Synthesis of cefazolin under linked, continuous flow conditions

Abstract

This invention relates to the linked-flow synthesis of cefazolin, an important first-line drug used for the prevention of primary infection in most surgical procedures. Rapid flow and efficient mixing of substrates within a suitable flow reactor allowed for rapid acquisition of the target compound without separation of intermediates. A flexible system design applicable to small to medium-scale synthesis was demonstrated, and optimal parameters for achieving synthesis were established. Synthesis on a 0.3 mol / hour scale was achieved with a space-time yield of 13.75 g / h dL of cefazolin and an isolation yield of 54%. The obtained substance had an acceptable impurity profile and could be purified by simple acid-base extraction and precipitation.

Description

[Technical Field]

[0001] This invention relates to the field of pharmaceutical antibiotics, and more particularly to the continuous flow synthesis of cefazolin. [Background technology]

[0002] Cefazolin sodium is a β-lactam antibiotic belonging to the first-generation cephalosporin, or more broadly, the cephalosporin group. It is known to be effective against both Gram-positive and Gram-negative bacteria and has a broad antibacterial spectrum. 1 Furthermore, cefazolin sodium is an important first-line drug used to prevent primary infections in most surgeries. 2 Due to its importance, cefazolin sodium is registered as one of the essential medicines designated by the WHO. 3 .

[0003] Cefazolins are primarily produced by the chemical derivatization of 7-aminocephalosporanic acid (7-ACA, 5). 7-ACA is obtained by the chemienzymatic hydrolysis of cephalosporin C isolated from fungi of the genus Acremonium, although its enzymatic synthesis is also widely studied. 4、5 Cefazolin was commercialized in 1971, but its importance continues to lead to widespread research. Furthermore, the demand for antibiotics containing cefazolin is increasing, and the use of this drug is on the rise worldwide. 6 Therefore, there is a growing urgency to manufacture such important drugs in locations close to medical facilities. 7Furthermore, since the demand for these products fluctuates greatly, it is important to flexibly manage the amount produced locally to meet the local demand. However, the essence and details of the manufacturing method are know-how. Thus, barriers to manufacturing due to new entry still exist, which is a problem that hinders the social requirement of stable supply of important pharmaceuticals and cannot be ignored. Therefore, the development of an efficient manufacturing method is important not only to supply a sufficient amount of drugs to meet global demand but also to maintain a stable supply of essential pharmaceuticals at the local level.

[0004] When manufacturing highly sensitive bioactive compounds such as antibiotics, it is necessary to isolate the manufacturing location and equipment to prevent contamination and ensure safety, and the air filtration equipment needs to be strictly managed. 8 Therefore, when starting the production of a new antibiotic, it is necessary to prepare a dedicated area for production, and a large amount of initial capital expenditure is required, such as the land and construction of the dedicated section and the cost of the isolation equipment. Therefore, in addition to the general problems described above, there are many problems to be solved, such as safety and economy, and it is difficult to introduce or reconstruct the production of highly sensitive antibiotics. Therefore, a major challenge in the research on the production of these drug molecule families is to develop a method that is low-cost, space-saving, highly safe, traceable, and reliable, departing from the conventional production methods.

[0005] The continuous flow synthesis of organic molecules has great advantages in the production of high-value-added compounds, including space-saving of experimental equipment, improvement of energy efficiency, and improvement of safety, compared with the conventional batch synthesis method. 9 There are several important elements in continuous flow synthesis organic chemistry that are particularly important advantages over the conventional batch method. First, it is to control high-speed and ultra-high-speed reactions by controlling the reaction time as the reaction space. 10 Another important aspect is the ease of safety management obtained by using small and sealed reaction spaces such as tubes and columns. 11Furthermore, it becomes possible to integrate multiple reaction steps into a single operation while meeting the requirement to minimize external exposure due to instability, high toxicity, and short lifespan. The latter two characteristics offer significant advantages in the production of highly active antibiotics. In particular, in linked-sequential flow synthesis, where multiple reactions are linked, contact between highly sensitizing compounds and workers can be reduced and cross-contamination with other compounds can be limited by isolating only one compartment during antibiotic synthesis. Moreover, the ability to arbitrarily control production volume is attractive to manufacturers. Not only does it reduce both cost and safety risks, but such next-generation systems are essential for realizing a sustainable and resilient society because they allow for the production of the required amount of compound only when needed, without the need for unstable intermediate stockpiles of drugs. Here, in order to take advantage of the benefits of linked-sequential flow synthesis, where two or more multi-step reactions are linked, it is necessary to avoid the purification of intermediate compounds. For this reason, it is important not only to link reactions but also to verify the interdependence before and after linking. This is because carryover of unreacted starting materials and by-products from upstream reactions can adversely affect subsequent reactions. Furthermore, in order to efficiently provide the final product in a pure form, it is also important to minimize carryover from upstream reaction steps.

[0006] There is still a need for the development of lower-cost, space-saving, highly safe, highly stable, and traceable methods for producing highly sensitizing antibiotics. [Overview of the project]

[0007] To achieve the above objectives, the inventors have conducted extensive research and have discovered that highly sensitizing antibiotics, particularly cefazolin, can be synthesized at low cost, in a space-saving, highly safe, highly stable, and traceable manner using a linked-continuous flow approach.

[0008] In other words, the present invention provides the following: [1] Formula 2:

[0009] [ka]

[0010] A continuous flow synthesis method for a compound represented by or a pharmaceutically acceptable salt, comprising the following steps: (i) In the presence of a base and water, 7-aminocephalosporanic acid (compound 5):

[0011] [ka]

[0012] 2-mercapto-5-methyl-1,3,4-thiazole (compound 6):

[0013] [ka]

[0014] React with the solution to form intermediate 3:

[0015] [ka]

[0016] The process of forming, and (ii) The intermediate is 1H-tetrazole-1-ylacetyl halide (compound 4):

[0017] [ka]

[0018] A method that includes a step of reacting with [a certain substance]. [2] The continuous flow synthesis method according to [1], wherein step (i) is carried out while flowing the base, water, compound 5, and compound 6 into the first reactor. [3] The continuous flow synthesis method according to [1], wherein step (ii) is carried out while the reaction mixture in step (i) is flowed from the first reactor to the second reactor connected to the first reactor, and while a solution of compound 4 is supplied to the second reactor. [4] The amount of compound 5 supplied per unit volume of the first reactor per unit time is

[0019]

number

[0020] (In the formula, F5: molar flow rate of compound 5; V) R1 (This shows the internal volume of the first reactor (R1).) The continuous flow synthesis method according to [1] or [2], wherein the flow rate is adjusted to be in the range of 0.01 to 1.00 mmol / min·mL. [5] The above F5 / V R1 The continuous flow synthesis method described in [4], wherein the concentration is 0.02 to 0.60 (mmol / min·mL). [6] A continuous flow synthesis method according to any one of [1] to [5], wherein compound 6 is further supplied to another reactor connected to the first reactor. [7] The base is Na3PO4, Na2HPO 4、 A continuous flow synthesis method according to any one of [1] to [6], wherein at least one selected from the group consisting of K3PO4, K2HPO4, K3PO4, triethylamine, and combinations thereof. [8] The continuous flow synthesis method according to [1], wherein an organic base is added to the reaction in step (i). [9] The continuous flow synthesis method according to [8], wherein the organic base is at least one selected from the group consisting of 2,6-lutidine, 3,4-lutidine, 2,4-lutidine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, tetrabutylammonium bromide, imidazole, 1,2-dimethylimidazole, 1-methylimidazole, 2,3,5,6-tetramethylpyrazine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 3-methyl-1-octylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, and combinations thereof.

[10] The continuous flow synthesis method according to any one of [1] to [9], wherein the solution of compound 4 comprises compound 4 dissolved in an aprotic polar solvent.

[11] The continuous flow synthesis method according to

[10] , wherein the aprotic polar solvent is selected from the group consisting of acetonitrile, acetone, tetrahydrofuran, and dichloromethane.

[12] A continuous flow synthesis method according to any one of [1] to

[11] , wherein step (i) is performed in the range of 60°C to 90°C.

[13] A continuous flow synthesis method according to any one of [1] to

[12] , wherein step (ii) is performed in the range of 10°C to room temperature.

[14] The pharmaceutically acceptable salt of the compound is a sodium salt], the continuous flow synthesis method according to any one of [1] to

[13] .

[0021] The inventors have succeeded in synthesizing cefazolin at low cost, in a space-saving manner, with high safety and stability, and traceability, using a linked-continuous flow approach.

[0022] The accompanying drawings incorporated herein and constituting part of this specification are illustrative of specific embodiments of the disclosure and do not limit the scope of the disclosure. The drawings are not to scale and are intended for use in conjunction with the following detailed description. [Brief explanation of the drawing]

[0023] [Figure 1]Figure 1 shows the synthesis route of cefazolin from 7-ACA(5). The step of obtaining intermediate 3 by the reaction of 7-ACA (hereinafter, "7-ACA" will also be referred to as "compound 5") with compound 6 corresponds to step (i), and the reaction of intermediate 3 with compound 4 corresponds to step (ii). [Figure 2] Figure 2 shows the screening of bases for thioesterification. "Base A" refers to the base used with compound 5, and "Base B" refers to the base used with compound 6. [Figure 3] Figure 3 shows the two-step linked, continuous flow synthesis of cefazolin 2. [Figure 4] Figure 4 shows the two-step linked continuous flow synthesis of intermediate 3. Concentrations a, b, and c are the concentrations of compound 6, K3PO4, and K2HPO4, respectively. [Figure 5] Figure 5 shows the revised flow settings and potential by-products. "Additives" can be used interchangeably with "organic bases". [Figure 6] Figure 6 shows the scale-up of the flow synthesis of cefazolin 2 and the derivatization of cefazolin sodium 1. [Figure 7] Figure 7 shows the synthesis pathway of cefazolin. [Figure 8] Figure 8 shows the base analysis for the thioesterification between 7-ACA(5) and MMTD(6). The equivalent weight of base 2, indicated as "x equivalent weight," is either 1.15 or 2.3. These correspond to Table 7. [Figure 9] Figure 9 shows the flow reaction for the synthesis of intermediate 3 using a single pump. [Figure 10] Figure 10 shows the investigation of a coupling-sequence flow reaction for the synthesis of cefazolin 2. [Figure 11] Figure 11 shows the optimization of the improved thioetherification reaction. The amounts of x, y, and z are shown in Table 4. [Figure 12]Figure 12 shows the study of an improved coupled-continuous flow reaction for the synthesis of cefazolin 2. The amount of 4-Cl, the concentration of y, and the tracking rate are listed in Table 5 as "stoichiometric value [equivalent]", "concentration [M]", and "v3 [mL / min]", respectively. [Figure 13] Figure 13 shows the purification of cefazolin 2 and the crystallization of cefazolin sodium 1. [Figure 14] Figure 14 shows the large-scale linked-continuous flow reaction for the synthesis of cefazolin 2. [Figure 15] Figure 15 shows a large-scale linked, continuous flow system. [Figure 16] Figure 16 shows a photograph of a large-scale synthesis module: (a) a large water bath, (b) stainless steel loop reactors (TR1aL, TR1bL, TR2L), (c) inline flow meters and pressure sensors, (d) thermocouple ports via tube connectors, and (e) T-shaped tube connectors. [Figure 17] Figure 17 shows the study on additives for linked and continuous flow reactions. [Figure 18] Figure 18 shows a batch analysis of solvent systems suitable for the amidation process. The "base" and "solvent" are listed in Table 16. [Figure 19] Figure 19 shows a control experiment of one-pot two-step synthesis of cefazolin 2 under batch conditions. [Modes for carrying out the invention]

[0024] In the following description, prior art features of the disclosed technology that are obvious to those skilled in the art will be omitted or described only briefly. References to various embodiments will not limit the scope of the claims appended herein. Furthermore, any examples described herein are not limiting and are intended to simply illustrate some of the many possible embodiments of the appended claims. In addition, certain features described herein can be used in combination with other features described herein in each of the various possible combinations and permutations. Those skilled in the art will know how to achieve other results not specifically disclosed in the examples or embodiments by using the present invention in combination with ordinary experiments.

[0025] Unless otherwise defined herein, all terms are to be interpreted in the broadest possible way, including not only the meaning implied herein, but also the meaning understood by those skilled in the art and / or the meaning defined in dictionaries, papers, etc. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as broadly understood by those skilled in the art in the disclosed field. It should also be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include multiple references unless otherwise specified, and the terms “include” and / or “including” used herein identify the presence of a described feature, element, and / or component, and do not exclude the presence or addition of one or more other features, processes, operations, elements, components, and / or groups thereof. Furthermore, methods, apparatus, and materials similar to or equivalent to those described herein may also be used in carrying out or testing the disclosed art.

[0026] Flow synthesis is a synthesis method in which raw materials and reactants are flowed into a flow channel system consisting of tubes with a diameter of several millimeters and a reactor (flow reactor) with grooves cut into metal, and the operations of reaction, extraction, and purification are carried out in a continuous manner. In this specification, "continuous flow" is also referred to as "linked continuous flow." In particular, a linked continuous flow system refers to a flow reaction system in which a single continuous flow reaction is connected in series, and two or more transformations are carried out as linked continuous flow reactions. In this method, chemical reactions are carried out in small amounts within the flow channel, so there is almost no risk of runaway reactions or explosions. In addition, because the reactions are carried out in connected flow channels, the introduction of impurities can be prevented. Furthermore, by hierarchically structuring the equipment based on conditions considered at the laboratory level, a seamless transition to industrial mass production can be achieved.

[0027] The continuous flow synthesis of the present invention (hereinafter referred to as "flow synthesis") comprises two steps: step (i) and step (ii). Step (i) of this synthesis method relates to reacting compound 5 and compound 6 in the presence of a base in the first reactor (R1) to obtain intermediate 3. Step (ii) of this synthesis method involves reacting the intermediate 3 obtained in step (i) with a solution of compound 4-X (where X is a halogen) in a second reactor (R2) connected to R1 to obtain compound 2. The halogen is selected from the group consisting of Cl, F, Br, and I.

[0028] Examples of reactors include microreactors or tubular reactors made from materials such as SUS (stainless steel), Hastelloy, resin, or glass. The first and second reactors used herein may be tube reactors (TRs). In a continuous flow method, if a third reactor, a fourth reactor, etc., are further used, these reactors may also be tube reactors. In this specification, "R1" is interchangeable with "TR1". "R2" is interchangeable with "TR2".

[0029] Examples of bases include, but are not limited to, at least one selected from the group consisting of Na3PO4, Na2HPO4, K3PO4, K2HPO4, triethylamine, and combinations thereof. In particular, phosphate buffering conditions are beneficial in avoiding undesirable decomposition of the starting compound 5. Organic bases such as triethylamine are also preferred, but inorganic bases may be preferred because they facilitate post-reaction work. Hereinafter, the base used with compound 5 will be referred to as "base A," and the base used with compound 6 will be referred to as "base B" (see, for example, Table 1 and Figure 2). In the combination of base A and base B, these bases may be the same or different. The ratio of base A to base B is, for example, 1:9 to 9:1, preferably 1:5 to 5:1, and more preferably 1:3 to 3:1. Base A and base B can each be used in amounts of 0.1 to 2.0 equivalents relative to compound 5 or compound 6.

[0030] Compound 5, Compound 6, Base A, and Base B can be combined in ratios such as 1:1:1:1 to 1:1:1:9.

[0031] To completely consume compound 5, compound 6 may be further supplied to another reactor connected to R1. The additional amount of compound 6 may be 0.1 to 1.0 equivalents relative to the compound 6 already supplied in step (i).

[0032] If necessary, an organic base may be added to the reaction in step (i) to accelerate the subsequent amidation reaction in order to prevent side reactions and improve the yield. The organic base can be used in an amount of 0.1 to 1.0 equivalent relative to compound 5 or compound 6. The amount of organic base used relative to compound 5 or compound 6 is, for example, 0.01 to 0.1 equivalents, preferably 0.01 to 0.05 equivalents.

[0033] The organic bases used in the present invention include, but are not limited to, at least one selected from the group consisting of 2,6-lutidine, 3,4-lutidine, pyridine, pyridine N-oxide, 4-dimethylaminopyridine (DMAP), N,N-dimethylaniline, tetrabutylammonium bromide (TBABr), triethylamine, N,N-dimethylaniline, imidazole, 1,2-dimethylimidazole, 1-methylimidazole, 2,3,5,6-tetramethylpyrazine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 3-methyl-1-octylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, and combinations thereof.

[0034] Compound 4-X can be used in an amount of 1.0 to 5.0 equivalents, preferably 2.0 to 3.0 equivalents, relative to compound 5. Increasing the amount of 4-Cl increases the desirable reaction with intermediate 3 and suppresses the formation of undesirable byproducts. Solvents used to prepare a solution of compound 4-X include, for example, aprotic polar solvents selected from the group consisting of acetonitrile, acetone, tetrahydrofuran, dichloromethane, water, and combinations thereof. Of these, water / acetonitrile is preferred. The ratio is preferably 1:2 to 1:8, more preferably 1:2 to 1:5, and particularly preferably 1:4.5 (see Table 2). The above ratio is very important because rapid mixing and the amount of cosolvent relative to water are expected to greatly affect amidation.

[0035] In continuous flow synthesis, in addition to the material composition, it is necessary to keep the molar flow rate per reactor volume F / V constant, which is an important indicator regardless of the concentration, mother liquor flow rate, and reactor size. Therefore, F5 / V R1 (F5 / V TR1 (Also known as) (Here, F5 is the molar flow rate of compound 5, and V R1The index (where F5 is the internal volume of the first reactor (R1)) should also be kept constant, and the amount of compound 5 supplied to the first reactor (R1) per unit volume per unit time should be F5 / V. R1 It is preferable to control the index to be between 0.01 and 1.00 (mmol / min·mL). More preferably, F5 / V R1 The index is 0.01 to 0.90, more preferably 0.01 to 0.80, more preferably 0.01 to 0.7, more preferably 0.02 to 0.7, and particularly preferably 0.02 to 0.60 (Table 5). F5 / V R1 If the index is less than 0.01, the system cannot produce a sufficient amount of product. On the other hand, F5 / V R1 If the index exceeds 1.00, the system cannot achieve a sufficient yield. In either case, sufficient productivity cannot be achieved. Also, F5 / V R1 Once the index is determined, the appropriate supply conditions for 4-Cl are automatically determined.

[0036] Examples of pharmaceutically acceptable salts of the compound represented by formula I include salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, and carbonic acid; salts with organic acids such as formic acid, acetic acid, propionic acid, trifluoroacetic acid, phthalic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and p-toluenesulfonic acid; salts with amino acids such as lysine, arginine, ornithine, glutamic acid, and aspartic acid; and salts with sodium, potassium, lithium, etc. Examples of salts include, but are not limited to, salts with alkali metals; salts with alkaline earth metals such as calcium and magnesium; salts with metals such as aluminum, zinc, and iron; salts with organic bases such as methylamine, ethylamine, diethylamine, trimethylamine, triethylamine, ethylenediamine, piperidine, piperazine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N-methylglucamine, and N,N'-dibenzylethylenediamine; and ammonium salts. Among these salts, salts with alkali metals such as sodium, potassium, or lithium are preferred, and sodium salts are more preferred.

[0037] The inventors have achieved continuous flow synthesis of cefazolin and have also scaled up the synthesis 30-fold (Figure 6). This was made possible by the characteristics of a linked system that enables a flow reaction, a well-controlled rapid reaction through efficient mixing, and the elimination of the need for intermediate separation. A flexible system design enabling small, medium, and large-scale reactions has been demonstrated, and optimal parameters for operating the system have been established. Furthermore, although performed manually in this study, acceptable purification was achieved by simple acid-base extraction and precipitation. The current process can be achieved in a closed system without external exposure from the supply of raw materials to the acquisition of crude cefazolin, and has the potential to be developed into a safer system with minimal risk of cross-contamination or human exposure. These results are expected to enable the local production of essential medicines in the required quantities on demand in the near future. Such a system is extremely important in modern society.

[0038] After the synthesis reaction is complete, the reaction mixture is subjected to standard procedures such as washing with a suitable solvent (e.g., ethyl acetate), acidification, and extraction with a suitable solvent (e.g., ethyl acetate) to obtain a solid product corresponding to compound 2. 1 ¹H-NMR analysis revealed that the solid yielded 13.75 g / h dL STY (space-time yield), showing reasonable agreement with the results of a small-scale investigation (see Table 5, entry 4). The solid obtained for purification was subjected to the usual procedures, extraction, and acidification described above to obtain purified compound 2. Further purification, for example, to a sodium salt, yielded the desired cefazolin sodium 1.

[0039] Next, the disclosed technology will be illustrated by the following examples. In no part of this specification are the uses of these and other examples for illustrative purposes only and do not limit the scope and meaning of the invention or the exemplary forms. Similarly, the invention is not limited to the specific preferred embodiments described herein. In fact, modifications and changes to the invention will be obvious to those skilled in the art by understanding this specification and can be made without departing from the spirit and scope of the invention. Thus, the invention is limited only by the conditions of the claims and the entire range of equivalents of the claims. [Examples]

[0040] 1H-tetrazole-1-ylacetyl chloride (4-Cl) solution In a three-necked round-bottom flask equipped with a magnetic stirrer, 1H-tetrazole-1-ylacetic acid (4) (1 equivalent) and THF as solvent were added under an Ar atmosphere. The solution was then cooled to 0°C and stirred. To the stirred solution, a catalytic amount of N,N-dimethylformamide was added using a syringe, followed by the dropwise addition of oxalyl chloride (1.5 equivalents) using a syringe. The reaction mixture was stirred at 0°C for 1 hour, then heated to room temperature and stirred for 2 hours. After the reaction was complete, the solvent was removed under vacuum to obtain 1H-tetrazole-1-ylacetyl chloride (4-Cl). The prepared reagent was used without purification. The acid chloride was dissolved in MeCN (0.0125~0.1 mol / L) to obtain a 4-Cl solution, which was used in the linked-continuous flow reaction.

[0041] Cefazolin linked and continuous flow synthesis (Figure 5 and Table 5) The 4-Cl acid chloride prepared as described above was dissolved in acetonitrile at the required concentration (0.0333 to 0.0667 mol / L) to obtain a 4-Cl solution, which was used in the linked-continuous flow reaction. A tubular reactor system (TR2; PTFE tube, inner diameter 2.0 mm × 1.0 mL) was prepared, and the reactor was immersed in a water bath. Next, the entire system was washed with acetonitrile using pump 3. A flow setup consisting of TR1a and TR1b was constructed.

[0042] As the mother liquor supplied from pump 1, aqueous solutions of 7-ACA(5) (0.2 mol / L, 1.0 equivalent), K3PO4 (0.2 mol / L, 1.0 equivalent), 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.2 mol / L, 1.0 equivalent), K2HPO4 (0.8 mol / L, 4.0 equivalent), and 2,6-lutidine (0.01 mol / L, 0.05 equivalent) were prepared. An aqueous solution of 6 supplied from pump 2 was also prepared, consisting of 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.2 mol / L, 0.5 equivalent), K3PO4 (0.1 mol / L, 0.25 equivalent), and K2HPO4 (0.4 mol / L, 1.0 equivalent).

[0043] Next, the two-stage system was operated and stabilized for 30 minutes. Then, the supply solution of pump 3 was replaced with 4-Cl solution, and its stream was combined with the outlet solution of the two-stage TR1 system using a micromixer. The flow rate of the 4-Cl solution was appropriately controlled to adjust the stoichiometry relative to 5 to 2-3 equivalents. After stabilizing the entire flow system for 30 minutes, the outlet solution was collected for 30 minutes. Next, the obtained solution was washed with AcOEt and acidified to approximately pH 2.0 (confirmed with test paper) with a 10% HCl aqueous solution. The acidified solution was extracted with AcOEt and H2O, the organic phase was dried with Na2SO4, filtered, and the solvent was removed under vacuum to obtain the crude product. The yield of cefazolin 2 was obtained in DMSO-d6. 1 The calculation was performed by 1H-NMR (internal standard: 1,1,2,2-tetrachloroethane). The crude product was isolated by flash column chromatography (CH2Cl2 / MeOH = 10 / 1~5 / 1 + 1% AcOH), and the corresponding product 2 was obtained in the specified yield.

[0044] result: A standard synthetic strategy for obtaining cefazolin(2) from commercially available compounds is a two-step process. 4,5This is a three-component coupling reaction consisting of thiadiazole thioetherification of cephalosporan core by 6 and subsequent amidation by the activated tetrazole-1-acetic acid derivative (4) (Figure 1). Therefore, the inventors began continuous flow studies with the base-accelerated substitution reaction of 7-ACA (5) and 2-mercapto-5-methyl-1,3,4-thiadiazole (MMTD, 6) in an aqueous medium. The initial flow setup consisted of two plunger pumps, namely pump 1 and pump 2, to supply 5 and 6 respectively. Both starting materials were dissolved in water with 1.0 equivalent of base. The two streams were merged in a T-mixer and transferred to a 1.0 mm × 10 m (7.85 mL) PTFE tube reactor TR1, which was heated to 80°C in a water bath. In initial studies to screen for suitable bases, both substrates were flowed at 0.15 mL / min, resulting in an estimated residence time of 26.2 minutes (Table 1).

[0045] [Table 1] a Space-time yield (STY) of intermediate 3. b Bases 5, 6, A, and B were supplied together in a single aqueous solution using a single pump system.

[0046] Therefore, a 30-minute adjustment period was provided to stabilize the flow reaction, followed by a 30-minute sampling period to determine the product yield. The total flow rate was 0.3 mL / min, and the system was able to reliably collect approximately 9.0 mL of solution without being affected by pressure loss.

[0047] Based on accumulated knowledge regarding cefazolin synthesis, phosphate buffering conditions were expected to be beneficial in avoiding undesirable degradation of the starting material, 7-ACA5, and the results shown in Table 1 demonstrate this effect. When using sodium phosphate or potassium phosphate / hydrogen phosphate systems, a yield of 70% and a space-time yield of 8.0 mmol / h dL were obtained. Similar yields were obtained with organic bases, but inorganic bases may be preferred due to easier post-reaction handling. The yield of intermediate 3 was unsatisfactory, and it may be possible to further improve the yield by using other additives such as Lewis acids or by using excess nucleophiles or electrophiles, but it was important that no unwanted reagents were carried over to the next step. This not only makes the purification of the final product difficult but can also induce side reactions with activated tetrazole-1-acetic acid derivative 4, potentially further complicating the product mixture. The yields achieved in this study were comparable to those of known cefazolin synthesis. 12 For these reasons, the inventors decided to accept this result and proceeded to the next amidation step.

[0048] The output from the two pump setups mentioned above was diverted to the amidation step, but a 7.85 mL tubular reactor TR1 was inserted between pump 1 and a T-shaped mixer, and another 2.0 mm × 1 m (3.14 mL) tubular reactor TR2 was connected to the mixer outlet. A mother liquor consisting of a mixture of 7-ACA (5, 0.05 M), MMTD (6, 0.05 M), K3PO4 (0.05 M), and K2HPO4 (0.20 M) in water (H2O) was flowed at 0.3 mL / min using pump 1, and the prepared tetrazole-1-acetyl chloride (TAACl, 4-Cl) acetonitrile solution was supplied from pump 2 at the desired flow rate.

[0049] First, the results of linked-continuous flow amidation using different equivalent amounts of acid chloride (prepared from the corresponding acid and oxalyl chloride using a small amount of DMF) were compared, and no significant difference was observed between the use of 4 equivalents and 2 equivalents; therefore, the use of 2 equivalents of acid chloride was selected as the standard condition (Table 2, entries 1 and 2). The effect of the water / acetonitrile ratio of the final mixed solution was investigated by fixing the equivalent amounts of 5 / 6 and the acid chloride 4-Cl at 1:2, and then varying the chloride concentration and flow rate. This parameter is extremely important as rapid mixing and the amount of cosolvent relative to water were expected to greatly affect the amidation. When a more diluted 0.0125 M acetonitrile solution of 4-Cl was supplied at a faster flow rate of 2.4 mL / min, the two-step yield from 5 improved to approximately 60% (Table 2, entries 2-4). The total flow rate of the mixed solution to TR2 was estimated to be 2.7 mL / min, with a residence time of 70 seconds; rapid mixing in a T-shaped mixer may have been advantageous for the reaction. These results prompted consideration of faster supply of the mother liquor to increase productivity (Table 2, entries 5-9); supplying the mother liquor at 0.8 mL / min yielded a satisfactory yield of 65% at 12.89 g / h dL STY (entry 8). The short estimated residence time of TR1 at 9.8 minutes was also beneficial to the results. The inventors also tested the use of a 2x concentrated mother liquor (5) because increasing the mother liquor supply rate to 0.9 mL / min resulted in a decrease in yield. This attempt was successful in maintaining other parameters and improved the STY value to approximately 25-26 g / h dL; the addition of 2,6-lutidine slightly improved yield and reproducibility, and target 2 was consistently produced in a yield of approximately 65% ​​(entry 11). The actual role of 2,6-lutidine is unclear, but it was speculated to activate 4-Cl and promote amidation. Furthermore, considering the similar effects of tetrabutylammonium bromide and tetramethylammonium bromide as shown in SI, these additives may function as phase-transfer catalysts.Furthermore, the inventors also attempted to reduce the amount of acetonitrile solvent used to produce the acid chloride 4-Cl, and found that using a concentrated solution of 4-Cl at a slower flow rate of 3.6 mL / min reduced solvent consumption while obtaining the same yield and STY (Entry 12). According to the results shown in Entry 12, the final composition of the amidation solution was water (H2O) / acetonitrile = 1:4.5, which indicates that the residence time of approximately 26-43 seconds, rather than the H2O / acetonitrile ratio, was the main factor in the good reactivity in the second amidation step. This two-step synthesis was considered to be significantly superior to the conventional batch method in terms of efficiency. Although not optimized, when the inventors attempted to synthesize cefazolin(2) by a one-pot two-step reaction in batch mode, the yield of the desired product was only 4%. The one-pot, two-step reaction consisted of two consecutive reactions: the reaction of 7-ACA(5) with MMTD(6, 1.0 equivalent) in the presence of 2.15 equivalents of K3PO4, and direct amidation with 2 equivalents of 4-Cl. The crude product after normal processing. 1 The 1H-NMR spectrum showed that compound 2 was produced in 4% yield. Furthermore, when isolated compound 3 was reacted with 4-Cl in the presence of K3PO4 under conditions similar to those of Schotten-Baumann, compound 2 was produced in 56% yield. This was likely related to mixing efficiency. In the batch reaction, when a solution of 4-Cl was added to a basic aqueous solution containing compound 3, the 4-Cl reacted with water and decomposed.

[0050] [Table 2] a The composition of the mother liquor was 5 / 6 / K3PO4 / K2HPO4 = 1:1:1:4. b1 Determined by 1H-NMR analysis. c Space-time yield of cefazolin 2. The combined reactor volume of TR1 and TR2 was used for the calculation. d This is equivalent to 4.0 equivalents of 4-Cl. e This is equivalent to 2.0 equivalents of 4-Cl. f 2,6-Lutidine (0.05 equivalents) was added to the mother liquor as an additive. gThis is equivalent to 2.25 equivalents of 4-Cl. R : Residence time; v1: Flow rate of process (i); and v2: Flow rate of process (ii).

[0051] Based on the results obtained up to this stage, the basic parameters and conditions for further investigation were extracted and summarized in Table 3. Here, in addition to the material composition, it is necessary to fix the important indicator F / V (molar flow rate per unit reactor volume); for example, for the thioetherification reaction, F5 / V obtained as a function of these parameters regardless of concentration, mother liquor flow rate, or reactor size. TR1 The indicator must remain constant. The inventors of this invention have found that residence time (t R ), reactor capacity (V TR1 Table 3 shows the interdependence values ​​of other parameters such as ), flow rate (v1), and concentration 5 (C5). Furthermore, once the initial reaction conditions are determined, the appropriate supply conditions for 4-Cl are automatically determined. Based on the results of the amidation process, to maintain acceptable reactivity, F3 / V TR2 An index of 0.051 mmol / min·mL is considered beneficial.

[0052] [Table 3] a Based on the results of entry 11 in Table 2. b 5V TR1 This is an index that indicates the amount of 5 supplied per unit volume in this process (F5; molar flow rate of 5). c F 3 / V TR2 This is an index that shows the theoretical pass rate of 3 per unit volume, which is the same as the F5 value, in this amidation process. R : Duration of stay.

[0053] Before scaling up the synthesis of cefazolin(2) and proceeding to the final purification to obtain cefazolin sodium(1), the formation of a by-product (by-product A in Table 6) was investigated. According to the HPLC chromatograms of the crude solutions in the latter half of Table 2, the inventors found that a significant amount of by-product, likely containing a carboxylic acid moiety within its structure, was formed in the second step (Table 6). This by-product made separation of the target product by acid-base extraction significantly more difficult. Furthermore, there were some issues with the reproducibility of the target product yield under the above conditions, indicating the need for further improvement of the reaction conditions at this stage. Chromatograms of the TR1 outlet solution under specific conditions, indicating the presence of a considerable amount of unreacted 7-ACA(5), revealed that the initial process was incomplete, providing a clue that the by-product might be an amidation product of unreacted 5 and 4-Cl. To suppress the formation of this by-product, the inventors considered adding MMTD(6) in two steps during the initial thioetherification step (Table 4).

[0054] [Table 4] a 0.05 equivalents of 2,6-lutidine were added to the solution containing 5.

[0055] When the composition of the substance in the additional solution 6 was fixed at 6 / K3PO4 / K2HPO4 = 1:0.5:2 and the equivalent amount of additional 6 relative to 5 in the mother solution was examined, the yield of 3 was slightly improved in all cases compared to Table 1 (Table 4). When 0.5 equivalents of 6 were added to the newly inserted 7.85 mL tube reactor TR1b, the intermediate was obtained in 76% yield (Table 4, Entry 1), and the same level of improvement was observed in the mother liquor containing 2,6-lutidine (Entry 4). Although the insertion of TR1b sacrificed STY, this change did not affect the productivity per unit period, so the inventors decided to prioritize the improvement of the impurity profile. The application of this new system to a linked, continuous flow was carried out as shown in Figure 5. However, the first attempt unfortunately ended in failure, resulting in the production of a small amount of 2 (Table 5, Entry 1). Therefore, based on previous experience, the inventors decided to shorten the residence time in TR2 as the acetonitrile / water mixing ratio increased. These tests increased the yield by 2–50% (Entry 3). The inventors confirmed that the formation of the problematic by-product likely occurred due to the reaction between 5 and 4-Cl and could be suppressed by using the new system, but the appearance of another by-reaction product (by-product B in Table 6) became apparent under these conditions. Careful investigation of this new by-reaction revealed that intermediate 3 was unexpectedly N-acetylated by the reaction of intermediate 3 with the acetate anion residue from thioetherification. The inventors hypothesized that this problem could be overcome by accelerating the rate in the amidation step and found that several additives other than 2,6-lutidine had this capability. However, ultimately, it was decided to use 3.0 equivalents of the acid chloride 4-Cl instead of 2.0 equivalents in this step (Entry 4). The yield of 2 obtained with the improved reaction system reached 60% at 13.90 g / h dL STY on a relatively clean crude HPLC chromatogram (Table 6). The inventors hypothesized that increasing the amount of 4-Cl would promote the desired reaction with 3, thereby suppressing the formation of undesirable by-products.

[0056] [Table 5] a Conditions other than 4-Cl supply: Mother liquor from pump 1; 5(0.2M) / 6 / K3P04 / K2HPO4=1:1:1:4, 2,6-lutidine 0.01M, 0.8 mL / min (v1). Additional 6 from pump 2; 6(0.2M) / K3P04 / K2HPO4=1:0.5:2, 0.4 mL / min (v2). b 5 equivalents of 4-Cl. c An index that shows the theoretical amount of TR3 passing through per unit volume per hour. d1 Determined by 1H-NMR analysis. e The space yield of 2 was calculated using a total reactor volume of 18.84 mL. f 2,6-Lutidine was not added to the mother liquor. R : residence time; and v3: flow rate.

[0057] Using these improved conditions, the inventors ultimately investigated a 30-fold scale-up of the synthesis of cefazolin(2) using a two-stage linked, continuous flow system, as shown in Figure 6. The stoichiometry of the mother liquor was determined as shown in Table 3, the main substrate concentration was maintained at 0.2 M, and the mother liquor was flowed at a flow rate of 25 mL / min. The mother liquor also contained 0.05 equivalents of 2,6-lutidine. To maintain the F / V1 index at approximately 0.0204 mmol / min·mL, a TR1a reactor with a capacity of 211.95 mL (inner diameter 3.0 mm × 30 m) was used. L The following was used. The estimated F / V1 index was 0.0235 mmol / min·mL, which is considered a negligible difference considering the residence time. An additional 0.5 equivalents of 6 and base were supplied at the same concentration and half the flow rate from another pump. The mixed solution at a flow rate of 37.5 mL / min was collected with the 4-Cl flow using a micromixer. To maintain a 4-Cl / 5 ratio of 3.0 and an H2O / acetonitrile ratio of 1:8, a 0.05 M 4-Cl solution was supplied at a flow rate of 300 mL / min; therefore, the total flow rate was 337.5 mL / min. TR2 L The inventors used a 141.3 mL tube to maintain the F / V3 index below 0.31 mol / h dL. The inventors used pump 1 L and pump 3 LA flow meter was installed on the outlet side, and five pressure sensors were installed throughout the flow path to monitor the flow rate and confirm that there were no problems with the flow path during operation. In addition, nine thermocouples were inserted into the system in preparation for sudden clogging (Figure 6), but no such abnormalities occurred during operation. The two-stage reaction was carried out for more than 10 minutes, excluding the pre-adjustment period. First, the inventors collected the outlet solution for 1 minute for analysis, and then collected it for another 10 minutes for separation. After the previous sample was washed with ethyl acetate, acidified, and extracted with ethyl acetate as usual, the inventors obtained solids corresponding to a yield of 56% of 2 ( cru We were able to obtain (2) using internal standards. 1 ¹H-NMR analysis revealed that the solid was cefazolin(2), with a yield of 57% based on the theoretical concentration. In fact, cefazolin(2) could be separated from the recovered NMR sample with a yield of 54%, thus the yield based on the weight of the solid and the crude yield. 1 The results were consistent with the H-NMR data. This result indicated 13.75 g / h dL STY, showing reasonable agreement with the results of the previous small-scale study (Table 5, Entry 4). For the sample obtained for purification, the inventors first performed the above-mentioned standard procedure, and then extracted it into the aqueous phase using a sodium bicarbonate solution. Acidification with an aqueous HCl solution yielded a precipitate, which was 7.2 g of purified 2( pur This corresponds to (2). Crude solids cru Based on the amount of 2, the purification yield was 59%. Further purification with sodium salt conversion yielded the desired cefazolin sodium (1). pur It was obtained in yields of 2 to 59%. The final cefazolin sodium 1 had a sufficient purity of approximately 98%.

[0058] [Table 6] a Details of the HPLC analysis are shown in Tables S4-S9. b Relative holding time. c Relative peak area. dThe peak of byproduct A was small enough that it almost overlapped with the shoulder of the adjacent peak of 6, and could not be distinguished as an independent peak. e The structures of by-products A and B are presumed to be as follows.

[0059] [ka]

[0060] Example 1: Details of the entire experiment 1 H-NMR spectrum and 13 ¹³C-NMR spectra were recorded using a JEOL ECX-600 spectrometer in DMSO-d6 (2.50 ppm and 39.5 ppm) and D2O (4.79 ppm). High-performance liquid chromatography (HCM) analysis was recorded using a SHIMADZU SPD-20AV. GL Science ODS columns (10 mm, 4.0 × 150 mm) were used for HPLC analysis. 7-ACA was purchased from Tokyo Chemical Industry Co., Ltd., BLDpharm, and Combi-Blocks. 2-Mercapto-5-methyl-1,3,4-thiadiazole was purchased from Tokyo Chemical Industry Co., Ltd. and Combi-Blocks. 1H-tetrazole-1-acetic acid was purchased from Zhejiang Hailan Chemical Group Co., Ltd. K3PO4, K2HPO4, Na3PO4, Na2HPO4, Et3N, N,N-dimethylformamide, and MeCN were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. 2,6-Lutidine was purchased from Sigma-Aldrich. Tetrahydrofuran was purchased from Kanto Chemical Co., Ltd. Oxalyl chloride was purchased from Tokyo Chemical Industries, Ltd.

[0061] For the linked-continuous flow reaction system, FLOM's Intelligent Pump UI-22-110 (plunger pump) or TACMINA's Q-5-6R-UP-S (diaphragm pump) were used for small-scale studies. Unless otherwise specified, two types of pumps with different drive systems were used without distinction. For scale-up synthesis, TACMINA's Q-100-6T-PS (diaphragm pump), TACMINA's XPL-1-STSX-XWX (diaphragm pump), and TACMINA's XPL-1-XTCX-XWX (diaphragm pump) were used. Advantec Toyo Co., Ltd.'s constant temperature bath TBN402 was used for heating the material. Keyence's clamp-on flow sensor FD-XS1 was used as the flow sensor, and Keyence's FD-XA1 was used as the flow sensor controller. Surpass Industries Co., Ltd.'s pressure sensor SVS-1 / 4-1MS was used as the pressure sensor. For temperature measurement, we used the ST-1 thermocouple from Amnis Corporation. For verification and recording of linked and continuous flow reaction data, we used the GL840-M midi logger from Graphtec Corporation.

[0062] Example 2: Flow synthesis of intermediate 3 from 7-ACA5 and MMTD6 A PTFE loop (tube reactor, TR1, 1.0 mm inner diameter x 10 mL) was heated to 80°C in a water bath. One end of the tube was placed in a receiver flask, and a T-connector was attached to the other end. Two pumps, pump 1 and pump 2, were filled with H2O, and the ends of the tubes from the pumps were connected to the T-connector to establish a flow system. H2O was flowed through the reaction loop from both pumps at a rate of 0.15 mL / min each to flush the entire system. Next, the feed solutions of pumps 1 and 2 were replaced from H2O to a solution containing [for pump 1, 7-ACA(5) (0.1 mol / L, 1.0 equivalent) and base 1 (1.0 equivalent) in H2O], and [for pump 2, 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.1 mol / L, 1.0 equivalent) and base 2 (1.15 of 2.23 equivalents) in H2O], and each was run at 0.15 mL / min. After stabilizing the system for 30 minutes, the outlet solution was collected for 30 minutes. The collected solution was washed with AcOEt and then acidified with an aqueous HCl solution. The pH was confirmed to be approximately 2.0 using pH test paper. The resulting solid was collected by vacuum filtration and washed with H2O, and then with acetone. The residue was vacuum dried to obtain the corresponding product 3 in 47-70% yield (shown in Table 7).

[0063] [Table 7]

[0064] Example 3: Flow synthesis of intermediate 3 from 7-ACA using a single pump TR1 (PTFE tube loop, 1.0 mm inner diameter × 10 mL) was warmed to 80°C in a water bath. One end of the tube was placed in a receiver flask, and the other end was connected to the outlet of pump 1 via a one-way connector. The entire system was washed by flowing H2O from the pump into the reaction loop at a rate of 0.3 mL / min. Next, the supply solution to the pump was replaced with H2O of 7-ACA(5) (0.05 mol / L, 1.0 equivalent), K3PO4 (1.0 equivalent), 5-methyl-1,3,4-thiadiazole-2-thiol(6) (0.05 mol / L, 1.0 equivalent), and K2HPO4 (2.23 or 4.0 equivalents), and flowed into the reactor at 0.3 mL / min. After stabilizing the system for 30 minutes, the outlet solution was collected for 30 minutes. The collected solution was washed with AcOEt, then acidified with an aqueous HCl solution, and the pH was confirmed to be approximately 2.0 using pH test paper. The resulting solid was recovered by vacuum filtration and washed with H2O, followed by acetone. The residue was vacuum-dried to obtain the corresponding product 3 in 70-71% yield (shown in Table 8).

[0065] [Table 8]

[0066] Example 4: Coupling and continuous flow synthesis of cefazolin Preparation of 1H-tetrazole-1-ylacetyl chloride (4-Cl) solution A magnetic stirrer was attached to a three-necked round-bottom flask, and 1 equivalent of 1H-tetrazole-1-ylacetic acid (4) and THF as a solvent were added under an argon (Ar) atmosphere. The mixture was then cooled to 0°C and stirred. A catalytic amount of N,N-dimethylformamide was added to the stirred solution via syringe. Next, 1.5 equivalents of oxalyl chloride were added dropwise to this mixture via syringe. The reaction mixture was stirred at 0°C for 1 hour, then heated to room temperature and stirred for a further 2 hours. After the reaction, the solvent was removed under vacuum to obtain 1H-tetrazole-1-ylacetyl chloride (4-Cl). The prepared reagent was used without purification. The acid chloride was dissolved in MeCN (0.0125~0.1 mol / L) to obtain a 4-Cl solution, which was used in the subsequent linked-continuous flow reaction.

[0067] Linked and continuous flow reaction using a unique flow system TR1 (PTFE tube loop, 1.0 mm inner diameter x 10 mL) was warmed to 80°C in a water bath. One end of the tube was placed in a receiver flask, and the other end was connected to the outlet of pump 1 via a one-way connector. Water (H2O) was flowed from the pump into the reaction loop at a rate of 0.3 mL / min to wash the TR1 system.

[0068] Meanwhile, a PTFE loop (tube-type reactor, TR2, inner diameter 2.0 mm × 1.0 mL) was submerged in a water bath, and one end of the tube was placed in another receiver flask. The other end was connected to the outlet port of a T-type micromixer α-600 manufactured by Nakamura Choko Co., Ltd. An outlet tube from pump 2, filled with acetonitrile, was connected to one of the inlet ports of the micromixer, and the other inlet port was capped with a plug. Next, this TR2 system was washed with acetonitrile.

[0069] A substrate solution consisting of 7-ACA(5) (required concentration), K3PO4 (1.0-5 equivalents), 5-methyl-1,3,4-thiadiazole-2-thiol(6) (1.0-5 equivalents), K2HPO4 (4.0-5 equivalents), and optionally 2,6-lutidine (0.05-5 equivalents) was prepared with H2O and started flowing into TR1 at the required flow rate. After maintaining the supply of this mother liquor for 30 minutes, the supply of the mother liquor was briefly stopped, and the end of the tube was connected to the unplugged inlet port of the micromixer of the TR2 system. Simultaneously with restarting the supply of the mother liquor, the outflow of 4-Cl (required concentration) in acetonitrile from pump 2 was started at the required flow rate. The linked and continuous flow system was stabilized for a further 30 minutes, after which the outlet solution of TR2 was collected for 30 minutes. The reaction solution was washed with AcOEt, acidified with 10% aqueous HCl solution, and then extracted with AcOEt and H2O. The organic phase was dried with Na2SO4, filtered, and the solvent was removed under vacuum. The yield of product 2 was calculated to be 44–65% by 1H-NMR in DMSO-d6 (internal standard: 1,1,2,2-tetrachloroethane) (shown in Table 2).

[0070] Example 5: Improvement for flow synthesis of intermediate 3 The previous TR1 (PTFE tube loop, inner diameter 1.0 mm × 10 mL) was used as TR1a, and the mother liquor and reactor system were prepared in the same manner as in Example 4.

[0071] Meanwhile, another PTFE loop (tube-type reactor, TR1b, inner diameter 1.0 mm × 10 mL) was prepared, and one end of the tube was placed in a receiver flask. The other end was connected to a T-shaped mixer.

[0072] An outlet tube from pump 2, filled with water (H2O), was connected to one of the mixer's ports, and the other port was capped with a plug. Next, the TR1b system was washed with water (H2O). A substrate solution consisting of 7-ACA(5) (0.2M), K3PO4 (0.2M), 5-methyl-1,3,4-thiadiazole-2-thiol(6) (0.2M), K2HPO4 (0.8M), and 2,6-lutidine was prepared with H2O as needed and flowed through TR1a at a flow rate of 0.8 mL / min. After maintaining the supply of this mother liquor for 30 minutes, the supply of mother liquor was briefly stopped, and the end of the tube was connected to the inlet port of the T-connector of the TR1b system with the plug removed. Simultaneously with restarting the supply of mother liquor, an additional 6 (as needed) aqueous solution containing the required amounts of K3PO4 and K2HPO4 was started flowing from pump 2 at a flow rate of 0.4 mL / min. The linked, continuous flow system was stabilized for a further 30 minutes, after which the outlet solution of TR2 was collected for 30 minutes. The collected solution was washed with AcOEt, acidified with an aqueous HCl solution, and the pH was confirmed to be approximately 2.0 using pH test paper. The resulting solid was collected by vacuum filtration and washed with H2O, followed by acetone. The residue was vacuum dried to obtain the corresponding product 3 in 69–76% yield (shown in Table 4).

[0073] Example 6: Improvement for linked and continuous flow synthesis of cefazolin The preparation of the 4-Cl acid chloride was shown in Example 4. The acid chloride was dissolved in acetonitrile at the required concentration (0.0333 to 0.0667 mol / L) to obtain a 4-Cl solution, which was used in the linked-continuous flow reaction. The TR2 (PTFE tube, 2.0 mm inner diameter × 1.0 mL) system was prepared in the same manner as shown in Example 4. The reactor was submerged in a water bath, and the entire system was washed with acetonitrile using pump 3. An improved two-step flow setup for the production of intermediate 3, consisting of TR1a and TR1b, was constructed as shown in Example 5.

[0074] Aqueous solutions of 7-ACA(5) (0.2 mol / L, 1.0 equivalent), K3PO4 (0.2 mol / L, 1.0 equivalent), 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.2 mol / L, 1.0 equivalent), K2HPO4 (0.8 mol / L, 4.0 equivalent), and 2,6-lutidine (0.01 mol / L, 0.05 equivalent) were prepared as mother liquor supplied from pump 1. Aqueous solutions of 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.2 mol / L, 0.5 equivalent), K3PO4 (0.1 mol / L, 0.25 equivalent), and K2HPO4 (0.4 mol / L, 1.0 equivalent) were prepared as additional supplies of 6 supplied from pump 2.

[0075] Next, the two-stage system was operated as shown in Example 5 and stabilized for 30 minutes. Then, the feed solution of pump 3 was replaced with a 4-Cl solution, and its stream was combined with the outlet solution of the two-stage TR1 system using a micromixer. The flow rate of the 4-Cl solution was appropriately controlled to adjust the stoichiometry of 5 to 2-3 equivalents. After stabilizing the entire flow system for 30 minutes, the outlet solution was collected for 30 minutes. The obtained solution was washed with AcOEt, acidified with a 10% HCl aqueous solution, and the pH was confirmed to be approximately 2.0 using pH test paper. The acidified solution was extracted with AcOEt and H2O, the organic phase was dried with Na2SO4, filtered, and the solvent was removed under vacuum to obtain the crude product. The yield of cefazolin 2 was obtained in DMSO-d6. 1The yield was calculated by 1H-NMR (internal standard: 1,1,2,2-tetrachloroethane) and was 60% trace. The crude product was isolated by flash column chromatography (CH2Cl2 / MeOH = 10 / 1~5 / 1 + 1% AcOH) and the corresponding product was obtained in yields of 2~57% (shown in Table 5).

[0076] Example 7: Conversion from cefazolin to cefazolin sodium The solution of the reaction products synthesized by linked-continuous flow synthesis was back-extracted with AcOEt and H2O. The separated aqueous phase was acidified with a 10% HCl aqueous solution and extracted with AcOEt and H2O. Next, the organic phase was back-extracted with a NaHCO3 aqueous solution. The aqueous phase was acidified with an 80% acetic acid aqueous solution, and the pH was confirmed to be approximately 5.0 using pH test paper. Charcoal (1.0 w / w%) calculated based on the NMR yield shown in the continuous flow synthesis of cefazolin) and alumina (2.0 w / w%) calculated based on the same charcoal addition method) were added to the acidified aqueous phase, heated to 40°C, stirred for 15 minutes, and the charcoal and alumina were filtered out. The filtrate was checked with pH test paper and further acidified to approximately 1.0 with a 10% HCl aqueous solution. The resulting solid was collected by vacuum filtration and washed with a small amount of acetone. The residue was vacuum-dried, and cefazolin(2) was obtained from the flow reaction in 60% yield.

[0077] To the cefazolin (2) obtained by the above method, NaHCO3 (1 equivalent) and H2O (1M) were added. Next, four times the amount of 2-propanol was added to the aqueous cefazolin solution. The solution was left in a cool, dark place. The resulting solid was collected by vacuum filtration, washed with 2-propanol, and then vacuum dried to obtain sodium cefazolin (1) in 88% yield.

[0078] Example 8: Final coupling and continuous flow reaction for large-scale cefazolin synthesis The preparation of the 4-Cl acid chloride is shown in Example 4. The acid chloride was dissolved in acetonitrile at a concentration of 0.05 mol / L to obtain a 4-Cl solution, which was used in the linked-continuous flow reaction.

[0079] Pump 1 LAs mother liquor supplied from the pump, aqueous solutions of 7-ACA(5) (0.2 mol / L, 1.0 equivalent), K3PO4 (0.2 mol / L, 1.0 equivalent), 2-mercapto-5-methyl-1,3,4-thiadiazole(6) (0.2 mol / L, 1.0 equivalent), K2HPO4 (0.8 mol / L, 4.0 equivalents), and 2,6-lutidine (0.01 mol / L, 0.05 equivalents) were prepared. Pump 2 L As aqueous solutions for the additional 6 supplies supplied from the source, 2-mercapto-5-methyl-1,3,4-thiadiazole (6) (0.2 mol / L, 0.5 equivalents), K3PO4 (0.1 mol / L, 0.25 equivalents), and K2HPO4 (0.4 mol / L, 1.0 equivalent) were prepared. TR2 L A new system (stainless steel tube, inner diameter 3.0 mm × 10 mL) was prepared using the same method as shown in Example 6. Some specific notes are provided below. The location of the equipment is shown in S9 or Figure 6.

[0080] #TACMINA XPL-1-XTCX-XWX pump 3 L It was used for that purpose. #Two thermocouples to TR3 L It was attached via an internal T-shaped connector (Figure 19(d)). #The other two thermocouples are connected via a T-connector TR3 L It was attached to the outside. #Micromixer and TR2 L Between and, and pump 3 L Two pressure sensors were installed in the line between the micromixer and the other components. #Pump3 L The flow rate of the stream was monitored through an in-line flow meter. #The measuring instrument was connected to the data logger.

[0081] TR1 consists of two stainless steel tube loops with an inner diameter of 3.0 mm and a length of 30 m. L A system was created. One stainless steel tube loop was used with TR1a L Specify as such, and connect one end to pump 1 LConnected to it. The other end was connected to the other loop designated as TR1b via a T-shaped connector. L The other port of the T-shaped connector was connected to Pump 2. L TR1 L The entire system was placed in a water bath and heated to 80 °C. The end of TR1b L was connected to the micromixer of the TR2 system. Some specific considerations are described below. The positions of the devices are shown in S9 or Figure 6.

[0082] #TACMINA XPL-1-STSX-XWX was used for Pump 1. L #TACMINA Q-100-6T-P-S was used for Pump 2. L #Two thermocouples were equipped via the T-shaped connectors inside TR1a L and TR1b respectively. L #The other thermocouple was installed via a T-shaped connector between Pump 1 L and TR1 L #Two pressure sensors were installed in the line between Pump 1 L and TR1 L and between TR1b L and the micromixer. #The flow rate of the stream from Pump 1 L was monitored through an in-line flow meter. #The measuring instruments were connected to a data logger.

[0083] Start the TR1 L system for the synthesis of Intermediate 3, stabilize it for 20 minutes, and discharge the outlet solution during the stabilization process from the system. Next, operate the supply of the 4-Cl solution by Pump 3 L at 300 mL / min. The entire connected and continuous flow system was stabilized for 5 minutes. Next, fractionate the outlet flow solution for 1 minute ( 1 ​​​​A fraction from another 10-minute flow (used in the precipitation step) was collected separately (for confirmation of H-NMR yield and isolation yield). The recovered reaction solution was washed with acetic acid for 1 minute, acidified with 10% hydrochloric acid (HCl) aqueous solution, and the pH was confirmed to be approximately 2.0 using pH test paper. The acidified solution was extracted with AcOEt and H2O, the organic phase was dried with Na2SO4, filtered, and the solvent was removed under vacuum to obtain the crude product. The yield of cefazolin (2) was compared with DMSO-d6. 1 The yield was calculated by 1H-NMR (internal standard: 1,1,2,2-tetrachloroethane) and was 57%. The crude product was isolated by flash column chromatography (CH2Cl2 / MeOH = 10 / 1~5 / 1 + 1% AcOH), and the corresponding product was obtained in yields of 2~54% (1.22 g).

[0084] To the cefazolin (2) obtained by the above method, NaHCO3 (1 equivalent) was added and dissolved in H2O (1 mol / L). Four times the volume of 2-propanol was added to the resulting solution. The solution was left in a cool, dark place. The resulting precipitate was collected by vacuum filtration and washed with 2-propanol. The residue was vacuum dried to obtain cefazolin sodium (1) from the cefazolin precipitate in 59% yield. Next, cefazolin sodium (1) was analyzed by HPLC (mobile phase: 800 mL of phosphate buffer (Na2HPO4:NaH2PO4=1:19) / 200 mL of MeOH, sample dissolved in 0.1 mol / L phosphate buffer pH 7.0), and the purity was 97.952%.

[0085] Example 9: Reaction data under large-scale linked and continuous flow conditions Figure 15 shows the reaction data under large-scale linked and continuous flow conditions.

[0086] Example 10: Test data on the effects of additives

[0087] [Table 9-1] [Table 9-2] aIn this case, the yield of cefazolin (2) was determined to be 50% after normal operation. This result is shown in Table 5, entry 3.

[0088] Example 11: HPLC analysis of reaction solution from linked-continuous flow reaction

[0089] [Table 10] By-product A: Amidation of 7-ACA(5) and TAACl(4-Cl). By-product B: N-acetylation intermediate 3.

[0090] The HPLC analysis results for the reaction solution of entry 3 in Table 5 are as follows:

[0091] [Table 11]

[0092] [Table 12]

[0093] Example 12: HPLC analysis of cefazolin(2) and its sodium salt The results of the HPLC analysis of cefazolin (2) and cefazolin sodium (1) are as follows.

[0094] [Table 13]

[0095] [Table 14]

[0096] [Table 15]

[0097] Example 13: Investigation of a batch of solvent systems suitable for the amidation process

[0098] [Table 16] a1 Determined by 1H-NMR analysis.

[0099] Before carrying out the amidation reaction in a flow system, a preliminary investigation of the reaction solvent system was conducted. Since thioetherification is carried out in a basic aqueous solution, we focused on water-miscible solvent systems. As shown in Table 16, although the basic conditions differ, acetonitrile was found to be the most suitable cosolvent. THF could not completely dissolve the acid chloride. Organic solvent systems such as DCM were also investigated, but intermediate 3 did not dissolve even in acidic form, and the yield of the target product was approximately 30%.

[0100] Comparative Example 1: Controlled one-pot two-step synthesis of cefazolin (2) under batch conditions Since the current linked-continuous flow reaction is essentially a one-pot process, a one-pot batch cefazolin synthesis was performed for comparison. The applicant did not optimize this process. As a standard procedure, thioetherification was carried out in aqueous solution under potassium phosphate base conditions, and then a base was added to this solution, followed by the amidation reaction by dropwise addition of an acetonitrile solution of the acid chloride 4-Cl. However, with this approach, crude 1 Only 4% of the corresponding amide, identified by 1H-NMR, was produced.

[0101] Example 14: Characteristic data of the product and intermediate NMR peak of (6R,7R)-7-amino-3-(5-methyl-1,3,4-thiadiazole-2-ylthiomethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]octo-2-ene-2-carboxylic acid (intermediate 3) 1 H-NMR (600MHz, D2O+NaHCO3)d(ppm): 4.85 and 4.59 (2H, ABq, J=5.0Hz, CH2), 4.34 and 3.78 (2H, ABq, J=13.7Hz, CH2), 3.64 and 3.26 (2H, ABq, J=17.9Hz, CH2), 2.58 (3H, s). (Figure 30). 13 ¹³C-NMR (150 MHz, D₂O + NaHCO₃) d (ppm): 170.9, 170.2, 168.9, 167.0, 131.8, 118.4, 62.8, 59.1, 39.0, 27.1, 15.5. (Figure 31). References for peak identification: Numata, M.; Minamida, I.; Yamaoka, M.; Shiraishi, M.; Miyawaki, T. Japan Patent 1976, JPS5111782.

[0102] NMR peak of cefazolin sodium (1) 1 H-NMR (600MHz, DMSO-d6) d(ppm): 9.53 (1H, d, J=8.5Hz, NH), 9.38 (1H, s, tetrazole CH), 5.54 (1H, dd, J=4.8, 8.5Hz, CH), 5.40 and 5.35 (2H, AB q, J=16.5Hz, CH2), 4.98 (1H, d, J=4.8, CH), 4.51 and 4.35 (2H, ABq, J=12.4Hz, CH2) 3.61 and 3.39 (2H, ABq, J=17.2Hz, CH2), 2.65 (3H, s, CH3). (Figure 32). 13 ¹³C-NMR (150 MHz, DMSO-d6) d(ppm): 166.0, 165.7, 165.5, 164.2, 162.7, 154.2, 133.8, 115.4, 58.5, 57.0, 49.2, 37.0, 26.6, 15.3. (Figure 33). References for peak identification: Sivakumar, B.; Parthasarathy, K.; Murugan, R.; Jeyasudha, R.; Murugan, S.; Saranghdar, RJSci.Pharm., 2013, 81, 933.

[0103] The above is merely to illustrate the principles of disclosure. The embodiments described herein are not limiting, but merely describe some of the many possible embodiments of the appended claims. Those skilled in the art will readily recognize various modifications and changes without following the exemplary embodiments and uses illustrated and described herein, and without departing from the true spirit and scope of the following claims.

[0104] All references cited and / or discussed herein are incorporated herein by reference in the same manner as if each reference were incorporated by reference individually.

[0105] literature 1. T. Nishino, Y. Yokota, T. Tanino, Jpn. J. Chemother. 1980, 28 S-1, 58-82. 2. DL Bratzler, EP Dellinger, KM Olsen, KTM Perl, PG Auwaerter, KM Bolon, DN Fish, LM Napolitano, RG Sawyer, D. Slain, JP Steinberg, RA Weinstein, Am. J. Health-Syst. Pharm. 2013, 70, 195-283. 3. WHO Model List of Essential Medicines - 22 nd List, 2021, who.int / publications / i / item / WHO-MHP-HPS-EML-2021.02 4. (a) A. Palomo-Coll, A. L. Palomo-Coll, C. P. Nicolau, Tetrahedron 1985, 41, 5133–5138. (b) R. Fernandez-Lafuente, J. M. Guisan, M. Pregnolato, M. Terreni, Tetrahedron Lett. 1997, 38, 4693–4696. (c) I. Estruch, A. R. Tagliani, J. M. Guisan, R. Fernandez-Lafuente, A. R. Alcantara, L. Toma, M. Terreni, Enzyme Microb. Technol. 2008, 42, 121–129. (d) M. Qiu, CN101696215B. (e) X. Wang, B. Wang, W. Yu, X. Yang, Y. Wenig, CN102617607B. (f) Z. Wu, W. Yu, X. Yang, S. Tao, CN 1102321101B. (g) G. Yang, W. Xin, T. Fei, H. Chuanshan, W. Yongjin, L. Fengxia, S. Hongbin, D. Fumin, F. Meiju, CN 104910188A. (h) F. Miaoquing, Z. Yeqing, S. Bin, X. Lei, Z. Xuwei, M. Quingshuang, Z. Baishui, W. Lei, CN 105541870B. (i) F. Miaoquing, W. Fengzhe, M. Bin, Z. Baishui, Y. Yuping, M. Quingshuang, CN 110396103B. 5. (a) M. Kostadinov, A. Nikolov, N. Tsoneva, N. Petkov, Appl. Biochem. Biotechnol. 1992, 33, 177-182. (b) OH Justiz, R. Fernandez-Lafuente, JM Guisan, P. Negri, M. Pregnolato, M. Terreni, J. Org. Chem. 1997, 62, 9099-9106. (c) JI Won, CG Kim, JH Kim, JH Lee, YJ Jeon, Appl. Biochem. Biotechnol. 1998, 69, 1-9. (d) P. Bonomi, T. Bavaro, I. Serra, A. Tagliani, M. Terreni, D. Ubiali, Molecules 2013, 18, 14349-14365. (e) L. Wang, AV Sklyarenko, D. Li, AI Sidorenko, J. Zhao, J. Li, SV Yarotsky, Bioprocess Biosyst. Eng. 2018, 41, 1851-1867. (f) AV Sklyarenko, IA Groshkova, AI Sidorenko, SV Yarotsky, Appl. Biochem. Microbiol. 2020, 56, 452-464. 6. EY Klein, TP Van Boeckel, EM Martinez, S. Pant, S. Gandra, SA Levine, H. Goossens, R. Laxminarayan, Proc. Nat. Acc. Sci. 2018, 115, E3463. 7. In 2019, the supply of cefazolin sodium in Japan was temporarily suspended due to quality problems with the API imported from overseas: H. Honda, S. Murakami, Y. Tokuda, Y. Tagashira, A. Takamatsu, Clin. Infect. Dis. 2020, 71, 1783-1789. 8. (a) U. S. Food and Drug Administration, Non-Penicillin Beta-Lactam Drugs: A CGMP Framework for Preventing Cross-Contamination, 2022, https: / / www.fda.gov / regulatory-information / search-fda-guidance-documents / non-penicillin-beta-lactam-drugs-cgmp-framework-preventing-cross-contamination-0. (b) International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, ICH Harmonised Tripartite Guideline, Good Manufacturing Practice Guide for Active Pharmaceutical Ingredients Q7, 2000. 9. (a) X. Y. Mak, P. Laurino, P. H. Seeberger, Beilstein J. Org. Chem. 2009, 5 No. 19. (b) D. Webb, T. F. Jamison, Chem. Sci. 2010, 1, 675-680. (c) J. Wenger, S. Ceylan, A. Kirchning, Chem. Commun. 2011, 47, 4583-4592. (d) J. Wenger, S. Ceylan, A. Kirchning, Adv. Synth. Catal. 2012, 354, 17-57. (e) I. R. Baxendale, J. Chem. Technol. Biotechnol., 2013, 88, 519-552. (f) T. Tsubogo, T. Ishikawa, S. Kobayashi, Angew. Chem. Int. Ed., 2013, 52, 6590-6604. (g) J. Pastre, D. L. Browne, S. V. Ley, Chem. Soc. Rev. 2013, 42, 8849-8869. (h) W.-J. Yoo, H. Ishitani, Y. Saito, B. Laroche, S. Kobayashi, J. Org. Chem. 2020, 85, 5132-5145. 10. (a) J. Yoshida, Flash Chemistry - Fast Organic Synthesis in Micro Systems, WILEY-VCH, Weinheim, 2008. (2) J. Yoshida, A. Nagaki, T. Yamada, Chem. Eur. J., 2008, 14, 7450-7459. (c) J. Yoshida, Chem. Rec., 2010, 10, 332-341. (d) J. Yoshida, Y. Takahashi, A. Nagaki, Chem. Commun., 2013, 49, 9896-9904. 11. (a) CJ Mallia, IR Baxendale, Org. Process Res. Dev. 2016, 20, 327-360. (b) M. Movsisyan, EIP Delbeke, JKET Berton, C. Battilocchio, SV Ley, CV Stevens, Chem. Soc. Rev. 2016, 45, 4892-5136. (c) N. Kockmann, P. Thenee, C. Fleischer-Trebes, G. Laudadio, T. Noel, React. Chem. Eng. 2017, 2, 258-280. 12. K. Kariyone, H. Harada, M. Kurita, T. Takano, J. Antibiot. 1970, 23, 131-136. [Industrial applicability]

[0106] Continuous flow synthesis methods have the potential to enable closed systems without external exposure, and are likely to be developed into safer systems with minimal risk of cross-contamination or human exposure.

Claims

1. Formula 2: 【Chemistry 1】 A continuous flow synthesis method of a compound represented by or a pharmaceutically acceptable salt thereof, comprising the following steps: (i) 7-Aminocephalosporanic acid (Compound 5): 【Chemistry 2】 in the presence of a base and water, is reacted with a solution of 2-mercapto-5-methyl-1,3,4-thiazole (Compound 6): 【Transformation 3】 to form Intermediate 3: 【Chemistry 4】 and (ii) reacting the intermediate with 1H-tetrazol-1-ylacetyl halide (Compound 4): 【Transformation 5】 A method comprising the steps of.

2. The continuous flow synthesis method according to claim 1, wherein step (i) is carried out while flowing the base, water, Compound 5, and Compound 6 into a first reactor.

3. Step (ii) is carried out while flowing the reaction mixture in step (i) from the first reactor to a second reactor connected to the first reactor and while supplying a solution of Compound 4 to the second reactor. The continuous flow synthesis method according to claim 1.

4. The amount of Compound 5 supplied per unit volume of the first reactor per unit time is [Math 1] (where F ５ : molar flow rate of Compound 5; V Ｒ１ : represents the internal volume of the first reactor (R1).) adjusted so that it is in the range of 0.01 to 1.00 mmol / min·mL. The continuous flow synthesis method according to claim 1.

5. Said F ５ / V Ｒ１ The continuous flow synthesis method according to claim 1, wherein the concentration is 0.02 to 0.60 (mol / min / mL).

6. The continuous flow synthesis method according to any one of claims 2 to 5, wherein Compound 6 is further supplied to another reactor connected to the first reactor.

7. where the base is Na ３ PO ４ 、Na ２ HPO ４、 K ３ PO ４ 、K ２ HPO ４ 、K ３ PO ４ The continuous flow synthesis method according to claim 1, which is at least one selected from the group consisting of triethylamine and combinations thereof.

8. The continuous flow synthesis method according to claim 1, wherein an organic base is added to the reaction in step (i).

9. The organic base is at least one selected from the group consisting of 2,6-lutidine, 3,4-lutidine, 2,4-lutidine, pyridine, 4-dimethylaminopyridine, N,N-dimethylaniline, tetrabutylammonium bromide, imidazole, 1,2-dimethylimidazole, 1-methylimidazole, 2,3,5,6-tetramethylpyrazine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 3-methyl-1-octylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride, and combinations thereof. The continuous flow synthesis method according to claim 8.

10. The continuous flow synthesis method according to claim 1, wherein the solution of Compound 4 contains Compound 4 dissolved in an aprotic polar solvent.

11. The continuous flow synthesis method according to claim 10, wherein the aprotic polar solvent is selected from the group consisting of acetonitrile, acetone, tetrahydrofuran, and dichloromethane.

12. The continuous flow synthesis method according to claim 1, wherein step (i) is performed in the range of 60°C to 90°C.

13. The continuous flow synthesis method according to claim 1, wherein step (ii) is performed in the range of 10°C to room temperature.

14. The continuous flow synthesis method according to claim 1, wherein the pharmaceutically acceptable salt of the compound is a sodium salt.

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