An Efficient Chemoenzymatic Process for Preparation of Ribavirin

Ribavirin is an important antiviral drug, which is used for treatment of many diseases. The pilot-scale chemoenzymatic process for synthesis of the active pharmaceutical ingredient Ribavirin was developed with 32% overall yield and more than 99.5% purity. The described method includes the chemical synthesis of 1,2,4-triazole-3-carboxamide, which is a key intermediate and enzymecatalyzed transglycosylation reaction for preparation of the desired product. 1,2,4-Triazole-3-carboxamide was synthesized from 5-amino-1,2,4-triazole-3-carboxylic acid by classical Chipen-Grinshtein method. Isolated from E. coli BL21(DE3)/pERPUPHHO1 strain the purine nucleoside phosphorylase was used as a biocatalytical system. All steps of this process were optimized and scaled.


Introduction
Antiviral properties of 1--D-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide or Ribavirin were described in 1972 for the first time [1].Since that moment it is used in the treatment of influenza, severe respiratory syncytial virus (RSV) infection, and Lassa fever virus infection [1].In combination with interferon-, it is a current standard for hepatitis C virus (HCV) therapy [1].This disease was determined by World Health Organization (WHO) as one of the most important problems of health care.Chronic form of HVC is the main reason for causing liver cirrhosis and hepatocellular carcinoma [2].There are 150 million infected people in the world and every year their number increases by about 3-4 millions [3].Therefore, the development of the reliable and effective technology for Ribavirin production is a topical problem.
That is why microbiological methods are considered more efficient.Nucleoside phosphorylase (NP) plays the key role in these methods.It catalyzes transfer of sugar moiety from one heterocyclic base to another.The bacterial [9,14] or isolated enzymes are used for nucleoside synthesis [17,18].The main disadvantages of this approach are low productivity and high costs of enzymes and starting materials.
Our technology of Ribavirinsynthesis combines the microbiological method for generation of the target structure with the chemical synthesis of key intermediates.This approach gives high level of reliability and efficiency while producing less toxic waste.and were determined in DMOS- 6 . 13C NMR spectra were recorded at 125 MHz.Chemical shifts are reported in parts per million relative to the peak of tetramethylsilane (TMS) (0.00 ppm).Melting points were measured on Mel-Temp 3.0.(7).To a suspension of 8.25 kg (64.5 mol) 5-amino-1,2,4-triazole-3-carboxylic acid 5 in 50.0 L water 1.9 L concentrated hydrochloric acid was added.The suspension was heated at 70 ∘ b to dissolve.The solution was cooled to room temperature and 30 kg of ice was added.Further ice-cold solution of 8.75 kg (126.8 mol) sodium nitrite in 19.0 L of water was added.Following the addition the reaction mixture was stirred 30 min at 0-10 ∘ b and 40 min at room temperature.The resulting suspension was filtered and washed in ice water (2 × 250 mL) to give 11.25 kg of diazonium salt 6, which was used in the next step without further purification.To a suspension of 3.75 kg (21.25 mol) diazonium salt 6 in 25.0 L of methanol 10 g of sodium borohydride was added at 0-4 ∘ b (exothermic).The reaction mixture was cooled to 0-4 ∘ b and procedure was repeated two times.After completion, the reaction mixture was refluxed 5 min, cooled to 0-2 ∘ b over 20-25 min, and filtered.The precipitate was washed with 6.5 L of cool methanol and 4.75 kg (65%) of 1,2,4-triazole-3carboxylic acid 7 was obtained, as a white solid, mp 136-137 ∘ b. 1 X NMR (500 MHz, DMOS- 6 ) : 8.67 (1H, s), 14.01-14.52(1H, bs). 13b NMR (125 MHz, DMOS- 6 ) : 148.5, 151.9, 165.6.Found, %: b 31.44;X 2.79; N 36.98.Calculate, %: C 31.87; H 2.67; N 37.16.
After completion, reaction mixture was stirred at room temperature for over 48 h.The resulting suspension was filtered and washed with 3.5 L cool methanol.Product was obtained as a white solid, yield 4.68 kg (88%), mp 185-186 ∘ b.   , 4).20.4 g of potassium dihydrophosphate was dissolved in 10 L of distilled water.The pH of the solution was adjusted to 7.0 with 5 N solution of potassium hydroxide.To the buffer solution 7.8 g (0.07 mol) 1,2,4-triazole-3-carboxamide 8, 42.5 g (0.15 mol) guanosine 9, and 30 mL of PNP (15 mg/mL, 52 ea/mg of protein) solution were added.The reaction mixture was thermostated at 52-55 ∘ b over 7 d.The resulting solution was concentrated at reduced pressure to 1/2 of volume and cooled to 4-8 ∘ b.The precipitated guanine with guanosine traces were filtered out.The filtrate was passed through ion exchange resin (eluted with water).Solvent was evaporated at reduced pressure and 12.2 g (71%) of ribavirin was obtained with 98.6% purity.After recrystallization from ethanol, 11.1 g (65%) of ribavirin was obtained, as white solid, mp 175-178 ∘ C. Purity is 99.5% by HPLC (Figure 1 and Table 3).

Results and Discussion
It was previously reported that 1,2,4-triazolecarboxylic acid esters are not PNP substrate [20].Therefore, only 1,2,4triazole-3-carboxamide 8 (TCA) could be used as a free heterocyclic base in Ribavirin synthesis.Thereby our first goal was to find out the most convenient and efficient way for chemical synthesis of TCA 8, which could be used in industry.
After analysis of the literature we have chosen the classical Chipen-Grinshtein method [21], represented in Scheme 2. The entire process is suitable for kg-scale preparation.The yields of intermediates at each stage were not less than 65%, and the overall yield of TCA 8 was 49%.
There are two described approaches to microbiological synthesis of Ribavirin 4 from TCA: enzymatic (exogenic) [9] and fermentative (endogenic) [8].In keys of exogenic approach all the components of the reaction systems should be added separately.Transglycosylation reaction, that is, transfer glycoside moiety from nucleoside (donor) to free purine base (acceptor), is an example of this kind of reactions.
During endogenic catalysis the substrate should be added to bacterial culture [8].
Transglycosylation reaction (Scheme 3) was chosen, because it is better studied and easy to control.Purified enzyme, the purine nucleoside phosphorylase (PNP), which was isolated from Escherichia coli (E.coli) BL21(DE3)/pER-PUPHHO1 strain, was used as a biocatalytical system.
Natural nucleoside guanosine (Guo) 9 was chosen as ribose donor, because of sedimentation of product guanine (Gua) 10 during the reaction, causing the equilibrium shift in favor of the required product.In addition, Guo 9 is commercially available, which is an important fact for the industrial application.The optimal ratio of Guo 9 to TCA 8 was also studied.The results are shown in Table 1.
In accordance with this data, the 97% conversion of TCA 8 into the Ribavirin 4 can be achieved when using 2 mol of Guo 9 per one mol of 8. Further increase of Guo 9 excess did not improve the conversion rate.In addition, comparison with described biocatalysts is shown in Table 2.The process was scaled from 0.5 L volume to 20 L volume.In order to achieve 97% conversion the reaction time was extended from 120 to 168 h.Residual TCA 8 was removed during chromatographic purification and recrystallization.Typical chromatogram of the obtained Ribavirin is shown in Figure 1 and Table 3. TCA 8 (0.434%, RT 16.180 min) and Guo 9 (0.026%, RT 8.351 min) is main impurity.

International Journal of Chemical Engineering
The use of soluble enzyme is the main limitation for industrial application of this process.Therefore, further efforts will be focused on the development of immobilized enzyme.Recently, Rivero et al. reported successful immobilization of E. coli ATCC 12407 in agarose matrix [16].Moreover, immobilization on fused silica Open Tubular Capillary [22], aldehyde-agarose [23], and MagReSyn epoxide microspheres [24,25], in calcium alginate, agar, and kcarrageenan matrix [18], was described in the literature.Thus, we believe, that the immobilisation of our enzyme is possible.

Conclusions
To conclude, we have found an efficient combined chemoenzymatic method of Ribavirin synthesis from commercially available materials.We have adapted previously reported laboratory protocol of 1,2,4-triazole-3-carboxamide chemical synthesis to kilograms scale.Purity of the final product is sufficient to use as an active pharmaceutical ingredient.The reported approach is superior in comparison with the earlier chemical methods and approaches.In the future, we plan to immobilize the enzyme for further improvement of the technology.

2. 1 . 2 International
Chemicals and Reagents.The starting reagents and solvents were obtained from Sigma-Aldrich Company and used

Table 2 :
The Ribavirin synthesis with different biocatalytical systems.