Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction

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A Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction of Artemisinic Acid Martin Philipp Feth, Kai Rossen, and Andreas Burgard Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/op300347w • Publication Date (Web): 14 Jan 2013 Downloaded from http://pubs.acs.org on January 19, 2013

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Organic Process Research & Development

A Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction of Artemisinic Acid Martin P. Feth 1,*, Kai Rossen 1 and Andreas Burgard 1,* 1

Sanofi-Aventis Deutschland GmbH, Chemical and Biotechnological Development (C&BD) Frankfurt Chemistry, Industriepark Höchst, Building G 838, 65926 Frankfurt am Main * Corresponding authors Email address: [email protected] [email protected]

MANUSCRIPT

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Graphical Abstract

A successful PAT-approach by Raman spectroscopy for the diastereoselective reduction of artemisinic acid (AA) to dihydroartemisinic acid (DHAA) using diimide is presented.


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Abstract In this study an attractive route for the diastereoselective synthesis of dihydroartemisinic acid (DHAA) starting from artemisinic acid (AA) is presented. Diimide was used as reducing agent, which was generated by two different methods: 1) by the reaction of hydrazine monohydrate and hydrogen peroxide and 2) by the reaction of hydrazine monohydrate and oxygen. Both methods were found to be suitable for the diimide reduction of AA showing full conversion and a high diastereoselctivity. Due to advantages in the crystallization step of DHAA the second option for generation of diimide was chosen for the pilot plant scale-up. The reaction and the crystallization process development as well as the batch production in pilot plant was monitored and controlled using dispersive Raman spectroscopy as PAT-tool. Three DHAA batches in kg-scale were successfully produced by the reaction of artemisininic acid, hydrazine monohydrate and a gas mixture of nitrogen and oxygen (containing 5% v/v oxygen) in 2-propanol at 40°C. Excellent yields of > 90% (including the crystallization, isolation and drying step) as well as high diastereoselectivities (≥ 97:3) of the products were achieved by the elaborated pilot plant manufacturing processes.

Keywords Artemisinic acid (AA), Dihydroartemisinic acid (DHAA), diimide, diastereoselective reduction, Raman spectroscopy, PAT


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1. Introduction

Malaria is a tropical disease and common in Africa, South East Asia, and South America. Approximately 300-500 million people are infected with malaria, making it one of the world’s major infectious diseases. In 2006, an estimated 1.5 to 2.7 million deaths resulted from malaria and most of the deaths occurred in children under five years old. Disease control is hampered by the occurrence of multi-drug resistant strains of the parasite Plasmodium falciparum. Therefore it is an important world health objective to develop new anti-malaria drugs, and alternative methods of producing anti-malaria drugs. One of these anti-malaria drugs is artemisinin [1,2] as depicted in Scheme 1.

Artemisinin is a sesquiterpene lactone endoperoxide which is a component of the traditional Chinese medical herb Artemisia annua. It has been utilized for controlling symptoms of fever in China for over 1000 years. The production of artemisinin can be accomplished through several annua

routes: [3,4]

For

instance,

by

extracting

artemisinin

from

Artemisia

, or by extracting the biosynthetic precursor molecule artemisinic acid from

Artemisia annua and then synthetically converting this molecule in several synthetic steps to artemisinin.

In 2006, scientists from Amyris Inc. and the University of California, Berkeley developed a fermentation process with engineered yeast to produce high titres of artemisinic acid [5]. With these new developments, semi synthetic methods involving the use of a biosynthetic precursor like artemisinic acid are considered to be cost-effective, environmentally friendly, high quality, and reliable sources of artemisinin, see Scheme 2. Semi synthetic artemisinin is obtained from artemisinic acid in three chemical steps [6].


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One of the critical steps during the production of artemisinin is the regio- and diastereoselective reduction of artemisinic acid to diastereomeric dihydroartemisinic acid (DHAA), refer to Scheme 2. The current SANOFI production process for DHAA from artemisinic

acid

is

performed

via

a

RuCl2[(R)-dtbm-Segphos](DMF)2

catalyzed

hydrogenation with a diastereomeric ratio of at least 95:5 (also refer to supplementary information, chapter 8). In this work the authors present the process development for an alternative synthesis route for the production of DHAA starting from artemisinic acid.

Carbon-carbon double bonds generally can be reduced with diimide as reducing agent [7,8,9,10]. D.J. Pasto and R.T. Taylor Volker Kraft et al.

[11]

[7]

described various methods for generating diimide. In 2011

disclosed the synthesis of dihydroartemisinic acid and corresponding

esters by diimide generation

from hydroxylamin-O-sulfonic acid (HOSA) and sodium

methanolate, from hydroxylamine and HOSA, from hydroxylamine and ethylacetate, from dipotassium azodicarboxylate and from hydrazine hydrate and hydrogen peroxide. When oxygen is used as an oxidant for the diimide formation from hydrazine hydrate, only the use of air (contains 21% by volume of oxygen) or pure oxygen has been described. The diastereomeric ratio in the obtained dihydroartemisinic acid (DHAA) and corresponding esters was at least 95 to 5. The very good diastereoselectivity of the diimide approach is surprising considering that the postulated diimide “HN=NH” carries no chiral information (Scheme 3). Only the artemisinic acid molecule possesses the chiral information which should have a crucial impact on the diastereoselectivity of the diimide approach. However, theoretical calculations (quantum modelisation of the transition states using Gaussian 09) justified the preferred formation of the desired diastereomer DHAA(1) [12].

The successful use of diimide for the reduction of artemisinic acid to dihydroartemisinic acid (DHAA) prompted the authors to pursue with the development of a process which should be ACS Paragon Plus Environment

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scalable. At first the reduction of artemisinic acid to dihydroartemisinic acid (DHAA) using hydrazine hydrate and hydrogen peroxide in water as solvent was investigated with the aim to minimize the amount of the reactants forming the diimide in situ (10 equivalents hydrazine hydrate and 6 equivalents hydrogen peroxide were originally used

[11]

to reduce artemisinic

acid to dihydroartemisinic acid in water). As second method for the generation of diimide the reaction of hydrazine monohydrate with oxygen was evaluated in this study and scaled-up to pilot plant scale. Due to its toxicity a major concern was to avoid any exposure to hydrazine hydrate (also during analytical investigations due to sample collection). Therefore dispersive Raman spectroscopy, which has been shown to be a powerful tool for the optimization and control of chemical reactions

[13]

and crystallizations

[14,15]

, was used as inline-tool for the

reaction/crystallization characterization, optimization and control. This PAT technique was also successfully implemented in the presented pilot plant production process.


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2. Results and Discussion

2.1 DHAA synthesis via diimide approach using H2O2 and hydrazine hydrate (lab scale)

The first synthesis trials for DHAA using hydrazine hydrate and H2O2 as diimide source were performed in pure water as solvent. Parameters like pH (8–11), equivalents of hydrazine hydrate (3-4.5 eq.) and H2O2 (3-4.5 eq.) as well as reaction temperature (40 or 50°C) were varied and the reaction rate, the reactant (AA) conversion, the product formation as well as the amount of H2O2 present in solution were monitored by Raman spectroscopy as response factors. Depending on the applied conditions reaction times between 6 and 23 hours were observed. The amount of the overhydrogenated product (THAA, tetrahydroartemisinic acid) at pH 8.5 - 11 was significantly higher than at pH 8.2 to 8.5 (up to 5 area-% instead of 1.1 area-% in the optimized case). Reaction temperature increase from 40°C to 50°C led to a decrease in reaction time from 22 hours to 6-8 hours. A major issue observed during the reaction optimization is foaming due to self-decomposition of H2O2 (detected by the fast decrease of the H2O2 Raman signal at 875 cm-1), especially at pH 8-11. A significant improvement could be reached by the addition of 2-propanol as defoamer to the reaction mixture. Furthermore by a controlled dosage of H2O2 to the reaction mixture the formation of foam could be reduced to a minimum. Also the reaction time could be reduced by the control of H2O2 dosing (down to 6 hours). In Figure 3a the Raman spectra of such an optimized reaction experiment (for reaction parameters: refer to Table 1) are shown. In the wavenumber range between 800 and 1000 cm-1 the experimental Raman spectra in dependence of time are presented. The signal at 875 cm-1 can be clearly assigned to the peroxide O-O-stretching vibration of hydrogen peroxide present in the reaction mixture. This band is ideal for the quantification of H2O2. Raman signals originating from diimide (e.g. 1529, 1583, 1598 and 1650 cm-1) as discussed in literature

[16,17,18]

for measurements at low temperatures in solid


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matrices could not be detected due to their weakness and/or their overlay by other signals. The weak Raman signals of hydrazine (1625, 1120 and 925 cm-1) as well as the hydrazinium monocation (966 cm-1) are also strongly overlain by the signals of AA, DHAA and 2-propanol and therefore could not be evaluated during the data analysis. The two spectra in the wavenumber range 1200-1700 cm-1 (Figure 3a) were calculated Raman spectra from a twocomponent MCR analysis of the reaction spectra. The two calculated spectra are identical with those of the reactant (AA) and product (DHAA) solution, respectively. The two Raman signals at 1635 and 1666 cm-1 present in the spectrum of artemisinic acid are the characteristic bands of the exo- and endo-cyclic –C=C-stretching vibrations. As during the diimide reduction of artemisinic acid to dihydroartemisinic acid the exo-cyclic double bond is reduced, the Raman signal at 1635 cm-1 decreases over time. Applying peak-height analysis this band is very well suited for the monitoring of the reactant consumption. In Figure 3b the fractions of reactant (AA) and product (DHAA) in dependence of the reaction time calculated by a twocomponent MCR analysis are presented. The t50 value was found at about 3 hours, the end of the reaction at ~ 6 hours. Included into the graph are the H2O2 dosage profile as well as the actual H2O2 concentration in solution and the equivalents of H2O2 consumed. From the plots it becomes evident that at the end of the reaction only ~ 0.2 eq. H2O2 of the in total 1.8 eq. H2O2 remain in solution. The remaining H2O2 content, however, turned out to be an important factor during the isolation step of DHAA by crystallisation. Even under the optimized reaction conditions with low H2O2 content present in the product solution only yellowish coloured, strongly agglomerated and poorly crystalline DHAA with yields < 60% could be isolated. Therefore the diimide reduction of artemisinic acid using H2O2 and hydrazine monohydrate was not favoured for pilot plant scale up.


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2.2 DHAA synthesis via diimide approach using oxygen and hydrazine hydrate (lab scale)

A further method to generate diimide is given by the reaction of hydrazine monohydrate with oxygen. However, the use of flammable solvents such as alcohols in chemical reactions in the presence of air having 21% by volume of oxygen is potentially very dangerous, particularly the use of pure oxygen. For alcohols such as ethanol or 2-propanol, the oxygen limit concentration at room temperature and atmospheric pressure is less than 9% by volume

[19]

.

Above the oxygen limit concentration, safe handling of flammable solvents is no longer ensured. Accordingly, on an industrial scale, synthetic air above the oxygen limit concentration is prohibited using flammable solvents. For instance, to ensure a sufficient safety margin, on the pilot plant and industrial plant, Sanofi has defined an oxygen concentration of 5% by volume as the maximum oxygen concentration admissible, and the use of flammable solvents for chemical reactions above 5% by volume of oxygen is not allowed. In the first reaction trials (proof-of-concept study, refer Table 1) air containing 20 % v/v oxygen was used. For the reaction optimization and process development the oxygen content in the air used was reduced to 5 % v/v. The reaction optimization process comprised testing of different reaction temperatures, solvents, equivalents of hydrazine monohydrate and gassing techniques. It turned out, that gas introduction via filter frits of heated chromatography columns was the optimal choice as reaction vessel for this type of reaction. Other tested techniques, like gas stirrers or jet loop reactors (refer to supplementary information), led to a significant degassing of the oxygen in the reaction mixture, which slowed down or even stopped the reaction as observed by Raman spectroscopy. Short-chain alcohols like methanol, ethanol or 2-propanol were found to be most suitable for the hydrogenation reaction of AA to DHAA (Table 1). For the scale-up non-toxic 2-propanol (bp. 82°C) was selected from the alcohols tested. During the reaction optimization process it was found that at least 2 equivalents of hydrazine have to be present for a complete conversion of ACS Paragon Plus Environment

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AA to DHAA. The last two rows in Table 1 represent the optimized reaction conditions. Full conversion of the reactant AA and a high diastereoselectivity of the product DHAA, which could be easily crystallized as free acid by the addition of 2N HCl to the reaction mixture, was observed by using these conditions. The fraction of the overhydrogenated product THAA was found to be comparable to the diimide approach using hydrogen peroxide and hydrazine monohydrate. In order to reduce solvent loss due to evaporation the reaction temperature was limited to 40°C.

2.3 DHAA synthesis via diimide route using oxygen and hydrazine hydrate (pilot plant scale)

Three batches of DHAA were successfully synthesized, crystallized and isolated in kg-scale in the pilot plant. Table 2 summarizes the reaction conditions, yields and some analytical properties of the batches. For the first two diimide reductions 3 equivalents of hydrazine monohydrate were used, the third reaction was performed with 2.5 eq. hydrazine monohydrate in order to test the influence on the reaction time in pilot plant scale. A typical set of Raman spectra collected during the diimide reduction can be found in Figure 4a. Again the consumption of artemisinic acid can be followed by the decrease of the exo-cyclic –C=Cstretching signal at 1635 cm-1. In the wavenumber range between 1200 and 1340 cm-1 increasing Raman signals were observed due to the formation of C-H-bonds. Figure 4b shows the comparison of calculated Raman spectra (2-component MCR analysis) of the first and the second batch. Both sets of spectra (reactant and product) are in good agreement proving the good reproducibility between two measurement cycles. In Figure 5a the MCRcalculated fractions of product and reactant in dependence of time are presented together with product formation calculated from the peak height decrease of the signal at 1635 cm-1 (-C=Cstretching signal). Both evaluation methods gave almost identical results, which indicates that a MCR analysis assuming 2 reaction components is a correct scenario. The black cycles in ACS Paragon Plus Environment

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Figure 5a indicate the change of the used air storage container (ASC) with a freshly prepared one (filled with premixed air containing 5 % v/v oxygen). In the first three hours reaction time of batch 1 the ASC was changed only once an hour. The reaction profile in this time frame shows that after the first 60 minutes the reaction rate has decreased to almost zero. At this point the oxygen concentration (measured by an oxygen probe) in the ASC was found to be about 2 % v/v (oxygen concentration limit for the diimide reduction). After changing the ASC the reaction rate again increased and slowed down again reaching the second hour of reaction time and so on. In order to optimize the reaction procedure it was decided on the base of these data to change the ASC more often. As the preparation time of a new ASC (evacuation, flushing and filling) took roughly 20 – 25 minutes, the containers could be changed about 3 times in an hour. This ASC exchange regime was applied on batch 1 after the fourth hour of reaction time and throughout for the batches 2 and 3. Figure 5b shows that about 90 minutes could be saved by optimizing the ASC exchange. In an industrial scale this issue will be avoided by a controlled direct mixing of pure nitrogen and oxygen to the desired gas mixture with an oxygen content of 5 % v/v. The end point of the reaction (reactant AA fully consumed) was determined by the absence of further spectral changes in the Raman signals at 1054 cm-1, 1272 cm-1 and 1635 cm-1. The sample taken after the end of the reaction, which was analyzed by HPLC, confirmed that no significant amounts of AA remained in the reaction solution (Conversion: >99%). Batch 3, which was synthesized using 2.5 eq. of hydrazine monohydrate, showed a reaction time of 12 hours compared to 10 hours for batch 2 (3 eq. hydrazine monohydrate). Thus 2.5 eq. hydrazine monohydrate are sufficient for the diimide reduction. By a further optimization of the gassing regime the reaction time using 2.5 eq. hydrazine monohydrate could be lowered to < 10 hours. Altogether a good correlation of the reaction profiles between laboratory and pilot plant scale was observed as depicted in Figure 6. After the reaction DHAA was crystallized by the addition of aqueous 2N HCl at temperatures between 10-15°C. Raman spectra collected during the crystallization process are ACS Paragon Plus Environment

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given in Figure 7a. Significant spectral changes were observed due to the formation of solid, crystalline DHAA. By following the Raman peak area change of the signal at 608 cm-1 or the peak position of the signal at 1668 cm-1 it is possible to “quantify” the extent of the crystallization process. The relationship between pH, amount of HCl added and the mentioned analyzed Raman signals are shown in Figure 7b. The crystallization point was observed at pH 6.7 for all three batches. The crystallization end points were determined by the absence of further spectral changes in the signal at 608 cm-1. For testing reasons the pH end points for the batches 1 and 3 were fixed at pH 5 whereas the pH endpoint of batch 2 was set at pH 6. For batch 2 a significant loss in yield was observed (81% instead of ~94%) accompanied by an increase in purity of the batch (refer to Table 2). However, pH 5 should be considered as final pH of the crystallization as the purity of the batches 1 and 3 is more than sufficient for the further synthesis of Artemisinin. In Figure 8 the X-ray diffraction pattern as well as a SEM picture of a typical pilot plant DHAA product is shown. The crystallinity was found to be excellent in all produced batches.

3. Conclusion

This study showed that the desired diastereomer of dihydroartemisinic acid (DHAA(1)), a key intermediate for the anti-malaria drug Artemisinin, can be conveniently synthesized in high yield (> 90%) by diimide reduction of artemisinic acid (AA) without the necessity of a further (chiral) catalyst as described by other authors in the literature

[20]

. Two different methods to

generate diimide have been tested at lab scale as possibilities for a later scale-up into pilot plant: 1) reaction of hydrazine hydrate and hydrogen peroxide and 2) the reaction of hydrazine hydrate and oxygen. It was shown that by using the first option complete conversion of AA to DHAA can be achieved. With the support of Raman spectroscopy as online analytical tool it was possible to reduce the required amounts of hydrazine hydrate and ACS Paragon Plus Environment

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hydrogen peroxide compared to the initially described process significantly [212]. However, the isolation of solid DHAA as free acid by crystallization from the reaction solution was challenging due to the presence of residual H2O2. The second option using oxygen and hydrazine monohydrate was found to be a safe, efficient and scalable way for the supply of diimide for the synthesis of DHAA from artemisinic acid. 2-propanol turned out to be the reaction medium of choice. Due to safety considerations the oxygen content in the used gas– mixture of nitrogen and oxygen was limited to 5 % v/v oxygen. For the industrialisation of the described process a switch from premixed air (5 % v/v oxygen) to an in-situ gas mixing unit (controlled by oxygen sensors) is suggested. Furthermore the process can be simplified by combining synthesis and crystallization in one vessel equipped with the gassing unit. As it has been shown Raman spectroscopy can be used for the process control either on pilot or production scale, thus no external sampling for analytics is needed. Handling of hydrazine monohydrate in production scale (storage, charging) can be performed in a fully closed system with pressure compensation pipes using specially designed, refillable plastic containers for storage (e.g. from LANXESS [21]). Residual hydrazine in the mother liquor can be easily destroyed by methods reported in the literature

[22, 23]

resulting in nitrogen as waste

(also refer to supplementary information, chapter 7). Overall this study showed that the diimide reduction of artemisinic acid is an attractive, safe and scalable synthesis technique for dihydroartemisinic acid which can be industrialized.


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4. Experimental Section

4.1 Chemicals Artemisinic acid (AA, CAS No. 80286-58-4) was purchased from Biovet (Sofia, Bulgaria). Hydrazine monohydrate (N2H4 · 1 H2O, CAS No. 7803-57-8, 98%) was obtained from ABCR GmbH & Co. KG, Karlsruhe, Germany. 30% hydrogen peroxide solution (H2O2, CAS No. 7722-84-1), 2-propanol (CAS No. 67-63-0), ethanol (CAS. No. 64-17-5, MEK denatured), potassium hydroxide, sodium metabisulfite, 2N aqueous HCl and 2N aqueous H2SO4 were purchased from Merck (Darmstadt, Germany) and other vendors. Premixed air in gas bottles containing 20 or 5 % v/v oxygen were obtained from Messer Group GmbH (Lenzburg, Switzerland).

4.2 Reaction, equipment and conditions for diimide approach using H2O2 in lab scale For a typical reaction 15 g artemisinic acid (AA) were suspended in a 500 mL 4 necked round bottom flask equipped with a KPG stirrer in a solvent mixture of 90 mL H2O and 22.5 mL 2propanol. Upon stirring at 150 rpm the suspension was heated to an inner temperature of 40°C. Subsequently 1.8 equivalents (eq.) hydrazine hydrate were added. After several minutes of stirring a clear solution with a pH of 8.1 was obtained. The pH was adjusted with 5N KOH to a final pH value of 8.4. The reaction monitoring by Raman spectroscopy using the MR dip probe was started. At reaction time tzero 0.9 eq. of H2O2 were added to the solution and linear dosage of further 0.9 eq. of H2O2 during 4 hours via a syringe pump (Havard Apparatus) was started. The pH was controlled throughout the reaction and adjusted with 5N KOH when it had decreased to a value of 8.1 (twice during the whole reaction). After 7 hours the reaction was finished and 0.13 eq. aqueous Na2S2O5 (sodium metabisulfite) solution were added to the cooled reaction solution (4°C) in order to remove the excess of H2O2. Solid DHAA was obtained by the addition of 51.5 g of 2 N H2SO4 and 60 g 2-propanol. The yellowish solid was ACS Paragon Plus Environment

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isolated by filtration and washed twice with 20 mL H2O. The product was dried at 40°C in vacuum. In the lab experiments yields of up to 58% were obtained. All products were analyzed by means of HPLC (purity and residual hydrazine content), GC-MS (diastereomeric ratio) and 1H-NMR spectroscopy (identity). The analytical results obtained complied with analytical data of reference compounds.

4.3 Reaction, and crystallization equipment for diimide approach using gas mixtures of nitrogen and oxygen in lab scale 15 g artemisinic acid were suspended in 45 mL 2-propanol (or ethanol) a heatable glass chromatography column equipped with a glass filter frit at room temperature. Subsequently 3 equivalents hydrazine hydrate were added. After some minutes the reaction mixture became a clear solution and then was heated to 40°C. Synthetic air containing either 20 or 5 % v/v oxygen was pumped in a closed-loop by a peristaltic pump (LS tubing pump from Masterflex with a pumping rate of 380 mL/min) from a 5 L glass storage vessel (ambient pressure) to the column plate (filter frit), then bubbled through the reaction solution and transferred back to the storage vessel. The air in the storage vessel was exchanged every 60 minutes with fresh synthetic air from a gas bottle. The reaction was monitored by Raman spectroscopy using the MR dip probe. Typical reaction times were in the range of 10 – 12 hours. After completed reaction the solution was transferred into a 500 mL four necked round bottom flask. After addition of further 5 mL 2-propanol and 65 mL water the pH was 8.3 – 8.5. The solution was cooled to 10°C and 2 N HCl was slowly added. At pH ~6.8 the first signs of turbidity were observed. At pH 6 the suspension was stirred for 30 min. The pH was further decreased by addition of 2 N HCl (total amount of 2N HCl: 35 mL) to the final pH 5. Further 30 mL water were added resulting in a 2-propanol-water ratio of 1:2. The suspension was stirred for further 2 hours at 10°C. The light yellowish DHAA was isolated by filtration, washed twice with 50 mL water and dried overnight at 40°C in vacuum. Yields > 90% were obtained. The dried ACS Paragon Plus Environment

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products were analyzed by HPLC (purity and residual hydrazine content), GC-MS (diastereomeric ratio) and

1

H-NMR spectroscopy (for details refer to supplementary

information). The analytical results obtained complied with analytical data of reference compounds.

4.4 Reaction and crystallization equipment for diimide approach using a gas mixture of nitrogen and oxygen in pilot plant scale (95 : 5 %v/v)

The pilot plant scale apparatus scheme for the diimide reduction using synthetic air with 5 % v/v oxygen is depicted in Figure 1a. In Figure 1b pictures of the equipment are shown. As reaction vessel a 49 L filter dryer “RoLab” from De Dietrich Process systems, Switzerland, was used. The MR Raman probe was connected to the filter dryer via the nitrogen gas inlet port of the dryer. For the reaction a clear solution containing 3 kg artemisinic acid and 1.92 kg hydrazine hydrate (3 eq.) in 9 L 2-propanol were transferred into the filter dryer. The synthetic air was pumped from the air storage container 1 or 2 by a delivery pump to the filter frit of the filter dryer, bubbled through the reaction solution (40°C) and then back to the storage container. After 15 to 30 minutes reaction time the used air storage container was exchanged with a freshly filled and prepared one. The end point of the reaction was determined by Raman spectroscopy, when no further spectral changes were detected. An end point sample was collected and analyzed by HPLC confirming the result of Raman spectroscopy (AA content < 1 area-%). For the crystallization of the product the reaction solution was transferred from the filter dryer into a 105 L vessel equipped with an anchor stirrer (refer to Figure 2a). The filter dryer was washed twice with 1 L 2-propanol. Subsequently 11 L water were added to the mixture. Then the solution was cooled to an inner vessel temperature of 10-13°C. The pH of the solution was monitored by an external pumping loop shown in figure 2a. The Raman MR probe was also ACS Paragon Plus Environment

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integrated into the pH loop (Figure 2b). As the desired temperature was reached the addition of 2N HCl was started. At pH ~6.7 the first signs of turbidity (crystallization) were observed. At pH 6 the suspension was stirred for 30 min (6.5 kg 2N HCl consumed). Then further 1.1 kg 2N HCl were added slowly followed by further stirring. The end point of the crystallization was determined by Raman spectroscopy (end point criterion: no further spectral changes in the signal at 608 cm-1). The solid DHAA was isolated by pressure filtration, washed twice with 10 L of water and dried at 40°C in vacuum. The dried products (three pilot plant batches were produced) were analyzed by means of differential scanning calorimetry (DSC; melting point determination), thermogravimetry and dynamical water vapour sorption gravimetry (TGA and DWVSG; residual solvent and surface adsorbed water), water content determination via Karl-Fischer titration, X-ray powder diffraction (XRPD; crystallinity and crystal phase), scanning electron microscopy (SEM; crystal shape determination), HPLC (purity and residual hydrazine content), GC-MS (diastereomeric ratio), MS Spectrometry and 1

H-NMR spectroscopy (for details refer to supplementary information, chapters 1 - 6 ).

4.5 Dispersive Raman spectroscopy

Dispersive Raman spectroscopy was performed on a Kaiser Optical Systems Ltd. (Ann Arbor, USA) RXN1 system equipped with a 785 nm Invictus laser diode (laser power: 400 mW) as source, an Andor iDus DV420-OE CCD camera (peltier-cooled, working temperature: -40°C) as detector and a MR Probe filtered probe head (dip probe). The installation of the Raman probe head in the pilot plant is shown in the figures 2 (reaction monitoring in filter dryer) and 3 (crystallization monitoring in external loop technique). The laser wavelength, frequency and intensity calibration of the system was performed using cyclohexane (as reference), neon emission lines and white light, respectively. Data acquisition and analysis were performed


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using the software packages IC Raman version 4.0 (Mettler Toledo, Greifensee, Switzerland), and PEAXACT Analyzer Toolbox 3.0.11 (S-PACT GmbH, Aachen, Germany). The spectra were recorded (IC Raman 4.0) in the wavenumber range between 1890 cm-1 and 100 cm-1 (Raman exposure time: 30 s) with a spectral resolution of 4 cm-1. For peak analysis by peak height or peak area integration method (IC Raman 4.0) each spectrum was baseline corrected (1st order baseline correction) and normalized to the peak height of the Raman signal at 1453 cm-1. The consumption of the reactant (artemisinic acid) during the diimide reduction was determined from the peak height decrease of the –C=C– stretching signal [24] at 1635 cm-1 (geminal). The peak height of each spectrum was calculated using a two point baseline with supporting points at 1580 cm-1 and 1700 cm-1. The reactant consumption (RCAA) was calculated as the ratio between peak height at the selected time point and the total peak height (corrected by the background peak height). The product formation (PFDHAA and THAA)

is the difference PFDHAA and THAA = 1 – RCAA. The quantification of H2O2 in solution was

performed by peak integration method of the peroxide stretching vibration

[25]

at 875 cm-1

using an one-point calibration at tzero (addition of a known amount of H2O2). The peak area at tzero was determined (peak integration range: 854 – 899 cm-1; single point baseline with supporting point at 910 cm-1) and correlated with the added amount of H2O2 (in equivalents, thus for the following time points the concentration of H2O2 present in solution (Eqsolution) can be calculated). As the dosage velocity of H2O2 is known it is possible to calculate the total amount of H2O2 (Eqtotal) added and the amount of H2O2 consumed (Eqtotal – Eqsolution). Crystallization monitoring was performed by following the spectral changes at 1737 cm-1 (signal increase, -C=O stretching signal carboxylic acid, monomer), 1700 cm-1 (signal increase, -C=O stretching signal carboxylic acid, dimer), 1669 cm-1 (signal shift, –C=C– stretching signal), 664 cm-1 (signal increase) and 610 cm-1 (signal increase; peak area determination in the wavenumber range 602 – 615 cm-1 using a single point baseline with a supporting point at 590 cm-1). ACS Paragon Plus Environment

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Multivariate Curve Resolution (MCR) analysis was performed in the program package PEAXACT. The raw Raman data sets were baseline corrected in this case by a linear fit subtraction without smoothing. A vector standardization on the data was applied in the spectral region between 900 – 2000 cm-1 (diimide reduction via O2 method) or 1000 -1700 cm-1 (diimide reduction via H2O2 technique and product crystallization). In all investigated cases a two component analysis was found to be the correct choice for the number of components. For the MCR analysis the following constraints were applied: calculation of nonnegative spectra and non-negative concentrations, calculation of unimodal concentration profiles and the sum of concentration was set to 1 (closure criteria). As convergence option the maximum number of iterations was set to 3000 with a convergence tolerance of 1·10-6.

4.6 X-Ray Powder Diffraction (XRPD)

XRPD measurements at ambient conditions were performed in reflection mode on a Bruker D8 Advance (Bruker AXS GmbH, Karlsruhe, Germany) equipped with a Ge(111) monochromator and a linear PSD detector over an angular range of 2θ = 2–60°. Cu Kα1 radiation (λKα1 = 1.54060 Å, U = 40 kV, I = 40 mA) was used for the measurements. The DHAA powders were prepared on Si sample holders.

4.7 Scanning Electron Microscopy (SEM)

SEM pictures were acquired on a Hitachi tabletop microscope TM-1000 (Hitachi HighTechnologies Europe GmbH, Krefeld, Germany). For the SEM measurements all samples were coated with gold using a SCD 005 sputter coater from Bal-TEC (Leica Mikrosysteme GmbH, Wetzlar, Germany). ACS Paragon Plus Environment

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Acknowledgement

The authors would like to thank the following colleagues from Sanofi for their strong support of the project: a) the laboratory team: Franceska Fischer, Claus Keleschovsky, Michael Spitzenberg, Harald Starke and Ursula Seitz-Felix; b) the pilot plant team: Alexander Putz, Willi Scharf and Dirk Techentin; c) the analytical team: Dr. Corina Hunger, Dipl.-Ing. Antonius Fischer, Dr. Hans-Jürgen Pletsch, Dr. Jürgen Schäfer, Dr. Peyman Sakhaii, Hans-Peter Sperzel and Dr. Chiara Zorn. d) for pilot plant safety aspects: Dr. Lars Bierer, Dipl.-Ing. Jörg Jurascheck, Dr. Christoph Tappertzhofen and Dr. Michael Weigerding.

Supporting Information Available This information is available free of charge via the Internet at http://pubs.acs.org/.


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References [1]

World Health Organization. World Malaria Report 2005, 2005, WHO, Geneva.

[2]

RBM/UNITAID/WHO Artemisinin Conference, Hanoi, Vietnam – 2nd/3rd November 2011.

[3]

Wallaart, T.E., Pras, N., Beekmann, A.C., Quax,W.J., Planta Medica 2000, 66(1), 57-62.

[4]

Abdin, M.Z., Israr, M., Rehman, R.U., Jain, S.K., Planta Medica 2003, 69(4), 289-299.

[5]

Ro, D.-K., Paradise, E.M., Ouellet, M., Fisher, K.J., Newman, K.L., Ndungu, J.M., Ho, K.A., Eachus, R.A., Ham, T.S., Kirby, J., Chang, M.C.Y., Withers, S.T., Shiba, Y., Sarpong, R., Keasling, J.D., Nature 2006, 440(7086), 940-943.

[6]

Dhainaut, J., Dlubala, A., Guevel, R., Medard, A:; Oddon G., Raymond, N., Turconi,, J., Sanofi, WO2011026865

[7]

Pasto, D.J., Taylor, R.T., Organic Reactions, Hoboken, New York, United States, 1991, 40, 91-155.

[8]

Aylward, F., Sawistowska, M., Chem. and Ind. 1962, 484-91

[9]

Imada,Y., Iida, H., Naota, T., J. Am. Chem. Soc. 2005, 127, 14544-14545

[10]

Smit, C., Fraaije, M.W., Minnaard, A.J., J. Org. Chem. 2008, 73, 9482–9485.

[11]

Kraft, V., Kretzschmar, G., Rossen, K., Sanofi, WO2011030223

[12]

Rossen, K., Castro, B., Ricci, G., et al, to be published in Angew. Chem. Int. Ed..

[13]

Johnson, G.L., Machado, R. M., Freidl, K. G., Achenbach, M. L., Clark, P. J., Reidy, S. K., Org. Process Res. Dev. 2002, 6(5), 637–644.

[14]

Pataki, H., Csontos, I. Nagy, Z.K., Vajna,B., Molnar, M., Katona, L., Marosi, G., Org. Process Res. Dev., early view, 2012.

[15]

Feth, M.P., Nagel, N., Baumgartner, B., Bröckelmann, M., Rigal, D., Otto, B., Spitzenberg, M., Schulz, M., Becker, B., Fischer, F., Petzoldt, C., Eur J Pharm Sci. 2011, 42(1-2), 116-129.

[16]

Bondybey, V.E., Nibler, J.W., J. Chem. Phys. 1973, 58(5), 2125-2134.

[17]

Pouchan, C., Dargelos, A., Chaillet, M. , J. Mol. Spec. 1979, 76, 118-130.

[18]

Kempera, M.J.H., Buck, H.M., Can. J. Chem. 1981, 59, 3044-3048.

[19]

Brandes, E., Möller, W. (Eds.), Gase - Safety Characteristic Data, Volume 1: Flammable Liquids and Gases, 2008, Wirtschaftsverlag NW, Verlag für neue Wissenschaft GmbH, Bremerhaven, Germany.


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[20] [21] [22] [23]

[24] [25]

Page 22 of 35

Smit, C., Fraaije, M.W., Minnaard, A.J., J. Org. Chem. 2008, 73, 9482–9485. LANXESS AG, Kaiser-Wilhelm Allee 40 51369 Leverkusen. Hepp, H., Jacobi, G., VGB Kraftwerkstechnik 1985, 65(2), 163-171. Kuethe, J.T., Childers, K.G., Peng, Z., Journet, M., Humphrey, G.R., Org. Pro. Res. Dev. 2009, 13, 576–580. Poshyachinda, S., Kanitthanon, V., Spectro. Chim. Acta 1994, 50, 2011-2017. Vacque, V., Sombret, B., Huvenne, J.P., Legrand, P., Suc, S., Spectro. Chim. Acta 1997, 53, 55-66.


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Table 1: Summary of the laboratory scale batches using diimide reduction of artemisinic acid (AA) to dihydroartemisinic acid (DHAA).

Diimide method / Reaction medium H2O2 / H2O / 2-propanol 20% O2 / ethanol 5% O2 / ethanol 5% O2 / 2-propanol 5% O2 / 2-propanol

Equivalents N2H4·1 H2O / H2O2

Reaction time

pH

Temperature

1.8 / 1.8

6-7h

8.2

40°C

3/-

20h

n.d.

40°C

3/-

24h

n.d.

40°C

2.5 / -

9h

n.d.

50°C

3/-

11h

n.d.

40°C

Yield /%

Yields and analytical results GC analysis / area-% DHAA DHAA THAA (1) (2) 98.8 0.1 1.1

H2O2 / H2O / 2-propanol

58

20% O2 / ethanol

86

98.9

0.3

0.6

5% O2 / ethanol

90

97.6

0.3

1.9

5% O2 / 2-propanol

80

96.6

0.4

2.8

5% O2 / 2-propanol

91

97.6

0.4

2.0

Work-up to isolate DHAA difficult (semicryst. solid) Crystallization by addition of aq. 2N HCl Crystallization by addition of aq. 2N HCl Crystallization by addition of aq. 2N HCl Crystallization by addition of aq. 2N HCl


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Table 2: Summary of the pilot plant scale batches using diimide reduction (5 % v/v O2 + hydrazine hydrate) of artemisinic acid (AA) to dihydroartemisinic acid (DHAA).

Pilot Plant Batch No. 1 2 3

Reaction Time /h

Equivalents N2H4·1 H2O

3 3 2.5 Yield Amount /% / kg

Reaction temperature / °C

11 10 12 GC analysis / area-% DHAA DHAA THAA (1) (2) 95.6 1.6 2.8

40 40 40 Crystallization Final pH

1

95.4

2.9

2

81.0

2.5

97.4

0.7

1.9

6

3

93.7

2.8

96.2

1.4

2.4

5


5

2

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H O O O H O O Scheme 1: Structure of Artemisinin.


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Scheme 2: Chemical conversion from glucose to Artemisinin.


4

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H

H

"HN=NH"

H

+

H

+

H

H

H

H

HOOC

HOOC

HOOC

HOOC

DHAA (1)

DHAA (2)

THAA

Scheme 3: Reduction of Artemisinic acid to Dihydroartemisinic acid using Diimide.


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a) Filter dryer „RoLab“ (Reaction vessel, 49 L)

Air storage container 1

Raman spectrometer RXN1 MR probe head

Vacuum pump

Gas scrubber

Air storage container 2

Delivery pump

Gas bottle N2 with 5% v/v O2

b)

Figure 1: a) Apparatus scheme for diimide reaction in pilot plant and b) pictures of the used equipment.


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a) HCl storage vessel on balance

Gear pump

Mother liquor collection vessel

Raman spectrometer RXN1 MR probe

(90 L, anchor stirrer)

pH meter Crystallization vessel (105 L, anchor stirrer)

Pressure filter (Isolation, 63 L)

b)

Figure 2: a) Apparatus scheme for DHAA crystallization in pilot plant and b) picture of the installed Raman probe head in the external loop.


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a)

b)

Figure 3: a) Raman spectra of reaction monitoring (diimde formation by the reaction of H2O2 and hydrazine), MCR calculated component spectra of the monitored reaction and b) 2-component MCR analysis of the reaction and H2O2 concentration plots (equivalents vs. time).


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a)

b)

Figure 4: a) Overlay of Raman spectra during the reaction of artemisinic acid with diimide (hydrazine hydrate and air with 5 % v/v oxygen) to DHAA and b) MCR component spectra 1 (product) and 2 (reactant) calculated by a MCR analysis of two sets of reaction spectra (pilot plant batch 1 and 2).


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a)

b)

Figure 5: a) Comparison of the Raman data analysis of pilot plant batch No. 1 performed by MCR and peak-integration method and b) Comparison of the MCR Raman data analysis results of batch No. 1 and batch No. 2 (product formation).


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Figure 6: Comparison of the conversion rate between laboratory and pilot plant scale.


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a)

b)

Figure 7: a) Overlay of Raman spectra during a pilot plant crystallization of DHAA and b) plot of the Raman data analysis (peak integration and peak position) in dependence of time, pH value and added amount of 2N HCl.


12

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a) 27000 26000 25000 24000 23000 22000 21000 20000 19000 18000 17000 16000

Lin (Counts)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 2

10

20

30

40

50

6

2-Theta - Scale

b)

Figure 8: a) XRPD pattern of the solid product (DHAA) and b) SEM picture of the crystalline DHAA.


13

Pilot Plant PAT Approach for the Diastereoselective Diimide Reduction

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