,J=8.2Hz,1H),5.28(d,J=2.2Hz,1H),5.08(t,J=9.0Hz,3H),4.87(t,J=20.3Hz,3H),4.62–4.50(m,4H),4.36(s,2H),4.26(q,J=7.4,6.8Hz,3H),4.15(d,J=14.8Hz,1H),3.96(d,J=15.2Hz,1H),3.79(d,J=9.5Hz,1H),3.63(d,J=2.2Hz,1H),3.21–3.09(m,2H),3.01(t,J=6.9Hz,2H),2.89(d,J=13.3Hz,1H),2.12–2.07(m,2H),1.13(d,J=6.3Hz,14H). 13 C NMR(101MHz,CDCl3)δ172.90,172.01,170.04,168.66,163.41,159.05,156.72,155.90,144.06,143.88,141.46,137.20,135.99,134.87,133.45,132.90,130.78,129.21,128.91,128.73,128.35,128.26,128.12,127.86,127.19,126.70,125.35,125.24,120.13,120.09,117.11,110.70,110.40,108.66,103.66,103.43,79.62,77.48,77.16,76.84,75.38,67.47,67.23,64.53,57.79,54.55,53.56,50.97,50.63,49.01,47.29,45.42,42.39,38.91,30.84,29.69,28.33,23.62,19.80,11.50.HRMS:(ESI,m / z);calculated for C 54 H 59 FN6O10 Na + [M+Na] + :993.4169,found:993.4163.

[0130] Example 2

[0131] Synthesis of polypeptide fragment compound 8-1

[0132] Synthesis of compound 4-2:

[0133]

[0134] n-BuLi (5.87 mL, 14.67 mmol, 2.2 eq.) was dissolved in anhydrous THF (10 mL) and added to the reaction flask, which was then cooled to -78 °C. Commercially available compound 4-1 (2.00 g, 6.67 mmol, 1.0 eq.) was dissolved in anhydrous THF (50 mL) and slowly added dropwise to the reaction flask, maintaining the reaction temperature at -78 °C with stirring for 1 hour. Anhydrous DMF (1.29 mL, 16.68 mmol, 2.5 eq.) was added to the reaction flask, and the reaction temperature was maintained at -78 °C with stirring for another 1 hour. The experiment was monitored using TLC column chromatography. When the starting material disappeared, water was added to quench the reaction. The reaction solution was concentrated under reduced pressure using a vacuum water pump, and the aqueous phase was extracted with ethyl acetate (2 × 100 mL). The organic phase was washed with saturated brine (50 mL) and dried over anhydrous Na₂SO₄. After filtration and concentration under reduced pressure, the crude product was obtained. Following column chromatography, the purified white solid product 4-2 (1.26 g, yield 75.8%) was finally obtained. TLC: R f =0.2 (silica gel, ethyl acetate / petroleum ether = 1:5).

[0135] Synthesis of compound 4-4:

[0136]

[0137] Hydroxylamine hydrochloride (335 mg, 4.82 mmol, 2.0 eq.) was dissolved in EtOH (20 mL), added to a reaction flask, and then Et3N (1.00 mL, 7.23 mmol, 3.0 eq.) was added. The mixture was stirred at room temperature for 30 minutes. Then, compound 4-2 (600 mg, 2.41 mmol, 1.0 eq.) was added, and the mixture was stirred at room temperature for 1 hour. The experiment was monitored by TLC column chromatography. When the starting material spot disappeared, the reaction solution was concentrated under reduced pressure using a vacuum water pump, dissolved in water, and the aqueous phase was extracted with ethyl acetate (2 × 100 mL). The organic phase was washed with saturated brine (50 mL) and dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the crude product was obtained. After column chromatography, the purified white solid product (570 mg) was finally obtained. TLC: R f =0.2 (silica gel, ethyl acetate / petroleum ether = 1:4).

[0138] The product obtained in the previous step was dissolved in a mixed solvent of AcOH (15 mL) / EtOH (15 mL), and Pd / C (50 mg) was added. The mixture was then aerated three times using a vacuum water pump to remove all O2 from the reaction system. The mixture was stirred under H2 conditions for 6 hours. The experiment was monitored using TLC thin-layer column chromatography until the starting material was completely eliminated. The mixture was then filtered with diatomaceous earth, and the filtrate was concentrated to obtain the crude product, which was directly used in the next step.

[0139] Compound 4-3 obtained in the previous step was dissolved in ACN (30 mL), and propylene bromide (0.30 mL, 2.50 mmol, 1.2 eq.) was added. The mixture was then refluxed at 80 °C for 48 hours. The experiment was monitored by TLC thin-layer column chromatography. When the starting material spot disappeared, the reaction solution was concentrated under reduced pressure using a vacuum water pump to obtain the crude product. After column chromatography separation, the purified colorless oily product 4-4 (310 mg, total yield of three steps 40.4%) was finally obtained. TLC: R f =0.2 (silica gel, dichloromethane / methanol = 20:1).

[0140] Synthesis of compounds 4-6:

[0141]

[0142] Compound 4-4 (250 mg, 0.79 mmol, 1.0 eq.) was dissolved in DMF (10 mL, 0.08 M). The reaction flask was cooled to 0 °C in an ice bath. Compound 4-5 (125 mg, 0.94 mmol, 1.2 eq.), DIPEA (0.33 mL, 1.88 mmol, 2.4 eq.), and HATU (358 mg, 0.94 mmol, 1.2 eq.) were added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour. The experiment was monitored by TLC column chromatography. When the starting material disappeared, a saturated NaHCO3 aqueous solution was added to quench the reaction. The mixture was extracted with ethyl acetate (2 × 50 mL), and the organic phase was washed with water (3 × 50 mL) and saturated brine (50 mL). The solution was dried over anhydrous Na2SO4. After filtration and concentration under reduced pressure, the crude product was obtained. After column chromatography, the purified white amorphous product 4-6 (281 mg, yield 82.4%) was finally obtained. TLC: R f =0.2 (silica gel, ethyl acetate / petroleum ether = 1:4).

[0143]

[0144] Compounds 4-6 (194 mg, 0.45 mmol, 1.0 eq.) were dissolved in a mixed solvent of THF (3 mL) / MeOH (3 mL) / H₂O (3 mL). The reaction flask was cooled to 0 °C in an ice bath, and LiOH (32 mg, 1.35 mmol, 3.0 eq.) was added. The reaction was maintained at 0 °C and stirred overnight. The experiment was monitored by TLC column chromatography. When the starting material disappeared, the organic phase was concentrated under reduced pressure and evaporated to dryness. The pH was adjusted to 5-6 with 1 M HCl aqueous solution in an ice bath. The aqueous phase was extracted with ethyl acetate (2 × 50 mL), and the organic phase was washed with saturated brine (50 mL) and dried over anhydrous Na₂SO₄. The crude product was obtained by concentration under reduced pressure and used directly in the next step.

[0145] The product obtained in the previous step was dissolved in a mixed solvent of TFA (2 mL) / DCM (10 mL) and stirred at room temperature for 2 hours. The experiment was monitored by TLC thin-layer column chromatography until the starting material was completely eliminated. Then, the reaction solution was concentrated under reduced pressure using a vacuum water pump to obtain the crude product, which was used directly in the next step.

[0146] The product obtained in the previous step was dissolved in a mixed solvent of 1,4-dioxane (5 mL) / H2O (5 mL). The reaction flask was cooled to 0°C in an ice bath. Na2CO3 (72 mg, 0.68 mmol, 1.5 eq.) and Fmoc OSu (167 mg, 0.50 mmol, 1.1 eq.) were added. After removing the ice bath, the mixture was allowed to rise naturally to room temperature and stirred for 6 hours. The experiment was monitored by TLC column chromatography. When the starting material spot disappeared, the reaction solution was concentrated under reduced pressure using a vacuum water pump, then diluted with water (15 mL), and the pH was adjusted to 3-4 with 1M HCl aqueous solution in an ice bath. The aqueous phase was extracted with ethyl acetate (2 × 50 mL), the organic phase was washed with saturated brine (50 mL), and dried over anhydrous Na2SO4. After filtration, the crude product was concentrated under reduced pressure. After column chromatography, the purified white solid product 8-1 (197 mg, total yield of three steps 81.2%) was finally obtained. TLC: R f =0.2 (silica gel, dichloromethane / methanol / formic acid = 40:1:0.01). 1 H NMR(400MHz,Chloroform-d)δ10.33(s,1H),7.73(d,J=7.6Hz,2H),7.54(d,J=7.6Hz,2H),7.37(t,J=7.6Hz ,2H),7.28(t,J=7.7Hz,2H),7.21–6.93(m,4H),5.81–5.68(m,1H),5.24(d,J=2.2Hz,1H),5.09–5.04(m,1H ),4.96(dt,J=22.5,8.8Hz,2H),4.51(d,J=31.4Hz,2H),4.37(t,J=7.3Hz,1H),4.17(d,J=6.9Hz,1H),3.43 –3.29(m,2H),3.18(t,J=8.0Hz,1H),2.90–2.51(m,6H),1.99(q,J=7.9,7.2Hz,2H),1.61(h,J=7.8Hz,2H). 13 CNMR(101MHz, CDCl3)δ177.0,172.1,156.5,143.9,141.3,137.7,137.0,135.4,129.4,129.0,128.0,127.7,127.0,126.5,1 25.1,125.0,120.0,115.8,115.0,66.6,53.5,48.5,47.2,42.2,35.7,30.7,29.6,27.8,27.2.HRMS: (ESI, m / z); calculated for C 33 H 36N2O5Na + [M+Na] + :563.2516,found:563.2512.

[0147] Example 3

[0148] Solid-phase synthesis.

[0149] As per the instruction manual Figure 1 As shown, the solid-phase synthesis process includes the following steps.

[0150] A is equipped with resin

[0151] Weigh 50 mg of 2-Cl-Trt resin (sample loading capacity 0.565 mmol / g, subsequent reaction equivalence will be based on this) and add it to a solid-phase synthesis tube. Add 2 mL of dry DCM and react on a shaker for 30 minutes. Filter the liquid through the bottom sintering core. Mix the first polypeptide fragment 1-6 (compound 1-6 prepared in Example 1, 25 mg, 0.0258 mmol, 1.0 eq.) with DIEA (10 μL, 0.0515 mmol, 2.0 eq.), dissolve in 2 mL of dry DCM, and add to the synthesis tube. React on a shaker for 12 h. Drain the liquid, add 2 mL of DCM / MeOH / DIEA (17:2:1) solution for end-capping, react on a shaker for 1 h, drain the liquid, and wash successively with DCM and DMF.

[0152] B Fmoc removal

[0153] Add 2 mL of piperidine / DMF (1:4) solution to the synthesis tube, shake for 1 hour, and wash three times with DCM and DMF in sequence.

[0154] Linkage of C amino acid fragments

[0155] Following steps A and B, five amino acid fragments—fragment 5-1, Fmoc-Ala-OH, fragment 8-1, fragment 4-7, and fragment 4-10—were sequentially linked. Fmoc-Ala-OH was a commercially available protected Fmoc compound, and fragment 8-1 used compound 8-1 prepared in Example 2. The other fragments used the following compounds as starting materials.

[0156]

[0157] The ligation reactions were performed by mixing amino acids / HATU / DIEA (4 eq / 4 eq / 8 eq) in 2 ml of DMF solution and adding the mixture to the synthesis tube. Each amino acid was reacted for 2 h. After the reaction, the cells were washed sequentially with DCM and then DMF. The Fmoc fragment was removed before ligating the next fragment.

[0158] After connecting all the fragments, compound II-a-1 was obtained;

[0159]

[0160] Compound II-b-1 was obtained by removing Fmoc protection following step A;

[0161]

[0162] D-fraction

[0163] Add 2 mL of lysis buffer DCM / AcOH / TFE (8:1:1) solution to the synthesis tube, react for 30 minutes, collect the filtrate, repeat three times, combine the filtrates into a round-bottom flask, and concentrate under reduced pressure to obtain compound II-c-1.

[0164]

[0165] Example 4

[0166] Liquid phase cyclization 1 (close the right side).

[0167] The product II-c-1 obtained in the previous step was dissolved in a mixed solvent DCM (100 mL) / DMF (5 mL), and HATU (9 mg, 0.0230 mmol, 1.2 eq) and DIEA (17 μL, 0.0958 mmol, 5.0 eq) were added. The reaction was carried out at room temperature for 2 hours, and the experiment was monitored by TLC column chromatography. When the starting material spot disappeared, the reaction was quenched by adding saturated NaHCO3 aqueous solution. The mixture was extracted with ethyl acetate (2 × 20 mL), and the organic phase was washed with water (3 × 20 mL) and saturated brine (20 mL). The mixture was dried over anhydrous Na2SO4. After filtration, the crude product was concentrated under reduced pressure. After column chromatography, the purified white amorphous product 7-3 (15 mg, total yield 37%) was obtained. TLC: Rf = 0.2 (silica gel, dichloromethane / methanol = 20:1).

[0168] Example 5

[0169] Liquid-phase cyclization 2 (intermediate cyclization).

[0170] Synthesis of compound 7-4:

[0171]

[0172] Compound 7-3 (25 mg, 0.016 mmol) was dissolved in DCM (5 mL). Zhan (1 B) (1 mg) was added at room temperature, and the reaction temperature was raised to 45 °C and stirred overnight. The experiment was monitored using TLC (thin-layer chromatography). When the starting material disappeared, the reaction solution was concentrated under reduced pressure using a vacuum water pump to obtain the crude product. After column chromatography separation, the purified yellow solid product 7-4 (20 mg, yield 81.3%) was obtained. TLC: R f =0.2 (silica gel, dichloromethane / methanol = 20:1).

[0173] Example 6

[0174] Synthesis of compound 1-1:

[0175]

[0176] Compound 7-4 was dissolved in MeOH (5 mL), and ethyl acetate solution of hydrochloric acid (1 mL) and Pd / C (5 mg) were added. After mixing and stirring for 30 minutes, the mixture was ventilated three times using a vacuum water pump to remove all O2 from the reaction system. The mixture was then stirred overnight under H2 conditions. The experiment was monitored using TLC thin-layer column chromatography until the starting material was completely eliminated. The mixture was then filtered through diatomaceous earth, and the filtrate was concentrated to obtain the crude product, which was used directly in the next step.

[0177] The product obtained in the previous step was dissolved in a mixed solvent of TFA (1 mL) / DCM (5 mL) and stirred at room temperature for 6 hours. The reaction was monitored by LCMS until the starting material was completely eliminated. Then, the reaction solution was concentrated under reduced pressure using a vacuum water pump to obtain a crude product, which was used directly in the next step.

[0178] The product obtained in the previous step was dissolved in ACN (5 mL), and the reaction flask was cooled to 0 °C in an ice bath. Compounds 1-5 (5 mg, 0.02 mmol, 1.2 eq.), DIPEA (0.02 mL, 0.11 mmol, 5.0 eq.), and HATU (9 mg, 0.02 mmol, 1.2 eq.) were added. The ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour. The reaction was monitored by LCMS until the starting material was completely eliminated. After adding IPAc (20 mL), a white solid precipitated. The precipitate was filtered, washed with IPAc, and collected. The precipitate was then purified by HPLC to obtain the purified trifluoroacetate as a white solid product. This was then converted to the hydrochloride salt of the final product 1-1 (15 mg, overall yield of four steps: 41.5%) using a hydrochloric acid-methanol solution. 1H NMR(400MHz,Methanol-d4)δ7.48–7.37(m,1H),7.33(s,1H),7.21(d,J=26.1Hz,4H),7.12(d,J=18.3Hz,2H),7.05–6.97(m,4H),6.90(d,J=7.8Hz,4H),5.35(d,J=5.2Hz,1H),4.71(s,2H),4.69–4.31(m,2H),4.28(s,2H),4.26–4.20(m,5H),4.15(t,J=15.0Hz,1H),4.09–3.97(m,1H),3.86–3.81(m,1H),3.80(s,5H),3.72(s,1H),3.69–3.42(m,2H),3.12(s,16H),3.10(s,8H),3.07–2.98(m,1H),2.98–2.75(m,3H),2.70(s,1H),2.58(d,J=19.5Hz,1H),2.23(dd,J=23.0,7.4Hz,5H),2.04(t,J=6.3Hz,3H),1.94(s,1H),1.83–1.75(m,7H),1.68(q,J=7.3Hz,4H),1.48(s,3H),1.39(d,J=9.0Hz,2H),1.29(s,6H),1.17(d,J=5.0Hz,4H),1.10–0.97(m,2H),0.91(d,J=5.4Hz,3H). 13C NMR(201MHz,MeOD)δ176.2,175.7,175.4,174.4,173.8,173.4,171.5,171.1, 170.8,170.5,170.0,169.8,159.9,158.1,139.6,139.2,137.5,136.7,133.2 ,132.1,131.4,130.8,130.6,130.3,130.1,129.8,129.6,128.8,128.3,114.8,112.5,111.8,110.2,104.9,81.8,68.8,68.7,67.4,64.1,57.2,55.5,54.6 ,53.3,51.8,51.4,49.2,49.0,48.9,48.8,48.7,48.6,48.5,47.2,46.7,45.5,43.6,40.6,38.4,36.8,36.5,36.2,36.0,35.2,34.6,32.8,30.6,30.5,30.5 ,30.4,30.4,30.2,30.1,28.8,28.5,27.9,26.7,26.5,26.4,25.9,25.8,23.5 ,23.4,23.4,21.7,21.4,20.4,19.7,17.6,17.0.HRMS:(ESI,m / z);calculated for C 82 H 110 FN 14 O 15 + [M] + :1549.8254,found:1549.8256.

[0179] Examples 1-6 illustrate a method for synthesizing compound 1-1. Those skilled in the art can use this method to synthesize other compounds with similar polycyclic structures through simple substituent changes. Since CN112313243A has disclosed the pharmaceutical uses of this type of compound, its synthesis is industrially useful.

[0180] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for synthesizing a compound of formula I or a pharmaceutically acceptable salt thereof, said compound of formula I having the following structure: in: R 1 Selected from: (a)-H; or (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for: (i)-H; (ii)–NH2; (iii)-N + H3; (iv)-N + (CH3)3; (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3; (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3; (vii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C For -O-(CH2) 3-4 -N + (CH3)3; and (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N + (CH3)3; (aiii) The following part: R 2 Selected from: (a)-H; and (b)-(CH2)zR 14A Where: z is 1-6, and R 14A Selected from: (i)-H; (ii)–NH2; (iii)-N + H3; (iv)-N + (CH3)3; (v)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3; (vi)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B For: -NH2; -N + H3; -N(CH3)2; or -N + (CH3)3; (vii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C -O-(CH2)3-4-N + (CH3)3; and (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N + (CH3)2R 14ca , where R 14ca For –CH3 or –(CH2) 1-4 -OCH3; (aiii) The following part: (aiv) The following part: Where R 14Cb and R 14Cc The range is 1 to 4; A is selected from: -CH2CH2- or -CH=CH-; R 8 Selected from: C1-6 alkyl groups, wherein the C1-6 alkyl group can be -OR 8a Instead, the R 8a Selected from H and straight-chain, branched, or cyclic alkyl groups with no more than 6 carbon atoms; X is selected from H, F, Cl, or Br; The method comprises the following steps: (1) Preparation of intermediate compound II-a, wherein intermediate compound II-a has the following structure: In formulas II-a and II-b, the sphere represents the solid phase support; R 1a Selected from: (a)-H; or (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for: (i)-H; (ii)–NH-amino protecting group; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B It is a -NH-amino protecting group; (iv)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B It is a -NH-amino protecting group; (v)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai) The following part: (2) The Fmoc protecting group was removed to obtain intermediate compound II-b, which has the following structure: (3) The solid support is removed to obtain intermediate compound II-c, which has the following structure: (4) The intermediate compound II-c undergoes a two-step cyclization process to obtain the intermediate compound II-d, which has the following structure: (5) Optionally, the C=C double bond formed in step (4) is reduced to obtain intermediate compound II-e, which has the following structure: (6) Using intermediate compounds II-d or II-e, prepare compounds of formula I or their pharmaceutically acceptable salts.

2. The method according to claim 1, characterized in that, R 1 Selected from: (a)-H; or (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for: (i)-H; (ii)-N + (H3C)3; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B -N + (CH3)3; R 2 Selected from: (a)-H; and (b)-(CH2)zR 14A Where: z is 1-6, and R 14A Selected from: (i)-H; (ii)–NH2; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B For: or -N + (CH3)3; (iv)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C For -O-(CH2) 3-4 -N + (CH3)3; and (viii)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai)–O-(CH2)2-N + (CH3)3; (aii)-N+(CH3)2R 14ca , where R 14ca For –CH3 or –(CH2) 1-4 -OCH3; (aiii) The following part: R 8 For -CH3 or the following part: Where R 8a It is -H, or a straight-chain, branched, or cyclic alkyl group with up to four carbon atoms.

3. The method according to claim 1, characterized in that, The compound of formula I is compound 1-1. G - This represents a pharmaceutically acceptable anion.

4. The method according to claim 1, characterized in that, In step (1), the solid support is 2-Cl-Trt resin.

5. The method according to claim 4, characterized in that, In step (1), HATU and DIEA reagents were used to generate amide bonds through a condensation reaction, and compound II-a was obtained by solid-phase synthesis.

6. A method for preparing compound II-c, characterized in that: The compound II-c has the following structure. in, R 1a Selected from: (a)-H; or (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for: (i)-H; (ii)–NH-amino protecting group; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B It is a -NH-amino protecting group; (iv)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B It is a -NH-amino protecting group; (v)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai) The following part: R 8 Selected from: C1-6 alkyl groups, wherein the C1-6 alkyl group can be -OR 8a Instead, the R 8a Selected from H and straight-chain, branched, or cyclic alkyl groups with no more than 6 carbon atoms; X is selected from H, F, Cl, or Br; The method includes the following steps: (1) Preparation of intermediate compound II-a, In formulas II-a and II-b, the sphere represents the solid phase support; (2) The Fmoc protecting group was removed to obtain intermediate compound II-b, which has the following structure; (3) Remove the solid support to obtain intermediate compound II-c.

7. Compounds II-a or II-b have the following structure:

8. The compound according to claim 7, characterized in that, Compound II-a is selected from compound II-a-1; Compound II-b is selected from compound II-b-1; 9. Use of compound II-a or compound II-b in the manufacture of a compound of formula I or a pharmaceutically acceptable salt thereof, wherein said compound I, compound II-a, and compound II-b have the following structures:

10. The application according to claim 9, wherein the compound of formula I is: G - This represents a pharmaceutically acceptable anion; The compound II-a is: The compound II-b is:

11. The synthetic method of compound II-e, The method includes: (1) Use compound II-c to synthesize compound II-d; (2) Use compound II-d to synthesize compound II-e; The compounds II-c, II-d, and II-e have the following structures in, R 1a Selected from: (a)-H; or (b)-(CH2)zR 14A Where: z is 1-6, and R 14A for: (i)-H; (ii)–NH-amino protecting group; (iii)-NH-C(O)-[(CH2)2-O-]2-(CH2)2R 14B , where R 14B It is a -NH-amino protecting group; (iv)-NH-C(O)-[(CH2) y12 -O-]2-(CH2) y13 R 14B Where: y12 and y13 are not both 2 and are independently 2 to 4; and R 14B It is a -NH-amino protecting group; (v)-NH-C(O)-(CH2) y R 14C Where y = 1 to 6 and R 14C for: (ai) The following part: R 8 Selected from: C1-6 alkyl groups, wherein the C1-6 alkyl group can be -OR 8a Instead, the R 8a Selected from H and straight-chain, branched, or cyclic alkyl groups with no more than 6 carbon atoms; X is selected from H, F, Cl, or Br.

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