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Amalgamation of Synthetic Biology and Chemistry for High Throughput Non-Conventional Synthesis of Antimalarial Drug Artemisinin Dharmendra Singh, Derek McPhee, Christopher J. Paddon, Joel Cherry, Ghanshyam Maurya, Ganesh Mahale, Yogesh Patel, Neeraj Kumar, Subhash Singh, Brajesh A Sharma, Lavkesh Kushwaha, Satinder Singh, and Ashok Kumar Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.6b00414 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on March 2, 2017

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Organic Process Research & Development

Amalgamation of Synthetic Biology and Chemistry for High Throughput Non-Conventional Synthesis of Antimalarial Drug Artemisinin

Dharmendra Singh†, Derek McPhee‡, Christopher J Paddon‡, Joel Cherry‡*, Ghanshyam Maurya†, Ganesh Mahale†, Yogesh Patel†, Neeraj Kumar†, Subhash Singh†, Brajesh Sharma†, Lavkesh Kushwaha†, Satinder Singh† and Ashok Kumar†* †

Chemistry Research and Development, Plot Number 123-AB, Ipca Laboratories Limited, kandivali Industrial Estate, Kandivali West, ‡

Mumbai, 400067, India. Research and Development, Amyris Inc. 5885 Hollis Street, Ste. 100, Emeryville, CA 94608, USA.

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Graphical Abstract


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ABSTRACT: Development of a cost-effective process for the production of artemisinin, the precursor of all artemisinin-derived drugs, the first-line treatment for malaria, has been a long pursued endeavor. The breakthrough achievement of coaxing genetically engineered yeast to express Artemisia annua genes for the commercial production of artemisinic acid, an advanced intermediate in the synthesis of artemisinin, has yet to fully realize an affordable malaria treatment for the poor owing to the lack of a cost-effective chemical conversion into artemisinin. We describe herein a commercially feasible and pragmatic synthesis of artemisinin from amorpha-4,11-diene, an early stage intermediate produced in 2-fold higher molar yield than engineered yeast cells can process into artemisinic acid. The key to this novel approach is an exceedingly effective functionalization of the isopropenyl group of amorphadiene via endoepoxyamorphadiene to give dihydroartemisinic acid, which on esterification followed by oxidation and cyclicization furnishes pure artemisinin in the yield approximating 60%.

KEYWORDS: Malaria, artemisia annua, amorphadiene, endoepoxidation, artemisinic acid, artemisinin



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INTRODUCTION

Almost half of the world’s population is at risk for malaria, a disease caused by Plasmodium spp. In 2013, US $2.7 billion was spent for malaria treatment and control, far less than the US $5.1 billion calculated to be required to achieve the global targets for malaria control and elimination.1,2,3 Artemisinin Combination Therapies (ACTs) are recommended by WHO as the first-line treatment for uncomplicated malaria and are adopted in 79 of 88 Plasmodium falciparum malaria endemic countries.4,5 These co-formulated drugs include a fast-acting artemisinin derivative and an anti-malarial drug of another class. The main limitation in making this life-saving treatment feasible worldwide has been the high price of artemisinin, which makes it unaffordable to the most vulnerable population.6 Reducing the cost of production of the drug substance artemisinin, could decrease treatment cost and save lives currently being lost to malaria every year.6-8 Artemisinin is extracted from the Chinese medicinal plant Artemisia annua, which is primarily cultivated in China and Vietnam. However, its supply and quality varies depending on the climatic conditions, geographical area and growing practices, the planting being strongly influenced by market dynamics.6 During the last decade, the price of artemisinin has been highly erratic due to the lack of synchrony between demand forecasts and production.9 Between 2003 and 2004, the price of this natural product bounced from just over US $350.00/kg to as high as US $1000.00/kg, and following considerable subsequent price variability has retreated to less than US $200.00/kg in 2016.7,10,11 Hence, there is a dire need to reduce, as well as stabilize, the price of artemisinin to ensure a consistent supply of anti-malarial ACT medication for those in need. Several efforts have been made in the past to develop a total synthesis12,13,14 for the production of artemisinin, but none has been able to deliver an efficient and cost effective alternative to plant extraction of the drug.15 Interestingly, an effective yet scalable alternative to agro-based production of artemisinin, based on insights into the natural biosynthesis pathway16, was developed by Keasling et al. wherein key metabolic pathway genes of Artemisia annua were transformed into yeast to produce amorpha-4,11-diene ACS Paragon Plus Environment

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(AD) and artemisinic acid (AA), the sesquiterpene precursors to artemisinin.17 The natural synthesis of artemisinin starts with farnesyl pyrophosphate (FPP) which is converted to an early intermediate, AD, by the enzyme amorphadiene synthase (ADS).18-20 AD is further oxidized to AA by cytochrome P450 monooxygenase (CYP71AV1) via its corresponding alcohol and aldehyde. The aldehyde is converted to dihydroartemisinic aldehyde (DHAAA) by a double-bond reductase. Conversion of DHAAA to (11R)dihydroartemisinic acid (DHAA) is catalysed by an aldehyde dehydrogenase. In the presence of sunlight, DHAA gives the endo-peroxy tetracyclic product artemisinin.21-24 Amyris Inc. (https://amyris.com/) has since created a yeast strain containing the necessary plant genes coupled to an upregulated terpene pathway that produces high levels of both AD and AA. This modified strain furnishes high titers (> 25 g/L) of AA.24 Despite the fact that the conversion of DHAA to artemisinin in the presence of light has been known for more than a decade and exploratory studies carried out by various groups have developed detailed understanding of the chemistry of all the steps involved in this transformation16,23, a cost effective workable and scalable synthesis of artemisinin has eluded organic chemists so far. Notably, methods for the transformation of DHAA to artemisinin using classical chemistry12-14,25, light catalysed continuous flow reaction26-28, as well as standard photochemistry29 are well documented. Even though the latter was employed for commercial production by Sanofi, none of the methods has been able to deliver the cost reduction in artemisinin production that was originally anticipated. Our commitment to provide effective, yet affordable, anti-malarial therapy to the entire world inspired us to attempt to use AD, an early intermediate being produced at 2-fold higher molar concentrations than AA during production of AA by engineered yeast.24,30 Herein we describe the results of this effort, a cost effective chemical process utilizing available simple reagents that is not only amenable to commercial scale-up, but also evade a costly photochemical process.31



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RESULTS

Since AD is an intermediate in AA biosynthesis, has the desired stereo-chemical orientation of functional groups required for its transformation into artemisinin and is amenable to scale up to a titer of more than 120 gm/liter, if desired, AD was deemed both useful and preferable to AA to develop an economically viable synthesis for artemisinin. AD was also considered to be an ideal starting material for artemisinin synthesis by Amyris, the producer of this sesquiterpene, and the approach adopted by them to functionalize AD at the exocyclic double bond, while perhaps the most logical32, was deemed to be too costly for industrial scale-up as it required use of an expensive reagent, 9-borabicyclo[3.3.1]nonane (9-BBN), to perform a hydroboration to give the desired alcohol in reasonable yields and diastereoselectivity; thus making the process practically and economically unappealing. Another route, also from Amyris, involving regio- and stereo-selective epoxidation of the exocyclic double bond of AD over the endocyclic alkene using an epoxidizing agent, with the help of a metalloporphyrin or metallosalen catalyst offered an attractive alternative32, but was also found to be expensive and ultimately impractical. Starting with the firm belief that AD was the best choice to meet the cost commitments to make ACTs accessible to all needy, we initiated our studies by examining methods for the selective functionalization of the isopropenyl group in the presence of a more reactive endocyclic double bond, and came upon the idea of converting the reactive double bond to its corresponding epoxide and then using endo-epoxy AD (3) as a less reactive version of AD for the synthesis of the target molecule. Being electronically rich, the endocyclic double bond of AD (2) was expected to undergo regioselective conversion to its epoxide with electrophilic reagents like meta-chloroperbenzoic acid (m-CPBA), eliminating the possibility of subsequent side reactions due to its presence. The protection of active functional groups to avoid undesired chemical transformations and the recovery of the original groups after removal of the protecting groups is common practice in the synthesis of complex organic molecules.33,34


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The idea of protecting the reactive double bond of 2 by epoxidation and subsequent functionalization of the isopropenyl group is well conceivable, but we also envisioned that its success would depend on four major positive outcomes, namely, availability of an epoxidation method which would give more than 99% regioselectivity in favour of the endocyclic double bond and high yields; availability of conditions for all the steps which are not detrimental to the stability of an epoxide (the protecting group); selective and quantitative recovery of the double bond at the original position from the epoxide and lastly but most important of all, the availability of cheap yet environmentally friendly reagents to perform the task in order to keep the synthesis cost low enough to make ACTs affordable to the poor. Since the first and foremost critical requirement for the success of this designed approach was the efficient regeneration of the double bond from the epoxide used as a double bond protecting group, a few quick reactions were performed to epoxidize 2 with m-CPBA, which produced a 80:5:15 mixture of endo-(3), exo- and diepoxyamorpha-4,11-diene, respectively. Without purification the combined epoxy-AD mixture was next subjected to de-epoxidation using known methods. Conditions using lithium metal not only proved helpful in achieving the “proof of concept”, but also regenerated in almost quantitative yield compound 2 of purity similar to the starting material used in the epoxidation reaction.33,34 This success in making 3 with reasonably good regioselectivity and its de-epoxidation leading to quantitative recovery of the double bond compound invigorated us to dedicate more efforts to finding an efficient yet practical method to epoxidise 2. The use of m-CPBA as an epoxidizing agent to synthesize epoxy-AD (3) was an obvious choice, given the success of our scouting reactions, however, the observed regioselectivity towards the endocyclic double bond of 2, giving around 80% yield of the target (as assessed by 1H-NMR) appeared to be too low to meet our objective. Another reason for not considering mCPBA was the very high cost of this reagent, which makes it unattractive for a commercial application. Other reagents and conditions were then examined and eventually we zeroed in on the green as well as most economical epoxidising agent, H2O2 (Table 1).



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a

Table 1: Epoxidation of amorpha-4,11-diene (AD)

Entry Reagent 1

2

3

mCPBA (1.26 eq)

Catalyst

Solvent

T (oC) Conversion (%) Yieldb (%)

Nil

DCM

0-5

99

80

4-chlorophenol

55-58

98

94

Nil

30 - 34 >99

H2O2 (50% aq.)

NaOAc

(1.0 eq)

(0.1 eq)

H2O2 (50% aq.)

HCOOH

(2.5 eq)

(0.25 eq)

a

96

b

Amorpha-4, 11-diene taken on 100% Basis. Yields were calculated based on the relevant peaks in the 1H NMR spectrum with reference to internal standard [quinol]. mCPBA: meta-chloroperoxybenzoic Acid, DCM: dichloromethane, H2O2: hydrogen peroxide, NaOAc: sodium acetate, HCOOH: formic acid.

It was heartening that with little effort we were able to find conditions giving excellent regioselectivity towards the endocyclic double bond (≥ 99%) and almost quantitative yields of 3. Although highly regioselective epoxidations of electronically rich double bonds in similar molecules have been previously reported35, the selectivity achieved using H2O2 in the presence of HCOOH36,37 as catalyst giving >99% yield is quite significant. Another merit of this combination is that the use of even a large excess of H2O2 (2.5 eq) does not give more than 2% of the diepoxy byproduct, making it easily operable at large scale (Figure 1).


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Figure 1: Chemical Conversion of amorphadiene to dihydroartemisinic acid. Chemical conversion of 2 to 8 is achieved by epoxidation of 2 to 3 leading to inactivation of active double bond; followed by chlorination to 4 and oxidation of 4 to 5 and subsequently to 6, using mild oxidizing agents. 6 is reduced to 7 with diimide generated in-situ from hydrazine hydrate with the help of hydrogen peroxide. Finally 7 is deoxygenated with lithium metal to give DHAA (8).

After attaining good yields and purity in the synthesis of 3, we turned our attention towards the desired functionalization of the isopropenyl group. A reaction like hydroboration is perhaps the best option as it achieves two steps, i.e. reduction of double bond and introduction of an alcohol group at the right position to give the corresponding dihydroartemisinic methyl alcohol in one step, provided the reaction proceeds with high diastereoselectivity. However, we decided not to pursue this route in light of the studies previously carried out by Amyris on AD using 9-BBN, perhaps the best possible choice of hydroborating agent for this purpose, as it does not give more than 91% diastereoselectivity and is relatively expensive, thus rendering the overall process economically unviable. The option of C-H functionalization of the allyl group by maintaining the double bond intact appeared practical and rewarding enough to pursue halogenation under free radical conditions using N-chloro/bromosuccinimide as well as SO2Cl2 in the presence of peroxides, but these approaches proved futile. Owing to the economics of the reaction, we chose to focus on chlorination and decided to again attempt this reaction using sodium/calcium hypochlorite38 or trichloroisocyanuric acid (TCCA)39 as chlorine substitutes to chlorinate the side chain under ionic conditions. Calcium hypochlorite (bleach) under aqueous and TCCA under non-aqueous conditions both delivered chloroepoxy-AD (4) in exceptionally high yields (Table 2). a

Table 2: Conversion of epoxide to chloroepoxy-AD

Entry Reagent

Acid

Solvent


4

NaOCl (1.4 eq)

HCl

DCM: H2O (20:5)

10-15

85

75

5

Ca(OCl)2 (1.4 eq)

HCl

DCM: H2O (20:5)

0-5

97

90

6

TCCA (0.36 eq)

Nil

cyclohexane

25-30

>99

94

a

b

1

Epoxide taken on 100% basis. Yields were calculated based on the relevant peaks in H-NMR spectrum with reference to internal standard [quinol]. NaOCl: sodium hypochlorite, DCM: dichloromethane, Ca(OCl)2: calcium hypochlorite, TCCA: trichloroisocyanuric acid.



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Under ambient conditions, TCCA is not only safe, stable and inexpensive but gives easily recyclable cyanuric acid as a byproduct; thus offering a green alternative to chlorine or other agents viz. calcium/sodium hypochlorite. In line with our expectations the transformation of 4 to the corresponding epoxy acid (6) via the corresponding aldehyde (5) was found to be comparatively easy, but still required a lot of hard work to replace the known NMO40 driven reaction by DMSO41 as a reagent for converting 4 into 5 in quantitative yield (Table 3). Table 3: Conversion of chloroepoxy-ADa to aldehyde

Base

7

NMO (3.0 eq)

Na2CO3 (3.0 eq) DMSO (10 Vol)

50-55

> 95

90

8

NMO (3.0 eq)

Na2CO3 (3.0 eq) DMF (10 Vol)

50-55

>97

92

9

DMSOc

K2HPO4 (2.5 eq) DMSO (20 Vol) 80-85

>99

95

a

Solvent


Entry Reagent

b

chloroepoxy AD taken on 100% Basis. Yields were calculated based on the relevant peaks in 1H-NMR spectrum with c reference to internal standard [quinol]. DMSO used as both solvent and reagent. NMO: N-methylmorpholine-N-oxide, Na2CO3: sodium carbonate, DMSO: dimethyl sulfoxide, DMF: dimethylformamide, K2HPO4: dipotassium hydrogen phosphate

NMO is expensive and its use in the synthesis of 5 from 4 was found to inflate the cost of the product considerably, while offering no yield advantage. Since we had started working on this approach only after establishing the feasibility of regenerating the endocyclic double bond by removing the epoxy group from 3 to get a quantitative yield of 2, we were confident in being able to regenerate the double bond at the original position in 6 to synthesize AA but unexpectedly this pursuit failed to achieve the desired results. This led us to carefully examine the reason(s) for this unforeseen result by isolating and characterizing all the products in the de-epoxidation step and discovering that apart from removal of the epoxy group, the conditions used were also favourable for reducing the exocyclic α,β-unsaturated double bond.34 Since that happened without any observable diastereoselectivity, the reaction yielded a mixture of both the isomers of DHAA (8) along with the desired product i.e. AA.


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This understanding turned out to be handy for quickly solving the problem and reframing the synthesis, which involved saturation of the α,β-unsaturated double bond 42 of 6 to give epoxy-dihydroartemisinic acid (7) prior to the removal of the epoxy group, resulting in a good conversion efficiency and high yield of 8, an advanced intermediate in the chemosynthesis as well as biosynthesis of artemisinin (1). Working backwards, the key to this novel strategy was the protection of the more reactive double bond of 2 in ≥ 99% regioselectivity leveraging the isopropenyl side chain for the intended modifications. Although removal of the epoxy group established in 2 was unsuccessful when attempted with 6, the purity and yields of all the intermediates and more importantly the stability of epoxy group in all the intermediates during subsequent modifications leading up to the synthesis of 8 supported the endeavor very well. The steps right from conversion of 2 till the formation of 7, when performed in-situ, circumvent the isolation of intermediates and offered better control. The conversion of 8 to 1 via the hydroperoxide (10) of methyl ester (9) was performed using commercially available inexpensive chemicals under conditions developed earlier by Amyris 43 with minor modifications, thus obviating the need for photoreactors (Figure 2).

Figure 2: Chemical conversion of dihydroartemisinic acid to artemisinin. Conversion of 8 to 9 is accomplished by esterification with the help of dimethyl sulphate. Compound 9 is reacted with singlet oxygen, generated in-situ from hydrogen peroxide in the presence of sodium molybdate to give 10. The subsequent Hock rearrangement in the presence of benzenesulfonic acid, indion 225H and triplet oxygen yields the desired product, artemisinin (1).

In order to keep the transformation costs in check, we decided to use indion 225H cation exchange resin, a cheaper alternative to the dowex 50WX8-200 resin used by Amyris, and benzenesulphonic acid as catalyst in the step involving triplet oxygen. Increasing the duration of singlet as well as triplet oxygen exposure ACS Paragon Plus Environment


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improved the conversions of the starting material and intermediates; thus ultimately giving close to 60% isolated yield of the desired product. The previous best yield for this conversion at temperature range 025oC is 45% as reported by Amyris. A simplistic synthesis of 1 from 8 at ambient temperature, circumvented esterification and generated 1O2 with 30% H2O2 in presence of catalyst Na2MoO4 and NaOH in MeOH/H2O. Intermediate in presence of acid catalyst and triplet oxygen yielded 1 in 41% overall yield.45 Highest

overall yield of 61% has been reported by Zhang et al.,46 albeit at operating temperature of -78oC (Table 4). Table 4: Transformation of DHAA to artemisinin: a comparison with existing methods

S. No. Process 1

2

Reagent

Catalyst

Solvent

T (oC) Acid/Base

Yielda (%)

EtOC(O)Cl

TPP

DCM

-15

CF3CO2H

55%

Nil

DCA

toluene

20-25

CF3CO2H

46%

SOCl2/MeOH

dowex 0-25 50WX8-200 toluene resin petroleum ether 25 (bp 60-90oC)

Photochemical Conversion (Sanofi)44 Photochemical Conversion 27

3

4

(Seeberger et al.) Chemical Conversion 24

Li2MoO4, H2O2

(Amyris)

Chemical Conversion 45

(Wu et. al.)

Chemical Conversion (Zhang et. al.)46 With esterification

5

H2O2

TESCl, H2O2, CrCl

-

IPA

-78

CHCl3

- 40

H2O2, NaOCl, NaOH

-

Chemical Conversion

Me2SO4, H2O2

indion resin

DCM,

(Ipca, this work)

Na2MoO4

225H

MeOH

Without esterification

6

Na2MoO4,

a

0-25

Cu (II) triflate

45%

CF3CO2H

41%

PTSA

61%

Cu (II) triflate PhSO3H

60%

60%

Isolated pure product yield. EtOC(O)Cl: ethyl chloroformate, TPP: tetraphenylporphyrin, CF3CO2H: trifluoroacetic acid, DCA: 9, 10-dicyanoanthracene, Li2MoO4: lithium molybdate, SOCl2: thionyl chloride, MeOH: methanol, dowex 50WX8-200 resin: strong acid cation exchange resin with 8% cross-linking, TESCl: triethylsilyl chloride, CrCl: chromium chloride, PTSA: ptoluenesulfonic acid, IPA: isopropyl alcohol, NaOCl: sodium hypochlorite bleach, NaOH: sodium hydroxide CHCl3: chloroform, Me2SO4: dimethyl sulfate, Na2MoO4: sodium molybdate, indion 225H resin: strong acid cation exchange resin containing sulphonic acid groups, PhSO3H: benzenesulphonic acid


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We believe that it should be possible to get even higher yields and therefore we continue to work on developing a better understanding of the complete transformation and viable alternative to metal-mediated epoxide deoxygenation. To summarize, ACTs represent the prime therapy for malaria3 and the biggest concern in implementing this life saving treatment regimen worldwide has been its cost, due to which it remains unaffordable by the most vulnerable population.6,47 Since the proposed synthesis starts from 2 and only requires cheap and environmentally friendly reagents and conditions, this breakthrough strategy is anticipated to bring the cost of artemisinin to US$≤100/kg, placing malaria treatment within the reach of the masses and also reducing the number of deaths caused by malaria across the globe every year.48,49

DISCUSSION

The novel approach to produce AD and AA in genetically engineered microorganisms has provided a viable alternative to reduce dependency on Artemisia annua plant cultivation for the manufacture of ACTs. Although the conversion of AA to artemisinin via a mixed anhydride of DHAA using photochemical peroxidation as the key step was implemented at a commercial scale by Sanofi31, but it failed to deliver a cost effective malaria treatment, mainly due to high capital intensiveness of the process. Low economical viability of Sanofi’s semi-synthetic’ artemisinin fueled sale of its manufacturing plant based at Garessio to Bulgarian company Huvepharma.(50) A photochemical process which uses energy-intensive liquid CO2 as co-solvent with toluene/EtOH and an exotic porphyrin based photocatalyst (TPP-Amb) claims to provides an environmentally friendly solution29, but does not appear to help in improving the yield over existing methods, nor ultimately the cost of production. In one opinion, cost-effective production is of fundamental importance for an antimalarial drug rather than cost intensive green chemistry and is not addressed by this greener alternative.51 The continuous flow synthesis from Seeberger26-29 also uses a photochemical transformation of DHAA but without converting it to the active ester and claims to be a scalable alternative to Sanofi’s process, but it ACS Paragon Plus Environment


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does not appear to be cost effective as the reported yield of 1 from 8 is only 39% and it requires column chromatography to deliver pure product. The chemical synthesis of 1 from 8, evading conversion to active ester, with 30% H2O2, catalyst Na2MoO4 and NaOH/MeOH-H2O at ambient temperature resulted in overall yield limited to 41%.45

Given the fact that yeast is relatively more tolerant to AD than AA and the former can thus be produced in higher titers, AD represents a good starting material for a practical synthesis of the drug substance.30 Selective epoxidation to get exo- versus endo- isomers, the key to this approach, was found to be quite challenging in practice and the one with 9-BBN which gave reasonable selectivity, turns out to be a costly option due to the high cost contribution of this reagent and in part also due to the formation of the undesired diastereomer.52 The way to achieve this feat by inactivating the endocyclic double bond of AD by developing a highly regioselective epoxidation and the effective deprotection of epoxy group in the scheme using easily available inexpensive chemicals and leveraging the isopropenyl side chain functionalization allows the intended modifications at negligible cost. The purity of intermediates and stability of endo-epoxide during subsequent molecular modifications supported the endeavour very well. Chemical synthesis of artemisinin via proposed route offers both excellent quality as well as conversion efficiency, resulting in higher yield whilst remaining cost effective. This has the potential to make ACT combination therapies reachable to the masses and alleviate the suffering of the millions affected worldwide by malaria, a deadly disease.

EXPERIMENTAL SECTION The structure of the intermediates and end products was confirmed by proton (1H), carbon (13C) nuclear magnetic resonance (NMR) and mass was confirmed by mass spectroscopy. Purity of batch 1 and batch 2 of end product was assessed by HPLC. Proton magnetic resonance spectra were determined in chloroformd1 (CDCl3) unless otherwise stated. Splitting patterns are indicated as follows: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad peak.


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High-resolution mass spectrometry (HRMS) data was acquired using Thermo SCIENTIFIC Q Exactive Orbitrap LC-MS system.

Synthesis of 1-naphthaleneacetic acid, 1,2,3,4,4a,5,6,8a-octahydro-α,4,7-trimethyl-, (αR,1R,4R,4aS, 8aS) [DHAA- Compound 8] from naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4,7- dimethyl-1-(1methylethenyl)-,(1R,4R,4aS,8aR) [AD- Compound 2] AD (232.56 gm, 1.138 mole) was added to formic acid (13.4 gm, 0.291 mole) at 25oC and reaction mass was warmed up to 30oC. H2O2 (50% aqueous solution) (195.4 gm, 2.872 mole) was added drop wise to it over a span of 12 hr and reaction mass was maintained at 30-34oC for 25 hr. Upon completion of reaction, the reaction mixture was cooled to 5-10oC; followed by addition of water and adjustment of pH to 7-8 using 5% NaOH solution. Reaction mass was extracted with cyclohexane (837 mL). Trichloroisocyanuric acid (91.2 gm, 0.392 mole) was added slowly at 25-30oC over a span of 30 min to the cyclohexane layer containing 3 and the reaction was maintained under stirring for 6-7 hr. Upon completion, the reaction mass was cooled to 0 to -5oC and stirred for an hr followed by filtration and subsequent washing with cyclohexane (3 x 93 mL). Filtrate was then washed with water and brine solution. Solvent was distilled out under vacuum at 40-45oC. 4 thus obtained was degassed under vacuum at 40-45oC and to it added DMSO (4651 mL), K2HPO4 (451.2 gm, 2.59 mole) and KBr (2.33 gm) under stirring at 25-30oC. Temperature was then raised to 80-85oC and the reaction mass stirred at the said temperature for 40-45 hr. Upon completion of reaction, solvent was distilled out under vacuum at 80-85oC. Reaction mass was quenched with water (1395 mL) and extracted with toluene (698 mL). Organic layer was washed with water and then with brine solution. Solvent was distilled out under vacuum at 40-45oC and oily mass containing 5 was degassed for 2-3 hr. Reaction temperature was lowered to 30oC, followed by addition of isopropyl alcohol (1395 mL) and further cooling to 0-5oC. NaClO2 solution (116.3 gm, 1.286 mole NaClO2 + 930 mL water) and sulfamic acid solution (39.53 gm sulfamic acid, 0.407 mole + 465 mL water) were added simultaneously to the above obtained isopropyl alcohol solution of 5, whilst maintaining pH of 3.7, over a period of 3.5 hr at



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said temperature. Reaction mass was then maintained for 1 hr at 0-5oC. Upon completion of reaction, pH was raised to 9-10 with 25% NaOH solution and Isopropyl alcohol was distilled out under vacuum at 3035oC. Reaction mass thus obtained was extracted with toluene (3 x 233 mL). Aqueous layer was acidified with dil HCl till pH of 3.5 is attained, followed by extraction with ethyl acetate (698 mL). Organic layer was washed with water and brine solution. Solvent was distilled out under vacuum at 40-45oC. Crude mass was degassed at 55oC under vacuum for 2-3 hr to give crude 6 and then cooled to 26oC. Isopropyl alcohol (930 mL) was added to the reaction flask and the reaction temperature was raised to 30-32oC followed by simultaneous addition of hydrazine hydrate (72% in aqueous solution) (127.93 gm, 1.84 mole) and hydrogen peroxide (50% in aqueous solution) (109.3 gm, 1.607 mole), over a span of 3.5 hr. The reaction mass was maintained for 6-7 hr at said temperature. Upon completion of reaction, water (1860 mL) was added to the reaction mass. The pH of the reaction mass was adjusted to 3.0 with diluted HCl (16% solution in water) (349 mL) at 25-30oC. Reaction mass was then filtered and washed with water till pH turns to neutral. The light yellow solid product, 7, was vacuum dried at 40-45oC (214.88 gm, 74.82% yield). To the latter in 1, 2-dimethoxyethane (1302 mL) was added biphenyl (65.58 gm, 0.4253 mole) and lithium metal (35.82 gm, 5.1614 mole) under nitrogen atmosphere. The reaction mass was heated to 8085oC and maintained for 10 hr. Upon completion, the reaction mixture was cooled to 0-5oC and water was added drop wise spanning an hr; followed by stirring for two hr at 20-25oC. Toluene (651 mL) was added to it with continuous stirring for over 30 minutes. After settling down, the aqueous layer was separated and was washed with toluene (186 mL). The aqueous layer thus obtained was cooled to 10-15oC and pH of the reaction mixture was adjusted in between 3.5 to 4 with dilute HCl (16% solution in water). Aqueous layer was extracted with DCM (698 mL) and DCM layer was washed with water and brine solution. The solvent was removed by distillation out under vacuum at 40-45oC and the crude mass on drying at 45-50oC gave DHAA as light yellow solid (179.07 gm, 66.57% yield). The purity of DHAA as assessed by 1H NMR was found to be ≥90%. This light yellow solid DHAA was directly used in the next step for conversion to artemisinin.


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Synthesis of 3,12-epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one, octahydro-3,6,9-trimethyl, (3R,5aS,6R,8aS,9R,12S,12aR) [artemisinin- Compound 1] from 1-naphthaleneacetic acid, 1,2,3,4,4a,5,6,8a-octahydro-α,4,7-trimethyl-, (αR,1R,4R,4aS,8aS) [DHAA- Compound 8] DHAA (139.53 gm, 0.5959 mole) was added to a solution of NaOH (23.72 gm, 0.59 mole) in water (419 mL) at 20oC, followed by stirring for 30 minutes to obtain a clear solution. Tetra butyl ammonium bromide (1.39 gm, 1% w/w) and dichloromethane (419 mL) were added to it followed by addition of dimethyl sulfate (67.01 gm, 0.53 mole) slowly; drop wise, over a span of 1.5 hr. The mixture was then stirred for 5.0 hr at 20oC.The utilization of the starting material was monitored by HPLC. Upon completion of the reaction, the contents were allowed to settle down followed by separation of organic layer. The latter was washed with water (2 x 279 mL) and concentrated under reduced pressure to give DHAA methyl ester as a brown coloured oily mass. Methanol (698 mL) followed by sodium molybdate (28.88 gm, 0.14 mole) was added to it whilst maintaining the temperature of the reaction mass at 25oC. H2O2 (50% aqueous solution) (303.72 gm, 4.4645 mole) was then added drop wise to it over a span of 8 hr during which the temperature is maintained at 30oC. Reaction mixture was further stirred for 1 hr and the progress of the reaction was monitored by HPLC. Post completion, water (698 mL) and dichloromethane (698 mL) were added to the reaction mixture. After stirring for another 30 minutes, the contents were allowed to settle down resulting in separation of the organic layer. Aqueous phase was extracted with dichloromethane (140 mL) and the combined organic layer was washed with water (3 x 279 mL). The pooled organic phase containing hydroperoxide derivative of DHAA methyl ester (10) thus obtained was directly used for the next stage without isolation and purification. In another round bottom flask, benzenesulfonic acid (5.4 gm, 0.0341 mole) and suspension of indion resin (22.32 gm, 16% w/w) in dichloromethane (837 mL) was saturated with oxygen by passing O2 gas at 0°C and to this was added above obtained pooled organic layer containing hydroperoxide, drop wise over a span of 30-40 minutes. Oxygen gas continuously purged at 0°C for 5 hr and disappearance of hydroproxide was monitored by HPLC. Post disappearance of hydroperoxide, reaction temperature was raised to 15°C



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Page 18 of 25

with continued O2 gas purging for 2 hr. The mixture was stirred at 25-30°C for another 20 hr and filtered to remove the solids. The filtrate was washed with saturated sodium bicarbonate solution (279 mL) and the organic layer thus obtained was concentrated under reduced pressure to yield crude artemisinin. Methanol (558 mL) was added to the crude artemisinin and temperature was raised to 55oC and kept under stirring for 1.0 hr. The clear, solution thus obtained was cooled gradually to 0oC to -5oC and stirred at the said temperature for 2.0 hr to give white crystals which were collected by filtration and subjected to washing with methanol (70 mL) and drying under vacuum at 35-40oC to give artemisinin- batch 1 (85.67gm, 51.40% yield). Second batch of crystalline product (14.96 gm, 8.98% yield) was obtained by passing the mother liquor through a SiO2 plug and normal processing to augment the overall yield of artemisinin from DHAA to ~60%. Overall yield of artemisinin from AD approximate 40%. HPLC analysis of artemisinin- batch 1, AM2/AM2/16260 P2, revealed purity of 98.91% and that of batch 2, AM2/AM2/16260 Crop II, revealed purity of 98.76%. The Melting point of artemisinin batch 1, as AM2/AM2/16260 P2, was found to be 152.6 oC - 153.7 oC and that of batch 2, as AM2/AM2/16260 Crop II, was 153 oC - 154 oC.53 Specific Optical Rotation of artemisinin batch 1: [α]20D = +76.55 [10mg/mL in ethanol]. Specific Optical Rotation of artemisinin batch 2: [α]20D = +76.51 [10mg/mL in ethanol].53

Characterization of Intermediates and End Product Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4,7-dimethyl-1-(1-methylethenyl)-, (1R,4R,4aS,8aR) (Compound 2) 1

H NMR (400 MHz, CDCl3) δ 5.10 (s, 1H), 4.91-4.89 (s, 1H), 4.68 (s, 1H), 2.61-2.56 (m, 1H), 2.04-1.88

(m, 3H), 1.77 (s, 3H), 1.73-1.69 (m, 1H), 1.64 (s, 3H), 1.61-1.51 (m, 2H), 1.49-1.39 (m, 1H), 1.37-1.25 (m, 3H) 1.06-0.97 (m, 1H) and 0.93-0.91 (d, J = 6.36 Hz, 3H).

13

C-NMR (100 MHz, CDCl3) δ 147.9, 134.5,

120.9, 109.8, 47.7, 41.9, 37.6, 35.4, 27.9, 26.5, 26.1, 25.8, 23.6, 22.6 and 19.8, HRMS, heated electrospray ionization (HRMS (HESI)): m/z calculated for C15H24 +H [M+H]: 205.1958, found: 205.1952. Naphth[1,2-b]oxirene, decahydro-1a,4-dimethyl-7-(1-methylethenyl)-, (3aS,4R,7R,7aS) ( Compound 3) ACS Paragon Plus Environment

Page 19 of 25

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1


H NMR (400 MHz, CDCl3) δ 4.94-4.93 (m, 1H), 4.74 (s, 1H), 2.63 (s, 1H), 2.13-2.06 (m, 2H), 1.83-1.62

(m, 7H), 1.38-1.20 (m, 7H), 1.16-1.01 (m, 2H) and 0.89 (d, J = 6.36 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 147.1, 110.5, 59.4, 57.5, 46.5, 40.2, 38.7, 34.9, 29.7, 27.3, 24.9, 23.5, 22.7, 22.2 and 19.2, HRMS (HESI): m/z calculated for C15H24O +H [M+H]: 221.1900, found: 221.1899. Naphth[1,2-b]oxirene,

decahydro-1a,4-dimethyl-7-(1-chloromethylethenyl)-,(3aS,4R,7R,7aS)

(Compound 4) 1

H NMR (400 MHz, CDCl3) δ 5.34 (s, 1H), 5.04-5.03 (m, 1H), 4.29-4.11 (dd, J = 60.40, 11.74 Hz, 2H),

2.55-2.49 (m, 2H), 2.10-2.08 (m, 1H), 1.85-1.62 (m, 4H), 1.37-1.11 (m, 9H), and 0.91-0.89 (d, J = 6.36 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 146.7, 115.5, 59.2, 57.7, 47.8, 41.5, 39.9, 38.3, 34.7, 29.6, 26.9, 24.8, 23.5, 22.1 and 19.1, HRMS (HESI): calculated m/z for C15H23ClO +H [M+H]: 255.1510, found: 255.1515. Naphth[1,2-b]oxirene-7-acetaldehyde, decahydro-1a,4-dimethyl-α-methylene-, (3aS,4R,7R,7aS) (Compound 5) 1

H NMR (400 MHz, CDCl3) δ 9.57 (s, 1H), 6.27-6.27 (d, J = 1.22Hz, 1H), 6.19 (s, 1H), 2.80.2.75 (m, 1H),

2.47 (s, 1H), 2.10-2.08 (m, 1H), 1.83-1.78 (m, 2H), 1.67-1.59 (m, 2H), 1.46-1.25 (m, 8H), 1.18-1.08 (m, 1H), 0.91-0.90 (d, J = 5.87 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 194.4, 151.7, 134.7, 59.4, 57.6, 39.8, 38.6, 38.4, 34.7, 29.5, 26.5, 24.7, 23.5, 21.9 and 19.1, HRMS (HESI): calculated m/z for C15H22O2 +H [M+H]: 235.1693, found: 235.1694. Naphth[1,2-b]oxirene-7-acetic

acid,

decahydro-1a,4-dimethyl-α-methylene-,

(3aS,4R,7R,7aS)

(Compound 6) 1

H NMR (400 MHz, CDCl3) δ 7.08 (br, 1H), 6.50 (s, 1H), 5.65 (s, 1H), 2.79-2.74 (m, 1H), 2.57 (s, 1H),

221-2.19 (m, 1H), 1.84-1.77 (m, 2H), 1.69-1.61 (m, 3H), 1.40-1.04 (m, 8H), 0.91-0.89 (d, J = 5.87 Hz, 3H).

13

C-NMR (100 MHz, CDCl3) δ 171.5, 142.0, 126.9, 59.3, 57.9, 41.2, 39.9, 38.7, 34.8, 29.4, 27.2,

24.8, 23.5, 22.0 and 19.1, HRMS (HESI): calculated m/z for C15H22O3 -H [M-H]: 249.1496, found: 249.1504. Naphth[1,2-b]oxirene-7-acetic acid, decahydro-α,1α,4-trimethyl-, (αR,3aS,4R,7R,7aS) (Compound 7) 1

H NMR (400 MHz, CDCl3) δ 8.40 (br, 1H), 2.66 (s, 1H), 2.62-2.53 (m, 1H), 2.04-2.02 (m, 1H), 1.86-1.61

(m, 5H), 1.35-1.18 (m, 9H), 1.11-0.98 (m, 3H), 0.86-0.85 (d, J = 6.36 Hz, 3H).

13

C-NMR (100 MHz,

CDCl3) δ 182.6, 58.3, 57.8, 42.7, 42.2, 40.2, 38.1, 34.8, 29.5, 28.6, 24.8, 23.5, 22.2, 19.0 and 15.4, HRMS (HESI): m/z calculated for C15H24O3 -H [M-H]: 251.1653, found: 251.1657.



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1-Naphthaleneacetic

acid,

1,2,3,4,4a,5,6,8a-octahydro-α,4,7-trimethyl-,

Page 20 of 25

(αR,1R,4R,4aS,8aS)

(Compound 8) 1

H NMR (400 MHz, CDCl3) δ 10.89 (br, 1H), 5.13 (s, 1H), 2.55-2.49 (m, 2H), 1.99-1.83 (m, 3H), 1.69-

1.52 (m, 6H), 1.46-1.41 (m, 2H), 1.30-1.28 (m, 1H), 1.22-1.20 (d, J = 6.85 Hz, 3H), 1.18-1.08 (m, 1H), 1.03-0.93 (m, 1H) and 0.89-0.88 (d, J = 6.36 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 183.5, 136.0, 119.3, 43.6, 42.2, 41.7, 36.3, 35.2, 27.7, 27.4, 26.6, 25.8, 23.8, 19.7 and 15.1, HRMS (HESI): m/z calculated for C15H24O2 +H [M+H]: 237.1849, found: 237.1849. 1-Naphthaleneacetic acid, 1,2,3,4,4a,5,6,8a-octahydro-α,4,7-trimethyl-, methyl ester, (αR,1R,4R,4aS, 8aS) (Compound 9) 1

H NMR (400 MHz, CDCl3) δ 5.10 (s, 1H), 3.64 (s, 3H), 2.51-2.43 (m, 2H), 1.94-1.74 (m, 3H), 1.63-1.50

(m, 6H), 1.44-1.35 (m, 1H), 1.25-1.20 (m, 2H), 1.12-1.10 (d, J = 6.85 Hz), 1.11-1.02 (m, 1H), 0.99-0.92 (m, 1H), 0.85-0.83 (d, d, J = 6.85 Hz).

13

C-NMR (100 MHz, CDCl3) δ 177.8, 135.8, 119.4, 51.3, 43.9,

42.1, 41.7, 36.4, 35.2, 27.6, 27.4, 26.6, 25.7, 23.8, 19.7 and 15.1, HRMS (HESI): m/z calculated for C16H26O2 +H [M+H]: 251.2006, found: 251.2017. 3,12-Epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one, octahydro-3,6,9-trimethyl-, (3R,5aS, 6R,8aS,9R,12S,12aR) (Compound 1) Artemisinin batch -1(86.57 gm) as AM2/AM2/16260 P2. 1

H NMR (400 MHz, CDCl3) δ 5.85 (s, 1H), 3.41-3.34 (m, 1H), 2.46-2.38 (m, 1H), 2.07-1.96 (m, 2H), 1.91-

1.85 (m, 1H), 1.80-1.73 (m, 2H), 1.50-1.36 (m, 6H), 1.20-1.18 (d, J = 7.34 Hz, 3H), 1.10-1.03 (m, 2H), 1.00-0.98 (d, J = 6.11Hz, 3H).

13

C-NMR (100 MHz, CDCl3) δ 172.1, 105.4, 93.7, 79.5, 50.0, 44.9, 37.5,

35.9, 33.6, 32.9, 25.2, 24.8, 23.4, 19.8 and 12.6, HRMS (HESI): m/z calculated for C15H22O5 +H [M+H]: 283.1540, found: 283.1550.

3,12-Epoxy-12H-pyrano[4,3-j]-1,2-benzodioxepin-10(3H)-one, octahydro-3,6,9-trimethyl-, (3R,5aS, 6R,8aS,9R,12S,12aR) (Compound 01) Artemisinin batch -2 (14.96 gm) as AM2/AM2/16260 Crop II. 1

H NMR (400 MHz, CDCl3) δ 5.78 (s, 1H), 3.31-3.24 (m, 1H), 2.36-2.29 (m, 1H), 1.99-1.90 (m, 2H), 1.80-

1.78 (m, 1H), 1.70-1.67 (m, 2H), 1.39-1.28 (m, 6H), 1.12-1.10 (d, J = 7.34 Hz, 3H), 1.01-1.00 (m, 2H), 0.92-0.90 (d, J = 6.11 Hz, 3H). 13C-NMR (100 MHz, CDCl3) δ 172.0, 105.3, 93.6, 79.5, 49.9, 44.8, 37.4, 35.8, 33.5, 32.8, 25.1, 24.8, 23.3, 19.8 and 12.5, HRMS (HESI): m/z calculated for C15H22O5 +H [M+H]: 283.1540, found: 283.1538.


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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. HPLC data, 1H NMR and 13C NMR spectra and Mass Spectra of both batches of terminal product, compound 1 (PDF)

1

H NMR and 13C NMR spectra and Mass Spectra of compound 2 to compound 9 (PDF)

1

H NMR and 13C NMR spectra of compound 10 (PDF)

AUTHOR INFORMATION

Corresponding Authors *E-mail: [email protected]; [email protected] ORCID Ashok Kumar: 0000-0001-9075-8816 Satinder Singh: 0000-0002-5889-0654 Notes All Amyris Inc. authors possess shares or stock options in Amyris and thus have a financial interest. Ashok Kumar and Dharmendra Singh possess shares or stock options in Ipca and thus have a financial interest.



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Amalgamation of Synthetic Biology and Chemistry ... - ACS Publications

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