An improved Synthetic Process of Two Key Intermediates and Their Application in the Synthesis of Li ﬁ tegrast

Benzofuran-6-carboxylic acid 2 and 2-( tert -butoxycarbonyl)-5,7-dichloro-1,2,3,4-tetra-hydroisoquinoline-6-carboxylic acid 21 are two key intermediates for the synthesis of li ﬁ tegrast ( 1 ). The present study aimed to obtain li ﬁ tegrast from the key intermediates of 2 and 5,7-dichloro-2-(2,2,2-tri ﬂ uoroacetyl)-1,2,3,4-tetrahydroisoquinoline-6-carbox-ylic acid ( 31 ), which had the same core structure as 21 . In this study, the synthetic routes of 2 and 31 were explored. 2 and 31 were synthesized from 4-bromo-2-hydroxybenzaldehyde ( 25 ) and 2-(2,4-dichlorophenyl)ethan-1-amine ( 28 ), with the yields of 78 and 80%, respectively. The route avoided the harsh reaction conditions of generating 2 in a previous study and could more ef ﬁ ciently achieve the core structure of 5,7-dichloro-1,2,3,4-tetrahydroisoquinoline-6-carboxylic acid. Besides, the hydrolysis reaction conditions of preparing li ﬁ tegrast were also optimized. In this work, li ﬁ tegrast was obtained from 2 and 31 with high purity ( > 99.9%) and an overall yield of 79%, which was higher than the reported yield of 66%


Introduction
Dry eye disease (DED), also known as keratoconjunctivitis sicca, is a multifactor disease (e.g., low blink rate) characterized by decreased tear secretion or increased tear evaporation while affecting the optical tables and tear glands. 1 DED is classified into aqueous-deficient DED and evaporative dry eye as the major two subtypes. Studies have shown that DED affects approximately 5 to 50% of the global population. 2 With the increase in age, the prevalence of DED is gradually increased, and the incidence of DED in Asian people is higher than that of other people. 3,4 Lifitegrast is a novel small-molecule integrin antagonist that can competitively block the binding of lymphocyte function-associated antigen-1 and intercellular adhesion molecule-1, [5][6][7][8][9][10][11] thus affecting the activation of T cells and the release of cytokines (proteins). It is a new chemical entity developed by Shire Development LLC and approved by the Food and Drug Administration in July 2016 for the treatment of the symptoms and signs of DED. It provides a new option for patients with DED. 12 With the acceleration of the pace of life, a large number of people have the symptom of DED in China, shown in recent survey reports. So, it would be of great interest to develop a more economical route for process development researchers.
A reported method for the synthesis of lifitegrast is illustrated in ►Scheme 2, which starts from the amidation reaction of compounds 3 and 21 (Boc-4). 13 After removing the Boc group in a HCl solution, the resulting compound 23 is subjected to amidation with 2 to provide 24, which undergoes ester hydrolyzation to give the target compound 1. However, there are some challenges in the reported method: (1) the reaction conditions for the preparation of intermediate 2 were harsh and difficult to operate; (2) the yields of intermediate 4 were low and the process is difficult to scale up; and (3) the degradation of lifitegrast occurred in hydrolyzation, so the conditions should be controlled strictly. In this manuscript, we are dedicated to solving the problems mentioned above.
Intermediate 2 can be obtained in two ways (►Scheme 3). 13,14 One starts from commercial 2-(2-bromoethyl)-1,3dioxolane 5, through a substitution reaction, Wittig olefination reaction, cyclization reaction, and amide hydrolysis reaction to give intermediate 2 (route A, ►Scheme 3). However, the reaction time of the route A was too long, and the operation of the reaction was complex. The other way employed the inaccessible and expensive 6-hydroxy-2(3H)-benzofuranone 9 as the key material, which was converted to intermediate 2 through five steps, including a substitution reaction, a reduction reaction, dehydration, carbonyl insertion, and an ester hydrolysis reaction (route B, ►Scheme 3). Unfortunately, the operation is complicated, and the reaction time is long, while the introduction of carboxyl groups requires palladium acetate in the fourth step, which may have heavy metal palladium residual problem.
Compound 18 was a derivative of intermediate 4. Zeller et al achieved 18 (Ph 3 C-4) from two routes (routes C and D, ►Scheme 4) using 3,5-dichlorobenzaldehyde (14) as a starting material. 13 As for route C, the second step involves cyclization to construct the tetrahydroisoquinoline ring at high temperature in a sealed tube without solvent. This step required a high reaction vessel and had a risk of explosion. In addition, the yield of the first step was only 35%, which severely limited the practicability of the route. Route D also had some insufficiency, including complex operations that are not conducive to actual production. Besides, it would be dangerous to reduce the isoquinoline ring of compound 20 under high-pressure hydrogen (the third step). Route D not only had a low yield but also used expensive catalysts. Thus, it is crucial to explore a method to construct the tetrahydroisoquinoline ring more reasonably and economically.
As for the preparation of lifitegrast, intermediate 3 coupled with compound 21 (Boc-4) under HATU and triethylamine in N,N-dimethylformamide. After removing the Boc group to get compound 23, it was coupled with compound 2 to get compound 24, followed by hydrolysis to get the final product compound 1. During the last step, there were three impurities (►Fig. 1; impurity 1, 2, and 3) produced when 2 mol/L sodium hydroxide was used for hydrolysis or hydrogenation with Pd/C to remove benzyl. 6 In this article, we will focus our effort on solving the synthetic problems given bellow: (1) avoid the harsh reaction conditions of compound 2; (2) design a more efficient route to synthesize compound 4; (3) optimization of the hydrolysis condition of compound 1 to improve the final product's purity.

Synthesis of Intermediate 2
We designed a novel synthetic route to obtain 2. First (►Scheme 5, route E), compound 25 and bromoacetic acid are coupled to afford 26 through Williamson ether synthesis. Compound 26 underwent a ring-closure reaction to give 6bromobenzofuran 27, which was reacted with CO 2 in the presence of n-butyllithium to give the target product. However, 26 was obtained with an unacceptable yield of 30%, despite several attempts to optimize the reaction conditions, and 25, as a starting material, remained even though the reaction time lasted for 24 hours at 75°C. To solve this problem, 26 was prepared through etherification of 25 with methyl 2-chloroacetate as the initial step to get 25-1 followed by its ester hydrolysis (►Scheme 5, route F,). Fortunately, all the efforts were effective, as the yield of 26 was improved, reaching 90%.
The substrates of the Pictet-Spengler reaction were screened (►Table 1). When R ¼ H (compound 28), no satisfactory results were achieved (►Table 1, entry 1). When R ¼ t-butoxycarbonyl, (a Boc-protecting group), there was also no reaction. Then, some strong electron-withdrawing groups were tried, with acceptable yields (►Table 1, entries 3-5). Considering the convenience of removing the protective groups, we finally chose trifluoromethyl as the protective group for the subsequent reaction. [15][16][17] Scheme 5 Novel synthesis methods of intermediate 2.
Scheme 6 Novel synthesis methods of intermediate 31.  The condition of the Pictet-Spengler reaction was further optimized. The ratios of concentrated sulfuric acid to acetic acid (V:V), as well as the reaction time, were screened (►Table 2). We found that when the proportion of concentrated sulfuric acid was too low, the reaction time would be prolonged or it would not react (►Table 2, entries 4 and 5). When the ratio is too high, the reaction time would be shortened, but the raw material may be decomposed, affecting the yield (►Table 2, entry 1). Finally, the volume ratio of 3:2 was selected as the optimal condition (►Table 2, entry 2).

Synthesis of Lifitegrast (1)
We noticed that the original synthesis process has a problem to be resolved: compound 32 (►Fig. 2), which is a byproduct of the reaction between compound 4 and HATU, is difficult to be converted to amide ester 22 and the yield was only 70%.
So, we contributed to increasing the yield of 22 and reducing the reaction time.
Condensing reagents, the equiv. amounts of HATU and triethylamine of the reaction were screened. We found the yield of 22 was still lower and the reaction time was longer when the amount of catalyst was increased (►Table 3, entries 8 and 9). Using a base is necessary for the reaction to achieve a better yield of 22. While we tried to change the equiv. of the base, a high yield of 22 (98%) could be provided in a short reaction time with the 5 equiv. base and 1.0 equiv. HATU (►Table 3, entry 4). Finally, under the improved reaction conditions, the yield of 22 was improved from 75% 13 to 98%. Meanwhile, the reaction time was reduced to 5 hours.
As for the synthesis of the target product (1), the conditions for hydrolysis of benzyl ester derivative 24 were screened (►Table 4). In the beginning, Pd/C (►Table 4, entry 1) or sodium hydroxide solution (►Table 4, entry 2) was tried. Unfortunately, the desired products had a low purity and were contaminated with impurities 1-3, which could not be easily removed by recrystallization. Suffering from the above issues of the economy of Pd/C and the residue of heavy metals, a hydrolysis reaction was chosen to obtain the target product. Subsequently, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide at a concentration of 1 mol/L were screened. Our    data showed that the use of K 2 CO 3 solution (1 mol/L) was effective and should be selected as the optimal condition for debenzylation. Crude product 1 was purified by recrystallization of isopropanol and n-heptane (HPLC: >99.9%) to achieve lifitegrast.

Conclusion
In this work, we have developed a novel and practical method for the synthesis of 2 starting from 4-bromo-2-hydroxybenzaldehyde 25. The synthesis was accomplished in three steps with a total yield of 78%. In addition, we used 2-(2,4dichlorophenyl)ethylamine as a raw material to obtain the key intermediate 31 in three steps with a yield of 80%. With an optimized synthetic route in hand, lifitegrast was obtained a total yield of 79% (calculated by compound 21).
The total synthesis route of lifitegrast including the two key intermediates is shown in ►Scheme 7.

Experimental General Procedures
Unless specified otherwise, all starting materials, reagents, and solvents are commercially available (Shanghai Haoyuan Chemexpress Co., Ltd., Shanghai, China; Bidepharmatech Ltd., Shanghai, China). Thin layer chromatography (TLC) on silica gel GF254 was used to monitor the progress of all reactions. Melting points were obtained on a melting point apparatus (WRS-2A, Shanghai INESA Scientific Instruments Co., Ltd., Shanghai, China) and were uncorrected. The electrospray ionization (ESI) mass spectra were collected on a Waters ZQ2000 spectrometer (Waters, United States). Compound purity was determined by high-performance liquid chromatography (HPLC) (Waters e2695 HPLC, 2998 PAD, Waters Corp., Milford, MA, United States). Nuclear magnetic resonance (NMR) spectra were recorded in chloroform-d or dimethyl sulfoxide-d 6 on a 400 MHz or 600 MHz Bruker NMR spectrometer (Bruker Bio-Spin, Rheinstetten, Germany) with tetramethylsilane as the internal reference, and all chemical shifts were reported in parts per million (ppm). The purity of the products was measured by a HPLC-peak area normalization method using a Welch Ultimate XB C18 column (4.6 mm Â 250 mm, 5 µm) under the following conditions: mobile phase A (ACN) and phase B (0.05% TFA [v/v] water solution) with gradient elution of 0 to 50 minutes: 80 to 10% B; 50 to 51 minutes: 10 to 80% B; 51 to 60 minutes: 80% B. The flow rate was 1 mL/min; the column temperature was 30°C ; and the detection wavelength was 220 nm.