Improvement in synthesis of 3'-N-demethylazithromycin and its derivatives

FU Yan-jie(付艷杰)， SUN Li-quan(孫立權(quán))， FAN Xin-yuan(范新苑)，LIU Cong(劉聰)， YAO Guo-wei(姚國偉)， LUO Ai-qin(羅愛芹)

(School of Life Science，Beijing Institute of Technology，Beijing 100081，China)

Azithromycin is a 15-membered second-generation macrolide known as an azalide with amino group(aza)in the lactone ring.It has high activity against a variety of G+bacteria，mycoplasma，and chlamydia and legionella pneumophila.It is significantly more potent than either erythromycin or clarithromycin against Haemophilus influenza，and makes up the deficiency of macrolides against Haemophilus［1－2］.

Azithromycin is derived structurally from erythromycin A in 4 steps［3－6］.Intermediates and by-products are considered as the main impurities in its active pharmaceutical ingredients.3'-N-demethylazithromycin(1)is one of the important related substances(including 16 related substances in European pharmacopoeia and 13 related substances in US pharmacopoeia［7－8］)that often occurred in bulk azithromycin.It is also an intermediate for other impurities，such as 3'-N-demethyl-3'-N-formylazithromycin(2)and 3'-N，N-dedimethyl-azithro-mycin.

Fig.1 Structures of compounds 1 and 2

Although preparation of compound 1 was reported in several literatures，detailed pathway could not be found［9－10］.Since the revision of the standards of Chinese Pharmacopoeia in 2010，the related substances 1 and 2 become necessary materials for routine assay.However，they can’t be easily obtained from commercial market.In this article，we synthesized and isolated the azithromycin related substances 1 and 2 with azithromycin as the starting material.The target compounds 1 and 2 were synthesized with a good yield and high purity their structures were confirmed by1H-NMR and MS.

1 Experiments

1.1 Reagents and instruments

Azithromycin was provided by Zhejiang Guobang Pharmaceutical Company.All regents and solvents were purchased from Beijing Chemical Reagents Company that marked as grade of analytical purity without further purification.1H-NMR spectra were measured on an ARX400 instrument(Bruker)in CDCl3，with TMS as internal standard.Mass spectra were recorded on a ZAB-HS mass spectrometer.Waters HPLC system with two 515 pumps，waters 996 diode array detector and waters 717 autosampler were employed for sample analyses;Agela preparative HPLC system(Bonna-Agela Tech.)was used in the separation of the products.

1.2 Synthesis of 3'-N-demethylazithromycin(1)

As shown in Fig.2，azithromycin(0.50 g)was dissolved in 80%aqueous MeOH(10 mL)，and sodium acetate(0.30 g)was added.The solution was heated to about 50℃followed by adding iodine(0.17 g)in three batches in an hour in presence of UV irradiation.The reaction was monitored by HPLC and accomplished in 90 min.This solution was cooled at room temperature，and poured into 20 mL aqueous sodium thiosulfate pentahydrate solution(0.05 mol/L)and stirred for 10 min.The pH of mixture was adjusted between 9 and 10 by adding NH3·H2O and then extracted with 60 mL of CH2Cl2in three batches.Combined organic layers were washed with solution containing 20 mL of water and 5%ammonia solution，then evaporated under reduced pressure to yield white powers.The product was vacuum dried at 80℃ for 10 h.Crude compound 1 had a weight of 0.49 g.Compound 1 was purified by silica gel column chromatography.An excellent elute was determined that give good resolution between azithromycin and compound 1 with ether/EtOAc/isopropanol/Et3NH=30∶10∶1∶1.After the solute was concentrated，white solid wasobtained with purity of 96%(HPLC)and yield of 44%.It was observed that fine crystal could be acquired through recrystallization in acetone and water.

Compound 1:EI MS m/z 736.1(M+H+);1H-NMR(400 MHz，CDCl3，δ ppm)9.52(s，1H，9a-N-H)，5.15(s，1H，11-OH)，5.13(d，1H，H-1″)，4.70(dd，1H，H-13)，4.41(d，1H，1'-H)，4.25(m，1H，H-3)，4.08(dq，1H，H-5″)，3.67(m，1H，H-11)，3.63(d，1H，H-5)，3.55(m，1H，H-5')，3.34(s，3H，3″-OCH3)，3.18(dd，1H，H-2')，3.04(d，1H，H-4″)，3.02(s，1H，2'-OH)2.74(m，1H，H-2)，2.69(m，1H，H-10)，2.56(d，1H，H-9eq)，2.54(m，1H，12-OH)，2.48(m，1H，H-3')，2.42(s，3H，9a-NCH3)，2.36(d，1H，H-2″eq)，2.32(s，3H，3'-N-CH3)，2.25(s，1H 6-OH)，2.22(d，1H，4″-OH)，2.05(m，1H，H-9ax)，2.03(m，1H，H-8)，1.97(m，1H，H-4)，1.96(d，1H，H-7eq)，1.90(m，1H，H-14eq)，1.75(d，1H，H-4'eq)，1.58(dd，1H，H-2″ax)，1.46(m，1H，H-14ax)，1.33(d，3H，5″-CH3)，1.32(s，3H，6-CH3)，1.26(m，1H，H-7ax)，1.25(s，3H，3″-CH3)，1.24(m，1H，H-4'ax)，1.20(d，3H，5'-CH3)，1.18(d，3H，2-CH3)，1.10(d，3H，10-CH3)，1.09(s，3H，12-CH3)，0.98(d，3H，4-CH3)，0.91(d，3H，8-CH3)，0.89(t，3H，15-CH3).

1.3 Synthesis of 3'-N-demethyl-3'-N-formylazithromycin(2)

For synthesis of 3'-N-demethyl-3'-N-formylazithromycin(2)，we performed reaction between compound 1 and formic acid(HCOOH)in chloroform at 75℃.Compound 1(1.00 g)was dissolved in CHCl3(100 mL).This solution was heated to about 75℃followed by adding 0.8 mL formic acid in four batches at every half an hour interval. Temperature was maintained for 20 h.HPLC was used to monitor the reaction.The reaction solution was then cooled at room temperature，and the solution was dropped into 20 mL water containing 5% ammonia solution and stirred for 10 min.The mixture was separated and the water layers were extracted twice with 20 mL of CH2Cl2.The combined organic layers were washed with 20 mL water containing 5%concentrated ammonia solution.It was evaporated under reduced pressure to give white powers.The product was vacuum dried at 80℃ for 10 h and weighted about 0.98 g.The product was isolated by silica gel column chromatography and purified by productive HPLC，compound 2 was recrystallized in acetone and water，to obtain a purity of over 98%and a yield of 50%.

Compound 2:EI MS m/z 764.1(M+H+);1H NMR(400 MHz，CDCl3，δ ppm)8.10(s，1H，N-CHO)，5.10(d，1H，H-1″)，4.74(s，1H，11-OH)，4.68(dd，1H，H-13)，4.42(m，1H，H-1')，4.20(dd，1H，H-3)，4.05(m，1H，H-5″)，3.65(m，1H，H-11)，3.62(m，1H，H-5')，3.61(d，1H，H-5)，3.41(m，1H，12-OH)，3.32(s，3H，3″-OCH3)，3.06(m，1H，H-2')，3.04(t，1H，H-4″)，2.85(s，3H，3'-NCH3)，2.80(s，1H，2'-OH)，2.72(m，1H，H-2)，2.69(m，1H，H-10)，2.67(s，1H 6-OH)，2.53(m，1H，H-9eq)，2.43(m，1H，H-3')，2.35(s，3H，9a-N-CH3)，2.31(d，1H，H-2″eq)，2.23(d，1H 4″-OH)，2.05(m，1H，H-9ax)，2.03(m，1H，H-8)，2.02(m，1H，H-7eq)，1.88(m，1H，H-14eq)，1.71(m，1H，H-4'eq)，1.68(m，1H，H-4)，1.58(dd，1H，H-2″ax)，1.45(m，1H，H-14ax)，1.33(d，3H，5″-CH3)，1.32(s，3H，6-CH3)，1.26(s，3H，3″-CH3)，1.24(m，1H，H-7ax)，1.23(m，1H，H-4'ax)，1.22(d，3H，5'-CH3)，1.19(d，3H，2-CH3)，1.10(d，3H，10-CH3)，1.08(s，3H，12-CH3)，0.98(d，3H，4-CH3)，0.92(d，3H，8-CH3)，0.89(t，3H，15-CH3).

Fig.2 Synthesis of 3'-N-demethylazithromycin(1)and 3'-N-demethyl-3'-N-formylazithromycin(2)

2 Results and discussion

2.1 Influence of iodine

The results showed that iodine had remarkable influence on the yields in demethylation reaction of azithromycin.When the amount of iodine was equal to azithromycin at molar ratio，the yield of compound 1 was 64.02%.The value is highest among three cases when molar ratio of iodine and azithromycin is 0.5，1.0 and 1.5，as shown in Fig.3.It indicated that deficiency and excess of iodine could reduce the product.With in excess of iodine，a new by-product was produced as shown at retention time of about 4 min.More iodine did not participate in the demethylation reaction，but made the solution darker.In other words，formation of by-product decreased the yield of compound 1.It was observed that whether iodine was added once or three times did not influence reaction conditions.

2.2 Effect of sodium acetate

Fig.3 Chromatograms of the reaction solution using different amount of iodine in the preparation of compound 1

To determine the effect of sodium acetate，seven different concentrations of sodium acetate were examined.The molar ratios between sodium acetate and azithromycin.Fig.4 showed the results at 1，3，5，7，9，11 and 13 of the molar ratio between sodium acetate and azithromycin from outside to inside.The yields obtained from the integration are 38.4%，43.8%，57.0%，64.7%，64.6% ，64.7% and 62.7% at above rations，respectively.The results demonstrated that there was minor increase in production of compound 1 upon changing the molar ratio from 1 to 7，and then no significant variation was detected from 7 to 13(Fig.4).Therefore，sodium acetate with a molar amount concentration of 7 times of azithromycin was used in the experiments.

Fig.4 Chromatograms of the reaction solution using different amount of sodium acetate in the preparation of compound 1

2.3 Choice of formylation reagents

Compound 2 was synthesized when compound 1 reacted with formylating reagent.Albert［11］reported that formic acid anhydride that was obtained by reacting acetic anhydride and formic acid，had been used as a formylating reagent to prepare compound 2.However，our finding was different from it.After following the above described procedure the main product，we obtained 3'-N-demethyl-3'-N-acethyl-azithromycin rather than compound 2.Therefore，a convenient synthetic route，which was different from the previous literatures，had been developed by reacting compound 1 with formic acid in chloroform at 75℃in our lab.The finalproductwas analyzed by1H-NMR and Mass spectrometry，as shown in experimental data.

2.4 Effect of reaction temperature

Generally，reaction temperature and intensity of UV radiation are important parameters that significantly affect the yield of product indemethylation reaction of azithromycin.To find out the optimized temperature for synthesis of compound 2，reaction was carried out at three different temperatures，50，75，and 100℃.The yields of compound 2 were 66.3%，85.2 and 52.9%at 50℃，75℃ and 100℃，respectively(from a to c).The chromatograms results(Fig.5)illustrated that the target yield at 75℃ was 85.2%，which was the highest among the three cases.A by-product could be observed clearly at 100℃，so the yield of compound 2 was diminished.

Fig.5 Chromatograms of the reaction solution in the preparation of compound 2 at different temperatures

2.5 Isolation of two compounds

Synthesis of two products was analyzed by HPLC.The raw product was separated with silica chromatography with the elution liquid of ether/EtOAc/isopropanol/Et3NH=30∶10∶1∶1.Target compound was markedly differentiated between the reagents and by-products. However， compound 2 could not be easily separated from the reaction mixtures with silica column.Therefore，af-ter silica chromatographic treatment，the raw compound was purified by preparative RP-C18 HPLC.HPLC condition fortheseparation of product was investigated on the basis of the analytical method.Finally，small amount of compound 2 was received for characterization.

3 Conclusion

After optimization of reaction conditions，two related substances of azithromycin in US Pharmacopoeia have been prepared with yields of 44%and 50%and purity of more than 95%.Simultaneously two compounds were isolated by the mean of silica gel column and preparative HPLC.A further study on large scale production of the two compounds is still under progress.

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