Identification, Characterization, Synthesis, and Strategy for

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Identification, characterization, synthesis and strategy for minimization of potential impurities observed in the synthesis of solithromycin Zhihong Zhong, Chong Du, Zhonghua Luo, shuaihua Song, Gaohong Liao, Jia Yao, Siegfried Goldmann, and Zhongqing Wang Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.7b00201 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 19, 2017

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Organic Process Research & Development

Identification, Characterization, Synthesis and Strategy for Minimization of Potential Impurities Observed in the Synthesis of Solithromycin ‖,†

Zhihong Zhong

, Chong Du

‖,†

Goldmann , Zhongqing Wang*, †

, Zhonghua Luo , Shuaihua Song , Gaohong Liao , Jia Yao , Siegfried †

†

†

†

,

† §

†

HEC Research and Development Center, HEC Pharm Group, Dongguan 523871, P. R. China

§

State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co., Ltd, Dongguan

523871, P. R. China

1 ACS Paragon Plus Environment


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TOC Graphic:


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ABSTRACT：： Potential causes for the formation of synthetic impurities that are present in solithromycin (1) during the laboratory development are studied in the article. These impurities were monitored by HPLC, and their structures are identified on the basis of MS and NMR spectroscopy. In addition to the synthesis, characterization of these seven impurities, strategies for minimizing them to the level accepted by ICH are also described. KEY WORDS: solithromycin, clarithromycin, impurity, macrolide antibiotics



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Macrolide antibiotics, such like erythromycin, azithromycin and clarithromycin, have proven to be safe, and effective for use in treating human infectious diseases such as community-acquired bacterial pneumonia (CABP), urethritis and other infections. Because of the importance of macrolide antibiotics, there has been a growing recent interest in this area as exemplified by the new fourth-generation macrolide solithromycin (1), which is developed by Cempra Pharmaceuticals as the first fluoroketolide antibiotic that has recently completed phase III clinical trials and demonstrates potent activity against the pathogens associated with CABP, including macrolide- and penicillin-resistant isolates of S. pneumoniaeis1.Thus many efforts have been devoted to the synthesis of 12-3. Scheme 1. Reported Retrosynthetic Analysis of Solithromycin by Semisynthetic Routes.

The retrosynthetic analysis of solithromycin employed in the previously reported methods2 shown in Scheme 1 shows a similar synthetic strategy that is practically using the modified biosynthetic routes, except that Ian B. et al. has developed a full synthetic route for the synthesis of 13. Considering the convenience and efficiency for readily scalable process, the semisynthetic methods are still preferred by now. These


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biosynthetic routes started from erythromycin2a or clarithromycin2b-i mainly differ in the sequence of structure modification (Scheme 1). However, according to our early experimental studies, we considered to take the synthetic method2c outlined in Scheme 2 for the process development based on the commercial availability of raw materials and better yield. Meanwhile, the corresponding impurity profile of this substance was thus studied in details. Scheme 2. Reported Route for the Synthesis of Solithromycin.

Me Me

OMe Bz2O

HO Me O O Me

Me Me

O

Et3N Me

Me

O

OH OMe

O Me Clarithromycin

O

O

Me

HCl

O

Me

O O

Me

O

NMe2

BzO

O

OMe

Me Me

NMe2

Me

BzO Me Oxidtion

O

O

OMe

Me Me

N

O

O

FN(SO2Ph2)2

Me

O Me OH Me

O Me Me

OBz OMe

OBz OMe

Me

N3

N

Me

Me

7

O

Me O O Me

O Me

OBz OMe

Me

O

OMe

Me Me

O

N3

H2N

Me O O Me

Me Me

Me

BzO

N

Me

N

N3

NMe2

Me

DBU

Me

O

O

O

8

N3 O

CDI

Me O O Me

O

N

OMe

Me Me

O

Me

O

O

HO Me

BzO

O

OMe

Me Me

HO

Me

O

O

NMe2

Me

BzO

O

HO

HO

NMe2

Me

NMe2

Me O

O

Me

O Me

O Me

O

O 5

6

Me O 4 N

N NH2

N3 NMe2

Me

O

O Me

O Me

O Me

O O

O

F

Me

O

Me

CuI

Me Me

N

O

O

Me

Me

F

O

O

Me

Me

O Me

O O

OMe

O

Me

O Me

Me Me

N

O

O 3

MeOH

OMe

HO

O

BzO

OMe

Me Me

N

NMe2

Me

NMe2

Me

BzO

O

N N

H2N

N N

H2N

O Me

Me

O O

2

F

Me

Solithromycin (1)

It is well known that impurity profile of an active pharmaceutical ingredient is of fundamental significance for its efficiency and safety point of view and is now receiving special attention from regulatory authorities. To the best of our knowledge, the study toward the identification, synthesis, characterization and control of impurities in solithromycin, which will be of immense importance for process development chemists to understand the source of potential impurities during the synthesis of 1, was rarely reported in the literature to



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date. In light of this great attention was paid on the study of impurity profile with the purpose to isolate and identify all the possible impurities that could be formed during the process for 1. As the International Conference on Harmonization (ICH) guidelines4 dictate rigorous identification of impurities at levels of 0.1%, we were required to identify a total of seven impurities appearing in the sample of the substance 1. In this context, a comprehensive study has been given to describe the identification, synthesis, characterization and strategy for controlling all the seven impurities present in the laboratory batches of 1 using spectroscopic and spectrometric techniques. This study will help a synthetic organic chemist to understand the potential impurities in solithromycin synthesis and thereby obtain the pure compound. Results and Discussion

The identification and possible pathways for the formation of impurities are elaborated at first, and then the strategies we adapted for assessing and minimizing theses impurities to the level accepted by ICH are described as follows. First of all, LC-MS was used as a common approach to identify impurities in 1, by which the molecular weights for impurities A-G (Table 1) were observed. Their structures were late confirmed by HRMS, NMR and HPLC co-injection. These impurities are formed due to degradation (impurity A), incomplete reaction (impurities B, F, G), or side reactions (impurities A, C, D, E). During our study we realized that almost all the impurities listed in Table 1 are hardly removed from 1 due to solubility issues. Therefore, we decided to control them in the previous steps by modifying and optimizing the corresponding reaction conditions. Table 1. Impurities detected during the synthetic process Name

Structure

Relative Retention Time


a

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Impurity-A

0.563

Impurity-B

0.844

Impurity-C

1.070

Impurity-D

1.177

Impurity-E

1.185



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a

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Impurity-F

1.473

Impurity-G

1.534

Agilent HPLC; column: Waters X bridge C18 (4.6 × 150 mm), 5 µm; flow rate: 1.0 ml/min, λ: 235 nm;

injection vol: 5 µl; mobile phase-A: Methanol/ACN (1:5, v/v); mobile phase-B: 0.1% aqueous solution of ammonia/mobile phase-A (60:40, v/v); Run time: 60min; Solithromycin retention time: about 24.55 min, temperature: 40 oC. gradient( A:B, 0min: 0:100; 30min: 30:70; 45min: 80:20; 60min: 0:100; V/V) Impurities Identification and Plausible Pathways. For the study of impurity A, it is observed that it will increase when excess fluorinating reagent N-fluorodi(benzenesulfonyl)amine (NFSI) is used in the fluorination reaction. This impurity might be introduced by Polonovski-type reaction in the following way (Figure 1). Firstly intermediate 3 reacted with the fluorinating reagent to afford the corresponding quaternary ammonium salt, from which hydrogen fluoride was then removed under strong alkaline conditions to get unstable imine ion 9. Followed with the hydrolysis, the formaldehyde was cleaved to obtain the hemiacetal, which will form the impurity A in the sequent process. Considering the whole process (Scheme 2), impurity A might also be introduced in the oxidation step via Polonovski-type reaction5. And in the late study, we found that impurity A increased obviously under


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accelerated conditions (oxygen and temperature), which was also consist with our proposed mechanism.

Figure 1. Possible pathway to impurity A. The formation of impurities B, F and G might be due to the incomplete reaction of the intermediates 4, 3 and 2 respectively

6-8

. And the impurity C9 as the regioisomer of substituted (1, 4)-triazole 1, which consist

with a structurally related (1, 5)-triazole, might be introduced via competitive reaction of intermediate 3 and 3-ethynylaniline. Impurity D is the structural analogue of 1, where the fluorine atom has been replaced by chlorine atom. This impurity was believed arising from the related intermediate 11 that was formed in the hydrolysis reaction step for 5 via the chlorination of 4 by Cl+, which might be introduced via the oxidation of chloride ion (Figure 2).



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Figure 2. Possible pathway to impurity D. During the reaction of intermediate 3 and 3-ethynylaniline, trace amounts of I+ might be produced via oxidation with the copper iodide, which can then react with the copper complex 12 to afford the iodide 13 and then followed by de-protection to form impurity E10 (Figure 3). N

N

N N

H2 N N3

CuLn N

Me

NH 2 O

O

O

Me

CuI

O O

F

OMe O

O

Me

Me

Me O F

O

O

Me Impurity E

Me O O

Me

Me

O

3

OMe

O Me

O Me

Me Me

N O

O Me

Me O Me

Me Me

N

BzO

O

O

+ O Me

I

N

Me

BzO

O

OMe

Me Me

N

I

N

Me

BzO

O

N N

H 2N

F

Me

O

12

13

Figure 3. Possible pathway to impurity E. Impurities Synthesis and Control. After identification of possible pathways for the formation of the impurities, the characteristics of the impurities which can influence the quality and safety of the drug were reevaluated. Among them, impurity A is a very important one, for its analogues are generally involved in the macrolides antibiotics11-12. The reason is mainly due to the activity of the nitrogen atom in the desosamine sugar ring which is easily to lose electron and becomes active. This impurity is obtained by the reaction of 1 and diisopropylazodicarboxylate13 (DIAD), followed by purification with silica gel chromatography (Scheme 3).


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Scheme 3. Synthesis of impurity A Impurities B and F are synthesized with the same synthetic protocol of 1, while the intermediates 3, 2 are replaced with intermediates 4 and 3 respectively, and both in good yield and purity (Scheme 4, 5).

Scheme 4. Synthesis of impurity B N3

N3 NMe2

Me Me Me

N O

O

Me MeOH

O 3

F

OMe O

O

Me

O Me

O Me

O O

Me Me

N

Me

O Me Me

HO

O

OMe O

NMe2

Me

BzO

O

Me

O O

Me F O impurity F

Me

Scheme 5. Synthesis of impurity F The impurity C, taken as the regioisomer of 1, is another potential impurity in the drug substance. This impurity was reported to be synthesized by Rh catalyst method9, however we used a modified route to get it, which was almost the same as the synthetic protocol of 1, except that the copper iodide (I) was not added and further purification with silica gel (Scheme 6).



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N

NMe2

Me

NH 2

Me Me

N

O

Me

Me

N

O

O

O Me

O Me Me

90°C

O O

F

Me

O Me

O Me

O Me

O O

BzO

O

OMe

Me

F

Me

O

Me +

OMe

Me Me

N

O

O O Me Me

O

Me

MeOH

Impurity C

Me O O

2

3

NMe2

Me

BzO

O

OMe O

NH 2

NMe 2

Me

BzO

O

N N N

N N

H 2N N3

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O

F

Me

15

Scheme 6. Synthesis of impurity C The following work was taken for the confirmation of the structure of impurity C, which differs mainly in the substituted position of the triazole ring. Based on that the absolute configuration of 1 (Figure 4) had been characterized by single crystal X-ray diffraction (also see Figure S1) during our study, the compare and assignment of impurity C and 1 was performed (NMR) through proton-NMR spectra and confirmed by HMBC and HSQC spectrum as follow. For impurity C, C5 was identified as quaternary carbon atom in HSQC spectrum (Figure 9), and H4, H6, H8, H12 were correlated with C5 in HMBC spectrum (Figure 7), these indicated that the benzene ring was connected with triazole ring at C5 position.

Figure 4. The crystal structure of solithromycin by X-ray analysis.


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Figure 5. Proton NMR spectra for solithromycin and impurity C.

Figure 6. HMBC expansion where the most important correlations are shown for solithromycin.



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Figure 7. HMBC expansion where the most important correlations are shown for impurity C.

Figure 8. HSQC expansion where the most important correlations are shown for solithromycin.


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Figure 9. HSQC expansion where the most important correlations are shown for impurity C. Impurity D has almost the same structure as 1 except the F atom was replaced with Cl atom. We got this impurity from the reaction of intermediate 4 and N-Chlorosuccinimide (NCS), then followed the relevant sequence as described for the active pharmaceutical ingredient (API) synthesis (scheme 1) and lead to the formation of impurity D (Scheme 7). N N3 NMe 2

Me Me Me

N

O

O Me

O

O O

Me

Me

N NCS

Me

Me

O

H

NH 2

O

O

Me

Me

O Me

Me

Me

MeOH

O

O

Me

Me

O Me

Me Cl

Me

O 4

OMe

O

O O

Me Me

N

O

base

HO

O

OMe

Me

NMe 2

Me

BzO

O

OMe

O

NMe2

Me

BzO

O

N N

H 2N

N3

O O O

Me Cl

Impurity D

16

Scheme 7. Synthesis of impurity D. Impurity E is formed as a result of side reaction of 3 with copper iodide and 3-ethynyl-aniline. It is similar



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to 1 except that the triazole ring is additionally substituted by an iodide atom. For the synthesis of impurity E, iodine monochloride was opted to add in the preparation of intermediate 17, and then product was afforded followed by deprotection and purification with silica gel. (Scheme 8).

N

NMe2

Me

I

O

O Me

O Me

O

Me

O

F

Me Me

O

Me

MeOH

Me Me

N

O

CuI, ICl

HO

O

OMe O

NMe2

Me

BzO

N

OMe O

O

Me

O Me

O Me

O

O

I

NMe2

Me O

OMe

Me Me

N

N N

H2N

BzO

O

Me

NH2

N

N N

H2N

N3

Me

Me

O 3

F

Me

O Me

O

O

Me

O

Me

O O

17

F

Me

Impurity E

Scheme 8. Synthesis of impurity E.

Control During the process development for 1, we found that the process impurity A was the most critical one. And the corresponding fluorination reaction for 3 from 4 was thus optimized with the purpose to avoid the generation of 10 as the precursor of impurity A (Figure 1). After the base, reaction solvent and temperature were evaluated successively, the base 1, 8-Diazabicyclo [5.4.0] undec-7-ene (DBU) was considered as the preferred one (Table 2) with the reaction taken in DMF (Table 3) while the temperature is at -20 oC (Table 4). However, even with the acquired optimized reaction condition, the generation of 10 was still hard to avoid or control to lower than 1.0%, which might mainly due to the use of NFSI ( see Figure 1) and result in the content of impurity A more than even 0.15% in the final product. b

Table 2. The effect of base in the reaction Entry

base

10

4

3

1

Potassium tert-butoxide

1.98%

1.56%

89.15%

2

Potassium hydroxide

1.11%

51.71%

5.45% 93.14%

3

DBU

2.29%

N/A

4

Et3N

N/A

91.96%

N/A

5

DIPEA

N/A

90.19%

N/A

b

All reactions performed at -20 oC, and DMF as the solvent


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c

Table 3. The effect of solvent in the reaction Entry

c

solvent

10

4

3

1

DMF

2.29%

N/A

93.57%

2

DMAC

1.24%

N/A

91.81%

3

acetone

2.86%

3.30%

87.53%

4

ethyl acetate

0.35%

6.06%

86.02%

5

toluene

0.33%

12.36%

79.49%

6

DCM

1.52%

22.34%

65.59%

All reactions performed at -20 oC, and DBU as the base d

Table 4. The effect of temperature in the reaction Entry

temperature

10

4

3

1

-35℃

3.47%

0.12%

92.98%

2

-20℃

1.98%

N/A

93.41%

3

-15℃

2.29%

N/A

93.14%

4

-10℃

1.79%

N/A

92.40%

5

0℃

1.95%

0.27%

90.31%

6

10℃

2.87%

1.21%

88.70%

7

20℃

2.88%

3.34%

84.95%

d

All reactions performed at DMF as the solvent, and DBU as the base One approach to solve this problem is trying to improve the impurity removal capacity in the final step.

To our disappointment it effected little for the removal of impurity A by screening the recrystallization solvent system, which might be due to the structure similarity of impurity A and 1. Thus a new solution is given by converting impurity A to 1 via methylation reaction. When the normal transformation reaction for 3 was completed, formaldehyde and formic acid was added to the reaction mixture, stirred for 2 h, and then the impurity 10 could be transformed to 3. Under this strategy, the level of impurity A was successfully reduced to lower than 0.05% in final substance 1 (Scheme 9).



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Scheme 9. Methylation reaction The formation of impurities B and F are due to the residue of intermediates 4 and 3 via the reaction of intermediate 2 to 1 respectively. These two impurities could be achieved to lower than 0.10% in the final product by controlling the conversion of the corresponding transformation reaction according to the in process control (IPC). For the control of impurity C, the use of copper (I) iodide in the preparation of intermediate 2 was an important aspect to reduce the unwanted formation. And we found that if no copper (I) iodide was added in the reaction, the content of intermediate 15 was about 40%, and while 0.05%~0.15% w/w copper (I) iodide was added in the reaction, the content of 15 was lower than 0.10% accordingly. By this way the impurity C can be controlled lower than 0.05% in 1 (Scheme 6).Based on the study of the proposed mechanism of impurity D, it could be controlled by reducing the residue of sodium chloride (NaCl) in 5. When 5 was separated from reaction mixture via centrifugation, and then washed with water, the residue of NaCl could be controlled in a low level, thus the impurity D was reduced to lower than 0.05% in 1 accordingly. For the impurity E, when the corresponding azide-alkyne cycloaddition between 3 and 3-ethynyl-aniline in the presence of copper (I) was taken under nitrogen protection, impurity E could be reduced to lower than 0.05% in 1. And for the control of impurity G, which was relatively easy to remove in the final purification step, this one was less than 0.10 % in 1 by prolonging the corresponding reaction time moderately to make sure the reaction was completed totally. Conclusion In summary，seven observed and potential impurities of solithromycin have been identified, synthesized and characterized here, which will be useful for the better understanding of the synthetic pathway of an API. The origins of formation impurities A-G during the preparation of 1 were also discussed. In addition to this, the


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strategy for minimization of these impurities to the level accepted by ICH has also been demonstrated here. This information would be immensely useful for process chemists working in this area. EXPERIMENTAL SECTION All materials were purchased from commercial suppliers. Unless specified otherwise, all reagents and solvent were used as supplied by manufactures. NMR spectra were measured on a Bruker Avance 400 spectrometer or 600 spectrometer in the solvents indicated; chemical shifts are reported in units (ppm) by assigning TMS resonance in the 1H spectrum as 0.00 ppm, CDCl3 resonance in the 13C spectrum as 77.0 ppm. Coupling constants are reported in Hz with multiplicities denoted as singlet (s), doublet (d), triplet (t), quartet (q), dd (doublet of doublets); m (multiplets), and so forth. HRMS were performed on Fourier transform ion cyclotron resonance mass spectrometer. HPLC analysis were run on a Agilent 1200 apparatus, Waters XBridge C18 (4.6 × 150 mm, 5 µm), detected at 235 nm. Impurity A. To a solution of compound 1 (31.5 g, 37.1 mmol) and dichloromethane (350 ml) was added DIAD (9.3 g, 46.2 mmol). And the reaction mixture was stirred at ambient temperature for 24 h, then concentrated under reduced pressure to obtain the crude solid. The solid was purified using flash chromatography to afford impurity A (6.2 g, 96.62% purity) in 20% yield. 1H NMR (400 MHz, DMSO-d6) δ 8.36 (s, 1H), 7.03-7.09 (m, 2H), 6.92 (d, J=7.6 Hz, 1H), 6.52 (dd, J=1.2, 7.9 Hz, 1H), 4.75 (dd, J=2.4, 9.7 Hz, 1H), 4.37 (m, 2H), 4.24 (d, J=7.5 Hz, 1H), 3.93 (d, J=10.3 Hz, 1H), 3.57 (m, 2H), 3.38-3.51 (m, 2H), 3.33 (s, 1H), 3.13 (q, J=6.6 Hz, 1H), 2.87 (t, J=8.5 Hz, 1H), 2.48 (m, 1H), 2.40 (s, 3H), 2.36 (m, 1H), 2.25 (s, 3H), 1.82-1.87 (m, 3H), 1.69-1.74 (m, 3H), 1.58-1.66 (m, 1H), 1.48-1.51 (m, 4H), 1.22 (m, 6H), 1.15 (m, 6H), 0.89 (d, J=6.8 Hz, 3H), 0.82 (t, J=7.3 Hz, 3H); 13C NMR (150 MHz, DMSO-d6) δ 216.3, 201.8, 166.7, 156.1, 146.1, 138.8, 131.5, 129.4, 121.3, 120.8, 118.5, 115.7, 102.6, 97.8, 81.7, 79.9, 78.1, 78.0, 70.3, 67.5, 60.2, 58.2, 49.1, 48.8, 44.2, 42.2, 40.2, 38.8, 38.6, 33.2, 29.7, 27.1, 24.7, 23.7, 21.8, 20.6, 19.4, 17.4, 14.6, 14.5, 13.3, 10.3; HRMS [M+H]+ for C42H63FN6O10, calculated: 831.4662, found 831.4665. Melting range: 229 −232 °C. Impurity B. To a solution of compound 4 (12.0 g, 15.0 mmol) and dichloromethane (60 ml) were added m-APA (2.24 g, 19.1 mmol), CuI (0.38 g, 2.1 mmol) and DIPEA (0.46 g, 4.1 mmol). The reaction mixture was refluxed at 40 oC and stirred overnight. The conversion was monitored by HPLC. After completion, active carbon (5 wt %, 0.6 g) was added and stirred for 30 min. The residue was removed by filtration, and the



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filtrate was concentrated to afford a solid. The solid was treated with methanol (33 ml) and stirred at 65 oC. When the material was converted completely, water (17 ml) was added dropwise into the mixture, and then cooled to 0 oC for 2 h. The precipitated solid was filtered and further purified by recrystallization from methanol/water (1:2, v/v) twice to afford impurity B (2.8 g, purity 96.77%) in 22.97% yield. 1H NMR (600 MHz, CDCl3) δ 7.85 (s, 1H), 7.27 (d, 1.7Hz, 1H), 7.16-7.20 (m, 2H), 6.65 (m, 1H), 4.94 (dd, J= 10.4, 2.0 Hz, 1H), 4.43 (td, J= 7.1, 1.4 Hz, 2H), 4.27 (d , J=7.3 Hz, 1H), 4.23 (ddd, J=18.4, 11.3, 3.7 Hz, 2H), 3.87 (m, 1H), 3.77 (m, 1H), 3.67 (m, 1H), 3.58 (s, 1H), 3.54 (m, 1H), 3.17 (dd, J=7.3, 10.2Hz, 1H), 3.13 (q, J=6.9 Hz, 1H), 3.07 (m, 1H), 2.60 (s, 3H), 2.59-2.60 (m, 1H), 2.44 (m, 1H), 2.26 (s, 6H), 1.95-2.01 (m, 3H), 1.83 (dd, J=2.7, 14.5 Hz, 1H), 1.66-1.71 (d, J=21.4 Hz , 3H), 1.57- 1.61 (m, 2H), 1.48 (s, 3H), 1.37 (d, J=8.6 Hz, 3H), 1.33 (s, 3H), 1.31 (d, J=7.1 Hz, 3H), 1.24 (d, J=6.1 Hz, 3H), 1.16 (d, J=6.8 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.84 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.3, 203.8, 169.9, 147.9, 147.0, 131.9, 129.8, 119.9, 116.3, 114.8, 112.5, 104.1, 82.3, 79.9, 78.3, 77.5, 70.5, 69.8, 66.0, 60.6, 51.4, 49.96, 49.93, 47.8, 45.0, 42.8, 40.4, 39.7, 39.1, 28.5, 22.4, 21.3, 19.9, 18.5, 16.0, 14.8, 14.5, 14.0, 10.6; HRMS [M+H]+ for C43H66N6O10, calculated:827.4913, found: 827.4910. Melting range: 204 −206 °C. Impurity C. To a solution of compound 3 (25.5 g, 30.6 mmol) and toluene (60 ml) was added m-APA (4.3 g, 36.7 mmol). The reaction mixture was heated to 90 oC and stirred. The conversion was monitored by HPLC. After completion, the reaction mixture was concentrated to afford a solid, then DCM (150 ml )was added, after then hydrochloric acid (4.60 g) and water (120 g) added, stirred for 30 min, standing for 10 min to separate the organic layer. Sodium carbonate (3.25 g) and water solution (65 g) was added, stirred for 30 min, standing for 10 min to separate the organic layer, concentrated to afford a solid. Methanol (190 ml) was added, the reaction mixture was heated to 65 oC, the reaction was monitored by HPLC, after completion, evaporated under reduced pressure to obtain the crude solid (25 g). The solid was further purified using flash chromatography to afford impurity C (1.5 g, 94.92% purity) in 5.8% yield. 1H NMR (600 MHz, CDCl3) δ 7.63 (s, 1H), 7.24 (t, J=1.7 Hz, 1H), 6.70-6.75 (m, 3H), 4.83 (dd, J=10.4, 2.0 Hz, 1H), 4.35 (td, J=7.1, 1.4 Hz, 2H), 4.31 (d, J=7.3 Hz, 1H) 4.01 (s, 2H), 3.51-3.67 (m, 5H), 3.40 (s, 1H), 3.18 (dd, J=7.3, 10.2 Hz, 1H), 3.09 (q, J=6.9 Hz, 1H), 2.59 (m, 1H), 2.50 (s, 3H), 2.45 (m, 1H), 2.27 (s, 6H), 1.86-1.95 (m, 4H), 1.78 (d, J=21.4 Hz, 3H), 1.60-1.69 (m, 4H), 1.53 (d, J=13.9 Hz, 1H), 1.48 (s, 3H), 1.34 (s, J=8.6Hz, 3H), 1.31 (d, J=7.1 Hz, 3H),


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1.24-1.27 (d, J=6.1 Hz, 3H), 1.18 (d, J=6.8 Hz, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.87 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.6, 203.0, 166.5, 157.2, 147.4, 138.1, 132.9, 130.1, 128.2, 118.7, 115.9, 114.9, 104.4, 97.8, 82.2, 80.8, 78.7, 78.6, 70.5, 69.8, 65.9, 61.0, 49.3, 47.9, 44.7, 43.1, 40.9, 40.4, 39.7, 39.3, 28.3, 27.7, 25.4, 24.4, 22.3, 21.3, 19.9, 18.0, 15.2, 14.8, 13.9, 10.5; HRMS [M+H]+ for C43H65FN6O10, calculated: 845.4819, found: 845.4813. Melting range: 252 −255 °C.

Impurity D. To a solution of compound 4 (20.0 g, 24.5 mmol) , toluene (75 ml) and DMF (40 ml), cooled to -20 oC. Sodium tert-pentoxide and toluene solution (9.4%, 40.3 g, 34.4 mmol) added slowly, then chlorosuccinimide (3.45 g, 25.8 mmol) in DMF (20 ml) was added slowly. The conversion was monitored by HPLC. After completion, sodium carbonate (5 g, 47.2 mmol) and water (100 ml) solution was added, stirred for 1h, standing for 10 min to separate the organic layer, evaporated under reduced pressure to obtain the crude solid (20 g). Isopropanol (75 ml) added into, and heated to 70 oC, then water (40 ml) was added dropwise and cooled to 0 oC. The precipitated solid was filtered to yield the chlorideof compound 15 (15 g). To a solution the chloride of compound 15 (15 g, 17.7 mmol) and dichloromethane (75 ml) were added m-APA (2.48 g, 21.2 mmol), CuI (0.5 g, 2.65 mmol) and DIPEA (1.14 g, 8.84 mmol) successively. The reaction mixture refluxed at 40 oC and stirred overnight. The conversion was monitored by HPLC. After completion, active carbon (5 wt %, 1.5 g) was added and stirred for 30 min. The residue was removed by filtration, and the filtrate was concentrated to afford a solid. The solid was treated with methanol (33 ml) and heated to reflux. After completion, water (60 ml) was added slowly and cooled to 0 oC, then the precipitated solid was filtered to yield impurity D (11.6 g，98.45% purity) in 54.85% yield. 1H NMR (600 MHz, CDCl3) δ 7.80 (s, 1H), 7.28 (m, 1H), 7.15-7.20 (m, 2H), 6.65 (m, 1H), 4.82 (dd, J=2.6, 10.1 Hz, 1H), 4.43 (m, 2H), 4.36 (d, J=7.3 Hz, 1H), 4.02 (d, J=9.7 Hz, 1H), 3.71-3.82 (m, 4H), 3.53-3.66 (m, 3H), 3.52 (s, 1H), 3.19 (dd, J=7.4, 10.2 Hz, 1H), 3.13 (q, J=7.0 Hz, 1H), 2.62 (m, 1H), 2.57 (s, 3H), 2.48 (m, 1H), 2.28 (s, 6H), 1.98 (m, 2H), 1.96 (s, 3H), 1.87 (s, 2H), 1.67-1.70 (m, 4H), 1.52 (m, 4H), 1.43 (d, J=7.0 Hz, 3H), 1.32 (s, 3H), 1.24 (m, 4H), 1.18 (d, J=6.8 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.88 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.7, 201.3, 166.4, 157.3, 148.0, 147.0, 131.8, 129.8, 119.8, 116.3, 114.9, 112.5, 104.1, 82.3, 80.9, 80.2, 78.7, 74.2, 70.5, 69.8, 65.9, 61.3, 49.8, 49.3, 44.8, 42.9, 42.8, 40.4, 39.8, 39.3, 31.5, 28.3, 27.7, 24.4, 22.4, 21.3, 20.0, 18.0, 16.8, 14.9, 13.8, 10.6; HRMS [M+H]+ for C43H65ClN6O10, calculated: 861.4523, found: 861.4518. Melting range: 229 −231 °C.



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Impurity E. To a solution of compound 3 (12.0 g, 14.4 mmol) and acetonitrile (30 ml) added CuI (3.29 g, 17.3 mmol) and DIPEA (2.79 g, 21.6 mmol), the mixture cooled to 0 oC, and then iodine chloride(2.80 g, 17.3 mmol) and acetonitrile(15 ml) solution added dropwise, m-APA (2.24 g, 19.2 mol) added, then heated to 25 oC and stirred overnight. The conversion was monitored by HPLC. After completion, active carbon (5 wt %, 1.2 g) was added and stirred for 30 min. The residue was removed by filtration, and the filtrate was concentrated to afford a solid. The solid was purified using flash chromatography to give impurity E (3.0 g, 95.00% purity) in 21.42% yield. 1H NMR (600 MHz, CDCl3) δ 7.22-7.30 (m, 3H), 6.73 (m, 1H), 4.86 (dd, J=2.0, 10.3 Hz,1H), 4.47 (m, 2H), 4.36 (d, J=7.3 Hz, 1H), 4.06 (d, J=10.0 Hz, 1H), 3.72-3.75 (m, 1H), 3.65 (m, 2H), 3.53-3.58 (m, 3H), 3.43 (s, 1H), 3.34 (dd, J=7.3, 10.1 Hz, 1H), 3.13 (q, J=7.0 Hz, 1H), 2.59 (m, 1H), 2.55 (s, 3H), 2.52 (m, 1H), 2.04 (s, 6H), 1.95-1.98 (m, 3H), 1.79-1.85 (m, 2H), 1.76 (d, J=13.6 Hz, 3H), 1.71-1.73 (m, 3H), 1.58 (s, 2H), 1.51 (m, 1H), 1.33 (s, 3H), 1.31 (d, J=7.1 Hz, 3H), 1.28 (d, J=6.1 Hz, 3H), 1.25 (d, J=7.1 Hz, 3H), 1.19 (d, J=6.8 Hz, 3H), 1.01 (d, J=6.9 Hz, 3H), 0.90 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.7, 203.0, 176.9, 166.4, 157.3, 150.0, 146.7, 131.4, 129.4, 118.0, 115.4, 114.6, 103.8, 97.9, 82.3, 80.9, 78.7, 78.6, 70.0, 68.7, 66.0, 61.0, 50.6, 49.4, 44.7, 43.0, 40.9, 39.7, 39.6, 39.3, 29.5, 27.5, 25.3, 24.4, 22.3, 21.9, 19.9, 18.0, 15.2, 14.8, 13.9, 10.6; HRMS [M+H]+ for C43H64FIN6O10, calculated: 971.3785, found: 971.3780. Melting range: 248 −251 °C.

Impurity F. To a solution of compound 3 (20.0 g, 24.1 mmol) and methanol, the mixture heated to reflux. The conversion was monitored by HPLC. When the reaction was completed, water (85 ml) added dropwise, then cooled to 0 oC and stirred for 1h. The precipitated solid was filtered and further purified by recrystallization from methanol/water to yield impurity F (9.45 g, purity 97.43%) in 54% yield. 1H NMR (600 MHz, CDCl3) δ 4.87 (dd, J=2.1, 10.4 Hz, 1H), 4.31 (d, J=7.3 Hz, 1H), 4.08 (d, J=0.72, 10.5Hz, 1H), 3.68 (m, 1H), 3.50-3.59 (m, 4H), 3.43 (s, 1H), 3.30 (m, 2H), 3.19 (dd, J=7.4, 10.2 Hz, 1H), 3.10 (q, J=7.0, 13.9 Hz, 1H), 2.62 (m, 1H), 2.59 (s, 3H), 2.46 (m, 1H), 2.27 (s, 6H), 1.98 (m, 1H), 1.89 (dd, J=2.8, 14.5Hz, 1H), 1.79 (d, J=21.4 Hz, 3H), 1.59-1.69 (m, 6H), 1.54 (d, J=12.8 Hz, 1H), 1.50 (s, 3H), 1.36 (s, 3H), 1.32 (d, J=7.1 Hz, 3H), 1.25 (d, J=6.1 Hz, 3H), 1.21-1.25 (m, 1H), 1.19 (d, J=6.8 Hz, 3H), 1.01 (d, J=7.0 Hz, 3H), 0.89 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.7, 203.1, 166.4, 157.2, 147.9, 104. 4, 97.9, 82.1, 80.8, 78.71, 78.69, 70.5, 69.8, 66.0, 61.1, 51.1, 49.3, 44.8, 43.2, 40.9, 40.4, 39.7, 39.4, 28.3, 26.4, 25.4, 24.5, 22.3, 21.3, 19.9,


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18.1, 15.2, 14.9, 13.9, 10.6; HRMS [M+H]+ for C35H58FN5O10, calculated: 728.4240, found: 728.4239. Melting range: 163 −165 °C. Impurity G : Compound 2 was impurity G. 1H NMR (600 MHz, CDCl3) δ 8.04 (dd, J=1.1, 8.3 Hz, 2H), 7.80 (s, 1H), 7.58 (t, J=7.4 Hz, 1H), 7.46 (t, J=7.8 Hz, 2H), 7.28 (m, 1H), 7.15-7.20 (m, 2H), 6.65 (m, 1H), 5.03 (dd, J=7.6, 10.4 Hz, 1H), 4.85 (dd, J=1.9, 10.3 Hz, 1H), 4.53 (d, J=7.5 Hz, 1H), 4.41 (m, 2H), 4.09 (d, J=10.0 Hz, 1H), 3.72-3.76 (m, 2H), 3.58-3.64 (m, 2H), 3.41 (s, 1H), 3.30 (m, 1H), 3.06 (q, J=7.1 Hz, 1H), 2.84 (m, 1H), 2.61 (m, 1H), 2.53 (s, 3H), 2.26 (s, 6H), 1.93-1.97 (m, 3H), 1.81 (m, 1H), 1.72 (d, J=14.6 Hz, 3H), 1.67-1.71 (m, 4H), 1.60 (m, 1H), 1.45 (s, 3H), 1.41 (m, 1H), 1.34 (s, 3H), 1.28 (d, J=6.1 Hz, 3H), 1.18 (d, J=6.9 Hz, 3H), 1.05 (d, J=6.9 Hz, 3H), 0.99 (d, J=7.0 Hz, 3H), 0.84 (t, J=7.4 Hz, 3H); 13C NMR (150 MHz, CDCl3) δ 216.7, 202.6, 166.5, 165.3, 157.3, 147.9, 147.0, 133.0, 131.8, 130.4, 129.9, 129.7, 128.5, 119.8, 116.2, 114.8, 112.4, 102.0, 98.0, 82.2, 79.6, 78.7, 72.2, 69.4, 63.6, 61.1, 49.8, 49.3, 44.6, 42.9, 40.8, 40.7, 39.4, 39.2, 31.3, 27.7, 25.2, 24.4, 22.2, 21.1, 19.8, 18.0, 14.7, 13.8, 10.5; HRMS [M+H]+ for C50H69FN6O11, calculated: 949.5081, found: 949.5097. Melting range: 198 −200 °C.

Supporting Information 1

H and 13C NMR spectra for these impurities. X-ray structure information for 1. This material is available free

of charge via the internet at http://pubs.acs.org

Corresponding Author E-mail:[email protected]. Author Contributions ‖

Z.Z. and C.D. contributed equally to this work.

ACKNOWLEDGMENT We gratefully acknowledge financial support from the State Key Laboratory of Anti-Infective Drug Development (Sunshine Lake Pharma Co.,Ltd), (N0. 2015DQ780357). We thank Jiaxiang Sun and Meijuan Wang (both HEC pharm Co., Inc.) for NMR assistance, Jing Tian (HEC pharm Co., Inc.) for HPLC assistance.



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We also thank Dr. Ning Xi (HEC pharm Co., Inc.) for the fruitful discussion and kindly advice during the writing for this paper. The authors declare no competing financial interest. REFERENCES (1) Farrell DJ, Flamm RK, Sader HS, Jones RN. Results from the Solithromycin International Surveillance Program (2014). Antimicrob Agents Chemother. 2016; 60 (6):3662–3668.; (2) (a) David E. P, Patent WO. 2011/146829. 2011. (b) Chang, H. L.; Jonathan D, et al. Patent US.2004/080391. 2004.(c) Fernandes, P. B.; Chapel H ; et al. Patent WO. 2010/048599, 2009. (d) David E. P. US. Patent 2016/046660. 2016. (e) David, E. P, Manish K. P.; Keshav, D . Patent US 2010/0216731. 2010. (f) Mou, X.; Zhu, C.G.; Zhong, Q.C, Wu, M.; Huang, K.; Feng, L.C.; He, Y. Patent CN 106432383,2016. (g) Zhong, Z.H; Du,C.; Lin, W.;He, L.; Song, L.H; Luo, Z.H. Patent CN 104650166.2014. (h)

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Zhang, T.; Huang, Y.. Patent CN 105348341. 2016. (i) Wang, P.; Li, P.X.; Gu, X.Y. Patent WO. 2017/050032. 2017. (3) Seiple, Ian B.; Zhang, Ziyang; Jakubec, Pavol; Langlois-Mercier, Audrey; Wright, Peter M.; Hog, Daniel T.; Yabu, Kazuo; Allu, Senkara Rao; Fukuzaki, Takehiro; Carlsen, Peter N.; et al. Nature; 2016, 553(7603):338-345. (4) ICH Q3A (R2) Impurities in New Drug Substances, International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH): Geneva, Switzerland, October 2006 ICH Q3A Impurities in New Drug Substances, R2; InternationalConference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH): Geneva, Switzerland, October,2006. http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/Q3A_R2/Step4/Q3A_R2__ Guideline.pdf (5) Thomas Rosenau, Andreas Hofiner.; et al. Organic letters, 2004, 6(4):541-544. (6) Fernandes P B. Methods for treating gastrointestinal diseases: U.S. Patent 8,791,080[P]. 2014-7-29. (7) Liang C H, Yao S, Chiu Y H, et al. Bioorganic & medicinal chemistry letters, 2005, 15(5): 1307-1310. (8) Pereira D E, Schneider S E,. Patent WO 2016/144833, 2016. (9) Glassford I, Teijaro C N, Daher S S, et al. Journal of the American Chemical Society, 2016, 138(9): 3136-3144. (10) Carcenac Yvan, David-Quilot Franck.; et al. Synthesis, 2013, 45(5):633-638. (11) A.Brian Jones. J.org. Chem, 1992, 57(16): 4361-4367. (12) Starcevic. Kristina, Pesic.Dijana.; et al. European Journal of medicinal chemistry, 2012, 49: 365-378.


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